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56 th EASD Annual Meeting of the European Association for the Study of Diabetes

21-25 September 2020

Abstracts

Index of Oral Presentations

OP 01 Diabetes complications: new insights from cutting edge epidemiology

OP 02 News on the insulin secretion front

OP 03 Insulin sensitivity and biomarkers

OP 04 Central actions in diabetes

OP 05 Glucose-lowering therapies and the liver

OP 06 Uncomplicating the pathogenesis of diabetes complications in humans

OP 07 Smoke on the water: Is BAT still hot?

OP 08 Charting human beta cell failure in type 1 diabetes

OP 09 Novel agents in type 1 diabetes

OP 10 Developing better insulins

OP 11 From diagnostics to the end-stage of diabetic kidney disease

OP 12 NAFLD: Is it all about the liver?

OP 13 Diabetic retinopathy: see what's new?

OP 14 Taking the long view of diabetes

OP 15 Pregnancy in diabetes prediction and outcomes

OP 16 Signals and networks in beta cell failure

OP 17 Broken heart in diabetes

OP 18 Unlocking the potential of digital health

OP 19 Decoding the heritable basis of type 2 diabetes

OP 20 Feeding the pipeline: from drugs to surgery

OP 21 SGLT-2 inhibitors: at the heart of the matter

OP 22 New Treatments for NAFLD: Hope or Hype?

OP 23 Addressing potential new treatments of diabetic kidney disease

OP 24 Glucagon and hormones beyond

OP 25 Incretin based therapies

OP 26 Unusual forms of diabetes

OP 27 Macrovascular complications and beyond

OP 28 Linking inflammation to metabolism

OP 29 What's new in automated insulin delivery

OP 30 Understanding the mechanisms of diabetic kidney disease

OP 31 Novel aspects of diabetic neuropathy

OP 32 Reducing the burden of hypoglycaemia

OP 33 What exercise does

OP 34 Back to the future: risk markers in diabetes

OP 35 Diet: not only quantity matters

OP 36 On the road to human islet failure in type 2 diabetes

OP 37 A deep dive into the mechanisms of diabetes

OP 38 Triggers and drivers of beta cell failure in type 1 diabetes

OP 39 Gastro-entero pancreatic factors: organoids, mice and men

OP 40 New aspects of novel therapies

OP 41 Fatty matters

OP 42 Diabetes care is expensive

OP 43 Developing beta cells

OP 44 Modelling metabolism: lessons from animals

OP 45 Diabetic foot: new developments in wound healing

OP 46 Challenges in delivering diabetes care: new solutions

OP 47 Thinking about diabetes complications in the brain

OP 48 Insulin secretion in various subgroups

Index of Poster Sessions

PS 01 Diabetes and early death

PS 02 Living with chronic diabetes complications

PS 03 Micro- and macrovascular complications of diabetes

PS 04 Global view on diabetes complications

PS 05 Type 2 diabetes treatment IRL

PS 06 Unusual forms of diabetes

PS 07 Molecular insights into glucose abnormalities

PS 08 Pathophysiology of glucose homeostasis

PS 09 The inner workings of the pancreas

PS 10 Islets and antibodies in type 1 diabetes

PS 11 Markers and phenotypes of glucose traits

PS 12 Global aspects on the epidemiology of type 2 diabetes

PS 13 Risk factors for type 2 diabetes

PS 14 Prevalence of type 2 diabetes around the world

PS 15 Risk factors in type 1 diabetes

PS 16 Islet transplants revisited

PS 17 Islets in type 1 diabetes: new players

PS 18 Beta cells under stress

PS 19 To live and let die: a beta cell perspective

PS 20 Job description: insulin secretion

PS 21 Further down the road to human islet failure in type 2 diabetes

PS 22 Sitting and exercising does it all

PS 23 The ins and outs of carbohydrate metabolism

PS 24 Pregnancy: in vitro and in vivo studies

PS 25 Pregnancy: Epidemiology

PS 26 Pregnancy: Who is at risk?

PS 27 Incremental studies on gut hormones

PS 28 The fundamentals of insulin resistance

PS 29 Studies on insulin resistance

PS 30 Treatment of hyperglycaemia in pregnancy

PS 31 Pancreatic hormones

PS 32 Insulin secretion in mice and men

PS 33 Something more about obesity

PS 34 More about metabolism

PS 35 Inflammation in type 2 diabetes

PS 36 Models of prediabetes and diabetes

PS 37 Models of obesity and insulin resistance

PS 38 Lipid metabolism

PS 39 Adipokine signalling

PS 40 Drugs and environment in obesity

PS 41 Weight loss interventions

PS 42 Brain matters

PS 43 SGLT-2 inhibitors: clinical aspects

PS 44 Different aspects of SGLT-2 inhibitors

PS 45 Basic aspects of incretin-based therapies

PS 46 Clinical outcome of incretin-based therapies

PS 47 Glycaemic control and incretin-based therapies

PS 48 Various clinical aspects of incretin-based therapies

PS 49 Various aspects of nutrition and diet

PS 50 Oral therapies: metformin, sensitizers and other non-secretagogues

PS 51 Novel agents to treat diabetes and its consequences

PS 52 Novel glucose-lowering agents in type 2 diabetes

PS 53 Key issues in improving outcomes in people with diabetes, education and costs

PS 54 How to improve diabetes care

PS 55 The impact of new basal insulins

PS 56 Insulin therapy: real world studies

PS 57 Insulin therapy: fast acting insulin analogues

PS 58 The challenges of insulin therapy in type 2 diabetes

PS 59 Different aspects of insulin therapy

PS 60 The continued advance of continuous glucose monitoring

PS 61 Insulin pump therapy

PS 62 Automated insulin delivery

PS 63 The varied use of technologies in type 2 diabetes

PS 64 Novel applications of technology in diabetes

PS 65 Novel therapies to reduce hypoglycaemia

PS 66 Mechanisms and clinical consequences of hypoglycaemia in diabetes

PS 67 Emerging topics in hypoglycaemia

PS 68 Investigating diabetes distress and depression

PS 69 Aspects of quality of life and well being

PS 70 Digital health in type 2 diabetes

PS 71 Is telehealth the answer to improving care in diabetes?

PS 72 Predicting prognosis of diabetic kidney disease

PS 73 Clinical aspects of diabetic kidney disease

PS 74 The ROCK and role of experimental kidney disease

PS 75 New tools to view diabetic retinopathy

PS 76 Diabetic retinopathy: screening and intervention

PS 77 Focus on diabetic foot ulcers

PS 78 Hypertension and vascular disease

PS 79 Cure the pain of diabetic neuropathy

PS 80 Understanding clinical neuropathy

PS 81 From artificial intelligence to treatment of diabetic foot

PS 82 From biomarkers to genetics of diabetic kidney disease

PS 83 Treatment of NAFLD and diabetes: from food to pharmacology

PS 84 Mechanisms and prevalence of NAFLD

PS 85 Lipids everywhere: lipid metabolism in the liver and the heart

PS 86 All about coronary arteries and diabetes

PS 87 Lipids and glucose: not so good for the heart

PS 88 Cardiac complications: of mice, rats and cells

PS 89 Atherosclerotic complications: stemming from cells to the kidney

PS 90 Stiff arteries and how to avoid them

PS 91 Cardiac function and dysfunction

PS 92 Cardiovascular complications in humans through and through

PS 93 Diabetes and neoplasia

PS 94 Contemplating cognitive dysfunction in diabetes

PS 95 Endothelial cell, circulation and the heart

PS 96 Tradition? No! Non-traditional complications of diabetes

OP 01 Diabetes complications: new insights from cutting edge epidemiology

1

Circulating metabolites significantly improve the prediction of renal dysfunction in type 2 diabetes

M. Scarale1, S. De Cosmo1, C. Prehn2, F. Schena3, J. Adamski2, V. Trischitta4, C. Menzaghi1;

1Fondazione IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy, 2Helmholtz Zentrum, München, Germany, 3University of Bari, Bari, Italy, 4Sapienza University, Roma, Italy.

Background and aims: Chronic kidney disease (CKD), mainly indicated by a reduced glomerular filtration rate (GFR) remains one of the leading causes of reduced lifespan in patients with type 2 diabetes (T2D). Discovering novel biomarkers able to predict low GFR will help identify high-risk patients to be targeted to more aggressive and burdensome preventive and treatment strategies.

Materials and methods: We measured 181 serum metabolites by AbsoluteIDQTM p180 Kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) and investigated their association with eGFR (calculated with the CKD-EPI formula) in a discovery sample of 325 patients with T2D (116 cases and 209 controls with eGFR<60 and ≥70ml/min/1.73m2, respectively). A threshold p value of 2.8x10-4 (i.e. 0.05/181 following Bonferroni's rule) was used as statistical significance in a model comprising age, sex, smoking, BMI, HBA1c, diabetes duration, albumin-to-creatinine ratio (ACR) and ongoing treatments. Metabolites associated in the discovery sample were validated (threshold p value of 0.05/number of surviving validation metabolites) in a second cohort comprising 465 diabetic patients (166 cases and 299 controls for eGFR<60 or ≥70ml/min/1.73m2, respectively). Standardized values of each validated metabolites, weighted for the effect size (i.e. β) observed in the discovery sample, were then summed up in a metabolic score (MetScore) to be used as a GFR prediction tool. To this purpose, MetScore was used on top of an established clinical model (comprising sex, age, BMI, HbA1c and ACR) and then discrimination [∆ area under the Receiver operating characteristic (ROC) curve (AUC) and the relative integrated discrimination improvement (rIDI)] and reclassification [the category-free net reclassification index (cNRI)] measures were evaluated.

Results: Thirteen metabolites (six acylcarnitines, six biogenic amines and one amino acid) were independently associated to low eGFR [ORs range 2.2-5.1 for 1SD increase; p range 1.3x10-7 - 2.0x10-4] in the discovery sample. All of them but one (a biogenic amine) were validated in the replication sample [ORs range 1.7-3.6 for 1SD increase; p range 3.2x10-18 - 4.3x10-6, below the threshold of 0.05/12=4.2x10-3]. The AUC of the above-mentioned clinical model was 92.7%, 81.8% and 86.6% in the discovery, the replication and the pooled sample, respectively. The addition of MetScore on top of the clinical model improved both discrimination and reclassification measures in the discovery (Δ AUC=4%, p=1.4x10-3; rIDI=29%, p=2.0x10-11; ½cNRI=54%, p=1.5x10-14), the replication (Δ AUC=3.9%, p=1.6x10-3; rIDI=28%, p=3.8x10-8; ½cNRI=30%, p=2.2x10-10) and the pooled samples (Δ AUC=3.9%, p=4.0x10-6 ; rIDI=29%, p=2.2x10-17; ½cNRI=35%, p=1.9x10-8).

Conclusion: We have discovered and validated 12 metabolites that are strongly associated with low eGFR in patients with T2D. A MetScore comprising these 12 metabolites improves an established clinical prediction model of low eGFR in terms of both discrimination and reclassification. Encouraged by these findings we are now investigating the ability of MetScore to improve prediction of GFR decline in prospective cohorts of T2D, with the aim of improving risk stratification and, therefore, refining prevention efforts of kidney dysfunction in diabetic patients.

Supported by: Italian Ministry of Health RF-2013-02356459

Disclosure: M. Scarale: None.

2

Association between insulin-like growth factor binding protein-2 and insulin sensitivity, metformin and mortality in patients with newly diagnosed type 2 diabetes

M.R. Kristiansen1,2, J.S. Nielsen1,2, I. Brandslund3, D.A. Olsen3, J.V. Stidsen2, S.K. Nicolaisen4, R. Hjortebjerg2,5, J. Frystyk5,6;

1Danish Centre for Strategic Research in Type 2 Diabetes (DD2), Odense, 2Steno Diabetes Center Odense, Odense, 3IRS, Lillebaelt Hospital, Biochemistry and Immunology, Vejle, 4Department of Clinical Epidemiology, Aarhus, 5Department of Clinical Research, University of Southern Denmark, Odense, 6Department of Endocrinology, Odense University Hospital, Odense, Denmark.

Background and aims: Insulin-like growth factor binding protein-2 (IGFBP-2) is engaged in metabolism. Circulating concentrations of IGFBP-2 are positively correlated to insulin sensitivity. Overexpression of IGFBP-2 protects against obesity and diabetes in mice, and metformin increases IGFBP-2 gene expression, indicating that IGFBP-2 is a target of metformin action. Interestingly, IGFBP-2 appears to predict mortality independently of insulin sensitivity. This study aimed to investigate the association between indices of insulin sensitivity, metformin treatment and mortality in patients with newly diagnosed type 2 diabetes (T2D).

Materials and methods: In this cross-sectional study, we included newly diagnosed patients with T2D enrolled in the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) cohort. Patients were continuously enrolled from 2010 to 2018 throughout Denmark and followed using Danish healthcare registries. Unbound fractions of IGFBP-2 were determined in serum from fasting drug naïve (n=864) and metformin treated (≥ two prescriptions 6 months prior enrollment) patients (n=558) using an in-house assay developed on the Simoa platform. Values are given as medians (IQR). Association was analyzed using a Pearson’s regression/Cox regression. A multivariable model was used to adjust for age, BMI, and HOMA-S.

Results: A total of 1422 patients with median age of 64 (56;71), median BMI of 30 (27;34) and median diabetes duration of 0.9 (0.0;2.3) years were included. IGFBP-2 level was positively correlated with HOMA-S (R2=0.26 and p<0.005) and inversely correlated with c-peptide (R2=-0.18 and p<0.005). Both associations persisted following adjustments for age and BMI. The IGFBP-2 level in metformin treated patients was slightly lower (245 (174;406) ng/mL) than in drug naïve patients (274 (188;450) ng/mL) (p=0.026). A total of 460 patients suffered from one or more comorbidities from Charlson comorbidity index. Their IGFBP-2 levels were higher than patients with no comorbidity (321 (204;497) vs. 246 (173;394) ng/mL, p<0.001). During a median of 4.9 (3.9-5.9) years of follow-up, a total of 87 (6.12%) patients died. IGFBP-2 level was significantly higher at baseline in patients that died vs. not died (458 (259;665) vs. 254 (178;415) ng/mL, p<0.001). IGFBP-2 was associated with mortality with a hazard ratio(HR) (95% CI) per doubling in protein concentration of 2.0 (1.5;2.7), p<0.001. This association was not observed when analyzing patients without comorbidities but was significant in patients with other comorbidities (HR: 2.3 (1.7;3.3), p<0.001).

Conclusion: This is the first larger study to confirm that IGFBP-2 is associated with indices of insulin sensitivity but is not largely affected by metformin treatment. Interestingly, increased IGFBP-2 level is associated with high mortality rates, but the association was mainly driven by the presence of comorbidities at baseline.

Supported by: University of Southern Denmark and Region of Southern Denmark

Disclosure: M.R. Kristiansen: None.

3

Building clinical risk score systems for predicting all-cause and cardiovascular-specific mortality among type 2 diabetes patients

C.-S. Liu1, T.-C. Li2, C.-C. Lin1, C.-I. Li1;

1China Medical University Hospital, Taichung, 2China Medical University, Taichung, Taiwan.

Background and aims: No prior prediction model for mortality considered the effect of glycemic variability and blood pressure variability which have been broadly reported as the important clinical predictors of mortality, especially in diabetes patients. The aim of this study was to develop and validate risk score systems with considering the effects of glycemic and blood pressure variability on all-cause and cardiovascular-specific mortality in persons with type 2 DM.

Materials and methods: This is a retrospective cohort study consisting of 10,800 type 2 diabetic patients aged 30-85 years during 2003-2014. All participants were randomly allocated into two groups, derivation and validation sets in 2:1 ratio and were followed up until death, or August 2019. Cox proportional hazards regression were used to develop all-cause and cardiovascular-specific mortality prediction model. Prediction model performance was assessed by the area under the receiver operating characteristics curve (AUROC).

Results: Overall, 2,528 deaths were identified after a mean of 8.6 years of follow-up. The prediction accuracy, measured by AUROC, of 3-, 5-, 10- and 15-year all-cause mortality based on a model containing the identified traditional risk factor, biomarkers, and variability in fasting plasma glucose and HbA1c, and diastolic blood pressure variability were 0.79 (0.77-0.81), 0.79 (0.77-0.80), 0.81 (0.79-0.82) and 0.81 (0.80-0.82), respectively, in derivation set; and the corresponding values for cardiovascular-specific mortality were 0.86 (0.83-0.89), 0.83 (0.81-0.86), 0.82 (0.81-0.84) and 0.81 (0.80-0.83), respectively. The prediction accuracy in the validation set for all-cause mortality were 0.82 (0.78-0.85), 0.81 (0.79-0.83), 0.81 (0.80-0.83) and 0.81 (0.79-0.82), respectively, and for cardiovascular-specific mortality were 0.85 (0.80-0.89), 0.83 (0.79-0.86), 0.82 (0.80-0.85) and 0.81 (0.79-0.83), respectively.

Conclusion: Our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascular-specific and all-cause mortality in patients with type 2 diabetes.

Supported by: Ministry of Science and Technology of Taiwan

Disclosure: C. Liu: None.

4

Incident cardiovascular disease by clustering of favourable risk factors in type 1 diabetes. The EURODIAB Prospective Complications study

S. Soulimane1, Y.D. Vogtschmidt1,2, M. Toeller3, B. Balkau4, N. Chaturvedi5, J.H. Fuller6, S.S. Soedamah-Muthu1,2;

1Department of Medical and Clinical Psychology, Center of Research on Psychological and Somatic disorders (CoRPS), Tilburg University, Netherlands, 2Institute for Food, Nutrition and Health, University of Reading, Reading, UK, 3Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany, 4Clinical Epidemiology, Université Paris-Saclay, UVSQ, Inserm, CESP, Villejuif, France, 5Institute of Cardiovascular Science University College of London, London, UK, 6Department of Epidemiology and Public Health, EURODIAB, London, UK.

Background and aims: The incidence of cardiovascular diseases (CVD) is up to eight times higher in people with type 1 diabetes (T1D). Greater clustering of adverse risk factors is thought to contribute to excess CVD risks in type 2 diabetes, though not explored in T1D. The aim of this study was to examine a) CVD risk reduction for those in the most favourable third of individual risk factors compared to the least favourable two thirds, and b) CVD risk reduction by clustering of favourable CVD risk factors.

Materials and methods: We analysed data of 2086 participants from the EURODIAB Prospective Complications study, a European T1D cohort, recruited in 16 countries, between 1989-91; 51% were men, with a mean age of 32±10 years. We studied seven CVD risk factors, namely HbA1c, smoking, BMI, combined systolic and diastolic BP, LDL cholesterol, physical activity (PA) and diet (Table). Cox Proportional Hazards analyses were used to calculate hazard ratios (HR [95%CI]) of incident CVD, for each CVD risk factor (adjusted for age, sex, retinopathy), comparing those in the most favourable tertiles with the least favourable two tertiles. We then scored each individual by the number of risk factors for which they occupied the most favourable tertiles.

Results: There were 147 incident CVD cases, after a mean follow-up of 7.2±1.3 years. Multivariable Cox models showed that participants with the most favourable HbA1c<5.7% [39mmol/mol] had a 54% significantly lower CVD risk HR [95%CI]: 0.46[0.28,0.77] than the least favourable two tertiles; non-significant inverse associations were found with favourable BMI: 0.92[0.60,1.43], PA: 0.77[0.52,1.16], diet score: 0.68[0.34,1.36] and BP: 0.80[0.46,1.39]. No associations were found with smoking or LDL-cholesterol. Greater clustering of favourable CVD risk factors was associated with a lower risk of CVD in univariate models, with a significant linear trend. In multivariate models, the results were partly attenuated, with the lowest HR of 0.52[0.29, 0.94] in people with clustering of 3 favourable CVD risk factors (Table).

Conclusion: Greater clustering of favourable CVD risk factors was associated with a lower risk of incident CVD in people with T1D, with a dose-response relationship. HbA1c remained the most protective factor against CVD in T1D. Targeting combined risk factors could be more effective in preventing CVD risk than targeting single risk factors.

figurea

Supported by: Welcome Trust, the European Community and Diabetes UK

Disclosure: S. Soulimane: None.

5

Bidirectional association between type 2 diabetes and obstructive sleep apnoea: a meta-epidemiological study

T. Karagiannis1, E. Athanasiadou1, A. Tsapas1,2, E. Bekiari1;

1Clinical Research and Evidence-Based Medicine Unit, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2Harris Manchester College, University of Oxford, Oxford, UK.

Background and aims: Individual epidemiological studies suggest a complex relationship between type 2 diabetes and obstructive sleep apnea. We aimed to assess whether there is a bidirectional association between the two conditions by conducting a meta-analysis of longitudinal cohort studies.

Materials and methods: We included cohort studies that evaluated the association between type 2 diabetes and obstructive sleep apnea in either direction, published until January 2020. We pooled cohort-specific estimates by means of random and fixed effects meta-analyses and calculated odds ratios (ORs) with 95% confidence intervals (CIs), to measure the association of prevalent obstructive sleep apnea with incident type 2 diabetes and of prevalent type 2 diabetes with incident obstructive sleep apnea.

Results: Out of 1928 records identified through the search, 15 cohort studies were included in the meta-epidemiological analysis. Ten studies evaluated the association between prevalent obstructive sleep apnea and incident type 2 diabetes, one study assessed the association between prevalent type 2 diabetes and incident obstructive sleep apnea, while four studies evaluated a bidirectional association. Duration of study follow-up ranged between 2.7 and 22 years (median = 8 years). The random effects meta-analysis for prevalent obstructive sleep apnea and incident type 2 diabetes (335,056 patients) yielded an OR of 2.47 (95% CI, 2.05 to 2.97). Results were consistent in the fixed effects meta-analysis (Figure). Prevalent type 2 diabetes increased the odds of incident obstructive sleep apnea (409,707 patients), with an OR of 1.70 (95% CI, 1.15 to 2.53) and 1.85 (95% CI 1.76 to 1.95) for the random-effects and fixed-effects meta-analysis respectively. Meta-analyses of effect estimates adjusted for confounding factors were similar to those of the main analysis.

Conclusion: Pooled evidence from large cohort studies suggests that presence of obstructive sleep apnea at baseline is associated with increased risk for developing type 2 diabetes, while presence of type 2 diabetes is associated with increased risk for developing obstructive sleep apnea. Thus, effective management of either condition could prevent development of the other.

Figure. Odds ratio for developing type 2 diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apnea

figureb

Supported by: Greece and the European Social Fund (ESF)

Disclosure: T. Karagiannis: None.

6

Glycated haemoglobin, type 2 diabetes and the links to dementia and its major sub types: findings from the Swedish National Diabetes Register

C. Celis-Morales1, S. Franzén2, A.-M. Svensson3, N. Sattar1, S. Gudbjornsdottir2;

1Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK, 2Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden, 3Swedish National Diabetes Register, Gothenburg, Sweden.

Background and aims: Type 2 diabetes (T2D) has been associated with high dementia risk. However, the links to different dementia sub-types is unclear. We examined to what extent T2D associated with Alzheimer, vascular and non- vascular dementia incidence and whether such associations differed by glycaemic control.

Materials and methods: In this Swedish National Diabetes Register study, we included 378,299 patients with T2D and 1,886,022 matched controls. The outcomes were incidence of Alzheimer, vascular and non- vascular dementia. The association of T2D with dementia was stratified by baseline Glycated Haemoglobin (HbA1c) concentrations. Cox regression was used to study the excess risk of outcomes.

Results: The follow-up (median 6.8 years) 21,651 (5.7%) T2D patients and 98,723 (5.2%) controls developed dementia. The strongest association was observed for vascular dementia: here, patients with T2D had a HR of 1.36 [95% CI 1.03, 1.09] compared to controls. The association of T2D with non-vascular dementia was more modest (HR: 1.08 [95% CI 1.04, 1.12]). However, risk of Alzheimer was lower in T2D patients compared to controls (HR: 0.92 [95% CI 0.87, 0.98]). When the analyses were stratified by circulating concentrations of HbA1c a dose-response association was observed. Compare to patients with T2D with HbA1c <52 mmol/mol, those with HbA1c >87 mmol/mol had a higher risk of Alzheimer (HR: 1.34 [95% IC 1.03, 1.75]), vascular dementia (HR: 1.93 [95% IC 1.53, 2.42]) and non-vascular dementia (HR: 1.67 [95% IC 1.45, 1.91]). When a 3-years landmark analysis was conducted, the associations remained similar for vascular and non-vascular dementia but disappeared for Alzheimer’s diseases.

Conclusion: The association of T2D with neurodegenerative diseases differ by type of dementia. The strongest detrimental association was observed for vascular dementia. Moreover, T2D patients with poor glycaemic control have an increased risk of developing vascular and non-vascular dementia.

Disclosure: C. Celis-Morales: None.

OP 02 News on the insulin secretion front

7

What makes beta cells 1st responders, and are they temporally consistent?

V. Kravets, W.E. Schleicher, J.M. Dwulet, A.M. Davis, R.K.P. Benninger;

Bioengineering, University of Colorado, Aurora, USA.

Background and aims: Calcium (Ca2+) uptake drives glucose-stimulated insulin secretion from the pancreatic β-cells. Functional subpopulations of β-cells disproportionally control the oscillatory phase of Ca2+ uptake, which is disrupted with ageing and in diabetes. Less is known about β-cells which impact the 1st phase of Ca2+ uptake, disrupted in early diabetes. Here we determine whether “1st-responder” cells that lead the 1st phase of Ca2+ uptake are the same as “hub” cells that coordinate oscillatory Ca2+ (2nd) phase. We study what makes β-cell a 1st responder, and whether 1st responders are a transient state or a distinct temporally stable subpopulation.

Materials and methods: We used MIP-CreER GCaMP6s mouse model which expresses Ca2+-sensitive GFP specifically in β-cells. We performed simultaneous recording of Ca2+ dynamics and gap junction permeability in individual islets. We stimulated islets with glucose, Katp channel blocker glibenclamide, and KCl. Based on Ca2+ dynamics we defined the 10% of cells responding to the glucose stimulation sooner than the rest of the islet as “1st responders”, and the 10% of cells responding slower as “last responders”. We tested their temporal consistency over 1, 24, and 48 hours. We used laser ablation to remove specific cells from the islet. We performed computational modelling of the islet electrophysiology.

Results: We found that Ca2+ wave coordination of the 1st responders was not greater than the islet-average, and hence they are not overlapping with highly-coordinated “hub” cells. In fact, according to our gap junction permeability data, 1st responders had lower than average electrical coupling (p=0.0157). Furthermore, our computational model showed lower electrical coupling conductance in both 1st and last responders (p=0.0447, p=0.0279). This may be explained by our finding that 1st responders are located at the islet’s periphery (at 0.8 ± 0.1 of the islet’s radius). We found 1st responders to be consistent under glibenclamide stimulation: cells which respond first to the glucose remained in the 15th percentile of the time response distribution when stimulated with glibenclamide (SEM 10%). This is consistent with our computational results: 1st responders had lower Katp conductance (hence higher membrane depolarization probability) (p=0.0086). Glucose elevations with 1h period showed that 1st responders remained consistent: with reaction time within the 25% of the reaction time distribution for all cells. With an elevation period of 24 hours, their reaction time shifted to the second quartile of the distribution, and with 48 hours to the median. Unlike 1st responders, last responder cells were not consistent at any time interval. Ablation of the 1st responders dis-coordinated, but did not disrupt, the Ca2+ response of the islet. A different cell took over the role of the 1st responder post-ablation. This new 1st responder was a cell which originally, pre-ablation, was within a leading 7th percentile of the time response distribution (SEM 3%).

Conclusion: In conclusion, 1st responders are distinct from “hub” cell subpopulation, have higher membrane depolarization probability and are less strongly coupled to other cells. After the laser ablation of a 1st responder, new 1st responder taking on it’s role comes from a pool of original leading cells. While initially consistent over a short 1h period of time, 1st responders may be losing temporal consistency over longer time periods.

Supported by: NR01 DK102950, DK106412, JDRF 3-PDF-2019-741-A-N

Disclosure: V. Kravets: None.

8

Beta-arrestin 2 is absolutely required for the potentiation of insulin secretion by GIP

M.A. Ravier1, J. Obeid1, M. Leduc1, S. Costes1, P. Gilon2, S. Dalle1, G. Bertrand1;

1IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France, 2Université Catholique de Louvain, Brussels, Belgium.

Background and aims: The scaffold protein beta-arrestin2 (ARRB2) is known to uncouple G protein coupled receptors (GPCRs) from the G protein and to recruit new signaling pathways (such as the ERK1/2, PI3K, FAK⋯). In non beta cells, ARRB2 interacts with a wide range of GPCRs, but its interaction with the GIP receptor (GIPR) is still unclear. Our aim is to determine if ARRB2 is involved in the signaling of the GIPR in pancreatic beta cells.

Materials and methods: The experiments were carried out in beta cells from five-month-old Arrb2+/+ and Arrb2-/- male mice. cAMP production (CAMPS-EPAC), endogenous PKA (AKAR3) and ERK1/2 (EKAR) activations, [Ca2+] in the cytosol ([Ca2+]c ; Fura2-LR) and in the endoplasmic reticulum ([Ca2+]ER ; D4ER) were assessed by live cell imaging in mouse pancreatic beta cells. EPAC2 (EPAC2-GFP) recruitment beneath the plasma membrane was monitored by total internal reflection fluorescence microscopy. F-actin depolymerisation was evaluated by phalloidin staining (Alexa Fluor 488-conjugated phalloidin) and the phosphorylation of Focal Adhesion Kinase (FAK) by immunofluorescence.

Results: Insulin secretion from Arrb2-/- islets was reduced by 50% compared to Arrb2+/+ islets in response to GIP (100pM-10nM, p<0.01). When ARRB2 (ARRB2-GFP) was re-expressed in Arrb2-/- beta cells, insulin secretion in response to GIP was restored to a similar level than in Arrb2+/+ islets. Surprisingly, upon GIP stimulation (10pM-10nM), the cAMP production, PKA activation and EPAC2 recruitment were similar in Arrb2+/+and Arrb2-/- beta cells. Both [Ca2+]c and [Ca2+]ER remained comparable. Finally, the activation of ERK1/2 was also similar in Arrb2+/+ and Arrb2-/- beta cells. By contrast, the F-actin depolymerisation induced by 10nM GIP was significantly reduced (~25%, p<0.01) in Arrb2-/- beta cells. PI3Kγ and FAK have been reported to be involved in F-actin depolymerisation in response to GIP and glucose, respectively, and to be required for optimal insulin secretion. As expected, the PI3Kγ inhibitor (AS604850; 1μmol/l) reduced F-actin depolymerisation (~30%, p<0.01) by GIP stimulation in Arrb2+/+ beta cells, but no additional effect was observed in Arrb2-/- beta cells. Moreover, GIP-induced FAK activation was also reduced by 50% in Arrb2-/- beta cells.

Conclusion: Our study revealed that ARRB2 is required for the potentiation of insulin secretion by GIP, through F-actin depolymerisation probably via FAK activation and PI3Kγ recruitment, but independently from the canonical cAMP signalling (PKA and EPAC2) and the ERK1/2 pathway. Therefore, any variation in the expression of ARRB2, as observed in diabetic states, should functionally affect the incretin effect produced by GIP.

Supported by: Société Francophone du Diabete (SFD)

Disclosure: M.A. Ravier: None.

9

Pancreatic beta cell-selective deletion of the mitofusins 1 and 2 (Mfn1 and Mfn2) impairs glucose-stimulated insulin secretion in vitro and in vivo

G.A. Rutter1, E. Georgiadou1, T. Rodriguez2, C. Muralidharan3, M. Martinez3, P. Chabosseau1, A. Tomas1, G. Carrat1, A. Di Gregorio2, I. Leclerc1, A.K. Linnemann3;

1Cell Biology & Functional Genomics, Faculty of Medicine, Imperial College London, London, UK, 2National Heart and Lung Institute, Imperial College London, London, UK, 3Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, USA.

Background and aims: Mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cells. Although altered in subjects with type 2 diabetes, whether mitochondrial ultra-structure, and the proteins controlling the fission and fusion of these organelles, are important for glucose recognition, is unclear. Here, we generated mice with beta cell-selective, adult-restricted deletion of Mfn1 and Mfn2, essential for mitochondrial fusion, and studied the impact on insulin secretion and glucose homeostasis in vivo and in vitro.

Materials and methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites were crossed to transgenic animals carrying an inducible Cre recombinase under Pdx1 promoter control (PdxCreERT). Recombination was achieved by daily tamoxifen injections for one week. Islets were isolated and used for live beta cell fluorescence imaging of cytosolic (Cal520) or mitochondrial (R-GECO) free Ca2+ concentration and membrane potential (tetramethyl rhodamine methyl ester, TMRM) using spinning disc confocal microscopy (Nikon Ti2). Mitochondrial network characteristics were quantified using super resolution fluorescence (Zeiss LSM 780) and transmission electron microscopy. Intravital imaging was performed in mice injected with an adeno-associated virus to express the cytosolic Ca2+ sensor gCaMP6s selectively in beta cells under the control of the rat insulin promoter using multiphoton microscopy (Leica TCS SP8 DIVE). Blood flow through the islet was visualised simultaneously after injection of fluorescent albumin647.

Results: Mitochondrial length was sharply (to 77±0.9% of controls, p<0.0001) reduced in the Mfn1/2 KO mice and these animals displayed higher fasting glycaemia than control littermates at 11-12 weeks (8.6 vs 6.4 mmol/L, p>0.05) in vivo. An increase in circulating glucose levels was also observed (p<0.05 at 30 min and p<0.01 at 60 min) and was associated with a substantial (>five-fold) decrease in plasma insulin (5-15 min, p<0.0001) post-intraperitoneal glucose injection. Mitochondrial Ca2+ accumulation and membrane potential were significantly reduced (p<0.01) in response to high glucose in the KO animals. Examined by intravital imaging of the exteriorised pancreas, antiparallel changes in cytosolic Ca2+ and mitochondrial membrane potential, observed in control animals, were largely suppressed after Mfn1/2 deletion.

Conclusion: Mitochondrial fusion and fission cycles are essential in the beta cell to maintain normal mitochondrial bioenergetics and glucose sensing both in vitro and in the living mouse. Such cycles may be disrupted in some forms of diabetes to impair mitochondrial function and, consequently, insulin secretion.

Supported by: Wellcome; MRC; EU, Diabetes UK, NIH

Disclosure: G.A. Rutter: Employment/Consultancy; Sun Pharmaceuticals. Grants; Les Laboratoires Servier.

10

Unveiling the role of a mitochondrially-encoded tRNA-derived fragment in beta cell function

C. Jacovetti, V. Menoud, S. Gattesco, B. Bayazit, R. Regazzi;

Department of Neurosciences and Biomedical Sciences, University of Lausanne, Lausanne, Switzerland.

Background and aims: Mitochondria play essential roles in cellular energy production and contain their own genome that is transcribed to generate 11 mRNAs, 2 rRNAs and 22 tRNAs, all required for the synthesis of 13 protein subunits of the electron transport chain. Mutations in mitochondrially-encoded tRNAs strongly associate with diabetes. Interestingly, the cleavage of tRNAs has been recently shown to generate short non-coding RNA molecules with regulatory functions. Indeed, emerging evidence suggests that these tRNA-derived fragments (tRFs) are not by-products from random degradation, but functional molecules that modulate a number of cellular processes. However, very little is known about the role and the mode of action of tRFs. The aim of this project is to determine the role played by mitochondrially-encoded tRFs (mt-tRFs) in Beta-cell function.

Materials and methods: We used high throughput RNA-sequencing to search for mitochondrially-encoded pancreatic islet tRFs. Mitochondrial enrichment of mt-tRF relative to whole-cell lysate RNA preparations was confirmed by quantitative real-time PCR. The functional impact of selected mt-tRFs on mitochondrial function, insulin secretion, cell proliferation and survival was studied by modifying their expression in the insulin-secreting cell line INS832/13 and in dissociated rat islet cells. Real-time PCR was used to determine the signalling pathways controlled by the mt-tRFs.

Results: RNA-sequencing led to the identification of 3187 tRFs in primary islet cells of adult rats, two of which mitochondrially-encoded, mt-tRF-1 and mt-tRF-4. Mt-tRF1 is cleaved from tRNALeu(TAA), is 41 bp long and is enriched 2 times in the mitochondrial fraction (MF) of adult rat islets. Transfection of antisense oligonucleotides complementary to this fragment, reduced mt-tRF-1 level by about 90% in INS832/13 and dissociated rat islet cells. Inhibition of tRF-1 resulted in impaired insulin release in response to glucose but not to KCl without affecting cell survival in the presence or absence of proinflammatory cytokines. Interestingly, we found that mt-tRF1 is strongly down-regulated in the islets of newborn rats exposed to low-protein diet (LP) during foetal and postnatal life, a growth retardation model characterized by impaired insulin secretion, mitochondrial dysfunction and diabetes susceptibility at adulthood. Blockade of mt-tRF1 in rat islet cells resulted in an increase in the expression of the Uncoupling Protein 1 (Ucp1), which uncouples the mitochondrial proton gradient from ATP synthesis. In agreement with this finding, the level of Ucp1 and mt-tRF1 were found to be inversely correlated in the islets of LP newborn rats.

Conclusion: Blockade of mt-tRF1, a fragment derived from the mitochondrially-encoded tRNALeu(TAA), which is decreased in the islets of newborn rats kept on a low protein diet, suppresses glucose-induced insulin release, suggesting its potential contribution to Beta-cell dysfunction and diabetes susceptibility associated with neonatal deleterious environment. Our data could pave the way for the development of new small non-coding RNA-based strategies aiming at preserving an appropriate functional Beta-cell mass.

Supported by: SNF

Disclosure: C. Jacovetti: None.

11

Post-transcriptional co-regulation of insulin secretory granule proteins

J. Vasiljević1,2, D. Vasiljević3, C. Niehage4, C. Wegbrod1,2, K. Ganss1,2, A. Soenmez1,2, A. Friedrich1,2, B. Hoflack4, M. Selbach3,5, M. Solimena1,2;

1Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, 2German Center for Diabetes Research (DZD e.V.), Neuherberg, 3Max Delbrück Center for Molecular Medicine (MDC), Berlin, 4Biotechnology Center, Dresden, Germany, 5Charité – Universitätsmedizin Berlin, Berlin, Germany.

Background and aims: Once glucose-stimulated beta cells release insulin, they immediately activate insulin production to adjust their insulin stores. Notably, glucose stimulation initially enhances insulin biosynthesis without affecting its mRNA levels. Thus, post-transcriptional mechanisms are essential to retain insulin stores and beta cell responsiveness. This adaptation relies on interactions of RNA-binding proteins (RBPs) with regulatory sequences in mRNA untranslated regions (UTRs). Moreover, functionally related mRNAs can be post-transcriptionally co-regulated through elements recognized by the same RBPs. Such elements are conserved in mammalian mRNAs for insulin and for other secretory granule cargoes, e.g. PC1/3PC2 and ICA512/IA-2/PTPRN. We previously showed that these mRNAs are post-transcriptionally coordinated by RBPs, but an overview of the latter was missing.

Materials and methods: We combined in vitro RNA pull-downs and mass spectrometry to identify RBPs that bind to mouse Ins1, Ins2, spliced Ins2, PC2 and ICA512 mRNA 5’-UTRs in resting and glucose stimulated MIN6 cells. Mouse γ-tubulin mRNA 5’-UTR was used as a control.

Results: mRNAs for secretory granule cargoes share many RBPs that are enriched compared to the γ-tubulin mRNA. Notably, different sets of RBPs control these mRNAs in resting and stimulated cells. We discovered that heterogeneous ribonucleoprotein A2/B1 (hnRNP A2/B1) is a novel post-transcriptional regulator of insulin expression in MIN6 cells. Mouse, human and rat Insulin mRNAs include sequences homologous to hnRNP A2/B1 response elements (A2REs). hnRNP A2/B1 binding to the 5’-UTR of Ins1 mRNA harboring mutated A2REs was reduced. Hnrnpa2b1-/- MIN6 cells had lower Ins1 mRNA, insulin and proinsulin levels, and consequently lower insulin secretion. In resting MIN6 cells hnRNP A2/B1 was enriched in cytosolic punctae co-stained for Ins1 and Ins2 mRNAs and markers of stress granules, which store repressed transcripts. Upon stimulation the stress granules dissolved and hnRNP A2/B1 localized predominantly to the nucleus, while the Insulin mRNAs were dispersed in the cytoplasm. We are investigating if similar patterns occur in beta cells of healthy and diabetic patients.

Conclusion: We propose that in resting beta cells, specific RNA-protein interactions allow for the storage of mRNAs for insulin secretory cargoes into stress granules. Glucose stimulation remodels these interactions: stress granules dissolve, a different set of RBPs bind and coordinate mRNA translation, enabling a burst in insulin secretory granule production. To our knowledge this is the first report of stress granules in beta cells. Further experiments will expand on post-transcriptional mechanisms in beta cells and their potential perturbations in diabetes.

Supported by: German Center for Diabetes Research (DZD e.V.)

Disclosure: J. Vasiljević: None.

12

The mechanosensor Piezo1 mediates glucose sensing and insulin secretion in pancreatic beta cells

M. Barghouth1, Y. Ye1, Y. Wang2, C. Luan1, A. Karagiannopoulos1, L. Eliasson1, P. Rorsman3, E. Zhang1, E. Renström1;

1Lund University Diabetes Centre, Department of Clinical Sciences, Malmö, Sweden, 2Section for Surgery Lund University Department of Clinical Science, Malmö, Sweden, 3Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Background and aims: Defective insulin secretion in pancreatic β cells is a hallmark of all types of diabetes, resulting in chronically elevated blood glucose levels. The impact of blood flow and mechanotransduction in the regulation of insulin secretion are incompletely investigated. In vascular endothelial cells, mechanical shear forces induced by blood flow induce ATP release and trigger Ca2+ waves. Glucose elevation stimulates insulin secretion by the well-known triggering pathway but is also known to result in β cell swelling. These previous observations prompted us to investigate the role of mechanical forces for the physiological function of pancreatic β cells. Recently, Piezo1 was identified a mechanosensitive ion channel. Piezo1 activates and leads to an increasing inward membrane current partially carried by Ca2+. Whether this proportion of Ca2+ can trigger or accelerate glucose stimulated insulin secretion in human b-cell remains elusive. Here we investigated the role of PIEZO1 in β cells, especially its role in glucose-stimulated insulin secretion.

Materials and methods: RNA sequencing; Animal models (β-cell-specific Piezo1 knockout mice (RIP-Cre+Piezo1f/f)); In situ pancreas perfusion; IPGTT; Ca2+ imaging; Plasma Membrane Potential Measurement.

Results: We found PIEZO1 protein expressed in both α and β cells at comparable levels in both human and mouse islet cells. The glucose-induced increase of [Ca2+]i oscillations were reduced by 45% in RNA silencing (siPiezo1) INS-1 832/13 cells, but ineffective when glucose was replaced by the non-metabolizable sugar mannitol. Hypotonic swelling also elicited robust [Ca2+]i transients and membrane depolarization in normal INS-1 832/13 cells. By contrast, the elevation of [Ca2+]i and membrane depolarization were reduced markedly in the presence of the GsMTx4, (a Piezo1/ Piezo2 inhibitor), or in siPiezo1cells. Yoda1 (Piezo1 agonist) increased [Ca2+]i even under resting conditions (2.8 mM glucose) in INS-1 832/13 cells, rodent and human islets. In situ mouse pancreas perfusion showed that glucose stimulated insulin secretion almost 3-fold, while yoda1 increased it ~7-fold. By contrast, GsMTx4 reduced insulin release by >30%. β-cell-specific Piezo1 knockout mice exhibited impaired glucose tolerance and blood glucose post-IPGTT was significantly increased. Calcium imaging in β-cells from Piezo1 knockout mice revealed a more than 50% reduction in glucose-induced [Ca2+]i . In human β-cells inhibition of Piezo 1 by GsMTx4 reduced the amplitude of glucose-induced [Ca2+]i oscillations by ~80%. Yoda1 increased both [Ca2+]i and membrane depolarization under basal conditions in human β-cells. Percentage of secreted insulin was significantly suppressed by GsMTx4 at basal and stimulated conditions. RNA-seq analysis revealed that PIEZO1 gene expression was significantly increased in islets from donors with type-2 diabetes, suggesting a compensatory effect.

Conclusion: These results establish mechanotransduction as an important signaling modality in both rodent and human glucose-induced insulin secretion. Our data point to highlight the role of the mechanosensor Piezo1 channel as the molecular mediator of this effect.

Supported by: Swedish Research Council (2017-01090); Clinical research (ALF); Crafoord foundation. Grants;

Disclosure: M. Barghouth: None.

OP 03 Insulin sensitivity and biomarkers

13

Kinome profiling reveals impaired signalling in primary human skeletal muscle cells carrying a novel Finnish-specific AKT2 gene variant

N. Datta1,2, S. Mäkinen1,2, S. Rangarajan3, Y.H. Nyugen1,2, A. Latva-Rasku4, P. Nuutila4, M. Laakso5, H.A. Koistinen1,2;

1Minerva Foundation Institute For Medical Research, Helsinki, Finland, 2Department of Medicine, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland, 3PamGene International B.V., s-Hertogenbosch, Netherlands, 4Turku PET Centre, University of Turku, Turku, Finland, 5Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland.

Background and aims: Abnormalities in kinase-mediated signalling are involved in the pathophysiology of metabolic disorders, including insulin resistance and type 2 diabetes (T2D). A novel partial loss-of function AKT2 coding variant (p. Pro50Thr) is highly specific for Finnish population and is associated with increased fasting insulin concentrations, reduced insulin-mediated glucose uptake in the whole body and in several insulin sensitive tissues, and predisposition to T2D. Here, we explore the hypothesis that kinase networks in skeletal muscle cells are dysregulated in the carriers of p.P50T/AKT2.

Materials and methods: Primary muscle cell cultures were established from vastus lateralis muscle biopsies of the carriers and non-carriers of the p.P50T/AKT2. Myoblasts were differentiated into myotubes. A comprehensive analysis of tyrosine (PTK) and serine-threonine (STK) kinase signalling was performed in insulin-stimulated myotubes from 9 carriers and 8 non-carriers using the PamChip® kinome profiling system. This technology is based on the detection of phosphorylation of peptides by PTK or STK kinases that are active in the muscle cell lysates.

Results: Kinase profile comparison (PTK and STK heatmaps) identified multiple differentially phosphorylated peptides between carriers and non-carriers of p.P50T/AKT2. Analysis of Volcano plots, as a result of T-tests, revealed more than thirty proteins, including several cell surface receptors, that were significantly less phosphorylated in the variant carriers (p < 0.05). Predictive kinase analysis using the Upstream Kinase PamApp tool further demonstrated a large-scale impairment of multiple tyrosine and serine-threonine kinase activities in carriers. As examples, signalling of the non-receptor Src-family kinases (SFK), calmodulin-modulated kinases (CaMK), and different isoforms of protein kinase C (PKCs) were downregulated in p.P50T/AKT2 carriers.

Conclusion: Kinome profiling revealed multiple differences in the intricate kinase networks in skeletal muscle cells from carriers of p.P50T/AKT2 variant. These core differences may contribute to development of insulin resistance in carriers of p.P50T/AKT2.

Supported by: Academy of Finland, Diabetes Wellness Sverige, Finnish Diabetes Research Foundation

Disclosure: N. Datta: None.

14

In vivo, up and down hepatic modulation of interactions between ER and mitochondria impacts hepatic insulin sensitivity and steatosis

A. Beaulant, J. Ji-Cao, N. Bendridi, M.-A. Berger, H. Vidal, J. Rieusset;

CarMeN laboratory INSERM U1060, Lyon, France.

Background and aims: Understanding the molecular mechanisms of insulin resistance (IR) is essential for proposing new preventive/therapeutic strategies against type 2 diabetes (T2D). Among newly identified mechanisms, the communication between endoplasmic reticulum (ER)-mitochondria, at contact sites called mitochondria-associated membranes (MAMs), recently emerged as a key regular of glucose homeostasis in multiple tissues. In the liver, our team identified MAMs as key regulators of insulin action and reported hepatic organelle miscommunication in different murine models of T2D. However, this topic is subjected to controversy, in part because strategies used to modulate organelle tethering target different endogenous MAM proteins with other cellular functions outside of MAMs. Therefore, it is now crucial to determine the causative role of MAMs in the development of hepatic IR, using non-endogenously expressed spacer and linker. The aim of the study is to clarify in vivo the causative role of ER-mitochondria interactions in the development of hepatic IR and steatosis through genetic up and down modulation of MAMs in the liver of mice.

Materials and methods: Using adenovirus, we modulated ER-mitochondria interactions in the liver of mice by overexpressing either a spacer called FATE1 (Fetal and Adult Testis Expressed 1, expressed only in the testis), or an artificial fluorescent linker (RFP fused to the outer mitochondrial membrane (OMM) and to ER membrane). Ad-GFP was used as a control of Ad-FATE1, whereas Ad-RFP targeted only to the OMM was used as a control of Ad-linker (107 pfu/mice). Adenovirus-mediated dampening of MAMs was performed in the liver of lean mice (2 weeks), whereas reinforcing of MAMs was performed before feeding mice with either a standard diet (SD) or a high-fat and high-sucrose diet (HFHSD) for 4 weeks. Repercussions on ER-mitochondria interactions (in situ proximity ligation assays and transmission electronic microscopy), hepatic insulin signalling and lipid accumulation (oil red o staining), and on whole-body glucose homeostasis (glucose and insulin tolerance tests) were measured in vivo, whereas ER-mitochondria calcium exchange (microscopy using the mitochondria-specific calcium probe, 4mtD3CPV) was measured on primary mouse hepatocytes.

Results: As expected, the overexpression of FATE1 in the liver of mice significantly disrupted hepatic ER-mitochondria interactions and calcium exchange, whereas the overexpression of the linker reinforced them. The FATE1-mediated disruption of MAMs induced glucose intolerance without modifying whole-body insulin sensitivity, pointing a specific hepatic IR. In agreement, hepatic insulin signalling is altered in the liver of Ad-FATE1 mice compared to Ad-GFP mice, and is associated with hepatic steatosis. Conversely, the linker-mediated reinforcement of MAMs prevented HFHSD-induced glucose intolerance and hepatic insulin resistance, whereas no effect was observed under SD. Effects on lipid accumulation are under analysis.

Conclusion: Altogether, our data demonstrate that the modulation of ER-mitochondria interactions in mice liver controls hepatic insulin sensitivity and lipid accumulation, supporting a causative role of disrupted MAMs in hepatic metabolic alterations. Targeting MAMs could be a new preventive/therapeutic approach to fight against T2D.

Disclosure: A. Beaulant: None.

15

GDF15 mediates the metabolic effects of PPARβ/δ by activating AMPK

D. Aguilar-Recarte1,2, J. Pizarro-Delgado1,2, L. Peña-Moreno1,2, X. Palomer1,2, S.-J. Lee3, M. Vázquez-Carrera1,2;

1Pharmacology, University of Barcelona, Barcelona, Spain, 2Spanish Biomedical Research Center in Diabetes and Associated Metabolic Diseases (CIBERDEM), Madrid, Spain, 3The Jackson Laboratory and University of Connecticut School of Medicine, Farmington, USA.

Background and aims: The antidiabetic effects of peroxisome proliferator-activated receptor (PPAR)β/δ agonists mostly rely on the activation of AMP-activated protein kinase (AMPK). Interestingly, many of the actions of PPARβ/δ are similar to those of growth differentiation factor (GDF)15, a stress-response cytokine that improves fatty acid oxidation, glucose tolerance and insulin sensitivity. The aim of this study was to examine whether the beneficial effects of PPARβ/δ agonists on lipid-induced endoplasmic reticulum (ER) stress, inflammation and insulin resistance were dependent on GDF15.

Materials and methods: A neutralizing antibody against GDF15 or IgG were injected 3 days before sacrifice to mice fed a control or a high-fat diet (HFD) and treated for 3 weeks with vehicle or a PPARβ/δ agonist. A similar study was conducted in WT and GDF15-KO fed with an HFD and treated with the PPARβ/δ agonist. A group of mice were also treated with vehicle or recombinant GDF15. Finally, C2C12 myotubes were treated with different compounds or small interfering (si)RNAs to examine the mechanisms by which PPARβ/δ agonists increase GDF15 levels.

Results: Injection of the neutralizing GDF15 antibody prevented the improvement in glucose tolerance caused by the administration of the PPARβ/δ agonist in mice fed an HFD without changes in food intake and body weight. The GDF15 neutralizing antibody also abolished most of the changes caused by the PPARβ/δ agonist treatment in the levels of genes and proteins involved in fatty acid metabolism, ER stress, inflammation and the insulin signalling pathway in skeletal muscle and liver. The experiment conducted in GDF15-KO mice showed a similar trend, where the PPARβ/δ agonist antidiabetic effects were abolished or attenuated compared to the WT mice, confirming the implication of GDF15 in PPARβ/δ actions. Studies conducted with inhibitors and siRNAS in cultured myotubes demonstrated the implication of AMPK and p53 in the increase of GDF15 levels caused by the treatment with PPARβ/δ agonists. Treatment with recombinant GDF15 caused an increase in the phosphorylation levels of AMPK protein in cultured myotubes and skeletal muscle of mice.

Conclusion: Overall, the findings of the present study demonstrate that the increase in GDF15 levels caused by PPARβ/δ activation through AMPK and p53 prolongs the increase in phospho-AMPK levels, contributing to the reduction of ER stress, inflammation and insulin resistance.

Supported by: SAF2015-64146-R, Ministerio de Ciencia, Innovación y Universidades de España

Disclosure: D. Aguilar-Recarte: Grants; FPI Spanish Government Grant.

16

Serum Fetuin-B is positively related to metabolic syndrome and insulin resistance

S. Xue1, L. Li1, G. Yang2;

1Key Laboratory of Diagnostic Medicine (Ministry of Education) and Department of Clinical Biochemistry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 2Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Background and aims: Fetuin-B, as a new hepatokine or adiponectin, has been reported to impair insulin action in myotubes and hepatocytes of mice with hepatic steatosis and regulate glucose and lipid metabolism in humans. Metabolic syndrome (MetS) represents a cluster of metabolically related symptoms that includes abdominal obesity, insulin resistance (IR), hypertension and dyslipidemia. The purpose of this study was to (1) compare serum Fetuin-B levels and the key components related to IR between patients with MetS and the control subjects, (2) set up multiple intervention experiments to further explore the relationship among serum Fetuin-B, MetS and IR.

Materials and methods: A total of 377 Chinese women (185 healthy controls and 192 MetS patients) were recruited in this cross-sectional study. Serum Fetuin-B levels were examined by ELISA kit. The anthropometric examination (weight, height, waist circumference, hip circumference, blood pressure, FAT%) and biochemical varies (fasting and 2h post-OGTT glucose, insulin, HbA1c, TG, TC, HDL, LDL, FFA) were detected and recorded by professional in all participants. The insulin sensitivity and glucose tolerance were evaluated by euglycemic-hyperinsulinemic clamp (EHC) and oral glucose tolerance test (OGTT), and the drug intervention experiment of Liraglutide was performed to explore the effect of serum Fetuin-B in MetS longitudinally.

Results: Serum Fetuin-B levels were significantly increased in MetS patients compared with the healthy women (p<0.001). Serum Fetuin-B were positively related to WHR, FAT%, TG, FBG, FIns, HOMA-IR, VAI, LAP (all p<0.001) and BMI, HbA1c%, 2h-BG, 2h-Ins (all p<0.01). We demonstrated that TG and WHR were independently connected with serum Fetuin-B levels. Further investigation found that serum Fetuin-B showed a linear trend and independently correlated with MetS. The levels of serum Fetuin-B increased with the number of components of MetS (p for trend < 0.05). In the ROC curve, the best threshold for serum Fetuin-B to distinguish MetS was 3.87 mg/L. Furthermore, serum Fetuin-B levels were markedly elevated after glucose loading in the healthy group (p <0.001) and significantly increased in MetS women during the EHC (p<0.05). After six months of Liraglutide intervention, serum Fetuin-B levels in women with MetS statistically decreased following improvement of IR.

Conclusion: Serum Fetuin-B levels are significantly associated with the key components of IR and MetS via regulating glucose and lipid metabolism. Serum Fetuin-B may be a potential biomarker for MetS to predict outcomes and therapeutic responses.

figurec

Clinical Trial Registration Number: ChiCTR-IIR-16007901

Supported by: NSFC(81873658)

Disclosure: S. Xue: None.

17

Carnitine supplementation improves insulin sensitivity and skeletal muscle acetylcarnitine formation in type 2 diabetes patients

Y.M.H. Bruls1, Y.J.M. op den Kamp2, P. Veeraiah1, E. Phielix2, B. Havekes3, J.E. Wildberger1, M.K.C. Hesselink2, P. Schrauwen2, V. Schrauwen1,2;

1Department of Radiology and Nuclear Medicine, Maastricht University Medical Center +, Maastricht, 2Department of Nutrition and Movement Sciences, Maastricht University Medical Center +, Maastricht, 3Department of Internal Medicine, Division of Endocrinology, Maastricht University Medical Center +, Maastricht, Netherlands.

Background and aims: Type 2 diabetes patients are characterized by decreased insulin sensitivity and concomitant disturbances in glucose homeostasis. Insulin sensitivity correlates positively with MR-based skeletal muscle acetylcarnitine concentration, indicating lower acetylcarnitine levels in insulin resistant individuals. Recent evidence suggests that low free carnitine availability may play a role in reduced acetylcarnitine formation. Therefore, we investigated if carnitine supplementation elevates skeletal muscle acetylcarnitine formation and thereby improves insulin sensitivity and glucose homeostasis in type 2 diabetes patients.

Materials and methods: 32 type 2 diabetes patients followed a 12 week L-carnitine treatment (2970 g/day). Plasma free carnitine concentrations were measured to check compliance. A 2-step hyperinsulinemic-euglycemic clamp (10 vs. 40 mU/m2/min) with D-[6,6-2H2]-glucose tracer infusion was performed to assess hepatic and peripheral insulin sensitivity. Skeletal muscle acetylcarnitine concentrations were measured in vivo in the vastus lateralis muscle using a combination of T1 editing and long echo time (TE=350ms) proton magnetic resonance spectroscopy (1H-MRS) in rest and post exercise (30 minutes at 70% Wmax) to stimulate near maximum as a parameter for free carnitine availability. Intrahepatic lipid content (IHL) was quantified using 1H-MRS. All measurements were performed before and repeated after carnitine supplementation.

Results: Plasma free carnitine levels increased upon carnitine supplementation (from 35.6±1.3 to 54.7±1.7 μmol/L, p=<0.01) indicating good compliance. Hepatic (endogenous glucose (EGP) suppression) as well as peripheral (Δ rate of disappearance, ΔRd) insulin sensitivity improved upon carnitine supplementation (EGP suppression: 31.9 ±2.9 vs. 39.9±3.2%, p=0.020 and ΔRd 10.53±1.85 vs. 13.83±2.02 μmol/kg/min, p=0.005). Resting and post-exercise skeletal muscle acetylcarnitine concentrations were both elevated after carnitine supplementation (1.18±0.13 vs 1.54±0.17 mmol/kgww, p=0.008 and 3.70±0.22 vs. 4.53±0.30 mmol/kgww, p<0.001, respectively). Finally, a trend towards reduced plasma glucose levels (from 8.1±0.3 to 7.7±0.3 mmol/L, p=0.083) and IHL (from 14.7±2.6 to 12.8±2.2 %, p=0.095) was found after carnitine supplementation.

Conclusion: The current study reveals very pronounced effects of carnitine supplementation on insulin sensitivity, intrahepatic lipid content and concomitant fasting plasma glucose levels in type 2 diabetes patients. We demonstrated that carnitine supplementation increases acetylcarnitine concentration in muscle in the resting state and the capacity to form acetylcarnitine with exercise, which may be underlying the beneficial effect on insulin sensitivity. We are currently investigating whether certain characteristics, such as baseline acetylcarnitine concentration, are predictive for the strength of the metabolic response to carnitine supplementation.

Clinical Trial Registration Number: NCT03230812

Supported by: Ministry of Economic Affairs by PPP Allowance of the Top Sector Life Sciences & Health

Disclosure: Y.M.H. Bruls: None.

18

Remission of type 2 diabetes with return of insulin secretory function restores normal pancreas morphology

R. Taylor1, K.G. Hollingsworth1, J.A.M. Shaw2, N. Sattar3, M.E.J. Lean4, A. Al-Mrabeh1;

1Translational and Clinical Research Institute, Magnetic Resonance Centre, Newcastle University, Newcastle upon Tyne, 2Translational and Clinical Research Institute, Regenerative Medicine, Newcastle University, Newcastle upon Tyne, 3Institute of Cardiovascular & Medical Sciences, Glasgow University, Glasgow, 4School of Medicine, Dentistry and Nursing, Glasgow University, Glasgow, UK.

Background and aims: Pancreas volume is subnormal and the shape of the organ is abnormal in type 2 diabetes. If these abnormalities resulted from rather than led to the disease state, return of β-cell function during remission of T2DM would be expected to correct the abnormalities.

Materials and methods: Participants (n=64) in the Diabetes Remission Clinical Trial were studied over 2 years and compared with matched non-diabetic controls. Those who achieved HbA1c <6.5% (48 mmol/mol) and fasting blood glucose <7.0 mmol/l off all anti-diabetes medication, were classified as ‘Responders’. Magnetic resonance techniques were employed to obtain anatomical and fat fraction images of the pancreas. Pancreas volume, intrapancreatic fat content, and the irregularity of the pancreas borders were quantified using custom MR techniques. Insulin secretion was measured using the Stepped Insulin Secretion Test with Arginine (SISTA).

Results: At baseline, pancreas volume was 63.8±1.8 vs. 79.8±2.9cm3 in non-diabetic controls, p<0.0001). Pancreas volume was unchanged from baseline at 5 months post weight loss irrespective of remission (responders: 63.0±2.8 to 64.0±2.8 cm3, p=0.10; non-responders: 59.0±3.5 to 60.0±3.7cm3, p=0.32). At 24 months, volume had increased by 12.6±1.5cm3 in responders compared with 4.5±1.3cm3 in non-responders (p<0.0001). The pancreas borders were more irregular in diabetes compared with non-diabetic controls at baseline (Fractal Dimension 1.116±0.013 vs1.097±0.005, p<0.0001), but normalised in responders at 24 months (1.097±0.008 vs. 1.097±0.005, p=0.92). At 5 months after weight loss, 1st phase insulin secretion increased only in responders (to 0.11 [0.060 to 0.157] nmol/ml/min, p<0.0001 vs. baseline), maintained at 24 months (0.12 [0.060 to 0.175] nmol/ml/min, p<0.0001 vs. baseline). Responders lost 1.56±0.3% of intrapancreatic fat compared to 0.51±0.4% for non-responders (p<0.05). Plasma GDF-15 decreased in responders only, but IGF-1 increased and FGF-21 levels decreased after weight loss irrespective of remission.

Conclusion: These data demonstrate for the first time the reversible nature of the abnormal pancreas morphology during remission of type 2 diabetes, and identify potential regulatory factors. The low pancreas volume and irregularity in shape are likely to be a consequence rather than a cause of the disease state, potentially related to deficiency of post-prandial insulin secretion. Fat removal from the pancreas is closely associated with restoration of β-cell function, which may lead to secondary restoration of exocrine tissue mass via trophic and anabolic effects of insulin.

Supported by: Diabetes UK

Disclosure: R. Taylor: Employment/Consultancy; Wilmington Healthcare. Grants; Diabetes UK. Lecture/other fees; Lilly and Novartis.

OP 04 Central actions in diabetes

19

Genetic deficiency of CRP confers resistance to obesity and enhances insulin and leptin sensitivity

S. Qiu1,2, L. Li1, G. Yang2;

1Department of Clinical Biochemistry, College of Laboratory Medicine,Chongqing Medical University, Chongqing, 2Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Background and aims: As a member of the pentaxin protein family, C-reactive protein (CRP) is mainly synthesized and secreted by the liver and released into circulation in response to inflammation. In addition to serving as a traditional inflammatory factor, CRP is closely associated with the development of obesity, diabetes, and cardiovascular diseases serving as a metabolic and inflammatory marker. We hypothesize that CRP protein was directly involved in the regulation of energy and glucose metabolism, rather than just a surrogate marker, and that genetic deficiency of CRP will lead to resistance to obesity and insulin resistance.

Materials and methods: Rat CRP gene deletion model was use to investigate the effect of CRP on energy and glucose metabolism. The CRP null mutant rat were placed on either a normal diet or a high-fat diet. The phenotypic changes in body weight, glucose metabolism, insulin sensitivity, energy expenditure, and inflammation conditions were examined. The central impact of CRP deficiency on leptin and insulin hypothalamic signaling as well as glucose homeostasis were examined via intracerebral ventricular delivery of leptin and CRP plus glucose clamp studies in the wild type or CRP deficient rats.

Results: Here, we revealed that CRP deficiency rendered rat resistance to obesity and high blood pressure development, elevated energy expenditure, and enhanced locomotor activity. Glucose clamp studies revealed that deletion of CRP enhanced hepatic insulin signaling and actions. Systematic CRP deficiency also promoted the effect of central leptin on hepatic and skeletal muscle glucose metabolism, and enhanced central leptin-stimulated STAT3/Akt signaling, particularly under HFD-induced obesity and IR conditions. In contrast, reinstatement of CRP into the hypothalamus of the knockout rats attenuated the effects of central leptin signaling on insulin sensitivity and peripheral glucose metabolism. CRP deficiency increased the hypothalamic expression of POMC following ICV leptin treatment and allowed prolonged and sustained anorexic and weight-reducing effects. Moreover, CRP regulateed body weight, energy expenditure, glucose metabolism, and blood pressure for at least 12 months.

Conclusion: This study represents the first line of genetic evidence that CRP is not merely a surrogate blood marker for inflammation and metabolic syndromes but directly regulates energy balance, body weight, insulin sensitivity, and glucose homeostasis through direct regulation of leptin’s central effect and hypothalamic signaling.

Supported by: NAFC(No.81630021)

Disclosure: S. Qiu: None.

20

Protein tyrosine phosphatase 1B deficiency enhances leptin action to improve glucose homeostasis in IDDM treatment with leptin

Y. Ito1, R. Banno2, R. Sun3, H. Yaginuma3, K. Taki3, M. Sugiyama3, T. Tsunekawa3, H. Takagi3, H. Arima3;

1CKD Initiatives International Medicine, Nagoya University Graduate School of Medicine, Nagoya, 2Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya, 3Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Background and aims: There are several lines of evidence that either intraarterially or intracerebroventricularly administration of leptin could normalize glucose metabolism in the rodent of insulin-dependent diabetes mellitus (IDDM) models. As the mechanisms, leptin has been reported to act on the hypothalamic neurons to suppress gluconeogenesis in the liver and enhance glucose uptake in brown adipose tissue and skeletal muscle, resulting in lowering blood glucose levels. On the other hand, peripheral administration of leptin is known to has only a limited effect on improving hyperglycemia. Protein tyrosine phosphatase 1B (PTP1B) is key enzyme that negatively regulates leptin receptor signaling. We have previously reported that in PTP1B deficient mice, peripheral administration of leptin enhances leptin receptor signaling in the hypothalamus compared to control mice. To investigate the role of PTP1B in leptin action for treating IDDM by using PTP1B deficient mice (KO) and PTP1B inhibitors.

Materials and methods: To generate IDDM mice, we injected wild-type (WT) mice and PTP1B deficient (KO) mice once intraperitoneally with streptozotocin (STZ) or vehicle. We evaluated glucose metabolism in IDDM WT and IDDM KO mice. Next, we evaluated glucose metabolism in mice received two kinds of treatment. One is the peripheral administration of a high dose of leptin or vehicle, and the other is the central administration of a low dose of leptin or vehicle. Finally, we evaluated whether if peripheral combination therapy of a high dose of leptin and PTP1B inhibitor in IDDM WT mice improved glucose metabolism or not. In addition, the mechanisms in which leptin treatment improved glucose metabolism under PTP1B deficiency were also examined.

Results: We found that (1) while blood glucose levels of IDDM group were higher than those of non-IDDM group, glucose metabolism in IDDM PTP1B deficient (KO) mice was significantly improved compared to IDDM wild-type (WT) mice, (2) peripheral administration of a high dose of leptin significantly improved glucose metabolism in IDDM KO mice compared to IDDM WT, (3) central administration of a low dose of leptin significantly improved glucose metabolism in KO mice compared to WT mice, and (4) peripheral combination therapy of leptin and PTP1B inhibitor in IDDM WT mice improved glucose metabolism to the same levels as control mice. We also found that the phosphorylation of stat3 in the arcuate nucleus of hypothalamus following peripheral leptin administration was enhanced under PTP1B deficiency, and those improvements of glucose metabolism are at least partly due to the action via β-adrenergic receptors signaling.

Conclusion: In IDDM treatment with leptin, PTP1B deficiency and PTP1B inhibitor enhanced leptin action in the brain to improve glucose metabolism.

Supported by: The Japanese Society for Promotion of Science (2618K16225) and the Japan IDDM Network

Disclosure: Y. Ito: Grants; Sanwa Kagaku Kenkyusho, Kowa Pharmaceutical, MSD K.K., Dainippon Sumitomo, Kyowa Kirin Co. Ltd., Chugai Pharmaceutical Co. Ltd., Boehringer Ingelheim, Nihon Medi-Physics Co. Ltd. Lecture/other fees; Astellas Pharma, Daiichi Sankyo, Ono Pharmaceutical Company.

21

Investigating the involvement of hypothalamic de novo ceramide synthesis in resistin/TLR4 induced neuronal inflammation and insulin resistance

J. Guitton, S. Al Rifai, C. Alexandre, M. Taouis, Y. Benomar, H. Le Stunff;

Institut des Neurosciences Paris Saclay (Neuro-PSI), UMR9197 CNRS, Orsay Cedex, France.

Background and aims: In the context of obesity, the excess supply of fatty acids (FA) and ectopic lipid accumulation in non-adipose tissues causes functional impairments in several metabolic pathways leading to a phenomenon, known as “lipotoxicity” that promotes peripheral inflammation and insulin resistance (IR). Recently, the hypothalamus, a brain area involved in energy homeostasis, has also been reported as a target of lipotoxicity. Interestingly, it has been shown that accumulation of reactive lipid species, such as ceramide, in the hypothalamus induces central IR and impaired glucose homeostasis. Beside, in an over-nutrition environment, the hypothalamus is also subjected to changes in circulating factors originated from adipose tissue and immune cells. Among these factors, resistin is described as a key mediator linking obesity to IR. Recently, we have reported that central resistin, through hypothalamic TLR4, induces whole body IR and promotes neuronal inflammation. Interestingly, growing evidence supports an important role for TLR4 in FA-induced ceramide biosynthesis and peripheral inflammation and IR. In this context, the present study aims to investigate the potential involvement of hypothalamic de novo ceramides synthesis in resistin-induced neuronal inflammation and insulin resistance.

Materials and methods: Using mouse hypothalamic (mHypoA) and human (SH-SY5Y) neuronal cells, we analyzed the impact of resistin overexposure on insulin signaling, and on the expression levels of proinflammatory mediators and key enzymes driving ceramide biosynthesis. This was assessed by western blotting and RTqPCR analyses. Intracellular ceramide contents were also quantified by lipidomic analysis. Two pharmacological inhibitors, myriocin and TAK-242, were used to evaluate the involvement of ceramide de novo synthesis pathways and TLR4 signaling pathways in resistin-induced neuronal inflammation and IR. Additionally, C57BL6J and TLR4-deficient mice were treated with or without resistin through ICV route to evaluate the impact of central resistin infusion on hypothalamic inflammation and reactive gliosis as well as on the hypothalamic expression of enzymes involved in ceramide biosynthesis.

Results: In neuronal cells, we show that resistin overexposure induces neuronal inflammation and IR as evidenced by increased expression of IL6 (89.93% p<0.05), and inhibition of insulin-dependent phosphorylation of Akt (40.6% p<0.02). In addition, resistin treatment increases ceramide contents and the expression levels of a key enzymes driving ceramide biosynthesis (SPT1/2, CerS4 and DES1). Interestingly, pharmacological inhibition of TLR4 signaling (using TAK-242) and ceramide de novo synthesis (using myriocin), prevents resistin-dependent neuronal inflammation and IR. Next, we validated the effects of resistin in mice, and showed that central resistin infusion for 3 days markedly increases hypothalamic inflammation and reactive gliosis, as well as the expression of enzymes driving ceramide de novo synthesis in a TLR4-dependent manner.

Conclusion: Taken together, these findings reveal resistin/TLR4/ceramide as a new regulatory pathway of neuronal inflammation and IR. Targeting this signaling pathway may constitute a significant breakthrough to overcome obesity-induced hypothalamic inflammation, IR and related metabolic dysfunctions.

Disclosure: J. Guitton: None.

22

Central nesfatin-1 attenuates hepatic steatosis by suppression of hypothalamic endoplasmic reticulum stress

M. Mokou1, L. Li1, G. Yang2;

1Key Laboratory of Diagnostic Medicine (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 2Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Background and aims: It has been reported that central nesfatin-1 signaling regulates glucose metabolism and improves insulin resistance (IR) in the liver. However, its mechanism is poorly understood. Furthermore, it remains unresolved whether central nesfatin-1 can regulate liver lipid metabolism and its possible mechanism. The purpose of the present study is to investigate the effect of hypothalamic nesfatin-1 signaling on hepatosteatosis.

Materials and methods: Nesfatin-1 was infused into the intracerebroventricular (ICV) of NUCB2-/- rats for 14 days to explore the impact of central nesfatin-1 on energy expenditure and hepatic lipid metabolism. NUCB2-/- rats were infused tunicamycin and/or nesfatin-1 to assess the effects of pharmacological and genetic-induced ER stress in the hypothalamus on the activation of central nesfatin-1. The influence of up-regulation of TPTb1 on the central nesfatin-1 on hepatic lipid metabolism was evaluated. MK2206, a specific Akt inhibitor, was infused into NUCB2-/- rats to investigate whether Akt signaling is required for central nesfatin-1-regulated lipid metabolism. For investigating the effects of the vagus nerve and sympathetic nervous system on the central nesfatin-1 regulation of hepatic lipid metabolism, NUCB2-/- Rats were subjected to hepatic branch vagotomy (HVG) or sham surgery, or injected with saline or guanethidine.

Results: We found that nesfatin-1-infused NUCB2-/- rats had reduced food intake and increased energy expenditure compared to aCSF-infused NUCB2-/-rats. Chronic central treatment of nesfatin-1 inhibited the expression of genes related to fatty acid synthesis and ER stress and increased the phosphorylation level of Akt in the hypothalamus. Under a high fat diet, the co-infusion of ICV nesfatin-1 and Tunicamycin in NUCB2-/- rats increased hepatic TG content and the expression of genes regulating lipid metabolism, compared with ICV nesfatin-1 infusion alone. The overexpression of TPTb1 in the hypothalamus aggravates IR and counteracts the effect of central nesfatin-1, but does not lead to ES stress. Nesfatin-1-infused NUCB2-/- rats that underwent HVG attenuated the inhibition of central nesfatin-1 on liver lipid deposition, but there was no effect of guanethidine treatment. These data indicated that the vagus nerve mediated the role of ICV nesfatin-1 in hepatic lipid metabolism.

Conclusion: Our study reveals that central nesfatin-1 regulates hepatic lipid metabolism through a novel network including hypothalamic ER stress, PTP1B/Akt pathway, parasympathetic nervous system.

Supported by: NSFC(81670755)

Disclosure: M. Mokou: None.

23

Empagliflozin improves insulin sensitivity of the hypothalamus in humans with prediabetes

S. Kullmann1,2, R. Wagner3,2, J. Hummel1,2, C. Dannecker1,2, A. Vosseler3,2, L. Fritsche1,2, K. Kantartzis3,2, J. Machann3,2, H.-U. Haering2, A. Fritsche3,2, H. Preissl1,2, M. Heni3,2;

1Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Centre Munich at the University of Tuebingen, Tuebingen, 2German Center for Diabetes Research, Neuherberg, 3University of Tuebingen, Tuebingen, Germany.

Background and aims: Insulin action in the human brain reduces food intake, improves whole-body insulin sensitivity, and modulates adiposity. In most cases of obesity and diabetes, the brain becomes insulin resistant with impaired brain-derived modulation of peripheral metabolism. As treatment with the SGLT2-inhibitor empagliflozin not only improves glucose metabolism but also reduces body weight and cardiovascular risk, we hypothesized that improved brain insulin sensitivity could be involved.

Materials and methods: In this double blind study, 40 participants with prediabetes (according to ADA’s OGTT criteria) were 1:1 randomized to receive 25 mg empagliflozin qd or placebo (mean ± SD: age: 60 ± 9 years; BMI: 31.5 ± 3.8 kg/m²). Before and after 8 weeks of treatment, brain insulin sensitivity was assessed by functional MRI combined with the intranasal administration of insulin to the brain.

Results: In healthy persons, intranasal insulin administration significantly decreases cerebral blood flow in the hypothalamus. In the current study, volunteers with prediabetes were unresponsive to this, as insulin could not induce hypothalamic inhibition prior treatment. We identified a significant interaction between treatment and the hypothalamic response to insulin (p<0.05, corrected for multiple comparisons). Post hoc analyses showed that only participants on empagliflozin showed a significant insulin-induced decrease in hypothalamic blood flow after treatment. The group receiving placebo showed no such improvement.

Conclusion: Our current results corroborate insulin resistance of the human hypothalamus in humans with prediabetes. Treatment with empagliflozin for 8 weeks was able to restore hypothalamic insulin sensitivity; a favorable response that could contribute to the positive effects of SGLT2 inhibitors. These findings reveal that brain insulin resistance is treatable by pharmacological interventions with potential benefits for cognition, adiposity, and whole-body metabolism.

Clinical Trial Registration Number: NCT03227484

Supported by: BMBF to DZD

Disclosure: S. Kullmann: Lecture/other fees; Novo Nordisk. Other; The Study was supported by Boehringer Ingelheim through and independent Research Grant.

24

Brain insulin sensitivity is modulated by menstrual cycle

J. Hummel1,2, C. Benkendorff1,3, A. Vosseler1,3, L. Fritsche1,2, S. Kullmann1,2, A.L. Birkenfeld1,3, H. Preissl1,2, H.-U. Häring1,3, A. Fritsche1,3, A. Peter1,4, R. Wagner1,3, M. Heni1,3;

1Institute for Diabetes Research and Metabolic Diseases (IDM), Helmholtz Center Munich at the University of Tübingen, Tübingen, 2German Center for Diabetes Research (DZD e.V.), Neuherberg, 3Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital, Eberhard-Karls-University Tübingen, Tübingen, 4Institute for Clinical Chemistry and Pathobiochemistry,Department for Diagnostic Laboratory Medicine, University Hospital, Eberhard-Karls-University Tübingen, Tübingen, Germany.

Background and aims: Insulin action in the human brain modulates whole-body metabolism by increasing peripheral insulin sensitivity and suppressing endogenous glucose production. In brain insulin resistance, this modulatory function of the brain is impaired. Experimental evidence from rodents and first observations in humans suggest major differences in brain insulin action between women and men. We therefore investigated effects of brain insulin delivery on peripheral metabolism in the follicular and luteal phase of the menstrual cycle.

Materials and methods: Eleven natural cycling women (age: 19-29 years, BMI: 17.8 - 23.8 kg/m²) underwent four hyperinsulinemic-euglycemic clamps. On two days during both the follicular and the luteal cycle phase, participants received intranasal insulin spray and placebo 90 min after initiation of the 210 min clamp in randomized, blinded order. Insulin spillover into the blood was mimicked by an appropriate iv insulin bolus on placebo days.

Results: The phase of the menstrual cycle interacted with the type of spray on the subsequent change of glucose infusion rates (GIR) (p=0.01). During the follicular phase, more glucose had to be infused after insulin delivery to the brain as nasal spray compared to placebo administration (p<0.0001; β=0.40±0.06). This difference remained significant after adjustment for plasma glucose and insulin concentrations (p<0.0001). During the luteal phase, GIR were only transiently higher after nasal insulin (p=0.005; β=0.16±0.06), but this depended on the circulating glucose and insulin levels as the difference was no longer present after respective adjustments (p=0.1).

Conclusion: In the follicular phase of the menstrual cycle, insulin delivery to the brain improves peripheral insulin sensitivity in lean women, comparable to what has previously been described in lean men. However, this response was not detected in the luteal phase, suggesting brain insulin resistance. Brain insulin resistance could therefore contribute to the long known peripheral insulin resistance in the luteal phase of the menstrual cycle.

Clinical Trial Registration Number: NCT03929419

Supported by: BMBF to DZD

Disclosure: J. Hummel: None.

OP 05 Glucose-lowering therapies and the liver

25

Role of bile acids on glucose-lowering by metformin in type 2 diabetes

D.J. Sansome, S. Veedfald, C. Xie, M. Bound, J. Grivell, K.L. Jones, M. Horowitz, C.K. Rayner, T. Wu;

Adelaide Health and Medical Sciences (AHMS) building, The University of Adelaide, Adelaide, Australia.

Background and aims: Emerging evidence indicates a key role of the gastrointestinal tract in the glucose-lowering action of metformin. We recently reported that glucose-lowering by metformin is associated with greater plasma concentrations of glucagon-like peptide-1 (GLP-1), and stimulation of GLP-1 secretion appears integral to glucose-lowering by small intestinal administration of bile acids. Given that metformin inhibits intestinal bile acid reabsorption, we examined whether the glucose-lowering effect of metformin was attenuated when endogenous bile acids were excluded (by balloon occlusion and aspiration), or potentiated by intrajejunal infusion of taurocholic acid (TCA), in type 2 diabetes (T2DM).

Materials and methods: Thirteen diet-controlled participants with T2DM (12 male; 59.9 ± 3.0 years; BMI 30.4 ± 1.1 kg/m2; HbA1c 6.2 ± 0.2%; duration of known diabetes 3.6 ± 0.8 years) were each studied on four occasions in a randomised order, following an overnight fast. On each day, a multilumen catheter was inserted transnasally and positioned so that an occlusive balloon and aspiration channel were located in the distal duodenum, and an infusion channel in the proximal jejunum. Subsequently, the balloon was inflated (for exclusion and aspiration of endogenous bile) on two days, or left deflated. At t=-60 min, metformin (1 g in 50mL water) or placebo (0.9% saline) was infused into the jejunum over 5 min. Then TCA (4 g TCA in 240 mL 0.9% saline), or 0.9% saline (240mL) alone, was infused into the jejunum (120mL between t=-30-0 min, then 120 mL between t=0-120 min), with concurrent intrajejunal glucose infusion (2kcal/min) between t=0-120 min. “Arterialised” venous blood was sampled every 30 min between t=-60-180 min for measurement of plasma glucose. Data are means ± SEM. One-factor ANOVA with Bonferroni’s correction for post hoc comparisons was used for statistical analysis.

Results: Fasting plasma glucose did not differ between study days. Following intrajejunal glucose infusion, there was an overall treatment effect on the incremental area under the plasma glucose curve (iAUC) between 0-180 min (P < 0.0001). When endogenous bile was excluded, metformin reduced the plasma glucose iAUC compared to placebo (506 ± 39 vs 343 ± 51 mol/L.min; P = 0.014). When endogenous bile was included, supplementation with TCA had no effect on the plasma glucose response to metformin (iAUC 568 ± 38 vs 516 ± 46 mmol/L.min; P = 0.076). During metformin treatment, plasma glucose was lower when endogenous bile was excluded than when included (iAUC 343 ± 51 vs 516 ± 46 mmol/L.min; P=0.007).

Conclusion: Contrary to the hypothesis, endogenous bile was not required for glucose-lowering by metformin, nor did supplementation with exogenous bile acids augment the effect of metformin on plasma glucose, during small intestinal glucose infusion in T2DM.

figured

Clinical Trial Registration Number: ACTRN12619000299101

Supported by: Diabetes Australia and THRF Mid-Career Fellowship

Disclosure: D.J. Sansome: None.

26

Metformin acutely elevates lactate in the portal vein of humans

N. Rittig1, E. Sundelin2, H. Grønbæk3, N.K. Aagaard3, T. Sandahl3, G. Villadsen3, K. Brøsen4, N. Jessen1;

1Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus N, 2Department of Diabetes and Hormone Diseases, Aarhus University Hospital, Aarhus N, 3Department of Gastro- and Hepatology, Aarhus University Hospital, Aarhus N, 4Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.

Background and aims: Metformin is widely used worldwide, but its underlying mechanisms of action remain debated. Studies in rodents indicate that Metformin increases intestinal lactate concentrations. We aimed to investigate whether this also translates into humans.

Materials and methods: We included eight cirrhotic participants with a portosystemic shunt system that allowed us to place intravenous catheters in the portal vein. Venous blood from the portal vein and peripheral arterialized blood was obtained simultaneously before and consecutively 90 minutes following oral consumption of 1000 mg Metformin.

Results: Lactate concentrations increased with 23% {CI95%: 6 to 40%} in the portal vein and 2% {CI95%: -15 to 19%} in arterialized blood 90 minutes following Metformin treatment (see figure, two-way repeated measure ANOVA, sampling site x time interaction, p= 0.001). Serum and plasma concentrations of glucose, insulin, and C-peptide all decreased during the 90 minutes (main effect of time, p<0.05) with no difference between sampling sites.

Conclusion: Metformin increases lactate concentrations in the gut of humans. These results emphasize and support the notion that Metformin exert important effects in the intestinal tract that warrants further investigations.

figuree

Clinical Trial Registration Number: EudraCT ID 2017-001132-19

Supported by: We thank the Aase and Ejnar Danielsen Foundation for their support (10-002192)

Disclosure: N. Rittig: None.

27

Metformin increases GDF15 independent of plasma metformin exposure and its proposed action in the liver

K.J. Kolnes1, P.M. Møller1, R. Kruse1,2, M.M.H. Christensen3, A. Handberg4,5, K. Højlund1,2;

1Steno Diabetes Center Odense, Odense University Hospital, Odense, 2Department of Clinical Research, University of Southern Denmark, Odense, 3Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, 4Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, 5Department of Clinical Medicine, Aalborg University, Aalborg, Denmark.

Background and aims: The antidiabetic drug metformin increases plasma GDF15, and this may explain its ability to lower body weight in humans. The glucose-lowering effect of metformin has been suggested to involve inhibition of the mitochondrial respiratory complex I in liver cells. Although mitochondrial dysfunction induces both GDF15 and FGF21 by activation of the integrated stress response (IRS), the tissue responsible for the metformin-induced increase in plasma GDF15 and whether this includes increased secretion of FGF21 remain to be investigated. Here, we examined the effect of metformin on circulating GDF15 and FGF21 levels in the glycogen-depleted state of a prolonged fasting in vivo, and on the expression of GDF15 and FGF21 in human hepatocytes in vitro.

Materials and methods: In a randomized, crossover trial, 34 healthy individuals completed a 42-h fast twice, either with or without prior treatment with 1 g metformin twice daily for a week. Glucose metabolism was assessed for 6-hours using [3-3H]-glucose and indirect calorimetry, and blood samples were analyzed for serum GDF15 and FGF21 at the end of the tracer study, and for plasma metformin for additional 6 hours. Moreover, we tested the in vitro effects of metformin (2 mM) for up to 24-h on the expression of GDF15 and FGF21 in human hepatocytes (HepG2).

Results: Metformin increased glucose disposal (P < 8 x 10-13) due to increased glycolytic flux (P < 2 x 10-11). This was accompanied by increased hepatic glucose production (P < 3 x 10-13) caused by increased counter regulatory hormones (P < 0.05). Under these conditions, metformin increased serum GDF15 (607 ± 89 vs 1004±61 ng/mL; P<0.001), whereas serum FGF21 (146 ± 30 vs 156 ± 29 ng/mL; P=0.65) was unaltered. The change in serum GDF15 was not related to the maximal or area under the curve (AUC) concentrations of plasma metformin or changes in measures of glucose metabolism (see above). In contrast to the absent effect of metformin treatment on serum FGF21 in vivo, metformin treatment in vitro markedly increased mRNA levels of both GDF15 (4-fold; P<0.001) and FGF21 (12-fold; P<0.001) in human hepatocytes.

Conclusion: We demonstrate that the metformin-induced increase in serum GDF15 is dissociated from plasma metformin exposure and its proposed inhibitory action on hepatic glucose production. Moreover, the lack of a metfomin-induced increase in serum FGF21 in vivo despite metformin’s ability to increase expression of both GDF15 and FGF21 in human liver cells in vitro, support recent work indicating that the liver may not be the primary site of metformin action on GDF15 release.

Clinical Trial Registration Number: NCT01400191

Supported by: Novo Nordisk Foundation

Disclosure: K.J. Kolnes: None.

28

Acute effects of dapagliflozin on hepatic lipid- and glucose metabolism in humans

P. Wolf1, P. Fellinger1, H. Beiglböck1, L. Pfleger1, P. Krumpolec1, C. Barbieri2, A. Gastaldelli2, R. Marculescu1, S. Trattnig1, A. Kautzky-Willer1, M. Krssak1, M. Krebs1;

1Medical University of Vienna, Vienna, Austria, 2Cardiometabolic Risk Unit, Institute of Clinical Physiology, Pisa, Italy.

Background and aims: Recent studies indicate that administration of SGLT-2 inhibitors paradoxically increases endogenous glucose production (EGP), potentially counteracting the glucose lowering potency of these drugs. So far acute effects of SGLT-2 inhibition on hepatic glycogen, lipid and energy metabolism are unknown. Therefore we aim to investigate the impact of a single dose of dapagliflozin (D) or placebo (P) on hepatic glycogenolysis, lipid content (HCL) and mitochondrial activity (kATP).

Materials and methods: 10 healthy volunteers (CON:age30±3years;BMI24±1kg/m2; HbA1c5.2±0.1%) and 6 patients with type 2 diabetes mellitus (T2DM:age63±4years; BMI28±1.5kg/m2,HbA1c 6.1±0.5%) were investigated on two study days (CON-PvsCON-D/ T2DM-PvsT2DM-D) in a double blinded randomized controlled setting. 1H/13C/31P magnetic resonance spectroscopy was performed before (-120-30min), 90-180min and 300-390 min after administration of 10mg dapagliflozin or placebo. EGP was assessed by tracer dilution techniques.

Results: EGP was 25% higher following administration of dapagliflozin (p<0.001) and strongly correlated with glucosuria (P=0.867; p<0.01).Hepatic glycogen concentrations were comparable at baseline in CON (CON-P:227±20 mM vs CON-D:212±18 mM; p=n.s.) and T2DM (T2DM-P:200±10mM vs T2DM-D:196±9 mM; p=n.s). The observed decrease in glycogen was about five times higher in CON-P vs T2DM-P (-30±0.06% vs -6±0.02%;p<0.001) and non-significantly higher in CON-D vs T2DM-D (-20±0.06% vs -10±0.02%;p=0.591). Dapagliflozin had no impact on glycogenolysis in both groups. HCL and kATP were significantly higher in T2DM at baseline. HCL increased by 20% non-significantly in CON-D and T2DM-D. No significantly different changes in kATP between were observed during the study days.

Conclusion: The rise in EGP following SGLT-2 inhibition is mainly due to increased gluconeogenesis, but not due to accelerated glycogen breakdown. HCL and kATP are not affected by a single dose of dapagliflozin.

Clinical Trial Registration Number: NCT02558270

Supported by: Unrestricted research grant by AstraZeneca

Disclosure: P. Wolf: None.

29

Pleiotropic effects of sodium-glucose cotransporter 2 inhibitor versus sulfonylurea in patients with type 2 diabetes and non-alcoholic fatty liver disease

Y. Takeshita, Y. Kita, T. Takamura;

Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan.

Background and aims: We previously investigated the histological course of serial liver biopsy samples of patients with NAFLD in a real-world clinical setting. The clinicopathological analyses revealed that a reduction in HbA1c and the use of insulin independently contribute to the reduction in liver fibrosis scores during the course of nonalcoholic fatty liver disease (NAFLD) development (Diabetes Care 2010). These findings led us to hypothesize that glycemic control and insulin therapy ameliorate or protect against the histological progression of liver fibrosis in patients with NAFLD. To test this hypothesis, we aim to compare the effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors and sulfonylureas, which lower glucose levels with decreases and increases in circulating levels of insulin, respectively, in patients with type 2 diabetes.

Materials and methods: This study is a 48-week, one-center, open-label, randomized, parallel trial. Patients who satisfied the eligibility criteria were randomly assigned (1:1) to receive once-daily 20 mg tofogliflozin or 0.5 mg glimepiride. The sample size was calculated to be 14 in each group with a significance level of 0.05 and a power of 0.90. The design required 40 evaluable patients in this study. The primary endpoint of this study will be the improvement in liver histology between liver biopsies at baseline and after 48 weeks of treatment. I will announce the interim analysis (N=20) in this meeting. The study is being conducted with the approval of the Certified Review Board, in accordance with the guidelines of the Declaration of Helsinki.

Results: Recruitment into this study started in November 2015 and will end in September 2020, with 40 patients randomized into the two groups. The 20 study patients (mean age 55.3 ± 14.0 years, mean HbA1c 8.2 ± 0.7%, mean body weight 80.0 ± 18.0 kg, mean AST 52.8 ± 43.4 IU/L, mean ALT 74.3 ± 70.4 IU/L) included and randomized. After 48 weeks, the changes in steatosis score (from 2.0 ± 0.7 to 1.2 ± 0.4, P=0.008), grade score (from 1.7 ± 0.9 to 0.9 ± 0.8, P=0.004) and stage score (from 1.6 ± 1.1 to 1.1 ± 1.3, P=0.035) were significantly improved in the tofogliflozin group compared with the glimepiride group. HbA1c was significantly decreased in both groups (tofogliflozin: from 8.0 ± 0.5 to 6.9 ± 1.2 %, P=0.029;glimepiride from 8.4 ± 0.9 to 7.5 ± 0.6%, P=0.020). Bodyweight and liver enzymes were significantly decreased in the tofogliflozin group (BW from 78.7 ± 17.7 to 73.3 ± 18.3 kg, P=0.001, AST from 55.4 ± 41.8 to 27.2 ± 22.4 IU/L, P=0.006, ALT from 72.2 ± 60.5 to 33.1 ± 31.5 IU/L, P=0.013), but not in the glimepiride group (BW from 81.3 ± 19.2 to 81.9 ± 21.1 kg, AST from 49.7 ± 46.8 to 52.3 ± 37.6 IU/L, ALT from 76.4 ± 77.8 to 78.9 ± 68.7 IU/L). The lipid profile was not changed in both groups.

Conclusion: Both groups (tofogliflozin and glimepiride) equally lower the HbA1c levels. However, tofogliflozin not glimepiride significantly lower the body weight, liver enzymes, and histology scores.

Clinical Trial Registration Number: jRCTs 041180132, NCT02649465

Disclosure: Y. Takeshita: None.

30

A dietary intervention to alter insulin sensitivity, intramyocellular and hepatocellular lipids, postprandial metabolism, and body weight: a 16-week randomised trial

H. Kahleova1, K.F. Petersen2, G.I. Shulman2,3, J. Alwarith1, E. Rembert1, A. Tura4, M. Hill5, R. Holubkov6, N.D. Barnard1;

1PCRM, Washington, USA, 2Yale School of Medicine, New Haven, USA, 3Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, USA, 4CNR Institute of Neuroscience, Padova, Italy, 5Institute of Endocrinology, Prague, Czech Republic, 6School of Medicine, University of Utah, Salt Lake City, USA.

Background and aims: Excess body weight and insulin resistance lead to type 2 diabetes and other major health problems. There is an urgent need for dietary interventions to address these conditions and for greater clarity in how dietary interventions work.

Materials and methods: Participants (n=244) were randomly assigned to an intervention group (n=122), which was asked to follow a low-fat vegan diet for 16 weeks or to a control group (n=122) making no diet changes for 16 weeks. Before and after the intervention period, body composition and visceral fat were measured by dual X-ray absorptiometry. Insulin resistance was assessed with the Homeostasis Model Assessment (HOMA-IR) index and predicted insulin sensitivity index (PREDIM). Thermic effect of food was measured by indirect calorimetry. In a subset of participants (n=44), hepatocellular and intramyocellular lipids were quantified by 1H magnetic resonance spectroscopy. Repeated measure ANOVA was used for statistical analysis.

Results: Body weight decreased by 6.4 kg in the intervention group and 0.5 kg in the control group (-5.9 kg [95% CI -6.7 to -5.0]; p<0.001). Thermic effect of food increased in the intervention group by 14.1% ([95% CI +6.5 to +20.4]; p<0.001). HOMA-IR index decreased (-1.3 [95% CI -2.2 to -0.3]; p<0.001), and PREDIM increased (+0.9 [95% CI +0.5 to +1.2]; p<0.001) in the intervention group. Hepatocellular and intramyocellular lipids decreased by 34.4% and 10.4% in the intervention group (p=0.002; and p=0.03, respectively). None of these variables changed significantly in the control group. The change in PREDIM correlated negatively with the change in body weight (r=-0.43; p<0.001). Changes in hepatocellular lipid and intramyocellular lipid correlated strongly with changes in insulin resistance (both r=+0.51; p=0.01). In both groups combined, changes in intramyocellular lipids correlated with fat mass changes (r=+0.51; p<0.05) and HOMA-IR (r=+0.52; p<0.05).

Conclusion: A low-fat plant-based dietary intervention reduces body weight by reducing energy intake and increasing postprandial metabolism, apparently due to increased insulin sensitivity resulting from reduced hepatocellular and intramyocellular fat.

Clinical Trial Registration Number: NCT02939638

Supported by: Yale Diabetes Center P30 DK-045735 and R01 DK-113984

Disclosure: H. Kahleova: None.

OP 06 Uncomplicating the pathogenesis of diabetes complications in humans

31

Small RNA-seq reveals a specific circulating miRNA signature linked to the type 2 diabetes complications

A. Abukiwan1, T. Fleming1, R. Thiele2, S. Kopf1, P. Nawroth1;

1Department of Endocrinology and Metabolism, Heidelberg University Hospital, Heidelberg, 2Heidelberg University, Heidelberg, Germany.

Background and aims: MicroRNAs (miRNAs) have been shown to play an important role in the pathogenesis of type 2 diabetes (T2D), and their circulating levels have appeared as potential biomarkers for the development and progression of the disease. However, few studies have examined whether a microRNA signature exists between the different diabetic complications and their multiple mechanisms including inflammatory response, cellular senescence, and glucose metabolism. The objective of this study was to identify whether such a signature exists.

Materials and methods: We used small RNA-sequencing analysis to identify differential plasma miRNAs in patients with type 2 diabetes (n=23), patients with type 2 diabetes with complications (n= 93), (nephropathy, neuropathy, liver fibrosis, and lung fibrosis), and healthy controls (n=31).

