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

Stockholm, Sweden, 19 - 23 September 2022

Abstracts

Index of Oral Presentations

OP 01 Diet: from plants to cans

OP 02 SGLT2 inhibitors: promiscuous pleiotropy

OP 03 Risk for CVD and CKD

OP 04 Walking with diabetes

OP 05 How to become a fat cell

OP 06 Intracellular regulation of insulin release

OP 07 Finding a phenotype for diabetic kidney disease

OP 08 Cardiovascular disease mechanisms: something new on the table?

OP 09 Fighting diabetes with tubes, scanners, and catheters

OP 10 Beta cells: protecting what is precious

OP 11 Adipose tissue profiling and cardio-metabolic risk

OP 12 GWAS and more

OP 13 Beyond type 1 and type 2

OP 14 Exercising your tissues in shape

OP 15 Preserving kidney function

OP 16 Flames and scars in the liver

OP 17 Toying with monitoring: from Present Continuous to Future Perfect

OP 18 Cross-talk communication in the pancreas

OP 19 GLP1 agonists: from here to eternity

OP 20 NAFLD and treatment

OP 21 Retinopathy future vision

OP 22 Giving birth with diabetes

OP 23 Are we too slow to outlaw the low?

OP 24 How to burn energy

OP 25 Lipid in and out of the liver

OP 26 The dark side of diabetes

OP 27 Improving your insulin sensitivity: lessons from human studies

OP 28 Desirable diets

OP 29 Saving sweet souls

OP 30 Novel ways of beta cell replacement

OP 31 Diabetes: size matters

OP 32 Pain or no pain?

OP 33 Therapy outside the box

OP 34 Insulin signalling, novelties from the petri dish!

OP 35 Different pathways involved in killing the beta cell

OP 36 Central aspects of diabetes

OP 37 Insulin deficiencies and cardiovascular disease

OP 38 It is always a D-D-Day: diabetes, digital, device

OP 39 Too little sugar is also bad: understanding hypoglycaemia

OP 40 When the clock ticks

OP 41 Viruses and diabetes: more than COVID-19

OP 42 Moving towards the beta cell plasma membrane

OP 43 Microvascular cocktail

OP 44 Grading insulin therapy: simple, simpler, the safest

OP 45 Newer agents, better outcomes

OP 46 Profiling human diabetes risk

OP 47 Novel mechanistic insights in peripheral insulin sensitivity

OP 48 How we loose our beta cells

Index of Short Oral Discussions

SO 01 Epigenetics rules

SO 02 Pregnancy and diabetes

SO 03 Food, drinks and spices

SO 04 Starting with autoimmunity

SO 05 Genetics of type 2 diabetes

SO 06 Sometimes one gene is enough

SO 07 SARS-CoV2 and other viruses

SO 08 Benefits of a healthy lifestyle

SO 09 Type 1 diabetes: from molecules to treatment

SO 10 Diversity in diabetes

SO 11 Novel biomarkers and risk factors

SO 12 Different facets of type 2 diabetes treatment

SO 13 It's getting complicated

SO 14 COVID-19 around the globe

SO 15 Calcium signalling in the islet: we are still learning

SO 16 A new niche to replace beta cells

SO 17 Intracellular signalling balancing beta cell survival and death

SO 18 Stressing the beta cell into dysfunction

SO 19 How complicated is type 1 diabetes?

SO 20 The other diabetes and islet function

SO 21 The fat burning the islets

SO 22 Exercise and diabetes: much to learn!

SO 23 Gestational diabetes and pregnancy

SO 24 Life with diabetes from conception to delivery

SO 25 Insulin sensitivity: lessons from cellular and animal models

SO 26 Novel markers and omics signatures

SO 27 If you cannot measure it, you cannot improve it: novel methods in diabetes research

SO 28 Understanding insulin sensitivity: lessons from the clinic

SO 29 Glucose homeostasis regulation beyond insulin

SO 30 Non-classical regulators of metabolism

SO 31 Gut feelings are good

SO 32 Latest drug avenues to treatment

SO 33 Insulin in action

SO 34 The hepato-skeletal impact on metabolic control

SO 35 Metabolic inflexibility and complications in humans

SO 36 Modelling obesity and type 2 diabetes

SO 37 Dietary and nutritional interventions

SO 38 SGLT2 inhibitors and renal outcomes

SO 39 Newer agents - cardiovascular outcomes

SO 40 Incretins: impact on BMI

SO 41 Incretins: basic science

SO 42 Clinical epidemiology and pharmacotherapy

SO 43 Glucose lowering agents

SO 44 Lessons from trials

SO 45 Beta cell function and glucose control

SO 46 Incretins everywhere

SO 47 Treatments, molecules and outcomes: a smorgasbord

SO 48 Hypoglycaemia: hip hip hooray yet to come

SO 49 Is newer (insulin) always better?

SO 50 Is longer better? Looking for different basal insulin approaches

SO 51 Even "old dogs" can learn new tricks

SO 52 Money isn't everything?

SO 53 Pumping, looping, freeing

SO 54 Making sense out of sensors and data

SO 55 From low to high and back: the many faces of insulin therapy

SO 56 Diversity of life with diabetes

SO 57 Type 1 diabetes: still the challenge number one

SO 58 Autonomic rhythm

SO 59 Getting a grip on nerves

SO 60 Preventing microvascular complications

SO 61 Saving the feet

SO 62 Brain, nerve, and heart interaction

SO 63 Diversity of the diabetic kidney

SO 64 New treatment avenues for the diabetic kidney

SO 65 Translating signals in the diabetic kidney

SO 66 Mechanisms of diabetic kidney disease

SO 67 Flames and scars in NAFLD: pathogenesis and therapy

SO 68 From brain circulation to cognitive dysfunction

SO 69 Type 1 diabetes: new findings and complications

SO 70 Circulating markers of cardiovascular risk

SO 71 Prevention and treatment of cardiovascular complications

SO 72 Diabetes dysmetabolism dialogues with the cardiovascular component

SO 73 Diabetes in the vessels

SO 74 Weighing risks of cardiovascular complications

SO 75 Emerging comorbidities in diabetes: clinical associations and mechanisms

SO 76 Cancer and type 2 diabetes: interconnections and mortality

SO 77 Disclosing fatty liver disease mechanisms

SO 78 Screening tools, lipids and novel biomarkers

SO 79 Focus on the heart and beyond

OP 01 Diet: from plants to cans

1

Diet and all-cause mortality in individuals with type 2 diabetes: a systematic review and meta-analysis of prospective studies

J. Barbaresko1, A. Lang1, E. Szczerba1,2, C. Baechle1,2, L. Schwingshackl3, M. Neuenschwander1,2, S. Schlesinger1,2;

1Institute for Biometrics and Epidemiology, German Diabetes Center, Düsseldorf, 2German Center for Diabetes Research (DZD), Munich-Neuherberg, 3Institute for Evidence in Medicine, University of Freiburg, Freiburg, Germany.

Background and aims: Type 2 diabetes (T2D) is a major health concern associated with several comorbidities and mortality. Dietary factors may influence the progression of diabetes; however, high-quality systematic reviews are lacking. Therefore, the aim was to systematically summarise and evaluate the evidence on dietary factors and the risk of all-cause mortality in individuals with T2D from observational prospective studies.

Materials and methods: A systematic literature search was conducted in PubMed and Web of Science up to September 2021 to identify prospective observational studies investigating any dietary factor (dietary patterns, food groups, macro- and micronutrients, and secondary plant compounds) in association with all-cause mortality in individuals with T2D. We conducted pairwise (high vs. low intake) and dose-response meta-analyses to calculate summary risk ratios (SRR) with corresponding 95% confidence intervals (CI) using random effects models. The inconsistency between the study results was assessed using I2. The certainty of evidence of the associations was evaluated by applying a validated tool.