Results: RNA-seq analysis identified 20 differential circulating miRNAs between the healthy andT2D patients, more than 54 miRNAs significantly deregulated between T2D and those with complications, and miRNA panel consisting of miR-200c-3p, miR-378c, miR-22-3p, let-7c-3p, miR-181d, miR-148a, miR-215-3p and, miR-9-5p had a distinct expression signature between T2D complications subgroups. GO analysis revealed that 50 cellular processes and pathways were significantly enriched by genes targeted by these 8 miRNAs. The reactive oxygen process was enriched (P < 0.0007) by more than 40 genes targeted by all 8 miRNAs. In addition, four other pathways, TGFß signaling, AGE-RAGE signaling pathway, cell cycle, and cellular senescence signaling, were also strongly enriched in significant nuclear processes including DNA damage, RNA transcription, and binding.

Conclusion: Our study identified a 8 miRNA signature capable of discriminating between all T2D complications and diabetes. Aberrant regulation of this miRNA signature, which is associated with underlying pathophysiology mechanisms, may contribute to the development of complications which arise in the T2D state.

Supported by: BMBF and SFB1118-A04

Disclosure: A. Abukiwan: Grants; BMPF and SFB1118-A04.

32

Downregulation of spingosine 1-phosphate receptor might be protective against vascular complications in people with long-term type 1 diabetes

T. Özgümüs1, T.J. Berg2, V. Lyssenko1;

1Department of Clinical Science, University of Bergen, Bergen, 2Institute of Clinical Medicine, University of Oslo, Oslo, Norway.

Background and aims: Individuals with long-term type 1 diabetes (T1D) who have remained largely free from vascular complications represent an important population for investigation of natural mechanisms protecting tissues and organs in the diabetic environment. The protective factors in these patients may provide insight for further improvement of current treatments or even prevention. In this study we investigated the underlying differences between people with long-standing T1D with and without vascular complications and age-matched non-diabetic individuals.

Materials and methods: The Dialong cohort included people with long-term T1D (n=62) and non-diabetic controls (n=34). We investigated differences in blood transcriptomic profiles using RNA sequencing method. The T1D patients were classified into non-progressors (NP, n=34) who have duration longer than 30 years without developing any complications and rapid-progressors (RP, n=28) who developed vascular complications (retinopathy, nephropathy or cardiovascular diseases) within 25 years of disease duration. RNA content of whole blood samples from patients were sequenced. The alignment of the transcripts and differential expression analysis were done using Kallisto 0.43.1 and DESeq2 1.24.0, respectively. P values were corrected for multiple testing using FDR method.

Results: In age-adjusted analysis, there were 22 genes found to be significantly differentially expressed between NP and RP groups. The downregulated genes in NP group consist of immune factors such as islet cell autoantigen (ICA1), RNASE3, CD33 and the receptor for sphingosine-1-phosphate (S1PR3). Between NPs and controls, there were about 600 genes found to be differentially expressed, of which 91% were downregulated in NPs. These genes were enriched in GO-BP term of regulation of transcription (padj = 4.3E-05). More than 1000 genes were found to be differentially expressed between RPs and controls, the downregulated genes (~71%) were enriched in same GO-BP term similar to NP vs controls (padj = 1E-11) while the upregulated genes (29%) were enriched in glycospingolipid metabolic process (padj=0.04). In a previous study of another cohort (PROLONG), we observed slightly reduced oxidative phosphorylation (OXPHOS) in the non-progressors compared with rapid progressors, which might have a protective effect via reduced mitochondrial function and reduced DNA damage. In the current study we found that the log-fold-changes of OXPHOS genes for the same comparison group are correlated with log-fold-changes in the previous study (cor=0.64, p<0.0001).

Conclusion: The overexpression of immune factors is associated with and may contribute to increased risk of vascular complications, while downregulation of spingosine 1-phosphate receptor might have protective effects against vascular complications in people with long-term T1D.

Supported by: UiB, VK, TMS, NN

Disclosure: T. Özgümüs: None.

33

The glycolytic by-product methylglyoxal is present in immune cells and may affect their recruitment

X. Zhang1, N. Hanssen1, A. Bektić1, M. van Oeteren1, J. Scheijen1, M. Streeter2, J. van de Gaar1, D. Spiegel2, K. Wouters1, C. Schalkwijk1;

1Department of Internal Medicine, Maastricht University, Maastricht, Netherlands, 2Department of Chemistry, Yale University, New Haven, USA.

Background and aims: The reactive dicarbonyl compound methylglyoxal (MGO), is mainly formed as a byproduct of glycolysis and is increased in diabetes. The formation and accumulation of MGO are linked to multiple cardiometabolic diseases. Immune cell activation is involved in cardiometabolic disease development and leads to a switch to glycolysis for their energy demand. Hence, immune cell activation may lead to formation of MGO. We investigated whether MGO is formed in immune cells and whether MGO affects immune cell function.

Materials and methods: MGO was measured in human blood fractions and circulating immune cell fractions using ultra-performance liquid chromatography-tandem mass spectrometry. Oral glucose tolerance tests (OGTT) were performed in 20 abdominal obese individuals, and a fluorescent probe was used to track MGO in immune cells using flow cytometry. In mice, we injected an iv bolus of 25μg highly purified MGO in 10 minutes, 24h or 72h prior to sacrifice. Immune cell numbers were studied in blood and tissues using flow cytometry and immunohistochemistry, respectively.

Results: In human, about 50% of whole blood MGO content (3.61±1.04 μM) was confined to leukocytes (2.02±0.7 μM), whereas MGO levels in plasma (0.12±0.03 μM) and platelets (0.007±0.004 μM) were very low. Purified leukocyte subsets showed a very high cellular MGO concentrations in lymphocytes (4409±1095μM) followed by monocytes (2804±2455μM) and granulocytes (1683±437μM). An increased glucose load during OGTT resulted in increased MGO levels in these immune cell fractions. In mice, injection of highly purified MGO resulted in a 7-fold increase of circulating neutrophils after 10 min compared to PBS injection, which normalized after 24h. We observed similar results in the liver with fast 9-fold increase of hepatic neutrophils 10 min post injection. CD11b expression (as a marker for neutrophils and macrophages) in the aorta showed a sharp decrease (19.9 fold) after 10 min, which normalized after 24h, suggesting a rapid release of the neutrophils adhered to the endothelium, or marginated neutrophil pool. MGO spiking also decreased circulating Ly6chi monocytes after 72h (- 40%), while macrophage numbers in the liver were increased (35.7±6.5 vs 24.2±6.8 macrophages per microscopic view).

Conclusion: MGO is present in a high concentration in lymphocytes, granulocytes, and monocytes, is further increased during an OGTT and may affect immune cell recruitment, possibly via vascular release of the marginated neutrophil pool.

Disclosure: X. Zhang: None.

34

Role of circulating Wnt1 inducible signalling pathway protein 1 (WISP1) in liver and adipose tissue fibrosis

O. Pivovarova-Ramich1,2, J. Loske1, S. Hornemann1, M. Markova1,2, N. Seebeck1,3, A. Rosenthal4, J. Raila3, R. Buschow5, V. Lange6, A.F.H. Pfeiffer1,7, N. Rudovich1,8, M. Ouwens2,9;

1German Institute of Human Nutrition, Nuthetal, Germany, 2German Center for Diabetes Research (DZD), Munich-Neuherberg, Germany, 3University of Potsdam, Institute of Nutritional Science, Nuthetal, Germany, 4Clinic for Nutritional Medicine, Berlin, Germany, 5Max Planck Institute for Molecular Genetics, Berlin, Germany, 6Helios Klinikum Berlin-Buch, Berlin, Germany, 7Charité-Universitätsmedizin Berlin, Berlin, Germany, 8Spital Bülach, Bülach, Switzerland, 9German Diabetes Center, Duesseldorf, Germany.

Background and aims: We recently identified Wnt1 inducible signaling pathway protein 1 (WISP1) as a novel proinflammatory adipokine that was associated with visceral obesity and insulin resistance in humans. Wnt signaling plays an active role in determining tissue fibrosis and remodeling in different pathological conditions. A role of WISP1 in the development of fibrosis was demonstrated in mouse lung and kidney, but whether it has an effect in liver and adipose tissue fibrosis associated with obesity and T2DM is unknown. The aim of present study was to investigate the role of WISP1 in liver and adipose tissue fibrosis in severe obesity.

Materials and methods: Human liver, visceral (VAT) and subcutaneous (SAT) adipose tissue collected from 35 severely obese humans (BMI 42.5 ± 0.7 kg/m2, age 46.7 ± 1.8 y) during bariatric surgery were examined for WISP1, fibrosis and inflammation markers by quantitative real time PCR and histological image analysis. Plasma samples were analyzed by commercial ELISA assays. Hepatic stellate LX-2 cells were treated with human recombinant WISP1 (10, 100, 500 ng/ml) alone or in combination with 1 ng/ml LPS or 1 ng/ml transforming growth factor beta (TGF-β) for 24h.

Results: In the liver, WISP1 mRNA expression positively correlated with BMI and with the expression of fibrosis markers COL1A1 (r = 0.652; p<0.001), COL3A1 (r = 0.579; p<0.001), COL6A1 (r = 0.645; p<0.001), alpha-SMA (r = 0.380; p=0.029), TGFB1 (r=0.500, p=0.003), as well as TIMP1 (r = 0.554; p<0.001) and MMP9 (r = 0.526; p<0.001), two key enzymes in the regulation of extracellular matrix turnover. In adipose tissue, WISP1 expression was strongly correlated with TIMP1 expression in SAT and VAT (r=0.607, p<0.001 and r=0.343, p=0.043, respectively) and with α-SMA in SAT (r=0.406, p=0.015). Circulating WISP1 levels showed no association with BMI and no differences between subjects with or without NASH and between subjects with different NAFLD activity score as determined histologically. In LX-2 cells, exposure to WISP1 caused a dose-dependent induction of MMP-9 and MCP-1. WISP1 potentiated the TGF-β-mediated induction of COL3A1, TIMP1 and MCP-1 and showed no interaction with LPS treatment.

Conclusion: Our results showed a contribution of WISP1 in the development of obesity-associated fibrosis and inflammation in the liver and adipose tissue and thus characterized WISP1 as a potential target in obesity and diabetes therapy.

Clinical Trial Registration Number: DRKS00009509

Supported by: DZD OP-R, MO; EFSD NR; DDG OP-R

Disclosure: O. Pivovarova-Ramich: None.

35

Insulin resistance and altered fibrin clot properties in overweight individuals with type 1 diabetes: A potential mechanism for increased vascular complications?

N. Kietsiriroje, S.M. Pearson, R.A.S. Ariëns, R.A. Ajjan;

Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK.

Background and aims: Overweight individuals with Type 1 diabetes (T1D) have a higher risk of vascular complications by mechanisms that remain unclear. Given that fibrin clot characteristics can determine vascular risk, we investigated fibrin network formation and lysis in those with different degree of insulin resistance, measured as estimated glucose disposal rate (eGDR).

Materials and methods: A validated turbidimetric assay and confocal microscopy were employed to study plasma-derived fibrin clot properties in 41 patients with T1D. Fibrinogen concentration in all plasma samples was measured by Clauss technique. HbA1c, body mass index and presence of hypertension were used to calculate eGDR using the following formula: eGDR = 19.02 - [0.22 x BMI(kg/m2) - [3.26 x presence of hypertension(1=yes, 0=no)] - [0.61 x HbA1c(%)]. Patients were catergorised into 3 tertiles by eGDR values for the comparisons of clot maximum turbidity, clot lysis time and fibrinogen levels. To investigate the effects of quantitative and qualitative changes in fibrinogen on the fibrin network, plasma levels of the protein were measured, and clots were also made from plasma-purified fibrinogen of the individuals studied.

Results: Clot maximum absorbance, a measure of clot density, was highest in the low eGDR tertile compared to the middle (T2) and high tertiles (T3) (0.21±0.07, 0.17±0.05 and 0.16±0.05 AU, respectively; p = 0.02, T1 vs T3). Similarly, clot lysis time, an indicator of fibrinolysis potential, was longest in low eGDR tertile compared with middle and high (1016±398, 726±374 and 680±190 sec, respectively; p=0.01, T1 vs T3). Plasma fibrinogen levels were similar in the three tertiles of eGDR, however, clots made from concentrated fibrinogen purified from pooled plasma of patients with eGDR<7 were denser than those with eGDR>8 (17.3±1.8 vs 12.9±2.0 fibers/100μm; p=0.01).

Conclusion: Insulin resistance is associated with prothrombotic changes in fibrin clot phenotype in individuals with T1D, related, at least in part, to qualitative changes in the fibrinogen molecule. Altered fibrin clot properties may be one mechanism for the increased risk of complications in overweight individuals with T1D.

figuref

Supported by: Faculty of Medicine, Prince of Songkla University, Thailand

Disclosure: N. Kietsiriroje: None.

36

Chronic complications versus glycaemic variability, time in range and HbA1c in people with type 1 diabetes: sub study of the RESCUE-trial

A. El Malahi1, M. Van Elsen1, S. Charleer2, F. De Ridder1,3, K. Ledeganck3, B. Keymeulen4, L. Crenier5, R. Radermecker6, B. Lapauw7, C. Vercammen8, F. Nobels9, C. Mathieu2, P. Gillard2, C. De Block1,3;

1Endocrinology-Diabetology, University Hospital Antwerp, Edegem, 2Endocrinology, University Hospitals Leuven - KU Leuven, Leuven, 3Laboratory of experimental medicine and paediatrics, University of Antwerp, Antwerp, 4Diabetology, University Hospital Brussels, Brussels, 5Endocrinology, Université Libre de Bruxelles – Hôpital Erasme, Brussels, 6Diabetes, Nutrition and Metabolic disorders, CHU Liège, Liège, 7Endocrinology, Ghent University Hospital, Ghent, 8Endocrinology, Imelda Hospital, Bonheiden, Belgium, 9Endocrinology, OLV Hospital Aalst, Aalst, Belgium.

Background and aims: So far, HbA1c is the only metric of glucose control showing a strong association with chronic complications. However, it does not reflect short-term glycemic variability nor provides guidance in decreasing risk of hypoglycemia. More widespread use of continuous glucose monitoring (CGM) has changed the way people with type 1 diabetes (T1D) manage their glycemia by providing information about glycemic variability and time spent in different glucose ranges.

Materials and methods: Parameters that could have a link with diabetes complications were analyzed of 515 adults with T1D who entered the Belgian reimbursement system for real-time CGM (rtCGM): HbA1c, standard deviation (SD), coefficient of variation (%CV), time in range (TIR, 70-180 mg/dL), age, diabetes duration, BMI, and gender. Association between glucometrics from the first 2 weeks of rtCGM use and presence of the following diabetes complications at start were investigated with multiple logistic regression: composite microvascular complications (defined as presence of at least 1 of the following: peripheral or autonomic neuropathy, retinopathy, nephropathy), macrovascular complications, and hospitalization for hypoglycemia and ketoacidosis.

Results: Diabetes duration (OR=1.12, P<0.001) and TIR (OR=0.97, P=0.005) were independently correlated with composite microvascular complications. For nephropathy, diabetes duration (OR=1.08, P<0.001) and HbA1c (OR=1.65, P=0.012) were independently associated. For retinopathy it were diabetes duration (OR=1.14, P<0.001) and TIR (OR=0.96, P<0.001). For peripheral and autonomic neuropathy it were diabetes duration (OR=1.09, P<0.001; OR=1.08, P<0.001) and SD (OR=1.03, P=0.026; OR=1.035, P=0.015). Age (OR=1.08, P=0.003) and HbA1c (OR=1.80, P=0.044) were independently correlated with macrovascular complications. Only TIR (OR=0.97, P=0.021) was independently associated with hospitalization for hypoglycemia or ketoacidosis.

Conclusion: Shorter TIR was associated with the presence of composite microvascular complications, and with retinopathy in particular. A higher SD was linked to peripheral and autonomic neuropathy. For hospitalization due to hypoglycemia or ketoacidosis, TIR was the most important factor.

figureg

Clinical Trial Registration Number: NCT02601729

Disclosure: A. El Malahi: None.

OP 07 Smoke on the water: Is BAT still hot?

37

Blocking endothelial ROCK2 promotes fat browning and improves metabolic dysfunction

Y. Takeda1, K. Matoba1, D. Kawanami2, Y. Nagai1, Y. Kanazawa1, T. Yokota1, K. Ustunomiya3, R. Nishimura1;

1Division of Diabetes, Endocrinology, and Metabolism Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, 2Department of Endocrinology and Diabetes Mellitus, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku Fukuoka, 3Center for Preventive Medicine, The Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Japan.

Background and aims: Intraperitoneal fat accumulation is considered an important risk factor for the impaired glucose tolerance, dyslipidemia, and coronary heart diseases. However, underlying mechanisms the excess lipid storage are not fully understood. The small GTPase Rho and its downstream effector, Rho-kinase (ROCK), regulate various cellular functions, including organization of the actin cytoskeleton, cell adhesion and gene expression. Studies have shown that Rho/ROCK signaling is implicated in the pathogenesis of diabetic vascular complications. ROCK has two isoforms, ROCK1 and ROCK2. Systemic gene deletion studies in mice suggest that these isoforms have distinctive roles in regulating cellular function. In this study, we investigated specific roles of endothelial ROCK2 in the progression of obesity.

Materials and methods: To examine the in vivo role of endothelial ROCK2, we generated endothelial-ROCK2 knockout mice (ER2KO) by breeding ROCK2 floxed mice with mice expressing VE-cadherin-cre recombinase.

Results: ER2KO mice are resistant to both weight gain and glucose intolerance induced by high fat diet. White adipose tissue (WAT) weight was lower in ER2KO mice compared with wild-type mice. Histological analysis revealed that adipose droplets were smaller in ER2KO than wild-type mice. Browning, the conversion of WAT to a beige phenotype, activates thermogenic function, suppresses obesity and improves glucose and lipid metabolism. Interestingly, we observed an increase of mRNA expression of browning marker including PPARα, CIDEA, PRDM16, UCP1, and specific markers of M2 macrophages in WAT obtained from ER2KO mice, regardless of whether they had been fed a normal chow or high fat diet.

Conclusion: Endothelial ROCK2 regulates glucose and lipid metabolism by suppressing browning of WAT. ROCK2 could be an important therapeutic target against obesity and diabetes mellitus.

Disclosure: Y. Takeda: Grants; a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (to Keiichiro Matoba and Rimei Nishimura), the MSD Life Science Foundation (to Keiichiro Matoba), the Takeda Science Foundation (to Keiichiro Matoba), the Suzuken Memorial Foundation (to Keiichiro Matoba). Other; Keiichiro Matoba has received research support from Sanofi KK, Tanabe Pharma, and Takeda Pharmaceutical., Kazunori Utsunomiya has received research support from Terumo, Novo Nordisk Pharma, Taisho Pharmaceutical, Böehringer Ingelheim, Kyowa Hakko Kirin, Sumitomo Dainippon Pharma, and Ono Pharmaceutical as, Rimei Nishimura has received speaker honoraria from Astellas Pharma, Nippon Boehringer Ingelheim, Eli Lilly Japan KK, Kissei Pharmaceutical, Medtronic Japan, MSD, Novartis Pharma KK, Novo Nordisk Phar.

38

The essential role of the α4 for insulin signalling in metabolic regulation and maintenance of brown adipocyte

M. Sakaguchi, S. Okagawa, Y. Okubo, M. Igata, T. Kondo, E. Araki;

Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.

Background and aims: Current studies demonstrated that insulin sensitivity of adipose tissues is essential for the maintenance of the systemic metabolic state. We created the experimental system to investigate the role of insulin and IGF-1 signaling in the adult adipose tissues by an inducible adipocyte-specific gene targeting of IR and IGF1R in mice. The IR/IGF1R knockout specifically in adipocytes caused a severe metabolic disease state. Here, we searched the essential downstream targets of insulin signaling for the maintenance of adipose tissues.

Materials and methods: We used the adipocyte-specific inducible IR/IGF1R gene-knockout in the adult mice. We obtained the RNAs from the adipocytes of both white fat tissues (WAT) and brown fat tissues (BAT) before and after the metabolic disease state. To identify new components of IR/IGF1R signaling, we compared gene expression patterns in WAT and BAT, and identified the mRNAs markedly altered after the metabolic disease state. Among the mRNAs, α4, a protein phosphatase protector which regulates phosphorylation state of various target molecules, is decreased significantly after the deficit of IR/IGF1R signaling in BAT. Thus, we have created inducible adipocyte-specific α4 KO (Ai-α4 KO) mice with tamoxifen-inducible Cre-ERT2 transgene and investigated the role of α4 in the IR-mediated signaling for the maintenance of BAT.

Results: ShRNA mediated α4 knockdown (KD) altered insulin-stimulated phosphorylation status in BAT, decreased IRβ (Y1162/1163), IRS1 (Y612), and Akt (S473), with a mild change in ERK1/2 (T202/Y204) but increased ribosomal S6 protein (S235/236), indicating that α4 reduces S6 phosphorylation state. Once induced α4 KO in adipocytes, the mice revealed extensive losses of adipocytes in SC-WAT and BAT depots. Ai-α4 KO mice showed severe diabetes, with ectopic lipid accumulation in the liver and pancreatic islet hyperplasia. RNA-seq of adipose tissues showed a marked reduction of genes associated with mitochondrial fatty acid oxidation and increases with inflammatory cytokine pathways in Ai-α4 KO SC-WAT and BAT. The histological analysis showed a significant increase in stromovascular cells with an elevation of F4/80, TNF-α, and IL-6 and a rapid development of an apoptotic process with increased TUNEL and cleaved caspase-3. Ai-α4KO showed a marked reduction of BAT-associated functional genes as UCP1, PRDM16, Tfam, and PGC1α mRNA compared to the control. Consistent with the changes in mRNAs and the decrease in BAT mass, Ai-α4 KO mice showed the marked cold intolerance and impaired energy expenditure.

Conclusion: The results demonstrated that α4 is an essential component for insulin signaling to regulate the maintenance of the brown adipocytes.

Supported by: JSPS, MSD foundation, Takeda foundation, Astellas foundation

Disclosure: M. Sakaguchi: None.

39

Proof-of-concept for CRISPR/Cas9 gene editing in human primary preadipocytes: deletion of FKBP5 and PPARG and effects on adipogenesis and metabolism

P.G. Kamble1, S. Hetty1, M. Vranic1, K. Almby1, C. Castillejo-López2, X.M. Abalo1, M.J. Pereira1, J.W. Eriksson1;

1Medical Sciences, Uppsala University, Uppsala, 2Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Background and aims: The clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology has advanced the field of genome engineering. Yet, its applications to human adipose tissue are scarce. We aimed to establish the CRISPR/Cas9 method for gene knockout (KO) studies in isolated human primary preadipocytes. As a proof-of-concept, we deleted the glucocorticoid receptor modulating gene FKBP5 (FKBP51, a resultant protein) in preadipocytes and explored its role in adipogenesis and the context of glucocorticoid effects on insulin resistance in human adipocytes. As a method validation, we also knocked out the PPARG gene, a master regulator of adipogenesis.

Materials and methods: Subcutaneous adipose tissue was obtained from 8 non-diabetic women (age: 20-71 years; BMI: 24.2-43.1 kg/m2). After tissue digestion, isolated preadipocytes were used to knockout FKBP5 and PPARG genes with CRISPR/Cas9. The CRISPR components were delivered into cells as a ribonucleoprotein complex using electroporation. For each target gene, three different sgRNA were used. A non-coding sgRNA was used as a negative control. The results are presented for the sgRNA with the highest KO efficiency. The KO efficiency was checked by DNA sequencing, mRNA and protein levels. The effect of FKBP51 loss on adipogenesis was studied. Differentiated adipocytes from wild type (WT) and FKBP51 KO cultures were treated with dexamethasone (0.3 μM/24 h) to assess its effect on adipocyte glucose uptake and downstream transcriptional activity of glucocorticoid receptors.

Results: The mutation efficiency for top-performing sgRNA against the FKBP5 and PPARG genes was 91% and 63%, respectively. At the protein level, we found between 90-100% loss of FKBP51 and PPARG in the KO cultures than WT cultures. This was achieved without clonal isolation. The loss of FKBP51 did not affect adipocyte differentiation compared to WT. In contrast, the loss of PPARG prevented adipogenesis, which also served as a positive control. As expected, in WT cultures, dexamethasone treatment significantly reduced basal and insulin-stimulated (1000 μU/ml) glucose uptake by ~50% compared to untreated cultures (p<0.05, n=6). In contrast to WT cultures, the inhibitory effect of dexamethasone was abrogated in FKBP51 KO cultures (p=NS). The expression of a glucocorticoid target gene, CNR1 in response to dexamethasone was higher by ~50% in FKBP51 KO cultures than WT (p<0.05, n=6).

Conclusion: We report proof-of-concept for CRISPR/Cas9 gene editing in human primary preadipocytes by knocking out FKBP51, a chaperone protein modulating glucocorticoid receptor activity, as well as PPARG, being a good control for adipogenesis. Our data suggest that FKBP51 does not affect adipogenesis in humans in contrast to what has been shown in rodent models. Instead, it may affect glucocorticoid effects on adipocyte glucose metabolism and transcriptional activity of glucocorticoid receptors. This has potential implications for the glucocorticoid-induced insulin resistance and type 2 diabetes. Our method is simple, easy to adapt and enables the use of human primary preadipocytes over animal adipose cell models to assess the role of key genes and their products in development and metabolism of adipose cells.

Supported by: SDF, EXODIAB, EF, SSMR, UU ALF grants.

Disclosure: P.G. Kamble: None.

40

Understanding Mig-6 functions of brown adipose tissue in adaptive thermogenesis and systemic energy homeostasis

S. Choung1,2, J. Kim2, K. Joung2, H. Kim2, B. Ku1,2;

1Research Institute for Medical Sciences, Chungnam National University, Daejeon, 2Internal Medicine, Chungnam National University, Daejeon, Republic of Korea.

Background and aims: Obesity is a major risk factor for metabolic syndrome such as type 2 diabetes mellitus, dyslipidemia, non-alcoholic fatty liver, cardiovascular disease, and even some cancers. In contrast to white adipose tissue, well known to store energy, brown adipose tissue (BAT) governs thermogenic energy expenditure. Due to special ability to dissipate energy as heat, BAT has a therapeutic potential to combat obesity, diabetes and metabolic syndrome. Mitogen-inducible gene 6 (Mig-6) is a negative regulator of the epidermal growth factor receptor (EGFR) signal. Deletion of the Mig-6 hyper-activate EGFR signaling, leading to spontaneous tumor formation in skin, lung and other tissues. Because Mig-6 is as tumor-suppressor gene, most studies focus on cancer. Recently, we characterized that Mig-6 has an important role in the regulation of cholesterol homeostasis and bile acid synthesis in the liver. In previous study, we demonstrated the association between EGFR signaling and metabolic disorder such as NAFLD. However, the roles of Mig-6 in BAT remain poorly understood. In the present study, we investigated the metabolic role of Mig-6 in BAT.

Materials and methods: Immortalize brown adipocytes were transfected siRNA targeting Mig-6 after differentiation. We generated mice specifically enhancing Mig-6 in BAT using a genetic strategy based on the Cre-ROSA recombination. We fed normal chow or high fat to KI mice for 12 weeks. Body weights and food intake were measured weekly. We conducted GTT and ITT. KI mice were measured energy expenditure by using indirect calorimetry system. Tissues staining was performed. The measurement of biochemical parameters was performed using mice serum. Western blot and quantitative polymerase chain reaction (Q-PCR) performed to analyze related genes.

Results: Here we showed that the inhibition of Mig-6 decreases the expression of thermogenesis relative genes, UCP1 and Elovl3, in the BAT cell. Mig-6 KI mice showed better metabolic phenotypes, including improved glucose tolerance and reduction of body weight. In the accelerated obese condition, transgenic mice induced the improvement of glucose tolerance, fasting glucose level and lipid levels. Importantly, Mig-6 upregulated the expression of thermogenesis relative genes (UPC1, Pgclα, Cieda, PPARα, Elovl3), consistent with the increased UCP1 in the BAT of mice. Mig-6 KI mice on HFD improved insulin sensitivity, glucose tolerance and energy metabolism.

Conclusion: These results together suggest that Mig-6 controls systemic energy homeostasis by regulating UCP1 based adaptive thermogenesis.

Disclosure: S. Choung: None.

41

Oncostatin M inhibits browning of white adipose tissue via gp130 signalling

P.P. van Krieken1,2, T.S. Odermatt1,2, M. Blüher3, S. Wueest1,2, D. Konrad1,2;

1Pediatric Endocrinology and Diabetology, University Children's Hospital Zurich, Zurich, Switzerland, 2Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland, 3Helmholtz Institute for Metabolic, Obesity and Vascular Research (HI-MAG) of the Helmholtz Zentrum München at the University of Leipzig and University Hospital Leipzig, Leipzig, Germany.

Background and aims: Obesity is associated with low grade adipose tissue inflammation and locally elevated levels of oncostatin M (OSM), a member of the glycoprotein 130 (gp130) cytokine family. It was recently shown that OSM can impair the thermogenic function of brown adipose tissue (BAT), suggesting OSM contributes to the positive energy balance that underlies obesity. However, when we blocked OSM signalling using adipocyte-specific gp130 knockout mice (gp130Δadipo), levels of the main thermogenic marker uncoupling protein 1 (UCP1) were unchanged in BAT. To consolidate these results we aimed to further study the role of OSM in thermogenesis and white adipose tissue (WAT) browning.

Materials and methods: Protein and gene expression levels of UCP1 and other thermogenic markers were assessed in extracts from a subcutaneous adipocyte cell line, adipose tissue depots from control (gp130F/F) or gp130Δadipo mice fed either chow or a high fat diet (HFD), or subcutaneous WAT biopsies from a human cohort of lean and obese subjects. WAT browning was modelled in vitro by exposing mature adipocytes to isoproterenol subsequent to stimulation with various concentrations of OSM.

Results: In line with mouse data, OSM gene expression in human WAT positively correlated with BMI (r=0.156, p=0.043, n=170) and negatively with UCP1 expression (r=-0.295, p=0.012, n=71). Similar to our previous findings in BAT, Ucp1 expression remained unchanged in the epididymal WAT of HFD-fed gp130Δadipo compared to gp130F/F mice. However, inguinal WAT of gp130Δadipo mice exhibited significantly elevated levels of Ucp1 and other browning markers such as Cidea and Pgc-1αIn vitro, OSM treatment lowered isoproterenol-induced UCP1 protein and gene expression levels in subcutaneous white adipocytes in a dose-dependent manner. Mechanistically, preliminary data indicate that OSM inhibits UCP1 expression ERK-dependently in white adipocytes.

Conclusion: Our data support the notion that OSM negatively regulates thermogenesis in adipose tissue, albeit in a fat depot dependent manner. Lowering OSM expression in (white) adipose tissue may be a beneficial strategy to treat obesity.

Supported by: SNSF grant no. 179344 and the Hartmann Müller-Stiftung (University of Zurich)

Disclosure: P.P. van Krieken: None.

42

Involvement of the Notch pathway and its ligand DNER in obesity-mediated inflammation of adipose tissues

J. Pestel, M. Robert, H. Vidal, A. Eljaafari;

CarMeN INSERM U-1060, Lyon, France.

Background and aims: During obesity adipose tissues (AT) are progressively infiltrated by inflammatory immune cells, leading to insulin resistance. Among them Th17 lymphocytes propagate inflammation, through secretion of IL-17A/F cytokines which bind to ubiquitously expressed receptors, and activate pro-inflammatory secretion by surrounding cells. Using a co-culture model with adipose-derived stem cells harvested from obese individuals (obASCs) and activated mononuclear cells (MNCs), we have demonstrated that obASCs are involved in Th17 cell polarization and subsequent inhibition of adipogenesis and insulin response. Because the Notch pathway is known to regulate helper T cell differentiation, and DNER a non-canonical NOTCH ligand, to regulate adipogenesis, this led us to investigate the role of NOTCH and DNER in obASCs-mediated inflammation.

Materials and methods: With this aim, DAPT (an inhibitor of the Notch pathway) was added or not to phytohemagglutinin A (PHA)-activated co-cultures of human obASCs and MNCs. In addition, siRNA targeting Dner (siDner) were transfected in ob-ASC, or not. Cytokine expression and secretion, were measured by RT-qPCR and ELISA, respectively. Finally, wild type (WT) and DNER knockout mice (DNERKO) were fed with a hypercaloric or chow diet for 16 weeks. BMI and glycaemia were monitored, and AT were harvested to measure pro-inflammatory cytokine expression by RT-qPCR.

Results: As expected, PHA-activated co-cultures of human ob-ASCs with MNCs enhanced IL-17A, but reduced TNFα secretion. DAPT significantly inhibited IL-17A secretion, and restored TNFα production, demonstrating thus the role of the NOTCH pathway in the polarization of T cells towards the Th17 cell subset. DNER expression increased upon inflammatory conditions in obASCs, i.e. when obASCs were co-cultured with activated MNCs. ob-ASC transfected with siDNER partially inhibited IL-17A secretion by human T lymphocytes. In the N Tac mouse model, DNER expression positively correlated with body weight and glycemia (p<0.05)and increased in subcutaneous and visceral AT of obese mice, as compared with lean mice. Finally, in N Tac mice fed with a hypercaloric diet, IL-17A did not increase in AT, but AT inflammation was assessed by increase of TNFα, IL-6, and IFNγ expression in both visceral and sub-cutaneous AT. Interestingly, DnerKO-mice demonstrated lower TNFα, IFNγ and IL6 expression in subcutaneous AT (p=0.0132; 0.0449 ; 0.0311, respectively), and lower IFNγ expression in visceral AT ( p=0.062), as compared with WT mice.

Conclusion: Our results demonstrate the involvement of the Notch pathway and its ligand DNER in human obASCs-mediated Th17 cell polarization, in vitro. In our experimental mouse model, we observed an involvement of DNER in obesity-mediated inflammation of adipose tissues, and a link between DNER expression and glycemia. Therefore, altogether our results suggest a role for DNER and NOTCH in obesity-mediated AT inflammation and metabolic alterations.

Disclosure: J. Pestel: None.

OP 08 Charting human beta cell failure in type 1 diabetes

43

111 In-exendin spect imaging suggests presence of residual beta cells in patients with longstanding type 1 diabetes

M. Boss1, I. Kusmartseva2, W. Woliner- van der Weg1, L. Joosten1, M. Brom1, M. Béhe3, C.J. Tack4, O.C. Boerman1, M.J.R. Janssen1, M. Atkinson5, M. Gotthardt1;

1Radiology, Nuclear Medicine and Anatomy, Radboudumc, Nijmegen, Netherlands, 2Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, USA, 3Paul Scherrer institute, Villingen, Switzerland, 4Internal Medicine, Radboudumc, Nijmegen, Netherlands, 5University of Florida, Gainesville, USA.

Background and aims: There is increasing evidence for the presence of residual, dysfunctional beta cells in patients with type 1 diabetes (T1D). Confirming the existence of such non-functioning beta cells in living subjects has been hampered by the lack of methods capable of quantifying beta cell mass (BCM) in vivo in humans. Pancreatic uptake of 111In-exendin (targeting the GLP-1 receptor), quantified by single photon emission computed tomography (SPECT) provides such a method. We hypothesized that T1D patients have considerable remaining BCM and therefore, should have detectable 111In-exendin uptake in the pancreas, even without or only low insulin secretion.

Materials and methods: 10 T1D patients and 10 matched healthy controls underwent quantitative SPECT following injection of 111In-exendin after which pancreatic tracer uptake was determined. In addition, immunohistochemical analysis of human pancreatic sections from organ donors with longstanding T1D was performed to assess GLP-1R expression as well as presence of insulin and glucagon.

Results: Pancreatic uptake of 111In-exendin was above background levels in 6/10 individuals with T1D. Strikingly, in 5 T1D patients, pancreatic tracer uptake was comparable to uptake levels in healthy controls. Pancreatic uptake of 111In-exendin was independent of stimulated C-peptide levels (<0.03 nmol/L in 8/10 T1D patients). In some individuals with long type 1 diabetes duration and undetectable C-peptide level, immunohistochemistry demonstrated the presence of extremely limited number of islets with residual insulin positive beta cells, some of which were GLP-1R positive. GLP-1R expression was also detected in insulin and glucagon negative islet cells.

Conclusion: Multiple individuals with T1D show measurable uptake of 111In-exendin. These data were corroborated by immunohistochemistry demonstrating the presence of GLP-1R-positive/insulin-positive as well as GLP-1R-positive/ insulin-negative/glucagon-negative islet cells in pancreas samples of longstanding T1D patients. The detected radiotracer uptake could indicate the presence of residual dysfunctional beta cells or, alternatively, GLP-1R expression on other endocrine cell types transdifferentiating into a beta cell-like type. Additional data are needed to clarify the origin of the radiotracer signal. The presence of a residual pool of dysfunctional beta cells has important implications for treatment of T1D, since these cells have the potential for functional restoration. In sum, exendin imaging could provide a valuable tool to further elucidate the complex pathophysiology of diabetes.

figureh

Clinical Trial Registration Number: 2013-004268-76

Supported by: INNODIA (IMI2-JU, grant agreement 115797)

Disclosure: M. Boss: None.

44

Comparative analysis of human pancreatic islets after type 1 diabetes, LADA and type 2 diabetes manifestation

A. Joerns1, S. Lenzen1,2;

1Hannover Medical School, Hannover, 2Institute of Experimental Diabetes Research, Hannover Medical School, Hannover, Germany.

Background and aims: During diabetes manifestation pancreatic islets show changes in beta cells and changes in cytokine expression of the immune cell infiltrate. Therefore a comparative analysis was performed between islets from T1DM, LADA, T2DM patients, and age-matched, non-diabetic subjects.

Materials and methods: By double immunofluorescence staining the immune cell types and beta cells stained by insulin or other glucose recognition markers were determined. The gene expressions of different cytokines in the islet infiltrate and caspase 3 as well as PCNA in the beta cells of human non-diabetic and the different diabetic (T1DM, LADA, T2DM) human pancreases were performed by in situ PCR analysis. Gene expression of the parameters was quantified in the infiltrating immune cells and beta cells with a computer-assisted method.

Results: The non-diabetic control islets of human subjects showed only few macrophages with a slight increase in islets of pancreases from T2DM patients. The islet immune cell infiltrate was mainly composed in the T1DM and LADA by CD4 and CD8 T cells as well as CD68 macrophages. In the infiltrated islets of the LADA pancreas there was a more pronounced increase of CD68 macrophages in relation to CD8 T cells as compared to the T1DM situation. Compared to the overall immune cell islet infiltration in the T1DM pancreas a heterogeneous infiltration pattern was observed in the LADA pancreas. Around one third of the analysed LADA pancreatic areas showed any signs of infiltration. Immune cells in the islet infiltrate of T1DM and LADA pancreases expressed all main pro-inflammatory cytokines, especially IL-1β and TNF-α with a threefold shift of the ratio between IL-1β and TNF-α in the infiltrated area of the human LADA pancreases. At the same time, the anti-inflammatory cytokine IL-10 showed a higher gene expression in the immune cell infiltrate of the LADA as compared to T1DM pancreases. All analysed pro- and anti-inflammatory cytokines revealed no gene expression in the few immune cells in the T2DM and non-diabetic situation. Caspase 3 expression was more increased in human T1DM than in LADA pancreases when compared to the beta-cells in the control islets or islets from non-diabetic and T2DM pancreases. The gene expression of the proliferation marker PCNA increased more in islets of LADA than of T1DM pancreases.

Conclusion: It can be concluded therefore, that LADA is a milder form of autoimmune diabetes in patients of an advanced age. The functional implications of these studies for developing prevention therapy strategies in T1DM including LADA are to eliminate the activated immune cell infiltration pattern with their specific pro-inflammatory cytokine profile in the islets to allow survival and regeneration of intact beta cells.

Supported by: DFG Jo395/2-2

Disclosure: A. Joerns: None.

45

Differential expression of inflammation-related genes in the pancreases of patients with two distinct endotypes of type 1 diabetes

F. Torabi1, P. Leete2, R. Wyatt2, J. Vadakekolathu3, D. Boocock3, M.D. Turner3, S.J. Richardson2, N.G. Morgan2, M.R. Christie1;

1School of Life Sciences, University of Lincoln, Lincoln, 2Institute of Biomedical and Clinical Sciences, University of Exeter, Exeter, 3School of Science & Technology, Nottingham Trent University, Nottingham, UK.

Background and aims: Autoimmune responses in Type 1 Diabetes (T1D) commonly appear within the first five years of life, but the age at which disease develops varies greatly from early childhood to late adulthood suggestive of different rates of disease progression. Morphological studies on pancreas samples from young people with T1D have revealed different patterns of immune cell infiltration between patients diagnosed <7 years and those developing disease at an older age (>13y) suggesting a link between phenotype of inflammatory responses and age at onset. The cellular composition of the islet infiltrate differs primarily according to the number of CD20+ B-cells infiltrating the islet, and patients may be categorised as either T1D endotype 1 (T1DE1) or 2 (T1DE2), with the former having an earlier age at onset and greater CD20+ cell infiltration. We hypothesised that islet infiltrating B-cells, by acting as antigen presenting cells, drive the specificity and phenotype of T-cells within the islet inflammation and thereby intensity of beta cell destruction. The objective of this study was to investigate whether islet CD20+ B-cell infiltration in T1D is associated with differences in immune cell activation consistent with a more proinflammatory phenotype in T1DE1 than in T1DE2. This was achieved by applying state-of-the art gene expression technologies to identify differences in immunophenotypic markers in the pancreases of patients with each endotype.

Materials and methods: Numbers of CD20+ cells in individual islets on sections of fixed pancreas from T1D cases in the Exeter Archival Diabetes Biobank were quantified by immunohistochemistry and samples categorised as T1DE1 (mean >3 CD20+ cells per islet) or T1DE2. RNA was extracted from each section and RNA concentration and fragmentation were evaluated using a Bioanalyser. Samples where >40% of RNA fragments exceeded 300 nucleotides in length were analysed for the expression of a panel of 750 autoimmunity-related genes and 20 housekeeping genes using nCounter gene expression technology. Expression levels were normalised and differentially expressed genes between disease endotypes were identified using nSolver software.

Results: RNA samples from 8 T1DE1 and 7 T1DE2 cases were subjected to nCounter analysis and the expression of between 460 and 710 autoimmunity-related genes were above the limit of detection across these samples. Differential expression analysis identified a single gene, SERPINA1 which was dramatically over-expressed in cases with T1DE2 vs T1DE1. This gene encodes a protein with known cytoprotective and anti-inflammatory properties. By contrast, more than 50 genes were significantly over-expressed in T1DE1 vs T1DE2 samples including selected chemokines (CCL19, CCL21), receptors (CXCR4, IL7R) and genes related to antigen presentation and lymphocyte activation, including HLA-F, PTPRC and CD22.

Conclusion: These data support the proposal that the inflammatory milieu differs markedly in the islets of subjects with T1DE1 and T1DE2. They suggest that detailed analysis of inflammatory gene expression profiles will provide additional insights into the aetiopathological events leading to beta-cell loss in these two disease endotypes.

Supported by: JDRF

Disclosure: F. Torabi: None.

46

Defects in proinsulin processing vary during disease progression in type 1 diabetes

P. Leete, M.A. Russell, C. Ziller, S.J. Richardson, N.G. Morgan;

RILD Level 4, University of Exeter, Exeter, UK.

Background and aims: Evidence for beta cell heterogeneity is emerging and it is increasingly proposed that individual islets may be composed of specific sub-populations of phenotypically distinct beta cells. These have been defined principally by transcriptional analysis but, as expected, there is also evidence for heterogeneity of protein expression. Recently, such heterogeneity was reported during the analysis of proinsulin expression in islets of people with recent-onset (RO) type 1 diabetes, where the variation correlated with the extent of beta cell destruction, phenotype of islet inflammation and age at diagnosis. On this basis, two discrete endotypes of type 1 diabetes were proposed (known as T1DE1 and T1DE2). In the present study, we have investigated whether the differences in proinsulin processing seen close to the onset T1D in the two disease endotypes, also persist in the islets of subjects with longer duration (LD) disease.

Materials and methods: The distribution of proinsulin and mature insulin were examined by immunofluorescence techniques in 4um sections of pancreas from a total of 41 individuals (8 control subjects, 20 RO T1D (<1y); 13 LD T1D (>5y disease)). Staining was visualised via high pixel rate confocal microscopy, and samples were analysed in a blinded manner. The extent of co-localisation of proinsulin and insulin was estimated by measurement of the Manders Overlap Coefficient (MOC) using ImageJ software. The proportion of beta cells having aberrant proinsulin processing was estimated according to the staining profiles achieved. These were then compared between individuals with RO vs LD disease (both in T1DE1 and T1DE2) and controls.

Results: In control subjects, most beta cells displayed a pattern in which proinsulin was localised primarily in the peri-nuclear region, and this was independent of age (between 2-41y). By contrast, among children with RO T1DE1, proinsulin was distributed throughout the cytoplasm in most beta cells in >90% of residual insulin-containing islets. Surprisingly, however, this feature was not seen in subjects with >5y disease when the few remaining beta cells displayed little evidence of defective proinsulin processing. Subjects with RO T1DE2 contained two distinct sub-populations of islets. A minority (<30%) displayed a proinsulin processing pattern similar to that seen in the islets of children with RO T1DE1. However, in the second, larger, sub-population (~70% of the total) proinsulin was retained preferentially in the peri-nuclear region suggesting that the prohormone was processed correctly in most beta cells. Similar to the findings in T1DE1 at >5y duration, those with >5y T1DE2 disease also showed little evidence of aberrant proinsulin processing. More strikingly still, among these subjects, only a single population of islets was retained, in which proinsulin was restricted to the peri-nuclear region in all beta cells. Thus, unlike the situation in people with RO T1D (whether T1DE1 or T1DE2) the residual beta cells found in longer duration (>5y) disease appear to process insulin normally.

Conclusion: Taken together, these data imply that beta cells having aberrant proinsulin processing may be targetted selectively during the early phase of the autoimmune attack in human T1D. The sub-population of beta cells surviving beyond this period appear to process proinsulin efficiently, implying that the ability to process proinsulin may influence the susceptibility of individual beta cells to immune attack during the progression of type 1 diabetes.

Supported by: DUK/JDRF

Disclosure: P. Leete: None.

47

Inhibition of serpinB13 stimulates beta cell development via Notch signalling pathway and delays progression to insulin-dependent diabetes

J. Czyzyk, Y. Kryvalap;

University of Minnesota, Minneapolis, USA.

Background and aims: Methods for repopulating the pancreas with new insulin-producing cells have strong potential for therapy in diabetes. Recently, we have found that inhibition of serpinB13 - a protease inhibitor of cathepsin L (catL) - with mAb in mouse embryos leads to a robust increase in the number of pancreatic Ngn3+ progenitor cells, significant expansion of islet mass, and improved resistance to severe diabetes in adulthood. To unveil the molecular mechanism of the augmented Ngn3+ cell response following inhibition of serpinB13 during gestation, we focused on the Notch communication system - a critical signaling pathway for pancreatic development.

Materials and methods: We used a mAb to serpinB13 or IgG isotype control to stimulate mouse embryonic pancreas explants (E12.5), and examined Notch and Ngn3 expression in these in vitro cultures by flow cytometry and Western blotting. The human autoantibodies to serpinB13 in subjects previously enrolled in a DPT-1 clinical trials were measured using Luminex methodology.

Results: We found that serpinB13 is expressed and secreted by epithelial cells in murine embryonic pancreases. Moreover, inhibition of serpinB13 during embryogenesis caused protease-dependent cleavage of the extracellular domain of Notch1 receptor in the pancreas (p<0.0001). On the other hand, embryonic pancreases of mice with genetic deficiency of catL had significantly fewer Ngn3+ cells compared with wild type controls. The partial loss of the extracellular Notch was followed by decreased presence of active Notch intracellular domain (aNICD), a fragment of Notch that is critical for restraining endocrine cell development. Finally, our screening of children enrolled in DPT-1, for serpin B13 autoantibody (AA) revealed an inverse correlation of this AA with risk level for type 1 diabetes (T1D), as well as a positive association with longer diabetes-free interval. Importantly dialyzed sera from serpinB13 AA-positive subjects stimulated the output of Ngn3+ cells in the pancreas suggesting that this AA is functional. This effect was dependent on serpinB13AA rather than other serum factors as positive serum samples immunodepleted of this AA were no longer able to stimulate development of additional Ngn3+ cells.

Conclusion: Our data point to a novel function of serpinB13 in maintaining Notch receptor-mediated repression of pancreatic endocrine progenitors. Consequently, the perturbation of this effect of serpinB13 enables protease activity to partially dismantle Notch signaling, thereby allowing for more efficient development of Ngn3+ progenitors cells and a subsequent increase in islet mass. Our data also demonstrate that blocking serpinB13 has the potential to partially prevent, or at least slow down, the development of T1D both in the mouse and human.

Supported by: JDRF, ADA, NIH

Disclosure: J. Czyzyk: None.

48

Efficacy and safety of anti-interleukin (IL)-21 in combination with liraglutide in adults recently diagnosed with type 1 diabetes

C. Mathieu1, M. von Herrath2, S.C. Bain3, B. Bode4, J.O. Clausen5, K. Coppieters6, L. Gaysina7, J. Gumprecht8, T. Krarup Hansen9, C. Morales Portillo10, O. Mosenzon11, S. Segel12, G. Tsoukas13, T.R. Pieber14;

1Clinical and Experimental Endocrinology, UZ Gasthuisberg, University of Leuven, Leuven, Belgium, 2Novo Nordisk Inc., Seattle, USA, 3Swansea University Medical School, Swansea, UK, 4Atlanta Diabetes Associates and Emory University School of Medicine, Atlanta, USA, 5Novo Nordisk A/S, Søborg, Denmark, 6Novo Nordisk A/S, Måløv, Denmark, 7Kazan Federal University, Kazan, Russian Federation, 8Medical University of Silesia, Katowice, Poland, 9Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark, 10Endocrinology and Nutrition, Virgen Macarena Hospital, Seville, Spain, 11Diabetes Unit, Hadassah Hebrew University Hospital, Jerusalem, Israel, 12Novo Nordisk A/S, Aalborg, Denmark, 13Medicine, McGill University, Montreal, Canada, 14Medical University of Graz, Graz, Austria.

Background and aims: To evaluate the effect of anti-IL-21 and liraglutide, alone and in combination, compared to placebo, on preservation of β-cell function after 54 weeks of treatment in adults with recently diagnosed type 1 diabetes.

Materials and methods: This was a multicentre, double-dummy, double-blind, efficacy, safety and pharmacokinetic randomised control trial in adults recently diagnosed with type 1 diabetes and non-fasting C-peptide peak ≥0.2 nmol/L, comprising a 54-week treatment period followed by a 26-week observation period. Primary endpoint: area under the curve (AUC)0-4h for meal-stimulated C-peptide at week 54 relative to baseline.

Results: At week 54, combination treatment was associated with a significant improvement of 48% in meal-stimulated C-peptide secretion vs placebo (Table). The insulin requirement was significantly lowered by 0.13 U/kg, corresponding to 32% reduction. Trends for better glycaemic control and lower risk of hypoglycaemic episodes at 54 weeks compared to placebo were also observed. No safety concerns were identified. Treatment benefits were not sustained at 26 weeks after end of treatment.

Conclusion: Treatment with anti-IL-21 and liraglutide for 54 weeks was safe and resulted in sustained insulin secretion and lower insulin dose. Trends for improved glucose control and fewer hypoglycaemic episodes compared to placebo were observed.

figurei

Clinical Trial Registration Number: NCT02443155

Supported by: Novo Nordisk A/S

Disclosure: C. Mathieu: Non-financial support; Abstract supported by Novo Nordisk.4

OP 09 Novel agents in type 1 diabetes

49

Innodia master protocol for the evaluation of investigational medicinal products in children, adolescents and adults with newly diagnosed type 1 diabetes

D.B. Dunger1, S.F.A. Bruggraber1, A.P. Mander2, T. Tree3, P. Jaroslaw Chmura4, M.J. Knip5, A.M. Schulte6, C. Mathieu7;

1Box 116 Level 8, University of Cambridge, Cambridge, UK, 2Cardiff University, Cardiff, UK, 3King's College London, London, UK, 4University of Copenhagen, Copenhagen, Denmark, 5University of Helsinki, Helsinki, Finland, 6Sanofi Deutschland GmbH, Frankfurt, Germany, 7KU Leuven, Leuven, Belgium.

Background and aims: Currently Phase 2 drug development for T1D may be slowed by lack of information to inform drug dosage and mechanistic data to understand variable responses to an investigational medical product. Trials would also benefit from identification of biomarkers to permit more robust stratification of participants at baseline. The INNODIA consortium has established a European infrastructure to evaluate prospectively clinical data from people with new onset T1D combined with centralised analysis of clinical samples to determine rates of decline in beta cell function and identify novel predictive biomarkers. Built on the backbone of this study we have developed a Master Protocol (MP) to accelerate the delivery of Phase 2 studies.

Materials and methods: The protocol of the existing INNODIA study includes formal assessment of beta-cell function by mixed meal tolerance tests and home dried blood spot measurement of C-peptide, standardised sample collections for centralised assessment of immune function and a range of measurements of disease activity (beta-cell death, microRNAs) and samples for genomics, metabolomics, proteomics and lipidomics biomarker discovery. Pseudo anonymised clinical data collected through an eCRF and all of the laboratory data are stored in a central data warehouse allowing integrated data analysis.

Results: The INNODIA MP uses the same inclusion criteria, visit schedule, duration, sample collection for standardised efficacy and mechanistic studies. The inclusion criteria can be adapted to the requirements of specific interventions, but the clinical and mechanistic evaluations remain largely unchanged providing the potential to explore more detailed analysis of variability in response. Additional sample collection can permit essential study of toxicology and PK/PD. The MP is suited for adaptive trial design such as dose finding, dropping study arms, inclusion of additional treatments, options to share controls and potentially inclusion of data from the natural history cohort. The MP was submitted to the EMA Scientific Advice Working Party (SAWP) of the Committee for Medicinal Products for Human Use (CHMP) for Qualifications advice for novel methodologies in clinical drug development. The EMA SAWP supported the intended context of use and endorsed strategies going forward for planned clinical trials within INNODIA’s infrastructure.

Conclusion: We believe the INNODIA MP will improve the standardisation of Phase 2 studies and accelerate the evaluation of established, novel and re-purposed IMP’s alone or in combination with the aim of halting or reversing the decline in beta cell function in people with newly diagnosed T1D.

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Clinical Trial Registration Number: NCT03936634

Supported by: IMI2 H2020

Disclosure: D.B. Dunger: None.

50

The Simplici-T1 trial: activation of glucokinase by TTP399 improves glycaemic control in patients with type 1 diabetes

C. Valcarce1,1, J.L.R. Freeman1, I. Dunn1, C. Dvergsten1, K.R. Klein2, J. Buse2;

1vTv therapeutics, High Point, 2UNC Chapel Hill, Chapel Hill, USA.

Background and aims: Identification of adjunctive, oral pharmacotherapies to treat type 1 diabetes (T1D) has been limited by hypoglycemia and ketoacidosis. TTP399 is a liver-selective oral glucokinase activator. In type 2 diabetes (n=190), TTP399 was shown to reduce A1C vs placebo (PBO) −0.9% (P < 0.01).

Materials and methods: The Simplici-T1 trial is an adaptive, Phase 1b/2 Proof of Concept study designed to explore the safety and efficacy of TTP399 versus placebo in patients with T1DM. Safety, pharmacokinetics, and pharmacodynamics were established in a sentinel phase (n=5). In Part 1 (n=19) patients on insulin pump therapy and continuous glucose monitoring were randomized (1:1) to receive 800mg TTP399 or PBO once daily for 12 weeks. In Part 2 (n=85), TTP399 effect was examined in a broader T1D population. Insulin dose was optimized prior to randomization and throughout the 12 wk double-blind treatment period with specified pre- and post-meal targets (fasting plasma glucose: ~80-130mg/dL; post meal glucose: <180-200 mg/dL). Treat to target continued throughout the study.

Results: Safety, pharmacokinetics, and pharmacodynamics were established in a sentinel phase (n=5). In Part 1 (n=19), TTP399 reduced A1C (-0.7%, p=0.03), improved TIR 70-180 (12%, p=0.04), and reduced bolus insulin vs PBO in patients on insulin pump therapy and continuous glucose monitoring. In Part 2 A1C at randomization was 7.6% (SD 0.6). TTP399 significantly reduced A1C (trial product estimand: -0.32%, 95%CI -0.50, -0.13, p<0.01; ITT: -0.20, 95%CI -0.38,-0.02, p=0.03). A responder analysis (improved A1C; without severe hypoglycemia or increase in insulin dose; nor abnormal betahydroxybutyrate or lactic acid) revealed 42% TTP399 responders vs 12% PL (ITT: p=0.001, hierarchically controlled [HC]). Daytime TIR improved (ITT: 8%, 95%CI 1,15, p<0.01, HC). Treatment-emergent adverse events were numerically lower with TTP399. No safety signals were identified.

Conclusion: These data suggest that further development of TTP399 as adjunctive therapy in T1D is warranted.

Clinical Trial Registration Number: NCT03335371

Supported by: JDRF

Disclosure: C. Valcarce: Employment/Consultancy; vTv therapeutics employee.

51

Mechanism matter: preliminary evidence that activation of glucokinase by TTP399 does not increase plasma or urine ketones in type 1 diabetes

J.L.R. Freeman, I. Dunn, C. Valcarce;

vTv therapeutics, High Point, USA.

Background and aims: Attempts to develop new oral adjunctive type 1 diabetes (T1D) treatments to achieve tighter blood glucose levels have been hampered by an increased risk of hypoglycemia and DKA. TTP399 is an oral liver-selective glucokinase activator (GKA). TTP399’s mechanism of action is insulin-independent and thus may be suitable as an adjunctive treatment for T1D. In contrast to observations with SGLT inhibition, non-clinical and clinical data to date suggest that activation of GK by TTP399 should not significantly affect parameters linked to the increase in risk of DKA (ex: decrease in nocturnal glycemia and basal insulin or increases in glucagon.)

Materials and methods: The Simplici-T1 trial is an adaptive, Phase 1b/2 Proof of Concept study designed to explore the safety and efficacy of TTP399 versus placebo (PBO) in patients with T1DM. Safety, pharmacokinetics, and pharmacodynamics were established in a sentinel phase (n=5). In Part 1 (n=19) patients on insulin pump therapy and continuous glucose monitoring were randomized (1:1) to receive 800mg TTP399 or PBO once daily for 12 weeks. In Part 2 (n=85), TTP399 effect was examined in a broader T1D population (device use not required). Insulin dose was optimized prior to randomization and throughout the 12 wk double-blind treatment period with specified pre- and post-meal targets (fasting plasma glucose: ~80-130mg/dL; post meal glucose: <180-200 mg/dL). Treat to target continued throughout the study.

Results: After 12 weeks, TTP399 significantly reduced HbA1c (-0.3%, 95%CI [-0.5, -0.01], p<0.001 trial product estimand; -0.2%, 95%CI [-0.38, -0.01], p=0.03 ITT), and improved TIR (8%, 95%CI [0.78, 14.72] p=0.03). No incidents of severe hypoglycemia occurred in the TTP399-treated group and no DKA events were reported in the study. Moreover, no significant or clinically relevant increases in plasma β-hydroxybutyrate (BOHB) or urine ketones were observed in the TTP399-treated group compared to placebo (Figure 1) independent of significant reductions in insulin.

Conclusion: These results, support the hypothesis that activation of GK by TTP399 should not increase the risk of DKA and are consistent with results from a non-clinical minipig model of T1D in which dosing of a liver-selective GKA, while reducing/eliminating insulin, did not increase ketones and protected from DKA. Further mechanistic studies and studies of longer duration are needed to confirm these observations.

figurek

Clinical Trial Registration Number: NCT03335371

Supported by: JDRF

Disclosure: J.L.R. Freeman: Employment/Consultancy; vTv therapeutics employee.

52

Long-term follow-up study of type 1 diabetes patients previously treated with IMCY-0098 or placebo in young adults with recent-onset type 1 diabetes

N. Bovy1, C. Boitard2, P. Achenbach3, R.D. Leslie4, C. Dayan5, B. Keymeulen6,7, K.R. Owen8, V. Carlier1, M. Van Mechelen1, J. Van Rampelbergh1;

1Imcyse SA, Liège, Belgium, 2Institut Cochin, Paris, France, 3Helmholtz Zentrum München, München, Germany, 4Queen Mary University of London, London, UK, 5Cardiff University, Cardiff, UK, 6UZ Brussel, Brussels, Belgium, 7Belgian Diabetes Registry, Brussels, Belgium, 8University of Oxford, Oxford, UK.

Background and aims: Type 1 diabetes (T1D) is an auto immune disease for which no curative treatment currently exists. IMCY-0098 (human T1D-peptide) is an innovative immunotherapeutic technology consisting of a synthetic peptide containing an MHC class II epitope of proinsulin linked to a thioredox motif. It is an antigen-specific therapy leading to the generation of cytolytic memory CD4 T cells targeting the pathogenic auto-immune response whilst preserving overall immune competence of diabetic patients. The safety, clinical efficiency and immune responses induced by IMCY-0098 treatment were evaluated in the IMCY-T1D-001, a phase 1b double-blind, placebo-controlled, multi-centre study in young adults with recent-onset T1D. Herein, we present the results of a long-term follow-up (LTFU) study on these patients.

Materials and methods: This study involved a follow-up of 6 months after the end of the initial participation. At week 24 of the IMCY-T1D-001 study, patients were offered to participate to this LTFU study including additional assessments at week 36 and week 48 after the first administration of IMCY-0098. Mixed meal tolerance tests (MMTT) were performed at week 36 and week 48 and the daily doses of insulin treatment were recorded. PBMCs were also collected at week 48 to measure IMCY-0098 specific cytolytic CD4 response and beta-cells antigen specific effector T cell responses by flow cytometry. Data were analysed using a datamining approach with artificial intelligence-based Knowledge Extraction and Management (KEM) technology.

Results: 30 out of 41 patients were re-consented. All received 4 injections of IMCY-0098 or placebo during the main study. 11 patients refused participation or were lost to follow-up. Overall, the study provided evidence of long term (up to 48 weeks) safety and tolerability of the study drug. The safety analysis identified 24 TEAEs that were mild or moderate in intensity and not related to IMCY-0098, except for 1 event of hypoglycaemia that was judged as possibly related and resolved by the end of the study. The positive early clinical and immunological trends observed in the main study up to 6 months, were not confirmed at 48 weeks after start of the treatment or were less prominent. However, due to the low number of observations and in-group variability, the study was explorative and was not designed or powered to demonstrate efficacy.

Conclusion: Results of this LTFU clinical trial have confirmed the excellent safety profile of IMCY-0098 observed in the IMCY-T1D-001 study, reaching the primary study objective. The positive trend in clinical and immunological parameters improvement detected in the main study was not observed in the LTFU. The current data suggest that the right dosage or regimen (number and timing of injections of IMCY-0098) to elicit a significant and long-term effect is yet to be identified. Overall, the results of the main study and the LTFU are very encouraging and informative. Further exploration is needed in a next clinical trial already in preparation under the master protocol and the support of the European Consortium INNODIA.

Clinical Trial Registration Number: NCT04190693

Supported by: EU FP7 // DG06 Walloon Region

Disclosure: N. Bovy: None.

53

Golimumab preserves beta cell function and reduces insulin use and hypoglycaemia in youth with recently diagnosed type 1 diabetes: the phase 2 T1GER study

T. Quattrin1, M.J. Haller2, A.K. Steck3, E. Felner4, Y. Li5, Y. Xia5, J.H. Leu5, M.R. Rigby6, R. Zoka6, J.A. Hedrick6, F. Vercruysse7;

1Jacobs School of Medicine and Biomedical Sciences, University at Buffalo and JR Oishei Children’s Hospital Diabetes Center, Buffalo, USA, 2Department of Pediatrics, University of Florida, Gainesville, USA, 3Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, USA, 4Division of Pediatric Endocrinology, Emory University School of Medicine, Atlanta, USA, 5Janssen Research & Development, LLC, Spring House, USA, 6Janssen Research & Development, LLC, Horsham, USA, 7Janssen Research & Development, Beerse, Belgium.

Background and aims: Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive loss of pancreatic β cells. Golimumab is a human IgG1κ monoclonal antibody specific for tumor necrosis factor α. This study assessed whether golimumab preserves β-cell function in children and young adults with newly diagnosed stage 3 T1D.

Materials and methods: This Phase 2a, double-blind, placebo-controlled study randomized participants aged 6-21 years with newly diagnosed stage 3 T1D to receive subcutaneous golimumab (BSA-based dose if <45 kg; fixed dose if ≥45 kg) or placebo (2:1) for 52 weeks. The primary endpoint was C-peptide area under the curve (AUC) at Week 52 after a 4-hour mixed-meal tolerance test. Insulin use, HbA1c, hypoglycemia rates, and proinsulin/C-peptide ratios were assessed.

Results: 84 participants were enrolled (golimumab, n = 56; placebo, n = 28). The study was positive as mean (SD) 4-hour C-peptide AUC at Week 52 was 0.64 (0.423) and 0.43 (0.388) pmol/mL with golimumab and placebo, respectively (P<0.001; Figure). Golimumab-treated participants had lower insulin use, hypoglycemia rates, and proinsulin/C-peptide ratios versus placebo. Both groups maintained good glycemic control. Golimumab was well tolerated, without any new safety signals.

Conclusion: In this study, golimumab demonstrated the ability to preserve endogenous insulin production and improve clinical and metabolic parameters in children and young adults with newly diagnosed stage 3 T1D.

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Clinical Trial Registration Number: NCT02846545

Supported by: Janssen Research & Development, LLC

Disclosure: T. Quattrin: Employment/Consultancy; Janssen Research & Development. Other; Clinical Trial, Principal investigator of the Buffalo site: Janssen, Provention Bio, Inc, OPKO Biologics Ltd, Clinical Trial, Co-Investigator Buffalo site: Novo Nordisk, Boehringer Ingelheim Pharmaceutical, Eli Lilly and Company, AbbVie Incorporated, Rhythm Pharmaceuticals Incorporated, Sanofi Pharmaceutical.

54

Development of novel modulators of the GABAA receptor for diabetes therapy

J. Eckel1, B. Hasse2, B. Belgardt3, M. Hecht4, R. Wördenweber5, A. Piechot6, M. Roden7;

1KomIT - Center of Competence for Innovative Diabetes Therapy, Duesseldorf, 2Algiax Pharmaceuticals, Erkrath, 3Institute for Vascular and Islet Cell Biology, German Diabetes Center, Duesseldorf, 4vivo Science, Gronau, 5A&M Labor für Analytik, Bergheim, 6Taros Chemicals, Dortmund, 7Institute for Clinical Diabetology, German Diabetes Center, Duesseldorf, Germany.

Background and aims: Accumulating data suggest that the GABAergic system may play a key role in beta-cell survival and regeneration in both type 1 and type 2 diabetes. Therefore, the development of new positive allosteric modulators of the GABAA receptors (GABAA-Rs) provides an interesting strategy for an anti-diabetic therapy, although central nervous system side effects mediated by α1-GABAA-Rs are a potential concern. Here we synthesized and functionally analyzed a series of low molecular weight thioacrylamides (ThAcs), designed to both positively modulate GABAA-R signalling and show low blood brain barrier penetration.

Materials and methods: Beta-cell proliferation was monitored using EdU incorporation in a rat beta-cell line (INS-1E). Radio ligand competition assays and functional patch clamp experiments were conducted to study the interaction between ThAcs and GABAA-Rs.

Results: Beta-cell proliferation was increased by several ThAcs at nanomolar concentrations (i.e. ~82% increased EdU incorporation after 24 hours treatment with ThAc HK-4 at 100 nM). The most effective candidate, HK-4, was further characterized. In competition assays, we observed a dose-dependent affinity of HK-4 for GABAA-Rs in rat cortical neuron membranes. In electrophysiological patch clamp assays, we determined the modulating activity of HK-4 to individual human GABAA-Rs using HEK293 cells stably expressing different subunit combinations. HK-4 induced very strong potentiation of the current elicited by the natural ligand GABA on GABAA receptors containing combinations of α2β3γ2 (current ~300% of GABA alone), α3β3γ2 (~770%) and on α1β3γ2 (~580%) subunits, respectively. Minimal blood brain barrier penetration of HK-4 was verified by mass spectrometry-based analyses of brain and plasma samples of rats. Additionally, HK-4 shows no major toxicological effects as determined in a 90-day toxicity study with daily dosing, has a favorable pharmacokinetic profile and demonstrates good oral bioavailability.

Conclusion: Our data reveal the potential of HK-4 and other ThAcs for further development into anti-diabetic drugs.

Supported by: EU and EFRE.NRW

Disclosure: J. Eckel: None.

OP 10 Developing better insulins

55

Phase I study investigating the PD, PK and safety of AT247 in comparison to insulin aspart and fast insulin aspart

E. Svehlikova1, T. Augustin2, F. Lawrence3, D. Gerring3, S. Howell3, J. Jezek3, L. Zakrzewski3, C. Magnes2, T.R. Pieber1,2;

1Medical University of Graz, Graz, Austria, 2Joanneum Research, Graz, Austria, 3Arecor Limited, Cambridge, UK.

Background and aims: AT247, an ultra-rapid acting formulation of insulin aspart designed for faster absorption following s.c. injection, will lead to better postprandial glycaemic control and is key to the development of closed-loop pump systems.

Materials and methods: Plasma glucose and serum insulin concentrations were measured in 19 adult male participants with T1DM following a single s.c. dose (0.3 U/Kg) of insulin in a randomised, double-blind, cross over euglycaemic clamp study.

Results: Data presented as median; range. AT247 had a faster onset of glucose lowering effect than insulin aspart (IAsp) and fast insulin aspart (fast IAsp) (onset of action: 17; 12-30 vs. 37; 20-88 vs. 23;16-50 mins). The early glucose lowering effect was greater for AT247 than IAsp and fast IAsp (AUCGIR 0-60min: 212; 63-465 vs. 49; 0-303 vs. 91; 11-300 mg/kg; GIR=glucose infusion rate) (Fig 1A). Similarly, greater initial insulin exposure was seen for AT247 compared to IAsp and fast IAsp (AUC Insulin 0-60min: 111; 48-205 vs. 41; 12-147 vs. 66; 25-188 mU*h/L). In addition, offset of exposure occurred earlier for AT247 than IAsp and fast IAsp (time to late 50% Cmax Insulin: 173; 89-414 vs. 212; 106-389 vs. 221;106-441 mins). (Fig 1B). Comparisons are statistically significant with p<0.05. No safety signals were detected for AT247.

Conclusion: AT247 has a superior (left-shifted) time-action and time-concentration profile as compared to both IAsp and fast IAsp.

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Clinical Trial Registration Number: EudraCT: 2018-003934-34

Disclosure: E. Svehlikova: None.

56

Once-weekly basal insulin icodec offers comparable efficacy and safety vs once-daily insulin glargine U100 in insulin naive patients with type 2 diabetes inadequately controlled on OADs

J. Rosenstock1, M. Kjærsgaard2, D. Møller2, M. Hansen2, R. Goldenberg3;

1Dallas Diabetes Research Center at Medical City, Dallas, USA, 2Novo Nordisk A/S, Bagsværd, Denmark, 3LMC Diabetes & Endocrinology, Thornhill, Canada.

Background and aims: Reducing the number of insulin injections may mitigate the burden of insulin therapy in patients with diabetes and facilitate adherence. Insulin icodec* (icodec) is a novel insulin analogue with a terminal half-life of ~196 hours in development as the first once-weekly basal insulin.

Materials and methods: This 26-week, randomized, double-blind, double-dummy, treat-to-target, phase 2 trial investigated the efficacy and safety of once-weekly icodec vs once-daily insulin glargine U100 (IGlar U100) in insulin-naïve patients with type 2 diabetes (T2D) inadequately controlled (HbA1c 7.0-9.5%) with metformin ± dipeptidyl peptidase-4 inhibitors (DPP4i). Starting doses were 70 U weekly and 10 U daily, respectively, with weekly titration to a pre-breakfast self-measured blood glucose target of 3.9-6.0 mmol/L (70-108 mg/dL). Primary endpoint was change in HbA1c from baseline to week 26. Secondary endpoints included change in fasting plasma glucose (FPG) from baseline to week 26, weekly insulin dose during the last two weeks of treatment and hypoglycaemic episodes during the on-treatment period.

Results: Participants (n = 247) were randomized 1:1 to icodec (n = 125) or IGlar U100 (n = 122). Baseline characteristics appeared similar in both groups; mean age was 59.6 years, diabetes duration 9.7 years, BMI 31.3 kg/m2 and FPG 10.0 mmol/L. Mean baseline HbA1c was 8.1% and 8.0% for the icodec and IGlar U100 groups, respectively. At week 26, estimated mean HbA1c was 6.7% for icodec and 6.9% for IGlar U100. The estimated mean change from baseline was -1.33%-points for icodec and -1.15%-points for IGlar U100 (Figure). There was no statistically significant treatment difference for change in HbA1c from baseline to week 26 (estimated treatment difference [ETD] [95% CI]: -0.18% [-0.38; 0.02]). Estimated mean FPG at week 26 was 6.84 mmol/L (icodec) and 7.05 mmol/L (IGlar U100) (ETD [95% CI]:-0.22 mmol/L [-0.66; 0.23]). The estimated mean weekly insulin dose during the last two weeks of treatment was 229 U/week for icodec and 284 U/week for IGlar U100 (estimated treatment ratio [95% CI]: 0.81 [0.69; 0.94]). During the on-treatment period, observed rates of combined level 2 (<3.0 mmol/L or <54 mg/dL) and 3 (severe) hypoglycaemia were low (53 and 46 events per 100 patient years of exposure for icodec and IGlar U100, respectively) and were not statistically significantly different (p = 0.85). There were no unexpected safety findings.

Conclusion: Icodec is the first once-weekly insulin with similar glucose-lowering effects and safety profile to once-daily IGlar U100. Insulin icodec has the potential to improve treatment acceptance and facilitate T2D management in patients needing basal insulin.

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Clinical Trial Registration Number: NCT03751657

Disclosure: J. Rosenstock: Employment/Consultancy; Novo Nordisk. Grants; Novo Nordisk. Lecture/other fees; Novo Nordisk.

57

Incidence of significant changes in pulmonary function during a 2-year study with inhaled technosphere insulin

N.S. Zaveri1, M.C. Jones1, J.A. Krueger1, B.J. Hoogwerf2, A.L. Hoogwerf1, P.M. Morey1, D.M. Kendall1;

1MannKind Corporation, Westlake Village, 2Endocrinology and Metabolism (Emeritus), Cleveland Clinic, Cleveland, USA.

Background and aims: The impact of inhaled Technosphere Insulin (TI) on measures of pulmonary function remain an important component of clinical use. TI is an ultra rapid-acting prandial insulin delivered via deep pulmonary inhalation allowing for prompt absorption in the alveoli. Prior studies have demonstrated that, on average, changes in pulmonary function testing (PFT) are limited to small changes (~1%) in forced expiratory volume in 1 second (FEV1).

Materials and methods: This comprehensive post-hoc analysis examined the incidence of significant decline in FEV1 over two years in subjects with type 1 and type 2 diabetes treated with TI. Subjects were included if they had at least five FEV1 measurements (including baseline and month 24). FEV1 must have been ≥70% of predicted at baseline. At each time point, subjects were classified as having a significant change in FEV1 if there was a ≥15% decline from baseline.

Results: Of the 377 subjects, 331 (87.8%) had no measure with ≥15% decline in FEV1 at any time point. Of subjects who had at least one measure with a decline of ≥15%, the majority (n=38) experienced this decline on a transient basis (defined as occurring at two or fewer time points). Eight individuals (2.1%) demonstrated a persistent decline in FEV1 (occurring at three or more time points). Four subjects in the comparator arm (0.7%, n=586) had persistent decline.

Conclusion: In conclusion, persistent significant decline in FEV1 during the use of TI was uncommon.

figureo

Disclosure: N.S. Zaveri: Stock/Shareholding; MannKind Corporation.

58

Improved postprandial glucose control with Ultra Rapid Lispro (URLi) versus Lispro with continuous subcutaneous insulin infusion in type 1 diabetes

M. Warren1, J. Cho2, R. Liu2, J. Tobian2, D. Ignaut2;

1Physicians East Professional Association, Greenville, 2Eli Lilly and Company, Indianapolis, USA.

Background and aims: Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion. With multiple daily injections, it has shown superior postprandial glucose (PPG) control and non-inferior HbA1c reduction compared to Lispro. The efficacy and safety of URLi vs. Lispro were evaluated in this phase 3, 16-week, treat-to-target study, in adults with type 1 diabetes on continuous subcutaneous insulin infusion (CSII). Primary endpoint was HbA1c change from baseline, with multiplicity adjusted objectives for 1- and 2-hour PPG control after a test meal, and time spent in target range 3.9 - 10.0 mmol/L (70 - 180 mg/dL) (TIR).

Materials and methods: After a 2-week lead-in on Lispro, patients were randomised to double-blind URLi (N=215) or Lispro (N=217). Two-week blinded continuous glucose monitoring (CGM) sessions were conducted prior to randomisation, and at weeks 8 and 16. In addition, a standardised meal test was performed at randomisation and at week 16 to evaluate PPG control.

Results: Non-inferiority of URLi to Lispro on the change from baseline to week 16 in HbA1c was confirmed: least squares mean (LSM) difference 0.3 mmol/mol (0.02%) with 95% CI of -0.6 to +1.2 mmol/mol (-0.06 to +0.11%). Mean change in HbA1c was -0.7 mmol/mol (-0.06%) URLi and -1.0 mmol/mol (-0.09%) Lispro, with mean HbA1c at week 16 of 58.3 mmol/mol (7.48%) URLi and 58.0 mmol/mol (7.46%) Lispro (p=0.565). URLi was superior to Lispro in controlling 1- and 2-h PPG levels during the meal test (Figure). Compared to Lispro, URLi resulted in significantly less percent time in hypoglycaemia (<3.0 mmol/L [54 mg/dL]) over the nighttime and 24-hour period: LSM difference -0.97% and -0.52% respectively, both p<0.05. TIR and time spent in hyperglycaemia (>10.0 and >13.9 mmol/L [180 and 250 mg/dL]) were similar between groups. The incidence of treatment-emergent adverse events was higher with URLi (60.5% vs. 44.7%), primarily driven by infusion site reaction and infusion site pain. Infusion-site-related events were primarily reported as mild or moderate in severity and resolved during the study; however, 3.3% of URLi-treated patients discontinued treatment due to these events. The rate and incidence of severe hypoglycaemia and diabetic ketoacidosis was similar between groups.

Conclusion: URLi was efficacious, providing superior PPG control and an acceptable safety profile compared to Lispro when administered by CSII in patients with type 1 diabetes.

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Clinical Trial Registration Number: NCT03830281

Supported by: Eli Lilly and Company

Disclosure: M. Warren: Grants; Novo Nordisk, Eli Lilly and Company, Sanofi-Aventis, Gan and Lee, AstraZeneca, Amgen. Lecture/other fees; Novo Nordisk, Eli Lilly and Company, Sanofi-Aventis, AstraZeneca, Amgen. Non-financial support; Eli Lilly and Company.

59

Long-term safety and efficacy of intraperitoneal insulin infusion by implanted pumps in a large series of patients with type 1 diabetes and initial high glucose variability

N. Jeandidier1, B. Guerci2, E. Renard3, on behalf of EVADIAC study group;

1Endocrinology Diabetes and Nutrition, University Hospital, Strasbourg, 2Endocrinology Diabetes and Nutrition, University Hospital, Nancy, 3Endocrinology Diabetes and Nutrition, University Hospital, Montpellier, France.

Background and aims: Intra-peritoneal (IP) delivery is an alternative route for insulin therapy in patients with type 1 diabetes (T1D) presenting high glucose variability under subcutaneous insulin treatment. We assessed using a post authorization safety study data the long-term safety and efficacy of IP insulin therapy.

Materials and methods: Two hundred and sixty- two patients followed in 12 French and 1 Belgian university hospitals have been enrolled in a multinational, multicenter, observational, prospective cohort study of patients with T1D, treated with Insuman Implantable 400 IU/mL in Medtronic MiniMed implantable pumps. Visits occurred according to routine clinical practice for the use of an implantable pump; refill visits every 40-45 days and ad hoc visits related to complications of the insulin treatment regimen or pump. The primary objective of the study was to better characterize identified risks of severe hypoglycemia, hyperglycemia (HG) due to insulin underdelivery, pump jamming, pump dysfunction or catheter occlusion, pump pocket infection, abnormal healing at the surgical incision site after device implantation, and skin erosion. Data after a follow-up of 2.6±0.7 years have been analyzed, representing a cumulated experience of 837.7 patient-years (PY).

Results: The cohort includes 249 (800.8PY) long term (>6 months IP treatment at study entry) and 13 (36.8PY) short term users. Patient characteristics at inclusion were: 156F/106M, age: 56.5±11.1, BMI: 25.7±4.3, T1D duration: 35.3±12.0 years, HbA1c: 7.7±1.0% (61±8.6 mmol/mol). IP insulin was motivated by brittle diabetes in 67.9%, and frequent severe hypoglycemia in 27.9%. At study entry, comorbidities included: hyperlipidemia (64.5%), hypertension (58.0%), cardiovascular diseases (27.1%).and diabetic complications were reported in 72.9% of patients. Premature discontinuation occurred in 46 cases (17.6%): 7 (2.7%) directly due to AEs, 8 deaths (3.1%) none being directly linked to pump or insulin use, 15 (5.7%) due to pump unavailability after pump explant and 16 (6.1%) due to switch to another type of treatment. Incidences of severe hypoglycemia, HG due to insulin underdelivery, pump pocket infection and skin erosion were 9.3, 21, 1.3 and 0.5 per 100 PY, respectively. Among the cases of HG due to insulin underdelivery, one ketoacidosis was reported and surgical outcomes included 8 temporary and 5 definitive explants, and 40 catheter replacements Longer duration of IP experience was significantly associated with lower risk of hyperglycemic events.

Conclusion: Our study shows sustained efficacy of IP insulin on glucose control with a low incidence of severe hypoglycemia in these patients with multiple comorbidities and initial high glucose variability. Hyperglycemic episodes related to underdelivery events were limited and solved in most cases with no surgical intervention. This data supports the utility of IP insulin delivery from implanted pumps in T1D patients with major glucose control issues.

Clinical Trial Registration Number: ENCEPP/SDPP/10872

Disclosure: N. Jeandidier: None.

60

Evening oral insulin (ORMD-0801) glycaemic effects in uncontrolled type 2 diabetes patients

R. Eldor1, A. Fleming2, J. Neutel3, K. Homer4, M. Kidron5, J. Rosenstock6;

1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2Kinexum, Harpers Ferry, USA, 3Orange County Heart Institute and Research Center, Tustin, USA, 4Integrium, LLC, Tustin, USA, 5Oramed Pharmaceuticals, Jerusalem, Israel, 6Dallas Diabetes Research Center, Dallas, USA.

Background and aims: Oral insulin formulations are being actively pursued, given their projected benefits in the forms of more physiological responses, and improved acceptance and adherence. ORMD-0801 is a novel oral human insulin formulated to increase hepatic insulin exposure, and subsequently reduce hepatic glucose production and improve glycemic control, with minimal hypoglycemia risk. This study aimed to assess the impact of ORMD-0801 on glucose homeostasis in type 2 diabetes (T2DM) patients treated for 12 consecutive weeks.

Materials and methods: In this randomized, placebo-controlled, multicenter, phase 2b, 12-week study, 354 subjects with T2DM uncontrolled by metformin mono/combination therapy and with HbA1c levels ≥7.5%, received placebo or 8 mg, 16 mg or 32 mg (2x16 mg capsules) ORMD-0801, once daily (QD) at bedtime, or two (BID) or three times (TID) daily. Masked continuous glucose monitoring was performed during a 14-day run-in period and over the last 14 days of treatment.

Results: By the end of the treatment period, HbA1c levels had improved from baseline for most active treatment regimens, with largest improvements recorded in the 8 mg QD and BID cohorts. Glucose AUC improved from baseline following 8 mg or 32 mg QD and following BID treatments at all tested doses, and was most pronounced in patients receiving 8 mg QD at bedtime. No hypoglycemia events were reported in the 8 mg QD and 16 mg BID cohorts, while all other cohorts, had hypoglycemia rates significantly lower or similar to the placebo arm. Body weight remained stable in all treatment arms and frequency of drug-related adverse events in active cohorts was similar (TID: n=9) or significantly lower (QD cohorts: n=0-6 events; BID cohorts: n=0-3 events) than in the placebo cohort (n=7 events).

Conclusion: ORMD-0801 elicited clinically significant HbA1c reductions in poorly controlled T2DM patients on standard therapies and with mean HbA1c levels >8%, without increasing hypoglycemia rate or weight. The 8 mg QD regimen appeared most effective, warranting further studies to confirm the efficacy of this once daily evening dose.

figureq

Clinical Trial Registration Number: NCT02954601

Disclosure: R. Eldor: None.

OP 11 From diagnostics to the end-stage of diabetic kidney disease

61

Evaluation of the diagnostic performance of four creatinine-based glomerular filtration rate estimation equations in people with diabetes

N. Zafari1, M. Lotfaliany2, L. Churilov1, N. Torkamani1, R.J. MacIsaac1, E.I. Ekinci1;

1Melbourne Medical School, University of Melbourne, Melbourne, 2Deakin Institute for Mental and Physical Health and Clinical Translation, Geelong, Australia.

Background and aims: Accurate diagnosis and early intervention may help reduce diabetic kidney disease progression. Gold standard methods of assessing GFR are resource-intensive and invasive. Therefore, GFR estimation equations using serum creatinine, age, and sex are used in clinical practice as a surrogate of kidney function. However, studies of the performance of these equations in estimating kidney function in people with diabetes show contradictory results. We aimed to assess the diagnostic performance of four GFR equations (chronic kidney disease epidemiology collaboration (CKD-EPI), full age spectrum (FAS), revised Lund-Malmo (rLM), and modification of diet in renal disease (MDRD)) in people with diabetes.

Materials and methods: People diagnosed with type 1 or type 2 diabetes with at least one measured GFR (mGFR) by 99m diethylenetriamine-pentaacetic acid (DTPA) and one measured serum creatinine in Austin Hospital, Melbourne, Australia, were included in this observational study (1487 participants, 2703 measurements).

Results: Diagnostic performance of four GFR estimation equations were compared with DTPA mGFR corrected for sex and Brøchner-Mortensen coefficient using concordance correlation coefficient (CCC), the reduced major axis regression (RMAR) slope and intercept, the Bland-Altman mean difference and 95% limit of agreement (LOA), bias, precision, and accuracy (P10, P30 and P50). Among participants, 591 were women (40%), 1189 had type 2 diabetes (86%), mean age (standard deviation, SD) was 62 (14) years, mean (SD) creatinine level was 94.8 (43.1) μmol/l, and mean (SD) of mGFR corrected for sex and Brøchner-Mortensen coefficient was 65.7 (25.4) ml/min/1.73m2. The CCC was highest in rLM equation (CCC=0.83) followed by the FAS, CKD-EPI and MDRD (CCC=0.81, 0.78, 0.76, respectively). The RMAR (slope, intercept) was 1, 6.4 in MDRD, 1.03, 3.0 in FAS, 0.96, 10.9 in CKD-EPI and 0.84, 11.3 in rLM. The Bland-Altman mean difference was lowest in rLM (1.0 ml/min/1.73m2), while being more than 5-folds higher in FAS, MDRD, and CKD-EPI (5.2, 6.3, and 8.2, respectively). The Bland-Altman 95% LOA was the narrowest in rLM (54.1 ml/min/1.73m2) and widest in MDRD (65.8 ml/min/1.73m2). Bias, defined as the median difference between eGFR and mGFR was the lowest in rLM (1.4ml/min/1.73m2) and highest in CKD-EPI (8.1ml/min/1.73m2). Precision, defined as interquartile range of bias was highest in rLM (16.2 ml/min/1.73m2) and lowest in MDRD (18.1 ml/min/1.73m2). The rLM equation showed the highest accuracy (P10=39%, P30=83%, P50=96%) while MDRD with a P10= 32% and CKD-EPI with P30=73%, P50=90% ranked last in the row.

Conclusion: In people with diabetes, performance of estimation equations differed depending on the assessment criterion with the revised Lund-Malmo outperforming other equations on most criteria.

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Supported by: The study was funded by the University of Melbourne

Disclosure: N. Zafari: None.

62

Waist-height ratio and waist circumference are the best estimators of visceral fat in type 1 diabetes independently of diabetic nephropathy

S. Mutter1,2, E.B. Parente1,2, V. Harjutsalo1,3, A.J. Ahola1,2, C. Forsblom1,4, P.-H. Groop2,4, FinnDiane Study Group;

1Folkhälsan Research Center, Helsinki, 2Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, 3National Institute for Health and Welfare, Helsinki, 4Abdominal Center, Nephrology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background and aims: Rising rates of obesity in type 1 diabetes (T1D) further aggravate the already high risk of cardiovascular disease. Furthermore, obesity has also been causally linked to diabetic nephropathy (DN). Therefore, understanding the body fat distribution and its clinical implications are important. Although body composition can be analyzed by Dual-energy-X-Ray-Absorptiometry (DXA) scans, in clinical practice, there is a need for a simple and accessible tool such as anthropometric measures to estimate body composition. This study is the first to investigate body composition of individuals with T1D with regards to DN and to assess the ability of anthropometric measures to estimate visceral fat in this population.

Materials and methods: In this cross-sectional study from the nationwide Finnish Diabetic Nephropathy Study (FinnDiane), we analysed the body composition from 603 DXA scans of adults with T1D and visceral fat was measured by CoreScan. Individuals with end-stage renal disease were excluded. Linear regressions were separately performed for men (n=246) and women (n=357) with measures of body composition as the dependent and BMI, waist circumference (WC), waist-hip ratio (WHR) and waist-height ratio (WHtR) as the independent variables. The relevance ranking for each anthropometric measure was based on the z-statistics. DN was defined as an AER ≥ 20 μg/min or ≥ 30 mg/24h (albuminuria) in at least two out of three urine collections. In sub-group analyses, individuals with normal AER (171 men, 284 women) or albuminuria (75 men, 73 women) were analysed separately. Median group differences were assessed with permutation tests with 10,000 permutations.

Results: In our cohort, men were older than women (46.6 vs. 41.8 years, p=0.0008), had a higher systolic BP (140 vs. 127 mmHg, p<0.0001), but the same median BMI (25.9 kg/m2, p=0.87). The percentage of visceral fat mass to total body weight (VFM%) was higher in men (1.20 vs. 0.58%, p<0.0001). Women with albuminuria had higher WHtR (0.54 vs 0.49, p=0.0007), higher BMI (27.2 vs. 25.4 kg/m2, p=0.05) and higher VFM% (0.9 vs. 0.5%, p=0. 017) compared to those without albuminuria. Men with albuminuria had also a higher WHtR (0.55 vs. 0.50, p=0.0002), higher VFM% (1.5 vs. 1.0%, p=0.0013), but no differences in BMI (26.5 vs. 25.5 kg/m2, p=0.09). In men, overall, WHtR estimated VFM% best (z-statistics=21.4), followed by WC (z=19.3), WHR (z=15.6) and BMI (z=12.5). In women, WC (z=29.4) was the best estimator, closely followed by WHtR (z=28.0), BMI (z=19.5) and WHR (z=13.5). In both men and women, the anthropometric measures were ranked in the same order when the analyses were conducted separately in those individuals with and without albuminuria.

Conclusion: Individuals with T1D and DN, regardless of sex, have greater VFM%. Independently of DN and sex, WHtR and WC are the two best measures for VFM% estimation. From a clinical perspective, this study supports the routine measurement of the sex-independent WHtR in adults with T1D.

Supported by: Folkhälsan, Academy of Finland, Stockmann, Liv och Hälsa Soc., Novo Nordisk Fdn., Diabetes Res. Fdn.

Disclosure: S. Mutter: Grants; Folkhälsan Research Foundation, Academy of Finland (316664), Wilhelm and Else Stockmann Foundation, Liv och Hälsa Society, Novo Nordisk Foundation (NNF OC0013659) and The Diabetes Research Foundation.

63

Genetics of kidney complications in diabetes subtypes

D. Mansour Aly1, T. Tuomi2,3, L. Groop1, E. Ahlqvist1;

1Department of Clinical Sciences, Lund University Diabetes Centre, Malmö, Sweden, 2Finnish Institute for Molecular Medicine, Helsinki, Finland, 3Abdominal Center, Endocrinology, Helsinki University Central Hospital, Helsinki, Finland.

Background and aims: Diabetes is a major risk factors for diabetic kidney disease (DKD). High glucose levels are generally considered the most important risk factor. However, in a recent study where we clustered diabetes into five subtypes based on six clinical variables, the subtype with the highest insulin resistance, so called Severe Insulin Resistant Diabetes (SIRD), had the highest risk of developing DKD in spite of relatively low HbA1c. Our objective was to identify genetic variants associated with DKD and compare genetic associations in the subtypes to determine if the underlying mechanisms differ.

Materials and methods: Genome wide association analysis (GWAS) of the last eGFR during follow-up was performed using HRC imputed data from the Swedish ANDIS (All New Diabetics in Scania) cohort (N=9367) in all T2D individuals and within the SIRD subtype (N=1116). A genetic risk score (GRS) was calculated for CKD-specific SNPs and analysed by logistic regression using non-diabetic controls from the MDC study (N=2744).

Results: eFGR was strongly associated with the A allele of rs77924615 in the well-established UMOD-PDILT locus (BETA= 0.126, p=6.605x10-13) in T2d but not in SIRD (BETA=0.063, p=0.246). ). Instead, in SIRD eGFR was associated with the C allele of rs3770382 in the CTNNA2 gene (BETA=-0.218572, p= 5.5x10-8) which was not associated in all T2D. GRS analysis showed that CKD-GRSs were not associated with risk of developing SIRD (OR=1.026, p=0.47)

Conclusion: The results provide a first support for different genetic backgrounds of DKD in diabetes subtypes. . The reason could be that DKD is more dependent on insulin resistance in SIRD, whereas glucose is a main driver of disease in other subtypes. The lack of association between the CKD-GRS and SIRD supports that CKD is secondary to insulin resistance.

Disclosure: D. Mansour Aly: None.

64

Diabetic kidney disease phenotypes, mortality and incidence of vascular outcomes in a single-centre cohort with type 2 diabetes: a 13-year follow-up observational study

G. Penno1, M. Garofolo1, E. Gualdani2, D. Lucchesi1, R. Miccoli1, F. Campi1, P. Falcetta1, P. Francesconi2, S. Del Prato1;

1Department of Clinical and Experimental Medicine, University of Pisa, Pisa, 2Epidemiology Unit, Regional Health Agency, Florence, Italy.

Background and aims: Diabetic kidney disease (DKD) is a frequent and costly complication of diabetes and a leading cause of renal failure. Additionally, DKD is associated with a substantially increased burden of cardiovascular (CV) disease. In particular, non-albuminuric DKD has become the prevailing phenotype (PH) in patients with type 2 diabetes (T2D). However, it remains unclear whether its prognosis is poorer than that of other DKD PHs. We evaluated the relationship between different DKD PHs and incidence of major vascular events and all-cause mortality in subjects with T2D.

Materials and methods: This observational prospective cohort study enrolled 986 individuals with T2D in 2002-2004; subjects were followed-up for 12.9±2.7 years. Based on UACR and eGFR (CKD-EPI), each subject was classified as: no DKD (Alb-/eGFR-), albuminuria alone (Alb+/eGFR-), reduced eGFR alone (Alb-/eGFR+), or both (Alb+/eGFR+). Vital status was retrieved for all individuals on December 31, 2017 by interrogating the Italian Health Card Database; data for vascular events were available for 972 participants (98.6%), and were obtained, to the same date, in collaboration with the Regional Health Agency of Tuscany Region through hospital discharge registers (ICD-9-CM). Subsequently to the Kaplan-Meier (K-M) analyses, hazard ratios (HRs, 95% CI) for different outcomes associated with each DKD PH were assessed by unadjusted and adjusted Cox regressions.

Results: Out of 986 T2D, 779 (79.0%) had no-DKD, 144 had DKD1-2 (14.6%), 33 (3.3%) Alb-DKD and 30 (3.0%) Alb+DKD; thus, Alb-DKD accounts for 15.9% of all DKD and for 52.4% of all DKD stages ≥3. A gradually heavier CV risk profile in terms of traditional and non-traditional risk factors is distributed through the DKD PHs. Death from all-causes occurred in 230 individuals (23.3%, 18.0 x 1000 patient/years, PYs): 19.1% no-DKD, 33.3% DKD1-2, 36.4% Alb-DKD and 70.0% Alb+DKD (K-M log-rank 77.97, p<0.0001). After adjustments, HRs for death were 1.47 (95% CI 1.04-2.07) for DKD1-2, 1.22 (0.66-2.25) for Alb-DKD and 2.43 (1.46-4.06) for Alb+DKD. Major CV events occurred in 276 out of 972 subjects (28.4%, 25.2 x 1000 PYs): 25.3, 38.5, 43.8 and 43.3% through DKD PHs (K-M, p<0.0001); HRs were 1.37 (1.00-1.89) in DKD1-2, 1.73 (0.98-3.03) in Alb-DKD, to decrease to 1.11 (0.61-2.03) in Alb+DKD, due to competition with all-cause mortality. Coronary events occurred in 184 subjects (18.9%, 16.0 x 1000 PYs): 16.7, 25.9, 31.3, and 30.0% through DKD PHs (K-M, p<0.0001); HRs were 1.41 (0.96-2.07) in DKD1-2, 2.18 (1.11-4.30) in Alb-DKD, to decrease to 1.31 (0.46-3.70) in Alb+DKD. Hospitalization for heart failure occurred in 84 subjects (8.6%, 6.8 x 1000 PYs): 6.8, 14.7, 21.9 and 13.3% through DKD PHs (p<0.0001): HRs 1.91 (1.14-3.19) in DKD1-2, 2.40 (1.07-5.38) in Alb-DKD, and 1.31 (0.46-3.70) in Alb+DKD. ESRD occurred in 71 individuals (7.3%, 5.7 x 1000 PYs): 5.7, 10.5, 9.4 and 30.0% through DKD PHs (p<0.0001): HRs 1.79 (0.99-3.26) in DKD1-2, 1.28 (0.39-4.23) in Alb-DKD and 5.37 (2.46-11.72) in Alb+DKD.

Conclusion: In our cohort with a very long follow-up, the Alb-DKD PH does not have a higher risk of mortality, has a significant risk of CVD events, mainly coronary events, has the highest risk of hospitalization for heart failure, but a low risk of renal function decline to ESRD.

Disclosure: G. Penno: None.

65

Temporal trends in renal replacement therapy in people with and without type 2 diabetes: the Fremantle Diabetes study

W.A. Davis, T.M.E. Davis;

Medical School, University of Western Australia, Fremantle, Australia.

Background and aims: Most studies that have examined the relationship between diabetes and renal replacement therapy (RRT) have utilized administrative databases and/or have had limited/incomplete data. The aim of this study was to determine i) the incidence of RRT in two well-characterized community-based cohorts of people with type 2 diabetes (T2D) studied 15 years apart compared with matched cohorts without diabetes, and ii) whether incidence rate ratios (IRRs) for RRT by diabetes status have changed over time.

Materials and methods: The Fremantle Diabetes Study (FDS) Phase I (FDS1) and Phase II (FDS2) T2D cohorts and four randomly-selected, de-identified, age-, sex- and postcode-matched people without diabetes per FDS participant were followed from entry (FDS1 1993-1996, FDS2 2008-2011) until first occurrence of RRT or death or census at 5 years, whichever came first, through the validated Western Australian Data Linkage System. Five-year incidence rates (IRs) and IR ratios (IRRs) were calculated. Cox and competing risk regression models were generated to ascertain the cause-specific (cs) and subdistribution hazard ratios (HR) for incident RRT by type 2 diabetes status and FDS phase.

Results: The 13,995 participants had a mean±SD age of 64.8±11.5 years and 50.4% were males. Thirty-one (0.2%) required RRT before study entry and were excluded from analyses. During 66,120 person-years of follow-up of the remainder, 30 commenced RRT (Figure). For the T2D cohorts, the IRR (95% CI) for FDS2 compared with FDS1 was 2.85 (1.01-9.87). For the cohorts without diabetes the corresponding IRR was 5.98 (0.77-269). The IRRs for T2D versus no diabetes were lower in FDS2 than FDS1 (9.74 (3.84, 27.8) versus 20.5 (2.29, 968); P=0.54 by Breslow-Day test). In the Cox model which included adjustment for age as the timeline and with the FDS2 no diabetes cohort as reference, the highest csHRs (95% CIs) for RRT were in FDS2 participants with T2D (10.1 (4.20, 24.5)), followed by FDS1 T2D participants (3.17 (1.01, 10.0)) and FDS 1 no diabetes (0.16 (0.02, 1.28). Further adjustment for sex, Charlson Comorbidity Index and time from start of the respective phase reduced these to 7.17 (2.09, 17.7), 2.22 (0.69, 7.27) and 0.16 (0.02, 1.32), respectively, with modest modification after adjusting for the competing risk of death. The mean age when RRT commenced showed no statistically significant difference by FDS Phase or T2D status (Bonferroni-corrected P>0.05), but the oldest age at commencement was 69.9 years for FDS1 and 85.4 years for FDS2.

Conclusion: The use of RRT is infrequent but has increased over time in community-based Australians, especially in those with T2D, probably reflecting changes in eligibility criteria including age.

figures

Supported by: Raine Foundation, University of Western Australia; Australian NHMRC Project Grants

Disclosure: W.A. Davis: None.

66

Long-term mortality among kidney transplant recipients with vs without diabetes: a nationwide cohort study in the United States

J.L. Harding1, M.E. Pavkov2, Z. Wang1, S.R. Benoit2, N.R. Burrows2, G. Imperatore2, A. Albright2, R.E. Patzer1;

1Department of Surgery, Emory University, Atlanta, 2Division of Diabetes Translation, Centers for Disease Control and Prevention, Atlanta, USA.

Background and aims: Diabetes is the leading cause of end-stage kidney disease (ESKD). Once diagnosed with ESKD, the preferred treatment is kidney transplantation. However, little is documented about the role the two main types of diabetes (type 1 and type 2 diabetes (T1D and T2D)) play in modifying prognosis among transplant recipients. Therefore, in this study we compare long-term mortality among US kidney transplant recipients with T1D, T2D, and non-diabetic (ND) causes of ESKD.

Materials and methods: Between Jan 2000 and Aug 2018, we included 254,188 first-time single kidney transplant recipients aged ≥18 years identified from the US Renal Data System, a national registry of all US citizens treated for ESKD. Transplant recipients were followed from transplant date until 10 Aug 2018 or death date, whichever occurred first. Diabetes status was defined using ICD-9-CM and ICD-10 codes as indicated on form CMS2728. Mortality status was obtained from the Social Security Administration mortality register. Cox regression models computed risk of death associated with T1D and T2D relative to ND. Models were adjusted for age, sex, race, pre-ESKD nephrology care, ESKD duration, donor factors (type (living or deceased), age, race and sex), transplant failure, and comorbidities. Standardized mortality ratios (SMR) compared death rates between the transplant population (by diabetes status) and the year (2000-2017) and age-matched general US population, obtained from the National Center for Health Statistics. Trends in SMRs over time were assessed using Joinpoint regression and annual percent changes (APC).

Results: During the study period, a total of 72,197 (28.4%) deaths occurred over a median follow-up of 6.3 (IQR: 2.9-10.5) years. In adjusted models, relative mortality risk was highest among people with T1D (Hazard Ratio: 1.74, (95%CI: 1.65-1.83)) and then T2D (1.50 (1.45-1.54)), as compared to ND. Between 2000 and 2017, SMRs significantly declined for ND, T1D and T2D groups, Figure 1. However, in 2017 SMRs were 2.38 (2.31-2.45), 6.55 (6.07-7.06), and 3.82 (3.68-3.98), for ND, T1D, and T2D, respectively, indicating continued excess mortality risk among transplant recipients as compared with the general population.

Conclusion: In the US, mortality among people receiving a kidney transplant has declined since 2000 but remains approximately 2 to 7-fold higher compared to the general population with highest rates among diabetes-related ESKD. Additional research is needed to identify effective interventions to further reduce mortality in those with diabetes who receive a kidney transplant, especially those with T1D.

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Disclosure: J.L. Harding: None.

OP 12 NAFLD: Is it all about the liver?

67

Fatty liver, irrespective of ethnicity, is associated with reduced insulin clearance and hepatic insulin resistance in obese youths

D. Trico1,2, A. Galderisi3, A. Mari4, N. Santoro5, S. Caprio5;

1University of Pisa, Pisa, Italy, 2Sant’Anna School of Advanced Studies, Pisa, Italy, 3University of Padova, Padova, Italy, 4National Research Council, Padova, Italy, 5Yale School of Medicine, New Haven, USA.

Background and aims: Non-Alcoholic Fatty Liver (NAFL) is the most common chronic liver disease in Western countries and identifies people at high risk to develop metabolic and cardiovascular disease. Fatty liver has been associated with reduced endogenous insulin clearance (EIC) and with hepatic insulin resistance (HIRI), which are early features of type 2 diabetes. These relationships, however, might be differentially affected by the ethnic background, as populations of African ancestry typically have reduced intrahepatic fat content (HFF%) but impaired EIC. Therefore, the aim of this study was to evaluate whether intrahepatic fat accumulation contributes to impaired EIC and HIRI to the same extent in the three most prevalent racial and ethnic groups in the United States.

Materials and methods: We analyzed cross-sectional and longitudinal data from a large and well-characterized multi-ethnic cohort of overweight and obese adolescents (266 boys and 366 girls, age 13.4 ± 3.1 years, BMI z-score 2.1 ± 0.7). The HFF% was quantified by a validated magnetic resonance imaging (MRI) method at baseline and after a median follow-up of 2 years. Insulin secretion rate (ISR), EIC and HIRI were assessed during 3-hour, 9-point oral glucose tolerance tests (OGTTs) by modelling glucose, insulin, and C-peptide data.

Results: African Americans (n=172) exhibited the lowest HFF% (A) and a prevalence of NAFL less than half of Caucasians (n=229) and one-third of Hispanics (n=231) (B). Furthermore, African Americans had lower EIC (C) and glucose-stimulated ISR, but similar HIRI (F) and plasma insulin levels. EIC and HIRI declined across group-specific HFF% tertiles (D, G) and were markedly lower in individuals with NAFL (E, H) in all ethnic groups. Consistently, the HFF% correlated with EIC (std. β= -0.13, p=0.0003) and HIRI (std. β=0.17, p<0.0001), irrespective of the ethnic background, after adjustment for age, sex, ethnicity, adiposity, pubertal status, and plasma glucose levels. African Americans showed lower susceptibility to intrahepatic fat accumulation at follow-up. In fact, the prevalence of adolescents whose HFF% remained stable (change < ±1%) was two-fold higher in African Americans (52%) than in Caucasians (28%) and Hispanics (20%) (p=0.036). Nevertheless, changes in HFF% over time were associated with changes in EIC (r= -0.25, p=0.02) and HIRI (r=0.22, p=0.04) across all groups, without ethnic differences.

Conclusion: This study demonstrates that intrahepatic fat accumulation is associated with reduced EIC and HIRI in obese youths, irrespective of their ethnic background. Our data dissect the metabolic characteristics of populations of African ancestry and provide novel evidence about the pathogenetic role of liver steatosis in the development of hepatic metabolic abnormalities contributing to the etiology of type 2 diabetes.

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Clinical Trial Registration Number: NCT01966627

Supported by: EFSD Future Leaders Mentorship Programme, EFSD Rising Star Fellowship

Disclosure: D. Trico: None.

68

Prevalence of non-alcoholic steatohepatitis in a cohort of subjects undergoing bariatric surgery

E. Lembo, M.F. Russo, G. Mingrone;

Università Cattolica del Sacro Cuore - Sede di Roma, Rome, Italy.

Background and aims: Non-alcoholic steatohepatitis (NASH) represents one of the stages of fatty liver disease (NAFLD) with a very high risk to evolve in cirrhosis and hepatocellular-carcinoma. Currently, the only diagnostic method is a liver biopsy which, for economic reasons and for the risks associated with the procedure, is not always performed. Our aim is to find a non-invasive predictor that helps us to identify high-risk patients in whom to perform a liver biopsy.

Materials and methods: We evaluated a cohort of 309 subjects, aged between 19 and 69 years, with an average BMI of 49.23±8.63 kg/m2, who underwent liver biopsy during bariatric surgery. 31.8% of the subjects had type 2 diabetes mellitus (DM2) with an average HbA1c of 57±7 mmol/mol.Liver biopsy was classified according to Kleiner's NAFLD Activity Score (NAS) that has a range from 0 to 8: NAS≥ 3 is indicative of NASH. The most common non-invasive liver damage tests were calculated: NAFLD Fibrosis Score (NFS), AST/ALT ratio, AST to Platelet ratio (APRI), fibrosis 4 score (FIB4). Spearman's correlation analysis between the above-mentioned scores and NAS was performed. We also run a neural network analysis (ANN) to identify the predictors of NASH with the following variables: presence of DM2, HbA1c, HOMA IR index, OGIS, NFS, AST/ALT, APRI, FIB4.

Results: The prevalence of NASH in the 309 liver biopsies was 69.2%: 58.7% with NAS between 3 and 4, 10.5% with NAS between 5 and 6. No NAS ≥7 was found. In the sample of patients with DM2 the prevalence of NASH was 82.1%: 68.4% with NAS between 3 and 4 and 13.7% with NAS between 5 and 6. Spearman's correlation analysis on the whole sample between the non-invasive tests and the NAS shows rs equal to 0.302 with P<0.01 for APRI; rs equal to -0.205 with P<0.01 for AST/ALT; rs equal to 0.143 with P<0.05 for FIB-4. The ANN highlighted specificity >70% in excluding NASH (NAS 0-1 and 2). The specificity was 54% for NAS=3; 79% for NAS=4; 61% for NAS=5 and 90% for NAS=6.

Conclusion: DM2 remains an important risk factor for NASH independently of the severity of this latter. None of the non-invasive tests currently used, including NAFLD Fibrosis Score, shows a good correlation with the histological stages of NASH. Our statistical model well predicts the absence of NASH, but it is unable to discriminate among severity stages, apart for NAS 5.

figurev

Disclosure: E. Lembo: None.

69

Hepatic fibrosis but not steatosis is independently associated with diabetic kidney disease in non-obese patients with type 2 diabetes

D. Seo1, Y.-H. Lee2, S. Seo1, Y. Cho1, S. Ahn1, S. Hong1, Y. Choi3, B. Huh3, S. Kim1;

1Inha University School of Medicine, Incheon, 2Yonsei University College of Medicine, Seoul, 3Huh's Diabetes Center, Seoul, Republic of Korea.

Background and aims: Recent studies investigated the association between nonalcoholic fatty liver disease (NAFLD) and diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus (T2DM) but the results are inconclusive. Here we aimed to investigate the association between NAFLD and DKD in a large cohort of participants with T2DM.

Materials and methods: In this cross-sectional study, a total of 3,439 patients (1,710 men and 1,729 women) with T2DM were recruited from the Seoul Metabolic Syndrome cohort between 2003 and 2016. NAFLD was defined by ultrasonographic detection of steatosis in the absence of other liver diseases and advanced fibrosis was defined as FIB4 index < 1.45. DKD was defined as an estimated glomerular filtration rate (eGFR) of ≤60 mL/min/1.73 m2.

Results: Among the entire population (mean age 57.4 ± 10.3 years and duration of diabetes 7.7 ± 7.1 years), 1869 (54.3%) subjects had NAFLD and 754 (40.3%) subjects had advanced fibrosis among those with NAFLD. The prevalence of DKD was higher in those with advanced fibrosis than those with no NAFLD and liver steatosis (no NAFLD vs. liver steatosis vs. advanced fibrosis: 7.0% vs. 4.5% vs. 11.9%, p <0.001). After adjustment for potential confounders including age, gender, duration of diabetes, smoking, waist circumference, blood pressures, medications, Kitt, HgbA1c, triglyceride and HDL cholesterol, advanced fibrosis, but not liver steatosis, was associated with increased risk of DKD (odds ratio [OR] 1.75; 95% confidence interval [CI] 1.08-2.86; p = 0.034) in patients with body mass index (BMI) < 25 kg/m2, but no association was found in those with BMI ≥ 25 kg/m2.

Conclusion: This study suggests that advanced liver fibrosis, a severe form of NAFLD, was independently associated with increased risk of DKD in patients with T2DM and BMI < 25 kg/m2.

Disclosure: D. Seo: None.

70

Diagnosing at-risk NASH: NIS4 performances in patients with escalating number of metabolic risk factors

R. Hanf1, V. Ratziu2, S.A. Harrison3, S. Francque4, Q.M. Anstee5, N. Dam1, Y. Hajji1, A. Roudot1, J. Brozek1, B. Staels6, D.W. Hum7, P. Birman1, S. Hosmane7, P. Chaumat1, A.J. Sanyal8;

1GENFIT SA, Loos, France, 2Hôpital Pitié-Salpêtrière, Paris, France, 3Summit Clinical Research, San Antonio, USA, 4Department of Gastroenterology and Hepatology, Antwerp University Hospital, Antwerp, Belgium, 5Newcastle University, Freeman Hospital, Newcastle Upon Tyne, UK, 6Université de Lille, INSERM, Pasteur, Lille, France, 7GENFIT CORP, Cambridge, USA, 8Virginia Commonwealth University, Richmond, USA.

Background and aims: Metabolic risk factors or features of metabolic syndrome (MetS) are important risk factors associated with NASH. The aim was: i) to assess the association of metabolic risk factors and prevalence of at-risk NASH (i.e. NAFLD activity score ≥4 and Fibrosis stage ≥2) and ii) to assess overall diagnostic performance of NIS4, a recently developed non-invasive test for detection of at-risk NASH, in patients with escalating numbers of metabolic risk factors.

Materials and methods: Patients (N=2363) screened for the NASH RESOLVE-IT trial with centrally-read liver biopsy were scored for histological activity (NAS) and fibrosis. Data on metabolic risk factors as defined by the IDF were collected: central obesity, dyslipidemia, raised blood pressure and raised fasting plasma glucose or diagnosed type 2 diabetes (T2D). Chi2 test was used to compare prevalence of at-risk NASH in patients with ≥3 vs <3 metabolic risk factors and in patients with increasing numbers of metabolic risk factors. Univariate logistic regressions were performed for ranking of metabolic risk factors as predictors for at-risk NASH. Diagnostic performance of NIS4 to detect at-risk NASH (AUROC) in subpopulations was compared using DeLong test.

Results: Presence of ≥3 metabolic risk factors (N=1666) was significantly associated with higher prevalence of at-risk NASH as compared to patients with <3 risk factors (N=697) (59% vs 38%; OR=2.4 [2.01-2.88]; p<0.0001). Prevalence of at-risk NASH increased with the number of risk factors: 13% in patients without (4/32; comparator), 34% in patients with 1 (57/167; p<0.05), 41% in patients with 2 (202/498; p<0.01), 51% in patients with 3 (419/827; p<0.0001) and 68% in patients with 4 metabolic risk factors (569/839; p<0.0001). Univariate logistic regressions ranked raised FPG or diagnosed T2D as the metabolic risk factor most strongly associated with prevalence of at-risk NASH by p-value (OR=2.5 [2.12-2.96]; p<10-26), followed by raised blood pressure (OR=1.96 [1.6-2.39]; p<10-10), dyslipidemia (OR=1.73 [1.45-2.07]; p<10-8) and finally central obesity (OR=2.0 [1.35-2.96]; p<0.001). Among patients with NIS4 data (N=469), performance of NIS4 to identify at-risk NASH was comparable in patients with ≥3 (AUC=0.81) or <3 risk factors (AUC=0.85; p=0.29) and in patients with 1 (18/32; AUC=0.89), 2 (40/78; AUC=0.83), 3 (98/169; AUC=0.81) or 4 (99/184; AUC=0.79) metabolic risk factors (p>0.05 for all comparisons).

Conclusion: The prevalence of at-risk NASH increases with the number of metabolic risk factors. Impaired FPG or diagnosed T2D is the strongest predictors for at-risk NASH. NIS4 showed high diagnostic accuracy for at-risk NASH regardless of number of metabolic features, supporting the use of NIS4 to screen individuals with metabolic risk factors for at-risk NASH identification.

Clinical Trial Registration Number: NTC02704403

Disclosure: R. Hanf: Employment/Consultancy; GENFIT SA. Stock/Shareholding; GENFIT SA.

71

Role of patatin-like phospholipase domain-containing 3 gene for hepatocellular lipid content in the severe insulin-resistant diabetes cluster

O.P. Zaharia1,2, K. Strassburger1,2, B. Knebel1,2, Y. Kupriyanova1,2, Y. Karusheva1,2, M. Wolkersdorfer3, K. Bódis1,4, D. Markgraf1, V. Burkart1,2, J.-H. Hwang1,2, J. Kotzka1,2, H. Al-Hasani1,2, J. Szendroedi1,4, M. Roden1,4, GDS Group;

1German Diabetes Center, Düsseldorf, Germany, 2German Center for Diabetes Research (DZD), München-Neuherberg, Germany, 3Landesapotheke Salzburg, Salzburg, Austria, 4Division of Endocrinology and Diabetology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.

Background and aims: The rs738409(G) single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene associates with increased risk of nonalcoholic fatty liver disease (NAFLD) and its progression. As one of the recently-described diabetes subgroups (clusters) specifically relates to NAFLD, this study examined whether this SNP differently associates with hepatic lipid content (HCL) and tissue-specific insulin sensitivity in patients with recent-onset diabetes mellitus.

Materials and methods: A total of 917 participants of the German Diabetes Study (GDS) underwent genotyping, hyperinsulinemic-euglycemic clamp tests with stable isotopic tracer dilution and 1H magnetic resonance spectroscopy. Cluster analyses using age, BMI, glycemia and homeostasis model estimates (HOMA-IR, -B) and diabetes-related autoantibodies identified distinct diabetes subgroups.

Results: Across the study population, the G allele associated with increased HCL (β=0.36, p=0.01) and increased peripheral insulin sensitivity (β=0.06, p=0.03), independent of age, sex and BMI. The severe insulin resistant diabetes (SIRD) cluster had the lowest whole-body insulin sensitivity compared to severe insulin deficient (SIDD), moderate obesity-related (MOD), moderate age-related (MARD) and severe autoimmune diabetes clusters (SAID; all p<0.001). Interestingly, SIRD presented with higher prevalence of the rs738409(G) SNP compared to the other diabetes clusters and the glucose-tolerant control group (p<0.05). Also, HCL was higher in SIRD [13.6 (5.8;19.1)%] compared to MOD [6.4 (2.1;12.4)%, p=0.028], MARD [3.0 (1.0;7.9)%, p<0.001], SAID [0.4 (0.0;1.5)%, p<0.001] and the glucose tolerant group [0.9 (0.4;4.9)%, p<0.001]. Although the PNPLA3 polymorphism did not directly associate with whole-body insulin sensitivity or HCL in SIRD, the G allele carriers had higher circulating free fatty acid concentrations and greater adipose-tissue insulin resistance compared to non-carriers (both p<0.001).

Conclusion: Patients of the severe insulin resistant diabetes cluster are more frequently carriers of the rs738409(G) variant. The associated adipose tissue insulin resistance and excessive lipolysis likely accelerate NAFLD and diabetes progression in this cluster.

Clinical Trial Registration Number: NCT01055093

Supported by: BMBF, DZD

Disclosure: O.P. Zaharia: None.

72

Evaluation of determinants of hepatic insulin clearance: a Mendelian randomisation study

A. Lamprinou1, J. Machann2, F. Schick2, S.S. Eckstein3, C. Dalla Man4, R. Visentin4, N. Stefan1, A.L. Birkenfeld1, A. Peter5, H.-U. Häring6, A. Fritsche1, M. Heni1, R. Wagner1;

1Internal medicine IV-Endocrinology, Diabetology and Nephrology, University Hospital of Tuebingen, Tuebingen, Germany, 2Section on Experimental Radiology, Department of Diagnostic and Interventional Radiology, University Hospital of Tuebingen, Tuebingen, Germany, 3Institute for Diabetes Research and Metabolic Diseases of the Helmolz Centre Munich, University Hospital of Tuebingen, Tuebingen, Germany, 4Department of Information Engineering, University of Padua, Padua, Italy, 5Diagnostic Laboratory Medicine, Institut of Clinical Chemistry and Pathobiochemistry, University Hospital of Tuebingen, Tuebingen, Germany, 6Institute for Diabetes Research and Metabolic Diseases of the Helmolz Centre Munich h, University Hospital of Tuebingen, Tuebingen, Germany.

Background and aims: Besides insulin resistance, type 2 diabetes associates with decreased insulin clearance. Most insulin degradation occurs in the liver. The magnitude of this effect is closely related to systemic insulin sensitivity. As insulin resistance is often accompanied by fatty liver, we investigated whether there is a causal link between liver fat accumulation and hepatic insulin clearance (HIC). We also investigated the causal connection between features of the metabolic syndrome and HIC.

Materials and methods: We computed HIC using data from oral glucose tolerance tests in 3391 non-diabetic individuals and the “Oral C-peptide and Insulin Minimal Models”. Liver fat was quantified by 1H-MR-spectroscopy in 1211 participants. We performed Mendelian randomization analyses (MR-Egger) to test for causal determination of HIC by liver fat and potentially related traits using established single nucleotide polymorphisms (SNPs; 115 for liver fat, 155 alanine-aminotransferase, 37 insulin sensitivity, 38 insulin secretion, 72 fasting insulin, 5285 Body mass index, 270 waist circumference, 442 triglycerides, 620 HDL-Cholesterol, 193 C-reactive protein).

Results: HIC associated inversely with liver fat content (β=-0.1±0.03, p<0.003) and insulin sensitivity (β=0.6±0.03, p < 0.0001). Both liver fat content and HIC were independently associated with insulin sensitivity (β=-0.2±0.03 and β=0.1±0.02 respectively, both p < 0.0001). Neither liver fat content nor alanine-aminotransferase were causally linked to HIC (p=0.6 and 0.2, respectively), as indicated by the Mendelian randomization. BMI-related SNPs were causally associated with HIC (β=-0.2±0.02, p<0.001) but not waist circumference SNPs (β=-0.1±0.08, p=0.06). Both, genetically determined insulin sensitivity and fasting insulin were causally related to HIC (β=0.3±0.1, p=0.04 and β=-0.4±0.1, p=0.005 respectively). C-reactive protein and HDL levels were causally associated with HIC in the MR-Egger analysis, but in a direction opposite to the trait’s association with HIC (β=0.1±0.07, p=0.03 and β=-0.09±0.03, p=0.01 respectively).

Conclusion: Hepatic steatosis does not causally influence hepatic insulin extraction. Other components of the metabolic syndrome such as inflammation and low HDL cholesterol seem to compensate peripheral hyperinsulinemia by increasing hepatic insulin extraction.

Disclosure: A. Lamprinou: None.

OP 13 Diabetic retinopathy: see what's new?

73

Assessing retinopathy screening frequency in adolescents with type 1 diabetes using Markov model

A.S. Januszewski1, V. Velayutham2,3, P. Benitez-Aguirre2,3, M. Craig2,3, G. Liew2,4, Y. Cho2,3, A.J. Jenkins1, K. Donaghue2,3;

1University of Sydney, Camperdown, 2The Children’s Hospital at Westmead, Westmead, 3University of Sydney, Sydney, 4University of Sydney, Westmead, Australia.

Background and aims: Current ISPAD guidelines recommend diabetic retinopathy (DR) screening to start at age 11 yrs with Type 1 diabetes (T1D) duration of 2 to 5 yrs at 1-2 year intervals. There is a growing body of evidence suggesting that the DR screening in T1D can be performed less frequently.

Materials and methods: The time-course of DR progression was assessed by analysis of repeated retinal images from 2,169 adolescents (baseline (mean±SD): age 13.1±2.1 yrs, HbA1c 8.5±1.4%, T1D duration 5.4±2.9 yrs, follow-up time 5.3±3.6 yrs) with at least two assessments by seven-field stereoscopic retinal photography between 1990 and 2018. DR was graded using the Early Treatment Diabetic Retinopathy Study (ETDRS) scale. As DR screening results represent observations of a continuous-time process at arbitrary / nonregular intervals we have used Markov model to calculate probabilities of DR stage change over time, for age, duration and decade of diabetes diagnosis, sojourn time for each stage and hazard ratio (HR) of DR stage transition per 1% of HbA1c increase.

Results: DR progressed to severe non-proliferative (SNP) DR or worse in 11 of 2,169 adolescents (0.5%). Probability of transition from no DR to severe NPDR or above over 5 yrs was 0.1% and over 15 yrs 1.2%. Probability of transition from moderate NPDR to severe NPDR or above was respectively 16.1% and 40.9%. Sojourn time in no DR stage was 4.9yrs (2.9yrs with HbA1c>9.5% and 7.8yrs with HbA1c≤7.5%). HRs for transition per 1% HbA1c increase were: 1.25 (1.18-1.33) from no DR to minimal NPDR, 1.13 (1.05-1.23) from minimal to mild NPDR, 1.38 (1.20-1.59) from mild to moderate NPDR and 1.30 (0.95-1.78) from moderate to severe NPDR or above. Progression was greater for higher HbA1c, older age and longer duration at first screening and earlier calendar year of diabetes diagnosis (Table 1).

Conclusion: Our data supports the approach of less frequent DR screening in this group of adolescents with T1D, especially without any DR at initial screening, younger age and shorter duration, and lower HbA1c. Although progression of DR to advanced stages is rather slow in adolescents, impaired glycaemic control dramatically increases it.

figurew

Disclosure: A.S. Januszewski: None.

74

One-point HbA1c value does not always reflect current retinopathy while ΣexcessA1C, an index of total glycaemic exposure, does in type 1 diabetes: a DCCT/EDIC subgroup analysis

A. Hirose1,2, Y. Maeda1,3, M. Minami3, A. Goto4, K. H Sonoda5, S. Kitano2, Y. Uchigata6;

1Minami Diabetes Clinical Research Center, Fukuoka, 2Diabetes Ophthalmology, Diabetes Center, Tokyo Women's Medical University, Tokyo, 3Clinic Masae Minami, Fukuoka, 4Graduate School of Data Science, Yokohama City University, Yokohama, 5Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 6Medicine, Diabetes Center, Tokyo Women's Medical University, Tokyo, Japan.

Background and aims: Why do some diabetic patients with high HbA1c values (A1Cs) measured at given time points (one-point A1Cs) have no retinopathy, while others with low values do have it? It seems that one-point A1C does not always reflect current retinopathy. Previously we demonstrated that ΣexcessA1C, an index of total excess glycemic exposure, could substantially predict retinopathy in type 1 diabetes. To show this, it was crucial to study only patients with A1C data for the entire period after the onset of diabetes to exclude the unknown disturbing effect of metabolic memory. To compare the prediction capability for retinopathy development of one-point A1C with that of ΣexcessA1C, we performed subgroup analyses of the DCCT/Epidemiology of Diabetes Interventions and Complications (EDIC) database.

Materials and methods: We employed a window of one year for A1C and retinopathy data (target date +-0.5 year). A given time point at the end of X years after the onset of diabetes was denoted “yearX”. To examine only selected cases, namely those who had A1C data for the entire period of hyperglycemia, we studied patients of primary prevention with the shortest diabetes duration at DCCT baseline (≤14 months). We included only patients who had no missing data of retinopathy in all three periods (year5, year9 and year13). Retinopathy was evaluated by steps on the severity scale of the final Early Treatment Diabetic Retinopathy Study, and it was defined as development positive (DR+) if a patient was step 2 or higher; otherwise it was considered negative. ƩexcessA1C was calculated by adding all the values of each yearly A1C - 6.5 (%; = 48 mmol/mol) from year1 to a given year. To compare the prediction capabilities of A1C and ΣexcessA1C at year5, year9 and year13 for DR+, AUCs of the receiver-operating characteristic curves were calculated, and those of A1C and ΣexcessA1C were compared by the DeLong test. Two-sided P value<0.05 was considered significant. The timepoints of patients when they moved from DCCT into EDIC were examined.

Results: The 70 cases that fulfilled the criteria (mean duration: 12.2 months at year1) showed 33, 45 and 54 DR+ at year5, year9 and year13, respectively. At year5, both A1C and ΣexcessA1C showed moderate capabilities of prediction for DR+ at a similar level (AUC=0.6704 vs 0.7019, respectively; P=0.3493 for difference). But at year9, A1C showed little capability for prediction while ΣexcessA1C showed a substantial one, significantly better than A1C’s (AUC=0.5480 vs 0.7333, respectively; P=0.0001 for difference). The same was true at year13 (AUC=0.5816 vs 0.7830, respectively; P=0.0012 for difference). Most patients moved from DCCT into EDIC between year5 and year9, which was thought to be related to the changes in their glycemic controls.

Conclusion: One-point A1C does not always reflect current retinopathy, especially after the changes in glycemic control, while ΣexcessA1C does, even after these changes, since retinopathy results from long-term glycemic exposure after the onset of type 1 diabetes.

Disclosure: A. Hirose: Grants; Novartis Pharma K.K.

75

Cerebral small-vessel disease is associated with the severity of diabetic retinopathy in type 1 diabetes

M.I. Eriksson1,2, P. Summanen1,3, D. Gordin1,2, C. Forsblom1,2, S. Shams4,5, R. Liebkind6, T. Tatlisumak6,7, J. Putaala6, P.-H. Groop1,2, J. Martola4,8, L.M. Thorn1,2;

1Folkhälsan Research Center, Helsinki, Finland, 2Department of Nephrology, University of Helsinki and Helsinki University Hospital Abdominal Center, Helsinki, Finland, 3Department of Ophthalmology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 4Department of Radiology, Karolinska University Hospital and Karolinska institute, Stockholm, Sweden, 5Department of Radiology, Stanford University Hospital, Stanford, USA, 6Department of Neurology, Helsinki University Hospital, Helsinki, Finland, 7Department of Neurology, University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, Sweden, 8Department of Radiology, Helsinki University Hospital, Helsinki, Finland.

Background and aims: Cerebral small-vessel disease (SVD) is a common finding in neurologically asymptomatic individuals with type 1 diabetes. The retinal vasculature is thought to mirror the vasculature of the brain, but there are limited data on this association in type 1 diabetes. Our aim was to further study associations between severity of diabetic retinopathy (DR) and cerebral SVD in type 1 diabetes.

Materials and methods: We enrolled 191 participants with type 1 diabetes and 30 healthy age- and sex-matched control subjects (median age 40 [33-45] years; 53% female; mean HbA1c 66±12 mmol/mol, duration 22 [18-31] years) as part of the Finnish Diabetic Nephropathy Study. All participants underwent a clinical investigation, brain MRI, and fundus imaging, which were evaluated according to the diabetic retinopathy (DR) severity scale (ETDRS). Brain MRIs were assessed for signs of SVD (white-matter hyperintensities, cerebral microbleeds [CMBs], and lacunar infarctions) and analyzed in relation to DR severity.

Results: We observed SVD in 67 (35%), CMB in 45 (24%), white-matter hyperintensities in 44 (23%), and lacunes in 4 (2%) participants with type 1 diabetes. Of the controls, only three had SVD and none of them had signs of retinopathy. In type 1 diabetes, the participants with cerebral SVD had higher median ETDRS scores and a higher prevalence of proliferative DR than those without SVD (35 [20-61] vs 20 [20-35], p=0.035 and 34 vs 9%, p=0.002). Proliferative DR remained significant after adjustment for age, systolic BP, and albuminuria (OR 2.65 [95% CI 1.08-6.49]), however ETDRS was no more significant in the adjusted analyses. Participants with ETDRS score >35 had higher prevalence (40 vs. 17%, p=0.001) and number (p<0.001) of CMBs than those with ETDRS score ≤35, while no association was observed for white-matter hyperintensities or lacunes. Participants with CMBs had a higher median ETDRS score (35 [20-64] vs 20 [20-35], p=0.042) and higher prevalence of proliferative DR (29 vs 10%, p=0.002). The ETDRS scores increased by number of CMBs and was 20 (20-35) in participants with 0 CMB, 20 (20-45) in those with 1-2 CMBs, and 64 (43-70) in those with >2 CMBs (p=<0.001). An independent association with >2 CMBs was found for both the ETDRS score (OR 1.05 [95% CI 1.01-1.08], p=0.005) and proliferative DR (OR 8.81 [95% CI 2.01-38.5], p=0.004) in separate analyses adjusted for age, albuminuria, and systolic BP.

Conclusion: Presence of cerebral SVD on brain MRI, particularly CMBs, is associated with the severity of DR. Fundus imaging may serve as a mirror into the brain for assessment of SVD and may specifically reflect the burden of CMBs.

Supported by: Folkhälsan Research Foundation, Academy of Finland, Stockmann Foundation, EVO governmental grants

Disclosure: M.I. Eriksson: Grants; Medical Society of Finland.

76

Molecular and functional effects of methylglyoxal on human microvascular retinal cells

M. Aprile1, A. Leone2, F. Scognamiglio1, C. Nigro2, A. Nicolò2, C. Perfetto1, S. Cataldi1, V. Costa1, C. Miele2, A. Ciccodicola1;

1National Research Council (CNR), IGB-ABT, Naples, 2National Research Council (CNR), URT GDD IEOS, Naples, Italy.

Background and aims: One of the primary events of diabetic retinopathy (DR) is the functional impairment of microvascular cells. Methylglyoxal (MGO) - reactive glycolysis by-product - mediates hyperglycemia-induced alterations in macrovascular endothelial cells, but its effects on retinal microvascular cells have not yet been clarified. We evaluated MGO effect on human retinal endothelial cells (hRECs) in terms of cell viability, angiogenic-capacity and transcriptomic identity, aiming to define miRNA/mRNA networks and molecular determinants mediating MGO-induced glucotoxicity in DR.

Materials and methods: Commercially available primary hRECs were exposed to increasing doses of MGO at different times and cell viability was assessed by MTT assay. Migration ability (transwell assay) and angiogenic capacity (tube formation assay) were analysed in hRECs exposed to 500μM MGO for 72h. Transcriptome and miRNome of MGO-exposed hRECs were analyzed (in triplicate) by RNA-Seq (~60M paired-end reads/sample) and smallRNA-Seq (10M single-end reads/sample). TopHat v2 and RNA-SeqGUI were used for mapping and secondary analysis; KEGG, DAVID and Panther databases to infer biological significance. Small-RNAseq data were analyzed by iMir package and miRPathDB 2.0. Enrichment for transcription factors (TFs) binding sites in differentially expressed (DE) genes was inferred using public ENCODE ChIP-Seq data.

Results: hRECs exposure to 500μM MGO for 72h does not compromise cell viability but impairs both cell migration and tube formation capacity by ~30% (p≤0.05) and ~70% (p≤0.05), respectively. Transcriptome analysis revealed that MGO exposure increases expression of ~1200 genes encoding mainly transcriptional regulators (~25%, p=9.1e-46) and apoptosis-related genes (~7%, p=3.6e-6). Conversely, MGO exposure causes downregulation of ~500 genes encoding membrane glycoproteins (~36.5%, p=4.2e-12), cell cycle control factors (~13%, p=1.1e-19) and cell adhesion molecules (~11%, p=2.2e-9), including integrins (~5%, p=1.5e-6). Furthermore, enrichment of TF binding in promoters of DE genes indicated a robust network of structural proteins regulated by a small subset of TFs deranged in hRECs upon MGO exposure, such as such as Nrsf, Ctcf and Znf263. MiRNome analysis revealed ~70 DE miRNAs following MGO treatment. Interestingly, most of the DE genes involved in cell cycle, insulin, PI3K-Akt, FOXO, P53 (~60%) are predicted targets of DE miRNAs that potentially represent putative mediators of MGO-induced transcriptional perturbation. Experimental assays to address whether selected protein-coding genes and miRNAs mediate MGO-induced glucotoxicity in hRECs are in progress

Conclusion: Our work reveals for the first time MGO as mediator of hyperglycemia-induced damage in microvascular retinal cells by reduction of cell motility and angiogenic capacity, as well as by extensive perturbation of cell transcriptional program. MGO directly promotes the expression of transcriptional regulators, whereas suppressing factors involved in cell cycle and cell-cell interactions. Aiming to attenuate the pathogenic impact of MGO on DR progression we are also exploiting the possibility of targeting candidate mRNAs/miRNAs to restrain MGO-induced detrimental effects on hRECs.

Supported by: EFSD/Boehringer Ingelheim European Research Programme in Microvascular Complications of Diabetes

Disclosure: M. Aprile: None.

77

The role of AMPK in the mechanism of ischaemic retinopathy: an in vitro study

M.N. Dátilo, G.P. Formigari, J.B. Lopes de Faria, J.M. Lopes de Faria;

Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil.

Background and aims: Ischemic proliferative retinopathy is the late stage of retinopathies caused by different diseases, such as diabetes and retinopathy of prematurity. Retinal proliferative retinopathy is characterized by abnormal neovascularization induced by hypoxia and may lead to irreversible blindness, and a better understanding of how to control neovascularization remains an unmet medical need. AMP-activated protein kinase (AMPK) -a cellular energy sensor- is involved in intracellular homeostasis. Recent studies have demonstrated the role of AMPK in tumor progression and cerebral angiogenesis after ischemia via hypoxia-inducible factor-1α (HIF-1α) modulation. Polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are a structural component of the retina. Previous studies showed that DHA is implicated in the amelioration of diabetic retinopathy by inhibiting inflammation of retinal endothelial cells. Recent evidence has shown that DHA activates AMPK in several tissues, leading to an increase in glucose uptake and control of energy metabolism, thus preventing ischemic injury. The aim of this study is to investigate whether AMPK is involved in the early changes in endothelial retinal cells that are exposed to hypoxic conditions and the possible beneficial effects of AMPK activator compounds.

Materials and methods: Primary human retinal microvascular endothelial cells (ACBRI-181) (passage 6 to 8) were cultured in endothelial growth media with 10% of FBS. At 70% of confluence, the cells were exposed for 24h to dimethyloxalylglycine (DMOG, 400 μM)—a prolyl-hydroxylase inhibitor used as a chemical model of hypoxia—both with and without the presence of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (0.5mM) or DHA (50μM). Markers of hypoxia (HIF-1α, HIF-2α, and vascular endothelial growth factor [VEGF]), tight junction integrity (Zonula Occludens-1 [ZO-1]), and endothelial mesenchymal transition (vimentin) were evaluated by immunofluorescence. Cell migration assay was performed to assess endothelial cell function. AMPK activity was assessed by western blot.

Results: Cells exposed to DMOG treatment displayed increased expression of HIF-1α, VEGF, and nuclear translocation of HIF-2α (p<0.002 vs control group [CT]). DMOG promoted disruption of the tight junction ZO-1, upregulated vimentin expression, and increased cellular migration compared to CT (p<0.0001). Supplementation with either DHA or AICAR fully prevented the above abnormalities (p<0.0001). AMPK expression showed a reduction in Thr172 phosphorylation accompanied by a reduction in phospho-ACC (Ser79), indicating a decrease in AMPK activity under DMOG treatment compared to the CT group (p<0.04).

Conclusion: This set of experiments indicates that hypoxia decreases AMPK activity in retinal endothelial cells. Treatment with AMPK activators restores cellular homeostasis. Further translational studies are being conducted to evaluate the effects of AMPK activators in ischemic proliferative retinopathy.

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Supported by: FAPESP (14/22687-0) (18/07398-3) (16/16655-4)

Disclosure: M.N. Dátilo: None.

78

Effects of topical administration (eye drops) of semaglutide on retinal neuroinflammation and vascular leakage in experimental diabetes

R. Simó1,2, P. Bogdanov1,2, H. Ramos1,2, J. Huerta1, C. Hernández1,2;

1Diabetes and Metabolism Research Unit, Vall d'Hebron Research Institute, Barcelona, 2CIBERDEM, Madrid, Spain.

Background and aims: The beneficial effects of semaglutide (a long-acting GLP-1 analogue) on cardiovascular events were clearly shown in the SUSTAIN-6 clinical trial. However, a significantly increase in the rate of severe diabetic retinopathy (DR) complications was observed. Although this effect was attributed to a rapid decrease in blood glucose levels, a direct deleterious effect of semaglutide on the retina could not be ruled out. In order to address this issue we have performed an experimental study aimed at testing the direct effect of semaglutide on the early stages of DR by using eye drops. The rationale for using topical administration (eye-drops) of semaglutide is because with this route we have previously shown that GLP-1 analogues reach the retina without modifying blood glucose levels. Therefore, with this approach we can examine the direct action of semaglutide on the retina independently of glycemic values. On this basis, the aim of the present study was to evaluate the effect of semaglutide administered by eye drops on retinal neuroinflammation and early microvascular abnormalities in a db/db mouse model.

Materials and methods: Semaglutide (0.33 mg/ml; 5 μl twice/daily) (n=10) or vehicle (PBS; 5 μl twice/daily) (n=10) eye drops were administered directly onto the superior corneal surface of each eye using a micropipette in 10 week-old db/db mice. Ten non-diabetic mice (db/+) matched by age served as the control group. The treatment (semaglutide or vehicle) was administered twice daily for 15 days. Retinal analyses were performed by RT-PCR, Western blot, and immunohistochemistry. In addition, vascular leakage was examined by the Evans blue method.

Results: We found that semaglutide significantly reduced glial activation, as well as the retinal expression of NFKB, proinflammatory cytokines (IL-1β, IL-6, IL-18) and ICAM-1. In addition, semaglutide prevented the apoptosis of cells from the retinal ganglion layer and activated the AKT pathway, which is essential for the survival of retinal neurons. The effect of semaglutide in abrogating the diabetes-induced downregulation of the ratio phospho-Akt/total AKT suggests the activation of GLP-1R. Finally, semaglutide prevented the disruption of the blood-retinal barrier, thus significantly reducing vascular leakage.

Conclusion: Our results suggest that the topical administration of semaglutide is effective in preventing retinal neuroinflammation and early microvascular impairment induced by diabetes. These experimental findings point to a beneficial rather than a deleterious effect of semaglutide on the retina of subjects with diabetes.

Disclosure: R. Simó: None.

OP 14 Taking the long view of diabetes

79

Mapping polypharmacy and it's association with adverse health outcomes in the Scottish population with type 1 diabetes

A. Höhn, A. Jeyam, T. Caparrotta, S. McGurnaghan, J. O'Reilly, H. Colhoun, & on behalf of SDRN-Epi;

Institute of Genetics and Molecular Medicine, Diabetes Medical Informatics & Epidemiology Group, Edinburgh, UK.

Background and aims: The prevalence of polypharmacy has been rapidly increasing in most general populations, including the general population of Scotland. The association of polypharmacy with adverse health outcomes are well explored for general populations and particular disease groups, such type 2 diabetes (T2DM). However, little is known about the prevalence of polypharmacy and potential adverse clinical outcomes among individuals with type 1 diabetes (T1DM). We map polypharmacy over age, gender, and socioeconomic status, measured by the Scottish Index of Multiple Deprivation (SIMD), and examine the association of polypharmacy with falls, diabetic ketoacidosis (DKA), and hypoglycemia in the total Scottish population with T1DM.

Materials and methods: This study utilizes data from the Scottish Care Information-Diabetes (SCI-Diabetes) collaboration database, the comprehensive, national diabetes register for Scotland. These data were linked with information on hospital admissions provided by the Information Services Division (ISD) of the National Health Service (NHS) in Scotland. Using summary statistics, we describe the prevalence of major diabetic complications and the number of prescribed drugs by age, gender and SIMD at 2017 − 01 − 01 (baseline). To obtain the number of drugs for each individual at baseline, we counted all prescribed, different drugs according to the 5th level of the WHO ATC classification (level of chemical substances), not counting insulin and glucose. Using multivariate cox proportional hazards models, we examined how the number of drugs were associated with the first hospital admission for falls, DKA, and hypoglycemia within the subsequent 12-month period.

Results: We studied 28245 individuals alive and observable with T1DM in Scotland at baseline, of which 15731 were men and 12514 were women. The mean age of the study population was 42.31 years (sd : 18.32 years) and the mean diabetes duration was 20.64 years (sd : 13.87 years). On average, individuals were prescribed 4.07 (sd : 4.36) drugs. The proportion of individuals taking 5 or more drugs at baseline consistently increased with age, from 2.37% (95% CI: 1.91% - 2.85%) among individuals aged 0-19, to 28.88% (27.44% - 30.36%) among individuals aged 40-49, and 76.53% (68.22% - 85.09%) among individuals aged 80+. Controlling for gender, age, diabetes duration, SIMD, and health status, each additional drug was associated with a significant increase in the risk of hospitalization for falls, DKA, and hypoglycemia (Hazard Ratio (HR) for each additional drug: falls - HR: 1.056, p-value: < 0.001)/ DKA - HR: 1.049, p-value: < 0.001) / hypoglycemia - HR: 1.079, p-value: < 0.001)

Conclusion: Polypharmacy is common among the Scottish population with T1DM. The prevalence of polypharmacy rapidly increases with age as the incidence of diabetic complications and other major noncommunicable diseases increases. Evaluating potential benefits and harms of medication regimes is of substantial importance as each additional drug increases the potential risk of adverse clinical outcomes.

Supported by: Diabetes UK (17/0005627)

Disclosure: A. Höhn: None.

80

Insulin resistance at type 2 diabetes diagnosis, not impaired beta cell function, is associated with total mortality

J. Otten, B. Tavelin, S. Söderberg, O. Rolandsson;

Umeå University, Umeå, Sweden.

Background and aims: We investigated the separate effects of insulin resistance and beta cell function at the diagnosis of type 2 diabetes on the development of mortality and diabetes complications.

Materials and methods: This cohort study included 864 individuals with type 2 diabetes (median age 60 years) in whom fasting glucose and fasting C-peptide were measured at diabetes diagnosis. Insulin resistance was estimated by HOMA-%S and beta cell function by HOMA-%B. Four groups were created based on the median HOMA-%S and HOMA-%B values: group 1, high insulin resistance and preserved beta cell function; group 2, high insulin resistance and impaired beta cell function; group 3, low insulin resistance and preserved beta cell function; group 4, low insulin resistance and impaired beta cell function (reference group). Mortality and diabetes complications were registered with a follow-up of 15 years. The associations between the four groups and mortality/complications were estimated by Cox regression adjusted for gender and age at diabetes diagnosis in model 1, and also for smoking, hypertension, BMI, and total cholesterol in model 2. In the figure a Kaplan-Meier plot is displayed not including adjustments for confounding factors.

Results: Total mortality in the four groups is displayed in the figure. Both groups with high insulin resistance had higher total mortality (group 1: HR 1.58, 95% CI 1.06−2.36; group 2: HR 1.85, 95% CI 1.20-2.84) than group 4. Fasting C-peptide, as a continuous variable, was independently associated with total mortality (HR 1.29, 95% CI 1.11−1.49) and cancer mortality (HR 1.42, 95% CI 1.09−1.84).

Conclusion: Insulin resistance was an independent risk factor for total mortality. Thus, treatment of type 2 diabetes should focus not only on normalizing blood glucose levels, but also reducing insulin resistance.

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Supported by: Västerbotten County Council, Umeå University, Uman Genomics Ltd

Disclosure: J. Otten: None.

81

Mortality in community-based adults with type 1 diabetes and matched people without diabetes: the Fremantle Diabetes Study Phase I

T.M.E. Davis, W.A. Davis;

University of Western Australia, Fremantle, Australia.

Background and aims: There are few long-term comparative mortality data for community-based people with type 1 diabetes (T1D). The aim of this study was to analyse deaths and their causes in participants in the longitudinal observational Fremantle Diabetes Study Phase I (FDS1) and in matched people without known diabetes from the same urban Australian population of 120,000.

Materials and methods: Each member of the FDS1 T1D cohort was matched on age, sex and postcode with 4 randomly-selected, de-identified people without diabetes who were randomly selected from the electoral role. Participants in both groups were followed from the time of FDS1 entry or equivalent between 1993 and 1996 to the end of 2017 through the validated Western Australian Data Linkage System. All deaths were ascertained and their causes adjudicated based on UK Prospective Diabetes Study categories. Mortality rates (MRs) and MR ratios (MRRs) were calculated. Unadjusted and adjusted Cox regression models were generated to ascertain the hazard ratio (HR) for all-cause mortality by T1D status.

Results: The mean±SD age of the pooled T1D and no diabetes cohorts (n=605) at study entry was 43.1±15.3 years and 59% were male . The 121 participants with T1D had a median [IQR] diabetes duration of 12 [3-21] years and they were aged 29.5±11.6 years at diagnosis. During a total 12,541 person-years (mean 20.7±5.7 years) of follow-up, 55 (45.5%) of the T1D cohort and 88 (18.2%) of the cohort without diabetes died. The respective MRs (95% confidence interval [CI]) were 25.7 (19.4, 33.5) and 8.5 (6.8, 10.4) /1,000 person-years. The crude MRR (95% CI) was 3.04 (2.13, 4.31) (P<0.001). The 10-year age-specific MRRs are shown in the Figure. In the <35 years age-group, there were no deaths in T1D participants and 3 in the matched controls (suicide or drug misuse). The 35-44 years age group had the highest MRR (>20) with a declining trend to a non-significant two-fold increased risk for those aged 75-94 years. T1D was associated with a significant HR (95% CI) for all-cause mortality of 3.44 (2.45, 4.83) (P<0.001). With adjustment for age, sex and the Charlson Co-morbidity Index (excluding diabetes and its complications), the HR was modestly attenuated to 3.15 (2.20, 4.51) (P<0.001). The mean age (95% CI) at death was 6.6 (2.0, 11.2) years younger in those with T1D versus those without diabetes. The main cause of death was cardiovascular disease (CVD) in T1D (45.5% versus 32.7% in those without diabetes).

Conclusion: During a mean of over 2 decades of follow-up, community-based Australian adults with T1D died an average of 7 years before their matched counterparts without diabetes. Approaching half of the deaths in T1D were from CVD versus only one third in people without diabetes. These data highlight the risk of premature death and the need for optimal CVD risk reduction in people with T1D.

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Supported by: TMED

Disclosure: T.M.E. Davis: Grants; Raine Foundation/University of Western Australia.

82

Forty-year mortality among patients with first-time hospital-diagnosed overweight or obesity

S.B. Gribsholt1, D.K. Farkas2, R.W. Thomsen2, B. Richelsen1, H.T. Sørensen2;

1Aarhus University Hospital, Aarhus N, 2Aarhus University, Aarhus N, Denmark.

Background and aims: Obesity is a risk factor for chronic diseases that confer an elevated mortality risk compared to the general population. Data on the impact of hospital-diagnosed obesity on long-term mortality are limited. We investigated 40-year mortality among patients with hospital-diagnosed overweight or obesity.

Materials and methods: We used national registries covering the entire Danish population of 8,846,452 residents and all Danish general hospitals between 1979 and 2018. We identified all Danes with a first incident hospital-based overweight or obesity diagnosis (median age 45 years), and constructed an age-, gender-, and index date-matched general population comparison cohort. We computed mortality risk and mortality rate ratios (MRRs) with 95% confidence intervals (CIs), using Cox regression.

Results: We observed 68,506 deaths among 322,130 persons (2,987,320 person years) with a hospital diagnosis of overweight or obesity, and 253,897 deaths among 1,610,596 persons (16,316,670 person years) in the comparison cohort, corresponding to an all-time MRR of 2.02 (95% CI: 2.01-2.04). The mortality risk for patients with overweight or obesity was greatly increased in the first year following the first diagnosis code (2.5% versus 0.4% in comparisons, MRR=6.58 (95% CI: 6.37-6.79)) of follow up. Mortality risk then remained approximately doubled throughout follow-up (1-10y MRR=1.80 (95% CI: 1.78-1.82); 11-20y MRR=1.92 (95% CI: 1.89-1.96); 21-30y MRR=2.05 (95% CI: 2.00-2.11), and 31-40y MRR=2.12 (95% CI: 2.00-2.24)). Patients with overweight or obesity had markedly higher all-time mortality due to diabetes and other endocrine diseases (MRR=4.99 (95% CI: 4.80-5.19)) and cardiovascular diseases (MRR=2.67 (95% CI: 2.63-2.72)), but also due to respiratory diseases (MRR=2.30 (95% CI: 2.24-2.36)), infectious diseases (MRR=1.83 (95% CI: 1.79-1.88), and cancer (MRR=1.48 (95% CI: 1.44-1.53)). The 1-10-year MRR associated with overweight or obesity decreased over calendar time: from 2.02 (95% CI; 1.98-2.06) in 1977-1989 to 1.63 (95% CI; 1.59-1.68) in 2010-2018.

Conclusion: Patients with hospital-diagnosed overweight or obesity had a two-fold increased 40-year mortality, compared with the general population. We found evidence for a modestly decreasing excess mortality in patients with hospital-diagnosed overweight or obesity over the last decades.

Supported by: The Danish Council for Independent Research, The Novo Nordisk Foundation, The Central Denmark Regio

Disclosure: S.B. Gribsholt: None.

83

Time trends in deaths before 50 years of age in people with type 1 diabetes: a nationwide analysis from Scotland (2004-17)

J.E. O'Reilly, A. Jeyam, T.M. Caparrotta, S. McGurnaghan, P.M. McKeigue, H.M. Colhoun;

University of Edinburgh, Edinburgh, UK.

Background and aims: To examine whether rates of crude mortality, and mortality relative to the general population below 50 years of age have improved in recent years in those with type 1 diabetes.

Materials and methods: Individuals with type 1 diabetes and age < 50 years at any time during the period 2004-2017 in Scotland were identified using the national register (n= 27,935). Death data were obtained by linkage to General Registrar data. Indirect standardisation was used to calculate standardised mortality ratios (SMRs). Poisson regression was used to test for calendar time effects as incident rate ratios (IRR).

Results: There was a significant decline in mortality rate over time (IRR for calendar year adjusted for age, diabetes duration, level of social deprivation and sex = 0.983, 95% CI = 0.967-0.998, p=0.03) but the SMR remained approximately stable at 3.1 and 3.6 in men and 4.09 and 4.16 in women for 2004 and 2017 respectively. Diabetic ketoacidosis or coma (DKAoC) accounted for 20.8 % of deaths and the rate did not decline significantly during the study period (IRR=0.975, 95% CI 0.94-1.011, p=0.168); 79.3 % of DKAoC deaths occurred out of hospital. Circulatory diseases accounted for 27.6 % of deaths and did decline significantly (IRR=0.946, (95% CI 0.914-0.979, p=0.002).

Conclusion: Absolute mortality has fallen but the relative impact of type 1 diabetes on mortality below age 50 has not improved in fourteen years and remains high, particularly in women. Strategies to both improve the prediction and prevention of out of hospital acute diabetes deaths and to improve circulatory disease prevention are still needed.

Supported by: This study was supported by funding from the Diabetes UK (17/0005627)

Disclosure: J.E. O'Reilly: None.

84

Mortality in first- and second- generation immigrants to Sweden diagnosed with type 2 diabetes

L. Bennet1, R. Udumyan2, C. Östgren3, O. Rolandsson4, S. Jansson2, P. Wändell5;

1Lund University, Malmo, 2Örebro University, Örebro, 3Linköping University, Linköping, 4Umeå University, Umeå, 5Karolinska Institute, Stockholm, Sweden.

Background and aims: Non-western immigrants to Europe are at high risk for type 2 diabetes (T2D). In this nationwide study including incident cases of T2D, the aim was to compare mortality in first- and second generation immigrants with native Swedes.

Materials and methods: Patients living in Sweden diagnosed with a new-onset pharmacologically treated T2D between 2006 to 2012 were identified through the Swedish Prescription Drug Register. Patients were followed until December 31, 2016 for all-cause mortality (ACM) and until December 31, 2012 for cause-specific mortality (CSM). Analyses were adjusted for age at diagnosis, sex, year of diagnosis, socioeconomy, education, treatment and region. Comparisons were assessed using cox-regression analysis.

Results: In total, 169 300 individuals (129 533 (76.3%) native Swedes; 31 988 (18.9%) first-generation immigrants, and 7 799 (4.8%) second-generation immigrants with either one or both parents born outside Sweden) were diagnosed with T2D between 2006 and 2012 and fulfilled inclusion criteria. First-generation immigrants had lower ACM rate [hazard ratio (HR): 0.85, 95% CI 0.82 to 0.89] compared with native Swedes.The mortality was particularly low in persons born in the Middle East [0.45,0.40 to 0.51], Asia [0.56, 0.46 to 0.68], and Africa [0.88. 0.82 to 0.95]. Mortality rates decreased with older age at migration and shorter stay in Sweden, with the lowest rate in those originating from the Middle East living in Sweden <25 years [0.40, 0.34 to 0.46]. First-generation immigrants born in the Middle East (0.43; 0.30-0.62), and Asia (0.38; 0.19- 0.77) had lower cardiovascular disease related mortality rates compared with native Swedes. Middle Eastern immigrants further displayed lower cancer related mortality rate (0.59, 0.42 to 0.84) compared with native Swedes. Second generation immigrants displayed similar survival rates as native Swedes.

Conclusion: Our data indicate that in T2D patients, exposure to the Swedish environment seems to have a larger impact on mortality risk than region of origin. This study indicates protecting mechanisms on mortality related to the non-western environment.

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Supported by: Lund University (ALF Grants); VR (EXODIAB Linné grants); UHCR Örebro; Västerbotten County Council

Disclosure: L. Bennet: None.

OP 15 Pregnancy in diabetes prediction and outcomes

85

Risk of major congenital malformations, perinatal or neonatal death with insulin detemir vs other basal insulins in pregnant women with pre-existing diabetes: EVOLVE study

E. Mathiesen1, A.C. Alibegovic2, L. Husemoen2, P. Kelkar2, D.R. McCance3, H.W. de Valk4, P. Damm1, on behalf of the EVOLVE study group;

1University of Copenhagen, Rigshospitalet, Copenhagen, Denmark, 2Novo Nordisk A/S, Søborg, Denmark, 3Metabolic Unit, Royal Victoria Hospital, Belfast, UK, 4University Medical Centre Utrecht, Utrecht, Netherlands.

Background and aims: The EVOLVE study examined the risk of major congenital malformations and perinatal or neonatal deaths when using insulin detemir (IDet) versus other basal insulins in pregnant women with pre-existing diabetes.

Materials and methods: A prospective, non-interventional, multinational study in pregnant women with type 1 or type 2 diabetes treated with IDet or other insulin treatment. In the present analysis, 727 women using IDet during pregnancy were compared with 730 women using other basal insulin, mainly insulin glargine. The primary endpoint was the number of women completing ≥22 weeks of gestation without any of the following events: major congenital malformations, perinatal or neonatal deaths.

Results: At enrolment 86% of subjects had type 1 diabetes (mean age: 31 years; BMI: 26 kg/m2) and mean HbA1c was 7.1%. There was no difference between treatment groups in crude or adjusted risk difference for pregnancies without major congenital malformations, perinatal or neonatal deaths (Table).

Conclusion: In pregnant women with pre-existing diabetes, IDet was not associated with excess risk of major congenital malformations, perinatal or neonatal deaths vs other basal insulin.

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Clinical Trial Registration Number: NCT01892319

Supported by: Novo Nordisk

Disclosure: E. Mathiesen: Honorarium; Novo Nordisk; Lilly; Sanofi-Aventis.

86

Maternal obesity is associated with beta cell dysfunction and impaired insulin action already during early pregnancy

D. Eppel1, I. Rosicky1, J. Blätter1, G. Yerlikaya-Schatten1, C. Schatten1, P. Husslein1, W. Eppel1, A. Tura2, C.S. Göbl1;

1Department of Obstetrics and Feto-Maternal Medicine, Medical University of Vienna, Vienna, Austria, 2CNR Institute of Neuroscience, Padova, Italy.

Background and aims: Gestational diabetes mellitus (GDM) is widely accepted as a major reason for fetal overgrowth and metabolic disorders in newborns later life. However, in addition to these long-known effects of hyperglycemia, there is increasing evidence suggesting that the impact of maternal obesity on pregnancy may be of additional or even more importance. One possible explanation is that obesity can influence maternal and fetal metabolism already at the start of pregnancy even before GDM is diagnosed (i.e., between 24 and 28 weeks of gestation). There is sparse information available with the focus to elucidate the complex interaction between glucose homeostasis and the degree of maternal obesity during early stage pregnancy.

Materials and methods: In this cohort study, we prospectively included 40 pregnant women (17 normal weight, 16 overweight and 7 obese). A detailed metabolic evaluation was performed at the initial contact (between 12+0 and 15+6 gestational weeks) including a frequently sampled intravenous glucose tolerance test over 60 minutes to estimate parameters of insulin sensitivity and β-cell function such as the calculated insulin sensitivity index (CSI), acute insulin response to glucose (dAIRG) and the disposition index (DI), respectively. The amount of hepatic insulin action was additionally assessed by the quantitative insulin sensitivity check index (QUICKI) from static measurements of glucose and insulin concentrations.

Results: Maternal overweight and especially obesity were associated with significantly reduced hepatic and whole-body insulin action (median CSI: 7.5 vs. 5.19 vs. 1.33 10-4 min-1 [μU/ml]-1 for normal weight vs. overweight vs. obese mothers). As visualized in Figure 1, the acute insulin response after intravenous glucose administration was comparable between the groups (poverall = 0.825), suggesting that insulin secretion was inadequate to compensate for the higher amount of insulin resistance in obese mothers. As a consequence, the disposition index (CSI × dAIRG) was significantly lower in obese mothers, compared to normal weight mothers (p=0.001).

Conclusion: Obese mothers suffer from pronounced insulin resistance in early pregnancy. The reduced level of insulin action, however, is inadequately compensated by increased insulin release from the pancreatic β-cells, suggesting that subtle hyperglycemia is still present in high-risk patients already before GDM is diagnosed. Therefore, early alterations in glucose homeostasis could contribute to the impaired pregnancy outcome observed in obese mothers.

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Disclosure: D. Eppel: None.

87

Risk prediction of gestational diabetes by low-invasive prediction models at early pregnancy

G. Kotzaeridi1, J. Blätter1, D. Eppel1, I. Rosicky1, M. Mittlböck2, G. Yerlikaya-Schatten1, C. Schatten1, P. Husslein1, W. Eppel1, A. Tura3, C.S. Göbl1;

1Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria, 2Center of Medical Statistics, Informatics and Intelligent Systems, Section for Clinical Biometrics, Medical University of Vienna, Vienna, Austria, 3Metabolic Unit, CNR Institute of Neuroscience, Padova, Italy.

Background and aims: Sensitivity and specificity of risk factors for the prediction of gestational diabetes mellitus (GDM) can be considerably improved by use of clinical prediction models consisting of a statistical combination of several risk indicators (e.g. maternal age or history of GDM). Several prediction models for GDM are provided in the literature, however their clinical significance, as of yet, has not been thoroughly evaluated, especially with regard to application at early gestation. This study aims to assess the predictive accuracy of published low-invasive risk estimation models for the later development of GDM at early gestation.

Materials and methods: In this cohort study 1132 pregnant women (<16+0 weeks of gestation) underwent a broad risk evaluation and a routine laboratory examination at fasting condition. GDM status was assessed by use of a 75g OGTT at late second and early third trimester according to the IADPSG diagnostic criteria. Nine clinical prediction models were calculated according to the published literature.

Results: GDM was diagnosed in 239 cases i.e. 21.1% of the study participants. Although the analysed clinical prediction models were developed according to different diagnostic criteria for GDM, they showed a similar predictive accuracy with an area under the receiver operating characteristic curve (ROC-AUC) ranging between 64.5% and 72.9% (Figure 1). This corresponds to a moderate to fair predictive power, whereby most models showed a better predictive accuracy as compared to maternal age (ROC-AUC: 56.6%, 95%CI: 52.6 - 60.7) or pregestational BMI (ROC-AUC: 66.0%, 95%CI: 62.1 - 69.9). Unbiased recursive partitioning revealed that history of GDM and fasting plasma glucose had higher variable importance as compared to other variables used in the respective risk prediction models. As a result, the risk assessment tools containing those variables showed improved predictive performance. Of note, all analysed risk assessment tools could be significantly improved by including a static index quantifying the amount of insulin action, such as the homeostatic model assessment of insulin resistance (HOMA-IR) or the quantitative insulin sensitivity check index (QUICKI).

Conclusion: Established low-invasive risk assessment tools showed modest to fair accuracy to predict the later development of GDM. However, the studied prediction models showed better predictive accuracy as compared to traditional risk factors such as age and BMI. We observed the highest variable importance for fasting plasma glucose and history of GDM suggesting that these variables in addition to static measurements of insulin resistance should be included in future models.

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Disclosure: G. Kotzaeridi: None.

88

GLP-1 hypersecretion in gestational diabetes

L. Fritsche1,2, M. Heni3,2, S.S. Eckstein1,2, M.J. Winzenried3, J. Hummel1,2, A.L. Birkenfeld3,2, H. Preissl1,2, H.-U. Häring1, A. Peter1, A. Fritsche3,2, R. Wagner3,2;

1Institute for Diabetes Research and Metabolic Diseases, Helmholtz Zentrum Muenchen, Tuebingen, 2Deutsches Zentrum für Diabetesforschung (DZD), München, 3Medizinische Klinik Abt IV, Universitätsklinikum Tübingen, Tuebingen, Germany.

Background and aims: Incretins are crucial for an adequate insulin secretion in response to ingestions of nutrients. In patients with diabetes, prediabetes or a specific genetic background, this incretin effect is blunted. Data on incretin secretion and action during pregnancy are scarce. Here we investigated the incretin response during an oral glucose tolerance test (OGTT) in pregnant women with and without gestational diabetes.

Materials and methods: Pregnant women from the ongoing PREG study underwent 5-point OGTT with 75 g glucose. We measured plasma glucose, insulin and C-peptide at all time points and total GLP-1 at minute 0, 30 and 120. Indices of insulin secretion and GLP-1 increase were calculated from the difference at min 0 and 30. We used linear regression to analyze the relation of GLP-1 and glucose with insulin secretion.

Results: We examined 163 women during gestational week 26.8 (±2.0 SD), GDM was present in 30 (18.4%) women. Insulin secretion was significantly lower in women with GDM (p=0.04, adjusted for BMI, age, week of gestation, insulin sensitivity). GLP-1 levels increased after glucose intake with a peak at 30 min. GLP-1 levels at minute 30 and AUCGLP-1 was significantly higher in women with GDM (by ~20%, both p=0.03, adjusted for age, BMI and week of gestation). The GLP-1 increase was associated with insulin secretion only in GDM, but not in women with normal glucose tolerance. This association remained significant even after adjustment for increase of glucose and basal insulin levels (GDM group: p<0.001, non GDM: p=0.69).

Conclusion: Women with GDM had lower insulin secretion despite increased GLP-1 levels during OGTT. The more pronounced GLP-1 increase in women with GDM could be part of a compensatory mechanism counteracting GLP-1 resistance. In addition to incretin resistance, this phenomenon suggests a dysfunction of glucose stimulated insulin secretion.

Supported by: BMBF grant 01GI0925 to the German Center for Diabetes Research (DZD)

Disclosure: L. Fritsche: None.

89

Comparison of IADPSG with NICE criteria for diagnosis of gestational diabetes

H. Sagili, S. Todi;

Obstetrics and Gynaecology, JIPMER, Puducherry, India.

Background and aims: Different screening procedures and diagnostic criteria are being followed in the same as well as in different countries with no single standard criteria established for diagnosis of Gestational Diabetes Mellitus (GDM). So far, there are no studies in the Indian population comparing International Association of Pregnancy and Study Groups (IAPDSG) with National Institute for Health and Care Excellence (NICE) criteria. This study was carried out to compare IADPSG with NICE criteria for diagnosis of GDM and its influence on maternal and perinatal outcomes.

Materials and methods: This prospective observational study was conducted in the Department of Obstetrics and Gynaecology of a tertiary care institute in South India from March 2017 to October 2018. Six hundred eighty women with or without risk factors for GDM were recruited in the study and screened for GDM in first /second trimester. Women with preexisting diabetes mellitus or with fasting plasma glucose >126 mg/dl were excluded. All women underwent 75 gm Oral Glucose Tolerance Test and Glucose values were interpreted according to IADPSG and NICE criteria. The study subjects were then stratified into four groups namely Normal glucose tolerance, GDM by IADPSG positive NICE negative, GDM by IADPSG negative NICE positive and GDM by both IADPSG and NICE positive criteria. The participants were followed till delivery and maternal and perinatal outcomes were noted. The percentage agreement and discordance between the two criteria were calculated using kappa-statistics. The categorical data were compared using x2 or Fisher’s exact test and p value <0.05 was considered statistically significant.

Results: The overall prevalence of GDM in our study was 27.2 % by either IADPSG/ NICE criteria. 25.1 % and 11.6 % women were diagnosed as GDM using IADPSG and NICE criteria respectively. The level of agreement between the two diagnostic criteria was found to be poor in our study and was statistically significant (kappa value= 0.429; p value <0.001). Women testing IADPSG positive NICE negative had a higher risk of abortions, gestational hypertension, premature rupture of membranes, preterm delivery, caesarean section, congenital anomalies, meconium stained liquor and low Apgar score at one minute when compared to non GDM group. In addition, except for preterm delivery, women diagnosed as GDM by both IADPSG and NICE criteria had adverse outcomes such as preeclampsia, urinary tract infection and polyhydramnios. Women diagnosed as GDM by IADPSG negative NICE positive criteria had no significant adverse maternal or perinatal outcomes.

Conclusion: There was a higher pick up rate of GDM using IADPSG criteria when compared to NICE criteria. Women diagnosed as GDM with IADPSG positive NICE negative criteria had significantly higher incidence of adverse maternal and perinatal outcomes in contrast to women diagnosed as GDM by IADPSG negative NICE positive criteria. Hence, a higher fasting plasma glucose cut off value as proposed by NICE for diagnosis of GDM would lead to misidentification of some of the high risk pregnancies and thus depriving them of timely care and management. Women with substantial risk of maternal and perinatal outcomes are better identified by IADPSG criteria and would have been missed if NICE criteria had been used.Thus IADPSG criteria appears to be more robust than NICE criteria for diagnosis of GDM and its influence on adverse maternal and perinatal outcomes. Further large cohort studies with longer follow up are needed for agreement on adoption of either of the criteria.

Disclosure: H. Sagili: None.

90

Offspring of women with gestational diabetes: a 5 year follow-up

V. Bartakova1, B. Baratova1, K. Chalasova1, P. Janku2, K. Kankova1;

1Masaryk University, Brno, 2University Hospital Brno, Brno, Czech Republic.

Background and aims: Gestational diabetes mellitus (GDM) represents a risk factor for both mother and her offspring in short-term (perinatal morbidity) as well as long-term horizon (postpartum diabetes or foetal programming). A lot of studies focused at peri/postnatal complications and selected parameters of GDM mother’s offspring, however relatively few were designed as prospective. No such study focused on this topic in Czech Republic so far. The aim of our study was to ascertain possible anthropometric and developmental abnormalities and/or morbidity in offspring of GDM mothers compare to controls in a 5-year follow-up.

Materials and methods: The prospective study comprised 89 offspring-mother pairs, of those 26 with GDM and 63 controls. Following offspring parameters were evaluated: weight, length/high, blood pressure, resting heart rate, psychomotor development, morbidity, need for regular drug therapy, need for regular specialist doctor observation, status of vaccination, duration of breastfeeding. Following perinatal data were available: offspring weight (macrosomia), length of delivery, necessity of necessity of delivery induction, necessity of instrument usage, necessity of Caesarean section, Apgar score, Base excess, cord blood PH

Results: At the age of 12 and 18 months, offspring of GDM mothers had significantly worse speech abilities (didn’t say any word at 12 months of age and didn´t link words in 18 months of age, P=0.015 and P=0.009 resp., Chi-square test). Psychomotor development and school readiness test was borderline worse in GDM group at 5 years of age (P=0.048 for both, Chi-square test). Offspring of GDM mothers were more ill in their first 5 years of age and need hospitalisation (P=0.022, Chi-square test). Adverse perinatal outcomes had no significant influence on offspring psychomotor development or morbidity up to 5 years in both groups. Offspring of obese mothers had significantly worse speech abilities in 18months of age (P=0.034, chi-square test), a higher percentile weight-for-high as in 3 years (P=NS), as in 5 years (P=0.04, Mann-Whitney test).

Conclusion: This is unique prospective study focused on psychomotor development in a cohort of offspring of GDM mothers, comprised perinatal outcomes. Pilot results indicate certain differences in selected parameters in offspring of GDM mothers, especially in speech abilities and total morbidity. Moreover, were found a significant link of mother’s obesity and offspring adverse outcomes (increased adiposity and worse verbal language), however, validity is diminished by small number of obese respondents.

Supported by: Ministry of Health of the Czech Republic, grant nr. NV18-01-00046

Disclosure: V. Bartakova: None.

OP 16 Signals and networks in beta cell failure

91

Impact of hepatic or pancreatic tissue selective PCSK9-deficiency on pancreas morphology, insulin release and glucose metabolism

C. Perego1, A. Marku1, L. Da Dalt1, A. Galli1, A.L. Catapano1, D.G. Norata2,3;

1Università degli Studi di Milano, Milan, 2Dept of Pharmacological and Biomolecular Sciences (DiSFeB),Center for the Study of Atherosclerosis, Bassini Hospital, Cinisello B, Milan, 3Center for the Study of Atherosclerosis, Bassini Hospital, Cinisello B, Milan, Italy.

Background and aims: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is crucially involved in regulating plasma cholesterol levels by controlling LDL-R expression. Loss of function genetic variants are associated with lower LDL cholesterol, but higher plasma glucose levels and increased risk of T2D. Although the liver is the main contributor to circulating PCSK9, also the endocrine pancreas produces PCSK9, pointing to a possible direct role of this protein in this tissue. Pcsk9-KO mice show impaired glucose tolerance, which appears to be the consequence of decreased insulin secretion rather that insulin resistance. Aim of this work was to understand the contribution of selective liver and pancreatic PCSK9 production on beta cell physiology and glucose homeostasis.

Materials and methods: Conditioned liver (AlbCre/Pcsk9LoxP/LoxP) and endocrine pancreas (Pdx1Cre/Pcsk9fl/fl) Pcsk9-KO mice were generated and 20 weeks after, they were characterized for islet morphology, insulin release and glucose tolerance. Clonal beta cells (INS1E, βTC1 and RIN-5F) and human islets of Langerhans were used to verify the PCSK9 localization and impact on insulin content and release.

Results: Liver-specific Pcsk9-KO mice, as expected, lack detectable circulating PCSK9 protein, but express PCSK9 in the islets. They showed GTT curves and plasma glucose levels following fast and refeeding experiments similar to control littermates, paralleled by pancreatic islets comparable in size, organization and insulin content to those of littermates. Pancreas specific Pcsk9-KO mice present normal PCSK9 circulating levels but lack PCSK9 expression in pancreatic delta cells. The analysis of pancreas morphology revealed islets comparable in size to littermates but with a decreased insulin content. In line with these findings, insulin levels following fast and refeeding experiments were significantly lower than in littermates. While little or non-detectable amount of PCSK9 is found in beta cells of human isolated islets, a variable amount of PCSK9 expression is observed in rat and mouse beta cell lines. Immunostaining of human pancreas tissue sections confirmed the results and revealed a prevalent PCSK9 localization in delta cells. Studies are in progress to understand the role of pancreatic PCSK9 on insulin synthesis and secretion.

Conclusion: These data, suggest that pancreatic and not circulating (liver produced) PCSK9 plays a prevalent role in beta cell physiology. Ongoing studies are directed to shed light on the mechanisms connecting PCSK9 with beta cell dysfunction in diabetes but also to address the safety of anti-PCSK9 therapies which have been proposed to patients with severe hypercholesterolemia and/or very high cardiovascular risk.

Disclosure: C. Perego: None.

92

Multiple CRISPR/Cas9 genome editing reveals novel regulators of insulin secretion identified by single cell RNAseq

A. Lopez-Pascual1, A. Lindqvist1, J. Martínez-López2, N. Wierup1;

1Lund University Diabetes Centre, Lund University, Malmö, 2Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Perturbed islet function is a culprit in Type 2 Diabetes (T2D) and islet research is on the verge of taking a major leap forward thanks to cell type specific information on gene expression using single-cell RNA sequencing (scRNAseq). In a comprehensive analysis of which biological processes are affected in T2D, we identified T2D-affected Gene Regulatory Networks (GRNs) in beta cells. The GRNs contain node genes that we anticipate play important regulatory roles in beta cell function. Here we aimed to test the function of node genes, without a previously known function in beta cells, in a GRN of exocytosis genes.

Materials and methods: CRISPR/Cas9 gene editing was used to simultaneously mutate five node genes (Atraid, Atp6ap1, Epcam, Krtcap2and Tusc3). A plasmid carrying five guide RNAs, high fidelity spCas9 and GFP fluorescent marker, was transfected into INS-1 832/13 cells using Lipofectamine 3000. Cell were FACS-sorted five days post-transfection to obtain GFP+ cells. Single-cell colonies were plated and grown to obtain KO clonal lines. Gene expression of the five target genes was measured by qPCR as a quality control to confirm the efficiency of our protocol to generate KO genes. In parallel, GFP+ cells were plated to obtain pooled KO cell cultures. Gene expression of the five node genes, insulin (Ins1and Ins2) gene expression and glucose-stimulated insulin secretion were measured in pooled KO cells, KO clones and after siRNA-mediated silencing.

Results: GFP positive cells (5% of all viable cells) were grown for three months through clonal expansion. Of those, 30% were grown in single-cell colonies and divided to analyze gene expression of the five node genes. Gene expression analysis showed that, on average, 58% of all clone colonies were homozygous KO, 28% heterozygous KO and 14% wild-type for one of the genes. Thus, 6% of clones could have all five node genes mutated in both alleles (10 clones considering the number of colony-growing clones). After 5 months of cell culture post-transfection we obtained 15 KO clones growing at the same rate as wild-type cells. Pooled CRISPR-KO cells showed decreased expression of the five targeted genes (32-74%) and Ins1(43%), but had unaffected insulin protein content. Furthermore, pooled CRISPR-KO cells showed increased insulin secretion at basal glucose conditions (2.8 mM) with and without K+compared with wild-type cells. This effect was, however, not observed at 16.7 mM glucose. Single-gene siRNA silencing of AtraidEpcamand Krtcap2 resulted in increased insulin expression (Ins1and Ins2). Conversely, Tusc3 KD decreased Ins1gene expression. At 16.7 mM glucose Atraidand Krtcap2KD produced increased insulin secretion and at 16.7 mM glucose with IBMX (100 uM) the single-gene KD of all genes caused an increase in insulin secretion.

Conclusion: Our data suggest that Atraid, Atp6ap1, Epcam, Krtcap2and Tusc3are novel regulators of beta cell exocytosis, as well as insulin expression and secretion. Further studies are needed to elucidate the mechanistic background for these effects.

Supported by: EFSD/AstraZeneca Cellular Plasticity Programme, Novo Nordisk, SSR, DW Sverige, Fisiograf. Sällsk. Lund, Påhlsson

Disclosure: A. Lopez-Pascual: None.

93

Cask promotes the plasma membrane targeting of insulin granules via interaction with apba1, stxbp1 and npsh1

K. Zhang, Y. Wang;

Department of Endocrinology, Southeast University, Nanjing, China.

Background and aims: In our previous study, we demonstrated that knockdown of Calcium/calmodulin-dependent serine protein kinase (CASK) reduced the anchoring of insulin vesicles to cell membranes. In the current study, we re-evaluated the role of CASK and explored its interactions with other proteins involved in regulating insulin granule exocytosis.

Materials and methods: The endogenous CASK interactome in INS-1 cells during insulin secretion were measured by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS). GO analysis and Ingenuity Pathway Analysis (IPA) were carried out to explore the bioinformatic implication. CASK-specific small interfering RNA (siRNA), pEGFP-N2-Cask and pEGFP-IJ-Apba1-ΔCI plasmids were transiently transfected in INS-1 cells using the Lipofectamine 2000 reagent. Mice with conditionally CASK deleted in islets were constructed by Cre-loxp recombination system. During insulin secretion, the interaction and localization of CASK and CASK-interacting proteins in INS-1 cells or in islet of conditional CASK knockout mice were examined by Co-IP, confocal microscopy and subcellular fractionation analysis. Besides, we established lipotoxicity or glucotoxicity damage cell model and C57BL/6J diabetic mouse model to explore the correlation between diabetes and CASK as well as CASK-interacting proteins.

Results: According to the results of LC-MS/MS, 154 proteins with intensity ratio of IP/IgG>2 and/or IgG=0, IP>0 were considered as CASK-interacting proteins. We conducted bioinformatic analysis of these 154 proteins and finally focused APBA1 (Adapter protein X11 alpha), STXBP1 (Syntaxin binding protein 1) and NPHS1 (Nephrosis 1 congenital finnish type) for further analysis. The results of Co-IP confirmed that CASK, APBA1, STXBP1 could form a tripartite complex during insulin secretion. Silence or knockout of Cask decrease the interaction between APBA1 and STXBP1 and may thereby cause insulin release defects. In addition, CASK could bind to NPHS1, which is expressed on the surface of insulin vesicles. Besides, in INS-1 cells stimulated with high potassium, the fluorescence intensities of CASK, STXBP1 and NPHS1 in cell membranes were significantly enhanced. Subcellular fractionation analysis revealed that CASK and CASK-interacting proteins moved from the cytoplasm to the plasma membrane in INS1-1 cells during insulin secretion. Moreover, insulin secretion and expression of CASK and CASK-interacting proteins were significantly declined in islet isolated from high fat diet-induced diabetic mouse and in INS-1 cells treated with high glucose or palmitic acid or. Overexpression of Cask in INS-1 cells could increase the binding of CASK to APBA1, STXBP1 and NPHS1, and may thereby rescue the abnormal decrease of insulin secretion.

Conclusion: Above all, CASK promotes the plasma membrane targeting of insulin granules via interaction with APBA1, STXBP1 and NPHS1. Under pathological conditions of diabetes, the abnormal decrease of CASK and CASK-interacting proteins may result in insulin secretion defects.

Supported by: National Natural Science Foundation of China (No. 81570734 to Y.W.)

Disclosure: K. Zhang: None.

94

No evidence for intra-islet paracrine hormone actions of GIP or GLP-1 to support glucose-stimulated insulin secretion from rat islets

O. Cabrera1, J. Ficorilli1, J.L. Shaw1, F. Echeverri2, O.G. Chepurny3, C.A. Leech3, F. Schwede4, G.G. Holz3;

1Diabetes and Complications Research, Eli Lilly and Company, Indianapolis, USA, 2Biorep Technologies, Inc., Miami Lakes, USA, 3SUNY Upstate Medical University, Syracuse, USA, 4BIOLOG Life Sci. Inst., Bremen, Germany.

Background and aims: Although GIP and GLP-1 are secreted from intestinal K-cells and L-cells in response to nutrient ingestion, controversy exists concerning whether both of these incretin hormones circulate at concentrations high enough to directly stimulate pancreatic insulin secretion. It is also uncertain whether GIP and/or GLP-1 act as intra-islet paracrine hormones so that their release from islet alpha-cells leads to increased insulin secretion due to the stimulation of GIP receptors (GIPR) and GLP-1 receptors (GLP-1R) located on islet beta-cells. The aim of the present study was to investigate the relative importance of GIP and GLP-1 to these multiple processes of regulated insulin secretion.

Materials and methods: Perifusion studies of SD rat islets were performed in which glucose-stimulated insulin secretion (GSIS) was initiated and terminated by a step-wise change of the glucose concentration (G) from 2.8 to 16.7 to 2.8 mM. Perifusion assays were also performed by imposing a linear gradient of increasing concentrations of glucose (3 to 30 mM). For all such assays, test peptides in 2.8G were administered 20 min prior to and then during exposure to elevated concentrations of glucose. Insulin release was quantified by ELISA relative to islet DNA content.

Results: Rat islets perifused at 2.8G exhibited 1st and 2nd phase GSIS in response to 16.7G. Administered GIP and GLP-1 potentiated both phases of GSIS, whereas neither was effective after treatment with a cAMP antagonist (Rp-8-Br-cAMPS-pAB). Synthetic cAMP analogs that selectively activate PKA (6-Bnz-cAMP-AM) or Epac (8-pCPT-2'-O-Me-cAMP-AM) also potentiated 1st and 2nd phase GSIS. Both GIP and GLP-1 enabled cAMP-dependent insulin secretion to occur during 2nd phase GSIS, whereas 2nd phase insulin secretion stimulated by glucose alone was largely cAMP-independent. Contrary to prior reports, inhibitors of PLC, PKC, and PKD acted on their own to stimulate insulin secretion rather than inhibiting the action of GLP-1 to potentiate GSIS. In side-by-side comparisons using step-wise or gradient assays of GSIS, GIP was at least 3-fold more potent than GLP-1 to potentiate GSIS. Low concentrations of GIP (10-100 pM) potentiated GSIS, whereas no such effect of GLP-1 was detected. Insulin secretion in response to glucose alone was not reduced by the GIPR antagonist GIP(3-30) or the GLP-1R antagonist Ex(9-39). However, these antagonists blocked the actions of administered GIP and GLP-1 to potentiate GSIS.

Conclusion: GIP at concentrations approximating that found in the circulation after a meal (10-100 pM) is more effective than GLP-1 to potentiate GSIS from rat islets. Intra-islet actions of endogenous GIP or GLP-1 to support GSIS were not detectable using GIPR and GLP-1R antagonists. Thus, levels of bioactive GIP and GLP-1 within rat islets appear to be too low to allow for their paracrine hormone regulation of GSIS in vitro.

Disclosure: O. Cabrera: Other; Over Cabrera, Janice Shaw, and James Ficorilli are employees of Eli Lilly and Company.

95

The circadian clock nuclear receptor Rev-erbα is implicated in autophagy alteration and beta cell deficit under diabetogenic conditions

S. Costes1, D. Laouteouet1, M. Ravier1, M. Delobel1, G. Bertrand1, O. Villard2, C. Broca2, J. Mathieu2, A. Wojtusciszyn2, S. Dalle1, A. Matveyenko3;

1Institute for Functional Genomics, Montpellier, France, 2Institute for Regenerative Medicine and Biotherapy, Montpellier, France, 3Mayo Clinic, Rochester, USA.

Background and aims: Type 2 diabetes (T2D) is characterized by hyperglycemia secondary to pancreatic beta-cell deficit. Circadian disruption is considered as a risk factor for T2D. At the molecular level, circadian rhythms are controlled by Clock-Bmal1 with nuclear receptor Rev-erbα as a repressor. In contrast to Clock and Bmal1, Rev-erbα has received little attention in beta-cells. Importantly, in addition to its circadian function, Rev-erbα is a repressor of the autophagy degradation pathway, the latter being crucial for beta-cell health. Nevertheless, little is known about the clock genes/autophagy interplay that may contribute to beta-cell failure in T2D. Therefore, in the present study, we set out to address whether Rev-erbα-mediated inhibition of autophagy caused by diabetogenic stress is involved in beta-cell deficit. The objectives are: 1) To evaluate the impact of Rev-erbα overexpression on beta-cell integrity and autophagy. 2) To investigate whether the negative modulation of Rev-erbα could protect beta-cells from diabetogenic stressors.

Materials and methods: Experiments were performed with pancreatic beta-cell lines (rat INS-1E, human EndoC-βH1) and human islets. Rev-erbα protein levels were evaluated by western blot analysis. Levels of LC3-II (marker of autophagosome number) and p62 (also known as sequestosome-1) were used to monitor autophagic degradation and evaluated by western blot. Since p62-positive inclusions are an additional marker for defective autophagy, p62 was also detected by immunofluorescence. Apoptosis was evidenced by cleaved caspase-3 emergence. Glucose-induced insulin secretion was assessed by Homogeneous Time Resolved Fluorescence (HTRF) technology.

Results: Exposure of INS-1E cells to either glucotoxicity (30 mM glucose for 48h) or cytokines (mix of IL-1β, TNFα and IFNγ for 24h) resulted in robust induction of Rev-erbα expression (1.5-2 fold, p<0.05) and corresponded with impaired autophagy flux characterized by increased protein levels of p62 (1.5-2 fold, p<0.05). Consistent with these data, exposure of beta-cells and human islets to a Rev-erbα agonist (SR9009) was characterized by impaired autophagy/lysosomal degradation as shown by increased LC3-II and p62 levels (p<0.05). Importantly, p62-positive inclusions were almost exclusively detected in SR9009-treated dispersed human beta-cells. As a consequence, defective glucose-stimulated insulin secretion (70 % decrease, p<0.05) and increased beta-cell apoptosis (increased cleaved caspase-3, p<0.01 vs vehicle) were detected in SR9009-treated INS-1E cells and human islets. In contrast, pharmacological inhibition of Rev-erbα (antagonist SR8278) or its knock-down by siRNA protected beta-cells from deleterious effects of glucotoxicity (INS-1E and EndoC-βH1) or cytokines-induced inflammation (INS-1E and human islets) by attenuating beta-cell apoptosis (~30%, p<0.05).

Conclusion: Taken together, these data reveal for the first time an underexplored link between the core circadian clock nuclear receptor Rev-erbα, autophagy and beta-cell failure under diabetogenic conditions. These data also suggest a therapeutic potential of elaborating new Rev-erbα-based strategies to preserve a functional beta-cell mass in T2D.

Disclosure: S. Costes: None.

96

The bidirectional regulation of the Hippo pathway and autophagy in pancreatic beta cells

K. Annamalai1, S. Naik1, T. Yuan1, B. Lupse1, D.-S. Lim2, K. Maedler1, A. Ardestani1;

1University of Bremen, Bremen, Germany, 2Department of Biological Sciences, KAIST, Daejeon, Republic of Korea.

Background and aims: A hallmark of beta-cell failure is the impairment in autophagy, an intracellular self-degradative catabolic process, which plays an important role in the recycling of cellular components, cell viability, stress response, and homeostasis. Understanding the molecular mechanisms, which lead to defective autophagy and beta-cell failure is urgently needed for beta cell protection and repair. LATS2, the central kinase of the Hippo pathway, is hyper-activated in beta cells under diabetic conditions and its overexpression induced beta-cell death and dysfunction. Conversely, LATS2 deficiency in beta cells and primary isolated human islets as well as beta-cell specific LATS2 ablation in mice improves beta-cell viability, insulin secretion, and beta-cell mass and ameliorated diabetes development. Mechanisms of LATS induced beta cell destruction are rarely understood; here, we unravel the mutual regulation of LATS2 and autophagy in beta cells.

Materials and methods: Firstly, the effect of LATS2 in modulating autophagic flux was analyzed by overexpression and genetic inhibition of LATS2 in the presence or absence of late-stage chemical inhibitors of autophagy in both INS-1E cells and isolated human islets. Secondly, to investigate whether autophagy reciprocally regulates the endogenous protein level of LATS2, we used three different autophagy inhibitors, namely, the combination of leupeptin/NH4Cl, Chloroquine, and BafilomycinA1. To provide direct evidence for the autophagic degradation of LATS2, intact lysosomes were isolated from LATS2 overexpressing beta cells, and subsequent LATS2 localization was analyzed. Thirdly, in order to identify the specific type of autophagy pathway that may be involved in the degradation of LATS2, we selectively targeted ATG7 and LAMP2A proteins, major essential components of canonical macroautophagy and CMA, respectively. Lastly, autophagosomes were isolated from LATS2-overexpressing GFP-LC3 expressing beta cells to detect LATS2 localization in autophagosomes; furthermore, we investigated the direct interaction of LATS2 and LC3, a key component of the autophagosome membrane.

Results: LATS2 overexpression exacerbated beta-cell apoptosis and further impaired autophagic flux, as represented by the strong accumulation of autophagy markers LC3-II and P62 in both INS-1E cells and human islets, treated with autophagy inhibitors. Conversely, LATS2 silencing reduced beta-cell apoptosis and restored the defective autophagic flux. This indicates that LATS2 regulates defective autophagy induced apoptosis and autophagic flux. Reciprocally, autophagy inhibition robustly upregulated endogenous LATS2 protein levels demonstrating that LATS2 acts as a potential substrate for autophagy-mediated protein degradation. In support of this, LATS2 protein levels were highly enriched in isolated intact lysosomes. Moreover, we observed that selective inhibition of macroautophagy but not CMA induced LATS2 upregulation, that isolated autophagosomes showed strong accumulation of LATS2 protein levels and that LATS2 and LC3 directly interact.

Conclusion: This unravels the novel bidirectional role of the Hippo kinase LATS2 and autophagy in the regulation of beta-cell survival.

Supported by: JDRF

Disclosure: K. Annamalai: None.

OP 17 Broken heart in diabetes

97

Clustering of patients with type 2 diabetes and established CV disease for prediction of disease progression and MACE (SAVOR-TIMI 53 trial)

Y. Aoki1, B. Hamrén1, L.E. Clegg2, C. Stahre3, D.L. Bhatt4,5, I. Raz6, B.M. Scirica4,5, J. Oscarsson3, B. Carlsson7;

1Clinical & Quantitative Pharmacology, AstraZeneca, Gothenburg, Sweden, 2Clinical & Quantitative Pharmacology, AstraZeneca, Gaithersburg, USA, 3Late CVRM, BioParmaceutical R&D, AstraZeneca, Gothenburg, Sweden, 4Brigham and Women’s Hospital Heart & Vascular Center, Boston, USA, 5Harvard Medical School, Boston, USA, 6Hadassah University Hospital, Jerusalem, Israel, 7Research and Early Development, CVRM, BioParmaceutical R&D, AstraZeneca, Gothenburg, Sweden.

Background and aims: Ahlqvist et al. proposed to apply k-means clustering of five essential glycemic control-related variables to subgroup patients with adult onset diabetes. Others have reported similar results indicating that this methodology is robust in terms of identifying diabetes subgroups. We aimed to investigate if clustering of diabetes essentially according to the method Ahlqvist et al. can be applied to a patient cohort with type 2 diabetes and established CV disease to investigate the clinical utility of the clustering to predict risk for disease progression and MACE.

Materials and methods: We have used a subset of SAVOR (Saxagliptin cardiovascular safety Phase-IV clinical trial) dataset that was approved for the secondary use of data, including both active and placebo arms. SAVOR included patients with type 2 diabetes. Type 1 diabetes was an exclusion criterion and patients on insulin therapy were excluded to be able to calculate HOMA2. We focused on the patient subpopulation who have established CV disease at baseline. As a result, 4644 patients with a mean follow up of 2.1 years and mean diabetes duration of 8.69 years were included. We clustered the cohort essentially according to Ahlqvist et al. into four subgroups using k-means clustering with the following five covariates: HOMA2-IR, HOMA2-B, HbA1c, age at diagnosis, and BMI. Disease progression was determined as addition of new diabetic medications and cardiovascular event as the composite six-point MACE (cardiovascular death, myocardial infarction, Stroke, Hospitalization for heart failure, Hospitalization for unstable angina, coronary revascularization). The multivariate Cox proportional hazard model was used to quantify the relative risk of each endpoint for the clusters.

Results: Patients from the SAVOR trial were successfully clustered into four groups: severe insulin-deficient diabetes (SIDD) 17%, severe insulin-resistant diabetes (SIRD) 17%, mild obesity-related diabetes (MOD) 27%, mild age-related diabetes (MARD) 38%, replicating the overall patterns of the baseline covariate values as presented by Ahlqvist et al. The age and sex adjusted hazard ratios for the diabetes progression for SIDD, SIRD, MOD as compared to MARD were 2.68 (95% confidence interval, 2.29-3.13), 1.45 (1.23-1.70), 1.41 (1.20-1.65), respectively. Similarly, the age and sex adjusted hazard ratios for the six-point MACE were 1.34 (1.04-1.74), 1.33 (1.05-1.69), and 1.13 (0.89-1.43), respectively.

Conclusion: We show that diabetes subgroups defined by Ahlqvist et al. can be reproduced in a randomized controlled trial dataset of patients with type 2 diabetes with established CV disease. Diabetes progression was predicted by the clustering but there were no major differences with respect to MACE. Thus, we believe a more efficient algorithm to segment diabetes population with established CV disease is desirable in order to optimize the clinical care for the secondary prevention of MACE in patients with diabetes.

Disclosure: Y. Aoki: Employment/Consultancy; AstraZeneca. Stock/Shareholding; AstraZeneca.

98

Association of incident myocardial infarction with insulin resistance and liver fibrosis

C. Möser1,2, O.P. Zaharia1,2, M. Rothe1,2, J.-H. Hwang1,2, P. Bobrov3,2, V. Burkart1,2, F. Bönner4, C. Jung4, M. Kelm4, M. Roden1,5, J. Szendrödi1,5;

1Institute for Clinical Diabetology, German Diabetes Center, Düsseldorf, 2German Center for Diabetes Research (DZD), München-Neuherberg, 3Institute for Biometrics and Epidemiology, German Diabetes Center, Düsseldorf, 4Division of Cardiology, Pulmonary Disease and Vascular Medicine, Medical Faculty, Heinrich-Heine-University, Düsseldorf, 5Division of Endocrinology and Diabetology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.