Results: In total, we identified 97 studies and performed 38 meta-analyses. Moderate certainty of evidence was found for decreased all-cause mortality with higher intakes of fish (SRR per serving/week: 0.95; 95% CI: 0.92, 0.99; I2=0%; n=6 studies), whole grain (SRR per 20 g/d: 0.84; 95% CI: 0.71, 0.99; I2=0%; n=2), fibre (SRR per 5 g/d: 0.86; 95% CI: 0.81, 0.91; I2=0%; n=3) and n-3 polyunsaturated fatty acids (SRR per 0.1 g/d: 0.87; 95% CI: 0.82, 0.92; I2=0%; n=2). There was low certainty of evidence for an inverse association of vegetable consumption (SRR per 100 g/d: 0.88; 95% CI: 0.82, 0.94; I2=0%; n=2), and plant protein intake (SRR per 10 g/d: 0.91; 95% CI: 0.87, 0.96; I2=42%; n=3), as well as positive associations of egg consumption (SRR per 10 g/d: 1.05; 95% CI: 1.03, 1.08; I2=56%; n=7), and cholesterol intake (SRR per 300 mg/d: 1.19; 95% CI: 1.13, 1.26; I2=0%; n=2). For other dietary factors such as dietary patterns, other food groups, macro- and micronutrients, evidence was limited.

Conclusion: This meta-analysis showed that intake of fish, whole grain, fibre and n-3 polyunsaturated fatty acids may be inversely associated with all-cause mortality in individuals with T2D. There is limited evidence for other dietary factors and all-cause mortality in individuals with T2D and thus, more research is needed.

Disclosure: J. Barbaresko: None.

2

Appropriate consumption of different animal-based foods to reduce type 2 diabetes risk: an umbrella review of meta-analyses of prospective studies

A. Giosuè, I. Calabrese, G. Riccardi, O. Vaccaro, M. Vitale;

Clinical Medicine and Surgery, "Federico II" University of Naples, Naples, Italy.

Background and aims: Dietary recommendations for the prevention of type 2 diabetes (T2D) clearly indicate the most appropriate choices for plant-based foods; as for foods of animal origin, a limited consumption of all items is generally recommended. However, not all animal protein sources are equal; moreover, they are largely used worldwide. Therefore, we have reviewed data on the relationship between the consumption of various foods of animal origin and the incidence of T2D to support dietary recommendations for T2D prevention with updated and reliable scientific evidence on the appropriate choices for animal-based foods.

Materials and methods: The study is an umbrella review of dose-response meta-analyses of prospective cohort studies. A systematic search of the literature was conducted in PubMed, Web of Science, Scopus and Embase according to PRISMA guidelines. The methodological quality of each meta-analysis was evaluated trough AMSTAR (A Measurement Tool to Assess Systematic Reviews). For each food group, we meta-analyzed the risk ratios (RR) for T2D incidence reported in the primary studies included in the available meta-analyses. The quality of evidence was evaluated with a modified version of NutriGrade.

Results: 13 meta-analyses met the criteria for inclusion in the review with 175 summary RR on consumption of total meat (n=13), red meat (n=21), white meat (n=8), processed meats (n=24), fish (n=12), total dairy (n=21), full-fat dairy (n=14), low-fat dairy (n=15), milk (n=11), cheese (n=10), yogurt (n=10) and eggs (n=16) in relation to T2D incidence. There was a substantial increase in T2D risk with the consumption of 100 g/day of total meat (RR 1.20, 95% CI 1.13-1.27) or red meat (RR 1.22, 95% CI 1.14-1.30) or 50/day of processed meats (RR 1.30, 95% CI 1.22-1.39), with a moderate quality of evidence; also 50 g/day of white meat showed a positive relationship with T2D risk (RR 1.04, 95% CI 1.00-1.08). As for dairy foods, we found an inverse association for T2D incidence with the intake of 200 g/day of total dairy (RR 0.95, 95% CI 0.92-0.98), low-fat dairy (RR 0.97, 95% CI 0.93-1.00) or milk (RR 0.90, 95% CI 0.83-0.98), as well as 100 g/day of yogurt (RR 0.94, 95% CI 0.90-0.98); conversely, a neutral relationship emerged for 200 g/day of full-fat dairy (RR 0.98, 95% CI 0.93-1.03) or 30 g/day of cheese (RR 0.97, 95% CI 0.91-1.04), with a quality of evidence scored between moderate and low. Finally, the consumption of 100 g/day of fish and 1 egg/day showed a neutral association with T2D risk (RR 1.04, 95% CI 0.99-1.09 and 1.07, 95% CI 0.99-1.15, respectively), with low quality of evidence.

Conclusion: The scientific evidence we have extensively reviewed shows that the habitual consumption of dairy foods in moderate amounts - especially low-fat types, milk and yogurt - could be appropriate for the optimization of T2D prevention. Within this context, moderate amounts of fish and eggs could represent suitable substitutes for red and processed meats in most eating occasions.

figure a

Clinical Trial Registration Number: PROSPERO CRD42022306145

Disclosure: A. Giosuè: None.

3

Ultra-processed food consumption and risk of type 2 diabetes: results from three prospective cohort studies in the US

Z. Chen1, N. Khandpur1, C. Monteiro2, S. Rossato2, T. Fung1, J.E. Manson1, W. Willett1, E.B. Rimm1, F.B. Hu1, Q. Sun1, J.-P. Drouin-Chartier3;

1Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, USA, 2Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, Brazil, 3Department of Nutrition, Institut sur la Nutrition et les Aliments Fonctionnels (INAF), Faculté de Pharmacie, Université Laval, Québec, Canada.

Background and aims: Prospective evidence on the association between ultra-processed food (UPF) consumption and type 2 diabetes (T2D) risk remains limited. We aimed to prospectively examine associations between UPF intake and T2D in US men and women.

Materials and methods: We prospectively followed 62,583 women in the Nurses’ Health Study (NHS, 1984-2014), 88,633 women in the Nurses’ Health Study II (NHSII, 1991-2017), and 38,837 men in the Health Professionals Follow-up Study (HPFS, 1986-2016). Diet was assessed using validated food frequency questionnaires every 2-4 years. UPF were categorized according to the Nova classification. Associations with T2D were assessed using Cox proportional hazards models with adjustments for demographics, dietary and lifestyle factors, and medical history.

Results: During 4,784,680 person-years of follow-up, we documented 17,432 T2D cases. In multivariable-adjusted analyses, higher UPF intake was associated with a higher risk of T2D: the pooled hazard ratio (HR) comparing extreme quintiles of intake in servings of UPF per day was 1.29 (95% confidence interval (CI): 1.21, 1.37; Ptrend<0.0001). The association remained significant after further adjustment for overall diet quality, assessed using the Alternative Healthy Eating Index (pooled HR comparing extreme quintiles: 1.21, 95% CI: 1.13, 1.28; Ptrend<0.0001). The results were consistent across subgroups in analyses stratified by age, sex, BMI, diet quality and physical activity, as well as when symptomatic diabetes at diagnosis was used at the outcome.

Conclusion: UPF consumption is associated with a higher risk of T2D, independent of overall diet quality. These findings provide further support for the current recommendations of limiting UPF consumption as part of a healthy diet for the prevention of type 2 diabetes.

Disclosure: Z. Chen: None.

4

The association between plant-based diet indices and obesity and metabolic diseases in Chinese adults: longitudinal analyses from the China Health and Nutrition survey

B. Chen1, J. Zeng1, M. Qin2, W. Xu1, Z. Zhang3, X. Li4, S. Xu1;

1Evidence-Based Medicine Centre, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 2Department of Traditional Chinese Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 3Evidence-Based Medicine Centre, Daxing Hospital, Xi’an, Shanxi, 4College of Medicine, Wuhan University of Science and Technology, Wuhan, Hubei, China.

Background and aims: A wide range of health benefits are associated with consuming a diet high in plant-based foods. Diet quality can be accurately assessed using plant-based diet indices, however there is inadequate evidence that plant-based diet indices are linked to obesity, hypertension, and type 2 diabetes (T2D), especially in Chinese cultures who have traditionally consumed plant-rich foods.