Background and aims: Insulin resistance and non-alcoholic fatty liver disease associate with increased risk of cardiovascular disease and predict a worse outcome. The mechanisms underlying the link between the two disease disorders are yet unclear. We hypothesized that individuals suffering from their first acute myocardial infarction (MI) already feature not only lower whole-body insulin sensitivity (M-value) and myocardial ejection fraction (EF), but also higher hepatocellular lipids (HCL) and liver fibrosis estimates (MRE) than matched individuals without MI.

Materials and methods: We compared participants of the DISTEMI (DIabetes and ST-Elevation MI) Study at 6-12 weeks after MI (MI+; n=21, 81% male, 29% type 2 diabetes, age 61±8 years, BMI 26.8±2.8 kg/m2, HbA1c 5.8±1.0 %) with humans without MI (MI-; n=34, 71% male, 32% type 2 diabetes, age 57±8 years, BMI 27.8±3.1 kg/m2, HbA1c 5.5±0.6 %). Participants were matched for sex, age, HbA1c and BMI. Participants underwent a 75-g OGTT and a hyperinsulinemic-euglycemic clamp test to measure insulin sensitivity. The respiratory quotient (RQ) was measured by indirect calorimetry. HCL, MRE, EF, infarct size and microvascular obstruction (MVO) were measured with magnetic resonance spectroscopy (1H), elastography and imaging, respectively.

Results: MI+ had a lower whole-body insulin sensitivity compared to MI- (M-value: 7.3±2.0 vs. 9.0±3.1 mg*kg-1*min-1, p<0.05). HCL was comparable (2.9±2.8% vs. 5.7±6.7%) between groups, while MRE was higher in MI+ compared to MI- (2.5±0.4 kPa vs. 2.1±0.4 kPa, p<0.05). EF was lower in MI+ than in MI- (53±16% vs. 65±7%, p<0.05). In MI+, EF correlated negatively with infarct size (r=-0.91) and MVO (r=-0.82) as well as with HbA1c (r=-0.61), fasting blood glucose (r=-0.61) and urinary albumin (r=-0.67, all p<0.05). Furthermore, MI+ featured a positive correlation of EF with RQ under fasted conditions (r=0.73) and HDL cholesterol (r=0.68, both p<0.05).

Conclusion: Individuals with incident MI have lower whole-body insulin sensitivity, reduced ejection fraction and higher liver fibrosis estimates than humans without MI, which relate to worse glucose metabolic control and may contribute to increased risk of heart failure and re-infarction after the first MI.

Supported by: SFB 1116

Disclosure: C. Möser: None.

99

Ketone bodies acutely affect cardiac autonomic function in patients with type 2 diabetes

N.J. Jensen1, M. Nilsson1, N. Møller2, A. Sajadieh3, P. Kumarathurai3, J. Rungby1,4;

1Department of Endocrinology, Bispebjerg University Hospital, Copenhagen, 2Department of Endocrinology, Aarhus University Hospital, Aarhus, 3Department of Cardiology, Bispebjerg University Hospital, Copenhagen, 4Copenhagen Center for Translational Research, Copenhagen University Hospital, Bispebjerg and Frederiksberg, Copenhagen, Denmark.

Background and aims: Ketone bodies are important alternative fuels for the diabetic heart and improve left ventricular function in heart failure by increasing both stroke volume and pulse rate with little or no change in blood pressure. Further, ketone bodies may have beneficial effects in other organ systems in persons with diabetes. Increased cardiovascular mortality, often related to diabetes, is associated with cardiac autonomic dysfunction. Since ketone bodies may be part of our future therapeutic armamentarium, we aimed to investigate if a ketone body infusion acutely affects cardiac autonomic function, in particular heart rate variability (HRV), in patients with type 2 diabetes.

Materials and methods: In this randomised cross-over study, 17 patients with type 2 diabetes received i.v. ketone body (beta-hydroxybutyrate) and placebo (saline) infusion in a randomised order on two separate occasions. On both visit days blood glucose was clamped at 7.5 mmol/L and short term HRV (measuring sympatho-vagal balance) was assessed before treatment (Pre treat), during rest (REST), and during mental assignments (STRESS). Measures of HRV were SD of beat-to-beat (NN) intervals (SDNN), root mean square of successive differences in NN intervals (RMSSD), and power in high-frequency (HF) and low-frequency (LF) range.

Results: Beta-hydroxybutyrate levels increased during the ketone infusion (2.4 ± 0.6 mM vs. 0.1 ± 0.6 mM during placebo). Compared to placebo, ketone infusion increased heart rate by 13.7 ± 1.2 bpm (p <0.001) and decreased both vagally derived HRV (RMSSD and HF), and mixed sympathetic-parasympathetic derived HRV (SDNN and LF), p<0.001 for all. However, LF/HF ratio did not change significantly. The changes in HRV remained significant after adjusting for heart rate (p<0.05). Blood pressure did not differ between the two treatments.

Conclusion: Increasing beta-hydroxybutyrate levels by infusion during rest and stress from mental assignments, significantly increases heart rate and decreases all measures of HRV except LF/HF ratio indicating an acutely reduced vagal activity and likely also an increased sympathetic activity.

figureae

Clinical Trial Registration Number: NCT03657537

Disclosure: N.J. Jensen: None.

100

High urinary dimethylamine and low urinary citrate are associated with coronary artery disease in individuals with type 1 diabetes

A. Antikainen1,2, S. Mutter1,2, N. Sandholm1,2, C. Forsblom1,3, P. Würtz4, V. Harjutsalo1,5, P.-H. Groop2,3;

1Folkhälsan Institute of Genetics, Folkhälsan Research Center, Helsinki, 2Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, 3Abdominal Center, Nephrology University of Helsinki and Helsinki University Hospital, Helsinki, 4Nightingale Health Ltd, Helsinki, 5National Institute for Health and Welfare, Chronic Disease Prevention Unit, Helsinki, Finland.

Background and aims: Individuals with type 1 diabetes (T1D) have an increased risk of coronary artery disease (CAD) compared to the general population. Metabolomics of urine and blood may provide new insights into the disease mechanisms and tools for early identification of high-risk individuals. We therefore investigated whether urinary metabolites are associated with CAD in individuals with T1D.

Materials and methods: The study included 3,387 individuals with T1D (385 CAD cases) from the Finnish Diabetic Nephropathy Study. Individuals with end-stage renal disease or a CAD event prior to the baseline visit were excluded. Baseline urine samples were analyzed with nuclear magnetic resonance (1H NMR) and the measured metabolite concentrations were normalized by urine creatinine, log-transformed and standardized. All individuals were followed up until the first CAD event or the end of 2017 (mean of 14.1±5.0 years). Associations of 31 metabolites with CAD were evaluated with Cox proportional hazard models adjusted for sex, diabetes onset calendar year and presence of diabetic nephropathy. A Bonferroni corrected p-value of 0.0016 was considered significant. We also analyzed metabolite interaction differences between the cases and controls with correlation-based networks and assessed the statistical significances with network permutations.

Results: Dimethylamine (HR per SD of 1.19 [1.08-1.31], p=0.00039) and citrate (0.88 [0.81-0.95], p=0.00058) were significantly associated with CAD. In addition, we observed nominal associations for 3-hydroxyisobutyrate (0.84 [0.76-0.94], p=0.0029), pseudouridine (1.16 [1.04-1.29], p=0.0068), 3-hydroxyisovalerate (0.86 [0.78-0.96], p=0.0079) and xanthosine (1.13 [1.02-1.26], p=0.026). Correlation network analysis suggested rather similar between-metabolite correlation structures for those with and without CAD. However, dimethylamine and xanthosine were stronger correlated with each other in cases than in controls (rcases/controls: 0.40/0.13, p=0.0039), while the negative correlation between dimethylamine and creatinine was stronger for cases (rcases/controls: -0.40/-0.17, p=0.0065). Citrate was more strongly correlated with 1-methylnicotinamide among individuals with CAD (rcases/controls: 0.22/0.037, p=0.0051).

Conclusion: Urinary dimethylamine and citrate are associated with CAD in individuals with T1D. Urinary dimethylamine is a degradation product of asymmetric dimethylarginine, and thus linked to the nitric oxide pathway. Urinary citrate has been associated with calcium reabsorption from the proximal tubule and with arterial calcification. Finally, the correlation-network analysis provides links between CAD and kidney disease as metabolites in the dimethylamine sub-network (xanthosine, creatine, urea and 4-hydroxyhippurate) are related to kidney disease and uremia.

Supported by: Folkhälsan Research-, Wilhelm and Else Stockmann- and Liv och Hälsa Foundations, HUS Research Funds

Disclosure: A. Antikainen: None.

101

Liraglutide and vascular inflammation in type 2 diabetes as assessed by FDG-PET/CT: the LiraFlame study

R. Ripa1, E.H. Zobel2, B.J. von Scholten2, L.J. Diaz2, J.K. Jensen1, V.R. Curovic2, T.W. Hansen2, P. Rossing2,3, A. Kjaer1;

1Dept of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, 2Steno Diabetes Center Copenhagen, Copenhagen, 3University of Copenhagen, Copenhagen, Denmark.

Background and aims: The mechanism behind the cardiovascular protection in type 2 diabetes (T2D) observed with human glucagon-like peptide-1 receptor agonists (GLP-1 RA) is unknown. We hypothesized that treatment with the GLP-1 RA liraglutide, had a positive effect on vascular inflammation.

Materials and methods: In a double-blind trial, we randomly assigned 102 persons with T2D to liraglutide up to 1.8 mg or placebo once daily for 26 weeks. The primary outcome was 18F-fluorodeoxyglucose (18F-FDG) PET/CT assessment of change in vascular inflammation. Carotid and aortic FDG uptake was quantified as target to background ratio (TBR) using venous blood uptake as background. Active segment analysis including only vascular segments with TBR>1.6 was the prespecified primary endpoint and most diseased segment analysis was a secondary endpoint.

Results: Mean age was 66.4 (SD 8.2) years and 16% were women; median [IQR] diabetes duration was 10.9 [5.7; 18.2] years and mean HbA1c was 58.4 (10.1) mmol/mol; 17% reported a history of cardiovascular disease (CVD). Ninety-eight participants (96%) underwent PET/CT at both baseline and week 26. Baseline characteristics were balanced between the two groups. Liraglutide significantly reduced HbA1c [mean change liraglutide vs. placebo (95% CI): -5.1 (-8.0; -2.0) vs. -0.1 (-1.9; 1.7) mmol/mol, (p=0.006)] and weight [-3.7 (-4.8;-2.6) vs -0.2 (-0.8; 0.4) kg, (p<0.001)]. LDL-cholesterol and systolic blood pressure were unchanged (p≥0.40). Vascular inflammation was unchanged in the carotid arteries and aorta combined (Figure) [mean TBR change liraglutide vs. placebo (95% CI): -0.04 (-0.17; 0.08) vs -0.09 (-0.19; 0.01) in active segment (p=0.53) and -0.22 (-0.40;0.03) vs -0.24 (-0.44;0.04) in most diseased segment (p=0.87) analysis; analyses restricted to the carotid arteries showed similar results in active segment (p=0.96) and in most diseased segment (p=0.62). The exploratory analysis compared change in carotid inflammation in participants with (n=17) and without (n=81) a history of CVD (Figure). In the liraglutide group participants with CVD had a larger decrease in inflammation compared to participants without [-0.59 (-1.23; 0.05) vs -0.13 (-0.30; 0.04), p=0.04] in most diseased segment analysis. A similar difference was not seen in the placebo group [-0.17 (-1.17; 0.84) vs -0.30 (-0.49; -0.12), p=0.62]. Moreover, a borderline significant interaction (p=0.06) between treatment group and history of CVD was demonstrated for predicting change in carotid inflammation.

Conclusion: In this low to moderate risk population with T2D, liraglutide did not change vascular inflammation compared to placebo, however, data indicated a decrease in vascular inflammation in the carotid arteries in persons with CVD.

figureaf

Clinical Trial Registration Number: NCT03449654

Disclosure: R. Ripa: Employment/Consultancy; Novo Nordisk. Grants; Novo Nordisk.

102

Estimating CVD-free life-years with the addition of semaglutide in people with type 2 diabetes using pooled data from SUSTAIN 6 and PIONEER 6

J. Westerink1, K. Sommer Matthiessen2, S. Nuhoho2, U. Fainberg2, M. Lyng Wolden2, F. Visseren1, N. Sattar3;

1University Medical Center, Utrecht, Netherlands, 2Novo Nordisk A/S, Søborg, Denmark, 3Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK.

Background and aims: CVD is the leading cause of disability and death in people with type 2 diabetes (T2D). In a post hoc analysis of pooled data (POOLED cohort) from two phase 3, randomized cardiovascular outcomes trials, SUSTAIN 6 and PIONEER 6, the addition of the glucagon-like peptide-1 analogue semaglutide to standard of care (SoC) in people with T2D at high risk of CVD significantly reduced the risk of major adverse CVD events (3-point MACE: CV death, non-fatal stroke, non-fatal myocardial infarction). The purpose of this study was to estimate the effect of adding semaglutide to SoC on CVD-free life-years and 10-year CVD risk in patients with T2D by predicting individual patient-level risk of CVD events in the POOLED cohort using the Diabetes Lifetime-perspective prediction (DIAL) CVD risk model.

Materials and methods: The 3-point MACE hazard ratio from the POOLED cohort (N = 6480; HR = 0.76 [95% CI: 0.62-0.92]) was applied to the patient-level lifetime risk of CVD events derived from the DIAL model. CVD-free life-years and 10-year CVD risk were then calculated based on the age-specific risks of CVD events and non-vascular mortality, using standard actuarial methods. Both new and recurrent CVD events were considered. The DIAL model was validated by comparing the predicted and observed number of CVD events after 1 year. The DIAL model was previously developed using data from people with T2D in the Swedish National Diabetes Registry and validated across geographical regions.

Results: The DIAL model was considered valid for use in the POOLED cohort because the predicted number of CVD events at 1 year was within 5% of the number observed. Adding semaglutide to SoC was associated with a mean reduction in 10-year CVD risk of 20.0% (95% CI: 6.4-32.6%) and a mean increase of 1.72 (95% CI: 0.52-2.96) CVD-free life-years. The number of mean CVD-free life-years gained ranged from 0.62 to 2.91 years between age groups (Table). For a 60-year-old male with baseline characteristics matched to the average male from the POOLED cohort, adding semaglutide to SoC reduced 10-year CVD risk by 20.8% and provided 2.53 additional CVD-free life-years. The number of CVD-free life-years decreased when baseline age was increased.

Conclusion: The addition of semaglutide to SoC was associated with a gain in CVD-free life-years. This analysis helps to contextualize the results of cardiovascular (CV) outcomes trials and may help to inform clinical decision-making. Note: Data submitted for presentation at the European Society of Cardiology Congress, 29 August-2 September 2020, Amsterdam, Netherlands.

figureag

Clinical Trial Registration Number: SUSTAIN 6 (NCT01720446) and PIONEER 6 (NCT02692716)

Supported by: Funded by Novo Nordisk A/S

Disclosure: J. Westerink: None.

OP 18 Unlocking the potential of digital health

103

Mobile health application usage shows long-term improvement on blood glucose control

V. Eichinger, J. Kober, R. Biven, L. Schuster, J. Wrede;

Medical & Research, mySugr GmbH, Vienna, Austria.

Background and aims: Self-managing a chronic illness can be challenging for patients. On average, people with diabetes make up to 180 extra decisions on diabetes management per day. Therefore, the continuous usage of mobile health (mHealth) applications might help users control their blood glucose (BG) more efficiently. Previous real world data (RWD) analysis showed significant improvement in blood glucose control with the use of an mHealth application for people with type 1 diabetes (T1D) after six months. New RWD analyses were conducted to look at a potential sustainable, clinically relevant effect on diabetes self-management in engaged users after six and twelve months. Subpopulation analyses were conducted to compare impact on users with T1D and T2D, as well as different therapy types.

Materials and methods: This retrospective study applied the following inclusion criteria to users of an mHealth app: mean blood glucose (mean BG) ≥183mg/dl, representing an estimated HbA1c (eHbA1c) ≥8% at baseline, engaged logging behaviour ( ≥2 logs/day on ≥14 days per 30 days) and recurring app usage of at least 12 months. Changes in BG control (mean, standard deviation (SD) and eHbA1c) were analysed in the selected user group as well as in its subpopulations, focusing on diabetes and therapy types. Monthly data from the first log (t0) up to 6 months (t1) and 12 months (t2) was analysed.

Results: 5,751 users met the inclusion criteria; 50.11% with T1D, 45.30% with T2D and 4.59% with other or unknown diabetes types. Baseline mean BG was 218.56 ± 74.37mg/dl (eHbA1c 9.24%) at t0, dropping to 190.20 ± 63.66 mg/dl (eHbA1c 8.25%) at t1. At t2 a sustained effect with a mean BG of 189.64 ± 63.69mg/dl (eHbA1c 8.23%) could be shown. Data analysis showed clinically relevant decreases of mean BG / eHbA1c in all distinct subgroups, regardless of diabetes or therapy type, respectively. At t2 the mean BG decreased from 216.85 ± 88.09mg/dl (eHbA1c 9.18%) to 200.60 ± 81.87mg/dl (eHbA1c 8.62%) for people with T1D. For people with T2D the mean BG decreased from 219.99 ± 59.88mg/dl (eHbA1c 9.29%) at t0 to 180.24 ± 45.91 mg/dl (eHbA1c 7.91%) at t2. Further analysis of the T2D user group showed a decrease in mean BG from 220.20 ± 62.25mg/dl (eHbA1c 9.30%) at t0 to 186.81 ± 50.33mg/dl (eHbA1c 8.14%) at t2 for insulin-dependent pen-users. In comparison, the 643 users that belong to the subgroup of insulin-independent users, had the largest decrease in mean BG, from 219.55 ± 52.72mg/dl (eHbA1c 9.28%) at t0 to 157.46 ± 29.53mg/dl (eHbA1c 7.11%) at t2.

Conclusion: Our RWD shows sustainable improvement in the quality of blood glucose control of high risk populations (eHbA1c ≥8% at baseline) with T1D as well as T2D over twelve months. A clinically relevant decrease in eHbA1c (≥ 0.3% according to EMA guidelines) by 1.01% at twelve months of mHealth application usage is shown. In comparison, the improvement of eHbA1c (1.38%) in T2D is more than twice as strong as in T1D (0.57%). Moreover, we see different positive effects in the distinct subgroups of diabetes as well as therapy types. The analysed data indicate that insulin-independent people with T2D might benefit the most from using the self-management diabetes app. Their mean BG was shown to decrease by 28.28% and SD by 44.00%. Our calculation shows an average reduction in eHbA1c of 2.16%. Although this work indicates a strong positive impact of the usage of mHealth applications in diabetes therapy, further prospective studies are necessary to verify our findings.

Disclosure: V. Eichinger: None.

104

Glycaemic control among people with type 1 diabetes during lockdown against the SARS-CoV-2 outbreak in Italy

F. Boscari, B.M. Bonora, A. Avogaro, D. Bruttomesso, G.P. Fadini;

Department of Medicine, Unit of Metabolic Disease, University of Padova, Padova, Italy.

Background and aims: In late February 2020, due to the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the Italian Government closed educational and sport activities. In March, several areas underwent an almost complete lockdown. We report the impact of these restrictions on glucose control among people with type 1 diabetes (T1D).

Materials and methods: We included data on 33 individuals with T1D who were using the flash glucose monitoring (FGM) system and were remotely connected to the diabetes clinic. We retrieved information on average glucose, standard deviation, and percent time in hypoglycemia (<70 mg/dl), glucose range (70-180 mg/dl) and hyperglycemia (>180 mg/dl). We compared glycemic measures collected during lockdown to those collected before SARS-CoV-2 epidemic, and the periods immediately before lockdown.

Results: In 20 patients who stopped working, overall glycemic control improved during the first 7 days of lockdown as compared to the weeks before SARS-CoV-2 spread. Average glucose declined from 177±45 mg/dl (week before) to 160±40 mg/dl (lockdown; p=0.005) and standard deviation improved significantly. Time in range increased from 54.4% to 65.2% (p=0.010) and time in hyperglycemia decreased from 42.3% to 31.6% (p=0.016). The number of scans per day remained unchanged. In 13 patients who continued working, none of the measures of glycemic control improved during lockdown.

Conclusion: Despite limited possibility to exercise and incumbent psychologic stress, glycemic control improved during lockdown in patients with T1D who stopped working. Thus, slowing routine daily activities can have beneficial effects on T1D management, at least in the short term.

Disclosure: F. Boscari: None.

105

Real-time CGM usage and estimates of glycaemic control among individuals with type 1 or type 2 diabetes

R. Dowd, G. Norman, J.B. Welsh, T. Walker, A. Parker;

Dexcom, Inc., San Diego, USA.

Background and aims: Demographics and comorbidities are substantially different for individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D), as is the prevalence of continuous glucose monitoring (CGM) utilization. Because CGM lowers the risk of iatrogenic hypoglycemia, it is often reserved for people using antihyperglycemic drugs such as insulin and sulfonamides. CGM-related behaviors and glycemic parameters of people with T2D who use CGM are therefore of interest. We aimed to quantify and compare individuals with T1D and T2D with respect to their usage of CGM features and glycemic control.

Materials and methods: Data were from anonymized, US-based users of the Dexcom G6 CGM System (Dexcom, Inc., San Diego, CA) who had uploaded in the 2019 calendar year and were associated with a valid ICD-10 code indicating a diagnosis as either T1D (E10.X) or T2D (E11.X). Time in range (TIR) was defined as the percentage of sensor glucose values (SGVs) 70-180 mg/dL (3.9-10 mmol/L). Usage of CLARITY, a system for retrospective review of CGM data, was defined as logging in at least once in 2019. The G6 System includes a discretionary "Urgent Low Soon" (ULS) predictive alert that is enabled by default. Persistence was defined as the percentage of observed days in which ≥ 1 SGV was uploaded. "Followers" were individuals with real-time remote access to the CGM users' data. Rebound hyperglycemia was defined as any series of SGVs >180 mg/dL starting within 2 hours of an SGV <70 mg/dL; iatrogenic hypoglycemia was defined as any series of SGVs <70 mg/dL starting within 2 hours of an SGV >180 mg/dL.

Results: Data from 3,790 individuals with T2D and 5,426 individuals with T1D were available for analysis. Compared to individuals with T1D, individuals with T2D were older (51.6 vs. 33.2 years), had generally better control as gauged by TIR (62.0% vs. 57.4%) and the percentage of SGVs <70 mg/dL (1.1% vs. 2.5%), and less glycemic variability as gauged by coefficient of variation of CGM values (30% vs. 36%). Persistence was high, with ~88% of observed days including ≥1 uploaded SGV in both groups, as was the proportion of users who engaged with CLARITY (~90% in both groups) and enablement of the ULS feature (~96% in both groups). The proportion of people with at least one Follower was higher among those with T1D than among those with T2D (63.1% vs. 40.4%, respectively). Episodes of rebound hyperglycemia and iatrogenic hypoglycemia were more prevalent, more frequent, more durable, and more severe (as judged by area under the curve, AUC) among those with T1D than those with T2D (Table).

Conclusion: These data suggest that patients with T2D can benefit from real-time awareness of their glucose levels and from using CGM features to help manage their diabetes. The adequacy of glycemic control for CGM users with T2D was comparable to or better than that for CGM users with T1D.

figureah

Disclosure: R. Dowd: Employment/Consultancy; Dexcom, Inc.

106

Beyond BG testing: digital health and intelligent monitoring

D. Shearer1, K. Snow2, A. Iyer3, M. Peeples3;

1Lifescan, Malvern, 2Aetna/CVS Health, Boston, 3Welldoc, Columbia, USA.

Background and aims: Health apps empower people to manage their health while enabling healthcare professionals to monitor patient progress and make informed treatment decisions. The OneTouch Reveal Plus® app (Powered by BlueStar®) syncs blood glucose readings from the Verio Flex®(VF) meter and analyzes the data to provide real-time guidance to the user and clinical decision support to HCPs. This prospective study evaluated the adoption of OTRP. Aims: Evaluate the impact of the OTRP app on BG control through a pre-post study of A1C, average BG, incidence of hypoglycemia and hyperglycemia, medication adherence, health care utilization and cost. Additional analysis included participant characteristics and engagement.

Materials and methods: Aetna members with Type 2 diabetes, with an A1C of ≥ 7.5%, on any diabetes medication, were contacted electronically. Interested participants completed an on-line consent form and were offered OTRP app, and the VF meter for 6 months. 291 subjects activated OTRP. Participants used the VF meter per their provider and received usual care. They used OTRP guided by the in-app, AI-generated coaching. No face-face interactions occurred as per study design. Participants could contact customer care for technology support.

Results: In 67 completed participants, a paired analysis of Aetna data on those who completed 6 months with an A1C, demonstrated a positive trend toward a lower A1C compared to baseline. Of note was the reduction in users with an A1C greater than 8 from 25% to 13%; with an increase in those in the 7-8 category (increased from 28% to 42%). At month 6, a statistically significant drop in maximum and average BG values was noted (n= 8,779 BG) - Max BG from 311 mg/ dL at month 1 to 246 mg/dL at month 6 (p= 0.034). A decrease in fasting BG was noted at 6 months (p=0.030) - from 162 mg/ dL to 152 mg/dL. The proportion of non-insulin users stayed relatively flat from baseline to follow up indicating that improvement in A1C and average BG was probably not driven by changes in medication but rather by factors attributed to OTRP. Average engagement in OTRP was 30 times/wk/person, totaling 31,663 suggesting an effective mechanism to improve self-management. Ratio of Daily Active Users (DAU) to Monthly Active Users (MAU) was 0.5. Strong engagement was demonstrated by 64% of members with a ‘guided journey’ and almost half of participants engaged with in-app curriculum. Members on differing medication regimens demonstrated a balanced engagement with management of BG, food, medications, activity, sleep and education. A statistically significant drop in average (p=0.034) BG values was observed over this time period (n= 8,779 BG values) as was average fasting BG (p=0.030; n=2,443 BG values) which correlated with a positive trend towards lower A1c. Diabetes related and all-cause ER visits and costs decreased from baseline to follow-up by 55% (p-value=0.0231).

Conclusion: Use of OTRP was associated with significant improvements in glycemic control after 3 and 6 months. This study suggests that real-time availability of patient data can assist users and HCPs to improve glycemic control and shift from scheduled care to data-driven care. Leveraging technology for intelligent monitoring - beyond glucose testing to include lifestyle and psychosocial monitoring provides robust data for treatment. The higher frequency and breadth of engagement may be the “active ingredient” to influence outcomes and bend the cost curve especially as in-person interactions with HCPs are challenged by global pandemics.

Disclosure: D. Shearer: None.

107

Change in HbA1c with and without intermittent use of continuous glucose monitoring in adults with type 2 diabetes participating in a virtual diabetes clinic

J.E. Layne1, H. Zisser2, R.M. Bergenstal3,4, R.A. Gabbay5,6, N.A. Barleen1, A. Armento Lee2, R.F. Dixon1;

1Onduo LLC, Newton, 2Verily Life Sciences, South San Francisco, 3International Diabetes Center at Park Nicollet, Minneapolis, 4HealthPartners Institute, Minneapolis, 5Joslin Diabetes Center, Boston,

6Harvard Medical School, Boston, USA.

Background and aims: The Onduo Virtual Diabetes Clinic (VDC) for people with type 2 diabetes (T2D) combines a mobile app, remote personalized lifestyle coaching, connected devices and live video consultations with board-certified endocrinologists for medication management and prescription of real-time continuous glucose monitoring (rtCGM) devices for intermittent use. This retrospective analysis examined change in HbA1c in VDC participants who used rtCGM intermittently compared to those who did not use CGM.

Materials and methods: Adults ≥18 years of age with T2D who enrolled in the VDC program from February 2018 through April 2019 with baseline and follow-up HbA1c values at 6 months were included. The rtCGM group was required to have used CGM ≥30 days prior to the follow-up HbA1c measurement. Outcomes included within group change in mean HbA1c with and without rtCGM use. Between group comparisons for change in HbA1c stratified by baseline categories of >9.0%, 8.0 to 9.0%, 7.0 to <8.0%, <7.0% and by <8.0% and ≥8.0% were evaluated by a two-sample t-test.

Results: Overall, participants (n=612) were (mean±SD): 53.5±8.7 years of age, 61.1% female and 26.5% lived in a rural geography. Baseline HbA1c was 7.8%±1.7, 33.0% were on insulin and 23.5% on a sulfonylurea. Characteristics of rtCGM group (n=213) and the no CGM group (n=399) were similar. HbA1c decreased significantly in the rtCGM group and in the no CGM group by 0.9%±1.7 and 0.4%±1.3, respectively (both p<0.001). Between group changes in HbA1c stratified by CGM use and baseline HbA1c categories are presented in the Table. There was an approximately two-fold greater improvement in HbA1c with intermittent rtCGM use in participants not meeting ADA HbA1c treatment targets compared to those with no CGM use. When stratified by a baseline HbA1c ≥8.0%, a significant greater proportion of the rtCGM group compared to the no CGM group had a follow-up HbA1c that meet the Healthcare Effectiveness Data and Information Set (HEDIS) treatment target of HbA1c <8.0%, 73.6% vs 47.5%, respectively (p<0.001).

Conclusion: The results indicate that participation in the VDC for 6 months was associated with significant improvement in HbA1c in all participants with greater benefit observed in rtCGM users. In conclusion, the VDC has potential to support people with T2D and their clinicians between office visits by increasing access to specialist care and advanced diabetes technology including rtCGM for intermittent use.

figureai

Disclosure: J.E. Layne: Employment/Consultancy; Employee of Onduo, LLC, the study Sponsor.

108

Evaluation of the one year efficiency of the EDUC@DOM telemonitoring and tele-education programme for type 2 diabetic patients

N. Costa1,2, M. Mounié1,2, J. Martini3, C. Latorre1, J.-C. Buisson4, M.-C. Chauchard3, J. Delaunay3, S. Schiir-Bonnans3, S. Taoui3, B. Lepage5,2, H. Colineaux5,2, P. Gourdy3, L. Molinier1,6, H. Hanaire3, M.-C. Turnin3;

1Health Economic Unit - Medical Information Department, University Hospital of Toulouse, Toulouse, 2Umr 1027, National Institute for Health and Medical Research (INSERM) - Toulouse III University, Toulouse, 3Department of Endocrinology, University Hospital of Toulouse, Toulouse, 4National School of Electrical Engineering, Electronics, Computer Science, Hydraulics and Telecommunications, Toulouse, 5Department of Epidemiology and Public Health, University Hospital of Toulouse, Toulouse, 6Umr 1027, National Institute for Health and Medical Research, Toulouse, France.

Background and aims: Due to its high prevalence and cost of care, estimated at 19 billion euros in France in 2018, diabetes is a major public health issue. A glycemic imbalance generates chronic complications responsible for a significant costs increase. The objective of this study is to assess the efficiency at one year, in France, of the EDUC@DOM telemonitoring and tele-education program for type II diabetic patients.

Materials and methods: Clinical data are from a randomized controlled interventional trial. The costs of treatment were estimated from the French National Health Data System (SNDS). Health insurance perspective was taken into account. Direct costs and those resulting from absences from the workplace were included. The efficacy endpoint was a decrease in the level of glycated hemoglobin (HbA1c). Missing data were imputed using the multiple imputation method. Costs and efficiency were adjusted from a multi-level model. The incremental cost-effectiveness and the confidence ellipse were then estimated from predicted values and bootstrap samples.

Results: Two hundred and fifty-six patients were included in the analyzes. Health care costs and indirect costs were estimated at € 10,989 for the remote-monitored group and € 13,120 for the control group, resulting from a difference of around 20%. The average HbA1c level was 7.49 and 7.67, respectively. Once adjusted, and according to the confidence ellipse presented in figure 1, the remote monitoring procedure was estimated to be "cost saving".

Conclusion: The EDUC@DOM telemonitoring and tele-education program is cost-saving and allow the optimization of type 2 diabetic patient’s care and in particular their glycemic balance. This program could help prevent complications and decrease associated costs. Additional data will be required to obtain results over a longer time horizon to confirm these results.

figureaj

Clinical Trial Registration Number: NCT01955031

Disclosure: N. Costa: Grants; French Ministry of Health.

OP 19 Decoding the heritable basis of type 2 diabetes

109

The expression quantitative trait (eQTL) landscape of type 2 diabetes in 404 human islet samples

A. Piron1,2, L. Alonso3, I. Morán3, M. Defrance2, M. Guindo3, S. Bonàs4, J. Ferrer5, A.L. Gloyn6, J.L.S. Esguerra7, L. Marselli8, P. Marchetti8, D.L. Eizirik1, D. Torrents3, M. Cnop1, J. Mercader9;

1ULB Center for Diabetes Research, Université Libre de Bruxelles, Bruxelles, Belgium, 2Interuniversity Institute of Bioinformatics in Brussels (IB2), Université Libre de Bruxelles, Brussels, Belgium, 3Barcelona Supercomputing Center (BSC), Barcelona, Spain, 4Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain, 5Section of Epigenomics and Disease, Department of Medicine, Imperial College London, London, UK, 6Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK, 7Lund University Diabetes Centre, Lund University, Malmö, Sweden, 8Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy, 9Broad Institute of Harvard and MIT, Cambridge, USA.

Background and aims: Type 2 diabetes (T2D) results from progressive pancreatic beta cell failure, caused by genetic and environmental factors. How genetic variants lead to beta cell failure remains poorly understood. Here, we performed a cis-expression quantitative trait loci (eQTL) analysis of human islets to establish the link between genetic variants and gene expression. We leveraged existing and novel genome wide association studies (GWAS) to guide the selection of eQTLs implicated in T2D.

Materials and methods: eQTL analysis was performed on 404 human islet transcriptomes, genomes and metadata, brought together in the Translational human pancreatic Islet Genotype tissue-Expression Resource (TIGER, created in the H2020 project T2DSystems). The genomic data was imputed with four panels (1000 Genomes, GoNL, HRC and UK10K), and the results were integrated to increase the number of high quality imputed variants to be analyzed, improving the coverage of low-frequency variants and indels. RNA-sequencing data were analyzed per cohort with RSEM for quantification and normalization, PEER for hidden confounding factors and fastQTL for the eQTL analysis. The by-cohort eQTL results were meta-analyzed, limiting batch effects while increasing statistical power. Co-localization analyses with the DIAMANTE T2D GWAS meta-analysis was done with the coloc R package.

Results: Thousands of cis-acting eQTLs were mapped, including novel low minor allele frequency (MAF) variants. Notably, the large sample size and quality of imputation enabled us to identify for the first time an eQTL for the low frequency variant (MAF 0.02) nearby CCND2 that is associated with 50% reduced risk for T2D. The intersection of the eQTL data with GWAS results showed significant eQTLs in human islets for more than 80 of the previously described T2D lead variants. Among these, at least 39 were confirmed by co-localization. Of particular interest, we found co-localization for an eQTL and GWAS locus near IGF2BP2. This T2D risk allele is associated with lower IGF2BP2 expression in human islets; the association seems islet-specific as, according to GTEx, it is absent in other tissues except thyroid. The summarized transcriptomes, genetic variants and eQTL results are available on the open access TIGER portal (http://tiger.bsc.es).

Conclusion: We present the largest regulatory variation study in human islet that results in the identification of 39 cis-acting eQTLs, including novel variants, co-localizing with T2D GWAS results. These genetic variants and associated dysfunctional genes expressed in human islets are an invaluable asset to understand the genetics of T2D.

Supported by: T2DSystems. EU Horizon 2020, No 667191.

Disclosure: A. Piron: None.

110

Polygenic risk score in type 2 diabetes risk prediction: genomics to healthcare

H. Marjonen1, T. Paajanen1, K. Auro2, A. Haukkala3, H. Kääriäinen1, K. Kristiansson1, M. Perola1,3;

1Finnish institute for health and welfare, Helsinki, 2Negen Ltd, Helsinki, 3University of Helsinki, Helsinki, Finland.

Background and aims: Utilization of genomic data in the personalized risk assessment and prevention of common chronic diseases such as type 2 diabetes creates a unique opportunity for the modern personalized health care, enabling more targeted and cost-effective use of the limited health care resources. Nevertheless, implementation of genomic medicine in the current health care operational environment is still a challenge due to the small number of proof-of-concept studies on the validity of such approaches as well as due to lack of targeted professional training. To successfully implement genomic medicine in the everyday healthcare, evidence-based and well-defined strategies are urgently needed. Genomics to Healthcare (P6), coordinated by the National Institute for Health and Welfare (THL), is a large-scale national initiative aiming to prepare the Finnish health care system for the clinical utilization of genetic risk information.

Materials and methods: In our P5 FinHealth pilot study, we provided personalized information on the individual T2D disease risk for 3.400 volunteering Finnish participants. We used a polygenic risk score (PRS) containing up to 7 million genomic regions and validated it in whole genome genotyped population based FINRISK cohorts (N=20.000) using Cox regression models.

Results: Our validation process showed that T2D PRS significantly associates with future T2D disease risk (HR:1.5 per 1 SD PRS, p-value:<2*10-16). The top 8% of the FINRISK population who had the highest PRS had fourfold increased risk for T2D compared to those in the lowest 8% with onefold risk. Moreover, almost 30% of the individuals with BMI >35 and the highest PRS were diagnosed with T2D during ten-year follow-up. T2D incidents in BMI >30 group occurred at a younger age concurrently with higher PRS, and there was a seven-year difference in the onset of T2D between the highest and lowest PRS group.

Conclusion: Our pilot project indicated that PRS could be used in preventive health care of type 2 diabetes. We have now initiated a ‘Genomics to Healthcare’ project which expands the pilot by recruiting 100 000 Finnish participants for a study of selected preventable or treatable common diseases. We form PRSes for the disease endpoints, and validate the PRSs for prediction of future disease in a large Finnish prospective population sample collection. We then return the genetic risk information on given diseases to participants via a secure online portal and enroll high-risk individuals to randomized intervention studies. Follow-up of the health behavior and morbidity of the participants collects data through surveys and national health care registers. We participate in coordinated set of activities in development of required infrastructures and technological solutions as well as training and communications. Our large project provides valuable scientific evidence and evaluates the health-economic impact of utilizing genetic risk information in healthcare.

Clinical Trial Registration Number: NCT03650127

Supported by: Diabetes Research Foundation, Yrjö Jahnsson, Sydäntutkimussäätiö, Sitra

Disclosure: H. Marjonen: None.

111

Characterisation of the genetic discordance between body mass index and type 2 diabetes: a phenome-wide analysis

D.E. Coral Candelo, J. Fernández-Tajes, N. Tsereteli, P.W. Franks;

GAME Unit, Lund University, Malmö, Sweden.

Background and aims: Obesity is on the rise globally, and is a leading risk factor for T2D. However, it is very heterogeneous, with varying degrees of T2D risk within the same levels of BMI. Better classification may lead to improve outcomes of current preventive and therapeutic strategies. Moreover, by elucidating the mechanisms uncoupling obesity from T2D risk, new possible therapeutic targets may emerge. Leveraging the vast amount of genetic data produced to date may contribute to reach these goals while overcoming the obstacles imposed by common assumptions, biases and confounders present in observational studies. Our aim is to compare the phenome-wide association patterns of BMI-increasing genetic profiles that either concordantly increase or discordantly decrease T2D risk.

Materials and methods: Highly concordant and highly discordant SNPs between BMI and T2D were obtained from the latest GWAS for both conditions. Their standardized effect sizes (SES) across multiple traits in the phenome, metabolome, proteinome and transcriptome were retrieved from the online genomic repositories. After alignment to the BMI-increasing allele, these effects were organized into a SNP x Trait matrix. A hierarchical clustering technique, combining PCA and Random Forest algorithms was applied, retrieving the optimal number of clusters of traits, organized in order of importance, useful to distinguish a discordant from a concordant SNP. Posterior probabilities of colocalization with T2D were calculated for each gene using transcriptome results. Tissue, biological process, molecular mechanism and cellular component enrichments were evaluated. The predictive potential of GRSs informed by these findings were assessed in the UK Biobank dataset.

Results: 121 SNPs were found to be significantly associated with BMI and T2D. 18 were discordant and 104 concordant. A total of 1372 variables were included in the analyses (Phenome = 546, Metabolome = 233, Proteinome = 593). The most important difference between discordant and concordant SNPs in the phenome matrix was found in a cluster of traits led by hypertension (Mean discordant SES = -1.59, Mean concordant SES = 2.56), highly correlated with two clusters led by coronary heart disease and overall health status, respectively. The second most important cluster was led by physical activity-adjusted WHR (Mean discordant SES = -2.69, Mean concordant SES = 0.24). The model obtained from the phenome matrix had the highest classification performance (Matthews Correlation Coefficient, MCC = 0.79). Metabolome results showed differences in polyunsaturated fatty acids and lipid contents in VLDL, but with lower performance (MCC = 0.67). The model from the proteinome matrix was unable to correctly classify SNPs (MCC = -0.03). Two genes (CCDC92 and DNAH10) showed the strongest association within the discordant set in adipose tissue, both involved in cilia formation. A GRS of these 121 SNPs with weights derived from the clusters with high classification performance was highly associated with T2D in both the general and obese populations in UK Biobank (p < 1x1016).

Conclusion: The main difference between BMI-increasing genetic profiles that either discordantly decrease or concordantly increase T2D risk is found in hypertension risk and physical activity-adjusted WHR. These traits can be used to inform GRSs to better classify T2D risk in obesity. Molecular mechanisms behind the discordant profile appear to involve cilia formation in the adipose tissue.

Disclosure: D.E. Coral Candelo: None.

112

Polygenic scores, diet quality, and type 2 diabetes risk

J. Merino1,2, M. Guasch-Ferre3, J. Li3,4, W. Chung4,5, B. Ma4, L. Liang4,5, F.B. Hu3,4, J.C. Florez1,2;

1Diabetes Unit and Center for Genomic Medicine, Massachusetts General Hospital, Boston, 2Department of Medicine, Harvard Medical School, Boston, 3Department of Nutrition, Harvard TH Chan School of Public Health, Boston, 4Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, 5Department of Biostatistics, Harvard TH Chan School of Public Health, Boston, USA.

Background and aims: The burden of type 2 diabetes (T2D) is not equally arrayed, as susceptibility to diabetogenic lifestyle factors varies between populations and within individuals. The extent to which genetic profiles can be leveraged to identify individuals more likely to benefit from targeted dietary recommendations is unclear. Here we tested the hypothesis that the generation of novel polygenic scores for T2D can identify individuals more likely to benefit from following a healthy diet.

Materials and methods: We included a total of 35,759 participants in the Nurses’ Health Study and the Health’s Professional Follow-up Study for whom genotype and dietary data were available T2D genetic profile was quantified using either a global polygenic score comprising ~1 million variants, or pathway-specific polygenic scores denoting different T2D pathophysiologic processes including beta-cell dysfunction, pro-insulin secretion, liver dysfunction, obesity, and lipodystrophy. Diet quality was assessed by the Alternate Healthy Eating Index 2010 (AHEI-2010). Cox proportional-hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (95% CI) for T2D risk in each cohort after adjusting for demographic, clinical, and lifestyle characteristics.

Results: Over 891,746 person-years of follow-up, 4,433 participants developed T2D. The relative risk of incident T2D was 29% higher (95% CI, 1.25 to 1.33; P<0.001) per 1SD increase in global polygenic score after adjusting for confounders. Similar associations were found for all five pathway-specific polygenic scores (P<0.001). The relative risk of T2D was 11% higher per each 10 units decrease in AHEI-2010 (95% CI, 1.08 to 1.14; P<0.001). We observed a significant interaction between diet quality and liver dysfunction polygenic score on T2D risk that was consistent across all three cohorts (pooled Pinteraction<0.001). No additional interactions were observed for other polygenic scores (Pinteraction>0.008; 0.05/6 scores). Compared with individuals at low genetic risk for liver dysfunction and high diet quality, low diet quality was associated with a 20% increased relative risk of T2D among those at low genetic risk (95% CI, 1.04 to 1.39) and a 58% increased relative risk among those at high genetic risk (95% CI, 1.38 to 1.81).

Conclusion: These data indicate that genetic risk and low diet quality are each associated with the risk of T2D, and that the diabetogenic effect of an unhealthy diet is more pronounced among individuals at high genetic risk for liver dysfunction. Our findings have the potential to deliver clinical and public health benefit through enhanced capacity to predict response to behavioral recommendations.

Supported by: H2020-MSCA-IF- 2015-703787

Disclosure: J. Merino: None.

113

Body mass index and kidney function: a two-sample Mendelian randomisation analysis

A.D. Kjaergaard1, D.R. Witte2, A. Teumer3, C. Ellervik4;

1Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark, 2Department of Public Health, Aarhus University, Aarhus, Denmark, 3Institute for Community Medicine, Greifswald Medical School, Greifswald, Germany, 4Department of Pathology, Harvard Medical School, Boston, Boston, USA.

Background and aims: A recent meta-analysis in more than 5 million individuals showed that elevated body mass index (BMI) was independently associated with estimated glomerular filtration rate (eGFR) decline. Because observational studies are prone to confounding and reverse causation, we employed a two-sample bidirectional Mendelian randomization (MR) approach to assess causality and directionality of this association.

Materials and methods: We used publicly available summary statistics from published genome wide association studies. Genetic instruments included 97 SNPs for BMI and 147 SNPs for eGFR. Outcomes were baseline eGFR, eGFR decline over time, blood urea nitrogen (BUN), chronic kidney disease (CKD), microalbuminuria (MA) and urinary albumin-to-creatinine ratio (UACR) from the CKD Genetics (CKDGen) Consortium (up to 765,348 participants) and BMI from the Genetic Investigation of ANthropometric Traits (GIANT) consortium (up to 806,834 participants). As main analysis, the inverse variance weighting method was applied. Sensitivity analyses included weighted median, penalized median, weighted modal and MR Egger regression analyses.

Results: Genetically instrumented BMI was not associated with baseline eGFR levels. However, a one standard deviation (SD≈4.7 kg/m2) higher BMI was associated with eGFR decline (β=0.18 (0.05-0.31) ml/min/1.73m2/year), increased BUN (β=0.03(0.03-0.04) mg/dl), and increased risks of CKD (OR=1.21 (1.13-1.30)) and microalbuminuria (OR=1.17(1.10-1.24)). Interestingly, higher BMI was associated with increased UACR (β=0.15(0.10-0.21)) only in individuals with diabetes, but not in the general population. Genetically instrumented eGFR was not associated with BMI in sex-stratified or sex- combined analyses.

Conclusion: This study suggests that higher BMI is a cause of decreased kidney function, but not vice versa.

Supported by: Novo Nordisk unrestricted grant

Disclosure: A.D. Kjaergaard: None.

114

Hedgehog signalling as a determinant of human fat expansion and distribution

A.D. van Dam1, E.M. Toledo2, N.Y. Loh1, M.J. Neville1,3, K.E. Pinnick1, M. Todorčević1, R. Dumbill4, L.B.L. Wittemans5,1, C. Langenberg5, F. Karpe1,3, C. Christodoulides1;

1University of Oxford, Oxford, 2Novo Nordisk Research Centre Oxford, Oxford, 3Oxford NIHR Biomedical Research Centre, Oxford, 4Oxford University Hospitals NHS Foundation Trust, Oxford, 5University of Cambridge, Cambridge, UK.

Background and aims: The role of developmental pathways in the regulation of human fat distribution is still poorly characterised. This project explores the role of hedgehog signalling using a single-cell RNA sequencing approach in combination with large-scale genetics and adipocyte biology. To do this, we focused on single nucleotide variants within the hedgehog interacting protein (HHIP) locus (rs1812175, rs13146972), that are strongly associated with hip circumference adjusted for BMI (HIPadjBMI).

Materials and methods: Single-cell RNA sequencing was performed on stromovascular fractions from paired abdominal and gluteal adipose tissue of healthy volunteers from the Oxford Biobank. The GWAS association (rs1812175, near HHIP, for HIPadjBMI) was confirmed in a large cohort with DXA-quantified regional fat measurements (n=17,212, Oxford Biobank, Fenland and EPIC-Norfolk). Functional studies of HHIP promoter activity and HHIP knockdown were performed in immortalised and primary human abdominal and gluteal pre-adipocytes.

Results: Single-cell RNAseq detected robust HHIP expression predominantly in the pre-adipocyte cluster and limited expression in endothelial cells. We therefore pursued functional studies using human pre-adipocyte models. The rs1812175 HIPadjBMI-increasing allele was associated with increased gluteal fat mass (β=0.08, p=1.37-06, n=17,212). Carriers of the HIPadjBMI-increasing allele had larger adipocytes in both the abdominal and gluteal depots compared to BMI- and age-matched controls.Comparison of single-cell RNAseq data from carriers of the HIPadjBMI-increasing allele and controls recruited from the Oxford Biobank (n=30 subjects) will provide further insight into the signalling pathways that mediate the association between the HHIP locus and fat distribution, and will define the cell types in which HHIP expression quantitative trait loci (eQTLs) exist.The association signal denominated by rs1812175 comprises nine variants in high linkage disequilibrium (r2>0.6), all of which are located in non-coding DNA regions. Two of these variants, rs1355603 and rs13106087, lie within the HHIP promoter. In HHIP promoter reporter assays the rs1355603 HIPadjBMI-increasing allele displayed lower activity than the major allele. We therefore pursued knockdown of HHIP in primary gluteal pre-adipocytes, which led to enhanced adipogenesis.

Conclusion: This study provides genetic and functional data demonstrating that HHIP plays an important role in the regulation of human adipogenesis and regional adiposity. Reduction in HHIP signalling has the potential to affect human fat distribution in a metabolically favourable pattern.

Supported by: Novo Nordisk Postdoctoral Fellowship, CVON-GENIUS Postdoc Grant

Disclosure: A.D. van Dam: Grants; Novo Nordisk Postdoctoral Fellowship.

OP 20 Feeding the pipeline: from drugs to surgery

115

Multiple mechanisms of a novel long-acting glucagon analogue, HM15136, on weight loss in animal models of obesity

J. Lee, S. Lee, J. Kim, J. Lee, S. Lee, S. Bae, D. Kim, Y. Kim, I. Choi;

Hanmi Phaarm. Co., Ltd, Hwaseong-si, Republic of Korea.

Background and aims: Although many anti-obesity drugs have been utilized, their weight loss efficacy is still marginal compared with bariatric surgery. Several studies have demonstrated that glucagon plays an essential role in body weight management both via increase of energy expenditure and suppression of appetite, suggesting its potential application as an anti-obesity medication. In line with this, we previously observed that chronic treatment of the novel long-acting glucagon analog, HM15136, led to efficient body weight loss (BWL) in diet-induced obesity (DIO) mice. To further investigate the potential benefits of HM15136 in obesity, the present study compared the BWL effect with available GLP-1R agonists (GLP-1RAs), and investigated the underlying mechanism for efficient BWL by HM15136.

Materials and methods: For a BWL efficacy comparison between HM15136 and GLP-1RAs, either HM15136 or available GLP-1RAs (liraglutide, dulaglutide or semaglutide) were subcutaneously administered into DIO mice for 4 weeks. The human equivalent doses tested were HM15136 2.0 nmol/kg, and 3.9 nmol/kg once every 2 days (Q2D); liraglutide 50 nmol/kg twice-daily (BID); dulaglutide 2.7 nmol/kg Q2D; semaglutide 20.5 nmol/kg Q2D. To assess the appetite regulation-independent BWL, the BW change by HM15136 treatment was compared with liraglutide under pair-fed controlled condition in DIO mice. At the end of the treatment, white adipose tissue (WAT) samples were prepared and the expression levels of thermogenic markers were examined. To measure energy expenditure and respiratory exchange ratio (RER), each DIO mice was subjected to indirect calorimetry, followed by VO2 and VCO2 monitoring. To explore an additional mechanism for BWL by HM15136, oral lipid tolerance test (oLTT) was performed after single administration of HM15136 in normal mice.

Results: In DIO mice, chronic treatment of HM15136 showed greater BWL (-38.5% vs. vehicle) than GLP-1RAs such as liraglutide, dulaglutide, and semaglutide (-16.8, -2.5, and -11.0% vs. vehicle). Of note, unlike liraglutide, HM15136 treatment was associated with more BWL compared to cognate pair-feed group, indicating the appetite regulation-independent BWL by HM15136. As to the responsible mechanism, HM15136 not only significantly increased the expression of PGC-1α and UCP-1 in WAT, but also enhanced energy expenditure. However, this was not the case when liraglutide treated. Together with the reduced RER, these results suggest that HM15136 could induce WAT browning through which fat utilization and energy expenditure increases. In respect to additional BWL mechanism, blood triglyceride level during oLTT was significantly decreased by HM15136 treatment compared to vehicle group, which coincided with decreased blood bile acid and ApoB48. These results suggest that the inhibition of lipid absorption is involved, at least in part, in efficient BWL by HM15136.

Conclusion: Based on these results, HM15136 could be a potential therapeutic option for the management of obesity via favorable regulation of energy expenditure and lipid absorption in addition to appetite inhibition. Efficacy study of HM15136 in obese patients is ongoing to assess the clinical relevance of these findings.

Disclosure: J. Lee: None.

116

Tirzepatide, a dual GIP/GLP-1 receptor agonist, interrupts metabolic adaptation to dietary restriction

T. Coskun, W.C. Roell, L.S. O'Farrell, E.C. Beebe, A. Regmi, P.J. Emmerson, Z. Milicevic, A. Haupt;

Diabetes and Complications, Eli Lilly and Company, Indianapolis, USA.

Background and aims: Body weight management via dietary restriction (DR) and exercise has limited success in prevention of weight gain after the intervention ceases. Rapid weight loss is accompanied by reduction in metabolic rate as a compensatory mechanism to counter reduced caloric intake, hindering overall success—a phenomenon known as metabolic adaptation. Tirzepatide has demonstrated profound weight loss in clinical trials, and in preclinical studies it significantly increased energy expenditure, unlike selective GLP-1 receptor (GLP-1R) agonist, and reduced ad libidum food intake. In the studies presented here, we aimed to investigate metabolic regulation by tirzepatide and semaglutide in diet-induced obese (DIO) mice.

Materials and methods: We created a mouse model of DR, in which DIO mice were placed under scheduled feeding with each mouse initially receiving food equal to the observed ad libidum daily food intake (~3 g). After monitoring for a week in indirect calorimetry chambers (Phenomaster, TSE) mice were switched to 50% DR (~1.5 g). Vehicle, tirzepatide or semaglutide at 3 nmol/kg daily administration (QD) was initiated with DR for 14 days with a pair-fed group matched to tirzepatide group. Daily body weight and food intake was measured along with constant respiratory exchange rate (RER) and energy expenditure throughout the study. In a follow up study, tirzepatide-treated mice at 3 nmol/kg QD were also treated with a long-acting GIP (glucose-dependent insulinotropic polypeptide) receptor antagonist at a dose of 1000 nmol/kg QD to pharmacologically block tirzepatide treatment effect.

Results: Tirzepatide treated mice demonstrated the highest degree of weight loss compared with semaglutide, pair fed, and vehicle treated groups during DR (22.4±1.3%, 19.9±1.3%, 17.0±0.7%, 15.2±1.2% respectively). Metabolic adaptation was observed during DR demonstrated by reduction in energy expenditure (~14%) observed in the vehicle, semaglutide, and pair fed groups. However, energy expenditure in the tirzepatide group was maintained similar to levels prior to DR suggesting an interruption of metabolic adaptation. Additionally, only tirzepatide treatment increased fat utilization more than 30%, compared with vehicle during DR. The increased fat utilization observed with tirzepatide was reversed during DR by GIPR antagonist suggesting a key function of GIPR agonism.

Conclusion: These studies demonstrate tirzepatide uniquely maintains metabolic rate during caloric restriction, potentially via increased lipid oxidation. Tirzepatide may indeed interrupt metabolic adaptation, potentially improving both magnitude and durability of weight loss. While further studies are ongoing to identify molecular mechanisms driving these results, the findings presented here are helping to better elucidate key metabolic mechanisms by which tirzepatide exerts its profound reduction in body weight and the potential benefits of GIP/GLP-1 dual agonism.

Disclosure: T. Coskun: Employment/Consultancy; Eli Lilly and Company.

117

Safe and efficient delivery of liraglutide and FGF-21 using NH2-HPSNs nanoparticles in vivo and in vitro

S. Yang1,2, L. Li1, G. Yang2;

1Key Laboratory of Diagnostic Medicine (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 2Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Background and aims: Nanomaterials have attracted great attention because of their low toxicity and high carrying capacity. However, in the field of metabolic diseases, nanomaterials are rarely used as a treatment. Liraglutide (Lira) and Fibroblast growth factor 21 (FGF-21) have an improvement effect on insulin resistance and type 2 diabetes, however, there have been no reports of studies carrying both at the same time. The current study was designed to investigate the the effect of mesoporous silica nanoparticles (NH2-HPSNs) carry both peptide drugs and plasmids on metabolic diseases in vivo and in vitro.

Materials and methods: We first detected the ability of NH2-HPSNs to carry liraglutide and plasmid of FGF-21 (pFGF-21) using agarose gel electrophoresis. Next, the cytotoxicity of NH2-HPSNs and Lira was assessed by the cell count kit-8 colorimetric (CCK-8) assays in vitro. We then compared the transfection efficiency of NH2-HPSNs/pFGF-21 with that of lipofectamine 2000/pFGF-21 in Hepa1-6 cells in vitro. To evaluated the transfection efficiency of NH2-HPSNs/pFGF-21 in vivo, male C57BL/6J mice were injected with saline, NH2-HPSNs, pFGF-21, pFGF-21 with hydrodynamic delivery, and NH2-HPSNs/pFGF-21 via a tail vein. The biological efficiency of NH2-HPSNs transfection was tested by PCR and Western Blotting. Subsequently, Lira, pFGF-21, Lira+pFGF-21, NH2-HPSNs, NH2-HPSN/Lira, NH2-HPSNs/pFGF-21 and NH2-HPSNs/pFGF-21+Lira were separately injected into HFD-fed mice, an insulin resistance (IR) animal model, via a tail vein. At the same time, metabolic parameters and energy expenditure of each group was measured. We then performed glucose tolerance test and insulin tolerance test. Finally, we examined the expression of key gluconeogenesis and insulin signaling molecules in the liver of each group.

Results: We found that NH2-HPSNs can carry both liraglutide and pFGF-21 and about 90% of the cells were survived after incubated at 300μg/ml of NH2-HPSNs for 48 hours. The transfection efficiency of NH2- HPSN/pFGF-21 was higher than that of lipofectamine 2000/pFGF-21 in vitro. The mRNA and protein expression of FGF-21 in mice transfected by NH2-HPSNs/pFGF-21 was higher than those with the hydrodynamic delivery of pFGF-21. Importantly, HFD-fed mice treated with NH2-HPSNs/pFGF-21+Lira significantly reduced food intake, body weight and blood glucose, improved energy metabolism, and improved insulin resistance compared with other group. Futhermore, NH2-HPSNs/pFGF-21+Lira also reduced the expression of phosphoenolpyruvate carboxykinase (PEPCK) and up-regulated the phosphorylation levels of protein kinase B (AKT) and insulin receptor (InsR) in the liver of HFD-fed mice.

Conclusion: Our study showed that, compared with Lira +pFGF-21-treated HFD-fed mice, NH2-HPSNs/pFGF-21+Lira-treated HFD-fed mice significantly improved glucose tolerance, inhibited PEPCK activity, and promoted the phosphorylation of InsR and Akt.

Supported by: NAFC

Disclosure: S. Yang: None.

118

The impact of bariatric surgery on microvascular complications in patients with type 2 diabetes: a matched controlled population-based cohort study

P. Singh1,2, N. Adderley1, A. Subramanian1, K. Gokhale1, K.A. Toulis1, R. Singhal2, S. Bellary3,2, A. Tahrani1,2, K. Nirantharakumar1,2;

1University of Birmingham, Birmingham, 2University hospital Birmingham NHS trust, Birmingham, 3Aston University, Birmingham, UK.

Background and aims: Bariatric surgery in patients with Type 2 diabetes (T2DM) is associated with significant improvements in glycaemic control and vascular risk factors but data regarding the impact of bariatric surgery on the development of diabetes-related microvascular complications is limited. The aim of our study was to assess the impact of bariatric surgery on microvascular complications defined as diabetes-related foot disease (DFD), sight threatening diabetic retinopathy (STDR) and chronic kidney disease (CKD) in patients with T2DM and obesity

Materials and methods: A retrospective matched, controlled population-based cohort study of adults with T2DM between 1/1/1990 and 31/1/2018 using The Health Improvement Network (THIN), a database of primary care electronic records. Each exposed (had bariatric surgery) patient was matched on index date for age, sex and body mass index (BMI) to 2 controls (did not have bariatric surgery). DFD was defined as a composite of either foot ulcer, gangrene, deformity, amputation, moderate foot risk, high foot risk, peripheral vascular disease or peripheral neuropathy (DPN). STDR was defined as either pre-proliferative or proliferative retinopathy or maculopathy or retinopathy treatment or vision loss. CKD was defined as eGFR <60 ml/min/1.73m2 or albuminuria (ACR ≥3mg/mmol). We conducted Cox regression to analyse the time to event using STATA version15.

Results: 1126 exposed and 2219 control participants were included. Exposed and control group were very similar in baseline characteristics. For the whole cohort, mean (SD) age was 50 (9) years, 2261 (68%) were women, mean BMI was 46 (7.6) kg/m2. The median follow-up was 3.9 years (IQR 1.8-6.4). After adjusting for age, gender, baseline BMI, smoking, social deprivation (Townsend score), ethnicity, hypertension, T2DM duration, baseline HbA1c and medications (ACE inhibitors, lipid-lowering drugs and insulin), bariatric surgery was associated with reduction in incident combined microvascular complications (adjusted HR 0.63, 95% CI 0.51-0.78, p<0.001), DFD (adjusted HR 0.612, 95% CI 0.497-0.753, p<0.001), STDR (adjusted HR 0.66, 95% CI 0.44-1.00, p<0.001), and CKD (adjusted HR 0.63, 95% CI 0.51-0.78, p<0.001) . Examining separately, bariatric surgery was associated with reduction in incident DPN (adjusted HR 0.717, 95% CI 0.524-0.98, p= 0.037)

Conclusion: Bariatric surgery was associated with a significant reduction in incident diabetes-related microvascular complications, including foot disease, sight threatening retinopathy, neuropathy and nephropathy. Improved access to bariatric surgery may reduce the health and economic burden of T2DM.

figureak

Disclosure: P. Singh: None.

119

Improvement in plasma metabolomic profile and hepatic insulin resistance 7 years after Roux-en-Y Gastric Bypass (RYGB)

C. Barbieri1,2, F. Carli1, M. Gaggini1, S. Pezzica1, B. Astiarraga3,4, M. Palumbo4, E. Ferrannini1, S. Camastra4, A. Gastaldelli1;

1National Research Council, Pisa, Italy, 2Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy, 3University Hospital Joan XXIII, Tarragona, Spain, 4Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.

Background and aims: Roux-en-Y Gastric Bypass (RYGB) leads to significant weight loss and improvement in glycaemic control, insulin resistance (IR), beta cell function, and in many cases, to the remission of type 2 diabetes (T2D). The aim of this study was to evaluate the long term effects of RYGB on plasma aminoacid (AA) and lipid composition and the relationship with changes in IR.

Materials and methods: The cohort comprised 30 patients (15 diabetic, T2D and 15 non diabetic, ND before RYGB) for which we had measurement of insulin resistance (IR), AA and lipid composition at baseline and 7 years after RYGB.

We measured IR indexes, i.e., hepatic (Hep-IR=(endogens glucose production (EGP)xIns)) and adipose tissue (Lipo-IR=(RaGlycerolxIns) by the infusion of stable isotope tracers of [6,6-2H]-glucose and [2H5]-glycerol. AA composition and the index of liver damage [GSG=Glu/(Ser+Gly)] were measured by GC-MS and TAG profile by LC-MS QTOF. The percent of unsaturated fatty acid (FA) in TAG was determined by the number double bonds, (db) in each fatty acyl chain (degree of saturation TAG(0-2db)/TAG(3-6db)).

Results: We observed long term (7 ys) effects of BS in both ND and T2D including diabetes remission in all T2D patient. BMI (kg/m2) decreased from 50.5±1.4 to 35.7±1.5 kg/m2 (p<0.0001). After RYGB n=9 had a BMI< 30 kg/m2 (4T2D/5ND), n=11 had a BMI 30-40 kg/m2 (7T2D/6ND), n=8 had a BMI>40 kg/m2 (4T2D/4ND).BMI at 7ys correlated to TAG concentrations (p=0.02; rho=0.43) and to the degree of TAG saturation (p=0.002; rho=0.71). Degree of TAG saturation increased with BMI at 7ys and with previous history of T2D (figure). Patients ND with BMI<30 had lower saturation compare to T2D.Hep-IR decreased from 144.9±20.7 to 46.3±5.1 μmol/kg/min*mU/l (p=0.001), although it tended to increase with residual BMI. The degree of TAG saturation (ie TAG(0-2db)/TAG(3-6db)) correlated with Hep-IR (p=0.037; rho=0.43), fatty liver index (FLI; p=0.0012; rho=0.69) but not Lipo-IR.The concentrations of the aromatic AA Phe and Tyr that are mainly metabolized in the liver, were decreased after RYGB (Phe: 71.5±4.9 vs 55.1±2.7; Tyr 91.2±6.2 vs 64.5±4.1 μM; p<0.01). The concentrations of the branched chain AA (Val, Leu, Iso) decreased after RYGB (440.3±41.4 vs 302.9±17.7 μM; p<0.01) and also the GSG index of liver damage was decreased after RYGB (1.3±0.1 vs 0.3±0.0; p<0.01).

Conclusion: RYGB not only dramatically reduces body weight but also improves hepatic IR, lipid composition, and AA, especially hepatic metabolites. Saturation of fatty acid in TAG is a discriminant value in relation to BMI and previous presence of T2D.

figureal

Supported by: Finalizzata

Disclosure: C. Barbieri: Grants; #RF-2011-02348446.

120

Predictors of type 2 diabetes remission after bariatric surgery: findings from 10 years follow up study

D. Moriconi1, S. Guerrini1, A. Di Carlo1, M. Anselmino2, E. Ferrannini3, S. Taddei1, M. Nannipieri1;

1Department of Clinical and Experimental Medicine, University of Pisa, Pisa, 2Unit of Bariatric Surgery, AOUP, Pisa, 3CNR Institute of Clinical Physiology, Pisa, Italy.

Background and aims: There are few prospective studies with long-term follow-up evaluating the remission of type 2 diabetes (T2D) in morbidly obese patients underwent bariatric surgery. AIMS: To evaluate the impact of bariatric surgery on T2D at 10 years of follow-up and the predictive factors of remission.

Materials and methods: Prospective observational study started in 2006; 85 obese patients, 65 women, with T2D, 20 of whom underwent sleeve gastrectomy (SLG) and 65 underwent gastric bypass (RYGB). Patients were evaluated every 6-12 months with clinical examination and blood tests during a follow-up period of 10 years. T2D remission was defined on the basis of the ADA criteria (2017).

Results: Based on fasting blood glucose (<100 mg/dl) and HbA1c (<5.7%), a complete remission (CR) was found in 40% of pts, a partial remission (PR) in 31% while T2D persisted in 23% of patients 1-year after surgery. At the end of the 10-years follow-up, CR was present in 23% of patients, PR in 32% and T2D in 45%. Baseline BMI was similar between the 3 groups, while 1-year after surgery there was a lower reduction of BMI in patients in which T2D persisted (-10.0 vs -14.6 vs -15.1 kg / m ^ 2, in T2D, PR and CR, respectively, p = 0.0005). Between 1 and 10 years after surgery, however, no significant BMI variation was observed between groups. Dividing the patients according to the duration of diabetes (DD) it was observed that in pts with DD <5 years, CR was achieved in 58%, PR in 38% while 4% of patients had persistence of T2D 1-year after surgery. At the end of 10-years follow-up, in the same group of patients, RC of 38%, RP of 45% and only 17% of T2D were observed. In the group with DD ≥5 years, CR was achieved only in 12%, PR in 33% and persistence of T2D was present in more than half of the pts (55%) already at 1-year after surgery. At the end of 10-years follow-up, no patient was in CR, PR was 12% while 88% of patients had T2D. In a logistic regression analysis, adjusting for all the main covariates (age, sex, diabetes duration, baseline therapy, type of surgery, and d-BMI), diabetes duration and insulin therapy before surgery were the only predictors of long-term diabetes remission.

Conclusion: The short duration of T2D (<5 years) and the absence of insulin therapy before surgery are the predictors of long-term T2D remission. Weight loss was associated with T2D remission 1 year after surgery, but it had no impact on the long-term relapse of diabetes.

Disclosure: D. Moriconi: None.

OP 21 SGLT-2 inhibitors: at the heart of the matter

121

Cardiovascular outcomes of patients with type 2 diabetes treated with SGLT-2 inhibitors versus GLP-1 receptor agonists in real life

G. Fadini1, E. Longato2, B. Di Camillo2, G. Sparacino2, L. Gubian3, A. Avogaro1;

1Department of Medicine, University of Padova, Padova, 2Department of Information Engineering, University of Padova, Padova, 3Azienda Zero, Regione Veneto, Padova, Italy.

Background and aims: Sodium glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) protect patients with type 2 diabetic (T2D) from cardiovascular events, but no trial has directly compared their cardiovascular effects. We aimed to address this gap using real-world data.

Materials and methods: We performed a retrospective real-world study on a population of ~5 million inhabitants from a region in North-East Italy. We identified T2D patients who received new prescription of SGLT2i or GLP-1RA from 2014 to 2018. SGLT2i and GLP-1RA initiators were matched 1:1 by propensity scores. The primary outcome was a composite of all-cause death, myocardial infarction, and stroke (3-point major adverse cardiovascular events [MACE]). Secondary endpoints were each component of the primary endpoint, hospitalization for heart failure, revascularization, hospitalization for cardiovascular causes, and adverse events.

Results: From a population of 330,193 diabetic patients, we followed 8596 SGLT2i and GLP-1RA matched initiators for a median of 13 months. Patients in both groups were on average 63 years old, 63% males, and 18% had pre-existing cardiovascular disease. T2D patients treated with SGLT2i versus GLP-1RA, experienced a lower rate of 3P-MACE (HR 0.68; 95% C.I. 0.61-0.99; p=0.043), myocardial infarction (HR 0.72; 95% C.I. 0.53-0.98; p=0.035), hospitalization for heart failure (HR 0.59; 95% C.I. 0.35-0.99; p=0.048), and hospitalization for cardiovascular causes (HR 0.82; 95% C.I. 0.69-0.99; p=0.037). Adverse events were not significantly different between the two groups.

Conclusion: In the absence of dedicated trials, this observational study suggests that SGLT2i may be more effective than GLP-1RA in improving cardiovascular outcomes of T2D.

Clinical Trial Registration Number: NCT04184947

Disclosure: G. Fadini: None.

122

The effects of canagliflozin on heart failure and cardiovascular death by baseline participant characteristics: analysis of the CREDENCE trial

D. de Zeeuw1, C. Arnott2, J.-W. Li2, C.P. Cannon3, B.L. Neuen2, H.J.L. Heerspink1,2, B. Neal2,4, D.M. Charytan5, G. Bakris6, T.-H. Chang7, N. Rosenthal8, B. Zinman9, V. Perkovic2,10, M.J. Jardine2,11, K.W. Mahaffey7;

1Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands, 2The George Institute for Global Health, UNSW Sydney, Sydney, Australia, 3Cardiovascular Division, Brigham & Women’s Hospital and Baim Institute for Clinical Research, Boston, USA, 4The Charles Perkins Centre, University of Sydney, Sydney, Australia and Imperial College London, London, UK, 5Nephrology Division, NYU School of Medicine and NYU Langone Medical Center, New York, USA, 6Department of Medicine, University of Chicago Medicine, Chicago, USA, 7Stanford Center for Clinical Research, Department of Medicine, Stanford University School of Medicine, Stanford, USA, 8Janssen Research & Development, LLC, Raritan, USA, 9Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, University of Toronto, Toronto, Canada, 10The Royal North Shore Hospital, Sydney, Australia, 11Concord Repatriation General Hospital, Sydney, Australia.

Background and aims: Individuals with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) are at high risk for hospitalized heart failure (HHF) and these events are reduced by canagliflozin (CANA). We investigated whether the effect of CANA on HHF or cardiovascular (CV) death differs by key participant characteristics.

Materials and methods: CREDENCE randomized participants with T2DM and CKD to CANA or matching placebo. In this analysis, we assessed the effect of CANA on the prespecified secondary outcome of HHF/CV death by baseline characteristics. Hazard ratios (HRs) and 95% CIs were estimated with Cox regression models, with subgroup by treatment interaction terms added to test for heterogeneity.

Results: Of 4401 trial participants, 432 experienced a HHF/CV death event over a median follow-up of 2.6 years. Participants at higher risk included those with a history of CV disease or HF, lower eGFR, higher UACR and baseline use of loop diuretics. CANA reduced the risk of HHF/CV death by 31% in the overall population (HR 0.69, 95% CI 0.57, 0.83), with consistent effect across a broad range of participant subgroups including those at high risk (all Pinteraction>0.246; Figure). The effect of CANA on HHF alone (HR 0.61, 95% CI 0.47-0.80) was also similar across most key participant subgroups (all Pinteraction>0.10).

Conclusion: CANA consistently reduces the risk of HHF/CV death and of HHF in T2DM and CKD across a broad range of participant subgroups, including those with and without prior HF.

figuream

Clinical Trial Registration Number: NCT02065791

Supported by: Janssen Research & Development, LLC

Disclosure: D. de Zeeuw: Employment/Consultancy; AbbVie, Bayer, Boehringer Ingelheim, Fresenius, Janssen, Mitsubishi-Tanabe, Mundipharma.

123

Empagliflozin reduces myocardial glucose uptake in persons with type 2 diabetes: a randomised double-blind, placebo-controlled crossover study

K.M. Lauritsen1,2, L.C. Gormsen3, T.K. Hansen1,2, B.R.R. Nielsen4, M. Johannsen5, J. Hansen5, H. Wiggers4, H.E. Bøtker4, L.P. Tolbod3, N. Møller1,2, E. Søndergaard1,2;

1Department of Internal medicine and Endocrinology, Aarhus University Hospital, Aarhus, 2Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, 3Department of Nuclear Medicine and PET center, Aarhus University Hospital, Aarhus, 4Department of Cardiology, Aarhus University Hospital, Aarhus, 5Department of Forensic Medicine, Aarhus University Hospital, Aarhus, Denmark.

Background and aims: Sodium-glucose cotransporter 2 (SGLT2) inhibition reduces cardiovascular morbidity and mortality. SGLT2 inhibition increases ketogenesis, which may contribute to the beneficial effects by serving as a cardioprotective oxygen-sparing fuel (“the thrifty substrate hypothesis”). To test this hypothesis, we investigated the effect of empagliflozin (EMPA) on cardiac glucose and free fatty acid (FFA) utilization and oxygen consumption.

Materials and methods: 13 individuals with type 2 diabetes (3 women; HbA1c: 57±6 mmol/mol; age: 62 (53-70) years) were treated for four weeks with EMPA or placebo in a randomized double-blind, placebo-controlled crossover study. At the end of each treatment period, 24-hour blood pressure (n=13) and 48-hour continuous blood glucose (n=13) were recorded. Cardiac glucose uptake and cardiac palmitate uptake, oxidation and esterification were measured in the postabsorptive state after an overnight fast with 18F-FDG (n=11) and 11C-Palmitate PET/CT (n=10), respectively. Myocardial oxygen consumption and myocardial external energy efficiency were measured with 11C-acetate PET/CT (n=10).

Results: EMPA reduced 48-hour mean blood glucose (8.0 ± 0.9 vs. 9.4 ± 2.2, p<0.01) and 24-hour mean arterial pressure (88±5 vs. 92±8 mmHg (p<0.05)). EMPA increased circulating FFA (1.0±0.4 vs. 0.8±3 mmol/L (p=0.02)) and 3-hydroxybutyrate (130 ± 17 vs. 65 ± 8 μmol/L (p<0.01)) concentrations. EMPA reduced myocardial glucose uptake (MGU) (0.6±0.6 vs. 1.4±0.6 μmol/100g/min (p<0.001) (figure 1)). EMPA did not affect myocardial FFA oxidation rate (7.2±3.1 vs. 8.0±3.1 μmol/100g/min (p=0.56)), FFA esterification rate (1.3±0.7 vs. 1.1±0.6 μmol/100g/min (p=0.34)) or total FFA uptake rate (8.4±3.6 vs. 9.1±3.2 μmol/100g/min (p=0.76). EMPA did not change myocardial external energy efficiency (29.5±7.3 vs. 27.7±4.5 % (p=0.22)) or myocardial oxygen consumption (8.97 ± 1.11 vs. 9.77 ± 1.34 ml/100g/min (p=0.12)).

Conclusion: EMPA reduces postabsorptive myocardial glucose uptake by 57% but does not affect myocardial FFA utilization despite significantly increased levels of substrate in the form of circulating FFAs. EMPA treatment therefore appears to selectively channel myocardial substrate utilization from glucose towards other sources such as ketone bodies. However, this shift in myocardial substrate utilization does not appear to improve either myocardial external energy efficiency or myocardial oxygen consumption.

figurean

Clinical Trial Registration Number: EudraCT-number 2017-001779-22

Supported by: NN Foundation, HRF of Central DK Region, DC for Independent Region, DDA, RM

Disclosure: K.M. Lauritsen: None.

124

Effects of 6 weeks of treatment with dapagliflozin, a sodium-glucose co-transporter 2 inhibitor, on myocardial function and metabolism in patients with type 2 diabetes

J. Oldgren1, S. Laurila2,3, A. Åkerblom1, A. Latva-Rasku3, E. Rebelos3, H. Isackson1, M. Saarenhovi3, O. Eriksson4, K. Heurling4, E. Johansson4, U. Wilderäng5, C. Karlsson5, E. Ferrannini6, J. Oscarsson5, P. Nuutila3;

1Uppsala Clinical Research Center and Department of Medical Sciences, Uppsala University, Uppsala, Sweden, 2Heart Center, Turku University Hospital, Turku, Finland, 3Turku PET Centre, University of Turku and Turku University Hospital, Turku, Finland, 4Antaros Medical AB, Mölndal, Sweden, 5BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, 6Institute of Clinical Physiology, National Research Council, Pisa, Italy.

Background and aims: We aimed to explore early effects of dapagliflozin (DAPA) on myocardial function and metabolism in patients with type 2 diabetes (T2D) without heart failure (HF), which could help explain the reduced risk for HF hospitalization observed within a few months in sodium-glucose co-transporter 2 (SGLT2) inhibitor outcome trials.

Materials and methods: T2D patients with BMI ≥25 kg/m2, with left ventricular (LV) ejection fraction >50% and without HF, on stable metformin, and no other antidiabetic treatment, were randomized to placebo (n=26) or 10 mg/day DAPA (n=27) in a 6-week parallel group, double-blind study. Investigations at baseline and at 6 weeks included cardiac MRI, [11C]-acetate positron emission tomography (PET) (oxygen consumption and perfusion) of the heart and [18F]-FTHA PET (fatty acid uptake) of the heart and liver, and analyses of circulating biomarker levels. Placebo-adjusted changes in the per-protocol analysis set were analyzed by ANCOVA as least square means with 95% confidence intervals.

Results: Evaluable patients (placebo: n=24, DAPA: n=25; 53% males) had a mean (SD) age of 64.4 (7.2) years, BMI of 30.1 (3.7) kg/m2, HbA1c of 6.7 (0.6) %. Hypertension (75.5%) and dyslipidemia (57.1%) were common, while few patients had a prior cardiovascular event. At 6 weeks, body weight and HbA1c were decreased in the DAPA group vs placebo. Myocardial efficiency was not affected, but external LV work, total LV energy consumption and myocardial perfusion were reduced from baseline in the DAPA group, but not significantly vs placebo. No significant effects on LV sizes or volumes were observed, whereas left atrial volume was reduced in patients randomized to DAPA. Global radial strain decreased vs placebo, while global longitudinal and circumferential strain tended to increase by DAPA treatment. Myocardial fatty acid uptake was not affected, but hepatic uptake of fatty acids was increased by DAPA vs placebo. Plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were unaffected. No adverse events leading to study treatment discontinuation were reported.

Conclusion: This exploratory study in patients with well-controlled T2D without HF showed limited effects on myocardial fatty acid uptake, function and efficiency, but results indicate reduced heart work after 6 weeks of treatment with dapagliflozin.

Clinical Trial Registration Number: NCT03387683

Supported by: The study was funded by AstraZeneca.

Disclosure: J. Oldgren: Other; AstraZeneca, Bayer, Bristol-Myers Squibb, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, Roche Diagnostics, Sanofi.

125

Direct and acute metabolic effects of empagliflozin in diabetic mouse hearts: reduced lactate generation mediated through NHE-1 inhibition

C.J. Zuurbier1, L. Uthman1, D. Bakker1, S. Sari1, M.W. Hollmann1, N.C. Weber1, S.M. Houten2, H. Zhang1, R. Coronel3, M. van Weeghel4;

1LEICA, Anesthesiology, Amsterdam UMC, UvA, Amsterdam, Netherlands, 2Icahn Institute for Data Science and Genomic Technology, New York, USA, 3Experimental Cardiology, Amsterdam UMC, UvA, Amsterdam, Netherlands, 4Clinical Chemistry, Amsterdam UMC, UvA, Amsterdam, Netherlands.

Background and aims: Changes in cardiac metabolism and ion homeostasis precede and drive cardiac remodeling and heart failure development upon hemodynamic or metabolic overload. We previously demonstrated that SGLT2i’s have direct cardiac effects on ion homeostasis through inhibition of the sodium/hydrogen exchanger (NHE-1). Here we investigate whether the SGLT2i Empagliflozin (Empa) 1) possesses direct and acute cardiac effects on metabolism of the intact isolated diabetic heart, and 2) mediates cardiometabolic effects through inhibition of NHE-1 activity. Purpose: To study direct and acute metabolic effects of Empa in isolated diabetic mouse hearts and its dependency on NHE-1 activity.

Materials and methods: 11-14 wks db/db male hearts were Langendorff-perfused at constant flow for 35 min with (in mM) 5.5 glucose, 1.0 lactate, 0.1 pyruvate, 0.5 glutamine, 0.4 palmitate, 0.5 L-carnitine, 100 mU/L insulin and 5 nM epinephrine. A balloon was positioned in the left ventricle to monitor mechanical function of the heart. Three different series were examined: 113C glucose perfusions (n=16); 213C glucose + 10 μM Cariporide (specific NHE-1 inhibitor) perfusions (n=17), and 313C palmitate perfusions (n=13). Within each series, Empa treated hearts (1 μM Empa) were compared with vehicle-treated hearts (0.02% DMSO). At end experiment, hearts were immediately frozen and lysed for stable isotope analysis and metabolomics using LC-MS techniques. Hearts were also analyzed for phosphorylation status of AKT, STAT3, AMPK, ERK, and eNOS (n=8 per group).

Results: At baseline, before treatment, end-diastolic pressure (3 ±1 mmHg), Rate-Pressure-Product (49.697±6.573 mmHg.beats/min), +dp/dt (5607±858 mmHg/s), -dp/dt (4726±732 mmHg/s) and oxygen consumption (51±12 μmol/min/gram dry weight) were similar between control and Empa group, and were not differentially affected by 35 min Empa treatment. Empa treatment was also without effect on protein phosphorylation status. 35 min perfusion of Empa significantly decreased lactate labeling in the 13C glucose perfusions (13C labeling of lactate: 58 ± 2% vs 50 ± 3%, for vehicle and Empa, respectively; p=0.02) and trended to lower the total content of unlabeled and labeled glucose-6-phosphate (G6P 5.2 ± 0.8 AU vs 3.3 ± 0.4 AU, for vehicle and Empa, respectively; p=0.052), without changes in other glucose metabolic pathways. Cariporide mitigated Empa effects on lactate labeling and G6P. Empa was without effect on fatty acid oxidation, except for an increased labeling in α-ketoglutarate (13C labeling of α-KG: 79 ± 1% vs 86 ± 1% for vehicle and Empa, respectively; p=0.002).

Conclusion: The present study shows for the first time that the SGLT2 inhibitor Empagliflozin directly and acutely decreases cardiac lactate generation in diabetic hearts, with a trend for lower G6P, through an NHE-1 dependent fashion. Less lactate generation, indicating an improved energy status heart, and lower G6P content, which reduces the activation of cardiac growth programs, may contribute to the beneficial effects of SGLT2i’s on cardiac remodeling, hypertrophy and heart failure development.

Supported by: EFSD/Novo Nordisk Programme

Disclosure: C.J. Zuurbier: Grants; EFSD.

126

Effect of empagliflozin on the fibrosis biomarkers and left ventricular haemodynamics in patients with type 2 diabetes and chronic heart failure

D. Lebedev, A. Babenko;

Almazov national medical research centre, Saint Petersburg, Russian Federation.

Background and aims: Sodium glucose co-transporter 2 inhibitor, a widely used class of antihyperglycemic medications acting on inhibiting glucose reabsorption, is shown beneficial in reduction of heart failure hospitalization and cardiovascular mortality. However the mechanisms remain unclear. Aim of the study: To investigate the impact of empagliflozin on fibrosis biomarkers and left ventricular parameters in patients with type 2 diabetes mellitus (T2DM) and chronic heart failure with preserved ejection fraction (HFpEF).

Materials and methods: Thirty five patients with T2DM and HFpEF were enrolled in the study. Inclusion criteria were: females or males aged 40 to 75 years, glycated hemoglobin (HbA1c) 7.5-10.0%, stable antihyperglycemic therapy at least for 12 weeks. Exclusion criteria were: acute illness or infection, a cardiovascular event during the past 6 months, chronic heart failure NYHA III-IV, chronic kidney disease (estimated glomerular filtration rate (eGFR), according to CKD-EPI (eGFR < 45 mL/min/1.73 m2). Patients were received empagliflozin 10 mg during 24 weeks. Transthoracic echocardiography and laboratory tests such as glycated hemoglobin (HbA1c), creatinine, galectin-3, tissue inhibitor of metalloproteinase-1 inhibitor (TIMP-1), procollagen type I carboxy-terminal propeptide (P1CP), matrix metalloproteinase-9 (MMP-9), N-terminal fragment brain natriuretic peptides (NT-pro-BNP), ST-2 were done.

Results: No significant difference was observed in galectin-3, P1CP, MMP-9, ST-2 concentrations between the baseline and the end of treatment. There was an increase in TIMP-1 concentration after 24 weeks of treatment compared with baseline (215 ng/ml (186,5-234) versus 177 ng/ml (118,25-202,5), respectively, p=0,006). Left ventricular mass index (LVMI) significantly decreased from 126 g/m2 (95,5-154) to 111,1 g/m2 (94,8-150,0), (p = 0.043). However, after applying of Holm-Bonferroni correction this difference has become nonsignificant. There was no significant difference in end-diastolic volume (EDV), end-systolic volume (ESV) and end-diastolic volume index (EDVI). Positive correlation was observed between galectin-3 concentrations and EDV after 24 weeks of treatment (-0,532, р=0,002).

Conclusion: Empagliflozin in patients with T2DM and HFpEF did not affect left ventricular function, measured by echocardiography. Furthermore, empagliflozin treatment did not lead to significant changes in fibrosis biomarkers, except TIMP-1. Further research is needed to clarify obtained results.

Supported by: RSF № 17-75-30052

Disclosure: D. Lebedev: Grants; Russian Scientific Fund: grant number 17-75-30052.

OP 22 New Treatments for NAFLD: Hope or Hype?

127

Therapeutic effect of a novel long-acting GLP-1/GIP/Glucagon triple agonist (HM15211) in CDHFD-induced NASH and fibrosis mice

J. Choi, H. Jo, J. Kim, H. Kwon, J. Lee, S. Bae, D. Kim, S. Lee, I. Choi;

Hanmi Pharm.Co.,Ltd., Hwaseong-si, Gyeonggi-do, Republic of Korea.

Background and aims: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease characterized by steatosis and inflammation, which eventually results in fibrosis. In particular, advanced fibrosis due to NASH is associated with a high risk of liver-related mortality, becoming one of the main causes for liver transplantation. To date, there are, however, no pharmacological therapy approved. One major hurdle for the drug development is limited pathologic features of current animal models, in terms of clinical relevance. The choline-deficient and high fat diet (CDHFD) mice develop multiple aspects of NASH and fibrosis similarly with those in human, being increasingly recognized as a feasible disease model for the evaluation of NASH drug candidates. HM15211 is a novel long-acting GLP-1/GIP/Glucagon triple agonist, and developed for the treatment of NASH and fibrosis. Previously, HM15211 treatment reduced hepatic fat contents and fibrosis in various animal models of NASH and fibrosis. Here, we further explored its therapeutic efficacy in CDHFD mice.

Materials and methods: To induce NASH and fibrosis, the mice were fed with choline-deficient and high fat diet (CDHFD) for 14 weeks, and HM15211 was subcutaneously administered during last 6 weeks. Cilofexor (CIL, FXR agonist) was used as comparative control. At the end of treatment, the liver tissue samples were prepared, and the degree of hepatic steatosis and fibrosis was determined by measuring hepatic triglyceride (TG) and hydroxyproline contents, respectively. Additional liver tissue samples were subjected to H&E and Sirius red staining, followed by histological grading. Quantitative PCR analysis was performed to determine the hepatic fibrosis marker gene expression, and the blood levels of fibrosis surrogate markers were measured by ELISA. To further evaluate the potential therapeutic effect of HM15211 on advanced fibrosis, CDHFD mice were established with an extended induction period (up to 24 weeks).

Results: In CDHFD mice, HM15211 treatment significantly reduced hepatic TG (2.6 and -51.0% vs. vehicle for CIL and HM15211) and TBARS (oxidative stress marker) (11.0 and -81.4% for CIL and HM15211). In addition, HM15211 treated group was associated with more reduction in hepatic pro-inflammatory marker gene expression such as F4/80 (-1.6 and -30.2% vs. vehicle for CIL and HM15211) and IL-1β (-37.5 and -68.8% for CIL and HM15211). Histological analysis further demonstrated the more benefits of HM15211 in steatosis and inflammation improvement, resulting in greater reduction in NAFLD activity score (NAS) than CIL. For efficacy in fibrosis, HM15211 treatment consistently showed more reduction in hepatic hydroxyproline (-13.7 and -37.7% for CIL and HM15211) and marker gene expression such as collagen-1α1 (-37.5 and -72.3% vs. vehicle for CIL and HM15211). To further confirm the therapeutic benefits, CDHFD mice with an extended induction period (24 weeks) was administered with HM15211, and same beneficial effects on NASH and fibrosis improvement were also confirmed.

Conclusion: Based on these results, we propose that HM15211 might be a novel therapeutic option for NASH and fibrosis. Hence, more efficacy than FXR agonist in CDHFD mice further highlight the potential benefits of multi-targeting approaches of HM15211. Clinical studies in biopsy proven NASH patients are ongoing to assess the clinical relevance of these findings.

Disclosure: J. Choi: None.

128

The selective PPAR gamma modulator CHS-131 improves liver histopathology and metabolism in a biopsy-confirmed mouse model of non-alcoholic steatohepatitis and obesity

N. Perakakis1, A. Joshi1, N. Peradze1, K. Stefanakis1, G. Li2, M. Feigh3, G. Rosen2, M. Fleming2, C.S. Mantzoros1;

1Internal Medicine, Boston VA Healthcare system and Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA, 2Coherus Biosciences, San Francisco, USA, 3Gubra, Horsholm, Denmark.

Background and aims: CHS-131 is a selective peroxisome proliferator-activated receptor gamma (PPARγ) modulator that demonstrates dose-dependent antidiabetic effects with less side effects (i.e. fluid retention and weight gain) compared to thiazolidinediones in phase II clinical trials. The aim of this study was to investigate the effects of CHS-131 on metabolic parameters and liver histopathology in a diet-induced obese (DIO) and biopsy-confirmed mouse model of non-alcoholic steatohepatitis (NASH).

Materials and methods: Male C57BL/6JRj mice were fed AMLN diet (40% fat with trans-fat, 20% fructose and 2% cholesterol) for 33 weeks prior to liver biopsy procedure. Only animals with biopsy-confirmed steatosis (score ≥2) and fibrosis (stage ≥F1) were included and stratified into treatment groups (n=12-13) to receive for the next 12 weeks: 1) vehicle, 2) Low dose CHS-131 (10 mg/kg), 3) High dose CHS-131 (30 mg/kg). Metabolic parameters related to body composition and glucose homeostasis, liver histopathology, markers of liver function and liver, subcutaneous and visceral adipose tissue gene expression profiles were assessed.

Results: CHS-131 has no substantive effect on body weight, body composition (fat, lean or water mass) and energy intake in DIO-NASH mice with fibrosis. CHS-131, both in low and high dose, improved fasting insulin levels and insulin sensitivity in intraperitoneal insulin tolerance test. CHS-131 (high dose) resulted in 37% lower plasma levels of alanine transaminase, 29% of aspartate transaminase and 20% of total cholesterol. Both low and high doses of CHS-131 increased robustly plasma adiponectin levels (by 114% and 137% of mean levels respectively). Urea levels, as a marker of hydration, were not affected by CHS-131 treatment. CHS-131 (high dose) improved NAFLD liver histology activity score with impacts on lobular inflammation and hepatocellular ballooning. Additionally, CHS-131 exhibited trends to decreased markers of hepatic fibrosis (-28% for hydroxyproline, -24% for Col1a1, -21% for a-SMA, -18% for Galectin-3). DIO-NASH mice treated with CHS-131 demonstrated a shift to diacyl- and triacylglycerols with shorter chains in the liver and a partial restoration of the reduced levels of hepatic amino acids. Additionally, CHS-131 increased the expression of genes stimulating mitochondrial function (PGC-1a), fatty acid oxidation (Acox1) and browning (Ucp1Elovl3) and decreased expression of genes promoting fatty acid synthesis (Fasn), triglyceride synthesis (Mlxipl) and inflammation (F4/80, Ccl2) in adipose tissue.

Conclusion: CHS-131 can be an effective treatment in NASH by improving hepatic lipid composition, reducing lobular inflammation and hepatocyte ballooning and decreasing markers of hepatic fibrosis. The beneficial effects of CHS-131 on NASH are most probably achieved indirectly by changes in visceral and subcutaneous adipose tissue function, elevated adiponectin levels and systemic improvement of insulin sensitivity. Treatment with CHS-131 was not associated with common side effects observed in full PPARγ activators, such as water retention and weight gain.

Supported by: NiP was funded by DFG, Number 389891681 (PE2431/2-1). CSM and MF were funded by Coherus Biosciences

Disclosure: N. Perakakis: Employment/Consultancy; CSM is consultant for Coherus Biosciences. Grants; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) –389891681 (PE 2431/2-1) provided funding to NiP, Coherus Biosciences provided funding to CSM and MF. Stock/Shareholding; CSM is shareholder of Coherus Biosciences.

129

A direct AMPK activator reduces liver steatosis in a mouse model of NASH

K.M. Mather1, M.L. Boland1, E.L. Rivers2, A. Srivastava2, M. Schimpl2, P. Hemsley2, J. Robinson2, P.T. Wan2, J.L. Hansen1, J. Trevaskis1, D.M. Smith2;

1CVRM, AstraZeneca, Gaithersburg, USA, 2AstraZeneca, Cambridge, UK.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is estimated to affect 25% of adults and is highly associated with metabolic disease. NAFLD often progresses to non-alcoholic steatohepatitis (NASH), which has no cure or treatment, and can lead to cirrhosis and hepatocellular carcinoma. AMPK (5’AMP-activated protein kinase) activators have shown potential for treating NAFLD/NASH due to their effects on fatty acid inhibition and cholesterol synthesis. After evaluating and characterizing several AMPK activators, we selected a compound, herein referred to as C455, to evaluate this potential in a preclinical NASH study.

Materials and methods: X-ray crystallography demonstrated the binding of AMPK α2β1γ1 protein by C455 at the ADaM site. Pharmacokinetic studies were performed by oral and IV administration in C57LB/6 and CD-1 mice. HepG2 cells were used to measure activation by ACC phosphorylation with an EC50 of 81nM. Selectivity was determined testing 1uM, or a dose response curve of C455 against panels from Eurofins and Thermo Fisher of over 470 targets. After 14 weeks on a high fructose / high cholesterol (Amylin -NASH) diet, or low-fat control (LFD) male ob/ob mice were randomized into groups based on liver fibrosis (determined by biopsy 3 weeks prior), body weight (BW), plasma ALT, and body composition. Mice were dosed via oral gavage with vehicle or C455 at 3 or 30mg/kg daily for 6 weeks. On day 28 a fasting glucose tolerance test was preformed using 1.5g/kg glucose bolus. On Day 42 mice were euthanized. Blood, liver, and heart were collected for histological, biochemical, and gene expression measurements.

Results: C455 demonstrated selective activation of AMPK and was equally potent with both beta-isoforms α1β1γ1 7.8 nM +0.2 SEM n=10; and α1β2γ1 7.2 nM +0.1 SEM n=5. Pharmacokinetics determined a bioavailability of 45% and half-life of 3h. Overall the compound showed good permeability 26 (1x10-6 cm/s), low protein binding 2.6% free, and low hepatic metabolism < 3 μl/min/106, but moderate solubility 8.0μM, making it a good candidate for our study. After 6 weeks of treatment, plasma ALT and terminal liver weight were decreased 32% + 7.9% p=0.0257 and 22% + 3.3%p=0.0003 respectively in mice treated with 30mg/kg C455 vs. vehicle. This dose also decreased liver lipid from 33% to 23% + 1.8 p<0.0001, which was below levels seen in the LFD group. Liver fibrosis via collagen staining did not improve with treatment, but significant decreases in mRNA transcripts for Col1a1 61% + 4.2% p<0.0001, Col1a2 59% + 2.9 p<0.001, Col4a1 55% + 3.9 p<0.0001, and Timp1 55% + 3.1% p<0.0001, were seen. There was a dose dependent increase in heart weight relative to body weight 13.3% + 3.9% p=0.008, and increased expression of genes associated with cardiac hypertrophy at 30mg/kg, including Ankrd1 48.2% +10.2% p=.0099. Mice given 30mg/kg C455 also increased their cardiac glycogen storage by 394% + 123% p = 0.012 compared to vehicle treated mice.

Conclusion: Our results show systemic activation of AMPK in a diet-induced mouse model of NASH reduces plasma ALT, decreases liver lipid, and suppressed hepatic collagen gene expression but shows no histological improvement in fibrosis. Coupled with increased heart weight and glycogen storage, our data suggests that current small molecule activators of AMPK may not be viable therapies for the treatment of NASH-related fibrotic disease.

Disclosure: K.M. Mather: None.

130

Empagliflozin ameliorates obesity associated fatty liver disease by regulating Sestrin2-mediated AMPK-mTOR signalling pathway in obese mice

X. Sun1, N. Hou1, F. Han2, Y. Liu1, N. Huang1;

1Department of Endocrinology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 2Department of Pathology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, China.

Background and aims: Obesity is linked to an increased risk of nonalcoholic fatty liver disease, which is also called obesity associated fatty liver disease (OAFLD). Sodium glucose co-transporter-2 (SGLT2) inhibitors may be effective for improving OAFLD by improving metabolic profiles. Sestrin2, a novel stress-inducible protein that lacks kinase activity, has been shown to maintain hepatic energy homeostasis by AMPK/mTOR. However, whether sestrin2-mediated AMPK/mTOR is involved in the protective effects of SGLT2 inhibitor on OAFLD remains elusive. Therefore, the aim of this study was to determine whether empagliflozin, an SGLT2 inhibitor,could improve OAFLD by upregulating sestrin2-mediated AMPK/mTOR in high fat diet (HFD) induced obese mice.

Materials and methods: C57BL/6 and Sestrin2 knockout mice were fed with a normal-chow diet or an HFD with 12 weeks and then were treated with or without empagliflozin (10mg/kg) for another 8 weeks. Liver injury was evaluated by liver function test, histopathology, oil red o staining and masson's trichrome. Mitochondrial superoxide production was detected by MitoSOX probe. Sestrin2-AMPK/mTOR signaling pathways were determined by western blot.

Results: HFD mice showed significant increased body weight, fat mass, NEFA, and triglyceride levels and impaired glucose tolerance and insulin sensitivity (body weight, 49.5 ± 2.8 g vs. 33.8 ± 1.9 g; fat mass 17.6 ± 3.4 g vs.5.5 ± 0.8 g; AUC glucose 19194± 610 vs. 38739± 1750; AUC insulin 9617± 242 vs.5407± 326; P < 0.05). Treatment of HFD mice with empagliflozin reduced body weight, body fat mass, improved glucose tolerance and insulin sensitivity without improving lipid levels (body weight, 41.2 ± 1.5 g vs. 49.5 ± 2.8 g; fat mass 10.7 ± 2.6 g vs.17.6 ± 3.4 g; AUC glucose 27714 ± 1052 vs. 38739± 1750; AUC insulin 6263 ± 337 vs.9617± 242; P<0.05). HFD mice showed significant hepatic injury, lipid accumulation, fibrosis and mitochondria injury. Treatment of HFD mice with empagliflozin treatment significantly improved hepatic injury, lipid accumulation and fibrosis (P<0.05). Additionally, empagliflozin ameliorated mitochondrial superoxide production and mitochondria injury (P<0.05). Empagliflozin treatment significantly enhanced proteins of sestrin2 and phosphorylation of AMPK, but inhibited phosphorylation of mTOR (P < 0.05). These beneficial effects were partially attenuated in HFD-fed Sestrin2 knockout mice when treated with empagliflozin.

Conclusion: Our study indicates that empagliflozin improves OAFLD via regulating Sestrin2-mediated AMPK/mTOR signaling pathway from HFD induced obese mice. These findings provide a novel mechanism for hepatic protection of SGLT2 inhibitor on OAFLD.

Supported by: National Natural Science Foundation of China (81870593, 81600688)

Disclosure: X. Sun: None.

131

Effects of biliopancreatic diversion on non-alcoholic steatohepatitis: 5 years follow up

M.F. Russo1, E. Lembo1, A. Mari2, G. Mingrone1;

1Università Cattolica Del Sacro Cuore- Sede di Roma, Rome, 2Institute of Neuroscience - National Research Council, Padova, Italy.

Background and aims: Over the past 40 years, biliopancreatic diversion (BPD) has been widely used both as an effective treatment for morbid obesity and for the resolution/remission of its associated metabolic comorbidities. Our study aims to investigate the evolution of NAFLD and NASH after BPD intervention.

Materials and methods: 46 patients who underwent BPD between 2008 and 2013 with concomitant preoperative and postoperative liver biopsy were included in our study. Liver biopsy was classified according to the Steatosis-Activity-Fibrosis score (SAF score) proposed by Bedossa et al., and the NAFLD activity score (NAS) proposed by Kleiner. The SAF score evaluates steatosis from 0 to 3, the activity grade namely the unweighted addition of hepatocyte ballooning (0-2) and lobular inflammation (0-2) and fibrosis in stages from 0-4. The NAS score has a range from 0 to 8: NAS ≥ 3 is indicative of NASH. The most common non-invasive liver damage tests were calculated: NAFLD Fibrosis Score (NFS), AST/ALT ratio, AST to Platelet ratio (APRI), fibrosis 4 score (FIB4). The β-cell function was assessed from OGTT using a model describing the relationship between insulin secretion and glucose concentration as the sum of two components as demonstrated by Mari et al. Insulin resistance was measured by oral insulin sensitivity (OGIS), homeostasis model assessment of insulin resistance (HOMA-IR index) and quantitative insulin sensitivity check index (QUICKI). Biological, histological and clinical data were collected before and 5 years after surgery. P values were calculated from Wilcoxon signed-rank test analysis.

Results: At baseline patients age was 43(±9) years, with a BMI of 49.9(±6.6) kg/m2; 16 of them had type 2 diabetes mellitus (T2DM) with an average HbA1c of 50(±4) mmol/mol. After 5 years BMI was 31.9(±6.4) kg/m2 and only two subjects had still diabetes with an average HbA1c of 34(±14) mmol/mol. Insulin sensitivity indexes showed a significantly improvement after BPD; in particular HOMA-IR from 4.3(±2.5) to 2.7(±1.8) with P=0.011; OGIS from 360.3(±95.6) to 448.6(±69.8) with P=0.002. Total insulin secretion did not show a statistically significant difference after BPD. Also fasting insulin and fasting plasma glucose decreased significantly (P=0.023 and P=0.000 respectively). Regarding the predictive indices of NAFLD, we found a reduction of the NAFLD fibrosis score (pre-BPD -0.482±1.54/post-BPD -2.04±1.41 with P=0.000). We found also a statistically significant improvement of total cholesterol, HDL and LDL cholesterol and triglycerides.

Conclusion: Biliopancreatic diversion is a valuable treatment for NASH ameliorating the main metabolic, anthropometric and insulin sensitivity related variables. In fact, liver biopsies obtained before and at 5 years after surgery showed a clear improvement of NASH features.

figureao

Disclosure: M.F. Russo: None.

132

Triple therapy with pioglitazone/exenatide/metformin prevents hepatic fibrosis and steatosis in type 2 diabetes

O. Lavrynenko, M. Abdul-Ghani, M. Alatrach, C. Puckett, J. Adams, E. Cersosimo, N. Alkhouri, R.A. DeFronzo;

UTHSCSA, San Antonio, USA.

Background and aims: Patients with NAFLD and T2DM are at high risk of liver fibrosis. Pioglitazone and GLP1 RAs have shown efficacy against NAFLD. The EDICT trial compared the efficacy of Triple (Pioglitazone/ Exenatide/ Metformin) vs Conventional (Metformin/Glipizide/Insulin) Therapy in T2DM. The aim of the present study was to evaluate the effect of these two approaches on liver fibrosis scores (AST/ALT ratio, APRI, FIB-4, NFS) and hepatic fibrosis and steatosis using the FibroScan

Materials and methods: 144 newly diagnosed T2DM were randomized to receive Triple or Conventional Therapy to maintain HbA1c <6.5%. After 2 years baseline measurements and liver fibrosis scores were repeated; we also performed FibroScan to quantitate hepatic fibrosis and steatosis

Results: At baseline patients were well matched for age, BMI, HbA1c (8.8%) and LFTs. Neither therapy reduced any liver fibrosis score. Triple, but not Conventional, Therapy reduced the AST and ALT (p<0.001). The greatest AST and ALT reductions with Triple Therapy occurred in subjects in the highest AST and ALT tertiles at baseline. After 2 years, only 1 subject receiving Triple Therapy had a fibrosis score > 0, while 43% of Conventional Therapy subjects had a fibrosis score of F3/F4 (p<0.00001) (See Table). 87% of Conventional Therapy subjects had a steatosis score of S2/S3 vs 38% of Triple Therapy subjects (p<0.001) (See Table)

Conclusion: Both Triple (6.0%) and Conventional Therapy (6.7%) markedly reduced the HbA1c after 2 years, but only Triple Therapy (Pioglitazone/Exenetide/Metformin) reduced the AST and ALT. Liver fibrosis scores did not change in either group and were not useful in predicting response to therapy. Triple Therapy completely prevented fibrosis and reduced steatosis by >50 % vs Conventional Therapy.

figureap

Clinical Trial Registration Number: NCT01107717

Disclosure: O. Lavrynenko: None.

OP 23 Addressing potential new treatments of diabetic kidney disease

133

Once-weekly exenatide effects on EGFR slope and UACR as a function of baseline UACR: an EXSCEL trial post-hoc analysis

A.B. van der Aart1, L.E. Clegg2, R.C. Penland3, D.W. Boulton2, D. Sjöström4, R.J. Mentz5, R. Holman6, H.J.L. Heerspink1;

1Clinical Pharmacy and Pharmacology, University of Groningen, Groningen, Netherlands, 2Clinical Pharmacology & Quantitative Pharmacology, R&D, AstraZeneca, Gaithersburg, USA, 3Clinical Pharmacology & Quantitative Pharmacology, R&D, AstraZeneca, Boston, USA, 4Late-stage Development CVRM, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, 5Duke University and Duke Clinical Research Institute, Duke University School of Medicine, Durham, USA, 6Diabetes Trials Unit, University of Oxford, Oxford, UK.

Background and aims: GLP-1 RA effects on major kidney outcomes in unselected T2D patients at high cardiovascular (CV) risk are modest or neutral. However, GLP-1 RA may provide renal benefits in those at high risk of worsening kidney disease. We examined once-weekly exenatide (EQW) effects on eGFR slope and UACR change, as a function of baseline UACR, in a subset of EXSCEL participants.

Materials and methods: Of 14752 EXSCEL participants, eGFR slope was assessed in those with baseline UACR and ≥1 post-baseline eGFR (n=3503 [23.7%]) via mixed model repeated measures (MMRM) analysis (median follow-up 3.3 years). UACR percent change from baseline to first post-baseline measurement (median time 8.9 months) was assessed in those with baseline and ≥1 follow-up UACR (n=2828 [19.2%]) via ANCOVA of log-transformed UACR, with baseline UACR as a covariate.

Results: Participants with baseline UACR measurements were generally similar to the overall EXSCEL population, and balanced across treatment arms. EQW improved eGFR slope, compared with placebo, in patients with baseline UACR>100mg/g (+0.79 mL/min/1.73m2/year [95% CI 0.24-1.34]) and UACR>200mg/g (+1.32 mL/min/1.73m2/year 95% CI [0.57-2.06], but not at lower UACR thresholds (Figure A). No difference in EQW effect on eGFR was observed as a function of baseline eGFR, CV disease history, RAAS inhibitor use, or SBP. EQW, compared with placebo, reduced UACR by 28.2% in patients with baseline UACR>30 mg/g. This effect was consistent in subgroups with higher baseline UACR (baseline UACR>100 mg 22.5%; baseline UACR>200 mg 34.5%) (Figure B).

Conclusion: This post-hoc EXSCEL analysis suggests that EQW reduces UACR, with improvement in eGFR slope specifically in participants with elevated baseline UACR.

figureaq

Supported by: This analysis was funded by AstraZeneca

Disclosure: A.B. van der Aart: None.

134

Renoprotection with semaglutide and liraglutide: Direct or indirect effects?

J.F.E. Mann1, J.B. Buse2, T. Idorn3, L.A. Leiter4, R. Pratley5, S. Rasmussen3, T. Vilsbøll6, B. Wolthers3, V. Perkovic7;

1Friedrich Alexander University of Erlangen, Erlangen, Germany, 2University of North Carolina School of Medicine, Chapel Hill, USA, 3Novo Nordisk A/S, Søborg, Denmark, 4Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, Canada, 5AdventHealth Translational Research Institute, Orlando, USA, 6Steno Diabetes Center Copenhagen and University of Copenhagen, Copenhagen, Denmark, 7The George Institute, UNSW, Sydney, Australia.

Background and aims: The SUSTAIN 6 and LEADER cardiovascular (CV) outcome trials indicated that the glucagon-like peptide-1 analogues semaglutide and liraglutide may provide renal as well as CV benefits. This post hoc analysis investigated the degree to which the observed renoprotective effects could be mediated by HbA1c, systolic BP (SBP) and body weight (BW).

Materials and methods: SUSTAIN 6 (N=3297) and LEADER (N=9340) assessed CV, renal and safety outcomes for semaglutide and liraglutide vs placebo in patients with type 2 diabetes and high CV risk. A prespecified secondary outcome in these trials was a renal composite of new‑onset persistent macroalbuminuria, persistent doubling of serum creatinine, need for continuous renal-replacement therapy or death due to renal disease. We performed counterfactual mediation analyses of HbA1c, SBP and BW using absolute values at each trial visit. The direct contribution of semaglutide/liraglutide to time to first renal event was estimated assuming that the mediator values changed to those observed in the placebo group (from baseline to 2 and 3 years in SUSTAIN 6 and LEADER, respectively). In the adjusted model for HbA1c, both SBP alone and in combination with BW were included as confounders. Due to the limited number of events in SUSTAIN 6, 95% CIs could not be calculated.

Results: In SUSTAIN 6 and LEADER, the rate of a renal event was reduced by 36% (95% CI 12%,54%; p=0.005) and 22% (95% CI 8%,33%; p=0.003) in the semaglutide and liraglutide groups, respectively, versus placebo. HbA1c was estimated to mediate 26% and 25% (95% CI 7.1,67.3) of the benefits of semaglutide and liraglutide, respectively, whereas the contributions of SBP (22% and 9% [95% CI 2.8,22.7]) and BW (-8% and 9% [95% CI -7.9,35.5]) were smaller. In adjusted analyses, the contribution of HbA1c increased to 36% (SBP as confounder) and 30% (95% CI -4.5,81.1; SBP and BW as confounders) in the semaglutide and liraglutide groups, respectively.

Conclusion: The renal benefits of semaglutide and liraglutide appear mediated to a modest extent by changes in HbA1c, SBP and BW, and are therefore likely to be also driven by other, potentially direct, mechanisms.

Clinical Trial Registration Number: SUSTAIN 6 (NCT01720446) and LEADER (NCT01179048)

Supported by: Novo Nordisk A/S

Disclosure: J.F.E. Mann: Non-financial support; Abstract supported by Novo Nordisk.

135

Liraglutide improves obese-induced renal injury by alleviating uncoupling of glomerular VEGF-NO axis in obese mice

Y. Ma1, K. Li2, N. Hou1, F. Han3, X. Han1, X. Sun1;

1Department of Endocrinology, Affiliated Hospital of Weifang Medical University, Weifang, 2Department of Nephrology, Affiliated Hospital of Weifang Medical University, Weifang, 3Department of Pathology, Affiliated Hospital of Weifang Medical UniversityD, Weifang, China.

Background and aims: The uncoupling of glomerular vascular endothelial growth factor (VEGF) - nitric oxide (NO) axis is considered to be an important mechanism of obesity-related renal disease. We aimed to determine whether liraglutide, glucagon-like peptide-1 agonist, reduced urinary albumin excretion through improving uncoupling of glomerular VEGF-NO axis in diet-induced obese mice.

Materials and methods: Six-week male C57BL/6J mice were fed a normal-chow diet or a high-fat diet (HFD) for 24 weeks with or without liraglutide (200 μg/kg/d) by intraperitoneal injections for another 8 weeks. Blood biochemical and urinary albumin excretion were measured. The cortical tissue of the kidney and glomeruli were collected by using the sieving technique. Glomeruli VEGF and AMPK-eNOS pathway were determined by Western-blot. Glomeruli NO, renal heme oxygenase-1 activity and malondialdehyde were determined.

Results: Treatment of HFD mice with liraglutide reduced body weight gains (45.55±1.07g vs. 54.65±0.95g, p < 0.05), visceral fat (8.91±0.57g vs. 14.54±0.61, p < 0.05), perirenal fat (2.85±0.20g vs. 5.41±0.34g, p < 0.05) and significantly reduced FFA levels (1.02 ± 0.08 mmol/L vs. 1.71 ± 0.12 mmol/L, p < 0.05). Liraglutide significantly improved glucose tolerance and increased insulin sensitivity, which were impaired in HFD mice. The HFD mice had a higher 24h urinary albumin excretion levels compared to the control mice (45.72 ± 3.44 ug vs. 15.11± 1.09 ug, P < 05), which was reduced by 45.9% after liraglutide treatment (24.73 ± 2.12 ug vs. 45.72 ± 3.44 ug, P < 0.05). No significant difference in serum creatinine was found among the groups (P>0.05). Additionally, liraglutide significantly reduced glomerular VEGF levels and increased glomerular NO production (P< 0.05), indicating that the restoring of glomerular VEGF-NO axis. Treatment HFD mice with liraglutide reduced the glomerular hypertrophy and partly improved the increased proliferation of endothelial cells in glomeruli. Liraglutide treatment also enhanced glomerular AMPK and eNOS phosphorylation (P <0.05). HFD mice showed reduced heme oxygenase-1 activity and increased malondialdehyde levels, indicating the excessive oxidative stress (P < 0.05). However, liraglutide significantly enhanced renal heme oxygenase-1 activity and reduced malondialdehyde levels, recovering the oxidative-antioxidative balance (P < 0.05).

Conclusion: Liraglutide could reduce urinary albumin excretion and ameliorate renal injury by rectify uncoupling of the glomerular VEGF-NO axis and reduced abnormal proliferation of endothelial cells in HFD-induced obese mice. These findings provide a novel mechanism for protective effects of liraglutide on kidney in obesity.

Supported by: NSFC 81870593, 81600688, 81400829

Disclosure: Y. Ma: None.

136

Empagliflozin, either alone or in combination with linagliptin, restores autophagy and apoptosis regulators in the kidney in db/db diabetic mice

A. Korbut1, N. Muraleva2, Y. Taskaeva1, N. Bgatova1, V. Klimontov1;

1Research Institute of Clinical and Experimental Lymphology – Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 2Institute of Cytology and Genetics, Novosibirsk, Russian Federation.

Background and aims: Recent data indicate emerging role of autophagy and apoptosis in diabetic kidney disease. Therefore, these could be the therapeutic target in diabetes. Inhibitors of sodium-glucose cotransporter-2 (SGLT2) and dipeptidyl peptidase-4 (DPP4) are considered as promising agents in diabetic nephropathy, but little is known about the mechanisms of their protective activity. Thus, our study assessed the effects of SGLT2 inhibitor empagliflozin, either alone or in combination with DPP4 inhibitor linagliptin, on renal expression of autophagy and apoptosis regulators in a model of type 2 diabetes.

Materials and methods: Male 8-week-old db/db mice (BKS.Cg-Dock7m+/+Leprdb/J) were treated by vehicle, empagliflozin (10 mg/kg) or combination of empagliflozin and linagliptin (10 mg/kg of each) for 8 weeks. Non-diabetic heterozygous db/+ mice were acted as control. Plasma glucose, fructosamine, glycated albumin, insulin, leptin, creatinine, urinary albumin to creatinine ratio (UACR) and body composition were assessed at Week 0 and 8 of experiment. Renal structural changes were analyzed quantitatively from the light and transmission electron microscopy (TEM). LAMP-1, an autophagy marker, caspase-3 and Bcl-2, the apoptotic markers, were evaluated in renal cortex by western blot. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes in TEM images.

Results: The db/db mice were obese and hyperglycemic and demonstrated substantially elevated plasma leptin and insulin and increased fat mass (all p<0.0001). Plasma glucose, fructosamine, glycated albumin, and UACR remained elevated throughout the experiment in the vehicle group, but decreased in empagliflozin and empagliflozin-linagliptin-treated animals (all p<0.05). Empagliflozin, either alone or in combination with linagliptin, attenuated mesangial expansion, thickening of glomerular basement membrane and effacement of podocyte foot processes. Vehicle-treated db/db mice demonstrated higher renal expression of caspase-3 and decreased LAMP-1 and Bcl-2 as compared to db/+ mice. Weaker glomerular staining for beclin-1 and LAMP-1, and lower Vv of autophagosomes, autolysosomes and lysosomes in podocytes was revealed (all p<0.05). There was increase in Vv of podocyte autophagosomes, autolysosomes and lysosomes and areas of glomerular beclin-1 and LAMP-1 under empagliflozin and empagliflozin-linagliptin treatment (all p<0.05 vs. vehicle). Renal Bcl-2 was restored in actively treated animals (all p<0.05). Besides, LAMP-1 expression was enhanced in empagliflozin group (p=0.03). The protein content of caspase-3 was decreased significantly in the combination group only (p=0.008).

Conclusion: The data demonstrate that empagliflozin, either alone or in combination with linagliptin, restores autophagy and suppresses apoptosis in the kidney in a model of type 2 diabetes. These effects could contribute to preservation of glomerular structure and mitigation of podocyte injury. The data provide further explanation of renal protective effect of SGLT-2 and DPP-4 inhibitors in diabetes.

Supported by: RFMEFI62119X0023

Disclosure: A. Korbut: None.

137

Inhibition of lysine63 ubiquitination prevents in vitro and in vivo the progression of renal fibrosis in diabetic nephropathy

P. Pontrelli1, R. Menghini2, F. Conserva1, V. Casagrande2, M. Rossini1, A. Stasi1, C. Divella1, C. Cinefra1, S. Simone1, G. Pertosa1, A. Gallone3, M. Federici2, L. Gesualdo1;

1Department of Emergency and Organ Transplantation, University of Bari Aldo Moro, Bari, 2Department of Systems Medicine, University of Rome Tor Vergata, Rome, 3Dept. of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Bari, Italy.

Background and aims: Our group previously demonstrated that in Diabetic Nephropathy (DN) an accumulation of lysine63 (K63)-ubiquitinated proteins at tubular level is involved in the progression of renal damage, in particular renal fibrosis (PMID: 27881486; 29806072; 31388051). Current treatments do not provide complete renoprotection and targeted therapies that completely prevent fibrosis or delay its progression are still lacking. Aim of the present study was to evaluate the renoprotective effect of specific drugs and their combinations, including an inhibitor of K63 ubiquitination (K63Ub) and/or an anti-hypertensive agent, in vitro and in vivo in a murine model of DN.

Materials and methods: All in vitro experiments were performed on immortalized Proximal Tubule Epithelial Cells (HK2) pre-incubated with a specific inhibitor of K63Ub and/or with the ACE-inhibitor Ramipril. All in vivo experiments were performed on streptozotocin (STZ)-treated DBA/2J mice. Accumulation of K63 ubiquitinated proteins along with α-sma expression, as indicator of epithelial-to-mesenchymal transition (EMT), were analyzed through immunofluorescence and western blotting. In mice, K63Ub was evaluated by IHC, while renal fibrosis was evaluated by Sirius red and Collagen III expression. Urinary albuminuria was measured by ELISA.

Results: Both the K63Ub inhibitor alone and the association of the specific K63Ub inhibitor with Ramipril were able to block hyperglycemia-induced EMT in HK2 cells by significantly reducing α-sma expression (p<0.05) when compared with ramipril alone. To demonstrate the efficacy of these drug alone and/or in combinations in reducing the progression of renal damage in vivo we firstly confirmed the increased accumulation of K63 Ub proteins in DBA/2J STZ-treated mice (p=0.01). Interestingly, increased K63Ub in diabetic mice was also associated to increased tubular-interstitial fibrosis (p<0.05). Treatment of STZ-mice with the specific K63Ub inhibitor was able to reduce both K63Ub proteins accumulation and renal fibrosis, evaluated on kidney samples by IHC against Collagen III (p≤0.05) and by Sirius Red staining (p≤0.05) when compared to both untreated mice and mice treated with Ramipril alone. However, treatment with the K63Ub inhibitor alone did not reduce albuminuria (STZ-mice: 561.29±390.56; STZ+K63Ubinhibitor: 724.25±690.89; p=n.s.), while the drug combination including the specific K63Ub inhibitor and Ramipril, significantly reduced both K63Ub-related fibrosis (p≤0.05) and albuminuria (p=0.01), demonstrating an addictive and synergic effect of these molecules when used in combination.

Conclusion: We proposed and patented a novel combination of drugs that ameliorates both fibrosis and proteinuria in a model of DN. Novel treatment regimens could represent an important goal for reducing the incidence of ESRD related to diabetes complication.

Supported by: H2020-BEAtDKD-IMI2-2015-05-02

Disclosure: P. Pontrelli: None.

138

Nox5 enhances the progression of diabetic kidney disease independent of renox (Nox4)

J.C. Jha1, S. Urner2, A. Dai1, M. Cooper1, K. Jandeleit-Dahm2;

1Monash University, Melbourne, Australia, 2German Diabetes Center, Leibniz Center for Diabetes Research, Düsseldorf, Germany.

Background and aims: Renal oxidative stress plays an important role in the pathogenesis of diabetic kidney disease (DKD). Evidences suggest pathogenic roles for pro-oxidant enzymes Nox4 and Nox5 in animal models of DKD. Nox5 is present in mans but not in mice/rats and it appears that Nox5 could be a main culprit in the context of human DKD. Therefore, we aimed to examine the roles of Nox5 versus Nox4 and their relative contribution to renal pathology in DKD.

Materials and methods: We examined the expression of Nox5 and Nox4 as well as their interaction and ROS production in human kidney biopsies. In vitro, certain human renal cells were knockdown for Nox4 and Nox5 and were exposed to high glucose. In vivo, we examined the effect of Nox5 in the absence of Nox4 expression in STZ- diabetic mouse model. We developed rabbit models of DKD and Nox5KO rabbits.

Results: Increased expression of Nox5 and enhanced level of ROS was seen in human diabetic kidney biopsies compared to non-diabetic kidney. Nox5 shows the highest upregulation in human renal cells exposed to high glucose in comparison to other Nox isoforms. Silencing of Nox5 attenuated high glucose induced increased expression of markers of fibrosis, inflammation and putative elements via reduction in ROS formation. Nox5 appears to be upstream of Nox4 and that Nox5 inhibition downregulates Nox4, but not vice versa. In vivo, cell specific expression of Nox5 in both Nox4 KO and GKT137831 (a renal Nox4 inhibitor) treated diabetic mice demonstrated a 30-40% increase in albuminuria, mesangial expansion, renal fibrosis and inflammation as well as enhanced ROS production in comparison to diabetic mice not expressing Nox5. In addition, both high fat feeding and alloxan induced diabetic rabbits showed increased renal Nox5 expression in association with increased renal injury along with upregulation of CTGF, fibronectin and MCP-1 as well as enhanced renal ROS production.

Conclusion: These findings provide evidence that Nox5 plays a superior pathogenic role in DKD in comparison to Nox4. Therefore, targeting Nox5 may represent a better approach to treat and prevent DKD in human.

Supported by: NHMRC

Disclosure: J.C. Jha: None.

OP 24 Glucagon and hormones beyond

139

Identification of a gut-derived LEAP2 fragment as a novel insulin secretagogue

C.A. Hagemann1,2, C. Zhang2, T. Jorsal1, K.T. Rigbolt2, M. Falkenhahn3, S. Theis3, M. Christensen1,4, T. Vilsbøll1,5, N. Vrang2, J. Jelsing2, F.K. Knop1,5;

1Center for Clinical Metabolic Research, Gentofte Hospital, Hellerup, Denmark, 2Gubra ApS, Hørsholm, Denmark, 3Sanofi-Aventis, Frankfurt, Germany, 4Department of Clinical Pharmacology, Bispebjerg Hospital, Bispebjerg, Denmark, 5Steno Diabetes Center Copenhagen, Gentofte, Denmark.

Background and aims: Roux-en-Y gastric bypass (RYGB) surgery often leads to rapid remission of type 2 diabetes along with large and sustained body weight reduction. Unknown peptides with anorexigenic and/or anti-diabetic effects may contribute to these beneficial effects. In the present study, liver-enriched antimicrobial peptide 2 (LEAP2) was identified as a novel putative anti-diabetic therapeutic target by analysing gene expression in human enteroendocrine cells sampled before and after RYGB.

Materials and methods: Twenty morbidly obese individuals underwent upper enteroscopy with gut mucosal biopsy retrieval three months before and after RYGB in addition to a perioperative gut biopsy. Enteroendocrine cells were immunohistochemically identified by chromogranin A, isolated by laser capture microdissection and processed using next-generation RNA sequencing. The distribution of LEAP2 mRNA and peptide expression in mucosal biopsies were investigated using in situ hybridization and immunohistochemistry. Finally, the effect of LEAP2 on glucose-stimulated insulin secretion (GSIS) was evaluated in isolated human pancreatic islets and during a graded glucose infusion in a double-blinded crossover study in healthy individuals.

Results: LEAP2 mRNA expression in enteroendocrine cells was significantly upregulated in obese individuals after RYGB. In situ hybridization revealed that LEAP2 mRNA was expressed in the epithelial lining, whereas immunohistochemistry demonstrated a distinct labelling of enteroendocrine cells in the gut mucosa. Interestingly, an endogenous LEAP2 fragment significantly increased GSIS in human pancreatic islets, but in the chosen dose, it did not affect insulin secretion during a graded glucose infusion in healthy individuals.

Conclusion: We conclude that LEAP2 is significantly upregulated in human enteroendocrine cells after RYGB and that an endogenous LEAP2 fragment increases GSIS in human pancreatic islets. This suggest that gut-derived LEAP2 may contribute to the beneficial metabolic effects of RYGB.

Clinical Trial Registration Number: NCT03093298

Supported by: Innovation Foundation Denmark, Sanofi, Gubra

Disclosure: C.A. Hagemann: Employment/Consultancy; Christoffer A Hagemann is employed as an industrial PhD student in a joint venture between Gentofte Hospital, University of Copenhagen and Gubra. Grants; Sanofi, Gubra, Innovation Foundation Denmark. Lecture/other fees; Gubra and Sanofi (Filip K Knop). Stock/Shareholding; Niels Vrang and Jacob Jelsing are owners of Gubra.

140

Regulation of substrate choice contributes to the regulation of glucagon secretion from alpha cells in response to glucose

S.L. Armour1, M.V. Chibalina2, B. Davies3, P. Rorsman2, J.G. Knudsen1;

1Department of Biology, Univiersity of Copenhagen, Copenhagen, Denmark, 2Radcliffe department of Medicine, OCDEM, Oxford, UK, 3Wellcome centre for human genetics, University of Oxford, Oxford, UK.

Background and aims: Type 2 diabetes is a metabolic disorder resulting in dysregulation of both insulin and glucagon secretion. Whilst the metabolic control of insulin secretion from pancreatic β-cells is fairly well established, the regulation of glucagon secretion from α-cells remains debated. α-cells rely on fatty acid oxidation for ATP production when glucose is low, and oxidise only 15-20% of utilised glucose irrespective of extracellular concentrations. β-oxidation in α-cells is decreased in response to increased glucose concentrations. Unlike β-cells, α-cells express high levels of pyruvate dehydrogenase (PDH) kinase 4 (PDK4). PDK4 acts by phosphorylating PDH and this favours β-oxidation over glucose metabolism. Here we investigate the role of PDK4 and substrate choice in the regulation of glucagon secretion.

Materials and methods: Islets were isolated from wild type C57BL/6J (WT), α-ell specific PDK4 overexpressing mice (αPDK4KI) or littermate Controls carrying either Pdk4, Cre or neither inserts (Con). Live-cell imaging was used to measure α-cell specific ATP/ADP ratios in intact islets using viral infection with the fluorescent sensor PercevalHR under the control of the preproglucagon promoter. Mice expressing the calcium probe Gcamp3 specifically in α-cells was used to study calcium oscillations in α-cells from intact islets. Lastly, glucagon secretion was investigated. All experiments were performed in a physiological Krebs ringer buffer with the addition of glucose and/or a physiological relevant mix of fatty acids (FA) corresponding to the circulating concentrations of lineoleate, palmitate and oleate in WT mice.

Results: Increasing glucose concentration from 1 to 5mM glucose in the presence of 0.35mM FA in WT islets reduced intracellular ATP/ADP in α-cells by 12.7% (100±4.21% vs 87.29±4.09%; p<0.0001). This was associated with reduced frequency of calcium oscillations (1.99±0.19 vs 1.09±0.21 spikes/min; p= 0.0002) and glucagon secretion (0.41±0.074 vs 0.17±0.026% content; p=0.0017). When islets were exposed to FA, calcium oscillations increased by 27% relative to what was observed in the presence of glucose alone (1.56±0.21 vs 1.98±0.19 spikes/min; p= 0.0247). This effect did not correlate with an increase in glucagon secretion. To understand if entry of pyruvate into the TCA cycle was required for the observed effect of glucose on α-cell ATP production, we investigated dynamic changes in the ATP/ADP in αPDK4KI mice. Con islets responded in a similar way to WT islets, whereby increasing glucose from 1 mM to 5 mM in the presence of FA reduced intracellular ATP/ADP by 14.7% (100±4.21% vs 85.31±4.74%; p<0.0047). No such decrease was observed in α-cells from αPDK4KI islets (100±2.60% vs 97.34±3.97%; p=0.98). In addition, glucagon secretion was reduced by 30% when glucose was elevated from 1 to 5mM glucose in Con islets (1.52±0.47 vs 1.01±0.22% content; p=0.034) but not in αPDK4KI mice (1.60±0.50 vs 1.43±0.58 % content; p=0.092).

Conclusion: These data suggest that α-cells generate much of their ATP under hypoglycaemic conditions by β-oxidation of FA and that increasing glucose inhibits ATP production by inhibiting endogenous fatty acid oxidation. This highlights PDK4 and PDH as key regulators of glucagon secretion through regulation of substrate choice in the α-cell.

Supported by: Novo Nordisk Fonden

Disclosure: S.L. Armour: None.

141

12-hour glucagon infusion stimulates adipocyte lipolysis and inflammation in vivo in humans

X. Chen, L. Norton, R. DeFronzo, D. Tripathy;

The University of Texas Health Science Center at San Antonio, San Antonio, USA.

Background and aims: Combined glucagon and GLP-1 agonists are being proposed as new therapeutic agents for the treatment of obesity and diabetes. However, the long-term effect of hyperglucagonemia on adipose tissue metabolism and glucose homeostasis in vivo in humans is unclear. The aim of the present study was to evaluate the effect of 12-hour glucagon infusion on hepatic glucose production (HGP) and adipocyte metabolism in healthy individuals.

Materials and methods: Eight subjects with normal glucose tolerance (NGT, 5M/3F, age=35±5, BMI = 24±1) participated in 2-hour (75 gram) OGTT. Subsequently, subjects returned for visit 2 when they received a 12-hour (6 PM to 6 AM) glucagon infusion (6 ng/kg/min) with 3-3H-glucose and 14-C glycerol infusion followed by subcutaneous adipose tissue biopsy at 6 AM. Within 4-8 weeks of visit 2, subjects returned for a repeat study with infusion (6 PM to 6 AM) of normal saline.

Results: Plasma glucagon increased from 57±3 to 219±21 pg/ml. Plasma glucose increased transiently after the start of glucagon and declined to baseline levels at 6 AM. Plasma insulin levels increased significantly following glucagon compared to normal saline (20±7 vs 8±3 mU/L, p<0.05) and remained elevated at 6 AM. Basal HGP (3.2±0.1 vs 2.9±0.1 mg/kg/min, p<0.01) and fasting plasma FFA concentrations (0.70±0.1 vs 0.39±0.1 mM, p<0.01) were increased after 12-hour glucagon infusion despite increased plasma insulin levels, indicating severe hepatic and adipocyte insulin resistance. Lipolysis-related genes expression in adipose tissue biopsy were upregulated following 12-hour glucagon infusion (ATGL, 1.14±0.07 vs 0.77±0.03; HSL, 1.17±0.03 vs 0.9±0.13; MGL, 1.2±0.04 vs 0.82±0.2, all p<0.05). Plasma concentration of inflammatory markers were also increased after 12-hour glucagon infusion compared to normal saline (IL-1β, 0.65±0.05 vs 0.49±0.05 pg/mL; TNF-α, 2.03±0.23 vs 1.75±0.17 pg/mL, both p<0.05).

Conclusion: Collectively, these findings indicate that prolonged (12-hour) physiologic hyperglucagonemia causes marked hepatic and adipocyte insulin resistances, stimulates lipolysis and increases plasma FFA, and induces adipocyte and systemic inflammation.

Disclosure: X. Chen: None.

142

Hepatic steatosis and glucagon resistance develope in parallel resulting in hyperglucagonaemia and hyperaminoacidaemia

M. Winther-Sørensen1,2, K.D. Galsgaard1,2, A. Santos3, J. Pedersen1, A.S. Hassing2, M. Dall2, J.T. Treebak2, S.A.S. Kjeldsen1,2, F.K. Knop4, M.P. Werge5, P.L. Eriksen6, H. Vilstrup6, L. Gluud5, J.J. Holst1,2, N.J. Wewer Albrechtsen1,3;

1Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 2Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, 3Center for Protein Research, University of Copenhagen, Copenhagen, 4Center for Clinical Metabolic Research, University of Copenhagen, Hellerup, 5Gastrounit, University of Copenhagen, Hvidovre, 6Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.

Background and aims: Glucagon regulates hepatic glucose production, and increased glucagon signalling contributes to type 2 diabetes. In the liver-α cell axis, plasma levels of amino acids (AA) are regulated by glucagon-dependent ureagenesis, while AA stimulate glucagon secretion from the α cells. We hypothesised that non-alcoholic fatty liver disease (NAFLD) may impair hepatic glucagon actions, resulting in decreased ureagenesis and hyperaminoacidaemia and subsequent hyperglucagonaemia.

Materials and methods: We measured plasma concentrations of alanine and glucagon in 9 healthy controls and 35 patients with biopsy-verified NAFLD (steatosis: n=18; non-alcoholic steatohepatitis (NASH): n=17). We also evaluated urea formation in primary hepatocytes from ob/ob mice (n=3) and AA clearance in vivo in mice with hepatic steatosis (n=9-12), mice treated with a glucagon receptor antagonist (GRA, 25-2648, 100 mg/kg, n=14), and transgenic mice with 95% reduction in α cells (n=15). Finally, we performed RNA sequencing on livers from mice with hepatic steatosis (n=3) and glucagon receptor knock-out (Gcgr-/-) mice (n=5). Results are presented as mean±SEM.

Results: The glucagon-alanine index (the product of fasting levels of glucagon and alanine) was increased in patients with steatosis (3.8±0.5 pmol/l×mmol/l, p=0.02) and NASH (4.1±0.3 pmol/l×pmol/l, p=0.001) compared to controls (1.6±0.3 pmol/l×mmol/l, one-way ANOVA). Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed AA compared to control hepatocytes (AUC0-120 min: 35.5±4.0 vs. 51.5±2.8 nmol/μg protein×min, one-way ANOVA, p=0.04). Upon i.p. administration of mixed AA, AA clearance (reflected by incremental AUC (iAUC)), tended to be lower in mice with hepatic steatosis (iAUC0-20 min: 6.8±0.7 vs. 5.5±0.3 min×mmol/l, unpaired t-test, p=0.1). AA clearance was reduced in GRA vs. vehicle-treated mice (iAUC0-20 min: 33.1±2.8 vs. 27.2±2.1 min×mmol/l, one-way ANOVA, p=0.1) concomitantly with reduced production of urea (iAUC0-20 min: 25.3±3.7 vs. 39.8±3.9 min×mmol/l, one-way ANOVA, p=0.04). Likewise, mice lacking endogenous glucagon (loss of α cells) had reduced AA clearance (iAUC0-20 min: 46.9±4.2 vs. 39.4±5.0 min×mmol/l, one-way ANOVA, p=0.1) and lower plasma levels of urea (iAUC0-20 min: 23.6±5.4 vs. 37.5±2.5 min×mmol/l, one-way ANOVA, p=0.04). Transcriptomic comparison of mice with hepatic lipid accumulation and Gcgr-/- mice revealed an overlap of several down-regulated genes responsible for AA catabolism (Cps1Slc7a2, and Slc38a2) (FDR-corrected multiple t-tests, p<0.05 for all).

Conclusion: Our study suggests that hepatic steatosis and glucagon resistance develop in parallel resulting in hyperglucagonaemia and hyperaminoacidaemia. Disruption of the liver-α cell axis leading to hyperglucagonaemia may therefore link NAFLD to type 2 diabetes.

Supported by: NNF Tandem Program, NNF Project Endocrinology and Metabolism, NNF Excellence Emerging Investigator

Disclosure: M. Winther-Sørensen: None.

143

Inappropriate glucagon response is associated with early-postprandial glucose excursions in Japanese patients with type 1 diabetes

A. Ito, I. Horie, N. Abiru, A. Kawakami;

Endocrinology and Metabolism, Nagasaki University Hospital, Nagasaki, Japan.

Background and aims: Some of the patients with type 1 diabetes (T1D) develop severe glycemic variability leading to brittle diabetes which is associated with significant mortality and poor quality of life. The variability is believed to be tied with the duration of diabetes resulting in a complete depletion of endogenous insulin secretion. Recent studies indicates that α-cells might be potentially effect to the instability of glucose, however, little is known about the influence of α-cell on postprandial hyperglycemia. The study aimed to evaluate whether the glucagon response after meals could influence the postprandial glucose levels in the patients with T1D.

Materials and methods: We enrolled 34 patients with T1D, and 23 patients with type 2 diabetes (T2D) as a control. All participants underwent to a liquid a mixed meal tolerance test (MMTT) after an overnight fasting. Blood samples were drawn measuring plasma glucose, C-peptide and glucagon before and 30, 60, and 120min after the meal. T1D patients were treated with two-thirds of the dose of bolus insulin calculated using their insulin-to-carbohydrate ratio for the meal while all participants received basal insulin as per usual.

Results: Inappropriate glucagon secretions with a paradoxical increase after meals were observed in both T1D and T2D and glucagon levels elevated peaking 30min during MMTT. The glucagon levels (pg/mL) at 30min in T1D were significantly lower than those in T2D (48±32 vs. 65±45, p=0.015). In T1D, the changes in glucose levels from fasting to 30min were positively correlated with those in glucagon (ρ=0.4, p=0.019), but not with those in C-peptide. By contrast, the changes in glucose levels from fasting to 120min were negatively correlated with those in C-peptide, but not with those in glucagon. There were no significant differences in the glucagon concentrations at each time-point between the three respective divisions; sex (female or male), fasting C-peptide levels (<0.1 or ≥0.1 ng/mL), and duration from clinical onset of T1D (<5 or ≥5 years).

Conclusion: The early-phase postprandial hyperglucagonemia was observed in T1D and associated with postprandial glucose excursions, regardless of residual β-cell function and diabetes duration.

Clinical Trial Registration Number: UMIN-CTR000020156

Disclosure: A. Ito: None.

144

Neprilysin inhibition increases plasma glucagon concentrations in humans with possible implications for hepatic amino acid metabolism

S.A.S. Kjeldsen1, S. Zraika2, S. Mongovin3, L.H. Hansen4, D. Terzic5, P.D. Mark5, P. Plomgaard5, J.P. Gøtze5, The Liver-Alpha-Cell Axis Group, J.E. Hunt1, M.M. Rosenkilde1, G.H. Goossens6, E.E. Blaak6, J.J. Holst1, N.J. Wewer Albrechtsen1;

1Department of Biomedical Sciences, Copenhagen University, Copenhagen, Denmark, 2Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, USA, 3Medical Research Service of the Veterans Affairs Puget Sound Health Care Center, University of Washington, Seattle, USA, 4Copenhagen University, Copenhagen, Denmark, 5Department of Clinical Biochemistry, Copenhagen University, Copenhagen, Denmark, 6Department of Human Biology, Maastricht University, Maastricht, Netherlands.

Background and aims: Glucagon (gcg) regulates hepatic glucose and amino acid (AA) metabolism, and increased plasma gcg levels (hyperglucagonemia) contribute to diabetic hyperglycemia. Studies in pigs have suggested neprilysin (NEP), a metalloprotease, to metabolize exogenous gcg. We hypothesized that NEP contributes to gcg degradation.

Materials and methods: Healthy males were investigated during a mixed meal after a single dose of a NEP-inhibitor/angiotensin II receptor blocker (194 mg sacubitril / 206 mg valsartan), a DPP-4 inhibitor (sitagliptin, 2x100mg), these combined, or the meal alone (n=9 or 10). Long-term effects of sacubitril/valsartan were investigated in obese individuals (n=7) receiving sacubitril/valsartan for 8 weeks (194 mg sacubitril / 206 mg valsartan per day). To test whether NEP degrades gcg and diminishes its signaling, we performed mass-spectrometry and assessed gcg degradation products in cells transfected with the gcg receptor (gcgr). Finally, we investigated different mouse strains to evaluate mechanisms responsible for the changes in amino acid metabolism upon NEP inhibition (sacubitril, 0.7 nmol/g).

Results: In healthy males, sacubitril/valsartan increased postprandial gcg levels 2.7-fold (iAUC0-240 min, paired t-test, P=0.005) and this was not altered by the addition of sitagliptin (iAUC0-240 min, unpaired t-test, P=0.28). Sacubitril/valsartan lowered postprandial AA levels (tAUC0-240 min, paired t-test, P=0.01). In obese individuals, 8 weeks sacubitril/valsartan treatment increased fasting gcg levels (paired t-test, P=0.02) with no difference in fasting AA levels (paired t-test, P=0.63). NEP induced gcg degradation identified by mass spectrometry showed that NEP cleaves gcg in the C-terminus and that the resulting gcg fragments were unable to activate the gcgr. In non-sedated C57BL/6JRj female mice (n=9) NEP inhibition increased gcg levels (mixed effects analysis, t=10 min, P=0.02) and tended to increase AA disappearance (mixed effects analysis, t=15 min, P=0.08) and urea formation (iAUC0-180 min, unpaired t-test, P=0.08) after an AA challenge. A gcgr antagonist (Novo Nordisk; 25-2648, 100 mg/kg) abolished the increase in urea formation observed with sacubitril alone (iAUC0-180 min, unpaired t-test, P=0.01). Sacubitril increased exogenous gcg levels 1.9-fold (iAUC0-60 min, unpaired t-test, P<0.002, n=10) after a single injection of gcg (96 ng/g). In NEP deficient mice (n=10), fasting plasma urea (unpaired t-test, P=0.003) but not fasting gcg levels (unpaired t-test, P=0.57) were increased compared to controls.

Conclusion: NEP degrades gcg and thus inhibitors of NEP may result in hyperglucagonemia with potential metabolic perturbations on hepatic AA metabolism.

Clinical Trial Registration Number: 31074791 and 27542885

Supported by: Novo Nordisk Foundation

Disclosure: S.A.S. Kjeldsen: None.

OP 25 Incretin based therapies

145

Six-day subcutaneous GIP infusion increases glycaemic time in range in patients with type 1 diabetes

S.M.N. Heimbürger1,2, B. Hoe2,3, C.N. Nielsen2, N.C. Bergmann2, B. Hartmann4,4, J.J. Holst4,3, J. Størling5,6, T. Vilsbøll5,2, T.F. Dejgaard2, M.B. Christensen7,8, F.K. Knop2,5;

1Type 1 Diabetes, Steno Diabetes Center Copenhagen, Gentofte, 2Gentofte Hospital, University of Copenhagen, Hellerup, 3Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, 4University of Copenhagen, Copenhagen, 5Steno Diabetes Center Copenhagen, Gentofte, 6Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, 7Gentofte Hospital, University of Copenhagen, Gentofte, 8Department of Clinical Pharmacology, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark.

Background and aims: We have previously reported a glucagonotropic effect of glucose-dependent insulinotropic polypeptide (GIP) during insulin-induced hypoglycaemia in patients with type 1 diabetes (T1D). This prompted us to investigate the effect of a 6-day s.c. GIP infusion on glycaemic levels in patients with T1D

Materials and methods: In a randomised, placebo-controlled, double-blinded, crossover study, 20 men with T1D (age [mean ± SD] 26 ± 8 years, BMI 23.8 ± 1.8 kg/m2, HbA1c 51 ± 10 mmol/mol), diabetes duration 9.1 ± 3.9 years, plasma C-peptide < 200 pmol/l, underwent double-blinded continuous glucose monitoring (CGM) and 2 × 6 days with continuous s.c. GIP (6 pmol/kg/min) and placebo (saline) infusion, respectively, with an interposed 7-day washout period.

Results: GIP significantly increased daytime (06:00-23:59) time in range (3.9-7.8 mmol/l) by [mean ± SEM] 160 ± 74 min/day (p = 0.04). There were no significant differences in daytime time below range (<3.9 mmol/l, p = 0.8), time above range (>10 mmol/l, p = 0.19) (Figure), mean glucose or hypoglycaemic events (assessed by CGM). Compared to placebo, GIP increased hepatic fat content by 12.6 ± 4.2 percentage points (assessed by FibroScan®) (p = 0.007) , decreased 24-hour systolic and diastolic blood pressure by 5.8 ± 2.6 (p = 0.04) and 3.1 ± 1.3 mmHg (p = 0.03), respectively, and increased heart rate by 4.4 ± 2.0 beats per minute (p = 0.04) (Figure).

Conclusion: Compared to placebo, a 6-day continuous s.c. GIP infusion in patients with T1D increased glucose daytime time in range, hepatic fat content and heart rate, while decreasing blood pressure.

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Clinical Trial Registration Number: NCT03734718

Supported by: The Leona M. and Harry B. Helmsley Charitable Trust; #2018PG-T1D037

Disclosure: S.M.N. Heimbürger: Lecture/other fees; AstraZeneca.

146

Effects of tirzepatide, a novel dual GIP and GLP-1 receptor agonist, on metabolic profile in patients with type 2 diabetes

V. Pirro1, J.A. Willency1, J.M. Wilson1, K.D. Roth1, Y. Lin1, K.A.L. Collins1, G. Ruotolo1, A. Haupt1, C.B. Newgard2, K.L. Duffin1;

1Eli Lilly and Company, Indianapolis, 2Duke Molecular Physiology Institute, Durham, USA.

Background and aims: In a Phase 2 trial, tirzepatide (TZP) dose-dependently reduced HbA1c, body weight, and improved markers of insulin sensitivity in patients with type 2 diabetes. Branched chain amino acids (BCAA) are emerging biomarkers of obesity and insulin resistant state suggested to promote the accumulation of hepatic and muscle lipids and cardiac hypertrophy. To understand changes in fasting BCAAs levels and related metabolites with TZP, metabolomics profiling was conducted.

Materials and methods: Patients (N=316) were randomised to receive weekly subcutaneous TZP (1, 5, 10, 15 mg), dulaglutide 1.5 mg (DU), or placebo for 26 weeks. Fasted serum collected at baseline, 4, 12 and 26 weeks was extracted, diluted, and analysed by targeted metabolomics using a Sciex Triple Quadrupole 6500 mass spectrometer. Targeted metabolites were separated over 30 minutes by HILIC chromatography and detected by multiple reaction monitoring. Data were analysed using a mixed-model repeated measure statistical approach.

Results: At 26 weeks, a total of 45 metabolites, including acylcarnitines, amino acids and related metabolites, were significantly modulated by TZP 15 mg treatment compared to baseline, while 125 metabolites remained largely unchanged. TZP 15 mg also caused a temporary increase in acetylcarnitine (adjusted p=0.04 at week 12), but levels returned to baseline at 26 weeks. BCAAs, isovalerylcarnitine (a byproduct of leucine catabolism), glutamate, glycine, alanine and 2-hydroxybutarate (2-HB) levels were affected by TZP dose-dependently (Table). Leucine, isoleucine and valine significantly reduced from baseline with TZP 15 mg compared with placebo (fold change of -1.27, -1.23, and -1.16; adjusted p<0.01) and compared with DU (fold change of -1.22, -1.21, and -1.16; adjusted p<0.05). BCAA levels positively correlated with HOMA-IR for isoleucine (Spearman Correlation coefficient of 0.58; p=0.0011), leucine (0.49; p=0.009) and valine (0.46; p=0.015).

Conclusion: Our data reinforce previous observations that a dual GIP and GLP-1 receptor agonist has a greater impact on insulin sensitivity than a selective GLP-1 receptor agonist, thus helping to explain the superior effect on overall glycaemia. Future studies are needed to better understand the impact of BCAAs and related metabolites on cardiometabolic diseases and how TZP-driven changes compare to other forms of weight loss intervention such as bariatric surgery.

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Clinical Trial Registration Number: NCT03131687

Supported by: Eli Lilly and Company

Disclosure: V. Pirro: Employment/Consultancy; Eli Lilly and Company. Stock/Shareholding; Eli Lilly and Company.

147

Reduction of cardiovascular events by GLP-1 receptor agonists is explained by HbA1c reduction

M. Roosimaa1,2, A. Jõgis2;

1Endocrinology unit, North Estonia Medical Centre Foundation, Tallinn, 2University of Tartu, Tartu, Estonia.

Background and aims: Since FDA issued guidance for the assessment of cardiovascular (CV) safety of new antidiabetic medications, multiple cardiovascular outcome trials (CVOT) with sodium-glucose transport protein 2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) have provided an unexpected reduction in CV risk. Both medication classes have reduced HbA1c level compared to the use of placebo, differentiating them from trials with other agents such as dipeptidyl peptidase-4 inhibitors (DPP4i) where glycemic equipoise and no risk reduction was observed. Even though in DCCT and UKPDS trials better glycemic control did result in a lower rate of CV complications after prolonged follow-up, this finding has not been confirmed in ADVANCE, ACCORD and VADT trials where older patients with already established CV risk factors, a population more similar to patients in current CVOTs, was included. Therefore, the importance of glycemic control as a mediator of risk reduction in CVOTs has been questioned. While there is convincing data that better glycemic control is not mediating risk reduction with SGLT2i, it is not confirmed nor rebutted for GLP-1RA. This meta-analysis tries to assess whether better glycemic control can explain decreased CV risk seen with GLP-1RA.

Materials and methods: Absolute risk of primary outcome, HbA1c and number of patients at risk was extracted for both GLP-1RA and placebo groups at multiple time points from each published GLP-1RA trial (ELIXA, LEADER, SUSTAIN-6, PIONEER 6, Harmony Outcomes, EXSCEL, REWIND). The relationship between risk and glycemic control was assessed by two methods: 1) Cumulative glycemic exposure was calculated separately for placebo and GLP-1RA groups for each trial and correlated with observed absolute CV risk. 2) Differential glycemic exposure between GLP-1RA and placebo group was calculated for each trial and correlated with a published hazard ratio (HR).

Results: There was a clear linear correlation between increasing HbA1c exposure and CV risk both with GLP-1RA and placebo. Placebo group appeared to have a lower absolute risk with the same glycemic exposure, but this difference was lost after correction for patient age. The difference in glycemic exposure between GLP-1RA and placebo group had a strong correlation (R2=0,75) with reported hazard ratio and 95% confidence intervals of HR-s from all trials included risk reduction expected from HbA1c difference. Lowering of HbA1c by 1% decreased CV risk by 30% and this relationship was consistent within placebo groups, GLP-1RA groups and between groups.

Conclusion: The reduction of CV events in CVOTs with GLP-1RA can be explained by a reduction in HbA1c.

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Disclosure: M. Roosimaa: Lecture/other fees; Sanofi, Novo Nordisk, Boehringer Ingelheim, AstraZeneca.

148

Exploring potential mediators of the cardiovascular benefit of dulaglutide in REWIND

H. Gerstein1, H. Colhoun2, M. Riddle3, K. Branch4, M. Konig5, C. Atisso5, M. Lakshmanan5, R. Mody5, C. Hasenour5;

1McMaster University, Hamilton, Canada, 2University of Edinburgh, Edinburgh, UK, 3Oregon Health & Science University, Portland, USA, 4University of Washington, Seattle, USA, 5Eli Lilly and Company, Indianapolis, USA.

Background and aims: The REWIND trial showed that relative to placebo (PL), once weekly dulaglutide (DU) 1.5 mg reduced the incidence of a major adverse cardiovascular event (MACE-3; nonfatal myocardial infarction, nonfatal stroke, or cardiovascular (CV) death) in patients with type 2 diabetes with and without established cardiovascular (CV) disease (hazard ratio [HR] 0.88, 95% CI [0.79, 0.99]; p=0.026). DU also significantly reduced A1C, body weight (BW), and systolic blood pressure (SBP). In this post-hoc assessment, a mediation analysis was used to estimate the degree to which the effect of DU on these risk factors could statistically account for its effect on MACE-3.

Materials and methods: Data were analysed from 9,901 patients who had 1,257 first MACE-3 over 5.4 median years of observation. Those risk factors for which the updated mean on follow-up was significantly related to cardiovascular events were added to a separate Cox model that included DU allocation, the baseline value of the measurement and the updated mean of the variable as time dependent covariates.

Results: Only A1C satisfied this condition (Table), suggesting that BW and SBP did not mediate the effect of DU on MACE in this study population. The effect size of DU on the MACE outcome was attenuated by 36.1% after accounting for its effect on A1C (Table).

Conclusion: The results suggest most of the CV benefits of dulaglutide on MACE is not attributable to the A1C, BW, or SBP-lowering effects of dulaglutide.

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Clinical Trial Registration Number: NCT01394952

Disclosure: H. Gerstein: Employment/Consultancy; Abbott, AstraZeneca, Boehringer Ingelheim, Eli Lilly and Company, Merck, Novo Nordisk, Janssen, Sanofi, Kowa. Grants; Eli Lilly and Company, AstraZeneca, Merck, Novo Nordisk, Sanofi. Honorarium; AstraZeneca, Boehringer Ingelheim, Eli Lilly and Company, Novo Nordisk, Sanofi.

149

Cardiovascular (CV) and hypoglycaemia outcomes across age groups in people with type 2 diabetes in the CAROLINA trial

M.A. Espeland1, R.E. Pratley2, J. Rosenstock3,4, T. Kadowaki5,6, Y. Seino7,8, B. Zinman9, N. Marx10, D.K. McGuire4, K.R. Andersen11, M. Mattheus12, A. Keller12, O.E. Johansen11, on behalf of the CAROLINA investigators;

1Department of Biostatistics and Data Science, Wake Forest School of Medicine, Winston-Salem, USA, 2Translational Research Institute for Metabolism and Diabetes, Orlando, USA, 3Dallas Diabetes Research Center at Medical City, Dallas, USA, 4University of Texas Southwestern Medical Center, Dallas, USA, 5Department of Prevention of Diabetes and Lifestyle-Related Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 6Department of Metabolism and Nutrition, Mizonokuchi Hospital, Faculty of Medicine, Teikyo University, Kanagawa, Japan, 7Kansai Electric Power Medical Research Institute, Kobe, Japan, 8Kansai Electric Power Hospital, Osaka, Japan, 9Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada, 10Department of Internal Medicine I, University Hospital Aachen, RWTH Aachen University, Aachen, Germany, 11Boehringer Ingelheim Norway KS, Asker, Norway, 12Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany.

Background and aims: Older people with T2D have a high prevalence of comorbidities, frailty and polypharmacy. We investigated the effects of linagliptin 5 mg versus glimepiride 1-4 mg once daily across age groups in the CARdiovascular Outcome Study of LINAgliptin Versus Glimepiride in T2D (CAROLINA).

Materials and methods: CAROLINA recruited adults with relatively early T2D, HbA1c 6.5-8.5%, and elevated CV risk. Its primary outcome was CV death, non-fatal myocardial infarction, or non-fatal stroke (3P-MACE), with secondary outcomes including mortality and changes from baseline in HbA1c, weight and hypoglycaemia.

Results: Of 6033 patients (median age 64 [range 36-85]) years, 846 (14.0%) were ≥75 years. During median follow-up of 6.3 years, CV and mortality outcome rates did not differ between treatment groups, overall and across age categories (interaction p-values >0.05). After some initial differences, there was no meaningful difference in HbA1c between linagliptin vs glimepiride, overall and across age groups, but hypoglycaemia rates (Figure) were significantly lower and weight losses were significantly greater with linagliptin vs glimepiride, regardless of age group.

Conclusion: Results for CV and mortality outcomes were consistent across age subgroups, but linagliptin has a modest benefit in weight and a significantly lower hypoglycaemia burden than glimepiride, which are important safety considerations when selecting therapy for the elderly.

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Clinical Trial Registration Number: NCT01243424

Supported by: Boehringer Ingelheim & Eli Lilly and Company Diabetes Alliance

Disclosure: M.A. Espeland: Employment/Consultancy; Boehringer Ingelheim.

150

Glucagon-like peptide-1 receptor agonists reduce cerebral and cardiovascular events: real world analysis using the National Database of Japan

M. Koshizaka1, R. Ishibashi2, T. Ishikawa3, K. Goda4, J. Sato4, M. Kitsuregawa4, K. Yokote5, N. Mitsutake3;

1Department of Diabetes, Metabolism and Endocrinology, Chiba University Hospital, Chiba, 2Department of Endocrinology and Metabolism, Kimitsu Central Hospital, Kisarazu, 3Institute for Health Economics and Policy, Tokyo, 4Institute of Industrial Science, The University of Tokyo, Tokyo, 5Department of Endocrinology, Hematology, and Gerontology, Chiba University Graduate School of Medicine, Chiba, Japan.

Background and aims: Although several clinical trials have shown the pleiotropic effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs), similar evidence for Asian patients in real medical settings is limited. Using the National Database of Health Insurance Claims and Specific Health Checkups of Japan, we compared the effect of GLP-1RAs on cerebral and cardiovascular events with that of other diabetes drugs.

Materials and methods: The type 2 diabetes (T2D) patients who were prescribed GLP-1RAs from 2011 to 2014 were included in the GLP-1RA group (group=Positive), and those prescribed other diabetes drugs were included in the control group (group=Negative).The outcome was the onset of cerebrovascular events (stroke, cerebral bleeding) or cardiovascular events (acute myocardial infarction, angina, heart failure) that continued for >3 months. Patient history included age, sex, duration of diabetes treatment, use of drugs at the start of treatment (other antidiabetic, antihypertensive, lipid-lowering, antiplatelet, and anticoagulant drugs), and cerebrovascular, cardiovascular and diabetes-related complications. These were used as explanatory variables in the logistic regression analysis. Propensity score matching was performed using nearest neighbor matching without replacement. Caliper was defined as standard deviation of propensity score * 0.25. Matching was done in order of propensity score close to median. The period from the prescription of new medication to the outcome was calculated and analyzed using the Cox proportional hazard model.

Results: Among 9,180,887 T2D patients, 34,399 and 46,326 patients in group=Positive reported cardiovascular and cerebrovascular events, respectively. In group=Negative, 877,523 and 742,776 patients reported cerebrovascular and cardiovascular events, respectively. After propensity score matching, 38,424 and 29,370 patients from both groups were analyzed for cerebrovascular and cardiovascular events, respectively. There were 2,248 (5.9 %) and 2,559 (6.7 %) cerebrovascular events were reported in group=Positive and Negative, respectively (HR 0.76, 95% CI 0.72-0.80, P < 0.0001), moreover 3,931 (13.4 %) and 4,024 (13.7 %) cardiovascular events reported in groups=Positive and Negative, respectively (HR 0.83, 95% CI 0.79-0.87, P < 0.0001, Figure).

Conclusion: Japanese patients treated with GLP-1RAs had significantly fewer cerebral and cardiovascular events than those not treated with GLP-1RAs.

figureaw

Disclosure: M. Koshizaka: None.

OP 26 Unusual forms of diabetes

151

Young-onset type 2 diabetes in White Caucasians and African Americans in the USA: multi-morbidity trend at diagnosis and atherosclerotic cardiovascular disease risk

S. Paul, D. Koye, O. Montvida;

University of Melbourne, Melbourne, Australia.

Background and aims: The multi-morbidity at Type 2 Diabetes (T2DM) diagnosis and long-term Atherosclerotic Cardiovascular Disease (ASCVD) risk in young-onset T2DM among White Caucasian (WC) and African American (AA) are not well studied. The aims were to evaluate the temporal patterns of multi-morbidity at diagnosis and ASCVD risk by age groups in WC and AA.

Materials and methods: Using US diabetes population representative Centricity Electronic Medical Records, 505,336 WC and 101,104 AA incident T2DM patients within age groups 18-39 years, 40-49 years, 50-59 years and 60-70 years from 2000 to 2018 were identified, with mean 5 years follow-up. The prevalence trend of multi-morbidity (at least 2 of: ASCVD, micro-vascular disease, cancer, chronic kidney disease, BMI ≥ 35 kg/m2 ) at diagnosis was explored. Among those without ASCVD at diagnosis, the risk of ASCVD and MACE-3 (myocardial infarction, heart failure or stroke) were compared between AA and WC by age groups at diagnosis adjusting for appropriate confounders including age, sex, smoking status, BMI, non-macrovascular comorbidities, and stratifying by insulin use.

Results: The multi-morbidity prevalence trend has consistently increased from 11% to 20% over last 17 years in all age groups, with no difference between ethnicity. AA had significantly higher mean HbA1c (8.2%), BMI (39.5 kg/m2, 64% Grade 2+ Obese) in the youngest age group compared to older groups, while WC had similar risk factor distribution across all age groups at T2DM diagnosis. AA had higher systolic blood pressure (SBP) across all age groups compared to WC, overall SBP > 130 mmHg in AA / WC: 55% / 49%, but similar lipid distribution between ethnicity across age groups.Compared to WC, adjusted HR (95% CI) of ASCVD for AA in 18-39 years, 40-49 years, 50-59 years and 60-70 years groups were 1.17 (1.02, 1.34), 1.04 (0.96, 1.12), 0.96 (0.91, 1.00), 0.94 (0.90, 1.00) respectively. However, AA had significantly higher adjusted MACE-3 risk by 9-55% across all age groups.

Conclusion: The multi-morbidity has been significantly increasing among White Caucasians and African Americans across all age groups at T2DM diagnosis. While among those with T2DM diagnosed at 40+ years age there was no difference in ASCVD risk between ethnicity, the African Americans diagnosed at 18-39 years had higher ASCVD risk compared to White Caucasians. African Americans had higher 3-point MACE risk compared to White Caucasians across age groups.

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Disclosure: S. Paul: None.

152

Identification and mechanistic studies of a novel form of neonatal diabetes caused by YIPF5 mutations leading to pancreatic beta cell endoplasmic reticulum stress

F. Fantuzzi1,2, C. Demarez1, E. De Franco3, H. Ibrahim4, Y. Cai1, T. Sawatani1, H. Shakeri1, N. Pachera1, M. Lytrivi1, K. Patel3, M. Yildiz5, D.L. Eizirik1, T. Otonkoski4, A.T. Hattersley3, M. Cnop1;

1ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium, 2Medicine and Surgery, University of Parma, Parma, Italy, 3University of Exeter Medical School, Exeter, UK, 4Stem cells and Metabolism Research Program, University of Helsinki, Helsinki, Finland, 5Kanuni Sultan Suleyman Training and Research Hospital, Istanbul, Turkey.

Background and aims: We recently identified 6 patients from 5 families with neonatal/early-onset diabetes, microcephaly and epilepsy, caused by homozygous mutations in YIPF5 (4 different missense mutations and 1 in-frame deletion). YIPF5 is involved in endoplasmic reticulum (ER)-to-Golgi trafficking and is highly expressed in pancreatic islets. We presently used pluripotent stem cell (iPSC)-derived β-cells from patients, corrected isogenic controls or healthy controls to study the mechanisms leading to the disease.

Materials and methods: Peripheral blood mononuclear cells from 2 p(Ile98Ser) YIPF5-diabetic siblings and a healthy control were reprogrammed into iPSCs using Sendai virus vectors. Corrected isogenic iPSCs were generated by CRISPR-Cas9. iPSCs were differentiated into β-cells using a 7-step protocol. β-cell markers and function were assessed by immunocytochemistry, qPCR and ELISA. Apoptosis and ER stress markers were measured in β-cell organoids exposed for 48h to the ER stressors thapsigargin (1 μM) or tunicamycin (5 μg/ml).

Results: Control or corrected (iPSC-β) and YIPF5 mutant iPSC-derived β-cells (YIPF5-β) followed a proper developmental pathway as determined by SOX9NGN3PDX1 and NKX6.1 mRNA expression across the differentiation process. The yield of insulin-positive β-cells tended to be slightly lower in YIPF5-β than in iPSC-β (46±2% vs 38±2%, n=10). There was no difference in insulin secretion in response to high glucose (16.7 mM) or high glucose plus forskolin (10 μM). YIPF5-β had higher basal cell death than iPSC-β (18±3% vs 6±2%, p<0.05, n=8) and greater sensitivity to tunicamycin (25±3% vs 15±3% apoptosis, p<0.05). Tunicamycin and thapsigargin induced greater mRNA expression of the ER stress markers CHOP and BiP (respectively, 8-to-9-fold and 5-fold in YIPF5-β, p<0.05), and induced the pro-apoptotic Bcl-2 proteins DP5 and PUMA.

Conclusion: YIPF5 mutations cause a novel genetic subtype of diabetes resulting in impaired ER-to-Golgi trafficking. Using patients’ iPSC-derived β-cells, we show that the YIPF5 mutation compromises neither β-cell differentiation nor function, but sensitizes β-cells to basal and ER stress-induced apoptosis. This furthers our understanding of β-cell failure caused by YIPF5 mutations and will allow testing β-cell protective therapies.

Supported by: IMI INNODIA

Disclosure: F. Fantuzzi: None.

153

Prevalence and BMI of early-onset adult type 2 diabetes in a multiethnic population

J.D. Ranchagoda1, D. Johnston1, A. Majeed2, J. Valabhji3, E. Gregg2, S. Misra1;

1Department of Medicine, Imperial College London, London, 2School of Public Heath, Imperial College London, London, 3Imperial College Healthcare NHS Trust, London, UK.

Background and aims: The incidence of type 2 diabetes in young adults has increased rapidly in the U.K. These individuals have a higher and accelerated risk of diabetes complications and mortality. Paediatric studies of type 2 diabetes show that non-white ethnicities are disproportionately affected, and early onset is associated with more obesity than those at older ages. However, population studies of early adult-onset type 2 diabetes in diverse ethnic groups are lacking. We studied early-onset adult type 2 diabetes in white, South Asian (SA) and African-Caribbean (AC) individuals.

Materials and methods: In this cross-sectional study, cases with type 2 diabetes were accessed from an anonymised population dataset of 1,407,990 individuals, derived from general practice records in North West London. All cases coded with type 2 diabetes were included if their last clinical encounter occurred between April 2015 - December 2019. Early adult-onset type 2 diabetes was defined as an age at diagnosis of 18-45 yrs, irrespective of current age. We calculated: 1) proportion of cases of type 2 diabetes by decade of onset within each ethnicity and 2) mean BMI by ethnicity, comparing those currently aged 18-45 to those aged 55-79 yrs, with duration <5 yrs.

Results: Overall prevalence of type 2 diabetes among persons aged 9-99 in the sample was 6.5% (n=93.635); non-white ethnicities had higher prevalence; 3.4% (23,418 /688,568) of white, 10.1% (58,661/580,878) SA and 8.3% (11,556/138,544) of AC individuals. Analysis 1: The proportion of cases by decade of onset varied significantly by ethnicity (table); in white people, 3.7% of cases were diagnosed at age 18-34yrs, 12% at 35-44, 25.6% at 45-54 with a peak 28.8% at age 55-64 yrs. SA individuals had higher proportions of earlier diagnoses, with 8.2% of all cases diagnosed 18-34 yrs, 22.5% 35-44 with the highest, 31.2%, at 45-54. AC individuals also had higher proportions of earlier diagnoses; 7.2% 18-34 yrs, 18.6% 35-44, with the highest proportion (30.7%) aged 45-54. Analysis 2: The Mean BMI was significantly higher across all ethnicities in those currently aged 18-44 versus 55-79 yrs with <5 yrs duration of type 2 diabetes: in white individuals currently aged 18-45(n=563), BMI was 34.8kg/m2 vs 31.8 for 55-79 yrs (n=3921). SA people had significantly lower BMI than white, but similar trends; BMI 30.3 kg/m2 (18-45, n=3806) vs 28.6 (55-79, n=8090). In AC individuals, BMI was 33.7 (18-45, n=475) and 31.0 age (55-79, n=1595).

Conclusion: Early adult-onset type 2 diabetes disproportionately affects SA and AC ethnicities; it is twice as high in SA and 10% higher in AC ethnicities as white. In all ethnicities studied, mean BMI in those with early-onset type 2 diabetes, is significantly higher than older onset diabetes and for SA individuals the trend of developing type 2 diabetes at lower BMI than other ethnic groups, is maintained for those specifically presenting early in adulthood.

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Supported by: EFSD Future Leaders Mentorship Programme

Disclosure: J.D. Ranchagoda: None.

154

Islet cell autoantibodies status in patients with MODY phenotype

E. Romanenkova1, N. Zubkova1, A. Timofeev2, D. Laptev1;

1Endocrinology Research Centre, Moscow, 2Pirogov Russian National Research Medical University, Moscow, Russian Federation.

Background and aims: Autoimmune damage to pancreatic beta cells are important markers of type 1 diabetes. However, autoantibodies are present in other types of diabetes. It's known, the screening of monogenic forms of diabetes is based on the evaluation of clinical data included in MODY calculator and determine islet autoantibody status.We tested for the presence of islet autoantibodies in a subgroup of patients with high probability MODY followed by a screening for mutations in MODY-candidate genes.

Materials and methods: The autoantibodies (GADA, ZnT8, IA2) were measured in a cohort of 124 patients with MODY phenotype (clinical prediction model to determine the probability of MODY have given a >75% positive predictive value).‘Diabetes panel’ genes were sequenced using a custom Ion Ampliseq gene panel and PGM semiconductor sequencer (Ion Torrent). Interpretation of the sequencing results and assessment of the pathogenicity of sequence variants were performed according to the ACMG guidelines (2015).

Results: 8 patients (6.5%) were autoantibody positive (one or more autoantibodies): GADA (n=1) 4,3 ME/ml (normal range <1,0 ME/ml); IA-2 (n=5) 28-71 ME/ml (normal range <15,0 ME/ml); ZnT8 (n=3) 20-20,4 U/ml (normal range <15,0 U/ml). The median age at onset of hyperglycemia was 11.2 years [6.46-15.7]. The length of the disease at the time of the examination was 13.4 years [7.3-17.3]. The average level of glycated hemoglobin during the manifestation of the disease was 6.7% [5.9-7.3%]. All participants showed positive family history of diabetes with affected first-degree relatives at least in two generations.Using NGS, 8 pathogenic and probably pathogenic mutations were identified in the GCK (n=7) and HNF4A (n=1) genes. For missense variants in GCK gene have been described before (p.F150Y, p.V182M, p.G261R, p.R191Q) and 3 (p.G295V, p.I225T, p.Y273N) were novel. Heterozygous missense variants p.R290H in HNF4A gene was previously reported and ranked as pathogenic.All GCK-MODY2 patients were asymptomatic. Their clinical data were the following: mean age 10.6 year [6.5-14.4], SDS BMI -0.7 [-3.7-1.2], mean fasting plasma glucose 6.5 mmol/l 6.6 [5.9-7.7], and mean HbA1c 6.7% [5.9-7]. 5 subjects met criteria of diabetes and 2 patients met criteria of IGT (ISPAD, 2019). The patients with GCK gene mutations were successfully treated with low-carbohydrate diets.A 15-year-old patient with HNF4A mutation was almost asymptomatic. The OGTT results done due to his obesity were consistent with diabetes. HbA1c was 11.7%. The boy responded well to low doses of sulfonylureas.

Conclusion: In our study, islet autoantibody positivity was associated with MODY phenotype in 6.3% cases. Detectable levels of islet cell autoantibodies in patients with MODY phenotype challenges the use of autoantibodies as a universal negative marker of MODY.

Disclosure: E. Romanenkova: None.

155

Linagliptin as add-on treatment to glimepiride in patients with HNF1A-diabetes (MODY3): a randomised, double-blinded, placebo-controlled, crossover trial

A.S. Christensen1,2, S. Hædersdal1,2, J. Støy3, H. Storgaard1,2, U. Kampmann3, M. Seghieri1,4, J.J. Holst5,6, T. Hansen6, F.K. Knop1,2, T. Vilsbøll1,2;

1Steno Diabetes Center Copenhagen, Hellerup, Denmark, 2Center for Clinical Metabolic Research, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark, 3Steno Diabetes Center Aarhus, Aarhus, Denmark, 4Diabetes Unit, USL Toscana Centro, Florence, Denmark, 5Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 6Novo Nordisk Foundation Center for Basic Metabolic Research, The Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, Copenhagen, Denmark.

Background and aims: First-line treatment of hepatocyte nuclear factor 1-alpha HNF1A-diabetes (MODY3) is sulfonylurea. Sulfonylurea has limitations with respect to providing sustained glycaemic control and risk of hypoglycaemia. We hypothesised that a low dose of sulfonylurea in combination with the dipeptidyl peptidase 4 inhibitor linagliptin provides a more efficacious treatment with less glycaemic variability and hypoglycaemia in patients with HNF1A-diabetes compared to sulfonylurea monotherapy.

Materials and methods: In a randomised, double-blinded, cross-over trial, patients with HNF1A-diabetes (n=20, [mean ± SD]; age: 43 ± 14 years; BMI 24.3 ± 2.8 kg/m2; HbA1c 57.1 ± 7.3 mmol/mol) were randomly assigned to treatment with glimepiride+ 5mg linagliptin (16 weeks), wash-out (4 weeks) and treatment with glimepiride+placebo (16 weeks). Glimepiride was titrated in a treat-to-target manner aiming for fasting plasma glucose of 4.5-6.0 mmol/l without hypoglycaemia. Treatments were evaluated by continuous glucose monitoring (CGM), HbA1c and a mixed-meal test at baseline and at the end of each treatment period. The primary endpoint was difference between treatments in mean amplitude of glycaemic excursions (MAGE) and secondary endpoints included differences between treatments in coefficient of variation (CV) on CGM, HbA1c and hypoglycaemia.

Results: The glimepiride dose was significantly reduced ([mean difference, 95% CI] -0.7 [1.2 to 0.2] mg/day) with glimepiride + linagliptin compared with glimepiride + placebo. Compared to glimepiride + placebo, glimepiride + linagliptin showed an insignificant reduction in MAGE (-0.7 [-1.9 to 0.4] mmol/l) and significant improvements in CV (-3.7 [-7.1 to -0.3] %), HbA1c (-5.6 [-9.3 to -1.8] mmol/mol), body weight (-1.0 [-2.0 to -0.1] kg) and beta cell function (assessed by baseline-subtracted AUC for C-peptide:glucose during meal test (4.3 [0.6 to 7.9] nmol/l×mmol/l-1×min-1)). Incidence of hypoglycaemia (assessed by patient-reported episodes and CGM) was similar in the two treatment periods.

Conclusion: Linagliptin as add-on treatment to glimepiride in patients with HNF1A-diabetes improved glycaemic variability, control and reduced the daily glimepiride dose without increasing the risk of hypoglycaemia.

figureaz

Clinical Trial Registration Number: EudraCT No. 2017-000204-15

Supported by: Unrestricted grant from Boehringer Ingelheim

Disclosure: A.S. Christensen: None.

156

Investigating the contribution of the HNF-1αG319S gene variant to childhood-onset type 2 diabetes using beta cell and mouse models

C.A. Doucette1, T.S. Morriseau2, K. Hunt1, M. Fonseca2, C. Nian3, V.W. Dolinsky2, F.C. Lynn3;

1Physiology and Pathophysiology, University of Manitoba, Winnipeg, 2Pharmacology and Therapeutics, University of Manitoba, Winnipeg, 3Surgery, University of British Columbia, Vancouver, Canada.

Background and aims: Type 2 diabetes (T2D) in children continues to increase in Canada, with Indigenous youth in the province of Manitoba carrying the highest burden. The annual incidence of childhood-onset T2D is 25 per 100,000 children/year in Manitoba, ~20-fold higher than the national average. 40% of the youth who have T2D in the affected Indigenous communities harbor a private single nucleotide variant in the hepatocyte nuclear factor-1α (HNF-1α) gene that changes a glycine at codon 319 to a serine (“G319S”). Clinical observations describe reduced fasting insulin and less obesity in children with T2D who carry the G319S variant, suggesting that the variant drives early beta cell dysfunction; however, the mechanistic impact of this variant on beta cell function is difficult to explore in human populations; animal and cell models are needed.

Materials and methods: CRISPR/Cas9 gene editing was used to knock-in the g>a.955 nucleotide substitution into MIN6 clonal β-cells (“G319S-MIN6”) and C57/BL6 mice. Heterozygous mice were bred to yield littermates with different doses of the variant “S allele”, namely GG (wild type), GS (heterozygous) and SS (homozygous for the variant). In vitro studies included glucose-stimulated insulin secretion (GSIS) assays, gene expression analyses and assessment of mitochondrial fuel oxidation. In vivo studies included glucose and insulin tolerance test, pyruvate tolerance tests and measurements of plasma hormone levels.

Results: Surprisingly, under standard conditions, the G319S variant did not affect GSIS in MIN6 β-cells; however, basal insulin secretion (at 2.8mM glucose) decreased 3.2-fold relative to control cells (p<0.01). Under chronic lipotoxic stress (48hr palmitate exposure), unlike control cells, G319S-MIN6 cells maintained 15-fold GSIS (p<0.01), accompanied by a 2-fold increase in carnitine palmitoyltransferase-1A (Cpt1A) expression (p<0.05) and a doubling in the rate of fatty acid oxidation. 6-month-old G319S-expressing female mice fed a chow diet revealed glucose intolerance (p<0.05) and elevated hepatic glucose production (p<0.05), despite no changes in body weight or insulin sensitivity. 6-month old male mice maintained normal glucose tolerance.

Conclusion: The HNF-1αG319S variant appears to shift β-cell metabolism to promote fatty acid oxidation and may confer protection to a high-fat dietary intake, which was abundant in traditional Indigenous food sources. While it appears maladaptive in the modern environment, the reduced basal insulin secretion from G319S-expressing beta cells may promote improvement in the metabolic response to fasting in this population by enhancing the mobilization of fuels, i.e. via enhanced hepatic gluconeogenesis; however, in modern times, drastically altered lifestyles as a result of colonization may trigger early hyperglycemia development, particularly as a result of excessive carbohydrate consumption. Future studies will address whether low-carbohydrate, high-fat diets are protective in G319S-expressing mice and will assess the effectiveness of dietary interventions in this population of people.

Supported by: CIHR PJT-159633

Disclosure: C.A. Doucette: None.

OP 27 Macrovascular complications and beyond

157

Aortic stiffness, peripheral and central haemodynamics in patients with screen-detected type 2 diabetes: the ADDITION-Denmark study

E. Laugesen1, L. Bjerg2,3, S.T. Andersen2,4, A. Sandbæk2,3, M. Charles3,5, M.E. Jørgensen6,7, D. Witte2,3;

1Aarhus Universitetshospital, Aarhus N, 2Department of Public Health, Aarhus University, Aarhus C, 3Steno Diabetes Center Aarhus, Aarhus N, 4Danish Pain Research Center, Department of Clinical Medicine, Aarhus University, Aarhus C, 5Research Unit of General Practice, Aarhus University, Aarhus C, 6Clinical Epidemiology, Steno Diabetes Center Copenhagen, Copenhagen, 7National Institute of Public Health, University of Southern Denmark, Odense, Denmark.

Background and aims: Intensive multifactorial treatment has an impact on aortic stiffness in type 2 diabetes as we have shown in the five year follow-up in the ADDITION study. However, the long-term impact of multifactorial treatment on the hemodynamic system is not well understood. We studied the 5 year post-intervention (10 years post-randomization) effect of intensive multifactorial treatment compared with routine care on hemodynamic parameters among individuals in the Danish arm of the ADDITION-Europe trial.

Materials and methods: The ADDITION-trial is a population-based screening and intervention study conducted in patients with screen-detected type 2 diabetes aged 40-69 years. General practitioners were randomized to provide intensive multifactorial treatment (IT) or routine care (RC). An unselected subsample of 411 patients underwent central hemodynamic assessments including carotid-femoral pulse wave velocity (cfPWV) by applanation tonometry in 2016-2017 approximately 5 years after the intervention finished (10 years post-randomization). We did an intention-to-treat analysis using linear regression models. All models were adjusted for sex, age and heart rate. The effect of additional adjustment for mean arterial pressure on cfpWV was also evaluated.

Results: In total, 242 patients from the IT and 169 patients from the RC underwent assessment by applanation tonometry. The median cfPWV was 10.2 m/s in the RC and 9.6 m/s in the IT (Table 1). cfPWV was 0.51 m/s lower (95% CI −0.97 to −0.05) in the IT compared with RC. With further adjustment by mean arterial pressure the positive effect by IT was attenuated to 0.4 m/s (95% CI -0.90 to 0.08). The peripheral systolic blood pressure was 3.7 mmHg lower (95% CI -7.2 to −0.3) while the peripheral diastolic blood pressure was 2.0 mmHg lower (95% CI -3.9 to −0.2) in the IT. There was no difference in other hemodynamic measures.

Conclusion: Intensive multifactorial treatment of patients with screen-detected type 2 diabetes in general practice showed a prolonged positive effect on aortic stiffness. Differences previously observed at the end of the intervention period were maintained 5 years after the end of the intervention period (10 years post-randomization). The intervention effect could be mediated by effects on mean arterial pressure.

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Clinical Trial Registration Number: 20000183 and 1-10-72-63-15

Supported by: IDNC research programme, Steno Research & Innovation Fund 2015

Disclosure: E. Laugesen: Grants; dd.

158

CAPTURE: a cross-sectional study of the contemporary (2019) prevalence of cardiovascular disease in adults with type 2 diabetes across 13 countries

O. Mosenzon1, A. Alguwaihes2, J.L. Arenas Leon3, F. Bayram4, P. Darmon5, T. Davis6, G. Dieuzeide7, K.T. Eriksen8, T. Hong9, C. Lengyel10, N.A. Rhee11, G.T. Russo12, S. Shirabe13, K. Urbancova14, S. Vencio15;

1Hadassah Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel, 2King Saud University Medical City, Riyadh, Saudi Arabia, 3Centro de Atención e Investigación Cardiovascular del Potosí, San Luis Potosí, Mexico, 4Erciyes University, Kayseri, Turkey, 5Hôpital de la Conception, Marseille, France, 6University of Western Australia, Fremantle Hospital, Fremantle, Australia, 7Centro de Atención Integral en Diabetes, Endocrinologia y Metabolismo, Chacabuco, Argentina, 8Novo Nordisk A/S, Søborg, Denmark, 9Peking University Third Hospital, Beijing, China, 10University of Szeged, Szeged, Hungary, 11Novo Nordisk Health Care AG, Zurich, Switzerland, 12University of Messina, Messina, Italy, 13H. E. C Science Clinic, Yokohama, Japan, 14Diabetologická Interní Ambulance s.r.o, Ostrava, Czech Republic, 15Instituto de Ciencias Farmaceuticas, Goiás, Brazil.

Background and aims: There is a paucity of global and country-specific data on the prevalence of cardiovascular disease (CVD) in people with type 2 diabetes (T2D). The primary objective of CAPTURE was to estimate the contemporary (2019) prevalence of established CVD in people with T2D across 13 countries from five continents.

Materials and methods: CAPTURE was a multinational, cross-sectional, non-interventional study. Detailed, standardised demographic and clinical data were collected from adults aged ≥18 years with T2D attending a single routine healthcare visit in primary or specialist care between Dec 2018 and Sept 2019. Overall CVD prevalence estimates (across all 13 countries) were weighted to account for the size of the T2D population of each country. Data were analysed descriptively.

Results: In total, 9823 adults with T2D (primary care: 4502; specialist care: 5321) participated, with the following median (interquartile range, IQR) characteristics: age 64.0 years (56.0-71.0), diabetes duration 10.7 years (5.6-17.9) and HbA1c 7.3% (6.6-8.4) [56 mmol/mol (49-68)]; 45.5% were female. Overall CVD prevalence was 34.8% [32.7; 36.8]95% CI), with most (85.8%) categorised as atherosclerotic CVD (31.8% [29.7; 33.8]95% CI) (Table). Overall CHD prevalence was 17.7% [16.2; 19.3]95% CI, carotid artery disease was 8.4% [7.0; 9.7]95% CI and cerebrovascular disease was 7.2% [5.9; 8.4]95% CI. The overall prevalence of heart failure was 2.4% [2.1; 2.7]95% CI, driven by a relatively low prevalence in China (0.2% [0.0; 0.9]95% CI). Prevalence estimates were similar across primary and specialist care settings.

Conclusion: CAPTURE is the first multinational, cross-sectional study to estimate CVD prevalence in adults with T2D using standardised methodology. Our findings demonstrate that, in 2019, approximately one in three adults with T2D attending a primary or specialist healthcare visit had established CVD.

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Clinical Trial Registration Number: NCT03811288; NCT03786406

Supported by: Novo Nordisk

Disclosure: O. Mosenzon: Non-financial support; Abstract supported by Novo Nordisk.

159

Derived time-in-range is associated with MACE in type 2 diabetes: data from the DEVOTE trial

R. Bergenstal1, E. Hachmann-Nielsen2, K. Kvist2, J.B. Buse3;

1International Diabetes Center, Minneapolis, USA, 2Novo Nordisk A/S, Copenhagen, Denmark, 3University of North Carolina School of Medicine, Chapel Hill, USA.

Background and aims: There is a need to validate time-in-range (TIR; percentage of time with plasma glucose between 3.9 and 10.0 mmol/L (70-180 mg/dL) as a surrogate endpoint for long-term clinical outcomes.

Materials and methods: We used data from patients with 8-point glucose profiles (8pp) from the double-blind cardiovascular outcomes trial, DEVOTE. In total, 7637 patients with T2D and either established CVD or at high risk for CVD were included in the trial. The primary endpoint in DEVOTE was time to first MACE. The 8pp were collected at 1 year, 2 years and end-of-trial. Median length of follow-up was 2 years. For 5644 patients, 8pps with at least 7 points existed. Among the 681 major adverse cardiovascular events (MACEs) in DEVOTE, 360 were among patients with 8pps. Individual TIR was derived as the proportion of the 8pp within range. A Cox model was used to estimate the association between derived TIR and time to first MACE. Hazard ratios (HR) were estimated for patients with TIR>70% vs TIR≤70%, and for TIR>70% and TIR 50−70% vs TIR≤50%.

Results: Derived TIR was >70% for 65% of the patients. Estimated rate of first MACE was lower for TIR >70% and TIR 50-70% vs TIR≤50% (Figure) and for TIR>70% vs TIR≤70% (HR: 0.74 [0.60;0.91]95% CI; p<0.01). The associations were maintained when analyses were adjusted for baseline characteristics.

Conclusion: Derived TIR was associated with rate of first MACE for T2D patients in DEVOTE.

figurebc

Clinical Trial Registration Number: NCT01959529

Supported by: Novo Nordisk

Disclosure: R. Bergenstal: Employment/Consultancy; Novo Nordisk, Abbott Diabetes Care, Calibra, Eli Lilly, Hygieia, Roche, Sanofi, DexCom, Medtronic, United HealthCare, Onduo. Grants; Novo Nordisk, Abbott Diabetes Care, AtraZeneca, Calibra, Eli Lilly, Hygieia, Medtronic, Sanofi, Takeda, DexCom. Honorarium; Novo Nordisk, Abbott Diabetes Care, Boehringer Ingelheim, AstraZeneca, Calibra, Eli Lilly, Hygieia, Roche, Sanofi, Takeda, DexCom. Non-financial support; Novo Nordisk. Stock/Shareholding; Merck.

160

Genetic risk for coronary artery disease is comparable to the risk imposed by traditional risk factors in individuals with type 1 diabetes

R. Lithovius1,2, A. Antikainen1,3, S. Mutter1,3, E. Valo1,3, C. Forsblom1,2, N. Sandholm1,3, P.-H. Groop2,3;

1Folkhälsan Institute of Genetics, Helsinki, 2University of Helsinki and Helsinki University Hospital, Helsinki, 3Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, Finland.

Background and aims: Individuals with type 1 diabetes have a higher risk of coronary artery disease (CAD) than the general population. In order to improve the risk prediction in the general population, genetic risk scores (GRS) have been suggested to complement the traditional risk factors for the identification of high-risk individuals, and thus enabling earlier interventions. Whether the same GRS are able to identify individuals with high risk of CAD in type 1 diabetes (T1D) is not known. Therefore, we investigated the potential of such GRS in individuals with T1D, combined with traditional risk factors, and also the impact of pharmaceutical treatment.

Materials and methods: GRSs were calculated from 156 known CAD susceptibility variants from the general population for 2736 genotyped individuals with T1D in the Finnish Diabetic Nephropathy Study (cases/controls=408/2328). Sex, age, diabetes onset year, systolic and diastolic BP, total cholesterol, HDL-cholesterol, triacylglycerol, smoking status, HbA1c, BMI, WHR, eGFR and presence of albuminuria were recorded. Cox regression analyses were performed with the standardized clinical variables and the GRS. Individuals were also stratified into the highest and lowest score decile groups (10th and 90th percentiles) and into quintiles (20th and 80th percentiles). Hazard ratios (HR) for CAD were evaluated with Cox regression between the lowest and highest score groups adjusted for age, sex and diabetes onset year. Finally, analyses were repeated in those with (cases/controls=323/917) antihypertensive or lipid-lowering medication at baseline based on the Drug Prescription Register.

Results: In Cox regression, GRS (HR=1.30 [1.18-1.45], p=4.37x10-7), presence of albuminuria (HR=1.48 [1.30-1.68], p=1.33x10-9), HbA1c (HR 1.22 [1.10-1.36], p=0.0002) and systolic BP (HR=1.21 [1.08-1.36], p=0.0008), were strongly associated with risk of CAD. The highest decile GRS group had a significantly higher risk of CAD in comparison with the lowest decile (HR=2.77 [1.77-4.33], p=7.51×10-6). The risk-increase was more modest for the quintile groups comparison (HR=2.16 [1.56-2.98], p=3.18×10-6). In those taking medication at baseline, the HRs between top versus bottom decile GRS groups were slightly reduced but remained substantial (HR 2.51 [1.50-4.21], p=0.0005). The HR between the quintile groups showed a similar trend (HR 2.12 [1.46-3.06] p=6.68 ×10-6).

Conclusion: GRS for CAD based on the general population successfully identified individuals with high risk of CAD in T1D, especially in those with the highest genetic risk. Importantly, the genetic risk of CAD is comparable to the risk imposed by the traditional risk factors, suggesting that genetic risk is an important life-long risk factor and should be considered in individuals with T1D in clinical practice. The results also suggested that in those with the highest genetic risk antihypertensive or lipid-lowering medication seemed to attenuate the risk of CAD.

Supported by: Finnish Heart Foundation, Diabetes Research Foundation

Disclosure: R. Lithovius: None.

161

Prognosis in patients with atrial fibrillation and type 1 diabetes, type 2 diabetes and severe hypoglycaemia: a nationwide report

S. Karayiannides1, A. Norhammar2, L. Landstedt-Hallin3, L. Friberg3, P. Lundman4;

1Academic Specialist Center, Center for Diabetes, Karolinska Institutet, Stockholm, 2Department of Clinical Physiology, Capio St Görans Hospital, Karolinska Institutet, Stockholm, 3Department of Clinical Sciences, Karolinska Institutet, Stockholm, 4Department of Cardiology, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Atrial fibrillation (AF), diabetes mellitus (DM) and severe hypoglycaemia are all associated with an increased risk for mortality, cardiovascular complications and impaired cognition. Few studies have compared the risk for adverse events between type 1 DM (T1DM) and type 2 DM (T2DM). The aim of this nationwide study in patients with AF was to describe the prognostic differences between T1DM, T2DM and in DM with documented severe hypoglycaemia.

Materials and methods: We included all 309 611 adult patients in Sweden who received a non-valvular AF diagnosis between 1 January 2013 and 31 December 2014. Patients were followed for all-cause mortality until 27 March 2017 and for incident heart failure, myocardial infarction, ischaemic stroke and incident dementia until 31 December 2015. Information on comorbidities, events and medication was collected from nationwide registries. Cox proportional hazard regression was used to calculate HRs adjusted for age, sex, comorbidities, and medications.

Results: DM was present in 60 294 (19.5%) patients, of whom 2 221 (3.7%) had T1DM and 58 073 (96.3%) had T2DM. Severe hypoglycaemia was documented in 1 560 patients (12.2% of T1DM and 2.2% of T2DM patients). Patients with T1DM were generally younger (71.2 vs. 76.0 years), more frequently had previous myocardial infarction (31.6% vs. 26.4%), peripheral artery disease (19.8% vs. 11.0%) and chronic kidney disease (21.5% vs. 9.6%) and less frequently had heart failure (34.9% vs. 38.6%) than those with T2DM. Adjusted HRs (95% CI) for all-cause mortality in patients with T1DM and T2DM compared with patients w/o diabetes were 1.43 (1.31-1.56) and 1.24 (1.21-1.28), for myocardial infarction 1.85 (1.58-2.17) and 1.20 (1.12-1.28) and for ischaemic stroke 1.29 (1.07-1.55) and 1.06 (0.99-1.12), respectively. Presence of severe hypoglycaemia was independently associated with increased risk for incident dementia [1.84 (1.08-3.14) in T1DM, 1.48 (1.16-1.89) in T2DM] and all-cause mortality [1.60 (1.32-1.95) in T1DM, 1.48 (1.37-1.62) in T2DM] compared with patients w/o diabetes (figure).

Conclusion: In a nationwide population with AF, T1DM was associated with a more pronounced increase in risk for all-cause mortality and myocardial infarction than T2DM. Severe hypoglycaemia in both T1DM and T2DM was associated with a higher risk for incident dementia and all-cause mortality. Our results highlight the importance of risk factor optimization in patients with DM, regardless of type, to avoid cardiovascular complications and that the treatment goal of reducing severe hypoglycaemia is also essential in patients with AF and DM.

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Disclosure: S. Karayiannides: None.

162

Cardiovascular risk prediction equations for patients with type 2 diabetes derived in a population with comprehensive diabetes screening

R. Pylypchuk1, P. Drury2, B. Wu1, S. Wells1, R. Jackson1;

1Epidemiology and Biostatistics, University of Auckland, Auckland, 2Ministry of Health NZ, Wellington, New Zealand.

Background and aims: Patients with longstanding diabetes may have similar cardiovascular risk to patients with pre-existing vascular disease, however the increasing numbers of screen-detected patients over recent years are likely to be at lower cardiovascular risk. For such patients, cardiovascular risk management should be informed by predicted risk, yet most prediction equations have been derived in patients with longstanding diabetes and will thus tend to overestimate risk. We derived new equations in a contemporary cohort of diabetes patients in New Zealand, where widespread diabetes screening was introduced in 2012.