Materials and methods: The data came from the China Nutrition and Health Survey. Overall, 11,580 adult participants were enrolled between 2004 and 2006 and followed up until 2009 or 2015 (follow-up rate: 73.4%). Dietary intake was assessed across three 24-hour recalls, and two plant-based dietary indices (overall plant-based diet indice (PDI) and healthy plant-based diet indice (hPDI)) were calculated using China Food Composition Code and categorized into quintiles. The study's endpoints were overweight/obesity, hypertension, and T2D. The Hazard ratio (HR) and dose-response relationship were assessed using the Cox proportional risk model and restricted cubic splines.

Results: During the median follow-up period of more than ten years, 1270 (33.4%), 1509 (31.6%), and 720 (11.5%) participants developed overweight / obesity, hypertension, and T2D, respectively. The higher PDI score was linked with a reduced risk of overweight/obesity [HR: 0.71 (95% CI: 0.55-0.93), P-trend <0.001], hypertension [HR: 0.63 (95% CI: 0.51-0.79), P-trend <0.001], and T2D [HR: 0.79 (95% CI: 0.72-0.87), P-trend <0.001]. The hPDI score was inversely associated with overweight/obesity [HR: 0.79 (95% CI: 0.62- 0.98), P-trend = 0.02] and T2D [HR: 0.84 (95% CI: 0.75-0.93), P-trend = 0.001]. In the aged <55-year-old group, subgroup analysis indicated a significant negative association between PDI/hPDI and overweight/obesity, hypertension, and T2D.

Conclusion: The PDI and hPDI scores were very similar in application in Chinese populations, and our findings highlight that adherence to overall plant-based diet index helps to reduce the risk of T2D, obesity, and hypertension in Chinese adults who habitually consume plant-based foods, especially for those aged <55 year. Further understanding of how plant-based diet quality is associated with chronic disease will be needed in the future, which will help develop dietary strategies to prevent diabetes, hypertension, and related chronic diseases.

Supported by: The study was partly supported by the Young Talents Project of Hubei Provincial Health Commission, C

Disclosure: B. Chen: None.

5

Longitudinal serum branched-chain amino acids, lifestyle intervention and the risk of type 2 diabetes in the Finnish Diabetes Prevention study

J. Kivelä1, J. Meinilä2, M. Uusitupa3, J. Tuomilehto1,4, J. Lindström1;

1Department of Public Health and Welfare, Finnish Institute for Health and Welfare, Helsinki, Finland, 2Department of Food and Nutrition, University of Helsinki, Helsinki, Finland, 3Public Health and Welfare, University of Eastern Finland, Kuopio, Finland, 4Saudi Diabetes Research Group, King Abdulaziz University, Jeddah, Saudi Arabia.

Background and aims: Circulating branched-chain amino acids (BCAA) are associated with the risk of type 2 diabetes (T2D). We examined to what extent lifestyle intervention aiming to prevention of T2D interacts with this association and how BCAA concentrations change during the intervention.

Materials and methods: We comprised trajectory clusters by k-means clustering of serum fasting BCAA analysed annually during the four-year intervention by sandwich ELISA. We investigated whether the baseline BCAA, BCAA trajectories and BCAA change trajectories predict T2D in a median 11-year follow-up and whether BCAA predicts T2D differently in the intervention (n=198) and control group (n=196) participants of the Finnish Diabetes Prevention Study.

Results: Elevated baseline BCAA predicted the incidence of T2D in the entire study cohort (HR 1.04; 95% CI 1.01, 1.06) and control group (HR 1.06; 95% CI 1.03, 1.09), but not in the intervention group. BCAA concentration decreased during the first year in the whole cohort (-14.9 μmol/l [SD 58.5], p<0.001), with no significant difference between the intervention and control groups. We identified five BCAA trajectory clusters and five trajectory clusters for the change in BCAA. Trajectories with high mean BCAA levels were associated with an increased hazard ratio for T2D compared to a trajectory with low BCAA levels (trajectory with highest vs lowest mean BCAA, HR 3.99; 95% CI 1.46, 10.93). A trajectory with increasing BCAA levels had a higher hazard ratio for T2D compared with a decreasing trajectory in the intervention group only (HR 25.39; 95% CI 2.83, 227.62).

Conclusion: Lifestyle intervention modified the association of the baseline BCAA concentration and BCAA trajectories with the incidence of T2D. Our study adds to the accumulating evidence on the mechanisms behind the effect of lifestyle changes on the risk of T2D.

Clinical Trial Registration Number: NCT00518167

Supported by: Päivikki and Sakari Sohlberg f., Yrjö Jahnsson f., Juho Vainio f., the Academy of Finland

Disclosure: J. Kivelä: None.

6

Fasting ketone bodies and incident type 2 diabetes in the general population

T. Szili-Torok1, M.H. de Borst1, E. Garcia2, R.T. Gansevoort1, R.P.F. Dullaart1, M.A. Connelly2, S.J.L. Bakker1, U.J.F. Tietge3;

1Internal Medicine, University Medical Center Groningen (UMCG), Groningen, Netherlands, 2Laboratory Corporation of America Holdings (Labcorp), Morrisville, USA, 3Division of Clinical Chemistry, Karolinska Institutet, Stockholm, Sweden.

Background and aims: With a rising incidence and prevalence of type 2 diabetes, prevention strategies including identification of prospective biomarkers become increasingly relevant. Ketone bodies recently received a renewed interest in this respect; however, data on a relationship between these metabolites and diabetes risk are scarce. Therefore, we investigated in the present prospective study the association between fasting ketone bodies and type 2 diabetes incidence in the general population.

Materials and methods: This study from the PREVEND cohort included 3786 participants from the general population initially free of diabetes. Baseline fasting ketone body concentrations were measured by nuclear magnetic resonance spectroscopy.

Results: 276 participants (7.3%) developed type 2 diabetes during a median [IQR] follow-up of 7.3 [5.6-7.7] years. In Kaplan-Meier analysis sex-stratified ketone body levels were strongly positively associated with incident type 2 diabetes (log rank test, p<0.001), which was confirmed in Cox regression analyses adjusted for several relevant confounders including age, sex, BMI, diastolic and systolic blood pressure, hsCRP, HOMA-IR, total cholesterol, HDL cholesterol, triglycerides, serum creatinine, eGFR and urinary albumin concentrations (aHR per 1 SD increase [95 CI], 11.84 [5.55, 25.25], p<0.001). There was no significant interaction by sex. Further, individually 3-beta-hydroxybutyrate (13.27 [6.18, 28.53], p<0.001) and acetoacetate/acetone (3.87 [2.1, 7.13], p<0.001) were associated with incident type 2 diabetes. In sensitivity analyses including only metabolic syndrome-free individuals the conclusions did not change (9.18 [2.99, 28.22], p<0.001). The addition of ketone body levels to the Framingham diabetes risk score has resulted in an improved model fit (p<0.001).

Conclusion: Fasting plasma ketone body levels are strongly associated with incident type 2 diabetes in the general population independent of several other recognized risk factors. These results may have important implications for diabetes prevention including dietary strategies.

Clinical Trial Registration Number: MEC96/01/022

Supported by: This work was supported by the Center for Innovative Medicine (CIMED, FoUI-963234, to UJFT).

Disclosure: T. Szili-Torok: None.

OP 02 SGLT2 inhibitors: promiscuous pleiotropy

7

Effect of SGLT2 inhibitor dapagliflozin on skeletal muscle fatty acid metabolism in patients with type 2 diabetes

A. Gemmink1, Y.J.M. op den Kamp1, M. de Ligt1, B. Dautzenberg1, R. Esterline2, J. Hoeks1, V.B. Schrauwen-Hinderling1,3, S. Kersten4, B. Havekes5, T.R. Koves6, D.M. Muoio6, M.K.C. Hesselink1, J. Oscarsson7, 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, 4Human Nutrition and Health, Wageningen University, Wageningen, Netherlands, 5Internal Medicine, Maastricht University Medical Center, Maastricht, Netherlands, 6Medicine, Duke University, Durham, USA, 7BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.