Materials and methods: The PREDICT cohort study enrols participants when general practitioners use PREDICT software to assess patients’ cardiovascular risk in routine practice. The analyses were restricted to patients from the PREDICT cohort with known diabetes but no known prior vascular disease (diabetes sub-cohort), recruited between 2004 and 2016; 53% after 2010. Patient risk profiles were linked to national ICD-coded hospitalisations and deaths. New equations were developed including 18 pre-specified variables and predicting 5-year cardiovascular event rates using Cox proportional hazard models. Calibration and discrimination performance of the new equations was assessed and compared with two other New Zealand equations; one derived from the diabetes-only New Zealand Diabetes Cohort Study (NZDCS), and the other from the general population PREDICT cohort study, combining people with and without diabetes.

Results: The PREDICT diabetes sub-cohort recruited 46,652 participants (49% women) aged 30 to 74 years. Over a mean of 5.2 years follow-up (244,840 person-years) they experienced 4,114 first cardiovascular disease events (9% fatal) during follow-up (mean 5.2 years, 244,840 person-years). The new diabetes-specific equations included additional clinically relevant predictors not present in the comparison equations. Discrimination performance of the new equations (e.g. Harrell’s C for women = 0.73 [95% CI:0.72, 0.74]) was significantly better than equations derived from the NZDCS (0.69 [0.67, 0.70]) and from the general population PREDICT cohort study (0.68 [0.67, 0.70]). NZDCS equations overestimated CVD risk by 75-100% when applied to the PREDICT diabetes sub-cohort, whereas PREDICT general population equations were well calibrated.

Conclusion: Widespread screening identifies many patients with short-duration diabetes at low cardiovascular risk so that equations derived in a diabetes-specific cohort only a few years before widespread screening now overestimate risk by up to 2-fold. Moreover, the new equations, which include additional diabetes-specific and renal predictors, had significantly better discrimination than either the older NZDCS equations or the contemporary general population (non-diabetic) PREDICT equations derived from the same study. As population diabetes screening is introduced worldwide, clinicians will require new equations derived from contemporary diabetes-only cohorts with additional diabetes-specific predictors. Without more accurate equations, informed shared decisions on cardiovascular risk management in this important and increasingly heterogeneous patient population will not be possible.

Supported by: This study was funded by the Health Research Council of New Zealand

Disclosure: R. Pylypchuk: Grants; Health Research Council of New Zealand, Programme Grant.

OP 28 Linking inflammation to metabolism

163

Netrin-1 mediates adipose immune equilibrium and insulin resistance in type 2 diabetes via UNC5h2 receptor

H. Shi, M. Liu, Y. Qu, C. Li;

Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.

Background and aims: Netrin-1 is a neuron guidance molecule with reported pro-angiogenic and anti-inflammatory properties. We have previously reported a dual effect of Netrin-1 on pancreatic islet insulin secretion and immune modulation in high fat diet (HFD)/streptozotocin-induced diabetic mice. However, controversial studies have observed that administration of Netrin-1 promotes adipose macrophage retention and insulin resistance in HFD-induced obese mice via UNC5h2 receptor. In order to elucidate the exact impact of Netrin-1 in type 2 diabetes, we have used db/db mice to assess the role of Netrin-1 on insulin resistance and adipose tissue metabolism.

Materials and methods: Osmotic minipumps that release Netrin-1 (500 ng/day) or Netrin-1 with UNC5h2 Fc (1 μg/day), which blocks UNC5h2 receptor, for 4 weeks were implanted subcutaneously in male db/db mice (4 week-old, n=10). Fasting plasma glucose levels were measured weekly. Intraperitoneal glucose/insulin tolerance tests were performed before minipump implantation and after removal. RNA-seq was employed to examine changes of gene expression in visceral fat extracted from all groups of mice. Expressions of insulin receptor signalling components in visceral fat were assessed by western blotting. Serum levels of pro-inflammatory cytokines were quantified by ELISA.

Results: We observed decreased fasting plasma glucose levels from db/db mice treated with Netrin-1 (AUC: 77±6% over db/db control, p<0.05; 66±5% over Netrin-1+UNC5h2 Fc, p<0.05). This hypoglycaemic effect of Netrin-1 was abolished by UNC5h2 receptor inhibition since no change was shown in mice treated with Netrin-1+UNC5h2 Fc (AUC: 118±16% over control, p>0.1). Glucose tolerance was also significantly improved in mice administrated with Netrin-1 (AUC: 84±3% over control, p<0.05; 81±3% over Netrin-1+UNC5h2 Fc, p<0.05), which is dependent on UNC5h2 signalling, as mice treated with Netrin-1+UNC5h2 Fc were glucose intolerant (AUC: 103±4% over control, p>0.1). Similarly, Netrin-1-induced amelioration of insulin resistance was likely also mediated by UNC5h2 in db/db mice, as inhibition of UNC5h2 led to exacerbated insulin resistance (76±8% over control, p<0.05; 71±8% over Netrin-1+UNC5h2 Fc, p<0.05). However, Netrin-1-UNC5h2-regulated improvement of insulin resistance is independent of the insulin receptor-mediated signalling cascade, demonstrated by unaltered phosphorylation status of AKT and ERK in visceral fat obtained from Netrin-1 and Netrin-1+UNC5h2 Fc treated mice. RNA-seq analysis further revealed significant differences of mRNA expression profile in visceral fat from Netrin-1-treated animals when compared to control and Netrin-1+UNC5h2 Fc groups. Interestingly, the majority of genes with the most profound expression changes were those that mediate NF-κB signalling, chemokine signalling and cytokine signalling. Furthermore, though local and serum levels of pro-inflammatory cytokines TNFα and IL-1β were reduced in Netrin-1-treated mice compared to the other treatment groups, local and serum levels of chemokines MCP-1 and RANTES were both elevated, implicating more complex immunological regulations of Netrin-1 in visceral fat.

Conclusion: Our results indicate that Netrin-1 ameliorates insulin resistance via UNC5h2 receptor-mediated signalling activities. The Netrin-1-UNC5h2 signalling is likely to regulate adipose metabolism via its impact on visceral fat immune equilibrium modulation in type 2 diabetes.

Supported by: NSFC

Disclosure: H. Shi: None.

164

Hyperglycaemia epigenetically propels CD34+ hematopoietic stem cell differentiation toward more pro-inflammatory monocyte subpopulation

V. Vigorelli, S. Genovese, G. Pompilio, M. Vinci;

IRCCS Centro Cardiologico Monzino, Milan, Italy.

Background and aims: Diabetes mellitus (DM) is characterized by enhanced inflammatory state that promote and/or accelerate the molecular and cellular processes involved in the development of atherosclerosis. To this regard, clinical studies described abnormal elevation of intermediate (CD14++CD16+) inflammatory monocytes and alteration in macrophage polarization in DM individuals. Recent evidence suggests that hyperglycemia might preprogram CD34+ hematopoietic stem cells (HSCs) differentiation into more inflammatory cell populations. On these basis, the goals of our study were: i) to assess the in vitro effects of high glucose (HG) exposure on HSC phenotype and myeloid differentiation; ii) to determine if similar alterations might be at work in HSCs isolated from bone marrow (BM) of type 2 DM (T2DM) patients.

Materials and methods: CD34+ HSCs were magnetically sorted from cord blood (CB) of healthy subjects and BM sternal biopsies of T2DM patients ± coronary artery disease (CAD). CB-HSCs were expanded in normal-glucose (NG; with 30 mM mannitol) or HG (30 mM) serum-free medium plus cytokines and counted after 10, 20 and 30 days. The expression of p27, p21, IL6, TNFα, RELA/p65, KAT2B/PCAF genes and telomere length were assessed by qPCR; secreted cytokines by ELISA. Western Blot was used to evaluate acetylation of NFkB-p65 at lysine-310. H3K9me3 and RNA polymerase II recruitment to the RELA/p65 gene promoter were evaluated by ChIP-qPCR assay. NFkB-p65 nuclear translocation was evaluated by ImageStream whereas ROS and monocyte subpopulations were evaluated by flow cytometry after CellRox and CD14/CD16 staining respectively.

Results: In vitro HG exposure induced significant cell proliferation lowering, ROS production (n=4; p≤0.05), telomere shortening (n=4; p≤0.05) and up-regulation of p21 (n=8; p≤0.05) and p27 (n=7; p≤0.05) gene expression in CB-HSC. Interestingly, this senescent phenotype coincided with enhanced expression and secretion of inflammatory TNFα, IL6 cytokines (n=6; p≤0.01) and upregulation of NFkB-p65 transcription factor (n=10; p≤0.01). The analysis of the mechanisms involved in the regulation of NFkB-p65 expression and activation revealed the reduction of repressive epigenetic mark H3K9me3 at promoter level (n=6; p≤0.001) that correlated with increased RNA polymerase II recruitment. At the same time, the up-regulation of KAT2B/PCAF gene (n=10; p≤0.05), a histone acetyltransferase implicated in NFkB-p65 acetylation and activation, was associated with increased acetylation at lysine-310 (n=3; p≤0.05) and nuclear translocation of the transcription factor. Finally, once HG-HSCs were differentiated in vitro into myeloid lineage generated higher level of intermediate (CD14++CD16+) monocytes when compared to NG cells. Notably, BM-HSCs from T2DM-CAD patients displayed a similar senescent/inflammatory phenotype with significant higher expression of p27, NFkB-p65 and TNFα genes (n=8; p≤0.05) when compared to the control subjects. In addition, these cells, when differentiated in vitro into myeloid lineage, generated abnormal output of intermediate (CD14++CD16+) monocytes (n=4; p≤0.05)

Conclusion: Overall, our data show that hyperglycemia elicits intrinsic HSC alterations potentially responsible for the abnormal differentiation towards highly inflammatory monocyte subpopulations.

Supported by: Ministry of Health PE-2011-02348537; RC2019

Disclosure: V. Vigorelli: None.

165

Importance of tissue CCL5/CCR5 signalling on monocytic-MDSCs-derived macrophage polarisation and inflammation in epididymal fat of high fat diet-induced obese mice

P.-C. Chan, P.-S. Hsieh;

Department of Physiology & Biophysics, National Defense Medical Center, Taipei, Taiwan.

Background and aims: Obesity is characterized by chronic low-grade systemic inflammation, where myeloid cells play a critical role. Monocytic-MDSCs (M-MDSCs) have a CD11b+Ly6G-Ly6C+ phenotype, which are more similar to monocytes. CD11b+Ly6G-Ly6Chi cells (inflammatory monocytes) and CD11b+Ly6G-Ly6Clow cells (reparative monocytes) were recruited into the adipose tissue following proinflammatory state in adipose tissues and to insulin resistance. However, it remains unclear whether the augmented CCL5/CCR5 signal is important for the recruitment of M-MDSCs and monocyte subsets distribution in adipose tissue in the development of high fat diet (HFD)-induced obesity.

Materials and methods: Six-week-old male C57BL/6J wild-type (WT), CCL5-/- (CCL5-KO) and CCL5/CCR5-double KO (DKO) mice were fed a regular diet or an HFD (45% fat) for 20 weeks. Diet-induced pathophysiology was examined in mice after 20 weeks by quantitative real-time PCR, ELISA and flow cytometry. Co-cultures of bone marrow-derived MDSCs with condition medium (CM) from epididymal fat explant were performed.

Results: In the present result, the HFD feeding for 20 wks significantly increases body weight, HOMA-IR value, plasma and adipose tissue inflammatory cytokine levels and the increase in M1/M2 macrophage ratio along with the increases of CCL5 and CCR5 gene and protein expression in epididymal fat of wild type (WT) mice, which were attenuated in mice with CCL5 gene deletion (CCL5-KO). CCL5-KO mice exhibited decreased CD11b+Ly6G-Ly6Chi inflammatory monocytes and increased CD11b+Ly6G-Ly6Clow reparative monocytes in epididymal fat compared with WT mice. We performed intravenous injections of lentiviral vectors to deliver full-length CCL5 and CCR5 constructs to the epididymal fat. Notably, we observed a highly significant increase in CD11b+Ly6G-Ly6Chi inflammatory monocytes and decrease in CD11b+Ly6G-Ly6Clow reparative monocytes in CCL5 and CCR5-injected mice compared to empty vector controls. A similar trend was obtained in M1-like/ M2-like ratio. The results indicated that the CCL5/CCR5-dependent monocytic-MDSCs subsets distribute might involve into the pathogenesis of adipose tissue inflammation. To further determine the role of adipose tissue CCL5-mediated signaling in trans-differentiation of peripheral blood MDSCs into adipose tissue M1 or M2 macrophages, the condition medium (CM) using epididymal fat explant incubated for 24 hrs from HFD-fed WT and CCL5/CCR5-double KO (DKO) mice were cultured with isolated BM-derived MDSCs from WT mice. Our data showed that the co-incubation with CM from fat explant of HFD fed mice and isolated MDSCs significantly increased the ratio of MDSCs transformation into M1 macrophages, which failed to changes in those of CCL5/CCR5-DKO mice. In contrast, the number of the trans-differentiation of M2 macrophage from M-MDSCs were remarkably increased in CCL5/CCR5-DKO mice in comparison to WT mice.

Conclusion: It is suggested that that local CCL5/CCR5 signal pathway is crucial for the recruitment and transformation of blood M-MDSC into adipose tissue macrophages and subsequent inflammation reaction, which might further contribute to the development of obesity associated adipose tissue dysfunction and insulin resistance.

Supported by: MOST 108-2320-B-016-001

Disclosure: P. Chan: None.

166

Differences in biomarkers of inflammation between novel subgroups of patients with recent-onset diabetes

H. Maalmi1,2, C. Herder1,2, K. Strassburger1,2, O.-P. Zaharia1,2, J. Ratter1,2, Y. Karusheva1,2, K. Bódis1,3, W. Rathmann1,2, D. Markgraf1,2, V. Burkart1,2, J. Szendroedi1,3, M. Roden1,3;

1German Diabetes Center (DDZ), Düsseldorf, 2German Center for Diabetes Research (DZD), München-Neuherberg, 3Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.

Background and aims: A novel clustering approach identified five diabetes subgroups (clusters) in individuals with adult-onset diabetes. These subgroups have distinct progression trajectories of diabetes-related complications which may be explained by their different clinical profiles. Since inflammation is an established risk factor for diabetes-related complications, we aimed to characterize potential differences in biomarkers of inflammation between these diabetes subgroups.

Materials and methods: Serum levels of 74 protein biomarkers of inflammation were measured using proximity extension assay technology in 414 individuals with recent adult-onset diabetes from the German Diabetes Study (GDS) who were classified in five clusters based on data-driven analysis of six variables (age at diagnosis, body mass index, HbA1c, homeostasis model assessment of β-cell function and insulin resistance based on C-peptide, and glutamic acid decarboxylase antibodies). Pairwise differences between means of biomarkers of inflammation across the clusters were compared with generalised linear models before (model 1) and after adjustment (model 2) for the aforementioned variables used for the definition of the clusters. For both models, we used Tukey-Kramer and Bonferroni corrections (α=0.05/74=0.0007) to account for both pairwise and total multiple comparisons between clusters.

Results: The study participants were assigned to five clusters: mild age-related diabetes (MARD, 35%), mild obesity-related diabetes (MOD, 32%), severe autoimmune diabetes (SAID, 21%), severe insulin-resistant diabetes (SIRD, 9%) and severe insulin-deficient diabetes (SIDD, 3%). After adjustment for multiple testing, 23 biomarkers of inflammation had at least one pairwise cluster difference in model 1 (all p<0.0007). Biomarker levels were highest in the SIRD and lowest in the SIDD cluster. All biomarkers correlated with at least one of the variables used for the definition of the clusters (all p<0.05). After additional adjustment for these variables (model 2), 6 biomarkers (CASP-8, CCL20, CD5, EN-RAGE, IL-6, IL-17C) showed at least one pairwise difference between clusters (e.g. higher CASP8, CD5, EN-RAGE and IL-6 in SIRD compared to SIDD, all p<0.0007).

Conclusion: Novel diabetes subgroups show multiple differences in circulating levels of biomarkers of inflammation. Our data suggest a prominent role of inflammatory pathways in particular in the SIRD cluster.

Clinical Trial Registration Number: NCT01055093

Supported by: DZD

Disclosure: H. Maalmi: None.

167

Role of serum uteroglobin as new indicator for obesity and insulin resistance

K. Joung1, J. Kim1, S. Choung2, S. Kang3, H. Kim1,2, B. Ku1,2;

1Internal Medicine, Chungnam National University School of Medicine, Daejeon, 2Medical Science, Chungnam National University School of Medicine, Daejeon, 3Internal Medicine, Inje University Busan Paik Hospital, Busan, Republic of Korea.

Background and aims: Uteroglobin (UG), also called as secretoglobin family 1A member 1 (SCGB1A1), is known to be a multifunctional protein with anti-inflammatory properties. Although low-grade persistent inflammation is one of most important pathophysiology in type 2 diabetes mellitus (T2D), the role of UG in progression of T2D remains unknown. This study aims to investigate the relationship between serum UG levels and metabolic parameters in subjects with normal and abnormal glucose tolerance.

Materials and methods: A total of 240 subjects were enrolled in this study, and they were divided into three groups [80 with T2D, 80 with prediabetes (preDM) and 80 with normal glucose tolerance (NGT)]. Blood collection and 75-g oral glucose tolerance test were performed in the morning after an overnight fast > 8 hours. Fasting serum UG levels were measured using a quantitative sandwich enzyme immunoassay technique with an enzyme-linked immunosorbent assay kit (R&D systems, Inc., MN, USA). Demographic findings and serum UG were compared between three groups using ANOVA. And we investigated correlation between serum UG and clinical factors using linear regression analysis. We additionally recruited 20 subjects with T2D to evaluate whether serum UG levels are changed by metformin treatment

Results: Serum UG levels in T2D and preDM group was significantly lower than those of NGT group (p=0.013). In linear correlation analysis, serum UG was significantly correlated with age (r = 0.196, p = 0.002), BMI (r = -0.164, p = 0.030), HOMA-IR (r = -0.181, p = 0.005), HOMA-beta (r = -0.153, p = 0.019) and eGFR (r = 0.265, p < 0.001). A total of 20 subjects with T2DM were additionally enrolled and treated with metformin monotherapy. Serum UG levels before (14.43±0.97 ng/mL) and after 12 months (16.47±1.33 ng/mL) of treatment show significant difference (p = 0.043).

Conclusion: This study demonstrated the association between serum UG and various metabolic parameters in human. Serum UG are significantly related to insulin resistance and renal function, and metformin treatment in T2D patients restored serum UG levels to that of NGT subjects, possibly due to improved insulin resistance.

Clinical Trial Registration Number: 201509042

Disclosure: K. Joung: None.

168

Characterisation of natural killer cell subsets in adipose tissue of morbidly obese subjects associated with type 2 diabetes

M.E. Haugstøyl1,2, M. Cornillet3, N. Stiglund3, K. Strand1,2, G. Mellgren1,2, N. Björkström3, J. Fernø1,2;

1Department of Clinical Science, University of Bergen, Bergen, Norway, 2Hormone Laboratory, Haukeland University Hospital, Bergen, Norway, 3Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Systemic low-grade chronic inflammation represents a likely mechanistic link between obesity and type 2 diabetes (T2D). Inflammatory signaling in the adipose tissue (AT) seems to be of particular importance and several recent studies in mice have revealed natural killer (NK) cells as important players in the initial inflammatory events that take place. NK cells express a range of activating and inhibitory receptors that determine NK cell function, and the tissue environment of NK cells is also thought to give rise to their diversity. While tissue-resident NK cells have been identified in the liver, uterus and skin, much is still unknown about the identity of NK cell subsets in different human AT depots and how their composition associate with T2D. In this study, we have performed a deep phenotyping of NK cells in AT of morbidly obese subjects.

Materials and methods: Matched subcutaneous (SAT) and visceral (VAT) adipose tissue biopsies from patients undergoing bariatric surgery were enzymatically digested to isolate the immune cells and further analyzed using 27-color flow cytometry. The antibody panel included surface receptors to distinguish between circulating (CD56dimCD16+) and tissue-resident (CD56brightCD16-) NK cells and to determine NK cell function. Additionally, seven receptors identified through a broad surface proteome screening were included as novel AT-resident NK cell markers.

Results: We observed that tissue-resident NK cells from both SAT and VAT retained a maturation profile and effector phenotype similar to other peripheral tissues, with lower expression of perforin, granzyme B, and KIRs and with higher expression of NKG2A compared to circulating NK cells. Moreover, the AT-resident NK cells displayed a higher expression of CD26 (DPP4) and a lower expression of CD38 (cyclic ADP ribose hydrolase) compared to circulating NK cells, possibly reflecting an imbalance of the metabolism capacity. Interestingly, some markers revealed specific imprints of SAT and VAT on resident NK cells, with CCR5 being more expressed in VAT and Sialyl Lewis X more expressed in SAT, suggesting specific adaptions to the particular depots.

Conclusion: In this study, we have identified tissue-resident NK cell subsets in adipose tissue depots of morbidly obese subjects. Our findings suggest that the developmental process leading to the formation of a pool of resident NK cells in peripheral organs might be shared. However, it seems like the cells residing in SAT and VAT might face different nutritional and functional exposures that pressure them to adapt and metabolically reprogram. Use of clinical data has been approved by all patients and we are currently in the process of extracting these from the journals in order to investigate the relationship between NK cell subsets and T2D status. These data will be presented at the conference.

Supported by: Helse-Vest RHF

Disclosure: M.E. Haugstøyl: None.

OP 29 What's new in automated insulin delivery

169

Glycaemic outcomes and the importance of active insulin time in the Pivotal trial of the MiniMedTM Advanced Hybrid Closed-Loop (AHCL) system

A. Carlson1, B. Bode2, M. Christiansen3, S. Garg4, K. Kaiserman5, M. Kipnes6, D. Liljenquist7, A. Philis-Tsimikas8, R. Pop-Busui9, J. Reed10, J. Sherr11, D. Shulman12, R. Slover4, J. Thrasher13, AHCL Study Group;

1Park Nicollet International Diabetes Center, Minneapolis, 2Atlanta Diabetes Associates, Atlanta, 3Diablo Clinical Research, Walnut Creek, 4Barbara Davis Center for Childhood Diabetes, Aurora, 5SoCal Diabetes, Torrance, 6Diabetes and Glandular Disease Clinic, San Antonio, 7Rocky Mountain Diabetes and Osteoporosis Center, Idaho Fall, 8Scripps Whittier Diabetes Institute, San Diego, 9University of Michigan Health System, Ann Arbor, 10Endocrine Research Solutions, Roswell, 11Yale School of Medicine, New Haven, 12University of South Florida, Tampa, 13Medical Investigations Inc., Little Rock, USA.

Background and aims: The MiniMedTM AHCL system offers a 100 or 120mg/dL target set point (SP), autocorrect to 120mg/dL every 5 mins and have fewer Auto Mode exits, was evaluated in adults and adolescents with T1D.

Materials and methods: The 16-site, single-arm, in-home AHCL system trial enrolled 39 adolescents (14-21yrs) and 118 adults (≥22yrs) with T1D. After a 14-day baseline period using sensor-integrated pump, HCL feature or predictive low glucose feature, the AHCL feature was enabled with a 100 or 120mg/dL SP for ~45 days and then the other SP for ~45 days. The initial AIT at study start was 3-4 hours and adjusted per investigator discretion. Endpoints included safety events, changes in A1C, sensor glucose (SG), and percentage of time in (%TIR), below (%TBR) and above (%TAR) target range (70-180mg/dL; 3.9-10mmol/L).

Results: Auto Mode was used ≥95% of the time and autocorrection was 22% of daily bolus. Outcomes for the overall group and each age group at baseline and study period (100 and 120mg/dL SP), as well as during 100mg/dL SP use, are shown (Table). At the 100mg/dL SP, the AIT was shortened/lengthened/unchanged in 12%/1.4%/86.6% of participants, respectively. The %TIR at the 100mg/dL SP and AIT durations of ≤2, >2 to ≤3, >3 to ≤4 and >4 hours was 78.8±5.5% (n=29), 75.6±7.8% (n=76), 73.9±6.7% (n=65), and 70.6±7.9% (n=4), respectively. The %TIR at the 120mg/dL SP was 75.0±7.0% (n=26), 74.4±7.8% (n=76), 72.3±7.9% (n=74), and 68.1±10.7% (n=2), respectively. The %TBR (<70mg/dL; <3.9mmol/L) at the 100mg/dL SP was 2.6±2.0% (n=29), 2.9±2.3% (n=76), 2.8±2.3% (n=65), and 4.8±3.2% (n=4), respectively. The %TBR at the 120mg/dL SP was 1.9±1.9% (n=26), 2.0±1.6% (n=76), 1.7±1.3% (n=74), and 1.4±0.4% (n=2), respectively. The best results (SG of 141±8.8mg/dL, %TIR of 78.8±5.5% and %TBR of 2.6±2.0%; N=29) were in a subset using a 100 mg/dL SP and an AIT of 2 hrs. No DKA or severe hypoglycemia episodes occurred in the study period.

Conclusion: These data demonstrate that AHCL is safe, that it significantly improved A1C and SG, and that glycemic control can be optimized with target SP and AIT settings. Specifically, shortening the AIT improves %TIR without increasing %TBR.

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Clinical Trial Registration Number: NCT03959423

Disclosure: A. Carlson: None.

170

Increased time in range and sustained Auto Mode use in 670G hybrid closed-loop system users: real world experience in DIABETER

D. Mul1, A. Arrieta2, P. Dekker1, E. Birnie3, T.C.J. Sas1, H.-J. Aanstoot1, H.J. Veeze1;

1Diabeter, Rotterdam, 2Medtronic, Maastricht, 3UMCG, Groningen, Netherlands.

Background and aims: Current outcomes in type 1 diabetes require more automated systems that not only improve glucose regulation, but also reduce burden by reducing the need for constant focus on pump handling for patients. Time in Auto Mode (automated basal insulin delivery, based on CGM data) is a crucial factor for success of the Medtronic 670G hybrid closed-loop (HCL) system. In a recent study the time in Auto Mode of the 670G HCL system decreased from 74% to 35% over 12 months. DIABETER, a treatment and research centre for people with diabetes, introduced the 670G HCL system with a comprehensive and structured education and an extended support and follow-up program. The aims of this real-word data analysis were 1) to assess if our introduction program helps patients to maintain the appropriate percentage of time spent in Auto Mode and 2) to assess glucose metrics after start on the 670G system.

Materials and methods: We included 77 people with type 1 diabetes on pump or MDI (with or without glucose sensor monitoring) who switched to the 670G HCL system for clinical reasons and/or patient preference. We analysed data from 1-OCT-2018 until 13-MAR-2020 of patients who provided consent for use of their pump and CGM data for research purposes and who had at least 10 days of sensor data available at any time point of evaluation. The percentage time in Auto Mode was calculated at 1, 3, 6, 9 and 12 months after start on the 670G HCL system by dividing time in Auto Mode by total pump time. Glucose metrics (time below range [TBR: <3.9 mmol/L], time in range [TIR: 3.9 - 10 mmol/L] and time above range [TAR: >10 mmol/L]) were also calculated.

Results: Patient characteristics: mean (SD) age, 20.1 (10.2) years; mean (SD) diabetes duration, 11.8 (8.4) years; gender (% male), 49.4%. HbA1c was available for 73/77 patients. Mean (SD) HbA1c was 7.0 (0.7)%. Median percentage time in Auto Mode remained relatively stable over time at 84-95% (figure). Compared to the pre-Auto Mode phase, TIR increased (+11%) and remained stable while using Auto Mode. This was also reflected by decreases in TBR and TAR (-0.5%, -11%)(figure).

Conclusion: After starting on the 670G HCL system, the percentage time in Auto Mode is sufficiently high and remained high over time in the observed DIABETER patients, as opposed to previous reports. In addition, this results in a sustained improvement of glucose metrics, compared with the pre-Auto Mode situation, and is in line with ranges specified by international guidelines. Although further analysis is needed, our comprehensive education and extended support and follow-up program seems to help patients to stay in the Auto mode and thus facilitates optimal use of the 670G HCL system.

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Disclosure: D. Mul: None.

171

Automated insulin delivery in free-life shows better glucose control when used 24/7 vs evening and night in pre-pubertal children with type 1 diabetes: The Free-life Kid AP Study

E. Renard1,2, N. Tubiana-Rufi3, E. Bonnemaison4, R. Coutant5, F. Dalla-Vale6, E. Bismuth3, N. Faure4, N. Bouhours-Nouet5, A. Farret1,2, J. Place2, M. Breton7, Free-life Kid AP Study Group;

1Dept. of Endocrinology, Diabetes, CHU Montpellier, Montpellier, France, 2Institute of Functional Genomics, University of Montpellier, Montpellier, France, 3Dept. of Pediatric Endocrinology and Diabetology, APHP, Robert Debré Hospital, Paris, France, 4Dept. of Pediatric Medicine, CHU Tours, Tours, France, 5Dept. of Pediatric Endocrinology and Diabetology, CHU Angers, Angers, France, 6Dept. of Pediatrics, CHU Montpellier, Montpellier, France, 7Center of Diabetes Technology, University of Virginia, Charlottesville, USA.

Background and aims: Control of type 1 diabetes (T1D) is a daily challenge in children because of highly variable insulin needs. We assessed the safety and efficacy of automated insulin delivery (AID) in free-life in pre-pubertal T1D children while used 24/7 vs. evening & night (EN).

Materials and methods: One hundred and twenty-two pre-pubertal T1D children treated by insulin pumps (CSII) were enrolled in a multicenter prospective open label randomized control trial comparing glucose control with 24/7 or EN use of the hybrid Tandem t:slim X2 with Control-IQ AID system for 18 weeks. After a 3-week run-in phase for Tandem X2 pump and Dexcom G6 CGM training, AID was activated 24/7 or EN according to randomization. Primary outcome is %time spent in 70-180 mg/dl target range (%TIR); secondary outcomes include average CGM, %time below and above target range (%TBR and %TAR, respectively), and the same metrics during day-time and overnight.

Results: Patient characteristics at inclusion were: 49F/73M, age: 8.6±1.6, T1D duration: 5.2±2.3 years, CSII use: 4.6±2.5 years, HbA1c: 7.7±0.7% (61±5.3 mmol/mol). Except for 3 early drop-outs, 119 completed the trial. AID was effective for 93.6 and 50.9% of time on 24/7 and EN modes, respectively. The increase of %TIR was significantly larger on 24/7 vs. EN mode: 52.9±9.5 to 67.5±5.6 (+14.5%, CI: 12.4-16.7%) vs. 55.2±10.8 to 64.7±7.0 (+9.5%, CI: 7.4-11.6%), p<0.001. While mean %TBR was similarly reduced: 4.4 to 2.6 (24/7) and 4.6 to 2.7 (EN), mean %TAR decreased significantly more on 24/7 vs. EN mode: 42.9 to 29.8 (-12.2%, 95CI: 9.7-14.6%) vs. 39.7 to 32.3 (-7.4%, 95CI: 5.0-9.9%), p=0.008. Increased %TIR was superior on 24/7 vs. EN mode during daytime: 54.3±10.1 to 62.6±6.2 vs. 55.7±11 to 58.4±7.6, p=0.001, and reached 77.2% on average overnight on both modes. CGM levels (mg/dl) more significantly decreased on 24/7 vs. EN mode: 171.9±20.3 to 158.1±10.6 vs. 168.9±20.8 to 162.6±12.1, p=0.02, while % of patients with HbA1c level<7% (53 mmol/mol) moved from 14 to 36 (24/7) vs. 13 to 22 (EN), from baseline to week 18. SD of CGM levels (mg/dl) was also reduced significantly more on 24/7 vs. EN mode: 69.6±9.8 to 61.5±8.0 vs. 69.8±11.3 to 66.7±10.0, p=0.0001. %TIR was increased through the whole range of baseline HbA1c and %TIR levels and always more with 24/7 use. No ketoacidosis or severe hypoglycemia occurred during the study.

Conclusion: Our study demonstrates the safety and efficacy on glucose control of Tandem Control-IQ AID system used in free-life in pre-pubertal T1D children for both 24/7 and EN use. 24/7 use shows better performance than EN use, with 14.5% more time in range, or more than 3.6h/day, with no safety issue. The post-study extension for 18 weeks will assess the sustainability of 24/7 use of AID in the full study cohort.

Clinical Trial Registration Number: NCT03739099

Supported by: French MoH

Disclosure: E. Renard: Grants; French Ministry of Health. Non-financial support; Tandem, Dexcom.

172

Individual response of automated glycaemic control with the iLet bionic pancreas in the insulin-only vs bihormonal configuration with a stable glucagon analogue, dasiglucagon

J.S. Sherwood1, C.A. Balliro1, R.Z. Jafri1, L.E. Castellanos1, M. Hillard1, M. Sullivan1, R. Selagamsetty2, E. Greaux1, H. Zheng3, F. El-Khatib2, E.R. Damiano2, S.J. Russell1;

1Diabetes Research Center, Massachusetts General Hospital, Boston, 2Department of Biomedical Engineering, Boston University, Boston, 3Biostatistics Center, Massachusetts General Hospital, Boston, USA.

Background and aims: Reductions in plasma glucose are often achieved at the expense of increased hypoglycemia in patients with type 1 diabetes. We assessed the efficacy of the iLet™ bionic pancreas system in the bihormonal configuration delivering insulin and dasiglucagon versus the insulin-only configuration in adults with type 1 diabetes.

Materials and methods: We performed an outpatient, home-use, random-order, controlled trial we compared automated glycemic control with the iLet bionic pancreas in the insulin-only (IOBP) vs. the bihormonal (BHBP) configurations for one week each in adult subjects (n=10) with type 1 diabetes. Subjects used their typical insulin analog (lispro or aspart) during both arms of the study. During the BHBP period the iLet delivered micro-doses of dasiglucagon, a glucagon analog stable in aqueous solution (Zealand Pharma). The insulin control algorithm was identical in both configurations, was initiated solely based on each subjects’ body mass without any information regarding patients’ baseline insulin needs, and used a glucose target of 110 mg/dl. We used an autoregressive time series model to determine statistical significance for differences between arms for each individual subject.

Results: The group mean CGM glucose was lower in the BHBP arm vs. the IOBP arm (139±11 vs. 149±13 mg/dl, p=0.004) while the % of time with CGMG <54 mg/dl was nominally reduced (0.2%, IQR 0-0.4 vs. 0.6%, IQR 0.2-1.1%, p=0.11). Eight subjects had a significantly lower mean CGMG during the BHBP arm (p<0.05, mean improvement 12±7 mg/dl), while in the remaining two subjects there was no significant difference (nominal absolute difference 2±1 mg/dl). Eight of the ten subjects had a nominal reduction of the % of time <54 mg/dL in the BHBP arm (mean nominal difference -0.5%, range -0.1 to -1.0%), none of which were statistically significant.

Conclusion: In a trial comparing the BHBP and IOBP configurations of the iLet we found a significant benefit of adding dasiglucagon in eight of ten subjects, allowing each of them to achieve a lower mean glucose without increased rates of hypoglycemia.

Clinical Trial Registration Number: NCT 03840278

Supported by: Study was funded by Beta Bionics and Zealand Pharma donated the study drug, dasiglucagon

Disclosure: J.S. Sherwood: None.

173

First home evaluation of the Omnipod Horizon™ automated glucose control system in children with type 1 diabetes

G.P. Forlenza1, B.A. Buckingham2, A. Criego3, S.A. Brown4, B.W. Bode5, C.J. Levy6, T.T. Ly7, Omnipod Horizon Study Group;

1Barbara Davis Center for Diabetes, University of Colorado School of Medicine, Aurora, 2Department of Pediatrics, Division of Pediatric Endocrinology, Stanford University, Stanford, 3Department of Pediatric Endocrinology, Park Nicollet Clinic, International Diabetes Center at Park Nicollet, Minneapolis, 4Division of Endocrinology and Medicine, University of Virginia, Charlottesville, 5Atlanta Diabetes Associates, Atlanta, 6Icahn School of Medicine at Mount Sinai, New York, 7Insulet Corporation, Acton, USA.

Background and aims: The Omnipod Horizon™ System is a hybrid closed-loop (HCL) system consisting of a tubeless insulin pump with a control algorithm linked to a Dexcom G6 sensor. The system provides automated insulin delivery with customizable glucose targets from 110-150mg/dL, adjustable by time of day to allow therapy personalization. This study is the first outpatient safety and effectiveness evaluation of the system, including use at the higher targets of 130-150mg/dL.

Materials and methods: Participants aged 6-13.9y with T1D>6mo and A1C<10.0% used the HCL system at home for 14 days over the winter holidays with unrestricted eating and exercise (8 participants spent first 2 days in a hotel). Participants set protocol-determined higher targets of 130, 140, and 150mg/dL for 3 days each, then could freely choose their targets from 110-150mg/dL for the last 5 days. Primary outcomes were safety measures and percent time 70-180mg/dL for the 5 days of HCL use with free choice of target, as well as for the first 9 days of HCL use stratified by target glucose.

Results: Participants thus far (n=15) had a mean±SD age of 11±2y, T1D duration 5±3y, and A1C 7.7±0.9%. Glycemic outcomes are shown in the Table. During the free choice period, participants primarily chose the 110mg/dL (69% of study time), 120mg/dL (10%), and 130mg/dL (21%) targets. For 72 patient-days of HCL use during the free choice period, percent time from 70-180mg/dL was 64.1±10.0%. Percent time <70mg/dL was low: 0.9±1.2% overall and 0.5±0.5% overnight. At the 130, 140, and 150mg/dL targets, percent time from 70-180mg/dL was 63.4±7.9%, 64.2±11.6%, and 52.1±11.7%, respectively. Percent time <54 and <70mg/dL was low and tended to decrease with increased target. There were no severe adverse events.

Conclusion: The HCL system was safe and performed well in children with T1D when used at home for 5 days with free choice of target glucose, as well as when used with higher glucose targets. Participants were invited to continue in a 3mo outpatient study of the system, which is currently underway.

figurebg

Clinical Trial Registration Number: NCT04176731

Supported by: Insulet Corporation

Disclosure: G.P. Forlenza: Employment/Consultancy; Insulet, Medtronic, Tandem, Dexcom, Lilly. Grants; Insulet, Medtronic, Tandem, Dexcom, Lilly, Abbott.

174

Novel fully automated fiasp-plus-pramlintide artificial pancreas for type 1 diabetes: randomised controlled trial

M. Tsoukas1, D. Majdpour2, J. Rutkowski3, A. El Fathi3, J.-F. Yale1, N. Garfield1, L. Legault4, A. Haidar3;

1Medicine, Division of Endocrinology and Metabolism, McGill University, Montreal, 2Biomedical Engineering, McGill University, Montréal, 3Biomedical Engineering, McGill University, Montreal, 4Pediatrics, Division of Endocrinology, McGill University, Montreal, Canada.

Background and aims: Current artificial pancreas systems improve glycemic control in type 1 diabetes but still require manual entry of meal carbohydrate content, which is a burdensome, error-prone task. We aimed to alleviate this burden by developing a novel fully automated fiasp-plus-pramlintide artificial pancreas.

Materials and methods: We conducted a randomized crossover non-inferiority trial comparing (i) a fully automated fiasp-and-pramlintide artificial pancreas and (ii) fiasp-alone hybrid artificial pancreas with carbohydrate-matched meal boluses, in 23 adults with type 1 diabetes (age 35±15 years, HbA1c 8.1±1.4%). Fiasp and pramlintide were delivered using a novel dosing algorithm at a fixed ratio (10 μg/u) to mimic a co-formulation. Each participant completed two 24-hour inpatient interventions in which participants ate a self-selected snack (31g±8g), breakfast (51g±21g), lunch (74g±21g), and dinner (77g±22g). Half of the participants in the afternoon completed 40 minutes of moderate exercise. The primary outcome was time in target 3.9-10.0 mmol/L.

Results: The fully automated system achieved similar time in target (71%) compared to fiasp-alone hybrid system (75%, p=0.47), but with less insulin delivery (53u, 64u, p=0.0034) and less time <3.9 mmol/L (median 0%, 1.4%, p=0.039). The fully automated system achieved comparable time >10 mmol/L compared to the fiasp-alone hybrid system (27%, 22%, respectively; p=0.29), as well as comparable >13.9 mmol/L (6.5%, 5.7%, respectively; p=0.80) and time >16.7 mmol/L (1.8, 2.3, respectively; p=0.81). During the day, the fully automated system had a slightly higher time above >10 mmol/L compared to hybrid fiasp-alone system (5 hours, 3.6 hours; p=0.099) but similar time >13.9 mmol/L (1.3 hours, 1 hours; p=0.62) and similar time >16.7 mmol/L (0.48 hours, 0.36 hours; p=0.76). Non-mild nausea was reported by 3 participants (13%) with the fully automated system compared to 0 with the fiasp-alone system.

Conclusion: Our novel fiasp-and-pramlintide artificial pancreas is fully automated and non-inferior to the first-generation fiasp-alone hybrid artificial pancreas that requires carbohydrate counting

figurebh

Clinical Trial Registration Number: NCT03800875

Supported by: Diabetes Canada

Disclosure: M. Tsoukas: Grants; Eli Lilly and Company. Lecture/other fees; Novo Nordisk.

OP 30 Understanding the mechanisms of diabetic kidney disease

175

The protective effect of bone marrow mesenchymal stem cells on diabetic nephropathy modulated by macrophage polarisation

S. Wang, J. Xie, X. Yu;

Department of Endocrinology, Tongji Hospital, Huazhong University of Science & Technology, Wuhan, China.

Background and aims: The accumulation of M1 macrophages in kidney tissues plays an important role in the development of diabetic nephropathy (DN), while mesenchymal stem cells (MSCs) have been found to exert immune-modulatory characteristics. We aim to identify that MSCs may ameliorate DN by converting M1 macrophages to M2 macrophages in kidney, and further to explore the mechanism of MSCs on modulating macrophage polarization.

Materials and methods: C57BL/6J mice were used to induce DN models with a combination of high-fat diet and low-dose streptozotocin. The mice were treated with MSCs (5×105, once every two weeks for six times) or saline via tail vein. Blood and urine were measured. Periodic acid-Schiff staining (PAS) and immunostaining of renal tissues was performed. The gene expressions of pro-inflammatory and pro-fibrosis factors in renal tissues were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). In vitro study we explored whether MSCs and MSCs conditioned medium (MSC-CM) could modulate the macrophage polarization under inflammatory condition using flow cytometric analysis, RT-PCR, as well as Western Blot.

Results: Serum creatinine and urinary microalbumin to creatinine ratio were significantly reduced in DN group with MSCs treatment (p < 0.05). The PAS indicated that the glomerulosclerosis was dramatically attenuated (p < 0.05). The gene expression of inflammatory factors including fibronectin (FN) and transforming growth factor-β1 (TGF-β1) , interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were reduced (p < 0.05). Interestingly, immuohistochemtry showed the expression of CD163, an M2 maker, was extremely increased in kidney (p < 0.01). However, the expression of iNOS, an M1 marker, were significantly decreased (p < 0.001). In vitro study, the M2 makers such as arginase-1 (Arg1) and CD206 gene expression were markedly increased in MSCs and MSC-CM treatment group (p < 0.05). In contrast, the M1 makers such as TNF-α and iNOS gene expression were extremely decreased (p < 0.05). In addition, the protein expression of phosphorylation of p65 and inhibitor of nuclear factor kappa-B kinase-α (IKK-α) were significantly decreased in macrophage treated with MSCs and MSC-CM under inflammatory condition (p < 0.001).

Conclusion: Our results found that MSCs had the protective effect on DN by switching the macrophages phenotype from M1 to M2. MSCs could ameliorate the renal inflammation and fibrosis by inducing macrophage polarization via NF-κB signal pathway.

Supported by: NSFC

Disclosure: S. Wang: None.

176

Activation of the adiponectin receptor ameliorates glomerular inflammation and injury

S.H. Lindfors1, Z. Polianskyte-Prause1, E. Lehtonen2, J. Tienari3, H. Nisen4, T. Mirtti3,5, S.H. Lehtonen1,2;

1Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, 2Department of Pathology, University of Helsinki, Helsinki, 3Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, 4Department of Urology, Helsinki University Hospital, Helsinki, 5Research Program in Systems Oncology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Background and aims: Chronic low-grade inflammation contributes to the pathogenesis of diabetic kidney disease. One of the diabetes-related factors inducing chronic inflammation is increased serum levels of bacterial lipopolysaccharides (LPSs). Type 2 diabetes (T2D) associates with lowered serum levels of adipose tissue-derived adiponectin. As adiponectin has anti-inflammatory properties, its deficiency may support chronic inflammation and predispose to the development of glomerular injury. Thus, we investigated whether activating the adiponectin receptor with a specific agonist, AdipoRon, ameliorates glomerular inflammation in a mouse model of T2D, and studied in podocytes in vitro and in human glomeruli ex vivo whether AdipoRon protects against LPS-induced inflammation and injury.

Materials and methods: Mouse study: DBA/2J mice were fed with low fat diet (LFD), high fat diet (HFD), or HFD supplemented with AdipoRon, for 9 weeks. In vitro study: Differentiated human podocytes (AB8/13) were pre-treated with AdipoRon for 2 hours and exposed to Escherichia coli LPS for additional 1, 24 or 48 hours. Protein expression was studied by immunoblotting and TNFα secretion by ELISA. Apoptosis was studied by AnnexinV/7-AAD staining followed by flow cytometry and cell migration by a scratch assay. Ex vivo study: Human glomeruli were isolated from non-cancerous parts of surgical nephrectomies from 5 subjects without T2D and 4 patients with T2D, resuspended in culture media and treated with LPS or LPS and AdipoRon for 24 hours. Secreted cytokines were measured by multiplex ELISA.

Results: AdipoRon treatment reduced HFD-induced weight gain in mice. HFD-fed mice had elevated blood glucose and serum LPS levels while HFD-fed mice receiving AdipoRon did not differ from LFD-fed mice. HFD alone and with AdipoRon supplementation induced a mild increase in albuminuria, whereas AdipoRon treatment reduced HFD-induced glomerular hypertrophy. Immunohistochemical analyses showed that AdipoRon reduced HFD-induced glomerular expression of TGFβ, fibronectin, phospho-NFκB and F4/80, indicating downregulation of inflammation and fibrosis. Transmission electron microscopy analysis of mouse renal tissue revealed that AdipoRon reduced the thickness of glomerular basement membrane and effacement of podocyte foot processes. In cultured podocytes, AdipoRon prevented LPS-induced upregulation of phospho-NFκB, phospho-JNK and phospho-p38 expression, secretion of TNFα, migration and apoptosis. In isolated human glomeruli, LPS increased and AdipoRon reduced the secretion of inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, IL-18, TNFα and VEGF-A.

Conclusion: AdipoRon ameliorated inflammation and injury in the glomeruli of HFD-fed mice and in cultured podocytes and isolated human glomeruli stimulated with LPS. Our findings suggest that activating the adiponectin receptor by AdipoRon is a potent strategy to lower glomerular inflammation and thereby prevent inflammation-related renal injury in obesity and T2D.

Supported by: Finnish Cultural Foundation, Finnish Kidney Foundation, Finnish-Norwegian Medical Foundation

Disclosure: S.H. Lindfors: Grants; Finnish Cultural Foundation; Päivikki and Sakari Sohlberg Foundation, Finnish-Norwegian Medical Research Foundation; Finnish Kindey Foundation.

177

Long non-coding RNA MALAT1 mediates endothelial-to-mesenchymal transition and kidney fibrosis

R. Bijkerk1, A. Lafzi2, Y.W. Au1, W. Stam1, J.M.G. Duijs1, A. Koudijs1, E. Lievers1, T.J. Rabelink1, H. Kazan2, A.J. van Zonneveld1;

1Leiden University Medical Center, Leiden, Netherlands, 2Antalya International University, Antalya, Turkey.

Background and aims: Diabetic nephropathy (DN) associates with the development of renal interstitial fibrosis characterized by a loss of the microvasculature and myofibroblast formation. Endothelial cells (ECs) are important for maintaining a healthy microvasculature while ECs also provide a potential source for myofibroblasts through endothelial-to-mesenchymal transition (EndoMT). Here, we aimed to identify a role for long non-coding RNAs (lncRNAs), novel central post-transcriptional regulators, in ECs in the development of kidney fibrosis.

Materials and methods: We used VE-cadherin-ERT2;tdTomato mice to label and trace endothelial cells. We applied both the ischemia-reperfusion injury (IRI) and unilateral urethral obstruction (UUO) models followed by FACS sorting of the tomato-positive cells from healthy and diseased kidneys. Subsequently, we isolated RNA from these cells and profiled for lncRNAs, which identified the conserved MALAT1 as a potential key mediator of kidney fibrosis. Functional in vitro and in vivo studies were performed to assess the role of MALAT1 in EC function and kidney fibrosis.

Results: Upon kidney injury, we observed substantial co-localization of VE-cadherin-derived tomato positive signal with a-SMA staining, indicating that a significant portion (~15-20%) of myofibroblasts originated from ECs. We confirmed that ECs acquired a myofibroblast phenotype by using qPCR on FACS sorted tomato-positive cells showing reduced expression of EC markers CD31 and VE-cadherin while myofibroblast markers α-SMA and col1α1 increased. In UUO and IRI, we found 586 and 416 lncRNAs to be differentially expressed (>2-fold, p<0.05) in the VE-cadherin-derived tomato-positive cells, respectively. Using bioinformatics analyses to determine transcription factor motif-enrichment amongst differentially expressed lncRNAs we found strong enrichment for HMGA1 binding sites. Using ChIP-seq, we confirmed this enrichment of HMGA1 binding sites in the promoters of differentially regulated lncRNA, including MALAT1. In vitro, we demonstrated that MALAT1 disrupts EC homeostasis, as silencing MALAT1 resulted in increased barrier function, less leakage and less EndoMT. In vivo, we found that gapmer-mediated knockdown of MALAT1 abrogated kidney fibrosis. Lastly, we found both renal and circulating MALAT1 levels to be increased in DN patients compared to healthy controls.

Conclusion: We demonstrated that MALAT1 is important for endothelial cell function and may provide novel strategies to counteract the development of diabetic nephropathy.

Supported by: EFSD/Boehringer Ingelheim Clinical European Diabetes Research Programme and Dutch Kidney Foundation

Disclosure: R. Bijkerk: Grants; European foundation for the study of diabetes, Dutch Kidney Foundation (Kolff grant).

178

Rock1/ampk axis regulates the development of diabetic kidney disease via modulation of fatty acid utilisation

Y. Nagai, K. Matoba, Y. Takeda, T. Akamine, Y. Kanazawa, T. Yokota, K. Utsunomiya, R. Nishimura;

Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan.

Background and aims: The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic glomerulosclerosis. Accumulating evidence shows that renal dysfunction in diabetic patient is associated with abnormal fatty acid oxidation in the kidney. However, the interaction of Rho-kinase and fatty acid oxidation in diabetic kidney remain unclear. In this study, we aimed to investigate the contribution of Rho-kinase to fatty acid utilization in mesangial cells.

Materials and methods: 8-weeks-old mice were divided into the following groups: nondiabetic db/m mice, diabetic db/db mice, and db/db mice treated with the Rho-kinase inhibitor, fasudil. Fasudil was administered in drinking water (100 mg/kg/day). Fasudil treatment was continued for 16 weeks. Mice were euthanized at 24 weeks of age.

Results: Glomeruli isolated from type 2 diabetic db/db mice demonstrated decreased gene expression of fatty acid oxidation mediators such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), CD36, carnitine palmitoyltransferase 1A (CPT1A). Chemical inhibition of Rho-kinase restored expression of fatty acid oxidation-related genes in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed that Rho-kinase mediates phosphorylation of AMPK and thus increases the expression of PGC-1α. Extracellular flux analyzer demonstrated that Rho-kinase inhibition improves TGF-β-induced mitochondrial dysfunction. Furthermore, Rho-kinase inhibitor suppresses ROS production as a result of mitochondrial damage due to abnormal fatty acid metabolism. Knockdown by small interfering RNA against each Rho-kinase isoform, ROCK1 or ROCK2, showed that ROCK1 but not ROCK2 controls this metabolic machinery. Consistent with this result, mesangial cells isolated from ROCK1 deficient mice were protected from TGF-β-mediated downregulation of fatty acid metabolism. These observations indicate that ROCK1 is a key player in the development of diabetic renal injury.

Conclusion: Glomerular ROCK1 may be a potential therapeutic target for the treatment of diabetic kidney disease.

Disclosure: Y. Nagai: None.

179

The role of the Irisin-AMPK axis in the improvement of diabetic nephropathy in exercised rats

G.P. Formigari, M.N. Dátilo, J.M. Lopes de Faria, J.B. Lopes de Faria;

Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil.

Background and aims: Several studies suggest that a reduction in AMP kinase (AMPK) activity in diabetes could contribute to the development and progression of diabetic nephropathy (DN). Clinical and experimental studies also suggest that physical activity can improve the markers of DN. Moreover, physical activity activates AMPK in skeletal muscle to promote glucose uptake and stimulate the secretion of some hormones (i.e., miokynes), such as irisin. Various animal models of chronic kidney disease have recently shown that elevation in irisin could reduce kidney fibrosis. However, the mechanism by which physical activity improves DN is not well understood. Therefore, this study aims to investigate the contribution of the irisin-AMPK axis to the amelioration of DN in diabetic rats that were submitted to an exercise program.

Materials and methods: Wistar rats (eight weeks of age) were rendered diabetic through an intravenous injection of streptozotocin. Rats were allocated to three groups: control (CT, non-diabetic), sedentary diabetic (DM) and diabetic animals submitted to an exercise training protocol on a treadmill (DM+Exe) for eight weeks. At the end of the experimental period, the renal cortex and muscle (gastrocnemius) were harvested to assess kidney inflammation and fibrotic parameters, the activity of the AMPK and the expression of the irisin using Western blot and immunohistochemistry (IHQ) techniques. The AMP and ATP levels were determined using HPLC.

Results: The DM rats showed a decrease in body weight compared with the CT rats (p < 0.05). The fasting blood glucose (78 ± 10 mg/dL vs. 497 ± 68 mg/dL) and albuminuria (2.4 ± 0.3 mg/g vs. 3.3 ± 0.6 mg/g) were higher (P < 0.05) in the DM rats compared with the CT rats. Exercise did not modify the body weight or blood glucose levels. However, the DM+Exe rats displayed a decrease in albuminuria (3.3 ± 0.6 mg/g vs. 2.5 ± 0.7 mg/g, P = 0.04) and systolic blood pressure (161 ± 18 mmHg vs. 141 ± 18 mmHg, P = 0.03) compared with the DM rats. The kidney weight, glomerular volume and renal expression of the fibronectin and collagen type IV were higher in the DM rats compared with the CT rats (P < 0.05). Exercise training reduced the glomerular volume and kidney expression of the fibronectin and collagen type IV compared with the DM rats (P < 0.05). The exercised rats also showed a reduction in the following kidney inflammation parameters: the acetylation of NF-κB (p65), the expression of TNF-α, renal macrophage infiltration (IHQ for F4/80) and cleavage of caspase-1 when compared with the DM rats (P<0.05). Furthermore, exercise increased kidney phosphorylation of threonine 172 AMPKα (p-T172AMPK), the expression of sirtuin 1 (SiRT1) and the association of SiRT1 with NF-κB (p65) (P < 0.05). In the gastrocnemius, the DM rats showed a decrease in phosphorylation of serine79 ACC (p-S79ACC), in a marker of mitochondrial biogenesis (PGC1-α) and irisin compared with CT rats (P < 0.05). The exercised rats showed an increase in muscle AMPK activity, as assed by the p-T172AMPK and p-S79ACC, which is associated with an elevation in the AMP/ATP ratio and the expression of PGC1-α and irisin, compared with DM rats (P < 0.05).

Conclusion: These results indicate that exercise can attenuate markers of DN and kidney inflammation in diabetic rats, which may be mediated by cross-talk between the muscle and the kidney and is promoted by an elevation in irisin and the activity of AMPK.

Supported by: FAPESP (14/22687-0) and CAPES

Disclosure: G.P. Formigari: None.

180

Erasing metabolic alteration in proximal tubular cells under hyperglycaemic condition using inducible CRISPR/Cas9 PGC1a hESC-derived 3D kidney organoids

C. Hurtado del Pozo1, P. Prado1, A. Gavalda-Navarro2, E. Garreta1, N. Montserrat1;

1Institute for Bioengineering of Catalonia, IBEC, Barcelona, 2Universidad de Barcelona, Barcelona, Spain.

Background and aims: There are about 60 million people with diabetes in the European Region. Among all the complications, Diabetic Nephropathy (DN) is the leading cause of end stage renal disease and it can explain most excess mortality associated with diabetes. From all the kidney cells type, proximal tubular renal cells (PTC) represent one of the most vulnerable cell types in DN due to its high-energy demand. Due to the increasing evidences which suggest that the metabolic state of a cell contributes to disease development we hypothesize that diabetic nephropathy (DN) is promoted by the metabolic alterations (hyperglycaemia) occurring during kidney development mainly in PTCs. We propose that these metabolic alterations can be erased modifying the intracellular metabolic profile of the kidney cells in a mitochondrial metabolism-dependent manner.

Materials and methods: Kidney organoid differentiation: Differentiation protocol was developed in our lab. Isolation of tubular epithelial cells from organoids was performed by flow cytometry. Purification of Total RNA and Quantitative RT-PCR was performed using Tri-Reagent following manufacturer’s recommendations. cDNAs (25 ng/well) were used to quantify gene expression by Quantitative RT-PCR. Seahorse Cell Mito Stress Test was performed to measure Oxygen Consumption rate (OCR) in Kidney organoids and tubular renal cells .

Results: To check if hyperglycaemia has an effect in the development of PTC in kidney organoids, we cultured them under oscillatory glucose levels versus constant normal glucose for 7 days at day 16 of differentiation. Preliminary results showed that kidney organoids treated under oscillatory glucose had lower expression of PTC makers and lower number of PTCs than control kidney organoids analysed by RT-PCR and flow cytometry analysis, respectively. In order, to characterize PTCs from oscillatory glucose versus control kidney organoids, we isolated and cultured them in renal epithelial cell growth medium (normal glucose concentration) for a month. PTC isolated from diabetogenic kidney organoids showed higher oxygen consumption rate than PTC from control group. No changes were found in mitochondria copy number neither in oxidative phosphorylation (OXPHOS) complexes expression by western blot. However, we found lower expression of the mitochondrial master regulator PGC1a in PTC from diabetogenic organoids by RT-PCR and immunofluorescence.Into the light of the results we generated an inducible CRISPR/Cas9 engineered line for PGC1α. Overexpression of PGC1a during kidney organoid development showed higher expression of tubular markers such as SLC3A1 and AQ1 analysed by RT-PCR however we did not find differences at PTCs number by flow cytometry. To study deeply the effect of PGC1a under hyperglycaemic condition. PTC from PGC1a inducible CRISPR/Cas9 kidney organoids cultured under diabetogenic condition rescued the expression of PGC1a and the oxygen consumption rate of the PTC.

Conclusion: In conclusion, this preliminary work showed: 1) metabolic programming plays a role in the development of PTC from kidney organoids, in the context of hyperglycaemia 2) PGC1a inducible CRISPR/Cas9 kidney organoids could have a protective role under diabetogenic condition rescuing the PGC1a expression and the oxygen consumption rate of the PTC.

Supported by: Marie Skłodowska-Curie Individual Fellowships (IF) grant agreement no. 796590

Disclosure: C. Hurtado del Pozo: None.

OP 31 Novel aspects of diabetic neuropathy

181

Altered mitochondrial activity in the thalamus and somatosensory cortex in painful diabetic peripheral neuropathy

G. Sloan1, A. Anton2, D. Selvarajah3, I. Wilkinson2, S. Tesfaye1;

1Sheffield Teaching Hospitals, Sheffield, 2Academic Unit of Radiology, Sheffield, 3Department of Oncology and Human Metabolism, Sheffield, UK.

Background and aims: Painful diabetic peripheral neuropathy (pDPN) is common and often causes unremitting, distressing painful neuropathic symptoms. Unfortunately, current management of the disorder is inadequate because the disease mechanisms are not fully understood. We assessed cerebral cellular bioenergetics using phosphorus magnetic resonance spectroscopy (31P-MRS) to determine whether high energy phosphate metabolite levels are altered in the pain processing regions of the brain in pDPN.

Materials and methods: A total of 56 subjects, 44 with type 2 diabetes (12 no-DPN, 13 painless-DPN and 19 pDPN) and 12 healthy volunteers, underwent detailed clinical and neurophysiological assessments, and 31P-MRS brain imaging at 3-Tesla (Ingenia, Phillips Healthcare) with voxels placed over the right somatosensory cortex and the thalamus (TR 4s, TE 0.26ms, Voxel size 25 x 25 x 40mm3). The AMARES method, in jMRUI software was employed to calculate ATP to phosphocreatine (PCr) and inorganic phosphate (Pi) ratios (ATP:PCr & ATP:Pi), reflecting cellular bioenergetics (mitochondrial function).

Results: There was a significant group effect in the ATP:PCr ratio at the thalamus (p=0.039) and somatosensory cortex (p=0.024). The ATP:PCr at the thalamus was significantly higher in the pDPN group (0.50±0.06) compared to HV (0.44±0.04, p=0.022) and no-DPN (0.42±0.07 p=0.014). Moreover, the ATP:PCr ratio at the somatosensory cortex was significantly higher in pDPN (0.48 ±0.1) compared to HV (0.38 ±0.1, p=0.011). In addition, the ratio correlated with the Neuropathic Pain Symptom Inventory score at both brain regions.

Conclusion: Despite considerable research into the mechanisms of pDPN, our understanding remains limited. This study is the first to use 31P-MRS to analyse cerebral energetics in human-DPN and peripheral painful neuropathies. We demonstrated significantly higher ATP:PCr ratios in patients with pDPN in the somatosensory cortex and thalamus, which correlated with neuropathic pain symptom intensity. This could be indicative of increased cellular energy usage in pain processing regions of the brain as a result of continuous nociceptive inputs. Altered cerebral phosphorus metabolite ratios may serve as a biomarker of neuropathic pain in diabetes.

Disclosure: G. Sloan: None.

182

Symptoms of peripheral neuropathy early in type 2 diabetes are associated with higher risk of subsequent cardiovascular disease

L. Bjerg1,2, D.H. Christensen3, S.K. Nicolaisen3, J.S. Nielsen4,5, S.T. Andersen2,6, M.E. Jørgensen7,8, T.S. Jensen6,9, A. Sandbæk1,2, H. Andersen9, H. Bech-Nielsen4,5, H.T. Sørensen3, D.R. Witte1,2, R.W. Thomsen3, M. Charles1,10;

1Steno Diabetes Center Aarhus, Aarhus, 2Department of Public Health, Aarhus University, Aarhus, 3Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, 4DD2, Steno Diabetes Center Odense, Odense, 5Department of Endocrinology, Odense University Hospital, Odense, 6Department of Clinical Medicine, Danish Pain Research Center, Aarhus C, 7Clinical Epidemiology, Steno Diabetes Center Copenhagen, Gentofte, 8National Institute of Public Health, University of Southern Denmark, Odense, 9Department of Neurology, Aarhus University Hospital, Aarhus, 10Research Unit of General Practice, Aarhus C, Denmark.

Background and aims: Diabetic peripheral neuropathy (DPN) may be a determinant of subsequent cardiovascular disease (CVD) and mortality. We examined whether symptoms of DPN early in type 2 diabetes may act as a marker of later CVD and all-cause mortality.

Materials and methods: This cohort study linked clinical data from two Danish type 2 diabetes cohorts, the ADDITION-DK (inclusion period 2001-2006) and the DD2 (inclusion period 2009-2016), with data from Danish national registers. DPN was assessed at a median diabetes duration of 0 years in ADDITION-DK (=screen-detected diabetes) and at 4.6 years in DD2, using the Michigan Neuropathy Screening Instrument questionnaire (MNSIq) with a score ≥ 4 indicative of DPN. Using Poisson regression models, we compared the incidence of CVD and all-cause mortality during follow-up, according to initial DPN status. Analyses were adjusted for well-known CVD risk factors at baseline and fixed-effect meta-analyses were used to estimate combined results from the two cohorts.

Results: In total, 6,476 individuals were included in the analysis (NADDITION-DK = 1,448 and NDD2 = 5,028). At baseline, 189 (13.1%) individuals in ADDITION-DK and 818 (16.2%) in DD2 had DPN. The median follow-up was 11.4 years and 2.2 years, respectively. In ADDITION-DK, a total of 394 individuals experienced a CVD event (IRMNSIq<4: 27.4/1000 person-years (PY), IRMNSIq≥4: 50.9/1000 PY) and 253 died (MRMNSIq<4: 16.4/1000 PY, MRMNSIq≥4: 17.1/1000 PY). The corresponding numbers in the DD2 cohort were 480 (IRMNSIq<4: 41.1/1000 PY, IRMNSIq≥4: 77.2/1000 PY) and 127 (MRMNSIq<4: 11.4/1000 PY, MRMNSIq≥4: 13.7/1000 PY), respectively. After confounder adjustment, the combined estimate for excess CVD risk with DPN was 65% (IRR=1.65, 95% CI: 1.39-1.95) and for mortality 11% (MRR= 1.11, 95% CI: (0.82-1.49), compared to those without DPN (Table 1).

Conclusion: Symptoms indicating DPN early in type 2 diabetes are associated with a clearly elevated risk of subsequent CVD, beyond the risk carried by well-known CVD risk factors. DPN may act as a marker for undetected determinants of CVD and may contribute to early identification of type 2 diabetes individuals with high CVD risk.

figurebi

Clinical Trial Registration Number: 20000183 and 1-10-72-63-15

Supported by: NNF, IDNC Research program

Disclosure: L. Bjerg: None.

183

Associations of cardiac autonomic dysfunction with higher plasma lipid metabolites in recent-onset type 2 diabetes

G.J. Bönhof1,2, A. Strom1,3, K. Straßburger4,3, B. Knebel5,3, J. Kotzka5,3, J. Szendroedi1,2, M. Roden1,2, D. Ziegler1,2;

1Institute for Clinical Diabetology, German Diabetes Center (DDZ), Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, 2Division of Endocrinology and Diabetology, Medical Faculty, Heinrich Heine University, Düsseldorf, 3German Center for Diabetes Research (DZD), Munich-Neuherberg, 4Institute for Biometrics and Epidemiology, German Diabetes Center (DDZ), Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, 5Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center (DDZ), Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany.

Background and aims: Emerging evidence suggests that obesity and insulin resistance play a role in the development of diabetic cardiac autonomic neuropathy (CAN) characterized by reduced heart rate variability (HRV). We hypothesized that specific lipid metabolites are associated with diminished HRV in recent-onset type 2 diabetes rather than type 1 diabetes.

Materials and methods: We assessed the relationship of 127 biomarkers of lipid metabolism (11 acylcarnitines, 39 free fatty acids, 12 sphingomyelins, 56 phosphatidylcholines, and 9 lysophosphatidylcholines) in plasma using mass spectrometry with HRV indices in individuals with recent-onset type 1 (T1D) or type 2 diabetes (T2D) from the baseline cohort (known diabetes duration (DD) ≤1 year) of the German Diabetes Study (GDS; T1D/T2D [mean±SD]: n=100/206; age: 34.5±13.0/53.5±10.9 years; BMI: 24.6±4.2/31.8±6.0 kg/m²; DD: 204±95/195±88 days; HbA1c: 6.8±1.4/6.5±0.9 %). Four time domain and three frequency domain HRV indices were derived from NN intervals recorded during a 3-h hyperinsulinemic-euglycemic clamp.

Results: After adjustment for age, sex, BMI, smoking status, antihypertensive drugs, and lipid lowering drugs as well as Bonferroni correction including seven HRV parameters and the number of metabolites of the corresponding lipid class, standard deviation of all NN intervals (SDNN) was inversely associated with higher levels of three free fatty acids (myristic acid: r=-0.262, p=0.0002; palmitic acid: r=-0.278, p<0.0001; palmitoleic acid: r=-0.271, p<0.0001), six phosphatidylcholines (e.g. phosphatidylcholine diacyl (PC aa) C32:0: r=-0.345, p<0.0001; phosphatidylcholine acyl-alkyl C36:0 r=-0.286, p<0.0001), and two sphingomyelins (sphingomyelin C16:0: r=-0.242, p=0.0005; sphingomyelin C16:1: r=-0.302, p<0.0001), while standard deviation of adjacent NN invervals (SD) was inversely associated with six phosphatidylcholines (e.g. PC aa C30:0: r=-0.266, p=0.0001; PC ae C36:0: r=-0.271; p<0.0001) and one sphingomyelin (sphingomyelin C16:1: r=-0.292, p<0.0001) in recent-onset T2D. Among the frequency domain HRV indices, very low frequency (VLF), high frequency (HF), and low frequency (LF) power were associated with five (e.g. PC aa C32:0: r=-0.349), two (e.g. PC ae C36:0: r=-0.260), and one (PC aa C32:0: r=-0.295) phosphatidylcholines, respectively, in recent-onset T2D (all P<0.05). In contrast, no associations of lipid metabolites with HRV measures were noted recent-onset T1D.

Conclusion: Higher plasma levels of specific lipid metabolites are closely linked to cardiac autonomic dysfunction in recent-onset type 2 as opposed to type 1 diabetes, suggesting a role for perturbed lipid metabolism in the early development of CAN in type 2 diabetes.

Disclosure: G.J. Bönhof: None.

184

Effects of intensive risk factor management on cardiovascular autonomic neuropathy in type 2 diabetes: findings from the ACCORD clinical trial

R. Pop-Busui1, Y. Tang2, H. Shaw2, C.R. Bueno2, X. Sun2, J. Mitri2, M. Sambataro3, L. Sambado3, H.C. Gerstein4, V. Fonseca5, A. Doria2;

1Endocrinology & Diabetes, University of Michigan, Ann Arbor, USA, 2Joslin Diabetes Center, Boston, USA, 3Maria of Ca’ Foncello Hospital, Treviso, Italy, 4Endocrinology & Diabetes, McMaster University, Hamilton, Canada, 5Endocrinology & Diabetes, Tulane University, New Orleans, USA.

Background and aims: Cardiovascular autonomic neuropathy (CAN) is a common complication that independently predicts cardiovascular (CV) morbidity and mortality in persons with type 2 diabetes (T2D). The effects of preventive interventions on CAN remain unclear. We examined the effect of intensively targeting traditional risk factors for CAN, including hyperglycemia, hypertension, and dyslipidemia, in persons with T2D and high CV risk participating in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial

Materials and methods: CAN was defined as heart rate variability indices below the 5th percentile of the normal distribution (standard deviation of all normal-to-normal R-R intervals [SDNN] <8.2 ms and root mean square of successive differences between normal-to-normal R-R intervals [rMSSD] <8.0 ms). Of the 10,250 ACCORD participants, 71% (n=7,275) had valid CAN evaluations at study entry and at least once after randomization. The effects of intensive interventions on CAN were tested among these subjects by means of generalized linear mixed models.

Results: As compared to standard treatment, the intensive glycemia intervention significantly reduced CAN risk during the entire duration of the study (OR=0.84, 95% CI 0.75 - 0.94, p=0.003). This effect was present among individuals with no cardiovascular disease (CVD) history (OR= 0.73, 95%CI 0.63 - 0.85, p<0.0001) but not among those with a positive CVD history (OR=1.10, 95% CI 0.91 - 1.34, p=0.34) (p for interaction=0.001). Intensive BP therapy also decreased the odds of CAN (OR=0.75, 95% CI 0.63 - 0.89, p=0.001), with no evidence of heterogeneity based on CVD history or other clinical characteristics. Fenofibrate did not have a significant impact on CAN outcome (OR=0.91, 95%CI 0.78 - 1.07, p=0.26). No significant interactions were observed between treatment strategies

Conclusion: Our data confirm the beneficial effect of intensive glycemic therapy anddemonstrate, for the first time, a similar benefit of intensive BP control on CAN in T2D. They also suggest that a negative CVD history could be used as a criterion to select those T2D patients who would most benefit from intensive glycemic control for CAN prevention, whereas BP control is effective regardless of CVD history.

Clinical Trial Registration Number: NCT00000620

Supported by: NIHLBI, NIDDK

Disclosure: R. Pop-Busui: None.

185

Statin therapy and risk of polyneuropathy in type 2 diabetes: a population-based cohort study

D.H. Christensen1,2, F.P. Kristensen1,2, B.C. Callaghan2,3, S.T. Knudsen4, S.H. Sindrup2,5, E.L. Feldman2,3, L. Østergaard2,6, H. Andersen2,7, T.S. Jensen2,7, H.T. Sørensen1, R.W. Thomsen1;

1Department of Clinical Epidemiology, Aarhus University, Aarhus, Denmark, 2The International Diabetic Neuropathy Consortium, Aarhus University, Aarhus, Denmark, 3Department of Neurology, University of Michigan, Ann Arbor, USA, 4Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark, 5Department of Neurology, Odense University Hospital, Odensen, Denmark, 6Department of Neuroradiology, Aarhus University Hospital, Aarhus, Denmark, 7Department of Neurology, Aarhus University Hospital, Aarhus, Denmark.

Background and aims: Statins may potentially reduce the risk of diabetic polyneuropathy (DPN) due to anti-inflammatory and lipid-lowering effects. Statins have also been reported to be neurotoxic. We examined whether statin therapy had impact on the risk of DPN in individuals with type 2 diabetes.

Materials and methods: Using Danish medical databases we conducted a population-based cohort study. We identified all Danish incident diabetes patients during 2002-2016. We then excluded those aged below 30 years at the time of their first diabetes record, as they were possible type 1 diabetes patients. New statin users were defined as filling their first statin prescription in an exposure window extending from 180 days before to 180 days after their first diabetes record. Prevalent statin users were defined as filling statin prescriptions both during and before that period. DPN was identified using previously validated hospital discharge diagnosis codes. Follow-up started 180 days after the first diabetes record. Cox proportional hazard analysis was used to compute adjusted hazard ratios (aHRs) for DPN.

Results: The study cohort comprised 59,255 (23%) statin new users, 75,528 (29%) statin prevalent users, and 124,842 (48%) statin non-users; median follow-up time was 6.2 years [interquartile range (IQR) 3.4-9.6]. The incidence rate of DPN per 1000 person-years was almost similar in new users [4.0 events (95% confidence interval (CI): 3.8-4.2)], prevalent users [3.8 events (95% CI: 3.6-3.9)] and non-users [3.8 events (95% CI: 3.7-4.0)]. The aHR for DPN was 1.05 (95% CI: 0.98-1.11) in new users, and 0.97 (95% CI: 0.91-1.04) in prevalent users, compared with statin non-users. The null association persisted in on-treatment and propensity score-matched analyses, and in the subgroup analysis with additional adjustment for pre-treatment blood lipid levels. Stratification of the follow-up period revealed an increased DPN risk in new users during the first year of follow-up (aHR 1.31, 95% CI: 1.12-1.53). This vanished after ≥2 years of follow-up and may represent either a potential acute neurotoxic effect or a protopathic bias (i.e., early symptoms of yet undiagnosed DPN that trigger statin initiation).

Conclusion: This large cohort study suggested that among newly diagnosed type 2 diabetes patients, statin therapy was not associated with DPN risk. A small acute harmful effect cannot be excluded. This is outweighed by the substantial clinical effect of statins in cardiovascular disease prevention.

Supported by: Novo Nordisk Foundation Challenge Programme

Disclosure: D.H. Christensen: Grants; Novo Nordisk Foundation Challenge Programme.

186

Neuromodulation for treatment of painful diabetic neuropathy: a multicentre randomised controlled trial

E. Petersen, SENZA-PDN Investigators;

Neurosurgery, University of Arkansas for Medical Sciences, Little Rock, USA.

Background and aims: The World Health Organization (WHO) estimates there are 422 million people globally living with diabetes, resulting in US$1.7 trillion in direct and indirect costs. Approximately 20% of persons with diabetes will develop painful diabetic neuropathy (PDN), a chronic pain condition significantly impacting health-related quality of life (HRQoL). Current treatment options are ineffective for many patients; however, preliminary data suggest 10 kHz spinal cord stimulation (SCS) relieves pain and may improve sensation in refractory PDN patients.

Materials and methods: A prospective, multicenter, randomized, controlled trial (SENZA-PDN) with 216 subjects assigned 1:1 to 10 kHz SCS (Nevro Corp.) combined with conventional medical management (CMM) or CMM alone. Key inclusion criteria: PDN symptoms ≥12 months, lower limb pain ≥5cm (on 0-10cm visual analog scale [VAS]), and appropriate candidate for SCS. Key exclusion criteria: hemoglobin A1c >10%, daily opioid dosage >120mg morphine equivalents, and upper limb pain ≥3cm. Outcomes include pain, neurological function, HRQoL, sleep, satisfaction, and cost-effectiveness. Follow-up will last 24 months.

Results: Enrollment was completed from 2017 to 2019 with 430 candidates screened to randomize 113 subjects to 10 kHz SCS+CMM and 103 to CMM alone. The treatment arms were well matched across a variety of baseline characteristics, including age, sex, race, duration of diabetes and peripheral neuropathy, and hemoglobin A1c. There were no reported study-related adverse events (AEs) for the CMM group and 19 study-related AEs reported in the 10 kHz SCS+CMM group up to 3 months. Two AEs were categorized as serious: an infection resolved with conservative care and a wound dehiscence requiring explant. There were 2 procedure-related infections in the 10 kHz SCS+CMM group (1.8%). Per-protocol analysis revealed 5% of CMM (5/96) and 86% of 10 kHz SCS+CMM subjects (76/88) met the primary endpoint (p<0.001). At 3-month follow-up, there were differences in lower limb pain scores (Table 1), with 89% of 10 kHz SCS+CMM subjects deemed responders (≥50% pain relief) compared to just 7% of CMM subjects. Investigator-assessed sensory improvements were observed for 72% of 10 kHz SCS+CMM subjects versus 7% of CMM subjects. In addition, differences between treatment groups were observed across several HRQoL measures, such as impact of pain on sleep and patient global impression of change (Table 1).

Conclusion: SENZA-PDN is the largest RCT to-date of SCS management in PDN and will inform the treatment continuum. The primary endpoint was met with a significant proportion of subjects responding to 10 kHz SCS. These early results are promising for PDN patients who are refractory to conventional care. Study participant follow-up will continue for a total of 24 months with planned analyses for healthcare related costs and long-term clinical utility.

figurebj

Clinical Trial Registration Number: NCT03228420

Supported by: Nevro

Disclosure: E. Petersen: Grants; Nevro.

OP 32 Reducing the burden of hypoglycaemia

187

Nasal glucagon was efficacious in reversing insulin-induced hypoglycaemia without increasing risk of secondary hyperglycaemia

M. Giménez1, Y. Yan2, Q. Wang2, C. Child2, M. Zhang2;

1Hospital Clínic i Universitari, Barcelona, Spain, 2Eli Lilly and Company, Indianapolis, USA.

Background and aims: Nasal glucagon, a ready-to-use therapy for treatment of severe hypoglycaemia, contains 3-mg glucagon dry powder absorbed passively through nasal mucosa. We evaluated efficacy, pharmacodynamics, and safety of nasal glucagon compared to injectable glucagon in reversing insulin-induced hypoglycaemia in Caucasian and Japanese adults with type 1 diabetes (T1D) or type 2 diabetes (T2D).

Materials and methods: Post hoc analyses used data from 2 randomised, cross-over studies. Treatment success was defined as an increase in blood glucose to ≥3.9 mmol/L or an increase of ≥1.1 mmol/L from nadir blood glucose within 15 minutes of receiving glucagon. Blood glucose was measured every 5 minutes for the first 30 minutes, every 10 minutes up to 60 minutes, and at varied extended time intervals thereafter. Time to treatment success does not include reconstitution and preparation time for injectable glucagon in the control group. Pharmacodynamic data, including area under the curve above 7.8 mmol/L (∆7.8 AUC [1-4 hr]), were used to evaluate the risk of secondary hyperglycaemia. Tolerability was assessed with treatment-emergent adverse events and a nasal symptom questionnaire.

Results: A similar proportion of nasal glucagon (97.8% [131/134)] and injectable glucagon patients (97.0% [130/134]) achieved treatment success. Mean time to treatment success (for blood glucose increase) was 11.7 minutes for nasal glucagon and 10.4 minutes for injectable glucagon (p<0.001). The median time for both nasal and injectable glucagon was 10 minutes. Geometric least square mean maximal blood glucose (BGMAX) for nasal glucagon and injectable glucagon were 10.8 and 11.4 mmol/L (p<0.001), respectively. Blood glucose concentrations over time for nasal glucagon and injectable glucagon are presented in the figure. Nasal glucagon had significantly lower ∆7.8 AUC (1-4 hr) (p<0.001), with 42% reduction compared to injectable glucagon. Nasal glucagon had similar rates of nausea (19.1%) and vomiting (8.5%) versus injectable glucagon (28.8% and 11.5%, respectively), with higher rates of side effects related to nasal administration (headache [7.8% nasal glucagon, 5.8% injectable glucagon], upper respiratory tract irritation [6.4% nasal glucagon, 0.7% injectable glucagon]). Separate T1D and T2D analyses showed similar results as the T1D+T2D groups combined.

Conclusion: Nasal glucagon was efficacious and well tolerated in reversing insulin-induced hypoglycaemia in adults with T1D or T2D and did not increase the risk of secondary hyperglycaemia compared to injectable glucagon.

figurebk

Clinical Trial Registration Number: NCT03421379, NCT03339453

Supported by: Eli Lilly and Company

Disclosure: M. Giménez: None.

188

Counterregulatory responses to hypoglycaemia in totally pancreatectomised patients

M. Baekdal1,2, A. Lund1, M.P.A. Baldassare1,3, K. Rose1, J. Egholk1, M.B. Christensen1,4, C.P. Hansen5, J.H. Storkholm5, J. Faber6, B. Hartmann7,8, J.J. Holst7,8, T. Villsbøll1, F.K. Knop1;

1Center for Clinical Metabolic Research, Gentofte Hospital, Hellerup, Denmark, 2Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark, 3Department of Medicine and Ageing Sciences, D’Annunzio University, Cheti, Italy, 4Department of Clinical Pharmacology, Bispebjerg Hospital, Copenhagen, Denmark, 5Department of Surgery, Rigshospitalet, Copenhagen, Denmark, 6Department of Endocrinology, Herlev Hospital, Herlev, Denmark, 7Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark, 8Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Background and aims: We have previously shown that totally pancreatectomised (PX) patients secrete substantial amounts of glucagon (most likely from enteroendocrine cells) during an OGTT whereas these patients suppress circulating glucagon concentrations during i.v. glucose infusions. Here, we investigated the effect of insulin-induced hypoglycaemia on extrapancreatic glucagon secretion and other counterregulatory factors in PX patients and in healthy controls (CTRLs).

Materials and methods: On two separate days, 12 PX patients (age 65.5±5.5 [mean±SD] years; BMI: 23.8±3.6 kg/m2; HbA1c 65.1±8.3 mmol/mol (10.6±1.5%)) and 12 matched, healthy CTRLs (age 64.8±6.5 years; BMI: 24.5±2.9 kg/m2; HbA1c 34.9±2.8 mmol/mol (5.8±0.5%)) underwent 1) a 50-g OGTT with 1.5 g paracetamol (for assessment of gastric emptying) and 2) an insulin-induced hypoglycaemic clamp followed by a 30-minute recovery period and a subsequent 50-g OGTT with paracetamol. Blood was intermittently sampled throughout both experimental days.

Results: Plasma glucagon responses to OGTT (as assessed by baseline-subtracted AUC) were greater in PX patients compared to CTRLs (386±150 vs -340±50 min×pmol/l [mean±SEM], p=0.0001). During the hypoglycaemic clamp, PX patients did not increase plasma glucagon concentrations and, thus, glucagon responses to hypoglycaemia were higher in CTRLs (903±104 vs -21±16 min×pmol/l, p<0.001). Hypoglycaemia-induced responses of catecholamines, growth hormone and cortisol were similar in the two groups. Gastric emptying was unaffected by hypoglycaemia in CTRLs but was decelerated by hypoglycaemia in PX patients.

Conclusion: We show that insulin-induced hypoglycaemia, which powerfully stimulates glucagon secretion in controls, does not stimulate extrapancreatic glucagon secretion in PX patients. Counterregulatory responses of catecholamines, growth hormone and cortisol were intact in PX patients, but hypoglycaemia decelerated gastric emptying in these patients. Our results provide a mechanistic insight into the high risk of hypoglycaemia in PX patients.

Clinical Trial Registration Number: H-17014216

Disclosure: M. Baekdal: None.

189

Amp-activated protein kinase (AMPK) activator R481 amplifies the glucagon response to hypoglycaemia without worsening hyperglycaemia in diabetic rats

C. Beall1, A.M. Cruz1, P.G. Weightman Potter1, J.M. Vlachaki Walker1, Y. Malekizadeh1, K.R. Pye1, S.J. Shaw2, K.L.J. Ellacott1;

1University of Exeter Medical School, RILD Building, University of Exeter, Exeter, UK, 2Rigel Pharmaceuticals Inc., South San Francisco, USA.

Background and aims: Hypoglycaemia is still a frequent concern for people with Type 1 and advanced insulin-treated Type 2 Diabetes. Frequent episodes of hypoglycaemia leads to impaired awareness of and defective hormonal responses to subsequent hypoglycaemia. Activation of brain AMPK may be a therapeutic strategy to improve glucose counterregulation and prevent future hypoglycaemia.

Materials and methods: Glucose tolerance tests (2 g/kg ; intraperitoneally) were performed on male Sprague-Dawley rats with indirect (metformin-like) AMPK activators R481 (brain permeable) and R419 (non brain permeable ; 5-20 mg/kg ; i.p) ± autonomic nervous system (ANS) blocker hexamethonium (50 mg/kg ; i.p.) or AMPK inhibitor SBI-0206965 (3 mg/kg ; i.p.). A second and third cohort were examined for hypoglycaemia glucose counterregulation and insulin sensitivity using hyperinsulinaemic-hypoglycaemic and euglycaemic clamps, respectively. The effect of R481 on blood glucose levels in rats with streptozotocin (STZ ; 60-125 mg/kg)-induced diabetes was also examined.

Results: During glucose tolerance tests, R481 (5-20 mg/kg) acutely raised peak glucose levels without impairing glucose clearance. This was completely blocked by hexamethonium, indicating that R481 activates to autonomic nervous system, which did not occur with R419. AMPK inhibitor SBI-0206965 also significantly attenuated the effect of R481 on glycaemia. During hyperinsulinaemic-euglycaemic clamps, R481 did not alter the glucose infusion rate. C-peptide levels declined with hyperinsulinaemia but this was not altered by R481. During the hypoglycaemic clamps, R481 treated animals had a lower glucose infusion rate, mediated by significantly elevated plasma glucagon levels, without any change to the adrenaline response. In STZ diabetic rats, R481 did not worsen fasting hyperglycaemia.

Conclusion: These data indicate that central AMPK activation raises glycaemia by activating the autonomic nervous system. This was not mediated by reduced insulin sensitivity. During hypoglycaemia, R481 augmented the glucagon response to hypoglycaemia without altering the adrenaline response. Importantly, R481 did not worsen hyperglycaemia suggesting this intervention could be taken before the possible onset of hypoglycaemia i.e. in the postprandial period. Moreover, these data suggest that when augmenting whole body AMPK activity, central AMPK activation supercedes peripheral AMPK activation to raise blood glucose levels for use by the brain. Our data suggests this only occurs when glucose levels are low, i.e. during hypoglycaemia and not during hyperglycaemia.

figurebl

Supported by: JDRF, Diabetes UK, University of Exeter Medical School

Disclosure: C. Beall: None.

190

Limited impact of impaired awareness of hypoglycaemia and severe hypoglycaemia on inflammatory profile in people with type 1 diabetes

N. Ali, A.W.M. Janssen, B.E. De Galan, C.J. Tack, M. Jaeger, L. Van de Wijer, W. van der Heijden, R. ter Horst, P. Vart, A. van Gool, L.A.B. Joosten, M.G. Netea, R. Stienstra;

Radboud UMC, Nijmegen, Netherlands.

Background and aims: In people with type 1 diabetes, severe hypoglycemia (SH) is independently associated with an increased risk of cardiovascular events. Pro-inflammatory changes with pro-atherogenic capacity may explain this association. We investigated whether a history of SH or the associated presence of impaired awareness of hypoglycemia (IAH) was characterized by a pro-inflammatory profile in type 1 diabetes.

Materials and methods: We measured circulating inflammatory markers and pro-and anti-inflammatory cytokines production after ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) in a well-characterized cohort of individuals with type1 diabetes(n=239) and in people without diabetes(n=56). Data were corrected for confounders by using multivariate linear regression models.

Results: People with type1 diabetes had higher circulating concentrations of hs-CRP (0.91 [0.36-2.25] vs 0.52 [0.20-0.98] pg/ml, p<0.001 and IL-18BP (1746 [1304-2112] vs 1381 [1191-1807] pg/ml, p=0.001) than those without diabetes. In multivariate analysis, only the higher hs-CRP persisted. Neither circulating immune cells nor ex vivo cytokine levels produced by PBMCs in response to an extensive panel of stimuli differed in groups defined by awareness state or a history of SH, apart from elevated IL-18BP in people with compared to those without a history of SH (1524 (1227-1903) vs 1913 (1459-2408) pg/ml, p<0.001).

Conclusion: IAH or a history of SH in people with type 1 diabetes was not associated with altered inflammatory profiles, arguing against chronically elevated inflammatory activity mediating the increased cardiovascular risk associated with hypoglycemia. The finding of higher circulating concentrations of IL-18BP in individuals with a history of SH requires further investigation.

Clinical Trial Registration Number: NL54214.091.15, 2015-1930 and NL42561.091.12, 2012-550

Supported by: NWO, EFSD/AstraZeneca Macrovascular Programme 2015, Hypo-RESOLVE, JU, SGF

Disclosure: N. Ali: None.

191

Early response to hypoglycaemia in type 1 diabetes is dependent on profound brain connectivity changes in response to falling glucose levels

P. Jacob, M. Nwokolo, F. Zelaya, S.A. Amiel, O. O'Daly, P. Choudhary;

King’s College London, London, UK.

Background and aims: Resting state networks (RSNs) are networks of neurons that can be detected with neuroimaging as synchronous low frequency blood oxygen-level dependent (BOLD) signal oscillations at rest. We investigated connectivity in three RSNs in people without diabetes (ND) and people with type 1 diabetes (T1D) with either normal awareness of hypoglycaemia (NAH) or impaired awareness of hypoglycaemia (IAH).

Materials and methods: Fourteen ND, 15 NAH and 22 IAH participants, age, sex and gender matched, underwent a hyperinsulinaemic glucose clamp during which we studied changes in connectivity in 3 RSNs (default mode network (DMN), salience network (SN) and central executive networks (CEN)) between euglycaemia (5.0mmol/L) and onset of hypoglycaemia (2.6mmol/L) using BOLD fMRI. BOLD sequences were preprocessed and analysed in the CONN toolbox and regression analyses performed in SPM12. A p-value of <0.05 was considered significant and this was Bonferroni corrected with FDR for neuroimaging analyses.

Results: At this early timepoint ND symptom scores rose modestly (8.9 ± 4.2 to 13.4 ± 6.5, p = 0.057) despite a significant rise in adrenaline concentration (0.15 ± 0. 10 to 2.15 ± 2.05 nmol/L, p < 0.001). NAH had an early significant symptomatic response (11.3 ± 6.3 to 18.8 ± 9.3, p = 0.029) despite a reduced adrenaline response (0.19 ± 0.12 to 1.01 ± 0.79 nmol/L, p < 0.001). Our IAH group had no symptom response (10.3 ± 5.3 to 9.9 ± 5.3, p = 0.700) associated with a statistically significant but low magnitude adrenaline response (0.17 ± 0.10 to 0.43 ± 0.28 nmol/L, p < 0.001). In ND hypoglycaemia did not alter connectivity of the DMN at hypoglycaemia. However, there was increased connectivity of the SN with basal ganglia nuclei and the reduced connectivity of the CEN with the precuneus, a core node of the DMN.

In NAH, hypoglycaemia reduced connectivity of the DMN with parts of the sensory cortex, increased connectivity of the SN with the operculum and reduced connectivity of CEN with the calcarine cortex. There were no connectivity changes in any of the networks studied in IAH related to hypoglycaemia. Due to the heterogeneity of symptomatic responses in the NAH group we performed a regression analysis to identify regions of connectivity changes that correlated with the degree of adrenergic symptom responses. In response to hypoglycaemia adrenergic symptoms significantly correlated with connectivity between the DMN and the right anterior insula, the dominant node of the SN (p = 0.048). The adrenergic response was also correlated with SN connectivity to the sensory cortex (p = 0.042).

Conclusion: There were significant changes to connectivity of the DMN, SN and CEN in early hypoglycaemia in NAH and ND but not IAH. In people with NAH, adrinergic symptoms were associated with changes from resting (DMN) to the active SN allowing it to ascribe importance to sensory cortex signals. It is possible that prior hypoglycaemic exposure triggers changes in connectivity in early hypoglycaemia that underlie awareness. These changes in connectivity may be crucial for early hypoglycaemia behavioural responses in T1D and are lost in IAH.

Supported by: JDRF

Disclosure: P. Jacob: Grants; This project was funded by JDRF.

192

Hyperinsulinaemic-hypoglycaemic glucose clamps in human research: a systematic review of the literature

T. Wilbek Fabricius1, C. Verhulst2, B.E. de Galan2,3, U. Pedersen-Bjergaard1,4;

1Department of Endocrinology and Nephrology, Nordsjaellands Hospital, Hillerød, Denmark, 2Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, Netherlands, 3Department of Internal Medicine, Maastricht University Medical Centre, Maastricht, Netherlands, 4Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background and aims: The hyperinsulinaemic-hypoglycaemia glucose clamp technique has been developed to assess effects of and responses to hypoglycaemia under standardized conditions. Whether the methodology of clamp studies has been standardized is unclear. This systematic review examines how hyperinsulinaemic-hypoglycaemic clamps are performed and elucidates potential important differences.

Materials and methods: A literature search in PubMed and EMBASE was conducted. Articles in English published from 1980-2018, involving adults with or without diabetes were included. This systematic review is registered in PROSPERO.

Results: We included 354 papers. A single-step clamp was performed in 224 (63%) papers, with a mean duration of 79±61 minutes (range 5-660). We found a glucose nadir of 2.8±0.4 mmol/L (range 2.2-4.3). Divided according to IHSG's hypoglycaemia definition, we found that 71% had a nadir <3.0 mmol/L (mean 2.6±0.2 mmol/L, range 2.2-2.9), and 29% had a nadir >3.0mmol/L (mean 3.2±0.2 mmol/L, range 3.0-4.3). A stepped clamp, involving multiple consecutive levels of hypoglycaemia, was conducted in 127 (36%) papers, with the most frequent number of steps used being 4. The duration of the steps depended on the number of steps and ranged from 20-90 minutes, with a glucose nadir of 2.6±0.3 mmol/L (range 1.8-3.3). Eighty-three percent had a nadir <3.0 mmol/L (mean 2.4±0.2, range 2.2-2.9), and 17% had a nadir >3.0 mmol/L (mean 3.2±0.1, range 3.0-3.3). The remaining 3 articles reported single and stepped clamps on two consecutive days. There was considerable variation in insulin infusion rates in the studies, ranging from 0.25 to 12.0 mU·kg-1·min-1 or 15 to 160 mU·m-²·min-1, respectively. This corresponds to a 49-fold difference between the lowest and the highest rate used, when normalized to a person of average posture. Information about the glucose infusion rate was only given in 24% of the articles, with vastly different ways of reporting it. Twenty-nine percent of the articles reported glucose levels from whole blood. In 71% of the studies, a dorsal hand vein was used for glucose measurement, applying some form of hand warming to arterialize venous blood in 80% of these studies, but with the use of a heated box in only 66% of the studies.

Conclusion: Although the hyperinsulinaemic-hypoglycaemic clamp technique is considered to be the gold standard for experimental studies on hypoglycaemia, there is no uniform standard on how to perform these experiments. Methodological differences should be considered when comparing results between hypoglycaemic clamp studies.

Supported by: The Hypo-RESOLVE project

Disclosure: T. Wilbek Fabricius: Grants; The Hypo-RESOLVE project.

OP 33 What exercise does

193

Exercise changes neuronal processing of food cues in sedentary overweight and obese adults

L. Wagner1, S. Kullmann1,2, R. Veit1, P. Schneeweiss3, A. Nieß3, H. Preissl1,2, A.L. Birkenfeld1,2, A. Peter1,4, H.-U. Häring1,2, A. Fritsche1,2, A. Böhm1,2, C. Weigert1,4, M. Heni1,2;

1Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tübingen, Tübingen, 2Department of Internal Medicine IV, University of Tübingen, Tübingen, 3Department of Sports Medicine, University of Tübingen, Tübingen, 4Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicine, University of Tübingen, Tübingen, Germany.

Background and aims: Exercise has beneficial effects on metabolism and brain function and is therefore recommended to promote weight loss and achieve weight maintenance. However, little is known whether exercise has a direct influence on the neuronal processing of food stimuli to influence eating behavior.

Materials and methods: 21 participants (14 women; BMI 31±3.9 kg/m2; age 31 ± 9 years) underwent two functional magnetic resonance imaging (fMRI) sessions before and after an 8-week supervised exercise intervention (1 h cycling and walking training, 3 times per week). After intranasal insulin administration, 60 visual food cues (high- and low calorie) were presented in a randomized order during the fMRI measurements. Afterwards, participants were asked to rate the food pictures.

Results: After the exercise intervention, activity in the striatum (i.e. caudate) increased in response to high calorie pictures (pFWE-corr= 0.003; small volume corrected (SVC)). Food pictures that were rated as more desirable elicited a stronger activation in the insula cortex before compared to after the exercise intervention (pFWE-corr=0.03; SVC).

Conclusion: Exercise can significantly impact neuronal processing of food pictures, particularly in reward- and taste-associated brain regions. These regions are known to be insulin sensitive and vital for the control of food intake. In the current study, exercise resulted in a differential activation in reward-related brain regions when responding to palatable food cues. This may help explain the often observed weight-cycling effect after a weight loss intervention. Further studies are needed, though, to show whether the identified improved brain processes in response to exercise ultimately translate into weight loss maintenance.

Clinical Trial Registration Number: NCT03151590

Supported by: DZD e.V. 01GI0925

Disclosure: L. Wagner: Grants; German Center for Diabetes Research (DZD e.V. 01GI0925).