Background and aims: SGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on whole-body energy and substrate metabolism, which may be facilitated by altered muscle metabolism. This may be the consequence of the adaptive response to the loss of about 50-100g glucose per day in the urine, which can be regarded as a form of mild calorie restriction. Here, we investigated the effects of 5 weeks of dapagliflozin (10 mg orally once daily) treatment on skeletal muscle fat metabolism in type 2 diabetes patients.

Materials and methods: Twenty-six type 2 diabetes patients were randomized to a 5-week double-blind, cross-over study with 6-8 weeks of wash-out. 31P- and 1H-Magnetic resonance spectroscopy was used to determine intramyocellular lipid content (IMCL) and phosphocreatine (PCr) recovery rate. Muscle biopsies were analyzed for lipid droplet (LD) morphology, mitochondrial network integrity and mitochondrial-LD interaction with confocal microscopy. Furthermore, biopsies were analyzed for levels of acylcarnitines, amino acids and Krebs cycle intermediates, and gene expression levels of CPT1A and CPT1B. Results are presented as Least Squares Means (95% CI).

Results: IMCL content increased after dapagliflozin treatment (0.27 (0.21-0.34) vs. 0.33 (0.25-0.40)%, p<0.05) due to larger (0.25 (0.19-0.31) vs. 0.28 (0.20-0.36) μm2, p<0.05) and more LDs (0.015 (0.009-0.021) vs. 0.0018 (0.012-0.018) μm-2, p=0.09). Dapagliflozin increased levels of several long-chain acylcarnitine species, while acetylcarnitine levels (154.63 (131.24-178.03) vs. 114.56 (91.16-137.95) pmoles*mg tissue-1, p<0.001) were decreased. Dapagliflozin treatment reduced levels of several amino acids and Krebs cycle intermediates in skeletal muscle. PCr recovery rate (23.1 (20.7-25.5) vs. 23.1 (20.7-25.5) s, p=0.88), mitochondrial network integrity (1.59 (1.31-1.87) vs. 1.73 (1.33-2.12), p=0.44), and mitochondrial-LD interaction (13.45 (10.41-16.49) vs. 12.51 (9.70-15.32)%, p=0.20) were unaffected by dapagliflozin. CPT1A (1.33 fold, p<0.001) and CPT1B (1.13 fold, p<0.05) gene expression increased upon dapagliflozin treatment.

Conclusion: The increase in IMCL levels and changes in LD morphology mainly resemble changes induced by fasting. Changes in long-chain acylcarnitine and acetylcarnitine levels suggest enhanced fatty acid metabolism in skeletal muscle. Reduced amino acid levels and Krebs cycle intermediates suggest enhanced amino acid utilization for gluconeogenesis. Taken together, these findings indicate that dapagliflozin induces a change in skeletal muscle substrate metabolism favoring fatty acid oxidation and a reduced glycolytic flux without changes in mitochondrial function and mitochondrial-LD interaction.

Clinical Trial Registration Number: NCT03338855

Supported by: AstraZeneca

Disclosure: A. Gemmink: None.

8

The effect of SGLT2 inhibitor dapagliflozin on substrate metabolism in humans with prediabetes

A. Veelen1, C. Andriessen1, Y. Op den Kamp1, E. Erazo Tapia1, M. de Ligt1, J. Mevenkamp2, J. Jörgensen1, E. Moonen-Kornips1, G. Schaart1, B. Havekes3, J. Oscarsson4, V. Schrauwen-Hinderling2,1, E. Phielix1, P. Schrauwen1;

1Nutrition and Movement Sciences, Maastricht University, Maastricht, Netherlands, 2Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, Netherlands, 3Department of Internal Medicine, Maastricht University Medical Center, Maastricht, Netherlands, 4BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.

Background and aims: Metabolic flexibility is defined as the capacity to switch from fat oxidation in the overnight fasted state to glucose oxidation in the postprandial state. We recently showed that individuals with prediabetes have impaired 24-hour and nocturnal fat oxidation. Inhibition of sodium-glucose cotransporter 2 (SGLT2) results in glucosuria, causing an energy deficit, which could trigger a more fasted condition and reliance on hepatic glycogen. The objective of the current study was to determine whether dapagliflozin, a SGLT2 inhibitor, could elicit a more pronounced 24-hour and nocturnal fat oxidation, improve mitochondrial function and lead to enhanced overnight glycogen use in individuals with prediabetes.

Materials and methods: Fourteen individuals with prediabetes (BMI 30.3 ± 2.1kg/m2; age 66.3 ± 6.2 years) underwent 2-weeks of treatment with dapagliflozin (10mg/day) or placebo in a randomized, placebo-controlled, cross-over design. Outcome parameters include 24-hour respiratory exchange ratio (RER) and substrate oxidation measured by whole-room indirect calorimetry. Twenty-four-hour blood samples were collected to determine levels of several metabolites. Hepatic glycogen and hepatic lipid content and composition were measured in the morning by MRS, and ex vivo skeletal muscle mitochondrial oxidative capacity was measured by high-resolution respirometry.

Results: Dapagliflozin treatment resulted in a urinary glucose excretion of 36 gram/24-hour, leading to a negative energy balance. Twenty-four-hour blood glucose levels decreased upon dapagliflozin (AUC; p = 0.017), while 24-hour free fatty acids and nocturnal β-hydroxybutyrate were elevated (AUC; p = 0.002 and p = 0.012, respectively), indicating a more pronounced reliance on fat oxidation. Indeed, following dapagliflozin, 24-hour RER was lower (0.814 ± 0.006 versus 0.827 ± 0.004; p = 0.051), in line with an increased 24-hour fat oxidation (p = 0.033) and a reduced 24-hour carbohydrate oxidation (p = 0.041). Nocturnal fat oxidation was higher after dapagliflozin (p = 0.039). Coupled, and maximally uncoupled mitochondrial respiration upon lipid-derived substrates were higher after dapagliflozin (O2-flux: 68.2 ± 3.2 versus 64.6 ± 3.2 pmol/mg/s; p = 0.071 and 87.6 ± 5.4 versus 78.1 ± 5.5 pmol/mg/s; p = 0.007, respectively). No changes were observed in hepatic glycogen or lipid content and composition.

Conclusion: Dapagliflozin treatment for 2 weeks in humans with prediabetes improves 24h and nocturnal fat oxidation. Dapagliflozin treatment also had significant effects on 24h glucose and free fatty acid levels, and on nocturnal β-hydroxybutyrate levels. These data indicated a more pronounced fasted state, although no changes were observed in hepatic glycogen. In addition, dapagliflozin improved ex vivo skeletal muscle mitochondrial oxidative capacity. These results show that dapagliflozin in prediabetes individuals elicits metabolic health effects that may mimic the effects of calorie restriction.

Clinical Trial Registration Number: NCT03721874

Supported by: AstraZeneca

Disclosure: A. Veelen: None.

9

Metabolome analysis of the effects by SGLT2 inhibitor ipragliflozin and metformin on human metabolites, and relationship with clinical data in a randomised controlled study

M. Koshizaka, A. Tsukagoshi, R. Ishibashi, Y. Maezawa, K. Yokote;

Chiba University, Chiba, Japan.

Background and aims: A clinical study comparing the effects of SGLT2 inhibitor ipragliflozin (Ipr) with those of metformin (Met) on visceral fat area and glucose and lipid metabolism revealed Ipr reduced visceral fat. To investigate the mechanism, metabolome analysis of the effects of Ipr and Met on human metabolites was performed with the samples obtained in the clinical study.

Materials and methods: In total 103 patients with type 2 diabetes, with HbA1c >=7% and < 10%, and BMI >=22 kg/m2 were randomly assigned to receive Ipr 50 mg or Met 1000 mg. Metabolome analysis using blood samples before and 24 weeks after administration was performed to identify changed metabolites, and analyzed their correlation with the changes of clinical data.