194

Bicycling and all-cause mortality among individuals with diabetes

M. Ried-Larsen1, M.G. Rasmussen2, K. Blond3, L.B. Andersen4, N. Wareham5, S. Brage5, A. Grøntved2;

1Centre for Physical Activity Research, Rigshospitalet, Copenhagen, Denmark, 2University of Southern Denmark, Odense, Denmark, 3Bispebjerg and Frederiksberg Hospital, Copenhagen, Denmark, 4Western Norway University of Applied Sciences, Campus Sogndal, Sogndal, Norway, 5University of Cambridge School of Clinical Medicine, Cambridge, UK.

Background and aims: The risk of premature death from all-causes and cardiovascular causes is increased among persons with diabetes, with few effective preventive measures. The aim of the study was to investigate the association between time spent bicycling and all-cause and cardiovascular mortality among individuals with diabetes. Secondarily, to investigate the association between change in bicycling and all-cause and cardiovascular mortality.

Materials and methods: In this prospective cohort study, nested in the European Prospective Investigation into Cancer and Nutrition, a questionnaire-based survey was administered in 10 western European countries in 1992-2000 (1st examination). A follow-up survey (2nd examination) was administered (mean (standard deviation)) 5.3 (2.3) years after the 1st examination. Adults with self-reported or confirmed diabetes (N=9,207) at the first examination were included in the study. The primary and secondary outcomes were all-cause and cardiovascular mortality, respectively. The primary exposure was weekly time spent bicycling at 1st examination. A secondary exposure was change in weekly time spent bicycling from the 1st to the 2nd examination. Multivariable-adjusted Cox proportional-hazards models were used to estimate hazard ratios (HRs) and 95% CIs.

Results: During 128,860 person-years of follow-up 2,158 deaths from all-causes were registered. Compared to no bicycling at 1st examination (reference), the multivariable adjusted hazard ratios (95% CIs) for all-cause mortality were; 0.75 (0.59,0.95), 0.77 (0.66,0.90), 0.69 (0.58,0.82) and 0.76 (0.64,0.91) for >0 <60 min/week, >=60 <150 min/week, >=150 <300 min/week and >=300 min/week of bicycling, respectively. In the analysis of change (60,469 person-years of follow-up), 1,079 deaths from all-causes were recorded. Compared to persons reporting no bicycling at both examinations (reference), the multivariable hazard ratios (95% CIs) for all-cause mortality were; 0.88 (0.71,1.09), 0.69 (0.50,0.94), 0.66 (0.54,0.80) for persons who ceased, initiated and continued bicycling from the 1st to the 2nd examination, respectively. Inverse associations with cardiovascular mortality were also observed for increased weekly time spent bicycling at baseline and change in bicycling from the 1st to the 2nd examination.

Conclusion: Any bicycling confers with benefit among persons with diabetes after considering other physical activities, as well as other putative risk factors. As initiation of bicycling decreases risk of both all-cause and cardiovascular mortality among persons with diabetes, these findings suggest that bicycling could be considered as an addition to existing physical activity referral schemes to increase physical activity in the clinical care of diabetes.

Clinical Trial Registration Number: NCT04171557

Supported by: Trygfonden

Disclosure: M. Ried-Larsen: None.

195

High-intensity interval training combining biking and rowing markedly improves insulin sensitivity, body composition and VO 2 max in obesity and type 2 diabetes

M.H. Petersen1, M.E. de Almeida2,1, E.K. Wentorf2, N. Ørtenblad2, K. Højlund1;

1Steno Diabetes Center Odense, Odense University Hospital, Odense, 2Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark.

Background and aims: Physical activity is a cornerstone in the treatment and prevention of type 2 diabetes. However, the beneficial effects of endurance exercise training on insulin sensitivity are often modest (10-20%). Recent studies suggest that high-intensity interval training (HIIT) may be more effective, and that the involvement of more muscle groups may enhance the effect of exercise training. Our aim was to examine the effect of a whole body HIIT-protocol recruiting both lower and upper body muscles on insulin sensitivity, substrate metabolism, VO2max, body composition and glycemia.

Materials and methods: In 15 obese (BMI: 31±0.8 kg/m2) men with type 2 diabetes, and age-matched obese (n=15, BMI: 31±0.7 kg/m2) and lean (n=18, BMI: 24±0.4 kg/m2) healthy glucose tolerant men, the effect of 8-weeks supervised HIIT combining biking and rowing (3 sessions/week) were examined by DXA-scans, VO2max tests and euglycemic-hyperinsulinemic clamps combined with indirect calorimetry. HIIT-sessions consisted of blocks of 5 x 1 min exercise interspersed with 1 min rest, shifting between blocks on cycle and rowing ergometers, and with an increasing volume from two to five blocks during the 8 weeks.

Results: At inclusion, men with type 2 diabetes had 35-37% lower insulin sensitivity and ~13% lower insulin-mediated suppression of lipid oxidation compared with obese and lean individuals (all p<0.01). In response to the HIIT-protocol, insulin sensitivity increased 32-37% in lean and obese healthy men and 44% in men with type 2 diabetes (all p<0.01). No changes in resting or insulin-stimulated substrate metabolism or respiratory exchange ratios were seen in response to the HIIT-protocol in men with type 2 diabetes or in obese and lean controls. VO2max increased 10% in lean and obese healthy men and 15% in men with type 2 diabetes (all p<0.05). Fat mass was reduced by 1.6-2.3 kg in all 3 groups (all p<0.01), whereas fat free mass was increased 0.9-1.5 kg in obese men with and without type 2 diabetes (all p<0.05). There were no differences in the HIIT-induced improvements between the groups. HIIT reduced HbA1c 3.9 mmol/mol (p<0.05), and fasting plasma glucose 1.0 mmol/l (p<0.001) in patients with type 2 diabetes.

Conclusion: A HIIT-protocol recruiting both lower and upper body muscles efficiently improves insulin sensitivity, VO2max and body composition to the same extent in obesity and type 2 diabetes as in lean healthy individuals. In patients with type 2 diabetes, the HIIT-protocol also improved glycemic control.

Clinical Trial Registration Number: 17/31977

Supported by: Novo Nordisk Foundation

Disclosure: M.H. Petersen: Grants; Novo Nordisk Foundation, Sawmill owner Jeppe Juhl and wife Ovita Juhl Memorial Bursary 2016, Christenson-Cesons Family Fund, OUH PhD Fund for Operation Costs. Other; Scholarship from the Region of Sourthern Denmark, PhD scholarship from the faculty of University of Southern Denmark.

196

Differences in physiological responses to cardio-pulmonary exercise testing in adults with type 1 diabetes and healthy controls: a pooled analysis

M.L. Eckstein1, D. Pesta2, O. McCarthy3, D.J. West4, J. Yardley5, T. Zueger6, C. Stettler6, J. Boufleur Farinha7, M.C. Riddell8, L. Brugnara9, M. Roden2, H. Sourij1, R.M. Bracken3, P. Hofmann10, O. Moser1;

1Cardiovascular Diabetology Research Group, Medical University of Graz, Graz, Austria, 2German Diabetes Centre, Düsseldorf, Germany, 3College of Engineering, Swansea University, Swansea, UK, 4Newcastle University, Newcastle, UK, 5University of Alberta, Alberta, Canada, 6Inselspital Bern, Bern, Switzerland, 7Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 8York University, Toronto, Canada, 9CIBERDEM, Madrid, Spain, 10Institute of Sports Science, University of Graz, Graz, Austria.

Background and aims: People with type 1 diabetes (T1D) show alterations in oxygen economy and heart dynamics during incremental cardio-pulmonary exercise (CPX) testing, which are associated with elevated glycated haemoglobin (HbA1c) levels. Yet, a comprehensive assessment of the impact of T1D, its associated glycaemic control and specific T1D characteristics on functional capacity is missing. This study investigated the physiological response to CPX testing in people with T1D when compared to healthy controls and assessed if cardio-pulmonary and performance responses are associated with HbA1c and specific T1D characteristics.

Materials and methods: The analysis included cycle ergometer CPX datasets and participants and T1D characteristics from 692 people with T1D and healthy controls. Ventilatory threshold 1 (VT1) and ventilatory threshold 2 (VT2) were defined as the aerobic and anaerobic thresholds. In addition, the degree and direction of the deflection of the heart rate to performance curve (kHR) were calculated. A linear mixed-effects model with post-hoc tests was applied to assess changes in CPX parameters over VT1, VT2 and peak performance and compare changes in CPX parameters between groups while HbA1c and diabetes characteristics were only available for T1D. For kHR, linear regression modelling was performed (for all p<0.05).

Results: 347 people with T1D and 345 healthy controls were included (age: 34 ± 11 vs. 34 ± 12 years; BMI 24.6 ± 3.6 vs. 24.3 ± 3.5 kg/m2; 244 male vs 240 male) (p>0.05)(Table 1).

Conclusion: We showed differences over all physiological parameters and performance capacity between people with T1D and matched healthy controls. Physiological parameters and power output were not associated with HbA1c, yet were associated by c-peptide and total daily insulin dose (TDD). While heart rate dynamics were not associated with any T1D characteristics, controversially, levels of c-peptide and TDD were associated with lower power output. T1D duration was not associated with any physiological parameter and power output. The question arises if T1D per se or lower levels of physical activity instead of HbA1c and specific T1D characteristics alter functional capacity in people with T1D.

figurebm

Disclosure: M.L. Eckstein: None.

197

Bolus insulin dose depends on previous-day race intensity during 5 days of professional road-cycle racing in athletes with type 1 diabetes: a prospective observational study

M. Dietrich1, O. McCarthy2, M.L. Eckstein1, R.M. Bracken2, O. Moser1;

1Medical University of Graz, Graz, Austria, 2Swansea University, Swansea, UK.

Background and aims: Individuals with type 1 diabetes (T1D) often participate in extreme sports competitions and hence prove that T1D per se is not the sole determinant of being physically inactive. However, physical activity and exercise are associated with glycaemic disturbances. Therefore, the question arises of whether dysglycaemia around extreme sporting events frequently occurs in athletes with T1D. The aim of this study was to investigate glycaemic responses and therapy adaptations during a 5-day professional road-cycling race in athletes with T1D.

Materials and methods: 7 professional male cyclists with T1D (age: 28±4 years; BMI: 20.9±0.9 kg/m2; HbA1c: 7.3±0.6 % [56±7 mmol/mol]; diabetes duration: 10±6 years; all using multiple daily injections (MDI); peak oxygen uptake (VO2peak) 72±5 mL/kg/min) participated in a 5-day Union Cycliste Internationale (UCI) road-cycling race. During the 5-day period, cyclists used a real-time continuous glucose monitoring system (rtCGM) and smart bolus and basal insulin pens, whilst macronutrient intake was recorded daily. Data were assessed by means of repeated measures ANOVA/Friedman test with post-hoc testing for glycaemia, macronutrient intake and insulin doses for the 2-day pre-race and race periods. Data were stratified for pre-defined glycaemic ranges and night-/daytime. Associations between exercise physiological data and diabetes markers data were analysed by linear regression modelling (p<0.05).

Results: Although glycaemic variability significantly increased during the 5-day competition period when compared against the pre-race condition (mean difference [MD] 5.0±4.6%, p=0.03), night-time sensor glucose decreased during the 5-day competition period (MD -21±20 mg/dL [-1.2±1.1 mmol/L], p=0.04). The total daily bolus insulin dose was significantly altered over the 5-day competition period (p=0.01). Data followed a bell shaped curve in which bolus doses initially increased from day 1 to day 2 (p=0.04) then decreased from day 2 to day 5 (p=0.04). Basal insulin dose remained unchanged (p=0.64). Subsequent day bolus insulin doses were reduced to a larger extent when the previous-day race intensity was higher (p=0.04). Higher race intensities were associated with lower mean sensor glucose levels (p=0.03) and less time spent in severe hyperglycemia (>250 mg/dL [>13.9 mmol/L] (p=0.04) in the post-race period. Macronutrient intake remained unaffected during the 5-day competition period (p=0.06).

Conclusion: This is the first study evidencing a deterioration in glycaemic variability, yet an improvement in night-time glycaemia during 5 days of road cycle racing compared to pre-race conditions in professional cyclists with T1D. Intriguingly, bolus insulin dose was firstly increased and then latterly reduced, while carbohydrate intake and basal insulin dose remained the same. Exercise intensity during racing was the main determinant in explaining the altered sensor glucose levels.

Clinical Trial Registration Number: DRKS00019928

Supported by: Team Novo Nordisk

Disclosure: M. Dietrich: Grants; Team Novo Nordisk.

198

Plasma aminoadipic acid levels responded to acute and long-term exercise and correlated with insulin sensitivity, pancreatic fat content and C-peptide concentrations in men

S. Lee1, A. McCann2, P.M. Ueland2, C. Drevon3, K.I. Birkeland3;

1340101, P.O. Box 1085 Blindern, University of Oslo, Kongsvinger, Norway, 2BEvital, Bergen, Norway, 3340101, P.O. Box 1085 Blindern, University of Oslo, Oslo, Norway.

Background and aims: The lysine metabolite aminoadipic acid (2-AAA) is strongly associated with the risk of developing type 2 diabetes in observational studies, and may enhance insulin secretion from pancreatic β-cells. Physical activity may improve insulin sensitivity and prevent or delay the onset of type 2 diabetes, yet the relationship between 2-AAA and exercise is unknown.

Materials and methods: We collected blood, skeletal muscle and adipose tissue samples from 13 dysglycaemic and 13 normoglycaemic sedentary, middle-aged men who underwent two bicycle challenges, one before and one after 12 w exercise intervention. We measured plasma concentrations of 2-AAA, lysine and a panel of 13 novel diabetes-related plasma biomarkers, such as branched-chain amino acids, ketone bodies and tryptophan metabolites, using LC- and GC-MS/MS. In addition to mRNA sequencing of skeletal muscle and adipose tissue biopsies, insulin sensitivity was quantified by euglycemic hyperinsulinemic clamping, and body fat distribution and pancreatic fat content analysed by MRI/MRS.

Results: Among the investigated plasma biomarkers 2-AAA concentrations was the most enhanced in dysglycaemic vs. normoglycaemic men at baseline (1.6-fold, p=0.002), and was most strongly negatively associated with baseline insulin sensitivity (M) (β[95%CI]: -2.68[-4.38,-1.00] mg/kg/min, p=0.0031) and M-improvement after 12 w of exercise intervention (-1.50[-2.87,-0.13] mg/kg/min, p=0.033). These associations remained unaltered after adjustment for branched-chain amino acid levels. Furthermore, plasma 2-AAA levels correlated positively with amounts of visceral adipose tissue and pancreatic fat content, and also positively with plasma c-peptide concentration and HOMA-β. Plasma concentration of 2-AAA was also the most exercise responsive metabolite increasing 11.5% (p<0.043) after 12 w of exercise and by ~40% (p<0.001) after both bicycle challenges. Changes in plasma lysine levels were similar to 2-AAA, although less pronounced. Untargeted mRNA sequencing pathway analyses revealed supressed lysine and tryptophan metabolism in adipose tissue from dysglycaemic men, and enhanced lysine and tryptophan metabolism in muscle after 12 w of exercise in both groups. Plasma concentrations of 2-AAA correlated negatively with mRNA levels of DHTKD1, a rate-limiting enzyme in lysine metabolism, in both skeletal muscle and adipose tissue.

Conclusion: Plasma concentrations of 2-AAA were strongly associated with markers of insulin resistance and secretion in men, and responded to acute as well as long-term exercise intervention. Our data support a role for plasma 2-AAA in insulin sensitivity.

Clinical Trial Registration Number: NCT01803568

Disclosure: S. Lee: None.

OP 34 Back to the future: risk markers in diabetes

199

Environmental assessment of persistent glycaemic traits in a northern Swedish population

H. Pomares-Millan1, A. Poveda1, P.W. Franks1,2;

1Lund University, Malmö, Sweden, 2Nutrition, Harvard University, Boston, USA.

Background and aims: Epidemiological research has demonstrated the complex (e.g. additive, synergistic) interplay of multiple environmental risk factors in disease onset. Not all individuals exposed to shared environments will develop the disease at the same rate. The heterogeneity in the disease presentation is more evident in resilient individuals to ‘unfavourable’ environments, and also for those susceptible to a ‘favourable’ one. We aimed to investigate and characterise environmental risk factors and their association with the persistence of glycaemic traits.

Materials and methods: We investigated 18,908 Swedish participants without diabetes or cardiovascular disease in a subcohort from the Västerbotten Intervention program followed between 1999-2009. Venous and capillary blood samples were drawn after overnight fasting and 2 h after the administration of a 75-gram oral glucose load. Environmental exposures were assessed with health, socio-economic, quality of life, and food frequency questionnaires. Using an environmental-wide association approach, we prioritised risk factors associated with glycemic traits at Bonferroni-correction significance (P≤3.47×10-5). Retrieved exposures were modeled into a linear mixed model to obtain 95% prediction intervals (bootstrapping). Individuals outside the lower and upper bounds were defined as ‘persistently resistant’ and ‘persistently susceptible’, respectively. After data partition (train:70%; test:30%) we performed logistic regression (log OR); Random forest (RF) classification under 10-fold cross-validation was performed to distinguish variables associated with each subgroup. All analyses were undertaken in R software (v3.6.1).

Results: We identified 37 (out of 160 variables) shared environmental factors between the two traits at the specified significance level. Shared modifiable factors included: smoking, quality of life, fitness status, alcohol intake, and iron intake. Top non-modifiable predictors were: relatives with diabetes or heart disease, years smoking, and self-rated overall health (R2 ranged from 0.05 - 0.09). Two-hour glucose resistant (n= 2,323) and susceptible (n= 2,216), and fasting glucose resistant (n= 1,927) and susceptible (n= 2,019) individuals were assessed in logistic models (adjusted for BMI, age, age2, FFQ version, and total energy intake). Odds Ratio (95%CI) of average portion size of meat (g/day): 0.89 (0.82, 0.97); and iron intake (mg/day): 0.83 (0.69,0.97) had lower odds when compared with resistant individuals. At follow-up, smoking status was 36% (1.36 95%CI 1.06,1.75) higher in the susceptible group when compared with resistants. In Fasting glucose, a higher quality of life was associated with 22% less chance of being susceptible. The RF performance (AUC) in the testing dataset, for the first visit, was 60% and 62% in the follow-up, respectively, higher than the logistic regression models (57%).

Conclusion: Our findings suggest modifiable risk factors increased the odds of being susceptible in a long follow-up. Future epidemiological research will benefit from incorporating non-linear assessments when exploring risk factors in subgroups with persistent glycemic traits.

Supported by: Swedish Research Council, Swedish Heart-Lung Foundation, NASCENT

Disclosure: H. Pomares-Millan: None.

200

Mapping robust risk factors for the development of type 2 diabetes: a data-driven approach in Lifelines, a prospective cohort study in the Netherlands

T.P. van der Meer1,2, B.H. Wolffenbuttel1, C.J. Patel2;

1Endocrinology, University Medical Center Groningen, Groningen, Netherlands, 2Department of Biomedical Informatics, Harvard Medical School, Boston, USA.

Background and aims: Many risk factors have been identified for the development of Type 2 Diabetes (T2D), leading to different models for prediction. Yet, conventional approaches consider a limited number of factors and use techniques susceptible for effect overestimation and false-positive findings. Further, while weight, glucose, lipids, and blood pressure are known antecedents, the reporting of novel risk factors often do not consider the complex trajectories to develop overt diabetes.

Materials and methods: We aimed to assess 134 factors - to our knowledge the largest range to-date - from six domains (Biochemicals, Anthropometrics, Lifestyle, Medication, Quality of Life, Pre-determined) for T2D risk with a data-driven exposure-wide association study (XWAS) approach in the population-based Lifelines cohort study (n=96,534, 5-year follow-up). We then compared replicated risk factors between general and at-risk (i.e. pre-diabetes or cardiovascular disease) populations to glean equivalences between risk factors. Subsequently, we assessed independent contribution of replicated factors within and between respective domains with a statistical machine learning approach, lasso-regression.

Results: We were able to replicate 63 out of 134 factors, including 24 biochemicals, nine anthropometrics, 11 lifestyle factors, nine medicaments, seven quality-of-life indicators and three predetermined variables. After we removed 8,109 at-risk individuals (730 cases), all replicated factors but neutrophilic granulocytes remained nominally significant (p <0.05) and we were able to replicate 36/63 risk factors through the XWAS pipeline. Exclusion impacted hazard ratios of glycaemic traits and family history (decrease of 11-20%), and quality-of-life factors (increase of 10-33%) most. Biochemicals and anthropometric factors explained disease risk best (c-index: 0.877, 0.803). Lifestyle-related factors showed similar discrimination as medication and quality of life (0.752, 0.747, 0.742, respectively). Next to established factors, work-related and light-intensity activity, statin and H2-receptor blocker use, and dietary protein intake independently contributed to disease risk.

Conclusion: We identified a wide variety of risk factors and quantified their relative contribution to the development of T2D using a data-driven approach. Information on lifestyle and medication use can be of additional value in risk prediction models. In development of risk prediction models for T2D, we recommend a systematic approach to identify and replicate factors that are sensitive to the complex etiology of the disease.

Disclosure: T.P. van der Meer: None.

201

Plasma concentrations of methylglyoxal during an oral glucose tolerance test are associated with worse beta cell function: the CODAM and Maastricht studies

M.M.J. van Greevenbroek1, J.L.J. Scheijen1, C.J.H. van der Kallen1, P.C. Dagnelie1, S.J.P. Eussen2, C.D.A. Stehouwer1, C.G. Schalkwijk1;

1Dept. of Internal Medicine, Maastricht University, Maastricht, 2Dept. of Epidemiology, Maastricht University, Maastricht, Netherlands.

Background and aims: After a meal, methylglyoxal (MGO) concentrations in the circulation increase. MGO is a highly reactive dicarbonyl that is produced during glycolysis. MGO reacts with, among others, arginines in proteins leading to the formation of advanced glycation end products and is associated with the development of diabetic complications. In the beta-cell, proteins that mediate the fusion of insulin-containing vesicles with the cell membrane, such as VAMP2 and CD59, have arginines in their active site. We hypothesized that high MGO concentrations, particularly in persons with hyperglycaemia and/or diabetes, are associated with beta-cell dysfunction.

Materials and methods: Analyses were done in two independent observational cohorts, i.e. the Cohort on Diabetes and Atherosclerosis Maastricht (CODAM, n=473, 60±7 yrs, 24% impaired glucose metabolism [IGM], 19% type 2 diabetes [T2D]) and The Maastricht Study (n=2608, 60±8 yrs, 16% IGM, 23% T2D). A standard oral glucose tolerance test (OGTT) was performed in CODAM (0-30-60-120 min) and in The Maastricht Study (0-15-30-45-60-90-120 min). Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) were used to quantify MGO in EDTA plasma at time-points 0 and 120 min. The association of fasting and post-OGTT MGO with different aspects of beta-cell function was evaluated using multiple linear regression analyses. C-peptidogenic index (ΔCpeptide t30-t0//ΔGlucose t30-t0) was used as a simple measure of beta-cell function. Beta-cell total insulin secretion, glucose sensitivity, potentiation and glucose rate sensitivity were derived from mathematical modeling. Main dependent and independent variables were standardized. Analyses were adjusted for age, sex, glucose metabolism status, Matsuda index and fasting plasma glucose, as wells as for lifestyle factors, use of medication and metabolic risk factors.

Results: In the CODAM study, higher post-OGTT concentrations of MGO were significantly associated with worse beta-cell function as reflected by the C-peptide index (standardized beta: -0.24, 95% confidence interval (CI) [-0.32; -0.15], fully adjusted model). It was also significantly and in an adverse direction associated with total insulin secretion, beta-cell glucose sensitivity and beta cell potentiation. Associations of fasting MGO with beta cell function were weaker; the association with C-peptide index was -0.12 [-0.21; -0.03]) and the only other measure that reached statistical significance was beta-cell glucose sensitivity. These observations were confirmed in the Maastricht Study. In the latter, larger, study we performed additional analyses stratified on glucose metabolism status and the results showed that the observed associations of post-OGTT MGO concentration with worse beta-cell functions were substantially more pronounced in persons with T2D (-0.04 [-0.09; 0.00 in NGM, n=1586; 0.05 [-0.02; 0.12] in IGM, n=425; -0.21 [-0.28; -0.13] in T2D, n=597).

Conclusion: Post-prandial MGO excursions may contribute to beta-cell dysfunction.

Supported by: This work was supported by the DFN and ZonMW (Diabetes II breakthrough project).

Disclosure: M.M.J. van Greevenbroek: None.

202

Visualising heterogeneous islet autoantibody trajectories of children who develope type 1 diabetes from multi-site birth cohort studies

V. Anand1, P. Achenbach2, J.L. Dunne3, W. Hagopian4, B. Kwon1, M. Lundgren5, R. Veijola6, B.I. Frohnert7, the T1DI Study Group;

1IBM Research, Cambridge, USA, 2Helmholtz Zentrum München, München, Germany, 3JDRF, New York, USA, 4University of Washington, Seattle, USA, 5Department of clinical sciences, Lund University, Malmö, Sweden, 6University of Oulu, Oulu, Finland, 7University of Colorado, Denver, USA.

Background and aims: We investigated evolution of islet autoantibodies (IAs) prior to onset of T1D from 5 large-scale birth cohort studies. Our analysis revealed three distinct IA trajectories leading up to diagnosis of T1D.

Materials and methods: Of 24673 children from five prospective studies (DAISY, DiPiS, DIPP, DEW-IT, and BABYDIAB), 688 who were diagnosed with T1D and had 3 or more visits were included in this analysis. Hidden Markov Models were developed to label visit-level observation of each subject based on three IAs: GADA, IAA, and IA-2A. Interactive visualizations were then applied to explore model outcomes, identify IA evolution trajectories, and examine their clinical characteristics.

Results: Three trajectories were identified (figure 1) with a majority of children having multiple IA (Tr1: n=265) or IAA first (Tr2: n=282) at seroconversion; the minority seroconverted with GADA first (Tr3: n=131). The Tr3 group had seroconversion and T1D onset at an older age in months (58, 132) than Tr1 (42, 96) and Tr2 (31, 88), P < .01. Distribution of HLA DR/DQ differed between groups: higher DRX/X and lower DR3/4 in Tr3 (18%, 24%) than Tr1 (11%, 26%) and Tr2 (10%, 31%).

Conclusion: The three IA trajectories show distinctive antibody patterns, ages of seroconversion and T1D onset and HLA DR/DQ group distributions among them. Furthermore, heterogeneity is also shown within each trajectory in terms of progression time and needs further investigation.

figurebn

Supported by: 1-IND-2019-717-I-X, 1-SRA-2019-722-I-X, 1-SRA-2019-723-I-X, 1-SRA-2019-719-I-X, 1-SRA-2019-721-I-X

Disclosure: V. Anand: None.

203

The transcriptome of islets and exocrine tissue in subjects with long-standing type 1 diabetes

L. Granlund, M. Wahlhütter, A. Hedin, P. Seiron, O. Korsgren, M. Lundberg, O. Skog;

Immunology, Genetics and Pathology, Uppsala, Sweden.

Background and aims: Classically described as a disease affecting the beta cells, type 1 diabetes (T1D) has recently been recognized to entail the entire pancreas. This is due to frequent reports of reduced pancreas volume and exocrine dysfunction in some T1D patients. However, the morphology of acinar tissue has only been described in a limited number of studies, often on autopsy biopsies where autolysis of the tissue is expected due to release of exocrine enzymes. Description of the acinar tissue with molecular biological methods is even more limited. The hypotheses of this study were that acinar tissue has an altered morphology and transcriptome profile in pancreases in subjects with longstanding T1D when compared with non-diabetic subjects and that such alteration would be relative to the distance from islets. Also, the transcriptome of islets from subjects with longstanding T1D was compared with that in non-diabetic subjects.

Materials and methods: Biopsies from heart-beating organ donor pancreases from 7 subjects with longstanding T1D and 8 non-diabetic subjects were examined in this study. Histological examination and laser capture microdissection (LCM) were used to selectively study regions of acinar tissue adjacent to, and at various distances from islets. Islets were also studied using the same methodology. Transcriptome analysis was performed on the LCM extracted tissue using Ion AmpliSeq.

Results: Acinar atrophy was estimated based on nuclei density, which was not reduced closer to islets than further away from islets in T1D subjects. Neither was there any difference in acinar nuclei density between T1D- and non-diabetic subjects. Furthermore, no atrophy could be noted by visual morphological inspection. A limited number of differentially expressed genes (DEG) were found in T1D acinar tissue (50 DEG closer to islets, 16 DEG further away from islets, FDR < 0.1, FC > 2), suggesting a conserved transcriptome profile. After IHC staining for amylase, trypsinogen and lipase, amylase negative patches could only be found in one case in our cohort. In this case, the pattern was consistent on consecutive sections stained with three different antibodies targeting amylase, although the same areas were positive for trypsinogen and lipase. The amylase negative patches were randomly spread throughout the sections, appearing both adjacent to and at various distance from islets. No beta cells were detected in the T1D donors and 279 DEG (FDR < 0.1, FC > 2) in the islet tissue have been identified.

Conclusion: We conclude that several of the previously reported differences in acinar tissue of T1D pancreases could not be confirmed in our cohort and that the transcriptome of the exocrine pancreas appeared unaltered. Furthermore, amylase negative patches in non-diabetic pancreases were not as frequent as previously reported. This study was performed on biopsies obtained from pancreases treated as intended for clinical transplantation from heart-beating organ donors. This may explain some of the discrepancy as compared to earlier studies where biopsies often have been obtained from autopsies where autolysis likely was present in the pancreas. As expected given the different composition of endocrine cells in the two groups, many differently expressed genes in the islet tissue were discovered. Current analysis is ongoing to determine whether beta-cell specific genes are still expressed in the islets in subjects with longstanding T1D.

Supported by: VR, Barndiabetesfonden, Diabetes Wellness and the Nordic Network for Clinical Islet Transplantation

Disclosure: L. Granlund: None.

204

Cardiovascular health, genetic predisposition, and lifetime risk of type 2 diabetes in general population

K. Wang, M. Kavousi, T. Vortman, M. Ikram, F. Ahmadizar;

Epidemiology, Erasmus University Medical Center, Rotterdam, Netherlands.

Background and aims: Ideal cardiovascular health (CVH) is associated with lower risk of type 2 diabetes (T2D). However, data on lifetime risk of T2D incidence across different CVH categories are scare. Moreover, it remains unclear whether the impact of CVH on lifetime risk of T2D is modified by genetic predisposition to T2D.

Materials and methods: Using data from the prospective population-based Rotterdam Study, an ideal CVH score of 0-6 was calculated based on baseline measurements of body mass index, blood pressure, total cholesterol, smoking status, diet, and physical activity. Participants were categorized into poor (CVH score 0-2), intermediate (CVH score 3) and ideal (CVH score 4-6) cardiovascular health categories. Genetic predisposition to T2D was assessed by creating a Genetic Risk Score (GRS) composed of 403 common genetic variants so far identified for T2D. GRS was divided into tertiles. Incident T2D cases were determined during follow-up. In each CVH category, we used a modified version of survival analysis adjusted for the competing risk of death to estimate the remaining lifetime risk for T2D at 55, 65, and 75 years of age. We then estimated the lifetime risk of T2D based on GRS status in each CVH category.

Results: Among 6057 individuals free of T2D at baseline (mean (standard deviation) age, 69.1 (8.5) years; 58% female), 3427 (56.6%) were categorized as poor CVH, 1885 (31.1%) as intermediate CVH, and 745 (12.3%) as ideal CVH. During a follow-up time up to 14.7 years, 756 individuals developed T2D. At age 55 years, the remaining lifetime risk of T2D was 30.9% (95%CI: 28.5-33.4) for poor CVH, 26.5% (22.8-30.2) for intermediate CVH, and 18.2% (13.0-23.4) for ideal CVH. Individuals in ideal CVH category and at the lowest GRS tertile had a lifetime risk of 16.2% (7.3-25.1) for T2D, whereas those in poor CVH category and highest GRS tertile had a lifetime T2D risk of 35.7% (31.1-40.3). At age 55 years, the lifetime risk for T2D was 23.4% (18.6, 28.2) for poor CVH, 21.9% (15.9, 27.9) for intermediate CVH, and 16.2% (7.3, 25.1) for ideal CVH in the lowest GRS tertile; 29.7% (24.8, 34.6) for poor CVH, 22.5% (17.0, 28.0) for intermediate CVH, and 15.3% (6.1, 24.5) for ideal CVH in the second GRS tertile; and 35.7% (31.1, 40.3) for poor CVH, 29.2% (23.2, 35.1) for intermediate CVH, and 22.4% (12.9, 32.0) for ideal CVH in the highest GRS tertile.

Conclusion: At age 55 years, more favorable CVH was associated with lower lifetime risk for type 2 diabetes and was not counterbalanced by the genetic susceptibility for T2D. Our results highlight the importance of favorable cardiovascular health in preventing T2D among middle-aged individuals regardless of their genetic predisposition.

Disclosure: K. Wang: None.

OP 35 Diet: not only quantity matters

205

Circulating miRNAs as predictive biomarkers for effectiveness of dietary and exercise intervention for weight loss and metabolic improvement

M. Clemente-Postigo1,2, L. Coin-Aragüez1, J. Fernandez-Garcia1,3, J. Alcaide-Torres1,3, R. El Bekay4,3, M. Bernal-Lopez5,3, M. Macias-Gonzalez1,3, F.J. Tinahones1,3;

1UGC Endocrinologia y Nutricion, Hospital Universitario Virgen de la Victoria, Universidad de Malaga, Instituto de Investigación Biomédica de Málaga (IBIMA), Malaga, 2Department of Cell Biology, Physiology and Immunology, University of Cordoba, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)- Reina Sofia University Hospital, Cordoba, 3CIBER Fisiopatologia de la Obesidad y Nutricion (CIBERobn), Madrid, 4UGC Endocrinologia y Nutricion, Hospital Regional Universitario, Universidad de Malaga, Instituto de Investigación Biomédica de Málaga (IBIMA), Malaga, 5Internal Medicine Department, Regional University Hospital, Instituto de Investigación Biomedica de Malaga (IBIMA), Malaga, Spain.

Background and aims: Lifestyle modifications based on diet and exercise are raised as strategies for the treatment and prevention of obesity and related comorbidities. However, there are great heterogeneity in the type of weight loss interventions as well as high interindividual response variability. Then, strategies for predicting the individual response are required for improving intervention efficiency by personalized recommendations. microRNAs (miRNAs), small RNA particles which regulates gene expression, has been detected in the circulation and proposed as biomarkers for disease and treatment response. However, there are few studies analyzing the usefulness of circulating miRNAs (c-miRNAs) as predictive biomarkers for the response to lifestyle modifications. Furthermore, c-miRNAs has not been specifically analyzed regarding interventions based on Mediterranean diet, which has been associated with higher health-related quality of life. Thus, the aim of this study was to analyze the relationship of the response to hypocaloric Mediterranean diet and physical activity with c-miRNAs previously associated with Type 2 Diabetes (T2D) and obesity.

Materials and methods: 37 obese subjects (BMI>30kg/m2) underwent a hypocaloric Mediterranean diet together with increased physical activity and c-miRNA levels as well as biochemical and anthropometric parameters were determined before and at year 1. Participants were classified according to their 1-year weight loss in low-responders (LR) and high-responders (HR).

Results: There was a significant improvement in anthropometric and biochemical variables after intervention, but there were no differences between baseline and 1-year c-miRNA levels. However, HR subjects had lower baseline miR-130a and miR-150 levels than LR group (p<0.05). There were positive and significant (p<0.05) correlations between baseline miR-130a levels and weight at year 1 (r=0.334); baseline miR-150 levels and 1-year triglyceride levels (r=0.430) and HbA1c (r=0.404); baseline miR-142-3p and 1-year weight (r=0.58), BMI (r=0.482), glucose (r=0.333) and triglyceride (r=0.355) levels. In a lineal regression model baseline miR-150 levels were independently associated with weight loss at year 1. In silico enrichment analyses of miR-150 and miR-130a target genes showed an overrepresentation of adiposity-related pathways (white adipocyte adipogenesis, insulin signaling and lipid metabolism).

Conclusion: c-miRNAs could serve as predictive biomarkers for the interindividual response to dietary intervention based on hypocaloric Mediterranean diet and physical activity.

Clinical Trial Registration Number: ISRCTN89898870

Supported by: Juan de la Cierva (FJCI-2017-32194), PI-0092-2017, PI17/00855, CIBEROBN, Fondos FEDER

Disclosure: M. Clemente-Postigo: None.

206

Eating fast speed has a significant impact on postprandial glycaemic excursion in young healthy women: randomised controlled cross-over trial

S. Imai1, Y. Saito1, S. Kajiyama2,3, T. Miyawaki1, N. Ozasa4, S. Kajiyama5, Y. Hashimoto6, M. Fukui6;

1Kyoto Women's University, Kyoto, 2Kajiyama Clinic, Kyoto, 3Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, 4Kyoto University, Kyoto, 5Japanese Red Cross Kyoto Second Hospital, Kyoto, Kyoto, 6Kyoto Prefectural University of Medicine, Kyoto, Japan.

Background and aims: Epidemiological studies have shown associations between self-reported fast eating and diabetes, obesity, and other metabolic syndrome. However, interventional study of the effect of eating speed on glycaemic response has not been investigated. Our aim was to evaluate the acute effect of consuming test meals in different eating speed on glycaemic parameters by flash glucose monitoring system (FGM) in young healthy women.

Materials and methods: In this randomized controlled two-treatment cross-over within-subject trial, we compared postprandial glycaemic responses for varying eating speed over 2 days. Nineteen healthy women [20.8 ± 0.6 years, BMI 20.6 ± 1.9 kg/m2, HbA1c 34 ± 2 mmol/mol (5.4 ± 0.2%): mean ± SD] wore FGM for 6 days. Each participant consumed identical test meals in a different eating speed on the fourth and the fifth day. The test meals of breakfast, lunch, and dinner were consisted of boiled white rice, white bread, milk, vegetable, and frozen lunch boxes of gluten-meat steak and fried fish with vegetable with energy 1,757 kcal, protein 67.2 g, fat 41.3 g, carbohydrate 282 g, and energy ratio of protein, fat, and carbohydrate were 15%, 21%, and 64%, respectively. All participants consumed breakfast at 07:00, lunch at 12:00, and dinner at 18:00. The eating protocol was controlled as follows; in fast eating the participants consumed test meals in 10 min by mixture of vegetable, main dish, and carbohydrate, while in slow eating they consumed the identical test meals in 20 min by sequence of vegetable → main dish → carbohydrate on the fourth or the fifth day. The order of eating speed was random sequence prior to participants beginning the study. The daily glycaemic parameters were compared between 2 days of consuming identical meals in a different eating speed.

Results: The mean amplitude of glycaemic excursion (MAGE; 3.67 ± 0.31 vs. 2.67 ± 0.20 mmol/L, p < 0.01), standard deviation of glucose (SD; 1.18 ± 0.10 vs. 0.92 ± 0.06 mmol/L, p < 0.05), incremental glucose peak (IGP; breakfast 2.30 ± 0.19 vs. 1.71 ± 0.12 mmol/L, p < 0.01, lunch 4.06 ± 0.33 vs. 3.13 ± 0.28 mmol/L, p < 0.01, dinner 3.87 ± 0.38 vs. 2.27 ± 0.27 mmol/L, p < 0.001) , and incremental area under the curve for glucose of dinner (IAUC; 2h 256 ± 30 vs. 128 ± 18 mmol/L×min, p < 0.001, 3h 336 ± 34 vs. 200 ± 25 mmol/L×min, p < 0.01, 4h 373 ± 35 vs. 260 ± 31 mmol/L×min, p < 0.01) of fast eating were all significantly higher compared to those of slow eating.

Conclusion: The results of this interventional study suggest that the eating fast speed is associated with higher postprandial glucose concentrations and glycaemic excursion in young healthy women without diabetes.

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Clinical Trial Registration Number: 38684

Supported by: Kyoto women's University

Disclosure: S. Imai: None.

207

Acute metabolic effects of intermittent fasting in the morning compared to two different breakfasts among lean individuals

D. Tsilingiris, A. Tentolouris, I. Eleftheriadou, I. Anastasiou, O. Kosta, C. Dimosthenopoulos, A. Kokkinos, N. Katsilambros, N. Tentolouris;

1st Department of Propaedeutic Internal Medicine, Laiko General Hospital, Athens University Medical School, Athens, Greece.

Background and aims: It has been hypothesized that prolongation of the nocturnal low insulin state that is achieved through an early day fasting results in a greater mobilization of adipose tissue stores. The aim of this study was to investigate further this hypothesis in comparison with two different approaches of early day nutritional strategies.

Materials and methods: In this cross-over study, 10 lean healthy volunteers (7 females and 3 males, aged 28.6±4.3 years, mean BMI 22.9±1.4 kg/m2) underwent three 6-hour morning sessions after an overnight fast as follows: (a) fasting, (b) 500 kcal zero carbohydrate breakfast, and (c) 500 kcal Mediterranean-type breakfast. Fasting duration before the experiments was reported. Insulin resistance (HOMA-IR) was measured at baseline. Plasma glucose and insulin measurements as well as visual analog scales (VAS) for hunger were obtained every 30 minutes during the study. As index of adipose tissue mobilization, plasma beta hydroxybutyric acid (bHB) concentrations were used and measured via a colorimetric assay on an hourly basis. The trapezoidal rule was used to calculate the area under the curves (AUCs) during the study for all obtained parameters.

Results: The unadjusted AUC [bHB] was not significantly different among the three sessions (p=0.108). After controlling for session type, linear regression analysis demonstrated that the AUC [bHB] correlated positively with fasting duration (beta=0.416, p=0.018) and negatively with HOMA-IR (b= -0.398, p=0.024). The AUC [bHB], after adjustment for fasting duration and HOMA-IR, was significantly higher after session (a) vs (b) (p=0.021) and (a) vs (c) (p=0.008) , but it did not differ (p>0.05) between sessions (b) vs (c) (6.08±0.55 vs. 4.14±0.55 vs. 3.76±0.60 mmol/h/L, for sessions a, b and c respectively). The AUC [insulin] was significantly lower for session (a) vs (c) (p=0.001) and there was a trend to be lower in session (a) vs (b) (p=0.067) as well as between session (b) vs (c) (p=0.081), while the AUC [glucose] was similar among the three sessions (p=0.907). The AUC [VAS-hunger] was significantly higher in session (a) compared with either (b) or (c) (p<0.01) and similar between (b) and (c).

Conclusion: In young healthy lean individuals, a greater mobilization of adipose stores, lower insulin levels but higher hunger was achieved through intermittent fasting in the morning compared with either a zero carbohydrate or a Mediterranean-type breakfast intake. Carbohydrate restriction in the morning and a Mediterranean-type breakfast constitute equal choices in terms of adipose tissue mobilization and hunger suppression. Further studies are needed to examine the long-term metabolic effects of fasting in the morning.

Clinical Trial Registration Number: NCT04293003

Disclosure: D. Tsilingiris: None.

208

Manchester Intermittent versus Daily diet Diabetes App Study (MIDDAS). Pilot RCT comparing a continuous with an intermittent low energy diet in patients with type 2 diabetes

B.G. Issa1, M. Harvie2, S. Mcdiarmid1, R. Johnson1, A. Vyas1, A. Aglan3, H. Ruane2, A. Hulme1, K. Sellers2, L.A. Jones4, M.G. Jenkins4;

1Department of Endocrinology and Diabetes, Manchester University NHS Foundation Trust, Manchester, 2Prevent Breast Cancer Research Unit, The Nightingale Centre, Manchester University NHS Foundation Trust, Manchester, 3Greater Manchester Mental Health NHS Foundation Trust, Manchester, 4Oviva UK Ltd, London, UK.

Background and aims: Continuous low energy diets (CLED) providing 800 kcal/day can produce significant weight loss and remission from Type 2 diabetes (T2D). Intermittent low energy diets (ILED) may be an alternative low energy approach, supporting patient choice and adherence. This pilot RCT assesses the acceptability (uptake, retention) and efficacy (weight loss, HbA1c) of a CLED versus an isocaloric ILED (5:2 diet) in a remotely delivered, digitally-enabled programme for patients with T2D.

Materials and methods: Seventy-nine participants were randomised to CLED (n=40) (8 weeks of Optifast® 800 calorie diet followed by 4 weeks of food reintroduction) or ILED (n=39) (2 days of Optifast® 800 calorie diet and 5 days of a portion controlled Mediterranean diet for 28 weeks), followed by a maintenance phase up to 12 months. Participants received remote 1-to-1 high-frequency support from a multidisciplinary team including a dietitian, diabetes nurse, exercise specialist and psychologist via a smartphone app (features: logging of food/photos/weight/activity and goal-completion, plus video/message-based communication) and telephone calls. Weight and HbA1c were recorded at baseline, 6-months and 12-months.

Results: Baseline characteristics: 53%(m)/ 47%(f), mean+/-SD BMI 36.9(+/-5.8)Kg/m2 (CLED) and 35.8(+/5.8)Kg/m2 (ILED) and mean+/-SD HbA1c were 63.0(+/-13.7)mmol/mol (CLED) and60.3(+/-11.3)mmol/mol (ILED). At 6 months 4x CLED and 5x ILED had withdrawn, which increased to 10xCLED (25% drop out) and 12x ILED (31% drop out) at 12 months. HbA1c<48 was achieved in 67% (CLED) and 56% (ILED) at 6 months and maintained in 53% (CLED) and 48% (ILED) at 12 months. Mean(95% CI) weight losses(%) were -8.9(CI -10.4 to -7.3 )% for CLED and -8.1(-10.1 to -6.2)% for ILED at 6 months, and 6.5%( -8.1 to -4.8) and 5.9%(-7.9 to -3.8) at 12 months, respectively. Whilst higher volumes of CLED achieved ≥10% weight loss at 6 months (43% v 33%), volumes achieving this at 12 months were similar; 20%(CLED) v 19%(ILED). This trend matched the findings in HbA1c reductions, -14.6(-20.2 to -9.1))mmol/mol (CLED) and -10.9 (-14.1 to -7.7)(mmol/mol) (ILED) at 6 months, and -8.9mmol/mol (-13.5 to 4.3) and -8.3mmol/mol (-11.4 to -5.2) at 12 months respectively.

Conclusion: This shows the feasibility of both CLED and ILED dietary interventions for weight loss and reduction in HbA1c at 12 months, delivered through a remote, digitally-enabled type 2 diabetes programme. ILED may be an effective alternative to CLED. A larger RCT is needed to confirm this.

Clinical Trial Registration Number: 34397

Supported by: Nestle Health Sciences

Disclosure: B.G. Issa: Grants; Nestle Health Science.

209

The effect of dietary fiber on glycaemic control in patients with type 2 diabetes on metformin monotherapy

F. Tramontana, E. Maddaloni, S. Greci, G. Defeudis, R. Strollo, P. Pozzilli, N. Napoli;

Department of Medicine, Unit of Endocrinology and Diabetes, Campus Bio-Medico University of Rome, Rome, Italy.

Background and aims: The efficacy of increasing dietary fiber intake to ameliorate glycaemic control in patients with type 2 diabetes (T2D) is still controversial. In this randomized open-label comparator-controlled study we tested the effect of high-fiber diet and fiber supplement on glycaemic control in patients with T2D on metformin monotherapy. Changes in body weight and lipid profile were also evaluated.

Materials and methods: Seventy-eight T2D overweight and obese patients on metformin monotherapy were randomized 1:2:1 to 12 weeks intensive nutrition program to follow standard diet recommendations (SDR), high-fiber diet (HFD) or dietary fiber supplementation (FS). Dietary recommendations were reinforced in all groups every 4 weeks by study dieticians. HFD contained a minimum of 35 g of dietary fiber per day through the consumption of unfortified foods. The FS added 9.73 g of dietary fiber per day to the normal nutrition. Biochemistry, anthropometric measures and food frequency questionnaires to asses dietary fiber intake were collected at baseline and after 12 weeks.

Results: At baseline groups did not differ in terms of mean age, BMI, metformin intake, HbA1c, fiber and calorie intake (p> 0.05 for all). After three months, dietary fiber intake significantly increased in both HDF and FS group but not in SDR group (HFD: 19.8 ± 6.1 g vs 24.3 ± 6.8 g, p= 0.0001; FS: 17.5 ± 5.9 g vs 27.0 ± 6.2 g p< 0.0001; SDR: 22.8 ± 9.1 g vs 21.2 ± 6.4 g, p= 0.32). HbA1c significantly improved in all groups (SDR: 7.2 ± 0.4 % vs 6.7 ± 0.5 g, p< 0.001; HFD: 7.1 ± 0.5 % vs 6.6 ± 0.6 %, p< 0.0001; FS: 7.1 ± 0.5 % vs 6.8 ± 0.5 % p< 0.001). All SDR, HFD and FS interventions reduced mean body weight by 1.1 ± 2.1 Kg (p< 0.05), 2.1 ± 2.6 Kg (p< 0.0001) and 1.0 ± 1.8 Kg (p< 0.05), respectively. Changes in HbA1c and body weight did not differ among groups. A significant correlation between calorie intake and the reduction of HbA1c levels was seen across groups (r= 0.307 p<0.01). No significant correlation between dietary fiber intake and HbA1c levels was observed. Total cholesterol, HDL, LDLc and triglycerides did not significantly change in all groups.

Conclusion: Intensive nutrition education programs with monthly meetings similarly reduced HbA1c in all groups. Furthermore, our study suggested that rather than fiber intake, caloric restriction followed by moderate weight loss is the main driver for glycaemic improvement in overweight and obese patients with T2D.

Disclosure: F. Tramontana: None.

210

Association of daily carbohydrate intake with glycaemic control in adults with type 1 diabetes using a hybrid closed-loop system

V. Lehmann, T. Zueger, A. Zeder, L. Bally, M. Laimer, C. Stettler;

Department of Diabetes, Endocrinology, Nutritional Medicine and Metabolism, Inselspital Bern, University Hopital and University of Bern, Bern, Switzerland.

Background and aims: With the clinical implementation of hybrid closed-loop (HCL) systems, efforts are moving towards personalised medicine. However, sparse evidence exists on how individual carbohydrate (CHO) intake affects glycaemic control in type 1 diabetes (T1D), especially in those using an HCL system. The HCL-device MiniMed 670G (MM670G) requires CHO-input for mealtime bolus calculation while in “auto-mode”. We aimed at assessing glycaemic control as a function of individual daily CHO-intake.

Materials and methods: We screened data from 59 adults with T1D using the MM670G HCL system between 11/2018 and 10/2019 at our tertiary referral centre. CHO-intake (g/day) and CGM data were evaluated during a 30-day period before a routine visit. Only days with availability of ≥ 70% of CGM data, and with time in “auto-mode” ≥ 50% were included in the analysis. Mean individual, daily CHO-intake (MIDC) was assessed per patient. For each day, the relative deviation from MIDC (rMIDC) was calculated, and days were stratified into low, medium and high CHO-consumption (≤80%, 81-120% and >120% rMIDC, respectively). Glucose control was assessed using standard CGM metrics including time in target range (TIR, 3.9-10.0 mmol/L), time above target range (TAR, >10mmol/L), time below target range (TBR, <3.9 mmol/L), mean glucose, and coefficient of variation (CV). CGM readouts were additionally stratified by time in “auto-mode” (<80%, 80-90%, >90%). The three rMIDC groups were compared using ANOVA and associations between CHO-intake and CGM metrics were assessed using mixed linear models.

Results: Records from 36 patients (26 male, 10 female; age 36.9±13.5y; HbA1c 7.1±0.9%; diabetes duration 23.0±13.0y; BMI 26.5±3.6kg/m2) were included, providing a total of 810 days of data (22.5±6.7 days per patient). Average time on MM670G at time of analysis was 107±36 days. Mean time of sensor use was 96.1±6.2% and mean time in ‘’auto-mode” was 91.0±12.4%. Mean daily CHO-intake was 166.4±69.6g distributed over 5.7±3.2 meals per day. CGM-findings and average daily CHO-intake of the three rMIDC-groups stratified according to time in ‘’auto-mode” are displayed in table 1. Mixed linear models adjusted for time in ‘’auto-mode” showed a decrease in TIR of -1.1% (p<0.001) and an increase in TAR of 1.2% (p<0.001) for every 10% increase in relative, daily CHO-intake. There was no effect of daily CHO-intake on TBR (p=0.42).

Conclusion: Individual daily CHO-intake was inversely associated with glycaemic control in adults with T1D using the MiniMed 670G HCL-system. The effect appears more pronounced with higher time in ‘’auto-mode”, suggesting that carbohydrate restriction may facilitate glucose control in patients consistently using the ‘’auto-mode”.

figurebp

Disclosure: V. Lehmann: None.

OP 36 On the road to human islet failure in type 2 diabetes

211

Cross-sectional multi-omics insight from islet and plasma samples into the progression to type 2 diabetes in metabolically profiled pancreatectomised surgical donors

L. Wigger1, M. Barovic2,3, A.D. Brunner4, F. Marzetta1, E. Schöniger2,3, F. Mehl1, N. Kipke2,3, K. Simons5, M. Distler6, A.M. Schulte7, M. Mann4, M. Ibberson1, M. Solimena2,3;

1Vital-IT, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland, 2Paul Langerhans Institute Dresden (PLID), Helmholtz Center Munich, Dresden, Germany, 3German Center for Diabetes Research (DZD e.V.), Neuherberg, Germany, 4Max Planck Institute of Biochemistry, Martinsried, Germany, 5Lipotype GmbH, Dresden, Germany, 6Department of Surgery, University Hospital and Faculty of Medicine, TU Dresden, Dresden, Germany, 7Sanofi Deutschland GmbH, Frankfurt, Germany.

Background and aims: Type 2 diabetes mellitus (T2D) is caused by the complex interplay of genetic and environmental factors. Its key physiological phenotype is the inability of pancreatic islet beta cells to secrete insulin in amounts adequate to metabolic demand. We performed a comprehensive multi-omics analysis of the islet state in relationship to glycemic control by integrating clinical traits with multiple islet and preoperative peripheral blood omics datasets of metabolically profiled pancreatectomized patients (PPP) across the continuum of glycemic rise from healthy to overt T2D.

Materials and methods: We collected medical histories and laboratory data, e.g. preoperative fasting glucose, HbA1c and 2-hour glucose OGTT values, in a cohort of 133 PPPs at a university hospital and stratified them into subgroups using ADA-recommended criteria for diabetes and prediabetes. Surgical pancreatic tissue and preoperative peripheral blood samples were snap frozen immediately after collection. Laser capture microdissected (LCM) islets were analyzed by RNA sequencing and mass spectrometry-based proteomics. Blood plasma lipidomics analyses were performed by shot-gun and targeted mass spectrometry. Data analysis included differential abundance analyses, pathway overrepresentation analyses and a multi-omics approach combining RNA-Seq, lipidomics and clinical data.

Results: We identified 535 differentially expressed genes in islets of 39 T2D PPPs (adj. p ≤ .01; FC ≤ 1.5) compared to islets of 18 non-diabetic (ND) PPPs. After additional QC filtering, we found 40 genes that were differentially expressed in islets of PPPs with impaired glucose tolerance (IGT) versus ND islets. Weighted gene correlation network and consensus orthogonal partial least square (WGCNA and Consensus OPLS) analyses identified gene co-expression modules and lipids jointly associated with HbA1c levels. Extracted model features constitute a combined transcriptomic-lipidomic signature for beta cell decline in T2D. Genes from the signature modules were enriched, e.g., in pathways related to carbohydrate, protein and sphingolipid metabolism and AMPK signaling. We found Aldolase B to be a major discriminator between islets of T2D and ND patients by both RNA-Seq (adj. p = .0004) and proteomics analysis (p = 4.15 x 10-10) while its gene co-expression module correlated well with HbA1c levels (r = 0.67).

Conclusion: For the first time, a multi-omics approach integrating transcriptomics and lipidomics data by WGCNA and Consensus OPLS identified islet gene co-expression modules and plasma lipids that characterize the progressive increase of HbA1c, hence pointing to promising candidates with direct or indirect causal relationship with beta cell failure in T2D. Proteomics analysis of LCM islets provided corroborating evidence for gene regulation patterns found in our primary analysis.

Supported by: EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (RHAPSODY grant No 115881)

Disclosure: L. Wigger: None.

212

Integration of single-cell datasets reveals novel transcriptomic signatures of beta cells in human type 2 diabetes

E. Bosi1, L. Marselli1, C. De Luca1, M. Suleiman1, M. Tesi1, M. Cnop2, D. Eizirik3, M. Ibberson4, P. Marchetti1;

1Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy, 2Division of Endocrinology, Universite Libre de Bruxelles, Brussels, Belgium, 3Center for Diabetes Research, Universite Libre de Bruxelles, Brussels, Belgium, 4Swiss Institute of Bioinformatics, Lausanne, Switzerland.

Background and aims: Pancreatic islet β-cells are key to the onset and progression of type 2 diabetes (T2D). Given the heterogeneity of islet cell subpopulations, the advent of single-cell RNA sequencing to study β cells transcriptomes has been welcomed. However, the application of this technique has been underwhelming, as three independent studies focused on the differences showed no shared differentially expressed genes in T2D β-cells. Here, we performed an integrative analysis of data from available studies of human islets from T2D and non-diabetic donors to overcome confounding sources of variability and better highlight T2D β-cell transcriptomic signatures.

Materials and methods: Raw sequencing data was downloaded from 3 available studies. Reads were aligned using STAR against GRCh37 (ensembl annotation) to obtain gene read counts for each cell and to filter low quality cells. The use of Scanpy allowed to perform dataset integration with MNN and to identify the main cell types. Differences associated with T2D in β cells were identified using DESeq2 (gene expression) and Enrichr (gene set enrichment).

Results: Integrating the reads of 3 single cell studies we produced a dataset with 3046 single cells collectively expressing 27931 genes. Cell level analyses allowed to reduce dataset specific biases and divide cells into endocrine cell types.In T2D β-cells (n=801) we found 210 and 16 genes respectively up and down regulated (several of which not reported previously) identifying key pathways and functions (including defective insulin secretion, SREBP signaling, oxidative stress and apoptosis) that are enriched in cells from T2D donors. Using available literature and databases, we manually curated the associations between differentially expressed protein-coding genes (n=60) and T2D at different levels, including β cell failure mechanisms. For 35 genes it was possible to find a known relation with T2D, whereas the other 25 were not previously described. Of them, 16 have a function unknown while 9 could be ascribed to processes linked to β cell dysfunction.We also compared our results with previous micro-array data of β cells obtained by laser capture microdissection (LCM). Despite differences between the methods, we identified 6 shared genes over-expressed in both RNA-seq and LCM T2D β cells, mostly not described so far.

Conclusion: In this work we harmonised available single-cell transcriptomics datasets of human islets, creating an integrated dataset that will represent an important resource for the community. The analysis of β cells from this dataset allowed to identify differentially expressed genes previously undetected that might represent central components of β cell dysfunction in T2D.

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Supported by: RHAPSODY, INNODIA, T2DSystems,

Disclosure: E. Bosi: None.

213

Single cell transcriptomics of transplanted human islets

L. Chen1, A. Ahnmark1, X. Li1, A. Zhou1, J. Liu2, Q. Peterson3, B. Tyrberg4, M.S. Winzell5, B. Zarrouki1;

1Research and Early Development Cardiovascular, Renal and Metabolism (CVRM), BioPharma R&D, AstraZeneca, Mölndal, Sweden, 2Single Cell Sequencing Facility, Integrated Cardio Metabolic Centre (ICMC), Karolinska Institutet/AstraZeneca, Huddinge, Sweden, 3Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, USA, 4Cardiovascular and Metabolic Diseases, Institute de recherches Servier, Suresnes, France, 5AstraZeneca IPD-CA, South San Francisco, USA.

Background and aims: The single cell transcriptome of pancreatic endocrine cells has been extensively characterized in vitro by several groups. However, the metabolic profiling and transcriptomic understanding of human pancreatic endocrine cells in vivo is still elusive. Extensive in vitro manipulation and long-term culture may change the phenotype of cells and thereby change the transcriptomic profile. In this study, we utilize single cell RNA sequencing (scRNA-Seq) technology to study implanted human pancreatic endocrine cells transcriptomics in vivo. By using this in vivo model system, we aim to better understand the transcriptomic changes that may occur during islet isolation and in vitro culture. We also aim to build on this model to understand islet transcriptomics during metabolic stress and diabetes in the recipient animal to model human disease.

Materials and methods: The Scid beige mice (CB17.Cg-PrkdcscidLystbg-J/Crl) were used as recipients for the transplantations. About 1500IEQ (Islet equivalents) human islets were transplanted under the kidney capsule and stayed maintained for 22 weeks. After transplantation, human C-peptide level was monitored at week 10 and week 22 to determine the in vivo function of human the islets. The xenografts was were dissected by using customized surgical tools and cell dissociation. The dissociated cells were stained with a live cell indicator (Calcein AM) followed by a human cell surface marker (HLA-ABC). Live human cells were then sorted into 384-well plates and used for single cell RNA sequencing (Smart-seq2). The scRNA-Seq data was then compared to Sandberg’s published in vitro human islet data.

Results: Human C-peptide increased over time post transplantation (267±13pM at 10 weeks; 1875±79pM at 22 weeks). After dissociation with TrpLE select and staining with Calcein A plus human HLA-ABC, we succeeded in sorting out a fraction of live human islet cells. Subsequent scRNA-seq analysis confirmed insulin, glucagon, PPY and GHRL positive cells. A set of typical beta-cell transcription factors including IAPP, NEUROD1, PAX6, MAFA/B, PDX1 were identified. In common with healthy donors in Sandberg’s study, we identified 4820 overlapping genes. For example the genes that were reported to express in endocrine cells, such as CHGA, SCGN, G6PC2, PCP4, STMN2, LINC00643, SLC30A8, were also found in our study. In addition, we identified 105 additional islet genes, which are annotated as “enriched in endoplasmic reticulum membrane (GO:0005789)”, “apical plasma membrane (GO:0016324) and extracellular exosomes (GO:0070062)”.

Conclusion: Our study has successfully established a model to study human pancreatic endocrine cells transcriptomics in vivo in a humanized mouse model. Further application will be transplanting human islets or inducible pluripotent cells-derived alpha/beta cells into normal and diabetic Scid beige mice (HFD-induced) to identify key changes during long-term diabetes.

Supported by: AstraZeneca postdoc innovation fund

Disclosure: L. Chen: None.

214

Endoplasmic reticulum stress contributes to the loss of beta cell identity in human pancreatic islets treated with glibenclamide

C. Fernandez1, N. Tellez1, V. Gutierrez1, K. Rivera1, M. Nacher1,2, E. Montanya1,2;

1CIBERDEM, IDIBELL-Universitat de Barcelona, Barcelona, 2Endocrine Unit, Hospital Universitari Bellvitge, Barcelona, Spain.

Background and aims: Loss of pancreatic beta cell mass and beta cell dysfunction are central in the development of type 2 diabetes (T2DM). Reduced beta cell mass has been attributed to increased beta cell apoptosis, and more recently to beta cell dedifferentiation. Beta cell dedifferentiation has been described in response to chronic pathophysiological stress. Chronic closure of the KATP channel in rodent beta cells by exposure to the sulfonylurea glibenclamide, or by genetic manipulation results in impaired beta cell function, endoplasmic reticulum (ER) stress and loss of beta cell mass, that could account for the secondary failure to sulfonylurea treatment described in T2DM. We aimed to investigate whether chronic exposure of human islets to glibenclamide induces beta cell dedifferentiation, and the potential contribution of ER stress.

Materials and methods: Islets from human multi-organ donors (n=13, 30.8% female; 57.38±2.3 y.o.; BMI: 25.5±0.89) were cultured for one week at 5.5 mM glucose in the presence or not of Glibenclamide (1 μM) and the chemical chaperone 4-phenylbutyrate (PBA, 2.5 mM). Beta cell function was evaluated by Glucose-Stimulated Insulin Secretion (GSIS), at 2.8 mM (basal) and 20 mM (stimulated) glucose, insulin content by ELISA (DNA-corrected) and beta cell apoptosis by TUNEL assay. Beta cell dedifferentiation and ER stress were determined by differential gene expression analyses of beta cell identity, disallowed, progenitor-related, and ER stress markers using qRT-PCR (Taqman).

Results: Human islets exposed to glibenclamide showed beta cell dysfunction (stimulation index in control group (C): 5.05±1.32; Glibenclamide: 1.65±0.26, p=0.05) with increased basal insulin secretion (C: 0.87±0.15%; Glibenclamide: 1.88±0.51%, p=0.07) and similar stimulated insulin secretion (C: 4.5±1.6%; Glibenclamide: 3.01±0.82%, p=0.17). Insulin content was similar (C: 349.2±87.6; Glibenclamide: 267.0±78.7 ng Ins/μg DNA, p=0.21). Differential gene expression analyses showed downregulation of key beta cell transcription factors MAFA, MAFB, PDX1, NKX6.1, NKX2.2 and PAX6, as well as insulin, PCSK2 and the beta cell marker GLP1R (p<0.05). No differences were observed on disallowed gene transcripts (HK1, HK3, LDHA) or progenitor-related gene transcripts (ALDH1A3, NGN3, SOX9). Glibenclamide-cultured islets showed differential gene expression of ER stress markers: increased DDIT3, increased XBP1 spliced form and decreased WFS1 (p<0.05). Accordingly, beta cell apoptosis was significantly increased (C: 0.75±0.05%; Glib: 1.49±0.24%, p=0.04). Addition of PBA prevented glibenclamide-induced changes in gene expression of ER stress markers DDIT3XBP1s and WFS1 and the downregulation of the beta cell identity markers PDX1, NKX6.1, NKX2.2, PAX6 and GLP1R.

Conclusion: Chronic exposure of human islets to glibenclamide resulted in ER stress and loss of beta cell identity. ER stress relief by addition of PBA partially prevented glibenclamide-induced loss of beta cell identity, indicating that glibenclamide-induced loss of beta cell identity in human islets is mediated, at least partially, by ER stress.

Supported by: PI 16/00462 ISCIII, FEDER

Disclosure: C. Fernandez: None.

215

Deciphering glucocorticoid-mediated stress responses in the human pancreatic beta cell

A. Karagiannopoulos1,2, J. Ofori1,2, J.L.S. Esguerra1,2, L. Eliasson1,2;

1Islet Cell Exocytosis, Department of Clinical sciences - Malmö, Lund University, Lund, 2Lund University Diabetes Centre, Skåne University Hospital, Malmö, Sweden.

Background and aims: Glucocorticoids (GCs) are a class of steroid hormones that are widely prescribed due to their anti-inflammatory and immunosuppressant properties. However, their use comes with various metabolic complications such as steroid-induced diabetes mellitus. Although the role of glucocorticoids is established in insulin resistance, recent studies show that they can also directly impair insulin secretion from pancreatic β-cells. Here, we investigated the transcriptomic changes in human islets and beta cells upon GC treatment to elucidate the molecular mechanisms underlying GC-mediated β-cell dysfunction.

Materials and methods: We treated human pancreatic islet preparations (n=4) and EndoC-βH1 cells (n=4) with dexamethasone (2 uM). We then performed RNA-seq using Illumina-based platform to identify differentially-expressed genes. To screen for direct glucocorticoid receptor (GR) targets, a customized bioinformatics pipeline was developed, in which RNA-seq data were integrated with publicly available GR ChIP-seq data. An aggregate score similar to a p-value was generated for each gene indicating the probability of direct GR targeting.

Results: RNA-seq revealed both established and novel genes to be differentially regulated in GC-treated human islets (1507 genes) and EndoC-βH1 cells (3175 genes) (adj P value = 0.05). Integration with ChIP-seq data revealed many potential direct targets of the glucocorticoid receptor. We found ZBTB16 to be the top GR target in both the EndoC (aggregate score 0.00001) and the human islet set (aggregate score 0.00005). Additionally, the pipeline validated other properties of GR such as the preferential DNA binding to distal, intronic and conserved sites. Finally, the discovery of transcription factor (TF) binding motifs in non-canonical GR DNA-binding regions, as well as their evolutionary conservation, imply the involvement of co-regulating TFs in the GC-regulated gene transcriptional program in the beta cell.

Conclusion: In this project we identified potential direct GR gene targets and GR binding properties in the human pancreatic β-cell, revealing at the same time the important role of auxiliary transcription factors in the GC-dependent gene regulation. Overall, this study provides a better understanding of the way GCs could potentially disrupt the normal β-cell function that triggers the development of diabetes in patients undergoing GC treatment.

Supported by: VR-SSF Exodiab, SSF LUDC-IRC, Crafoord Foundation

Disclosure: A. Karagiannopoulos: None.

216

Mitochondrial STAT3 contributes to pancreatic beta cell adaptation in obesity

A. Schaschkow1, L. Pang2, S.A. Litwak2, E. Maillard3, F.M.M. Paula1, D.L. Eizirik1, P. Marchetti4, D.J. Gough5, E.N. Gurzov1;

1ULB Center for Diabetes Research, Brussels, Belgium, 2St Vincent’s Institute of Medical Research, Melbourne, Australia, 3Centre Européen d'Etude du Diabète, Strasbourg, France, 4Department of Clinical and Experimental Medicine, Pisa, Italy, 5Hudson Institute of Medical Research, Melbourne, Australia.

Background and aims: While reduction of β-cell mass and function can be observed in obese diabetic patients, most obese and insulin resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and function, but little is known about its role in β-cell adaptation in obesity.

Materials and methods: Human pancreas from organ donors with different body mass index (BMI) have been stained for STAT3 by immunofluorescence. To address the functional role of STAT3 in adult β-cells, we generated a tamoxifen-inducible STAT3 β-cell specific knockout model in MIP-CreERT transgenic mice (βSTAT3KO mice) and fed them with high fat diet before analysis. The EndoC-βH1 β-cell line and dispersed human islets were transfected with STAT3 siRNAs and mitochondrial respiration measured.

Results: In human organ donor pancreas (n=3-5), STAT3 was localized in the cytoplasm of β-cells and its expression correlated with BMI (p<0.05, lean vs obese). To study the role of STAT3 in β-cells in vivo, we treated βSTAT3KO homozygous, heterozygous and control mice with oral gavages of tamoxifen at 10 weeks of age to induce complete or 50% STAT3 gene deletion respectively. The mice did not show any metabolic phenotype after 14 weeks when fed a chow diet (n=5-9). We challenged βSTAT3KO and control mice with a high fat diet as model of obesity. Body weight, oxygen consumption, respiratory exchange rate, energy expenditure, food intake and ambulatory activity were similar in 12 weeks high fat fed βSTAT3KO and control mice (n=8-14). Interestingly, homozygous and heterozygous βSTAT3KO mice showed glucose intolerance in oral and intraperitoneal glucose tolerance tests when compared to controls (n=10-14, p<0.05). The serum insulin concentration in βSTAT3KO mice was 2.1-fold lower than in control mice after glucose administration (n=9, p<0.05). No difference was observed in percentage insulin area between βSTAT3KO and control mice, suggesting no changes in β-cell mass. qPCR analysis showed reduced (30-35%) expression of mitochondrial genes Nd4Nd5 and Cytb in FACS-purified β-cells from βSTAT3KO mice (n=4-5, p<0.05). Mitochondrial STAT3 was confirmed by colocalization studies in β-cells of high fat fed mice and in EndoC-βH1 cells. EndoC-βH1 cells with 80% knockdown of STAT3 have impaired mitochondria activity (n=4, p<0.05). The result was confirmed in 5 out of 6 dispersed human islet preparations, suggesting a mechanism for STAT3-regulated β-cell function.

Conclusion: Our results implicate STAT3 in mediating β-cell adaptation in obesity. We propose a novel role of STAT3 in the regulation of mitochondrial activity during glucose induced insulin secretion in β-cells.

Supported by: NHMRC - FNRS

Disclosure: A. Schaschkow: None.

OP 37 A deep dive into the mechanisms of diabetes

217

Pnliprp1 hypermethylation in human exocrine pancreas reveals a link between diabetes and pancreatic cancer

A. Khamis1,2, M. Canouil1, L. Marselli3, R. Boutry1, M. Suleiman3, N. Jonckheere4, A.M. Schulte5, M. Solimena6, A. Bonnefond1, I. Van Seuningen44, M. Ibberson7, A. Jörns8, S. Lenzen8, P. Marchetti3, P. Froguel1,2;

1Univ. Lille, CNRS, CHU Lille, Institut Pasteur de Lille, UMR 1283/8199 - EGID, Lille, France, 2Imperial College London, Section of Genomics of Common Disease, Department of Metabolism, London, UK, 3University of Pisa, Department of Clinical and Experimental Medicine, Pisa, Italy, 4Univ. Lille, CNRS, Inserm, CHU Lille, UMR9020 – UMR1277 – Canther – Cancer Heterogeneity, Plasticity and Resistance to Therapies, Lille, France, 5Sanofi-Aventis Deutschland GmbH, Diabetes Research, Frankfurt, Germany, 6Paul Langerhans Institute Dresden of the Helmholtz Center Munich at University Hospital Carl Gustav Carus and Faculty of Medicine, Dresden, Germany, 7Vital-IT Group, Swiss Institute of Bioinformatics, 1015, Lausanne, Switzerland, 8Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.

Background and aims: Type 2 diabetes (T2D) is a known risk factor of the mostly lethal pancreatic cancer, but the molecular mechanisms are unknown. The putative epigenetic effects of hyperglycaemia in the exocrine tissue has not yet been explored.

Materials and methods: Pancreatic exocrine tissue from 141 organ donors were isolated and subjected to a genome-wide DNA methylation analysis performed using Illumina’s Infinium ‘850K’ MethylationEPIC array. Genotyping of the same samples was performed using the Illumina Omni2.5M array and combined with methylation data to generate cis-mQTLs (cis-methylation quantitative trait loci) using a cis-window of 50 kb. Biological validation was performed using the rat exocrine cell line AR42J.

Results: We found one FDR-significant epigenetic association with T2D at the cg15549216 probe located within the gene body of the PNLIPRP1 gene (Pancreatic Lipase Related 1 Protein) (beta-value = 11.2 %; FDR = 0.02; unadjusted p-value = 2.1x10-8; standard error = 0.10). Based on the aggregation of this CpG with two adjacent CpGs (cg06606475 and cg08580014), nominally associated with T2D in the same locus, we identified a strong overall differentially methylated region (with a minimum FDR = 3.2x10-10). We showed that PNLIPRP1 expression was dysregulated in response to high glucose and insulin treatment of the AR42J cell line (35% downregulation; p<0.01). We further investigated a link between PNLIPPR1 and pancreatic cancer and found that the expression of PNLIPRP1 in two human cohorts was significantly decreased in human pancreatic tumour tissue compared to normal tissue (p<0.001). Finally, we combined our methylome analysis with genotyping data of the same samples and found 13 significant cis-mQTLs that co-localised with GWAS SNPs for T2D (including KCNJ11) and pancreatic cancer (ZNRF3and FAM91A1), demonstrating a pleiotropic effect of these variants.

Conclusion: We have identified epigenetic markers for T2D in the exocrine pancreas in genes relevant to pancreatic cancer pathophysiology, providing insight into the link between T2D and pancreatic cancer. Although future studies are needed to investigate the role of PNLIPRP1 in exocrine tissue within the context of T2D, our novel findings provide a foundation for future hypothesis development in clinical studies for prevention and treatment of pancreatic cancer.

Supported by: RHAPSODY IMI

Disclosure: A. Khamis: Grants; RHAPSODY.

218

Anti-insulin receptor antibodies improve hyperglycaemia in a mouse model of human insulin receptoropathy

G.V. Brierley1, H. Webber2, E. Rasijeff2, S. Grocott2, K. Siddle1, R.K. Semple1,3;

1Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, 2MRC Disease Model Core, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, 3Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK.

Background and aims: Biallelic loss-of-function mutations in the insulin receptor (INSR) usually lead to death in childhood or early adult life, and therapeutic options are limited. This study aims to build upon previous proof-of-concept studies in cell model systems and generate a novel mouse model of insulin receptoropathy. This mouse model was used to assess the effects of two thoroughly characterised murine anti-INSR monoclonal antibodies (83-7, 83-14) as surrogate agonists to provide targeted therapy for the treatment of severe insulin resistance arising from insulin receptoropathies.

Materials and methods: A novel mouse model of insulin receptoropathy was generated using adeno-associated virus (AAV) to deliver Cre to the liver of floxed Insr mice. After knockout of endogenous mouse Insr (L-IRKO), adenovirus (AdV) were used to ‘add-back’ mutant human Insr transgenes, enabling the shuttling of different mutant human receptors into the model. Utilising this approach, two mutant human INSR (S350L, D734A) and WT INSR were expressed in the liver of L-IRKO mice. Antibodies were administered twice weekly (10mg/kg) via intraperitoneal injection and glucose metabolism assessed after a five hour fast by oral glucose tolerance test (2g/kg).

Results: Viral vectors effectively transduced the liver of floxed Insr mice resulting in knockout of endogenous mouse INSR (L-IRKO+GFP) and ‘add-back’ of human INSRs. L-IRKO+GFP mice were glucose intolerant and hyperinsulinemic, which was corrected by the add-back of human WT INSR, but not mutant human INSR. Antibody treatment reduced hyperinsulinemia in both S350L and D734A INSR-expressing mice and improved glucose tolerance in D734A INSR-expressing animals. Antibody injection did not cause hypoglycemia in WT INSR-expressing animals. Antibody treatment lead to a downregulation of both WT and mutant INSR protein expression, attenuating its beneficial metabolic effects.

Conclusion: A novel mouse model of insulin receptoropathy was generated in which the therapeutic potential of anti-insulin receptor antibodies with partial agonistic activity could be tested. Antibodies improved glucose tolerance and reduced hyperinsulinemia in the acute model of insulin receptoropathy. However, results imply a narrow therapeutic window due to the effect of antibodies on receptor downregulation.

Supported by: Diabetes UK (15/0005304)

Disclosure: G.V. Brierley: Grants; Diabetes UK (15/0005304), Wellcome Trust (210752/Z/18/Z), MRC (MC_UU_00014/5).

219

Chromatin 3D interaction analysis of the STARD10 locus unveils FCHSD2 as a new regulator of insulin secretion

M. Hu1, I. Cebola2, G. Carrat1, S. Nawaz3, A. Khamis4, M. Canouil4, P. Froguel2, A. Schulte5, M. Solimena6, M. Ibberson7, P. Marchetti8, P. Gadue9, B. Hastoy3, H. McMahon10, G. Rutter1;

1Section of Cell Biology and Functional Genomic, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK, 2Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, UK, 3Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Oxford, UK, 4CNRS, CHU Lille, Institut Pasteur de Lille, University of Lille, Lille, France, 5Diabetes Research, Industriepark Höchst, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany, 6Molecular Diabetology and DZD, Paul Langerhans Institute, Dresden, Germany, 7Vital-IT Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland, 8Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy, 9Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA, 10MRC MRC Laboratory of Molecular Biology, Cambridge, UK.

Background and aims: Genome-wide association studies (GWAS) have identified more than 200 loci in the human genome associated with type 2 diabetes. We recently analysed a locus close to the STARD10 gene and mapped five causal variants within an islet enhancer cluster. Here, we aimed to understand how these variants affect enhancer activity, gene expression and beta cell function.

Materials and methods: EndoC-βH1 cells were used throughout. Promoter-luciferase reporter, chromatin conformation capture (3C), glucose-stimulated insulin secretion (GSIS) assay, membrane capacitance measurements and expression quantitative trait locus analysis (eQTL) were deployed. CRISPR/Cas9 genome editing was used to create the required mutations or deletions.

Results: We first analysed the enhancer regions at this locus. Of these, R2 displayed a 6-fold increase of luciferase activity compared with the empty vector [control, 1.26 ± 0.06 vs. R2, 7.82 ± 0.17; p<0.001; n=3]. Deletion of R2 using CRISPR/Cas9 reduced STARD10 (fold; Control 1 vs. dR2, 0.39 ± 0.07; p<0.001, n=3) and FCHSD2 (fold; Control 1 vs. dR2, 0.7 ± 0.06; p<0.01, n=3) gene expression, and lowering of GSIS (15/0.5 mM glucose, fold change: Control, 3.07 ± 0.11 vs. dR2, 1.72 ± 0.16; p<0.05; n=4). 3C assays demonstrated that the causal variants interact with R1 and R13 enhancers through chromatin looping. Deletion of the variant region (VR) reduced the chromatin interaction between R1 and R13, lowered STARD10 (fold: control 1 vs. dVR, 0.765 ± 0.03; p<0.01, n=3) and FCHSD2 (fold: control 1 vs. dVR, 0.859 ± 0.03; p<0.01, n=3) mRNAs and reduced GSIS (15/0.5 mM glucose, fold change: control, 2.00 ± 0.11 vs. dVR, 1.64 ± 0.06; p<0.05; n=4). Deletion of STARD10 reduced GSIS (15/0.5 mM glucose, fold change: Control, 2.56 ± 0.14 vs. STARD10-KO, 2.12 ± 0.19; p<0.05; n=4) while FCHSD2 deletion reduced basal insulin secretion (0.5/0.5 mM glucose, fold change: Control, 1 ± 0 vs. FCHSD2-KO, 0.78 ± 0.053; p<0.05; n=4). In preliminary measurements of capacitance changes, a non-significant tendency towards lowered exocytosis was observed after FCHSD2 inactivation. A nominal association by eQTL analysis was observed with rs1552224 and FCHSD2 expression in analysis of 103 samples from IMIDIA consortium (n=47 non diabetes, n=56 T2D, p=0.013). The association with STARD10 expression was similarly significant in these samples (p=2.98 x 10-4).

Conclusion: Both STARD10 and FCHSD2 contribute to disease risk at the STARD10 locus and might provide new therapeutic targets.

Supported by: Wellcome Trust Senior Investigator, MRC Programme grants

Disclosure: M. Hu: None.

220

Non-parallel roles of three disulfide bonds in proinsulin folding and pathogenesis of diabetes

Y. Yang, H. Shu, Y. Huang, X. Zhang, L. Ding, M. Liu;

Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin, China.

Background and aims: Proinsulin (PI) has three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11), which are critical for PI folding in the endoplasmic reticulum (ER). The B19-20 bond is considered as the most important and the first formed bond during oxidative folding of PI. Insulin gene mutations disrupting any one of the three disulfide bonds can lead to proinsulin misfolding and diabetes in humans. Diabetogenic effects of unpaired B7-A7 or A6-A11 bonds has been experimentally confirmed in mouse lines and transgenic pigs. However, transgenic expression of C(B19)G mutation did not impair glucose homeostasis and cause β-cell apoptosis in zebrafish, suggesting that diabetogenic effect caused by disruption of B19-A20 bond remain to be elucidated. In a program screening for monogenic diabetes in China, we identified PI-C(B19)Y mutation in a patient with neonatal diabetes. In this study, we aimed to characterize biological behavior and diabetogenic function of PI-C(B19)Y.

Materials and methods: We established Ins2 KI mouse lines heterozygous and homozygous for PI-C(B19)Y mutation in C57BL/6J background using CRISPR/Cas9 mediated genome editing. We monitored fasting blood glucose (FBG) and body weight (BW) of mice weekly and performed intraperitoneal glucose tolerance test (IPGTT) monthly. Serum insulin levels were detected by Elisa. Pancreatic islet cell composition were detected by immunohistochemistry and confocal immunofluorescence. The insulin content and cellular response in KI mouse islets were examined by western blotting. For in vitro experiments, PI-WT and different mutants that disrupt either B7-A7, or B19-A20, or A6-A11, respectively, were transiently expressed in HEK293T cells and Min6 cells. The oxidative folding, the ER export, degradation and the dominant negative effects of PI-WT or mutants were analyzed using SDS-PAGE under both non-reducing and reducing conditions.

Results: In the heterozygous KI mice, although intra-islets insulin content decreased, no significant differences in FBG, BW and IPGTT were observed compared with that of wild-type mice. However, in the homozygous KI mice, insulin content was further decreased, which led to glucose intolerance at about 2 months of age. We compared formation of abnormal disulfide-linked proinsulin complexes (DLPC) formed between PI-WT and mutants. We found that C(A7)Y and C(A6)Y formed large amount of DLPC with co-expressed PI-WT in the ER. However, C(B19)Y did not appear to form DLPC. The intracellular degradation rate of C(B19)Y was faster than that of C(A7)Y and C(A6)Y, suggesting that C(B19)Y is less stable in the cells. More importantly, due to its instability and less interactions with co-expressed PI-WT, C(B19)Y showed much less degree of dominant negative effect, which accounted for mild diabetes phenotype in KI mice.

Conclusion: PI-C(B19)Y mutant induced diabetes causes proinsulin ER entrapment, leading to decreasing insulin production from mutant PI. However, unlike other two PI mutants, C(A7)Y and C(A6)Y, that cause severe insulin deficient early onset diabetes, C(B19)Y has less significant dominant negative effect on co-expressed PI-WT, leading to mild late onset diabetes. This study not only uncovers molecular mechanism of diabetes caused by C(B19)Y, but also reveals non-parallel roles of PI disulfide bonds in PI folding and diabetes phenotypes.

Supported by: NNSFC, National Key R&D Program,Tianjin Municipal Science and Technology Commission of China

Disclosure: Y. Yang: None.

OP 38 Triggers and drivers of beta cell failure in type 1 diabetes

221

Presentation of insulin granule derived peptides on MHC I in Enterovirus-infected beta cells and type 1 diabetes

Z. Marinicova1, M. Ghosh2, K.-P. Knoch1, A. Petzold1, C. Wegbrod1, A. Sönmez1, R. Scharfmann3, S. Stevanović2, M. Solimena1;

1Paul Langerhans Institute Dresden, of the Helmholtz Center Munich, Dresden, Germany, 2Department of Immunology, University of Tübingen, Tübingen, Germany, 3Endocrinology, Metabolism and Diabetes, INSERM U1016, Institut Cochin, Paris, France.

Background and aims: Type 1 diabetes (T1D) results from autoimmune destruction of the insulin producing pancreatic beta cells. Enteroviruses (EVs), including Echoviruses, have been studied as possible factors for T1D onset and/or progression. Our lab found that EV infection decreases the stores of mature secretory granule (SG) cargoes such as insulin, ICA512/IA-2/PTPRN, PC1/3, PC2 and CgA, some of which are targets of autoimmunity in T1D. This depletion likely results from intracellular protein degradation, which is the main pathway to generate antigen peptides presented on MHC I. We hypothesize that EV-induced degradation of mature SG proteins alters the presentation of peptides thereof, hence possibly influencing the response of activated autoreactive CD8+ T-cells toward these antigens.

Materials and methods: 1x108 ECN90 human beta cells/replicate were infected with Echovirus 9 strain DM, which was isolated from a diabetic patient who died of ketoacidosis, under multiplicity of infection 0,003 plaque-forming units/cell. After a 48h incubation, several viral and cellular markers were assessed by western blotting. HLA I molecules were immunoisolated from infected and control ECN90 cells; eluted HLA I-bound peptides were identified by LC-MS/MS, and compared for their presence and abundance between infected and control ECN90 cells.

Results: Infection conditions were optimized based on a) detection of the viral protein VP1; b) cleavage of cellular factors eIF4G, PABP1, PTBP1, PARP and Cas3 to assess the stage of cell infection; c) levels of ICA512 and ChgA and their proforms to assess the size of SG stores; d) levels of HLA I and β2 microglobulin to confirm sufficient antigen presentation. About 500 unique HLA I presented peptides were found per replicate and condition with purity of 89% (peptides predicted to bind HLA alleles expressed by ECN90 cells). In total, we detected 23 peptides from known T1D autoantigens associated with SGs (e.g. insulin, chromogranin A, ICA512) in both conditions. The majority of them were predicted to bind HLA I alleles B4001 and A0201, while two viral peptides were found to bind B4001 and A0301 alleles. The distribution of unique peptides presented by infected ECN90 cells significantly differed from those presented by control cells as 54 unique peptides were present only in all infected samples and none of uninfected and 13 peptides were only found in uninfected cells.

Conclusion: Antigen presentation is altered in EV-infected ECN90 cells. We identified several novel autoantigen epitopes, including 4 novel A0201 restricted epitopes from IAPP, HSPA5, ICA512 and IA-2/PTPRN2, which will be characterized with pHLA tetramers for their reactivity with CD8+ T-cells from subjects with T1D and healthy controls.

Supported by: EU-IMI INNODIA, DZD e.V., DFG IRTG 2251: ICSMD

Disclosure: Z. Marinicova: None.

222

Diabetogenic CD4+ T-cells induce autoimmune diabetes in an interferon regulatory factor 4-dependent manner

T. Niri1, S. Akazawa2, M. Miwa3, M. Kobayashi4, N. Abiru1;

1Division of Advanced Preventative Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 2Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 3Department of Metabolism/Diabetes and Clinical Nutrition, Nagasaki University Hospital, Nagasaki, 4Center for Health and Community Medicine, Nagasaki University, Nagasaki, Japan.

Background and aims: Both acquired and innate immune system are essential for the development of autoimmune diabetes. We previously reported that haploinsufficiency of the transcription factor interferon regulatory factor 4 (IRF4) almost completely suppressed the onset of spontaneous diabetes in NOD mice. To clarify which immune cell types were responsible for the protection from autoimmunity by IRF4 deficiency, we established IRF4 gene-deleted BDC2.5 TCR transgenic NOD mice (BDC2.5Tg-NOD) or Rag1 knockout NOD mice (Rag1KO-NOD) and observed spontaneous progression of diabetes and conducted adoptive transfer experiments as follows.

Materials and methods: BDC2.5Tg-NOD and Rag1KO-NOD were crossed with IRF4 deficient NOD mice respectively to produce wild-type (wt)Irf4+/-Irf4-/--BDC2.5Tg-NOD and -Rag1KO-NOD. Then, wtIrf4-/--BDC2.5Tg-NOD were crossed with wtIrf4-/--Rag1KO-NOD respectively to produce wt, Irf4-/--BDC2.5Tg Rag1KO-NOD mice, and the incidence of spontaneous progression of diabetes was observed by consecutive blood glucose monitoring. In adoptive transfer experiments, firstly, naïve BDC2.5-TCR T cells (CD62L+CD4+ T cells) magnetically isolated from wtIrf4+/-Irf4-/--BDC2.5Tg-NOD were intravenously transferred into wt-Rag1KO-NOD. Secondly, naïve wt-BDC2.5-TCR T cells were transferred into wtIrf4+/-Irf4-/--Rag1KO-NOD recipients. Each experiments were followed by consecutive blood glucose monitoring in the recipient mice to evaluate incidence of diabetes.

Results: Irf4-/--BDC2.5Tg Rag1 KO-NOD were completely protected from the spontaneous progression of diabetes whereas wt-BDC2.5Tg Rag1 KO-NOD showed rapid diabetes-onset at 27 days of median duration after birth (p<0.01). In adoptive transfer experiments, the diabetes-onset was completely protected in wt Rag1KO-NOD recipients transferred with Irf4-/--BDC2.5 T cells (p<0.01), and suppressed up to 50% in those with Irf4+/--BDC2.5 T cells (p=0.03), respectively compared to those with wt donor-T cells. In contrast, no significant differences were observed in the cumulative incidence of diabetes among Irf4-/--, Irf4+/-- and wt-Rag1KO-NOD recipients transferred with wt-BDC2.5 T cells. The median duration of diabetes-onset in Irf4-/--Rag1KO-NOD recipients were 17 days after transfer with a significant delay compared to 13 days of median duration in wt-recipients (p<0.04). Irf4+/--Rag1KO-NOD recipients transferred with wt-donors developed delayed diabetes without statistically significance compared to wt-recipients.

Conclusion: IRF4 deficiency abrogates effector function of diabetogenic CD4+ T cells in gene-dose dependent manner and attenuates innate immune function to delay the onset of autoimmune diabetes. IRF4 predominantly controls adaptive immunity as well as it adjunctively promotes innate immune responses towards progression of diabetes in NOD mice.

Disclosure: T. Niri: None.

223

The role of NF-κB-inducing kinase (NIK) in beta cell-mediated inflammation in type 1 diabetes

P. Xiao1, T. Takiishi1, N.M. Violato1, G. Licata2, F. Dotta2, G. Sebastiani2, A.K. Cardozo1;

1ULB Center for Diabetes Research, Université Libre de Bruxelles (ULB), Brussels, Belgium, 2Dept. of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy.

Background and aims: In type 1 diabetes (T1D) β-cell destruction results from an aberrant inflammatory crosstalk between the β-cells and immune-cells mediated, partly, via activation of the transcription factor NF-κB. NF-κB signaling occurs via two major pathways called canonical and the alternative. The alternative pathway is characterized by stabilization of the NF-κB-inducing kinase (NIK) triggering p100 processing into p52, which dimerizes with RelB to regulate gene transcription. The canonical NF-κB pathway was shown to contribute to β-cell death in T1D, however, the role of the alternative pathway in T1D is unknown. Ligands that activate the alternative pathway (e.g. LIGHT, CD40L) are present in the serum of T1D patients and are involved in pathogenesis in non-obese diabetic mice. We previously identified that in vitro cytokine treatment, which induces β-cell death, promotes NIK stabilization and activation of downstream NF-κB signaling in rat β-cells. Thus, the aim of this study is to characterize the role of NIK in β-cell demise during T1D and its regulation on peripheral and local immune responses.

Materials and methods: To evaluate the role of NIK specifically in β-cells we developed a β-cell specific NIK KO mouse (NIKβKO). To verify if lack of NIK in β-cells affects its development and function we followed NIKβKO mice and wild type littermates (WT) up to 24 weeks. Mouse glycemia and bodyweight were measured weekly and intraperitoneal glucose tolerance tests (ipGTT) were performed at 12 and 24 weeks of age. Insulin content of the islets were measured when mice were euthanized. To induce T1D, mice were injected with multiple low doses of streptozotocin (MLDSTZ). Mice were sacrificed 14 and 45 days after the last streptozotocin injection. The metabolic parameters described above were measured weekly and (ipGTT) were performed at both endpoints. Moreover, to evaluate immune profiles, T cell and myeloid cell populations were analysed by FACS in blood, spleen and pancreatic draining lymph nodes (pLN) of mice sacrificed at day 14.

Results: Under physiological conditions lack of NIK didn’t affect β-cell development nor function. However, after MLDSTZ treatment, a significantly higher diabetes incidence in NIKβKO mice was observed (83% NIKβKO mice vs 40% WT, n=15-17, p<0.05). Moreover, NIKβKO mice had markedly worse glucose control compared to WT during an IPGTT (492 mg/dL NIKβKO vs 400 mg/dL WT, p<0.05, 15 min). Furthermore, our preliminary results shows that NIKβKO mice display a discreet reduction in the frequency of regulatory T cells (Tregs) (CD4+Foxp3+, 12.5%±5.6% NIKβKO vs 14.6%±4.8% WT) but noticeable higher frequencies of cytotoxic CD8+IFN-γ+ lymphocytes (55.0%±23.8% NIKβKO vs 38.2%±18.9% WT) in the pLN. Particularly, NIKβKO mice presented higher cytotoxic/Treg ratio (9.2±9.2 NIKβKO vs 3.8±4.0 WT) indicating local immune dysregulation in KO β-cells. Overall, the data suggest stronger inflammatory responses in β-cells that are KO for NIK, indicating a protective role for NIK in T1D.

Conclusion: Our new results unveiled NIK as a new player on the crosstalk between β-cells and the immune cells leading to T1D. Revealing the downstream players of these network may allow new targeted approaches to treat or prevent T1D development.

Supported by: Excellence of Science Grant – FNRS/FWO

Disclosure: P. Xiao: Grants; Excellence of Science Grant (EOS) Fonds National de Recherche Scientifique.

224

Ptpn2 is a pro-inflammatory cytokine regulator and novel player in the endoplasmic reticulum stress response in beta cells

B. Elvira Jimenez, V. Vandenbempt, E. Gurzov;

Signal Transduction and Metabolism Laboratory, ULB Center for Diabetes Research, BRUSSELS, Belgium.

Background and aims: Type 1 diabetes (T1D) results from autoimmune destruction of β cells. Previous studies have demonstrated that sustained inflammation induces endoplasmic reticulum (ER) stress in β cells, resulting in cellular dysfunction and eventually cell death. Available evidence suggests that protein tyrosine phosphatases play a key role in the break of tolerance and development of T1D. Our aim is to study the role of PTPN2, a candidate gene for T1D, in the stressed β cell in a pro-inflammatory environment and dysregulated signaling.

Materials and methods: PTPN2 was silenced by transfection of siRNAs in the human EndoC-βH1 cell line. The nuclear (45kDa) or ER (48kDa) isoforms of PTPN2 were overexpressed by adenovirus transduction. Transfected and transduced cells were treated with hIFN-γ (1000U/ml) in a pulse-chase experiment. ER stress was induced in transfected and transduced cells with thapsigargin (TG, 1μM) and cyclopiazonic acid (CPA, 75μM) for 48h. β-cell apoptosis was evaluated by Hoechst 33342/propidium iodide staining and caspase-3 activation. The effect of TG on intracellular calcium levels under basal condition was examined by Fura-2 dye in transfected cells.

Results: PTPN2 knockdown (>70%, p<0.001) increased pSTAT1 (p<0.001) at the timepoints 0, 2 and 4h and pSTAT3 (p<0.05) 2h vs control after hIFN-γ exposure. The mRNA levels of the chemokines CXCL9 and CXCL10 were increased at 24h after hIFN-γ treatment and PTPN2 knockdown: 2 fold, p<0.05, and 1.5 fold, p<0.05, respectively. Adenovirus-mediated overexpression of the nuclear (45kDa) but not the ER (48kDa) isoform of PTPN2 decreased hIFN-γ-induced pSTAT1 activation. PTPN2 silencing sensitized EndoC-βH1 cells to TG-induced apoptosis (47±3% in PTPN2-silenced vs 36±1% in control cells, p<0.01) and significantly increased expression levels of the ER stress markers CHOP, ATF3, ATF4, peIF2α and ER chaperone BiP (p<0.05, p<0.001). Overexpression of the 48kDa PTPN2 isoform (5.6 fold, p<0.001) protected EndoC-βH1 cells from TG-induced apoptosis (39±3% in 48kDa PTPN2-overexpressed vs 49±3% in control cells, p<0.05) but not the 45kDa PTPN2 isoform (46±3% in PTPN2-overexpressed vs 49±3% in control cells). We confirmed the results with a second ER stressor, CPA: 45±3% apoptosis in PTPN2-silenced vs 29±2% in control cells, p<0.001 and 24±1% apoptosis in 48kDa PTPN2-overexpressed vs 31±1% in control cells, p<0.05). PTPN2 silencing decreased the intracellular calcium levels (p<0.05), suggesting a mechanism for the 48kDa PTPN2 function in the ER in β cells.

Conclusion: The 45 kDa nuclear isoform of PTPN2 reduces hIFN-γ response in β cells via STAT1/STAT3-dependent signaling pathways. The 48kDa ER isoform of PTPN2 protects β cells from ER stress-induced signaling and apoptosis. Our results demonstrate isoform-dependent dissociation of the PTPN2 activity, and postulate PTPN2 as an important protective factor in β cells in inflammation and ER stress.