Results: Of them, 15 patients in the Ipr group and 15 in the Met group were analyzed, and there was no difference in the background between the groups.After 24 weeks, the visceral fat area significantly reduced in the Ipr group than in the Met group (-17.8% vs. -4.7%, P=0.015), as were subcutaneous fat area and body weight. Both HbA1c and blood glucose levels decreased by more than 7% and 14%, respectively. Red blood cell count and hemoglobin were increased in the Ipr group and decreased in the Met group. GOT, GPT and γGT were decreased in the Ipr group and increased in the Met group. Uric acid levels were decreased in the Ipr group. Triglycerides were decreased by more than 8% in both groups, total cholesterol (TC) was decreased in the Met group, LDL-C tended to be decreased in the Met group, and HDL-C tended to be increased in the Ipr group. The bone resorption marker TRACP5b was increased in the Ipr group and decreased in the Met group.As table, metabolome analysis showed that the increased metabolites after Met administration were methionine, glutamine, methyl-2-oxovaleric acid, hypotaurine, and the decreased metabolites were citrulline, indol-3-acetaldehyde, 1-methyl-4-imidazole acetic acid, octanoic acid, and hexanoic acid. Whereas inosine and N2-phenylacetylglutamine were the metabolites that increased after Ipr administration.Of them, methionine, methyl-2-oxovaleric acid, hypotaurine, citrulline, octanoic acid, and hexanoic acid were significantly different between the groups. Regarding to the correlation between metabolites and changes of clinical data, indole-3-acetaldehyde was correlated with HbA1c (r=-0.47), inosine with TRACP-5b (r=-0.75), muscle area (r=0.61) and HbA1c (r=-0.52), and N2-phenylacetylglutamine with BAP (r=0.79).

Conclusion: In Ipr, N2-phenylacetylglutamine, metabolite of phenylalanine, increased. Reportedly, phenylalanine reduced visceral fat. The patients treated with Ipr may reduce visceral fat by the mechanism of phenylalanine-N2-phenylacetylglutamine pathway. In Met, the changes in octanoic acid and hexanoic acid suggested that the β-oxidation of short-chain saturated fatty acids by mitochondria.

figure b

Clinical Trial Registration Number: UMIN000015170

Supported by: This study was funded by API. The funding source had no role in the study.

Disclosure: M. Koshizaka: Grants; Astellas Pharma Inc.

10

Effect of Dapagliflozin on renal and hepatic glucose kinetics in type 2 diabetes and NGT subjects

D. Tripathy1, C. Solis-Herrera2, X. Chen2, A. Hansis-Diarte2, R. Chilton2, R. DeFronzo2, E. Cersosimo2;

1Dept. of Medicine, University of Texas Health Science, STVHS, 2Dept. of Medicine, University of Texas Health Science, San Antonio, USA.

Background and aims: We previously have shown that both acute and chronic SGLT-2 inhibition increases endogenous glucose production (EGP). However, the relative contribution of liver versus kidney - responsible for the increase in EGP has not been identified.

Materials and methods: We assessed the effect of a single dose of Dapagliflozin or Placebo on renal glucose production in 13 T2DM (age=57.5±1.8 yrs, BMI=30±1.4 kg/m2) and 9 NGT (age 42±2 yrs, BMI= 30±1.1 kg/m2) subjects. Renal glucose production was measured using arteriovenous balance technique across the kidney combined with [3-3H] glucose infusion and PAH infusion (for determination of renal blood flow) before and 4 hours after administration of Dapagliflozin (10 mg) or Placebo; thus, each subject served as their own control.

Results: EGP increased following Dapagliflozin (DAPA) in both T2DM (2.00±0.11 to 2.43±0.15, P<0.05) and NGT (1.72±0.11 to 2.1±0.16, p<0.05), while it decreased after placebo in T2DM (2.02±0.12 vs 1.15±0.06) and NGT (2.10±0.2 vs 2.05±0.1) (both p<0.01, DAPA vs placebo). The fractional renal extraction of glucose (0.02± 0.004 vs 2.99±1.0, p=0.001 in T2DM, and 0.02± 0.004 vs 1.62± 1.4 in NGT, p=NS) and renal glucose uptake (0.067 ± 0.02 vs 0.347 ± 0.06 in T2DM and 0.08±0.02 vs 0.27 ± 0.08 mg/kg.min in NGT) were higher following DAPA vs placebo (p<0.05) and were entirely explained by the increase in glucosuria. There was a small, non-significant increase (0.065 & 0.032 mg/kg.min, respectively) in renal glucose production (RGP) following dapagliflozin in T2DM and NGT compared to the 0.45 mg/kg.min increase in total body EGP.

Conclusion: A single dose of Dapagliflozin significantly increases EGP which primarily is explained by an increase in hepatic glucose production.

Clinical Trial Registration Number: NCT02981966

Supported by: AstraZeneca

Disclosure: D. Tripathy: None.

11

Different effects of SGLT-2 inhibitors on subcutaneous and epicardial adipose tissue metabolome in severe heart failure subjects

B.J. Kasperova1, M. Mraz2,3, O. Kuda4, T. Cajka4, D. Hlavacek5, J. Mahrik5, S. Stemberkova-Hubackova1, I. Pleyerova1, K. Rosolova1, P. Svoboda1, P. Novodvorsky2,6, P. Ivak7, V. Melenovsky8, I. Netuka7, M. Haluzik2,3;

1Experimental Medicine Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 2Department of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 3Department of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Prague, Czech Republic, 4Institute of Physiology, Academy of Science of the Czech Republic, Prague, Czech Republic, 5Anestehsiology and Resuscitation Department, Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 6Department of Oncology & Metabolism, University of Sheffield, Sheffield, UK, 7Cardiovascular Surgery Department, Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 8Cardiology Department, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Background and aims: The exact mechanisms behind favorable metabolic and cardioprotective effects of SGLT-2 inhibitors (SGLT-2i) are still not fully understood. Here, we performed a complex metabolomic analysis of subcutaneous (SAT) and epicardial (EAT) adipose tissue of heart failure subjects treated with SGLT-2i in order to assess their impact on different fat depots and identify potential cardioprotective factors.

Materials and methods: Nine subjects with severe heart failure with reduced ejection fraction (NYHA III-IV) treated with SGLT-2i and 8 age-, BMI- and left ventricular ejection fraction-matched control subjects scheduled for heart transplantation or mechanical support implantation were included into the study. Eight SGLT-2i subjects and 5 control subjects had type 2 diabetes mellitus. A complex metabolomic analysis of SAT and EAT obtained during surgery was performed using liquid chromatography and mass spectrometry.

Results: SAT of SGLT-2i subjects showed marked increase in ketone bodies and a corresponding decrease of ketogenic triacylglycerols with medium-chain fatty acids suggesting enhanced ketogenesis typical for SGLT-2i use. In contrast, no such change was seen in EAT which, conversely, contained increased amount of long-chain triacylglycerols indicating significant differences in response to SGLT-2i treatment between these depots and a tendency to preserve EAT lipid content. Compared with control group, both SAT and EAT of SGLT-2i subjects consistently exerted surprisingly high levels of sphingolipids, especially sphingomyelins and ceramides, and ether-linked lipid species.

Conclusion: SGLT-2i treatment elicits different metabolic responses in SAT and EAT with SAT showing mainly accented ketogenesis, while the preservation of EAT suggests other functions including potential cardioprotection. The exact role of increased sphingolipids and ether-linked lipids in both adipose tissue depots remains to be elucidated.

Supported by: IKEM, IN 00023001, NV19-02-00118

Disclosure: B.J. Kasperova: None.

12

Empagliflozin-induced metabolic changes and cardiac function in patients with type 2 diabetes: a randomised cross-over MRI study with insulin as comparator

R. Thirumathyam1, G. Hall2, J.P. Gøtze2, J.J. Holst3, U. Dixen4, E.A. Richter5, N. Vejlstrup6, S. Madsbad1, P.L. Madsen7, N.B. Jørgensen1;

1Department of Endocrinology, Hvidovre Hospital, Hvidovre, 2Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, 3Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 4Department of Cardiology, Hvidovre Hospital, Hvidovre, 5Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, 6Department of Cardiology, Rigshospitalet, Copenhagen, 7Department of Cardiology, Herlev Gentofte Hospital, Herlev, Denmark.

Background and aims: Empagliflozin reduces cardiovascular risk in type 2 diabetes (T2D), possibly through improved cardiac function. Metabolic effects of empagliflozin include lowered glucose and insulin concentrations, elevated free fatty acids (FFA) and ketone bodies and have been suggested to convey the cardiovascular benefits of empagliflozin treatment. We aimed to evaluate the influence of these metabolic changes on cardiac function in patients with T2D.

Materials and methods: 17 subjects (13 M) with T2D, aged 58±3 years (mean±sem), BMI 32.9±0.9 kg/m2, HbA1c 52.4±2.4 mmol/L, TD2 duration of 8.9±1.3 years were treated with empagliflozin (E) and NPH-insulin (I) for 5 weeks in a cross-over design with 3 weeks of washout between treatments. Insulin was titrated to produce similar glycemic control as during empagliflozin. Patients were studied before and at the end of each treatment period. Metabolic changes were evaluated with fasting glucose, insulin, FFAs and 3-OH butyrate concentrations. Cardiac function with cardiac MRI during rest and chronotropic stress on two separate days without and with acute lowering of FFAs with acipimox. Cardiac endpoints were changes between treatments and washout (E vs WO or I vs WO) and between treatments (E vs I) in left ventricular peak filling rate (∆LVPFR) and left ventricular ejection fraction (∆LVEF).

Results: Fasting glucose was reduced from 8.7±0.5 mM during washouts to 7.6±0.3 mM on both treatments, while serum insulin was lower (E: 103±14; I: 141±16 pM (p<0.01)) and FFAs (E: 0.60±0.03; I: 0.50±0.05 mM (p=0.02)) and 3-OH butyrate higher (E: 0.27±0.03; I: 0.23±0.02 mM (p=0.03)) on empagliflozin compared with insulin treatment. Cardiac function was largely unchanged with any treatment and was not improved with empagliflozin compared to insulin in rest or during stress (∆LVPFR Rest: E vs WO: -38±19 (p=0.08);I vs WO: 8±25 ml/s; E vs I: -55±19 ml/s (p<0.01); ∆LVEF Rest: E vs WO: 2 ±1 (p=0.11), I vs WO: 1±1%, E vs I: -0±2%; ∆LVPFR Stress: E vs WO:-4±45, I vs WO : -16±28 ml/s, E vs I: -11±47 ml/s; ∆LVEF Stress: E vs WO: -1±2; I vs WO: -1±1%; E vs. I: 1±1%). Acipimox reduced FFAs by ~35% at all visits, without affecting ketone body concentration. Cardiac diastolic function was unchanged by acipimox administration, but LVEF was similarly reduced during both treatments (E: -6±2% I: -5.4±1%) under resting condition.

Conclusion: Neither empagliflozin nor insulin treatment improve cardiac function in patients with T2D. Treatment specific metabolic effects play no role for cardiac function under the studied conditions.

Clinical Trial Registration Number: EudraCT no. 2017-002101-35

Supported by: Boehringer Ingelheim

Disclosure: R. Thirumathyam: None.

OP 03 Risk for CVD and CKD

13

CSII is associated with lower NAFLD indices in patients with type 1 diabetes

L. Bozzetto, G. Della Pepa, E. Raso, R. Boccia, S. Gianfrancesco, A.A. Rivellese, G. Annuzzi;

Federico II University, Naples, Italy.

Background and aims: NAFLD is a raising concern also in type 1 diabetes (T1D) and is associated with micro and macrovascular complication. This study evaluated whether different ways of insulin administration (multiple daily injections [MDI] or continuous subcutaneous insulin infusion [CSII]) may affect NAFLD indices.

Materials and methods: We performed a cross-sectional study on 658 patients with T1D (37±13 years, 51% male, HbA1c 7.8±1.2%, body mass index 25±4 kg/m2) who had no history of excessive alcohol consumption or other secondary chronic liver disease, regularly attending the Diabetes Unit of an University Teaching Hospital. NAFLD was assessed by the Fatty Liver Index (FLI) and Hepatic Steatosis Index (HSI). Anthropometric, biochemical, and clinical parameters were retrieved by electronic records. Differences in NAFLD indices between patients on MDI or CSII were evaluated by univariate analysis, adjusted for possible confounders.

Results: Patients on CSII (n=259), compared with those on MDI (n=399), differed for gender distribution (men: 47% vs 55%, p=0.046), diabetes duration (22±11 vs 18±12; p<;0.001), prevalence of retinopathy (26% vs 18%, p=0.018), and nephropathy (15% vs 10%, p=0.035), respectively. According to univariate analysis adjusted for gender and diabetes duration, patients on CSII had a significantly lower HSI (36±5 vs 37±6; p=0.003), FLI (20±21 vs 25±24; p=0.003), waist circumference (85±12 vs 87±14 cm; p=0.047), triglycerides (76±44 vs 85±60 mg/dl; p=0.035), and insulin daily dose (0.53±0.22 vs 0.64±0.25 UI/kg body weight; p<;0.001).

Conclusion: Patients with T1D on CSII had better NAFLD indices, despite a longer diabetes duration and a higher prevalence of diabetes microvascular complications. Lower and more physiologically distributed daily insulin doses may have contributed to a better regulation of lipogenic pathways.

Disclosure: L. Bozzetto: None.

14

Increased prevalence of NAFLD in adults with glomerular hyperfiltration: a 8 year cohort study based on 147,162 Koreans

D.-J. Koo1, S. Park2;

1Division of Endocrinology and Metabolism, Department of Internal Medicine, Changwon Fatima Hospital, Changwon, 2Division of Endocrinology and Metabolism, Department of Internal Medicine, Kangbuk Samsung Hospital, Seoul, Republic of Korea.

Background and aims: Finding an indicator that can predict the development of nonalcoholic fatty liver disease (NAFLD) at an early stage is of clinical significance. Renal function and liver status are interconnected and share several physiological pathways. This study aimed to evaluate whether glomerular hyperfiltration (GHF) could predict the development of NAFLD and fibrosis progression.

Materials and methods: We performed a longitudinal cohort study of 147,479 participants who underwent comprehensive medical examinations annually or biennially from January 3, 2011 to December 31, 2018. Study subjects were the age group between 20 and 65 years of age without baseline kidney disease and NAFLD. Age- and sex-specific estimated glomerular filtration rate (eGFR) above the 95th percentile was defined as the GHF cutoff value, and eGFR values ​​between the 50th and 65th percentiles were used as reference groups. The primary endpoint of this study was the development of NAFLD diagnosed by abdominal ultrasonography, and fibrosis status was assessed by the NAFLD fibrosis score (NFS).

Results: During 598,745 person-years of follow-up (median, 4.6 years), NAFLD occurred in a total of 29,410 subjects. Subjects with baseline GHF had the highest hazard ratio (HR) for NAFLD (HR 1.21; 95% CI 1.14-1.29) and fibrosis progression (HR 1.42; 95% CI 1.11-1.82) after adjusting for confounding factors. The persistent GHF group during follow-up had the highest HR for NAFLD compared to the persistent non-GHF group (HR 1.31; 95% CI 1.14-1.51). Higher baseline eGFR percentile maintained a higher risk of NAFLD and fibrosis progression. These results were consistent in all subgroups and statistically more prominent in participants without diabetes.

Conclusion: In conclusion, this study demonstrated that glomerular hyperfiltration was associated with the development of NAFLD and fibrosis progression in relatively healthy young adults. Glomerular hyperfiltration may be used as a clinical surrogate marker for the early diagnosis of NAFLD regardless of obesity, insulin resistance and hypertension, especially in subjects without diabetes.

figure c

Disclosure: D. Koo: None.