Supported by: F.R.S-FNRS Charge de recherches and ERC consolidator grant

Disclosure: B. Elvira Jimenez: Grants; F.R.S-FNRS Charge de Recherches, ERC consolidator grant.

OP 39 Gastro-entero pancreatic factors: organoids, mice and men

225

Characterisation of human GLP-1 secreting cells after fluorescent tagging in primary ileal organoids

F. Reimann, D. Goldspink, V. Lu, E. Miedzybrodzka, C. Smith, R. Foreman, L. Billing, R. Kay, F. Gribble;

Addenbrooke's Hospital, University of Cambridge, Cambridge, UK.

Background and aims: Injectable Glucagon-like peptide-1 (GLP-1) receptor agonists are widely used in the treatment of type2 diabetes and obesity. Bariatric surgery profoundly exaggerates postprandial GLP-1 plasma excursions, suggesting an alternative therapeutic strategy of targeting endogenous GLP-1 pools located in distal intestinal L-cells, found scattered in and constituting <1% of the epithelium. Whereas our understanding of murine L-cell physiology has been enhanced by studying transgenic mice with fluorescently tagged L-cells, it has not been possible to study stimulus secretion coupling in human L-cells in detail. In this project we fluorescently tagged human L-cells in ileal organoids to study L-cell physiology.

Materials and methods: CRISPR-Cas9 was used to engineer a P2A ribosomal stutter sequence followed by the fluorescent protein Venus-sequence 3’ of the proglucagon coding-sequence in chromosome 2. Bulk RNAseq and LC-MS/MS were used to characterise L-cell transcriptome and peptidome, respectively, after FACSorting. GLP-1 secretion was measured by ELISA in organoid supernatants, single-cell Ca2+-dynamics were monitored after Fura2-loading and whole-cell patch recordings were used to assess electrophysiological activity in response to different agonists.

Results: Co-immunohistochemistry confirmed labelling of >90% of glucagon-expressing cells with Venus in human ileal organoids and vice versa. Addition of Notch- and MEK-inhibitors to standard IF-medium (IF*) boosted L-cell frequency ~5-fold from 0.4±0.1% (n=5) to 2.1±0.6% (n=3) of all cells. L-cells were enriched for other hormones, including peptideYY and neurotensin, by transcriptomic and peptidomic analysis. L-cell transcriptomic profiles were clearly different from non-fluorescent cells and broadly similar in IF (n=5) and IF* (n=3) medium. GPCR-mRNAs enriched in L-cells included the bile acid receptor GPBAR1, lipid sensing receptors FFAR1 and GPR119 and the vasopressin receptor AVPR1B. Agonists for these receptors stimulated GLP-1 secretion (mn±sem -fold increase relative to control (10 mM glucose): GPBAR1A [3 μM] 7.9±0.9 n=12; FFAR1 agonist AM1638 [10 μM] 6.1±0.5 n=19, GPR119 agonist AR231453 [100 nM] 3.3±0.2 n=6, AVP [10 nM] 4.9±0.7 n=10). AM1638 [10μM], AVP [10 nM] and KCl [70 mM] increased cytosolic Ca2+ (median (IQR; n) 1.08 (1.03-1.28; 56), 2.25 (1.45-3.04; 24) and 3.47 (2.08-5.67; 91) -fold, respectively. Human L-cells were electrically excitable and the interspike membrane potential (-60±1.6 mV) depolarised in response to glucose (10 mM, ΔVm 4.5±1.2 mV n=13) resulting in an increase in action potential frequency. This effect was more pronounced when cAMP levels were elevated, and in the presence of forskolin [10 μM] glucose-stimulated GLP-1 secretion was sensitive to inhibition of sodium-coupled glucose uptake with phloridzin (mn±sem -fold increase relative to control (10 mM glucose): fsk [10 μM] 5.9±0.3 n=6; fsk+phloridzin [5 μM] 4.3±0.1 n=6; p<0.001).

Conclusion: Human L-cells employ similar sensing machinery to their murine counterparts. The ability to label and maintain human L-cells in organoid culture opens new avenues to explore L-cell function and develop drugs targeting the human enteroendocrine system, either to stimulate L-cell secretion or to boost L-cell numbers.

Supported by: Wellcome Trust, MRC, BBSRC, LGC

Disclosure: F. Reimann: None.

226

Metabolic surgery recovers Ca 2+ dynamics across pancreatic islets in obese mice

E. Akalestou, K. Suba, L. Lopez-Noriega, E. Georgiadou, P. Chabosseau, I. Leclerc, V. Salem, G.A. Rutter;

Imperial College London, London, UK.

Background and aims: Metabolic surgery improves both glucose tolerance and insulin sensitivity in T2D but its impact on insulin secretion is difficult to monitor continuously and directly. The impact of surgery on β-cell function and the time course of this effect, remain unclear. To investigate the effect of metabolic surgery on β-cell function in vivo, we imaged Ca2+ dynamics prospectively and at the cellular level in islets engrafted into the anterior eye chamber.

Materials and methods: Ins1Cre mice were crossed to animals that express the genetic calcium indicator GCaMP6f behind a LoxPSTOPLoxP cassette. Isolated islets were engrafted into male C57BL6 mice maintained for 12 weeks on high fat/high sucrose diet (HFD). Mice were separated into Vertical Sleeve Gastrectomy (VSG) (n=7) and sham (n=6) groups. Islets were imaged in anaesthetised mice at post-operative weeks 4, 8 and 10 using a Nikon microscope equipped with a spinning disc, 488 nm laser and 20x/0.8 water immersion objective. Islets were categorised for wave activity when these were recurrent, showed a defined point of origin and covered> 75 % of the image plane. Glucose (3g /kg) tolerance and insulin and incretin secretion were assessed in parallel.

Results: The VSG group initially demonstrated substantial weight loss but regained pre-operative weight by week 10. However, VSG mice displayed significantly improved glucose tolerance (p<0.001) and insulin secretion (p<0.01), as well as increased basal (21.8pmol/L ±0.9) and post-prandial (135.5pmol/L ±35) GLP-1 secretion (p<0.01), when compared to sham mice. VSG improved coordinated Ca2+ activity, with 100% of islets observed exhibiting enhanced wave readouts 8 weeks post-surgery, while islet wave activity dropped to zero discernible coordinated islet Ca2+ dynamics by week 10 in the sham group. Moreover, the percentage of significantly connected cell pairs and correlation coefficient decreased vastly in the sham group at week 10, while the VSG group remained stable across the length of the study. Although percentage of pancreatic area occupied by β-cells was not changed between the two groups (0.65% ±0.5), α to β cell ratio was increased in the VSG group (p<0.01), indicating higher α-cell population.

Conclusion: Continuous imaging of islet function in the eye in vivo demonstrates that metabolic surgery leads to an increase in glucose-induced Ca2+ dynamics of individual islets in a time-dependent manner, likely to underlie increased insulin secretion.

Supported by: Wellcome Trust, Rosetrees Foundation, MRC, DUK

Disclosure: E. Akalestou: None.

227

Impaired insulin secretion via Wnt signalling induces diabetes in pancreatic cancer patients: insights from a prospective cohort study

M. Lee1, H. Park2, S. Kang2;

1Yonsei University College of Medicine, Seoul, 2Gangnam Severance Hospital, Seoul, Republic of Korea.

Background and aims: Pancreatic ductal adenocarcinoma (PDAC) patients are known to have a higher prevalence of new-onset diabetes but the mechanisms are largely unknown.

Materials and methods: We built a prospective cohort composed of 160 patients scheduled for pancreatectomy mainly as pylorus preserving pancreaticoduodectomy (PPPD) (72 PDAC patients, 88 non-PDAC patients). The patients underwent 75g oral glucose tolerance test before, and 2 weeks and 1year after pancreatectomy. Pancreatic tissues were obtained during surgical resection.

Results: Compared with non-PDAC patients, PDAC patients had a higher prevalence of new-onset diabetes (31.9% vs. 19.3%, p = 0.012), with a higher HbA1c level and decreased insulin secretory function. After PPPD, there was a consistently larger improvement of HbA1c in PDAC patients than in non-PDAC patients. Furthermore, unlike non-PDAC patients whose insulin secretion was consistent before and after PPPD, PDAC patients revealed a significant improvement in insulin secretion 1 year after PPPD, suggesting that a diabetogenic factor secreted from pancreatic cancer may induce hyperglycemia by suppressing the insulin secretion. In immunofluorescent staining of pancreatic tissue, PDAC patients had a higher β-catenin expression than that of non-PDAC patients. Pancreatic β-catenin expression positively correlated with hyperglycemia and negatively correlated with insulin secretion in PDAC patients only. The plasma level of Wnt5a, which was highly expressed in PDAC cells compared to normal ductal cells, positively correlated with pancreatic β-catenin level and was elevated in PDAC patients with new-onset hyperglycemia compared with non-PDAC patients. Treatment with Wnt5a significantly suppressed insulin release in response to glucose stimulation in isolated mouse islets.

Conclusion: In conclusion, the development of PDAC-induced hyperglycemia could result from an impaired insulin secretion by activated Wnt5a/β-catenin pathway in PDAC patients.

Supported by: Ministry for Health, Welfare & Family Affairs

Disclosure: M. Lee: None.

228

Effect of macronutrient composition and energy content on postprandial secretion of satiety hormones and next meal food intake

C. Martinussen1, M.S. Svane1, K.N. Bojsen-Møller1, B.V. Andersen2, O.J. Hulme3, D.V. Byrne2, H.R. Siebner3, K. Hermansen4, S. Gregersen4, J.F. Rehfeld5, B. Hartmann6, J.J. Holst6, S. Madsbad1;

1Dept. of Endocrinology, Hvidovre Hospital, 2Food Quality Perception and Society Team, iSense lab, Dept. of Food Science, Aarhus University, 3Danish Research Centre for Magnetic Resonance, Center for Functional and Diagnostic Imaging and Research, Hvidovre Hospital, 4Dept. of Endocrinology and Internal Medicine, Aarhus University Hospital, 5Department of Clinical Biochemistry, Rigshospitalet, 6Dept. of Biomedical Sciences and NNF Center for Basic Metabolic Research, University of Copenhagen, Denmark.

Background and aims: The satiating capacity of food depends on its energy content and macronutrient composition with proteins having a greater satiating effect per unit energy than carbohydrates and fats. It is unknown whether this is related to differences in secretion of appetite-regulating hormones from the gut and pancreas. We investigated the impact of 4 different preload meals with varying energy content and protein/carbohydrate (P/C) ratio on secretion of satiety hormones (glucagon-like peptide-1, GLP-1; Peptide YY, PYY; cholecystokinin, CCK; and insulin) and next meal food intake.

Materials and methods: In a randomized, single-blinded, cross-over design, 24 healthy male participants (age 24.9 range 20-40 years, BMI 23.2 range 20.9-24.9 kg/m2) ingested 4 different preload meals. A: 1679 kcal, P/C ratio 2.1 (HighPROHighCAL) B: 1680 kcal, P/C ratio 0.2 (LowPROHighCAL) C: 839 kcal, P/C ratio 2.1 (HighPROLowCAL) D: 840 kcal, P/C ratio 0.2 (LowPROLowCAL). The preloads consisted of meal replacement powder (Queal) mixed in water to which was added either Whey Protein Isolate (HighPRO) or Lactose Monohydrate (LowPRO). Paracetamol was added to all meals to assess gastric emptying rate. Blood was sampled before and frequently after the preload (at -15, -5, 20, 35, 50, 65, 100, 120, 140, 180 min; n=23). Subjective appetite and satiety were evaluated using visual analogue scores. After 180 min, an ad libitum meal was served to investigate the effect of the preloads on next meal caloric intake. Data were analyzed in linear mixed effects model with P/C ratio and energy content as independent variables. Hormone responses are presented as 3-h area-under-the-curve.

Results: Ad libitum food intake 3 h after the preloads did not differ significantly (HighPROHighCAL 1122 ± 306 kcal; LowPROHighCAL 1166 ± 266 kcal; HighPROLowCAL 1205 ± 284 kcal; LowPROLowCAL 1214 ± 276 kcal; mean ± SD). Preloads with high energy and protein content led to increased satiety and lower hunger scores compared with preloads with low energy and protein content after 3 h. Paracetamol absorption rate (gastric emptying) was comparable between the 4 preloads. Peak plasma glucose was lower after HighPRO vs. LowPRO (p<0.001) and greater after HighCAL vs. LowCAL (p<0.001). Plasma insulin (p<0.01), GLP-1 (p<0.01) and CCK (p<0.01), but not PYY (p=0.16), depended on the energy content of the preload (HighCAL > LowCAL) but none of the hormonal responses depended on the P/C ratio (p=0.68 for insulin, p=0.15 for GLP-1, p=0.46 for CCK, p=0.62 for PYY).

Conclusion: Postprandial secretion of satiety hormones was determined by meal energy content rather than macronutrient composition. The results suggest that factors other than acute gut hormone secretion explain the greater satiating capacity of proteins compared with carbohydrates.

Clinical Trial Registration Number: NCT03900130

Supported by: We thank Arla Food for Health for funding the OmniSaM project (The omnibus satiety metric)

Disclosure: C. Martinussen: None.

OP 40 New aspects of novel therapies

229

Effects of 5 weeks of treatment with dapagliflozin, a SGLT2 inhibitor, on energy metabolism in patients with type 2 diabetes

Y. Op den Kamp1, M. de Ligt1, B.D. Dautzenberg1, R. Esterline2, J. Hoeks1, V.B. Schrauwen-Hinderling1,3, B. Havekes4, J. Oscarsson5, E. Phielix1, P. Schrauwen1;

1Nutrition and Movement Sciences, Maastricht University, Maastricht, Netherlands, 2BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, USA, 3Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, Netherlands, 4Internal Medicine, Maastricht University Medical Center, Maastricht, Netherlands, 5BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.

Background and aims: To explore the effects of dapagliflozin (DAPA) on insulin sensitivity, 24h energy metabolism and skeletal muscle mitochondrial function in patients with type 2 diabetes (T2D).

Materials and methods: Twenty-six T2D patients with HbA1c between 42 and 75 mmol/mol, were randomized to a double blind, cross-over study. Examinations were done at the end of the 5-week treatment periods, separated by 6-8-week wash-out. 24h energy- and substrate metabolism was measured in respiration chambers, and blood was sampled at 7 time points. A two-step euglycemic hyperinsulinemic clamp (10 and 40 mU/m2/min) with infusion of [6.6-2H2] glucose to determine glucose rate of disposal (Rd), endogenous glucose production (EGP) and indirect calorimetry was performed. Intramyocellular (IMCL), intrahepatic lipid (IHL) content and resting and post-exercise (70% Wmax) muscle acetylcarnitine were analysed by 1H-Magnetic Resonance Spectroscopy (MRS). Phosphocreatine recovery upon exercise was measured by 31P-MRS and body composition by DEXA. Ex vivo mitochondrial respiration was measured in a muscle biopsy taken after overnight fast. Results are presented as the LSM (95% CI) difference between treatments.

Results: Evaluable patients (n=24) had a mean (SD) age of 64.2(4.6) years, BMI of 28.1(2.4) kg/m2, and HbA1c of 51.7(6.8) mmol/mol. Patients were on metformin or no antidiabetic therapy. DAPA decreased total body weight (-1.12 (-1.66, -0.58) kg, p<0.01). Rd was unaffected by DAPA, while fasting EGP increased (+2.27 (1.39, 3.14) μmol/kg/min, p<0.01), EGP upon insulin infusion was unchanged. A trend towards a larger increase in carbohydrate oxidation (+0.77 (-0.37, 1.91) μmol/kg/min, p=0.11) and a larger decrease in fatty acid oxidation (-0.28 (-0.65, 0.09) μmol/kg/min, p=0.13) upon insulin infusion was observed. 24h energy expenditure (-0.11 (-0.24, 0.03) MJ/day), sleeping metabolic rate or diet-induced thermogenesis were unaffected by DAPA. DAPA reduced RER during day- (-0.024 (-0.034, -0.014), p<0.01) and night-time (-0.033 (-0.046, -0.020), p<0.01). Day-time glucose was lower (p<0.01), while free fatty acids (p<0.01) and β-hydroxybutyrate (p<0.05) levels were higher upon DAPA. On placebo, urinary glucose loss was neglectable, whereas DAPA induced a 24h glucose loss of about 90g; rate of glucose loss was 50% lower during the night compared to daytime. IMCL increased upon DAPA (+0.06 (0.01, 0.11) %, p<0.05), whereas IHL decreased (-0.29 (-2.53, 1.94) %, p<0.05). DAPA had no effect on ex vivo mitochondrial respiration, phosphocreatine recovery rate or acetylcarnitine metabolism.

Conclusion: Five weeks dapagliflozin treatment in T2D patients had no effect on insulin sensitivity or energy expenditure, while 24h fatty acid oxidation was increased. A trend towards improved metabolic flexibility was observed. Intramyocellular lipids were increased and intrahepatic lipids decreased, but skeletal muscle mitochondrial function was not changed by dapagliflozin treatment.

Clinical Trial Registration Number: 2016-003991-27

Supported by: AstraZeneca

Disclosure: Y. Op den Kamp: None.

230

The SGLT2 inhibitor empagliflozin does not stimulate compensatory appetite responses in patients with excess adiposity and type 2 diabetes

M.J. Davies1,2, E.L. Baldry1,2, D.H. Bodicoat3, S. Chatterjee4, C.L. Edwardson1,2, L.J. Gray1, K. Khunti1, J.A. Sargeant1,2, D.J. Stensel5,2, D.R. Webb1,2, J.P.H. Wilding6, S.A. Willis5,2, T. Yates1,2, J.A. King5,2;

1Diabetes Research Centre, University of Leicester, Leicester, 2NIHR Leicester Biomedical Research Centre, Leicester, 3Simplified Data, Leicester, 4University Hospitals of Leicester NHS Trust, Leicester, 5School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, 6Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.

Background and aims: In patients with type 2 diabetes (T2D), SGLT2 inhibitors (SGLT2i) lower HbA1c and cause weight loss; however, observed weight change is less than predicted by modelling. This study tested the hypothesis that compensatory changes in appetite, and appetite-related hormones, explain this less-than-expected weight loss with SGLT2i.

Materials and methods: In a 24-week prospective, double-blind placebo-controlled trial, patients with overweight / obesity and T2D (age 30 - 75 years, BMI ≥ 25kg/m2 ) were randomised (1:1:1:1) to one of four treatments: 1) placebo; 2) empagliflozin 25mg/day [EMPA]; 3) placebo and diet-induced weight loss [DIET]; 4) empagliflozin 25mg/day plus diet-induced weight loss [EMPA+DIET]; and assessed at 0, 2, 6, 12 and 24 weeks. DIET and EMPA+DIET groups reduced energy intake by 1500kJ/day. The primary outcome was circulating total peptide-YY (PYY) concentrations over a 3-hour mixed meal tolerance test (33% of daily energy requirements) at 24 weeks. Secondary outcomes included circulating concentrations of acyl ghrelin, GLP-1, leptin, appetite perceptions (100 mm VAS), body composition (DEXA) and physical activity (accelerometery). Data were analysed using generalised linear models at each time-point comparing each group with placebo; adjusting for baseline, age and BMI. Generalised estimating equations (GEE) examined overall treatment effects across follow-up.

Results: 68 participants were randomised (median [IQR]; age 63 [57, 69] years; BMI 31.8 [29.2, 35.1] kg/m2; HbA1c 6.8 [6.6 -7.2]%; 35% female) with primary outcome data available for 61. Circulating concentrations of PYY were no different vs placebo in any treatment arm at 24 weeks (Table 1); but were elevated in EMPA at 12-weeks (P = 0.003). Circulating acyl ghrelin and GLP-1 were unchanged at all time-points; however, GEE showed that GLP-1 was higher in EMPA vs placebo (P = 0.016). Treatments had no effects on perceived huger or fullness. Lean mass was reduced in EMPA and EMPA+DIET vs placebo at 24 weeks (P ≤ 0.001), with accordant (but not significant) reductions in resting metabolic rate. GEE highlighted a reduction in daily steps with EMPA vs placebo (P = 0.038); but not in the other treatment arms.

Conclusion: Empagliflozin does not provoke obvious compensatory appetite or appetite-related hormone responses in patients with excess adiposity and T2D. Additional studies should explore the effects of SGLT2i on hedonic drivers of eating behaviour.

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Clinical Trial Registration Number: NCT02798744

Supported by: Funded by Boehringer Ingelheim and supported in-kind by the NIHR Leicester BRC

Disclosure: M.J. Davies: None.

231

Peripherally administered incretin peptides, including GIP, GLP-1 and a dual GIP/GLP-1 receptor agonists, activate several brain regions in anesthetised rats

J.M. Wilson1, L. Becerra2, K. Goetter2, K. Briley2, A. Novicki2, D. Cissell2, R.M. Smith1, M. Ai1, P.J. Emmerson1, Z. Milicevic1, A. Haupt1, T. Coskun1;

1Diabetes and Complications, Eli Lilly and Company, Indianapolis, 2Invicro, A Konica Minolta Company, Boston, USA.

Background and aims: Following peripheral administration of incretin peptides, activation of deep brain regions has been shown and may occur through neuronal relays. To characterise the central responses to peripherally administered (i.v.) incretin peptides, we used superparamagnetic iron oxide (SPIO)-based MRI to image the activation of multiple brain regions known to regulate appetite in anesthetised rats.

Materials and methods: Anesthetised Sprague-Dawley rats were positioned in the cradle of a Bruker 7.0T MRI system. Anatomic imaging was acquired with a T2-weighted sequence. For SPIO-MRI, a Multiple Gradient Echo sequence with in-plane resolution of 230x230 μm and 1 mm slices with a repetition time of 25.6 s was prescribed either transversely (for amygdala) or parasagittally (all other regions). For SPIO-MRI scans, Molday ION contrast agent (BioPAL Inc.) was administered through a tail vein at a dosage of 10 ml/kg and infusion rate of 2.5 mL/min 5 min after initiation of scan. After 20 minutes of continuously scanning, a placebo (buffer vehicle), acylated long-acting GLP-1 receptor agonist (LA-GLP-1RA), acylated long-acting GIP receptor agonist (LA-GIPRA) or acylated long-acting GIP/GLP-1 dual receptor agonist (LA-GIP/GLP-1RA) were administered at a dosage of 10 nmol/kg with an infusion rate of 0.16 mL/min. Images were acquired for an additional 20 minutes post infusion. Standard analysis was used to calculate voxel-wise maps of change in relative cerebral blood volume (rCBV) following the administration of each agent. Average rCBV for each anatomic region of interest was calculated from the maps.

Results: Treatment with each peptide led to increased rCBV in multiple brain regions including area postrema, hypothalamus, amygdala, cingulate cortex and retrosplenial cortex compared to vehicle.

Conclusion: This is the first study demonstrating that peripherally administered incretin peptides can activate brain regions which are known to regulate appetite via increased blood flow in rats. Further studies are needed to demonstrate the functional importance of these findings on the regulation of appetite or glucose control.

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Disclosure: J.M. Wilson: Employment/Consultancy; Eli Lilly and Company.

232

GIP infusion in patients with type 1 diabetes seems to attenuate postprandial glucose excursions after prandial insulin over-dose and physical activity

B. Hoe1,2, S.M.N. Heimburger1,3, L.S. Gasbjerg1,2, A.R. Lanng1,2, M.B. Lynggaard1, B. Hartmann2,4, J.J. Holst2,4, T. Vilsbøll3,1, A. Lund3,1, S. Engberg3, T.F. Dejgaard3,1, M.B. Christensen1,5, F.K. Knop1,3;

1Gentofte Hospital, Hellerup, 2University of Copenhagen, Copenhagen, 3Steno Diabetes Center Copenhagen, Gentofte, 4Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 5Department of Clinical Pharmacology, Bispebjerg Hospital, Copenhagen, Denmark.

Background and aims: The gluco-regulatory effects of the insulinotropic and glucagonotropic gut hormone glucose-dependent insulinotropic polypeptide (GIP) in type 1 diabetes (T1D) are unclear. We evaluated the effects of exogenous and endogenous GIP on plasma glucose excursions in a setting of prandial insulin over-dose and physical activity after meal ingestion.

Materials and methods: In a randomised, placebo-controlled, double-blinded, crossover study, 12 men with T1D (age [mean ± SD]: 26 ± 6.6 years; BMI: 23 ± 2.3 kg/m2; HbA1c: 48 ± 6.3 mmol/mol (6.5 ± 2.7 %); diabetes duration: 11 ± 5.5 years; plasma C-peptide: < 200 pmol/l) underwent three separate study days involving a liquid mixed meal test with 125 % of regular prandial insulin dose, 30 minutes of intermediate bicycling (60 minutes after mixed-meal), and 270 minute infusions of GIP, the GIP receptor antagonist GIP(3-30)NH2 and placebo, respectively.

Results: The GIP infusion attenuated postprandial plasma glucose excursions (Cmax - Cmin) by [mean ± SEM] 1.5 ± 0.5 mmol/l and 0.92 ± 0.56 mmol/l compared to GIP(3-30)NH2 and placebo, respectively (p = 0.03) (Figure). The amounts of infused glucose needed to avoid plasma glucose < 2.5 mmol/l were similar on all three study days (p = 0.13) (Figure).

Conclusion: In conclusion, GIP infusion seems to attenuate postprandial plasma glucose excursions without significantly increasing the need of glucose to avoid hypoglycaemia in patients with T1D.

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Clinical Trial Registration Number: H-18002707

Supported by: Helmsley Charitable Trust

Disclosure: B. Hoe: None.

OP 41 Fatty matters

233

Liraglutide accelerates the catabolism of apolipoprotein B100 containing lipoproteins (VLDL 1 , VLDL 2 , IDL and LDL) in patients with type 2 diabetes: an in vivo kinetic study

B. Vergès1,2, B. Bouillet1,2, A. Rouland1, S. Baillot-Rudoni1, P. Buffier1, E. Crevisy1, J. Petit1,2, L. Duvillard1,2;

1Hopital du Bocage, Dijon, 2INSERM, Dijon, France.

Background and aims: Dyslipidemia observed in type 2 diabetes (T2DM) is highly atherogenic and plays an important role in the increased cardiovascular risk in T2DM patients. Important features of diabetic dyslipidemia are increased levels of triglyceride-rich lipoproteins and small dense LDL particles which, all have the apolipoprotein B100 (apoB100) as major apolipoprotein. This prompted us to study the effect of the GLP1 agonist, liraglutide, on the metabolism of apoB100 containing lipoproteins.

Materials and methods: We performed an in vivo kinetic study with stable isotopes (L-[1-13C] leucine) in 10 T2DM patients featuring diabetic dyslipidemia (triglycerides ≥1.7 Mmol/L and/or HDL-cholesterol < 1.29 (F)/1.03 (M)), before and 6 months after the initiation of a treatment with liraglutide at a dose of 1.2 mg/day. Lipoproteins were separated by ultracentrifugation and apoB100 isolated by electrophoresis. ApoB100 isotopic enrichment was measured by mass spectrometer after separation of amino acids by gas chromatography.

Results: Six months after initiation of liraglutide treatment, significant reductions in the means of HbA1c (7.1±1.1 vs. 9.6±2.6 %, p=0.009), body weight (100.5±19.6 vs.104.9 ±19.6 kg, p=0.021), fasting triglycerides (1.761±0.37 vs. 2.48±0.69 Mmol/L, p=0.005), plasma apoB100 (0.93±0.13 vs. 1.09±0.11 g/L, p=0.011) were observed. A borderline significant decrease in LDL-cholesterol was also noted (2.75±0.56 vs. 3.04±0.47 Mmol/L, p=0.09). The kinetic study showed a significant increase in catabolism of VLDL1-apoB100 (4.72±2.23 vs. 3.28±1.12 day-1, p=0.013), of VLDL2-apoB100 (6.17±2.11 vs. 3.42±1.97 day-1, p=0.013), of IDL-apoB100 (5.27±2.77 vs. 3.74±1.85 jour-1, p=0.017) and of LDL-apoB100 (0.72±0.22 vs. 0.56±0.22 jour-1, p=0.005). Kinetic data showed that only the indirect catabolisms of VLDL1, VLDL2 and IDL were increased, indicating an acceleration of the catabolism of the VLDL-IDL-LDL cascade.

Conclusion: Treatment with liraglutide induces a significant acceleration of the catabolism of triglyceride-rich lipoproteins (VLDL1, VLDL2, IDL) and of LDL. This positive effect on lipoprotein metabolism may reduce vascular risk in T2DM.

Clinical Trial Registration Number: NCT02721888

Supported by: Novo Nordisk

Disclosure: B. Vergès: Grants; from Novo Nordisk.

234

Serine palmitoyl transferase 2 deficiency in mice hepatocytes induces changes in bile acids composition and improves glucose tolerance

J. Lallement1, E. Foppen1, F. Lachkar2, D. Rainteau3, G. Merlan4, F. Preitner5, A. Rebelo Pimentel5, M. Schiffano6, M. Croyal6, M. Krempf6, F. Foufelle2, T. Tordjmann4, H. Le Stunff1, C. Magnan1, C. Cruciani-Guglielmacci1;

1Université de Paris, Unité Biologie Fonctionelle et Adaptative (BFA) - UMR 8251 CNRS, Paris, France, 2Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, AP-HP, Hôpital Saint Antoine, Biochemistry Department, Paris, France, 3INSERM, Sorbonne Université, Université de Paris; Centre de Recherche des Cordeliers, Paris, France, 4INSERM U1174 Université Paris Sud, Orsay, France, 5. Mouse Metabolic Evaluation Facility, Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland, 6Plateforme de Spectrométrie de Masse du CRNH-O, UMR1280, Nantes, France.

Background and aims: Numerous studies have shown the role of ceramides as lipotoxic inducers, which could impair insulin pathway and cause insulin resistance, leading to type 2 diabetes (T2D). Recently, several studies suggest that ceramides could be relevant plasma biomarkers of T2D susceptibility. Ceramides are precursors for the predominant sphingolipids, which are components of cell membranes. Their de novo synthesis, very active in the liver, involves serine palmitoyltransferase 2 (SPT2), the rate limiting enzyme.

Materials and methods: In this study, we investigated the role of de novo ceramide synthesis in the liver on energy homeostasis. Therefore, using the cre-lox system, we generated mice lacking SPT2 in the liver (SPT2ΔHep). The SPT2ΔHep mice and their littermate controls (SPT2lox/lox) were fed with control diet or high fat diet (HFD) for 2 months, during which we measured metabolic parameters.

Results: Despite lower expression of liver spt2, we found higher concentration of ceramides in the liver of SPT2ΔHep mice associated with an increased sphingomyelin phosphodiesterase expression, and a decreased sphingomyelin content. These results suggested a compensatory mechanism from sphingomyelin hydrolysis. We find that SPT2ΔHep mice are protected against body mass gain induced by HFD and display a decreased body fat mass. Bile acid composition and content are modified in KO mice. : the hydrophobic bile acid pool is drastically decreased in SPT2ΔHep mice, leading to lipid absorption defect. In addition, the nuclear bile acid receptor Farnesoid X receptor (FXR) and its target genes are downregulated in intestine and liver of SPT2ΔHep mice. Moreover, SPT2ΔHep mice, fed with HFD, display a significantly enhanced glucose tolerance compared to the controls. This is not associated with an improved insulin sensitivity. However, the ability of glucose production after injection of gluconeogenic substrates is lower in SPT2ΔHep mice. We measured glycemia time course during a 24H fast, and SPT2ΔHep mice displayed a lower glycemia compared to controls after 5h of food deprivation. These results suggest a defect in glucose production or storage by the liver

Conclusion: Our data shows for the first time a potential compensatory mechanism of ceramide synthesis in the liver. Then, these results highlight the role of hepatic sphingolipid modulation on hepatic glucose production through bile acid composition change. We are currently investigating the role of FXR on glucose homeostasis in our model.

Supported by: IMI - Rhapsody

Disclosure: J. Lallement: None.

235

Difference in lipid metabolism between men and women: implications for the pathophysiology of type 2 diabetes development and remission

A. Al-Mrabeh1, S. Melhem1, S. Zhyzhneuskaya1, C. Peters1, A. Barnes2, A. Jesuthasan1, K.G. Hollingsworth1, N. Sattar3, M.E. Lean4, R. Taylor1;

1Translational and Clinical Research Institute, Magnetic Resonance Centre, Newcastle University, Newcastle upon Tyne, 2Human Nutrition Research Centre, Population Health Sciences Institute, Newcastle University, Newcastle upon Tyne, 3Institute of Cardiovascular & Medical Sciences, Glasgow University, Glasgow, 4School of Medicine, Dentistry and Nursing, Glasgow University, Glasgow, UK.

Background and aims: Women experience a greater increase in cardiovascular risk than men on developing type 2 diabetes, possibly linked to differences in hepatic lipoprotein metabolism. We compared sex for markers of hepatic lipid content and triglyceride export in the Diabetes Remission Clinical Trial (DiRECT).

Materials and methods: The Tyneside subset of DiRECT (34M/30F, 52.3±SD 8.0 years, BMI 35.2±4.6kg/m2) was studied at baseline and followed up to 24 months after weight loss (BMI 29.9±4.6kg/m2). Results were compared with non-diabetic controls selected to match the diabetes group for weight after weight loss (14M/13F, 55.8±6.0 years, BMI 29.7±3.8kg/m2). Intra-organ and abdominal fat were quantified by 3-point Dixon MRI, and hepatic VLDL-TG production was measured using a competitive blocking method. Plasma biomarkers were quantified using commercial kits.

Results: Liver and pancreas fat were lower in women than men within the non-diabetic group, (3.4±0.1 vs. 5.4± 1.1 %, p=0.005, and 4.7±0.4 vs. 7.6±0.5%, p=0.0006, respectively). No such difference was evident in diabetes at baseline (16.9±1.9 vs. 15.4± 1.9%, p=0.49, and 8.3±0.5 vs. 8.5±0.4%, p=0.83, respectively). After weight loss, plasma VLDL-TG fell much less in women than men (0.58±0.08 to 0.54±0.09 mmol/l, p>0.05 and 0.83±0.08 to 0.40±0.06 mmol/l, p=0.0001, respectively) despite similar falls in VLDL-TG production rates (559±33 to 475±37 and 554±39 to 419±28mg/kg/day). Women had a lower: VLDL-TG pool size (1894± 294 vs. 3176± 339mg diabetes; 924±349 vs. 2640±618mg controls), visceral adipose tissue (226.6±12.3 vs. 320.9±12.4cm2 diabetes; 92.8±16.0 vs. 287.3 ±19.7cm2 controls) and fasting glucagon (22.5±1.8 vs. 30.3 ±2.5 ng/mL diabetes; 9.2±1.7 vs. 19.2±1.9 ng/l controls) than men. They also had higher: subcutaneous adipose tissue, adiponectin, GDF-15, FGF-21, and leptin (all p<0.01). After weight loss, pancreas fat remained similar in women and men unlike non-diabetic controls. However, after weight loss, men and women remained significantly different in subcutaneous/visceral adipose tissues, GDF-15, FGF-21, leptin, and adiponectin (p<0.05 for all). In contrast, fasting NEFA, HDL cholesterol, and IGF-1 became similar between men and women (p>0.05 for all).

Conclusion: Women have lower liver and intrapancreatic fat than men in the non-diabetic state, but in type 2 diabetes these differences are lost. Overall, women appear to have more efficient mechanisms to clear VLDL-TG from blood which are impaired in type 2 diabetes. Sex needs to be taken into account when planning metabolic studies. Recognition of the inadequate clearance of VLDL-TG in women with diabetes may lead to specific therapeutic interventions.

Supported by: Diabetes UK

Disclosure: A. Al-Mrabeh: None.

236

Characterisation of seven HDL subspecies and their association with incident type 2 diabetes in PREVEND study

S. Sokooti Oskooei1, J.L. Flores-Guerrero1, L.M. Kieneker1, H.J.L. Heerspink2, M.A. Connelly3, S.J.K. Bakker1, R.P.F. Dullaart1;

1Internal Medicine, University Medical Center Groningen, Groningen, Netherlands, 2Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen, Netherlands, 3Laboratory Corporation of America® Holdings (LabCorp), Morrisville, USA.

Background and aims: While high concentrations of high-density lipoprotein (HDL) and HDL cholesterol are thought to be protective against type 2 diabetes (T2D), HDL particles vary in size, density, protein composition and function have been shown to associate differently with development of T2D. Also, associations between small, medium and large HDL subclasses with incident T2D have been inconsistent. A newly developed algorithm provides concentrations for seven HDL subspecies and, categorizes the HDL particles more specifically. The aim of our current study was to investigate seven HDL subspecies and evaluate their association with incident T2D.

Materials and methods: We included 4828 subjects of the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study without T2D at baseline. HDL subspecies with increasing size from H1P to H7P were measured using the LP4 algorithm of the Vantera NMR platform. Insulin resistance was determined by homeostatic model assessment index (HOMA-IR).

Results: Among the seven HDL subspecies, H1P, H3P, H4P, H6P, and H7P were inversely associated with HOMA-IR ([ r=0.058; P<0.001], [ r=0.118; P<0.001], [ r=0.168; P<0.001], [ r=0.265; P<0.001], and [ r=0.283; P<0.001] respectively), whereas H2P was positively associated with HOMA-IR [ r=0.205; P<0.001]. During a median follow-up of 7.3 years, 265 individuals developed T2D. In multivariable-adjusted Cox regression models, higher levels of H1P, H4P, H6P, and H7P were associated with a lower risk of developing T2D, independent of adjustment for baseline covariates, including age, gender, lifestyle, use of medication, BMI, and hypertension. Oppositely, higher levels of H2P were associated with an increased risk of developing T2D. The associations for H2P, H4P, H6P, and H7P remained, independent of further additional adjustment for glucose and triglycerides. Moreover, the association of H6P was not independent of HDL cholesterol. In the last model, with adjustment for all relevant variables, H2P was the only subspecies that remained significant, with a positive association with T2D development.

Conclusion: H2P, the predominant HDL subspecies, was positively associated with metabolic factors including HOMA-IR and triglycerides. Additionally, H4P and H7P levels showed the strongest inverse association with incident T2D, whereas H2P levels were positively associated with incident T2D, independent of clinical risk factors.

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Supported by: European Union’s Horizon 2020

Disclosure: S. Sokooti Oskooei: None.

OP 42 Diabetes care is expensive

237

Socioeconomic factors and obesity: Are they independently associated with prevalence of diabetes?

S. Liatis1, G. Touloumi2, N. Kalpourtzi2, I. Ioannidis1, S. Iraklianou1, A. Raptis1, A. Sotiropoulos1, A. Karakosta2, G. Karamanakos1, K. Makrilakis1;

1Hellenic Diabetes association, Athens, 22Department of Hygiene, Epidemiology & Medical Statistics, National and Kapodistrian University of Athens Medical School, Athens, Greece.

Background and aims: Due to population aging and diet habits change, prevalence of diabetes and obesity has increased in Greece during last decades. Financial crisis widened socioeconomic disparities, associated with both diabetes and obesity. We aimed to assess the independent association of socioeconomic indices and BMI with diabetes prevalence

Materials and methods: Data were derived from the health examination survey EMENO (National Study of Morbidity and Risk Factors), conducted in Greece during 2014-2016, in a random sample of the general adult (≥18 years) population. Diabetes was defined as fasting plasma glucose (FPG) ≥126mg//dL or HbA1c≥6.5% or taking antidiabetic medications or self-reported. The study design was taken into account in the statistical analysis whereas inverse probability weighting was applied to adjust for the differences between those with/without available FPG/HbA1c measurements.

Results: : Of 6006 EMENO participants, 4,393 (48.5% men; median age: 47.7 years) had available FPG/HbA1c measurements. The overall diabetes prevalence was 11.9% (95% CI:10.9-12.9). Univariately, the higher the educational level and the family income the lower was the diabetes prevalence; being overweight or obese were significantly associated with increased diabetes prevalence (Table). Multivariable analysis showed that diabetes prevalence was increased with age, was lower in women and in those living in rural compared to urban areas, and higher in those with dyslipidemia and family history. The association with the socioeconomic indices remained significant even after adjusting for the above-mentioned factors. Further adjustment for BMI did not practically alter these associations, whereas BMI categories remained a significant and independent risk factor (Table). Food insecurity or physical examination did not remain significant factors in adjusted analysis. .

Conclusion: In Greece, low socioeconomic status is associated with higher diabetes prevalence independently of BMI. Interventions should include obesity prevention measurements and targeted primarily to people of low educational and socioeconomic status

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Supported by: co-funded by the European Union (European Social Fund) and the Hellenic Diabetes Association

Disclosure: S. Liatis: None.

238

Estimation of hospitalisations cost savings deriving from a wider application of EMPAREG and LEADER inclusion criteria in the real world practice: data from AMD Annals

A. Da Porto1, V. Manicardi2, C.B. Giorda3, E. Manicardi4, A. Agliadoro5, R. Fornego6, A. Nicolucci7, A. Rocca8, M. Rossi7, G. Russo9, D. Mannino10, P. Di Bartolo11;

1Clinica Medica ASUFC, Udine, 2Coordinatore gruppo Annali AMD, Reggio Emilia, 3Metabolism and Diabetes Unit ASL 5, Turin, 4SOS Diabetologia, Reggio Emilia, 5SSD Diabetologia, Endocrinologia e Malattie Metaboliche ASL3, Genova, 6SSD Diabetologia e Malattie Metaboliche ASLTO4, Chiavasso, 7Coresearch, Pescara, 8UOS Diabetologia e Malattie Meboliche ASST Nord, Milano, 9Dipartimento di Medicina Clinica e Sperimentale, Università di Messina, Messina, 10UOC Diabetologia, Reggio Calabria, 11Rete Clinica di Diabetologia AUSL Romagna, Ravenna, Italy.

Background and aims: Aim of the study was to quantify the proportion of patients potentially eligible for the EMPA-REG and LEADER studies and to estimate the potential impact on reduction of MACE, HHF and corresponding cost savings resulting if the use of these treatments would have been extended to all eligible patients.

Materials and methods: In Italy, an initiative of continuous monitoring and quality improvement of diabetes care (AMD Annals) is in place since 2004 promoted by the scientific society of diabetologists (AMD). A network of diabetes centers periodically extracts anonymous data from electronic clinical records for the continuous monitoring of quality of care. The same selection criteria used for recruiting patients in EMPAREG-Outcome and LEADER study were applied to the AMD Annals population. Reductions in absolute risk of cardiovascular death and hospitalization for heart failure or myocardial infarction associated with the use of the drugs if all eligible patients had been treated, were estimated on the basis of rates shown in the EMPA-REG OUTCOME and LEADER trials. Hospitalization cost savings were estimated from national reference cost in 2018 for diagnosis related group codes (DRG).

Results: From the AMD-database including 468,940 patients seen in 222 Diabetes Clinics in 2016, 342,190 had all the data required for evaluating the eligibility for both EMPA-REG or LEADER study. 41,715 patients met the eligibility criteria for EMPAREG study and 139,637 for LEADER study. Although theoretically eligible, in real world setting only 2,161 patients (5.2%) were currently treated with empaglifozin and 4,823 (3.5%) with liraglutide. Estimate numbers of prevented events and potential cost savings are summarized in table 1.

Conclusion: Even if CVOTs results provide evidence that use of empaglifozin and liraglutide are associated with reduced cardiovascular morbidity and mortality, only a minimal portion of eligible patients is on treatment with these drugs in a real world setting. Our cost analysis suggests that a wider use of empaglifozin and liraglutide in patient meeting CVOT eligibility criteria would result in a significant reduction of CV event rate and subsequent relevant hospitalization cost saving.

figurebw

Disclosure: A. Da Porto: None.

239

Costs of diabetes complications: hospital based care and production loss for 392,200 people with type 2 diabetes and matched controls in Sweden

K. Steen Carlsson1,2, E. Andersson1, S. Persson1,3, N. Hallén4, Å. Ericsson5, D. Thielke4, P. Lindgren1,6, J. Jendle7;

1The Swedish Institute for Health Economics, IHE, Lund, Sweden, 2Department of Clinical Sciences, Malmö, Lund Unviersity, Lund, Sweden, 3Department of Clinical Sciences, Malmö, Lund University, Lund, Sweden, 4Novo Nordisk A/S, Copenhagen, Denmark, 5Novo Nordisk Scandinavia, Malmö, Sweden, 6Department of Learning, Informatics, Management and Ethics, Karolinska Institutet, Stockholm, Sweden, 7Örebro University, Örebro, Sweden.

Background and aims: The prevalence of diabetes has increased rapidly over the last decades worldwide. The risk of complications and medical consequences is well known and identified as key driver of costs. Less evidence on the impact of individual diabetic complications on the societal burden is available. The objective was to analyse costs of hospital-based health care and work absence related to individual macrovascular and microvascular complications of type 2 diabetes in Sweden in 2016.

Materials and methods: The study used data from a Swedish retrospective observational database cross-linking 20 years of individual-level data (1997-2016) from national population-based health, social insurance and socio-economic registers for 392,200 people with type 2 diabetes and matched controls (5:1). Diabetes status and presence of 19 types of complications were derived from years 1997-2016 while the costs of hospital-based care and of production loss due to diabetes complications were estimated for 2016. Regression analysis was used for comparison to controls, to attribute production loss to individual complications, and to account for joint presence of complications.

Results: Complications are prevalent and patterns complex in type 2 diabetes (Fig. 1). Use of hospital care for complications was higher compared to controls: 86,104 vs 24,608 outpatient visits per 100,000 persons and 9,894 vs 2,546 inpatient admissions per 100,000 persons (p<0.001) in 2016. 26% vs 12% had ≥1 hospital contact. The corresponding total costs of hospital-based care for complications were EUR 91,875 vs EUR 23,222 per 100 persons (p<0.001) and 75% were directly attributed to diabetes (EUR 689/person). Regression analyses distributed the costs of days absent from work across diabetes complications, basic type 2 diabetes effect and unattributed causes: diabetes complications amounted to EUR 2,165/person in 2016. Key drivers of costs of production loss were macrovascular complications angina pectoris, heart failure and stroke, and microvascular complications eye disease including retinopathy, kidney disease and neuropathy. Early mortality in working ages cost additional EUR 579/person and medications used in risk-factor treatment amounted to EUR 418/person.

Conclusion: The economic burden of complications in type 2 diabetes is substantial. Costs of productivity loss in this study were found to be greater than those of hospital-based care highlighting the need for considering treatment consequences in a societal perspective in research and policy.

figurebx

Supported by: Novo Nordisk A/S, Copenhagen Denmark to the Swedish Institute for Health Economics, Lund, Sweden

Disclosure: K. Steen Carlsson: Grants; Novo Nordisk A/S, Copenhagen Denmark to the Swedish Institute for Health Economics, Lund, Sweden.

240

Economic burden associated with diabetes technologies: a cross-national comparison of out-of-pocket expenses

T. Froment1, A. Thieffry2, H. Ballhausen3, M. Wäldchen1, S. O'Donnell1, B. Cleal4;

1University College Dublin, Dublin, Ireland, 2Technical University of Denmark, Copenhagen, Denmark, 3DeDoc Labs GmBH, Berlin, Germany, 4Steno Diabetes Center, Copenhagen, Denmark.

Background and aims: This study looked into the financial burden for those living with Type-1-Diabetes (T1D) in regards to Out-of-Pocket Expenses (OoPE) associated with diabetes technologies and insulin. This investigation highlights the costs of living with diabetes and identifies potential sources of social inequality in diabetes treatment, such as a technological divide. The primary aim is to understand the economic burdens for People with Diabetes (PwD) associated with accessing diabetes technologies and the extent to which these burdens are barriers to access for optimal treatment.

Materials and methods: Using the 2018 T1International “Type 1 Diabetes Access to Insulin and Supplies Survey”, a combination of both quantitative and qualitative methods were used to analyse the out-of-pocket expenses for diabetes technology in relation to health care coverage, with focus on continuous glucose monitoring (CGM), test strips, insulin and pump supplies.

Results: A total of 1048 answers representing 8 countries (Australia, Canada, Croatia, Germany, UK, Pakistan, South Africa and USA) were considered in this study. Quantitative analyses showed substantial variation of OoPE between countries, which appeared to be largely driven by the extent of healthcare coverage availability in each country. The majority of respondents reported partial healthcare coverage (77.7%, cross-country average). In half of the selected countries, participants with full healthcare coverage still reported OoPE. In the USA, substantial OoPE were reported both with respect to insulin and diabetes technologies. In contrast, the vast majority of UK respondents experienced virtually no OoPE associated with insulin. Manual curation of more than 390 qualitative responses shed light on the experience of PwD and provided valuable insights on the relationship between healthcare coverage and costs. This relates both to what is perceived as diabetes-related costs and expectations about what coverage should actually cover; a sliding scale from access to insulin to coverage for the latest technological innovation. Respondents in countries with universal healthcare coverage acknowledged the benefits of the system, even when the state did not cover every supply or the total cost of diabetes management. In the absence of public healthcare coverage, PwD applied a variety of methods to access diabetes medication and supplies wherever possible, with many relying on private health insurance and employment-based insurance plans, whereas some reported struggle accessing even the most basic necessities such as insulin and test strips. In terms of technology access, latest development were often reported as hardly accessible and/or unaffordable (i.e. CGM).

Conclusion: The study demonstrated inequalities for diabetes technologies within and across eight countries. Public healthcare coverage provided patient satisfaction more likely than privately led systems, as PwD can benefit from it and reduce their monthly OoPE. The analysis further uncovered that OoPE associated with hypoglycemia treatments and other medical supplies should not be ignored when discussing the financial burden of living with T1D.

Supported by: H2020-MSCA-RISE-2018

Disclosure: T. Froment: None.

OP 43 Developing beta cells

241

Real-time functional assessment and quality control of iPSC-derived human beta-like cells for diabetes modelling

M. Jaffredo1, N. Krentz2,3, B. Champon2, A. Clark4, S. Nawaz4, C. Duff2, S. Renaud5, A. Gloyn2,3, J. Lang1, M. Raoux1, B. Hastoy4;

1CBMN UMR 5248, Bordeaux, France, 2WTCHG, Oxford, UK, 3Stanford Department of Medicine, Stanford, USA, 4OCDEM & NIHR Biomedical Research Centre Churchill Hospital, Oxford, UK, 5IMS UMR 5218, Bordeaux, France.

Background and aims: Human β-like cells (BLC) derived from iPSCs hold promise for diabetes research and therapy, but their differentiation remains long, costly and challenging in terms of homogeneity and maturity. Current quality control focuses on expression of key genes providing minimal insight on function and on static stimulations of insulin secretion which vary due to heterogeneity. Here, we characterized BLC electrical behaviour with non-invasive multielectrode arrays (MEAs) to provide a standardized and automated functional quality control of BLC. To evaluate our system further, we analysed BLC from an established SLC30A8 loss-of-function diabetes model.

Materials and methods: BLC were generated in clusters (N=8) and monolayers (N=9) according to the Rezania protocol. Both single-cell (action potential, AP) and multicellular coupling signals (slow potential, SP) were recorded with MEAs and processed either offline, or online (when specified) with an electronic circuit dedicated to real-time analysis. Whole-cell patch clamp was used to measure ion currents and exocytosis (membrane capacitance).

Results: The majority of BLC preparations were electrically active: 87.5% of clusters and 88.9% of monolayers. 85.7% of electrically active clusters and 87.5% of monolayers generated both APs and SPs. The detection of SPs, an established β cell-specific coupling signal, revealed that mature BLC are electrically coupled. This was supported by the maximal Connexin-36 (GJD2) expression from the endocrine-like stage onward. We established a standardised functional quality control of BLC to successive stimuli. Upon glucose and forskolin, the electrical activity increased in 67% of monolayers (N=4/6) by 9.6±3.7-fold and in 40% of clusters (N=2/5) by 2.1±0.1-fold. Glibenclamide increased the activity in 33% monolayers (N=1/3) by 2.8-fold and in 67% clusters (N=2/3) by 2.7±0.2-fold. Monitoring BLC revealed that they were glucose-responsive for a short period (2-3 days). Furthermore, automatic online analysis of SPs and APs correlated well with glucose levels and with insulin secretion measured on the same cells. Application of Bay K8644, Cobalt and TTX as ion channel modulators showed the contribution of Ca2+ and Na+ voltage-gated channels in BLC’s electrical activity. Single cell patch clamp recordings from 3 independent differentiations revealed 48.9±7.9pA.pF-1 Na+ and 9.22±1.1pA.pF-1 Ca2+ current densities (N=20), similar to human primary β cells. The amplitude of exocytosis was of 497±200fF with a biphasic kinetics (N=14), the latter as described in mature β cells. Finally, assessment of the electrical activity of BLC derived from loss-of-function SLC30A8 CRISPR-edited iPSCs confirmed the improved glucose and forskolin responsiveness, corroborating recently published data on the protective effect against type 2 diabetes (N=2 independent CRISPR clones).

Conclusion: Our non-invasive system allows real-time dynamic functional assessment of BLC even in heterogeneous preparations. We show that mature BLC are electrically active and coupled. The MEA represents a valuable quality control of iPSC differentiations protocols for therapy and type 2 diabetes research.

Supported by: EFSD Albert Renold Travel Fellowship Programme, French Ministry of Research, ANR-18-CE17-0005, FEDER, Diabetes UK, Wellcome Trust

Disclosure: M. Jaffredo: None.

242

Intravital microimaging of human iPSC-derived surrogate islets in the anterior chamber of the eye

K. Zhao1, Y. Shi1, J. Yu1, L. Yu1, A. Mael2, A. Kolton2, T. Joyce2, J. Odorico2, P.-O. Berggren1, S.-N. Yang1;

1The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden, 2Regenerative Medical Solutions, Inc., Madison, USA.

Background and aims: Induced pluripotent stem cell (iPSC) technology enables engineering human iPSC-derived surrogate islets (hiPSC-SIs) from human subjects’ or patient’s own somatic cells. So far, hiPSC-SIs have not been used to treat patients with diabetes, primarily due to incomplete maturation in vitro into authentic pancreatic islets. To guide hiPSC-SIs in vivo to their full maturity where they timely and accurately secrete insulin in response to glycemic changes, we have established a way to microimage the in vivo dynamics and fates of islet hormone-expressing cells in hiPSC-SIs in a longitudinal and non-invasive manner. To make this possible, we have exploited the anterior chamber of the eye (ACE) of immunodeficient mice as a transplantation site and the cornea as a natural body window for microimaging hiPSC-SIs.

Materials and methods: hiPSC-SIs were produced in vitro from the undifferentiated hiPSC line NCRM-1 and immunocytochemically, flow cytometrically, and functionally characterized. The resultant hiPSC-SIs were transplanted into the ACE of NOD/scid/IL2Rgamma -/- immunodeficient mice. The engraftment, vascularization and backscattering signal of the intraocular hiPSC-SIs were longitudinally and non-invasively monitored by applying intravital microimaging.

Results: hiPSC-SIs generated in vitro resembled native human islets in size and shape. Most of cells in hiPSC-SIs were islet-hormone positive and displayed glucagon, insulin or somatostatin immunofluorescence. Once implanted into the ACE, hiPSC-SIs rapidly engrafted onto the iris. Thereafter, transplanted hiPSC-SIs underwent vascularization. When imaged at day 3 post-transplantation, functional blood vessels were sparsely seen in hiPSC-SI grafts. By 1 week after transplantation, blood vessels became significantly enriched within islet structures, and continued to increase in density through week 4 after transplantation and plateaued thereafter. Light scattering signals, reflecting the abundance of zinc-insulin crystals within insulin secretory granules, were also imaged and quantified. The light scattering signal of intraocular hiPSC-SI transplants progressively increased over the first month and plateaued 2 months after transplantation.

Conclusion: The present work establishes a unique research platform for intravital microimaging of hiPSC-SIs transplanted into the mouse ACE in a longitudinal and non-invasive manner. Importantly, the platform enables high spatiotemporal resolution dynamics of engraftment, vascularization and insulin granule density. This not only lays a solid foundation for monitoring in vivo hiPSC-SI maturation, but also offers a feasible and reliable means to screen compounds with clinical potential for promoting this cellular process.

Supported by: VR; ERC; SRP

Disclosure: K. Zhao: None.

243

Spatiotemporal expression pattern of adhesion G-protein coupled receptors in developing human pancreas reveals a role for GPR56 in developing islets

O.E. Olaniru1, K. Toczyska1, N. Guccio1, S. Giera2, X. Piao2, A.J. King1, P.M. Jones1, S.J. Persaud1;

1King's College London, London, UK, 2University of California, San Francisco, USA.

Background and aims: Adhesion G-protein coupled receptors (aGPCRs) play important roles in organ development but their role in islet development and function is poorly understood. We have now systematically characterised their expression in developing human pancreas and generated β-cell-specific GPR56 knockout mice to determine the effect of β-cell-specific deletion of GPR56 on islet function.

Materials and methods: The aGPCR transcriptome in human fetal pancreatic cells at 8 to 21 weeks post conception (wpc) was profiled by single cell RNA sequencing and qPCR. Immunohistochemistry of GPR56, the aGPCR with the highest fold increase during pancreas development, and its colocalization with endocrine progenitor markers, was investigated in human fetal and mouse embryonic pancreas sections. We generated transgenic mice with GPR56 deletion specifically in islet β-cells (GPR56-βKO) by crossing Ins1Cremice with LoxP-GPR56 mice flanking exons 4 to 6. Glucose tolerance tests were performed in lean GPR56-βKO mice and their wild type (floxWT) littermates at 8 weeks, and insulin secretion from isolated islets was determined by radioimmunoassay. Islet morphology of GPR56 global null mice and GPR56-βKO mice, with their appropriate littermate controls, was evaluated by immunoprobing for insulin, glucagon, BrdU and Ki67, and images were quantified by ImageJ.

Results: Transcripts encoding 29 aGPCRs were detected in developing human fetal pancreases and aGPCRs were dynamically expressed across the developmental timepoints, with GPR56 showing the highest fold increase at 17wpc. Unbiased clustering and t-SNE visualization of 7,369 human fetal pancreatic cells showed that GPR56 is expressed in endocrine, ductal and pancreatic progenitor clusters while GPR123, CELSR3 and VLGR1 expression are restricted to the endocrine progenitor cluster. Genotyping showed a 465kb LoxP band and the presence of Cre in GPR56-βKO mice, as expected, and GPR56 deletion was confirmed by Western blotting in isolated islets. There was no significant difference in the weights of GPR56-βKO mice and WT littermates at 8 weeks (floxWT: 24.5±1.9g,βKO: 25.5±1.4g, n=6, P>0.2), in pancreas size (weight;floxWT: 0.30±0.06g,βKO: 0.37±0.03g, n=3, P>0.2) nor in fasting glucose levels (floxWT: 8.4±0.7mM, βKO: 7.9±0.4mM, n=6, P>0.2). In addition, GPR56 deletion in β-cells did not significantly affect glucose tolerance (AUC;floxWT: 1,292±58.2, βKO: 1,114±81.2, n=7, P>0.2) nor insulin content (ng/islet;floxWT: 36.9±3.8, βKO: 34.4±2.0, n=4), but the stimulation index of βKO islets to 20mM glucose was 46% lower than in floxed islets. There was no change in islet morphology in GPR56 null mice or GPR56-βKO mice, but there was an altered α/β cell ratio with less β-cells (% β-cells/islet; WT: 68.5±0.8, KO: 54.8±3.0, n=3, p<0.05), and higher numbers of α-cells in GPR56 null islets at P9 (% α-cells/islet; WT: 17.7±0.9, KO: 33.7±2.8, n=3, p<0.01).

Conclusion: These data demonstrate that aGPCRs are dynamically regulated during human pancreas development and may be required at key stages of endocrine lineage decisions. Lean GPR56-βKO mice are phenotypically normal and show normal glucose tolerance, but GPR56 deletion is associated with altered α/β cell ratio. These mice will allow us to evaluate the requirement of β-cell GPR56 in β-cell development and compensatory responses of β-cells to metabolic dysregulation.

Supported by: Diabetes UK

Disclosure: O.E. Olaniru: None.

244

Regulatory role of tyrosine kinase 2 (TYK2) in human pancreatic endocrine differentiation

V. Chandra1, H. Ibrahim1, J. Kvist1, D. Balboa2, R.B. Prasad3, O.P. Dwivedi4, L. Groop4, D. Eizirik5, T. Otonkoski1;

1Faculty of Medicine, Helsinki University, Helsinki, Finland, 2Centre for Genomic Regulation (CRG), Barcelona, Spain, 3Department of Clinical Sciences, Diabetes and Endocrinology, Lund University, CRC, Malmo, Sweden, 4Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland, 5ULB Center for Diabetes Research, Brussels, Belgium.

Background and aims: Type 1 diabetes is a multifactorial autoimmune disease that results in the destruction of insulin producing pancreatic beta cells. One of the genes associated with T1D is TYK2 which encodes a JAK tyrosine kinase with critical roles in cytokine mediated intracellular signaling. Specific variants of TYK2 have been associated with either protection against or predisposition to T1D.

Materials and methods: To study the role of TYK2 in human pancreatic beta cell development we generated TYK2 knockout (KO) iPSCs using CRISPR-CAS9 and induced them to differentiate to pancreatic lineage.

Results: Interestingly, loss of TYK2 did not alter the pluripotency or the early pancreatic endoderm differentiation but severely compromised the emergence of endocrine progenitors. Transcript levels of key pancreatic transcription factors (PDX1 (55±17%, p≤0.05), NGN3 (65±13%, p≤0.01)), RFX6 (58±14%, p≤0.05) were significantly reduced in the TYK2-KO cells at the endocrine progenitor stage. TYK2-KO lines also showed low expression levels of NKX6.1, INS, GCG and SST compared to control lines. Additionally, selective inhibition of TYK2 activity using the inhibitor BMS-986165 decreased the number of NKX6.1/PDX1- positive endocrine progenitors suggesting a direct regulatory role of TYK2 in endocrine lineage commitment. In contrast to the wild-type controls, the TYK2-KO iPSC-derived pancreatic cells displayed no activation of STAT1 and STAT2 and showed impaired STAT3 activation in response to IFNα or stimulation by polyinosinic-polycitidilic acid, a viral infection mimetic. Stage specific transcriptomic analysis revealed the upregulation of KRAS (p = 1.06e-13) at all differentiation stages in the TYK2-KO cells. Interestingly, KRAS has been shown to antagonize endocrine neogenesis in the developing pancreas. Additionally, microarray data from human cadaveric islets showed a highly significant (p = 4.4e-14) negative correlation (r = -0.51) between TYK2 and KRAS expression.

Conclusion: These results provide evidence for an important role for TYK2 in pancreatic endocrine differentiation through the control of STAT and KRAS signaling.

Supported by: INNODIA

Disclosure: V. Chandra: None.

OP 44 Modelling metabolism: lessons from animals

245

Critical role of TRAPalpha in maintaining beta cell function and glucose homeostasis

X. Li, M. Wang, W. Feng, Y. Huang, X. Zhang, H. Shu, X. Xu, J. Sun, M. Liu;

Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin, China.

Background and aims: An endoplasmic reticulum (ER) membrane protein, translocon-associated protein alpha (TRAPα), is one of subunits of TRAP complex assisting secretory proteins translocation across the ER membrane. Genome-Wide Association Studies (GWAS) reveals that TRAPα is a type 2 diabetes-associated gene. We have recently reported that TRAPα is required for insulin biosynthesis in β-cells. However, the pathophysiological significance of TRAPα in maintaining normal β-cells function and glucose homeostasis remains unknown. By establishing a pancreatic β-cell specific TRAPα knockout (TRAPα βKO) mouse line, in this study, we aim to elucidate biological significance of TRAPα in β-cells and its role in maintaining glucose homeostasis.

Materials and methods: A TRAPα βKO mouse line was generated using CRISPR-Cas9-mediated genome editing. The TRAPα βKO mice and their littermate controls were fed with either regular chow or high-fat diet for three months. Body weight, fasting blood glucose, and plasma insulin were recorded weekly. Intraperitoneal glucose tolerance tests (IPGTT) and plasma insulin response to glucose challenge were performed monthly. Pancreatic islet morphology and cell composition, as well as the distribution of insulin, glucagon, and somatostatin were detected by immunohistochemistry and confocal immunofluorescence in paraffin-embedded pancreatic sections. The effects of TRAPα deficiency on the stability of other subunits of TRAP complex and intracellular insulin precursors (including preproinsulin and proinsulin) and mature insulin were examined by western blotting.

Results: Targeted disruption of TRAPα in pancreatic β-cells has no significant effects on the mouse body weight, fasting blood glucose, and fasting insulin in both regular and high-fat diet (HFD). However, IPGTT revealed that glucose tolerance and insulin secretion response to glucose were significantly impaired in the TRAPα βKO mice fed with regular chow, and these impairments were further aggravated in HFD fed mice. Consistent with these phenotypes, glucose-stimulated insulin secretion (GSIS) was markedly impaired in isolated islets from TRAPα βKO mice. Immunohistochemistry and confocal immunofluorescence showed that although insulin content was decreased, neither islet morphology nor cell composition was not significantly affected by TRAPα βKO. Western blotting revealed that deficiency of TRAPα in β-cells resulted in diminished other subunits of TRAP complex, including TRAPβ, TRAPγ, and TRAPδ, suggesting that TRAP complex was destabilized in the absence of TRAPα. Consistent with our recent report on the role of TRAPα in insulin biosynthesis in β-cell lines, we found that preproinsulin translocation was impaired along with markedly decreased intracellular mature insulin in the islets isolated from TRAPα βKO mice, supporting the notion that TRAPα is critical for maintaining β-cell function in vivo.

Conclusion: These results provide the first in vivo evidence that ablation of TRAPα leads to defects in insulin production and glucose intolerance. This study not only reveals pathophysiological significance of TRAPα in maintaining β-cell function, but also provides insight into pathogenesis of type 2 diabetes associated with impaired TRAPα function.

Supported by: National Natural Science Foundation of China 81830025, 81620108004, 81870533, 81800733, 81700720

Disclosure: X. Li: None.

246

Glucokinase haploinsufficiency ameliorates glucose intolerance by increasing beta cell mass in db/db mice

K. Omori1, A. Nakamura1, H. Miyoshi2, K. Takahashi1, H. Nomoto1, H. Kameda1, K. Cho1, Y. Terauchi3, T. Atsumi1;

1Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine, Hokkaido University, Sapporo, 2Division of Diabetes and Obesity, Faculty of Medicine, Hokkaido University, Sapporo, 3Department of Endocrinology and Metabolism, Yokohama City University School of Medicine, Yokohama, Japan.

Background and aims: Some clinical trials have shown that glucokinase activators (GKAs) improve glycemic control, but the effect is limited to a short-term period. One reason for the loss of efficacy may be the toxicity of GKAs toward beta-cells. In the present study, we aimed to investigate the effect of glucokinase haploinsufficiency in pancreatic beta-cells on glucose tolerance as well as beta-cell mass using a mouse model of diabetes.

Materials and methods: We obtained Leprdb/+ (db/+) mice and crossed them with glucokinase haploinsufficiency in pancreatic beta-cells (Gck+/−) mice, generating Gck+/−db/+ mice. We then crossed mice to generate Gck+/+db/+, Gck+/−db/+, Gck+/+db/db, and Gck+/−db/db mice. Glucose tolerance, beta-cell mass, and survival time were evaluated. Gene expressions in isolated islets were evaluated by microarray and quantitative real-time PCR analyses. Metabolome analyses were also performed on the isolated islets.

Results: Fed glucose levels in Gck+/−db/+ mice were higher than those in Gck+/+db/+ mice, consistent with previous findings, while those in Gck+/−db/db mice gradually decreased after 13 weeks of age and remained lower than those in Gck+/+db/db mice. Oral glucose tolerance tests revealed that glucose tolerance improved significantly in Gck+/−db/db mice compared with Gck+/+db/db mice. Beta-cell mass were significantly increased in Gck+/−db/db mice at 24 weeks of age compared with those in the other three mice (Gck+/−db/db 22.5±10.1 mg vs. Gck+/+db/+ 2.2±1.4 mg, Gck+/-db/+ 1.9±0.6 mg, and Gck+/+db/db 7.0±7.1 mg). Furthermore, the survival time of Gck+/−db/db mice was significantly longer than that of Gck+/+db/db mice. Pathway analyses on the microarray data demonstrated that oxidative stress-related genes were downregulated in islets isolated from Gck+/−db/db mice compared with those from Gck+/+db/db mice. Quantitative real-time PCR analyses revealed that the gene expressions of pyruvate carboxylase (Pcx), Nkx6.1, Mafa, Pdx1, Ki67, and Ccnd2 were increased in Gck+/−db/db mice compared with Gck+/+db/db mice. Meanwhile, oxidative stress-related genes including Atf3 and Cyba were decreased in Gck+/−db/db mice. In metabolome analyses, glycolytic intermediates and metabolites such as fructose 6-phosphate, pyruvate acid and lactic acid were decreased, while isocitric acid and ATP were increased in Gck+/−db/db mice. These results suggested that the metabolic pattern shifted from glycolytic pathway dominance, as observed in diabetic islets, toward TCA cycle and oxidative phosphorylation dominance.

Conclusion: The present results demonstrated that glucokinase haploinsufficiency in pancreatic beta-cells ameliorated glucose intolerance by increasing beta-cell mass in db/db mice. Amelioration of glucotoxicity by glucokinase haploinsufficiency may reduce oxidative stress, followed by increased expression of transcription factors related to maintenance and maturation of beta-cell function, and improved metabolic remodeling, resulting in augmentation of beta-cell proliferation. Glucokinase inactivation in beta-cells may be a potential strategy for treatment of type 2 diabetes.

Disclosure: K. Omori: None.

247

Stbd1 deficiency results in altered hepatic and cardiac glycogen levels and decreased glucose tolerance in mice

S. Kyriakoudi, A. Drousiotou, P.P. Petrou;

Biochemical Genetics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

Background and aims: Glycogen metabolism and endoplasmic reticulum (ER) stress are disturbed in diabetic patients and in experimental animal models of diabetes. Moreover, recent findings support that both hepatic glycogen levels and the extent of ER stress directly impact on the severity of pathological features related to type 2 diabetes (T2D). Starch binding domain-containing protein 1 (Stbd1) is a glycogen-binding protein which resides in the ER membrane with a poorly characterized role in glycogen metabolism. The aim of the current study is to evaluate the metabolic abnormalities of Stbd1-/- mice. This will provide new insights into the role of Stbd1 in glycogen metabolism and its potential role as a modulator of pathological conditions related to T2D.

Materials and methods: Quantification of hepatic and cardiac glycogen content was performed in randomly fed 6-months-old wild type (WT) and Stbd1-/- male mice on a C57Bl6 background, at 18hrs of fasting and 3hrs after refeeding following an overnight fast, by the enzymatic degradation of glycogen and the colorimetric determination of the liberated glucose. Western immunoblot was employed to assess Stbd1 expression levels in the liver of C57Bl6 male mice following i.p injection of the ER stress inducer tunicamycin (1mg/kg) for 24 hrs. OGTT was conducted by monitoring blood glucose levels at timed intervals following the administration of dextrose solution (2g/kg). Glucose measurements were performed in blood samples obtained from the tail tip using a hand-held glucometer.

Results: Randomly fed Stbd1-/- mice displayed significantly reduced glycogen content in the liver as compared to WT controls (p<0.001). Fasting resulted in a marked reduction of hepatic glycogen in WT and it’s near complete depletion in Stbd1-/- mice. In contrast to glycogen in the liver, cardiac glycogen was found to be increased in randomly fed Stbd1-/- mice (p<0.01). We found that fasting induced an opposite effect on cardiac glycogen content in WT and Stbd1-/- mice resulting in an increase in controls and a reduction in knockout animals (p<0.05). Our results further indicate that Stbd1-/- mice displayed impaired tolerance to fasting as evidenced by a significant reduction in body mass and decreased ability to defend blood glucose as compared to WT controls. Moreover, Stbd1-/- mice showed reduced ability to clear glucose from the blood as demonstrated by OGTT (p<0.05). We further identify Stbd1 as a target of the ER stress response as evidenced by the marked upregulation of Stbd1 in the liver following the i.p administration of tunicamycin (p<0.001).

Conclusion: Our results reveal for the first time metabolic aberrations in Stbd1-/- mice such as decreased glucose tolerance, impaired response to fasting and abnormalities in tissue glycogen content. In particular, the reduced liver and increased heart glycogen content displayed by randomly fed Stbd1-/- is also featured by patients with diabetes. Moreover, our findings strongly suggest a direct relationship between ER stress activation and Stbd1 expression. Interestingly, both ER stress and hepatic glycogen content were previously shown to influence metabolic manifestations related to T2D such as insulin resistance and glucose intolerance. Taken together the above data may suggest a potential modifying effect of Stbd1 on the metabolic abnormalities related to diabetes and further imply that Stbd1-/- mice could serve as a novel animal model for the study of T2D and related pathologies.

Supported by: RIF

Disclosure: S. Kyriakoudi: None.

248

Dynamic characteristics of high-fat diet model and its temporal transcriptomic landscape of interorgan crosstalk between islet and liver contributing to beta cell dysfunction

R. Gao1,2, H. Jiang2, Q. Fu2, Q. Zhang1, T. Yang2;

1Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK, 2Department of Endocrinology and Metabolism, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

Background and aims: Hyperinsulinemia and insulin resistance are co-existing characteristics of type 2 diabetes. However, the forerunner initiating the deleterious cycle remains elusive. The temporal transcriptomic landscape of islets (responsible for hyperinsulinemia) and liver (involved in insulin resistance) could provide new insights.

Materials and methods: The dynamic profile of glucose metabolism, islet architecture and secretion, insulin sensitivity and T cell subpopulations were monitored in C57BL/6N mice fed on a 60% high-fat diet (HFD) or control for 24 weeks. RNA-sequencing and transcriptomic analyses of islets and liver were respectively performed in quadruplicates at 4, 8, 12, 16, 20 and 24 weeks. To evaluate co-ordinated molecular interactions of islets and liver, we generated a massive matrix of Pearson correlation coefficients in weighted gene co-expression network analysis (WGCNA). Ingenuity Pathway Analysis was also applied to construct networks and identify major integrative hubs.

Results: HFD mice exhibited progressively impaired glucose homeostasis with evident hyperinsulinemia and first-phase insulin secretion defect since 4 weeks. Insulin, glucagon and somatostatin secretion in response to glucose or co-stimulated palmitic acid demonstrated a gradually deteriorated transition from islet dysfunction to failure. HFD islet morphology showed increased abundance of β-cell whose proliferation peaked at 4 weeks, with concomitant reduction in δ- and α-cell proportion. Ultrastructure of β-cell also presented decreased docked granules and deranged cristae of mitochondria. We identified impaired systemic insulin sensitivity from 12 weeks with variable time course in tissue-specific insulin action. Liver and skeletal muscle developed insulin resistance from 16 weeks, while adipose tissue initiated from 8 weeks. Our RNA-sequencing dataset outlined the impact of HFD on dynamics of molecular network in islets and liver at different stages. Correlation analyses of islet and liver modules with metabolic phenotypes illustrated that these two tissues jointly program β-cell compensatory adaption and irreversible impairment. Top scored networks combining the islet and liver transcriptomes showed potential interactions of genes implicated in cell cycle during 4 weeks, organismal development around 12 weeks, and immune cell trafficking at 24 weeks. To validate that immune and inflammatory responses might be involved in HFD-induced diabetes, we observed significant increase in the proportion of T helper 1 cells and T helper 17 cells, and decrement in regulatory T cells.

Conclusion: Our data provide a comprehensive landscape of crosstalk between islets and liver in diet-induced diabetes, linking to the development of islet dysfunction and insulin resistance.

figureby

Supported by: NFSC

Disclosure: R. Gao: None.

OP 45 Diabetic foot: new developments in wound healing

249

Extracellular vesicles derived from platelet-rich plasma accelerate dermal wound healing via activation of TGF-β1/Smad2 signalling pathway in a diabetic rat model

S. Rui1,2, L. Li1, W. Deng2, G. Yang3, D. Armstrong4;

1Key Laboratory of Diagnostic Medicine (Ministry of Education) and Department of Clinical Biochemistry, Chongqing Medical University, Chongqing, China, 2Bioengineering College and Department of Endocrinology, Chongqing University Central Hospital, Chongqing University, Chongqing, China, 3Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China, 4Department of Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, USA.

Background and aims: Dermal wounds have become an economic, social, and public health burden and need advanced treatment in diabetic patients. Our previous study has proven that platelet-rich plasma (PRP) contains an abundance of promotion-related factors secreted by platelets and promotes wound healing in diabetic foot patients, but the underlying mechanism is not clear. Extracellular vesicles (EVs) released from PRP play an important role in cell-to-cell communication. This study aimed to investigate the effects of PRP-derived extracellular vesicles on dermal wound healing and the underlying mechanism in a diabetic rat model.

Materials and methods: Extracellular vesicles were isolated from PRP using a gradient ultracentrifugation method. EVs were identified by transmission electron microscopy (TEM), dynamic light scattering, western blotting. In vitro, HUVEC, HaCaT, human dermal fibroblasts are used for cytological analysis. EVs uptake, cell migration, cell proliferation and effects on major signal transduction pathways were analyzed by immunofluorescence, wound-healing assay, flow cytometry, tubule formation assay, qRT-PCR, Western blotting. In vivo, we established a cutaneous wound model in streptozotocin-induced diabetic rats. Rats were randomly assigned to two groups of ten rats each: diabetic control group and PRP-EVs group. PRP-EVs were injected dispersively into the wound edge. The curative effects of PRP-EVs on inflammation and wound healing were observed and evaluated. Histology, immunofluorescence, and immunochemical analysis were performed for wound histological analysis.

Results: Our in vitro results showed that PRP-EVs stimulated the cell migration, proliferation of HaCaT, human dermal fibroblasts and HUVEC in a dose-dependent manner. Furthermore, PRP-EVs also promoted collagen synthesis of HDF via activation of TGF-β1/Smad2 signaling pathway and tube formation of HUVEC. Testing this system in a diabetic rat model, we found that this approach resulted in accelerated skin wound healing, remodeling, activated angiogenesis, and promotion of collagen maturity in vivo. The levels of expression of TGF-β1 and Smad2 mRNAs were significantly higher in the PRP-EVs treated group than in the control group (p < 0.05). The expressions of TGF-β1 and Smad2 proteins were also significantly upregulated in the PRP-EVs treated group than in the control group (p < 0.05).

Conclusion: We provide evidence of the probable molecular mechanisms underlying the PRP-EVs effect on the healing of chronic ulcers and could have infinite possibilities for future therapy.

Supported by: cstc2018jcyjAX0335

Disclosure: S. Rui: None.

250

Vasomotion analysis based on speed-resolved perfusion measurement as a method to investigate microvascular dysfunction in patients with type 2 diabetes

F. Iredahl1, E. Tesselaar2, R. Mirdell2, H. Jonasson3, S. Bergstrand3, F.H. Nyström1, C.J. Östgren1, I. Fredriksson3, M. Larsson3, T. Strömberg3;

1Department of Health, Medicine and Caring Sciences, Linköping University, Linköping, 2Department of Biomedical and Clinical Sciences, Linköping University, Linköping, 3Department of Biomedical Engineering, Linköping University, Linköping, Sweden.

Background and aims: Type 2 diabetes is associated with the risk of microvascular complications such as diabetic foot ulcers and diabetes nephropathy. Early detection of impaired microvascular circulation could be used to increase preventive action, before clinical complications appear. Vasomotion has been suggested to be the mechanism by which the microcirculation adjusts local tissue blood flow, maintaining local homeostasis. The aim of this study was to analyze vasomotion in healthy subjects and patients with type 2 diabetes with microalbuminuria (DMA) and no microalbuminuria (DNMA), respectively. We hypothesized that decreased vascular myogenic and endothelial activity would be observed in patients with type 2 diabetes as a sign of microvascular complication, particularly in signals related to perfusion in capillaries, venules and small arterioles.

Materials and methods: Speed-resolved microcirculatory perfusion (% red blood cells (RBC) × mm/s) divided into three speed regions: 0-1, 1-10 and above 10 mm/s, were assessed during baseline and after local heating of the foot with a new device integrating diffuse reflectance spectroscopy and laser Doppler flowmetry. Patients with type 2 diabetes (41 DMA defined as uACR ≥ 3.0 mg/mmol, and 34 DNMA) and 41 age-matched control subjects were included. For each speed region, blood flow variations related to respiration (0.15 - 0.6 Hz), myogenic activity in the vessel wall (0.05 - 0.15 Hz), sympathetic activity (0.02 - 0.05 Hz) and endothelial activity (0.008 - 0.02 Hz) was quantified using wavelet analysis. Speed regions below 1 mm/s are associated with capillary flow, 1-10 mm/s is foremost constituted from flow in venules and small arterioles, and above 10 mm/s are related to flow in larger arterioles and other larger vessels.

Results: At rest, blood flow variations associated with endothelial, myogenic and sympathetic activity was lower in DMA compared with controls in the 0-1 mm/s speed region (endo: 2.4 vs. 9.8 %RBC × mm/s, p = 0.002; myo: 7.6 vs. 14.6 %RBC × mm/s, p = 0.01; symp: 6.0 vs. 16.1 %RBC × mm/s, p = 0.001). After local heating, endothelial, myogenic and sympathetic activity was lower in DNMA compared with controls in the 0-1 mm/s speed region (endo: 5.8 vs. 13.2 %RBC × mm/s, p = 0.04; myo: 22.0 vs. 30.3 %RBC × mm/s, p = 0.01; symp: 10.8 vs. 20.3 %RBC × mm/s, p = 0.01).

Conclusion: Patients with type 2 diabetes show impaired microvascular vasomotion related to endothelial, myogenic and sympathetic activity compared to age-matched controls, specifically in low speed regions, both at rest and after local heating. Vasomotion analysis in combination with speed-resolved laser Doppler flowmetry seems to be a promising method for non-invasive observation of microvascular dysfunction.

Supported by: VINNOVA, NovaMedTech

Disclosure: F. Iredahl: None.

251

Macrophage phenotype in diabetic wound healing

I. Eleftheriadou1, A. Tentolouris1, I. Anastasiou1, D. Tsilingiris1, O. Kosta1, E. Tzeravini1, I. Pateras2, N. Tentolouris1;

11st Department of Propaedeutic Internal Medicine, Laiko General Hospital, Medical School, National & Kapodistrian University of Athens, Athens, 2Department of Histology-Embryology, Medical School, National & Kapodistrian University of Athens, Athens, Greece.

Background and aims: Diabetic foot ulcers (DFUs) are usually chronic wounds stalled in the inflammatory phase of the wound healing process. In the early inflammation phase pro-inflammatory (M1) macrophages clean the ulcer by phagocytosing bacteria and debris. As the inflammation resides macrophages undergo a transition to an anti-inflammatory and healing phenotype (M2 macrophages). Diabetic animal wound studies have shown delayed macrophage phenotype transition and increased M1/M2 macrophage ratio. The aim of our study was to examine the macrophage phenotype in the skin of patients with diabetes with and without DFUs.

Materials and methods: A total of 20 patients with diabetes (10 with chronic non-infected DFUs, 10 without DFUs) and 12 healthy controls were recruited. Forearm skin punch biopsies were obtained from all participants. In addition, punch biopsies from the borders of the ulcers were performed from patients with DFUs. CD64 (M1 macrophages) and CD163 (M2 macrophages) immunohistochemistry staining was performed in all biopsies.

Results: The number of CD64+ and CD163+ cells from the forearm biopsies differed significantly between the 3 groups of participants (p=0.001 and p=0.003, respectively); sub-analysis showed that patients with DFUs had significantly higher number of CD64+ cells [(5.8 (5.3, 6.4)] when compared with patients without DFUs [3.9 (3.1, 4.4)] (p=0.001) and healthy participants [3.4 (3.1, 4.5)] (p<0.001). Participants with DFUs and without DFUs had significantly higher number of CD163+ cells [6.5 (5.2, 7.5) and 7.0 (6.0, 7.6), respectively] when compared with healthy individuals [3.6 (2.7, 5.7)] (p=0.006 and p=0.002, respectively) in the forearm biopsies. The number of CD64+ and CD163+ cells did not differ between the forearm and foot of patients with DFUs.

Conclusion: There is increased inflammation in the skin of patients with DFUs when compared with patients with diabetes without DFUs and with healthy individuals. In the forearm and foot of individuals with DFUs similar macrophage phenotype was observed that is associated with a chronic pro-inflammatory state. This notion could suggest that increased inflammation in the skin of patients with diabetes either results in foot ulceration or impairs normal wound healing.

Disclosure: I. Eleftheriadou: None.

252

Additive effect of miRs-146a and 29a inhibition using in vitro and in vivo wound healing models of type 1 diabetes

M. Petkovic1, A.E. Sørensen1, E.C. Leal2, R.J. Willemoes1, H. Jenssen1, E.M. Carvalho2,3, L.T. Dalgaard1;

1Roskilde University, Roskilde, Denmark, 2University of Coimbra, Coimbra, Portugal, 3Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, USA.

Background and aims: Impaired wound healing among diabetic patients is a result of chronic inflammation, microvascular and macrovascular complications, leading to development of diabetic foot ulcers. The role of microRNAs miR-146a and miR-29a, both highly increased in diabetes, is not well characterized in wound healing. We aimed to evaluate the impact of miR-146a and miR-29a, on wound healing using an in vitro scratch essay followed by pathway analysis of proteomics data. In addition, the effect of topical dermal treatments of miR-146a and miR-29a inhibitors on wound healing kinetics and restoring the collagen structure during wound healing in diabetic mice was evaluated.

Materials and methods: Human keratinocyte cells (HaCaT) were cultured in high glucose DMEM medium. For scratch migration assays, HaCaT cells were transfected with a negative control (neg ctrl) or miR inhibitors targeting miR-146, miR-29a alone or in combination. Predicted target genes in skin were retrieved from TargetScan 7.1 and filtered using an mRNA expression array (E-GEOD-23006) and subsequently analyzed for Gene Ontology (GO) enrichment using DAVID. Diabetes was induced in C57BL/6 mice using low-dose streptozotocin injections for 5 consecutive days. Wound-healing kinetics were evaluated up to day 10. Collagen deposition was assessed by the Masson Goldner staining in mouse skin sections 10 days after full thickness wounding.

Results: MiR-146 inhibition (25pmol) caused slower scratch closure: 64.07+/- 8.87% remaining gap after 24hrs compared with miR-29 inhibition (25pmol) (43.57+/-10.86%) or the neg. ctrl (25pmol) (35.95+/- 9.08%) (p˂0.001). Combination of miRs-146a and -29a inhibitors (12.5pmol each) accelerated the scratch closure to 48.37+/- 13.07% remaining gap, 24h later relative to miR-146 inhibition (64.07+/- 8.87% remaining gap) (25pmol), p˂0.01. GO analysis indicated predicted target genes of miR-29a to be over-represented in the GO category 0030199~collagen fibril organization (p=0.045). MiR-146a and -29a inhibitors (2.5 nmol/day) accelerated the wound closure on day 8 (22.02+/-8.80) and day 9 (15.36+/-8.73) compared to negative controls (2.5nmol/day) (22.02+/-8.80) MiR-29a inhibition (2.5nmol/day) improved collagen deposition (63.06+/-10.37%), when compared to miR-146a inhibition (11.02+/-6.01%) and neg. ctrl (15.47+/-8.74%) (2.5 nmol/day), (p˂0.001).

Conclusion: Migration of HaCaT keratinocytes was the highest following the combined inhibition of miR-146a and miR-29a. Moreover, miRs-146a and -29a, dynamically regulated by wounding and differentially regulated under diabetic conditions improved collagen structure in an animal model of wound healing. These findings imply that manipulating the expression levels of miR-146a and miR-29a may improve healing outcome under diabetic conditions.

Supported by: EFSD

Disclosure: M. Petkovic: None.

OP 46 Challenges in delivering diabetes care: new solutions

253

HbA1c thresholds have substantial impact in screening procedures for those at risk of developing type 2 diabetes

R.S. Greiner1, A. Hill1, B.A. Knight1, T. McDonald1, B. Shields1, A.G. Jones1, L.R. Rodgers2;

1Exeter Clinical Research Facility, University of Exeter, Exeter, 2Institute of Health Research, University of Exeter, Exeter, UK.

Background and aims: We aimed to investigate the performance of screening procedures for identifying individuals at high risk of Type 2 diabetes (T2D) in identifying progression to T2D within 5 years. Screening is carried out to identify individuals for referral to a diabetes prevention intervention designed to reduce risk through weight management, diet and exercise change. Places are limited, so ensuring that they are given to those at the greatest risk of developing T2D is of paramount importance.

Materials and methods: The sample consisted of 3,469 participants from the Exeter 10,000 population cohort (non-diabetic at baseline). At baseline participants completed the Cambridge Risk Score (CRS) and Leicester Risk Score (CRS) and had their HbA1c measured. HbA1c results from routine clinical care in the following 5 years were recorded. Participants were considered to have developed T2D when their HbA1c rose above 47 mmol/mol (≥48 mmol/mol). Progression to T2D within 5 years was modelled using a flexible parametric survival model. NICE recommended cut offs of CRS ≥0.128, LRS ≥16, and HbA1c 42-47 mmol/mol were used to assess diabetes risk. We divided those at high risk in to categories of HbA1c 42-44 mmol/mol and 45-47 mmol/mol. Hazard ratios (HR) for risk of developing T2D within 5 years for these categories relative to those at low risk (HbA1c <42 mmol/mol) were calculated. We calculated the hazard ratio for those with a high HbA1c alongside a high risk score, compared to those with high HbA1c alone.

Results: The median (IQR) age of participants was 62 years (52, 68); BMI was 26.3 kg/m2 (23.8, 29.3); HbA1c was 39 mmol/mol (37, 41); and follow up was 51 months (27-60). 3.0% of participants progressed to T2D within 5 years (n=105). 21.9% of participants were classified as high risk for T2D (n=760) by HbA1c, and of those 10.9% progressed to T2D (n=83), compared to 0.8% of those classified as low risk (n=22). 17.0% had HbA1c 42-44 mmol/mol (n=588) and of those 6.0% developed T2D within 5 years (n=35). 5.0% of participants had HbA1c 45-47 mmol/l (n=172), and of those 27.9% developed T2D within 5 years (n=48). Those identified as high risk for T2D by HbA1c alone had 13.6 (95% CI: 8.5, 21.7) times the chance of developing T2D within 5 years as those with low risk. Within the high risk range there were different degrees of increase in risk; those with HbA1c 45-47 mmol/mol had 39.8 (95% CI: 24.0, 66.0) times higher risk compared to <42 mmol/l, whereas for those in the 42-44 mmol/mol range had a risk only 7.1 (95% CI: 4.2, 12.2) times higher. Among those with high HbA1c, both risk scores helped further separate those at greatest risk (HR=2.8 (1.6, 4.8) for LRS≥16 vs <16; HR=2.6 (1.5, 4.4) for CRS≥0.128 vs <0.128).

Conclusion: Individuals at high risk for T2D had a greater risk of developing T2D within 5 years compared to those identified as low risk. However, individuals with HbA1c 45-47 mmol/mol had higher risk of developing T2D within 5 years compared to those with HbA1c 42-44 mmol/mol, despite both being considered as high risk. Raising the threshold for identification of high risk individuals may help to ensure that places are allocated to those with the greatest need. The addition of a risk score, calculated using readily available data such as age and BMI, helped to identify those at greater risk, and the CRS and LRS performed similarly in this task.

Disclosure: R.S. Greiner: None.

254

Effects of patient-initiated visits in the diabetes outpatient clinic: 2 year RCT (DIATAST - the DIAbetes patient TAkes reSponsibiliTy)

N. Drojdahl Ryg1,2, J. Gram1,3, M. Haghighi1, C.B. Juhl1,2;

1Medical Department/Endocrinology, Hospital South West Jutland, Esbjerg, 2STENO Diabetes Centre Odense, Odense, 3Department of Regional Health Research- Hospital South West Jutland, Odense, Denmark.

Background and aims: Type 1 diabetes (T1DM) patients usually attend the outpatient-clinic with regular intervals decided by the health care providers. No previous studies have assessed possible consequences of exclusively patient-initiated visits in this patient population. The aim of the study was to examine the effects of patient-initiated visits in the diabetes outpatient clinic on 1) Patient reported experience measures (PREM), 2) Clinical diabetes variables and 3) Number of contacts to the outpatient clinics.

Materials and methods: Adults with T1DM for more than 6 months, who were internet users, were included. Patients with unstable diabetic complications, large increase in HbA1c within the past 6 months, or found non-eligible because of frailty were excluded. After informed consent and collection of baseline data, patients were randomized (1:1) to two years intervention with 1) Patient-initiated visits and push reminders every 3rd month (INT) or 2) Usual care with pre-scheduled visits (CON). The primary outcome was PREM evaluated by a self-designed questionnaire (5-point Likert scale). Questions were focused on 1) accessibility of the outpatient clinic, 2) the benefit of the consultation and, 3) overall patient satisfaction with the use of the outpatient clinic. Secondary outcomes included Hba1c and other clinical diabetes variables and use of resources in the outpatient clinic. Data were analyzed as intention to treat. Likert data were analyzed using linear logistic regression, continuous data by mixed model multilevel regression, resource use by Poisson regression, and dichotomous data by χ2-test. All data were corrected for age, sex, diabetes duration (+/- 5 years), and insulin administration (pump/injection).

Results: Of 849 patients screened, 596 were found eligible, and 357 accepted inclusion (INT: 178/CON: 179). After 2 years, more patients in the intervention group reported to be able to get an appointment when needed (p< 0.001). The INT group experienced more benefit of the consultations within group (p<0.04) and compared with CON (p=0.06). Similarly the INT group reported having fewer unnecessary visits (p<0.005) and being more involved in the content of the consultations compared to CON (p<0.01). Overall patient satisfaction was high in both groups at baseline and at 2y with no change from baseline to 2y between groups (p=0.17). The number of visits in the outpatient clinic during the 2 year study period were significantly lower in the INT-group (median 4 [IQR 3;6]) compared to CON (6 [5;8]) (p<0.001) covering visits both at physicians, nurses, and dieticians. Concurrently, there the was an increase in the number of telephone contacts (INT: 2 [1;4] /CON: 1 [0;3], p<0.001). Mean HbA1c (mmol/mol) was unchanged within and between groups (INT: 59.7(bl)/60.5(2y); CON: 59.7/59.3), p>0.5. Blood pressure, LDL-cholesterol, and albumin-creatinine-ratio likewise remained equal between groups.

Conclusion: The on-demand structure resulted in high and maintained or improved patient reported experience measures and no decline of in the quality of clinical diabetes care. The total use of outpatient clinic resources was reduced. Implementation of such a concept will potentially save resources that can be relocated to patient groups with special needs.

Clinical Trial Registration Number: NCT03083899

Disclosure: N. Drojdahl Ryg: None.

255

Monitoring perception of risk and disruption to medical supplies in people with type 1 diabetes during the covid-19 pandemic

S.N. Scott1, F.Y. Fontana2, T. Zueger1, M. Laimer1, C. Stettler1;

1Department of Diabetes, Endocrinology, Nutritional Medicine and Metabolism, University of Bern, Bern, Switzerland, 2Team Novo Nordisk Professional Cycling Team, Atlanta, USA.

Background and aims: The Centers for Disease Control and Prevention and World Health Organization have determined that Coronavirus Disease-19 (COVID-19) is a serious public health threat, stating that people with chronic medical conditions, including diabetes, are at a higher risk of experiencing complications. However, due to the rapid onset of the pandemic and changing situation, there is currently no data on the risk perception of people with type 1 diabetes (T1D) and whether they are experiencing disruption to healthcare and medical supply. We aimed to 1) gather real-time information on the challenges and perception of risks to people living with T1D during the COVID-19 pandemic and 2) develop a means to display the data in a clear and meaningful way.

Materials and methods: We designed an anonymous questionnaire using an open-access web-based platform (SurveyMonkey.com), which was widely distributed via social media. The survey covered questions relating to coronavirus infections, symptoms, incidence of hospitalization, risk perception and whether respondents have experienced interruption to medical supplies. Data were then analyzed descriptively and the mean population responses were summarized in a live electronic library using Microsoft Power BI (Figure 1).

Results: In the first 7 days of the study, there were 3361 survey responses from individuals in 82 countries (34% from Europe, 43% from America and the rest from Africa, Asia and Oceania). The majority of responses were collected from UK (15%) and USA (36%). 33% of respondents were men, 67% were women. 55% of respondents were in the 25-44 years age category and the average HbA1c of the entire cohort was 7.1±1.2%. The majority (>80%) perceived themselves to be in a good-to-excellent health condition. However, 80% perceived themselves to be exposed to a higher risk to complications if they contract COVID-19, compared to the average person without diabetes. The average risk perception was 88% in USA, 72% in Europe, 86% in Asia, 85% in Australia and 80% in Africa. 60% reported that the pandemic had affected their healthcare access. Insulin, continuous glucose monitors and fast-acting carbohydrates were the diabetes-relates supplies most difficult to access due to the COVID-19 pandemic.

Conclusion: Based on the present data from this large-scale, worldwide survey, we demonstrate an increase in risk perception of people with T1D related to COVID-19. Secondly, access to relevant healthcare services and/or medical supplies appears to have been significantly impaired for people with T1D so far during the pandemic. These issues and concerns need to be taken into account in the care of these patients. Interactive survey approaches such as this may help to address these challenges.

figurebz

Disclosure: S.N. Scott: None.

256

AMD Annals as a model for improving the quality of diabetes care in Italy

M. Rossi1, V. Manicardi2, G. Clemente3, S. De Cosmo4, R. Manti5, A. Rocca6, P. Pisanu7, A. Nicolucci1, A. Aglialoro8, D. Mannino9, P. Di Bartolo10, on behalf of AMD Annals Study Group;

1Center for Outcomes Research and Clinical Epidemiology, Pescara, 2AMD Annals Study Group Coordinator, Rome, 3CNR, Istituto di Ricerche sulla Popolazione e le Politiche Sociali (IRPPS), Fisciano (SA), 4Scientific Institute, S. Giovanni Rotondo (FG), 5ASL Turin 5, Turin, 6ASST Nord H. Bassini, Cinisello Balsamo (MI), 7Azienda Ospedaliero-Universitaria, Cagliari, 8ASL3 Genovese, Genova, 9AMD Past President, A.O. Bianchi Melacrino-Morelli, Reggio Calabria, 10AMD President, AUSL Romagna, Ravenna, Italy.

Background and aims: Diabetes management is complex and extends beyond glycemic control. A huge gap between ideal care (guidelines) and actual care exists. Standardized performance measures are considered a key strategy to assess quality of care in different settings. In Italy, an initiative of continuous monitoring and quality improvement of diabetes care (AMD Annals) is in place since 2004, promoted by the scientific society of diabetologists (AMD). Changes in AMD qua