15

Increased risk for microvascular outcome in NAFLD: a nationwide, population-based cohort study

T. Ebert1, L. Widman2, P. Stenvinkel3, H. Hagström4;

1Medical Department III - Endocrinology, Nephrology, Rheumatology, University of Leipzig, Leipzig, Germany, 2Division of Biostatistics, Karolinska Institutet, Stockholm, Sweden, 3Division of Renal Medicine, Karolinska Institutet, Stockholm, Sweden, 4Karolinska Institutet, Stockholm, Sweden.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is considered a multisystemic disease, as it is bidirectionally linked to other cardiometabolic disorders, such as type 2 diabetes (T2D). However, the long-term risk for microvascular outcomes in NAFLD is unclear.

Materials and methods: Using the outpatient part of the nationwide Swedish Patient Register, we identified all individuals with a first NAFLD diagnosis (N=4,943) and matched these (age, sex, and municipality) with up to ten reference individuals from the general population (N=44,606). Using population-based registers, we ascertained the development of microvascular diseases. The primary outcome was defined as a composite outcome of any diagnosis representative of microvascular disease (chronic kidney disease, retinopathy, and neuropathy). As secondary outcomes, we separately examined the risk of each specific microvascular outcome. Hazard ratios (aHR, adjusted for cirrhosis, T2D, hypertension, hyperlipidemia, malignant tumors) for time-to-event analyses were investigated by Cox proportional-hazards models.

Results: Median follow-up times was 5.0 years. The incidence rate of microvascular diseases was twice as high in patients with NAFLD (11.6 per 1000 person-years [95% confidence interval (CI)=10.5-13.0]) vs. reference individuals (5.8 per 1000 person-years [95%CI=5.6-6.1]). Kaplan-Meier estimates for the development of (A) microvascular diseases and (B) chronic kidney disease are shown in Figure 1. NAFLD was independently and positively associated with the development of microvascular diseases compared to non-NAFLD subjects (aHR=1.41 [95%CI=1.23-1.61]). When stratifying the analysis by follow-up time, sex, or age categories, results remain virtually unchanged.

Conclusion: NAFLD is positively associated with the development of microvascular diseases, independent of available confounders, e.g. T2D. Individuals with NAFLD should be screened for microvascular diseases in addition to macrovascular and metabolic diseases.

figure d

Supported by: EFSD Future Leaders Mentorship Programme, Novo Nordisk postdoctoral fellowship, Swedish Research Council, CIMED,ALF

Disclosure: T. Ebert: Grants; EFSD Mentorship Programme supported by AstraZeneca, Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet, Stockholm, Sweden, Karolinska Institutet Research Foundation, Swedish Kidney Foundation, Stiftelsen Stig och Gunborg Westman. Lecture/other fees; Sanofi, CME-Verlag, Santis.

16

Fatty liver index is an independent risk factor for all-cause mortality and major cardiovascular events in type 1 diabetes: a 10-year observational study

G. Penno1, M. Garofolo1, D. Lucchesi1, E. Gualdani2, P. Falcetta1, M. Giambalvo1, P. Francesconi2, S. Del Prato1;

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

Background and aims: Non-alcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction-associated fatty liver disease (MAFLD), has been associated with increased risk of death, with CVD as the most common cause of death in people with and without type 2 diabetes. Whether this also applies to type 1 diabetes (T1D) has not been yet reported.

Materials and methods: We prospectively observed 774 T1D (males 52%, 30.3±11.1 years old, diabetes duration (DD) 18.5±11.6 years, HbA1c 7.8±1.2%) to assess whether fatty liver index (FLI, based on BMI, waist, GGT and triglycerides), a proxy of NAFLD, predicts the risk of all-cause death and major CVD (combined endpoint of myocardial infarction, stroke, ischemic amputation or coronary, carotid and peripheral revascularizations).

Results: Over a median follow-up of 11 years (IQR 9.9-13.0), 57 out of 774 subjects died (7.4%) while 49 major CV events (6.7%) occurred in 736 individuals for whom incidence data were retrieved (95.1%). FLI score was <30 in 515 subjects (66.5%), ≥30-60 in 169 (21.8%), and ≥60 in 90 (11.6%). Mortality rate increased across FLI scores: 3.9, 10.1 and 22.2%, K-M log-rank 40.367, p<0.0001. In unadjusted Cox, with score <30 as reference, the risk of death increased in score ≥30-60 (HR 2.85, [95% CI 1.49-5.45], p=0.002) and even more in score ≥60 (HR 6.07, [3.27-11.29], p<0.0001). After adjustment for the Steno Type 1 Risk Engine (ST1-RE, inclusive of age, sex, DD, systolic BP, LDL-cholesterol, HbA1c, albuminuria, GFR, smoking and exercise), that enters first, HRs for death was 1.52 (0.78-2.97, p=0.222) for FLI ≥30-60 and 3.041 (1.59-5.82, p=0.001) for FLI ≥60. Inclusion of prior CVD among covariates modified HRs only slightly. The effect of FLI was unchanged when analysis was restricted to 733 subjects without prior CVD. Adjustment for the EURODIAB PCS Risk Engine (EURO-RE: age, HbA1c, WHR, ACR and HDL-cholesterol) instead of ST1-RE, confirms the independent role of FLI (HRs of 1.24 [0.62-2.48] for FLI ≥30-60 and 2.54 [1.30-4.95], p=0.007) for FLI ≥60, also after inclusion of prior CVD as a confounder, or when analysis was restricted to subjects without prior CVD. Incidence of major CV events increased across FLI scores: 3.5, 10.5 and 17.2%, K-M log-rank 29.16, p<0.0001. In unadjusted Cox, with score <30 as reference, risk of CV events increased in score ≥30-60 (HR 3.24, [95% CI 1.65-6.34], p=0.001) and in score ≥60 (HR 5.41, [2.70-10.83], p<0.0001). After adjustment for the ST1-RE, that enters first, HRs for death was 1.80 (0.90-3.61, p=0.096) for FLI ≥30-60 and 2.98 (1.45-6.13, p=0.003) for FLI ≥60. Inclusion of prior CVD among covariates modified HRs only moderately. The effect of FLI became weaker when analysis was restricted to 733 subjects without prior CVD. Adjustment for the EURODIAB PCS risk engine instead of ST1-RE, confirms the independent role of FLI (HRs of 1.49 [0.73-3.03] for FLI ≥30-60 and 2.44 [1.17-5.09, p=0.017] for FLI ≥60), also after inclusion of prior CVD as a confounder.

Conclusion: This is the first observational prospective study to demonstrate that FLI is associated with higher all-cause mortality and increased risk of incident major CV events in T1D, independently of validated risk engines based on established CV risk factors and diabetes-related variables.

Disclosure: G. Penno: None.

17

Sub-optimal glycaemic control and insulin resistance in young adults with type 1 diabetes increases platelet expression of P-selectin and phosphatidylserine

R.C. Sagar, S.M. Pearson, N. Kietsiriroje, M. Hindle, K. Naseem, R. Ajjan;

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

Background and aims: Patients with Type 1 Diabetes Mellitus (T1D) have increased risk of morbidity and mortality associated with earlier onset cardiovascular disease (CVD) and those with a combination of T1D and insulin resistance (IR) may be at even greater risk. Platelet hyperactivity has been described in diabetes and is associated with adverse cardiovascular complications. However, the factors driving pathological platelet function and the relative contribution of glycaemia and IR remain unclear. Our aim was to investigate the impact of glycaemic control and IR on platelet activation in young adults with T1D through use of four-colour multi-parameter flow cytometry.

Materials and methods: Patients aged 18-40 with T1D (>3 years since diagnosis) were recruited (n=23). Glycaemic control was evaluated using HbA1c and time in range (TIR) over 14 days using FreeStyle Libre Pro. Estimated glucose disposal rate (eGDR), a clinical marker of insulin resistance, was calculated using a validated equation. eGDR <8mg/kg/min confers IR status. Using whole blood, we applied 4-colour multiparameter flow cytometry to measure 3 distinct markers of platelet activation, both at rest and in response to activation with dual agonists Protease-activated receptor-1 (PAR-1) and Collagen related peptide XL (CRP-XL). Statistics were conducted via Prism v9.3.1.

Results: Mean age of participants was 24 (range 19-30) (69% males). All were on insulin therapy [1 with adjunctive metformin]. Mean HbA1c (SD) was 67mmol/mol (±14). Individuals were split into 2 equal groups based on 1) HbA1c (≥67 and <67 mmol/mol), 2) TIR (<50% vs >50%), 3) eGDR (<8 vs >8 mg/kg/min). Platelet data are expressed as mean(±SD) Mean Fluorescence intensity (MFI) of each marker. Those with HbA1c ≥67 expressed higher basal P-selectin MFI (641±290 vs 354±202; p=0.01) and following dual agonist stimulation (73345±62376 versus 25829±8607; p=0.02). Phosphatidylserine (PS) expression at the platelet surface was measured as a marker of procoagulant platelets. Basal PS expression appeared higher in those with HbA1c ≥67mmol/mol compared with <67 (1112±1037 vs 505±125; p=0.13). Following dual agonist stimulation, PS exposure was higher in those with HbA1c ≥67mmol/mol compared to those with lower HbA1c (21531±10214 vs 10858±3478; p=0.07). Patients were also stratified using TIR, with largely similar findings.Participants with eGDR <8mg/kg-1.min-1 showed a trend towards higher mean basal PS expression, but not P-selectin, compared with higher eGDR (PS 1216±1152 vs 458±163, p=0.09). Following dual agonist stimulation, those with eGDR <8mg/kg-1.min-1 had a greater activation induced PS exposure (23718±10200 vs 13649±6017; p=0.02), while P-selectin failed to show a statistical difference (57782±52949 vs 34991±30283; p=0.16).

Conclusion: In young adults with T1D, suboptimal glycaemic control appears to enhance platelet activation through increased expression of P-selectin and PS both basally, suggestive of elevated circulating platelet activity and following agonist stimulation, demonstrating a greater propensity to activate. The presence of low eGDR in this cohort may further exacerbate platelet activation, indicating both glycaemia and IR contribute to platelet reactivity in individuals with T1D.

Supported by: BHF Clinical Research Fellowship

Disclosure: R.C. Sagar: None.

18

Non-invasive fibrosis scores as prognostic biomarkers of liver and cardiovascular events and all-cause mortality in adults with type 2 diabetes in the UK: a longitudinal study

M. Jara1, Q.M. Anstee2,3,3, T.L. Berentzen1, L.M. Nitze1, M.S. Kjær1, K.K. Mangla1, J.M. Tarp1, K. Khunti4;

1Novo Nordisk A/S, Søborg, Denmark, 2Translational & Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, UK, 3Newcastle NIHR Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Trust, Newcastle, UK, 4Diabetes Research Centre, Leicester General Hospital, Leicester, UK.

Background and aims: Non-alcoholic steatohepatitis is associated with type 2 diabetes (T2D), life-threatening liver-related complications, increased liver-specific and all-cause mortality and cardiovascular (CV) disease. Biopsy-confirmed liver fibrosis is an important predictor of severe outcomes but biopsies are not scalable for widespread use. This real-world study investigated the prognostic utility of six non-invasive fibrosis scores on clinical outcomes in patients with T2D seen in routine general practice.

Materials and methods: Using a longitudinal cohort design, patients ≥18 years with T2D, ≥1 fibrosis score calculable from the UK Clinical Practice Research Datalink (CPRD) after 1 January 2001, no alcohol-related disorders and/or other chronic liver diseases in Hospital Episodes Statistics (HES), and/or no prescriptions of drugs inducing liver disease in CPRD were included. Patients were followed from date of inclusion until time of first clinical outcome event (liver-related hospitalisation or death [liver event], CV-related hospitalisation or death [CV event] or all-cause death) recorded in HES or Office for National Statistics Death Registration; database migration; 10 years’ follow-up; or 1 January 2020, whichever came first. Fibrosis-4 Index (FIB4), the score of focus, was categorised as low (<1.30), indeterminate (1.30-2.67) or high (>2.67) risk according to established cut-offs previously shown to be associated with fibrosis. Cumulative incidence functions were calculated and hazard ratios (HRs) were estimated using Cox proportional hazard models with calendar time as underlying timescale.

Results: In total, 17 793 eligible patients (55% male, median age 66.7 years) had T2D and measures available for FIB4 calculation. Among these patients there were 584 liver events, of which ascites (n=233), cirrhosis (n=113) and gastro-oesophageal varices (n=110) were the most common. Cumulative incidence proportions for an incident event after 10 years’ follow-up in the high FIB4 group were 16% (liver), 36% (CV) and 69% (death). HRs for patients in the indeterminate and high FIB4 groups vs the low-risk group indicated a significantly higher risk of all three event types (Table), also after adjustment for sex and age. For the other scores, HRs were also higher in patients with a high vs low score.

Conclusion: In this real-world population of patients with T2D and no other clinically recognised liver disease, the risk of a clinical event was significantly higher in patients with a high vs low FIB4 score, highlighting the prognostic potential of FIB4 (and other non-invasive fibrosis scores) in this population.

figure e

Supported by: Novo Nordisk

Disclsoure: M. Jara: Employment/Consultancy; Novo Nordisk A/S. Stock/Shareholding; Minority shareholder in Novo Nordisk A/S.

OP 04 Walking with diabetes

19

Prevalence, incidence and risk factors for Charcot foot in patients with diabetes: a nationwide Swedish study

G. Tsatsaris1, N. Rajamand Ekberg1, T. Fall2, S. Catrina1;

1Department for Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, 2Department of Medical Sciences, Molecular Epidemiology, Uppsala Universitet, Uppsala, Sweden.

Background and aims: Charcot foot (CF) is a serious complication of diabetes mellitus (DM) with potential disastrous consequences. Despite being reported for the first time in 1868, the epidemiology of this condition is still unknown since just a few studies are available. We report here the prevalence, incidence and risk factors for the CF in a nationwide Swedish retrospective register-based study of patients with DM.

Materials and methods: The prevalence and incidence of CF in patients with DM were established based on data from the Swedish National Patient Registry and the Swedish Prescribed Drug Register for the period 2006 - 2016. Risk factors of CF were assessed using logistic regression based on data from 2001 - 2016 from the Swedish National Diabetes Register in a matched design with a control diabetes group without CF (1:10). In order to examine the effect of HbA1c, diabetes duration, macro- and microalbuminuria, atherosclerosis and retinopathy on CF risk, direct acyclic graphs (DAGs) were used to select potential confounders to be included in the statistical analysis.

Results: 3449 patients with DM and CF were included in the study. The prevalence of CF in the total diabetic population increased during the observation period from 0.55% in 2006 to 0.79% in 2016, whereas the incidence remained stable under the same time period. The increase in prevalence was observed in both type 1 (T1D) and type 2 diabetes (T2D) patients, with higher prevalence in T1D (1.97% in 2016) than in T2D (0.60% in 2016). Subjects with T1D had a longer diabetes duration before developing CF compared to subjects with T2D (33.12 ± 13.39 years respective 14.64 ± 9.49 years). Female gender was identified as a risk factor for the development of CF in T1D (OR: 1.29, 95% CI: 1.14 - 1.45, p-value <0.001) but not in T2D (OR: 0.72, 95% CI: 0.64 - 0.81, p-value <0.001). Atherosclerotic disease was associated with increased risk for CF in both types of DM (T1D OR: 4.73, 95% CI: 3.54 - 6.32, p-value <0.001) (T2D OR: 6.14, 95% CI: 5.12 - 7.36, p-value <0.001). Diabetes duration, HbA1c, pre-existing diabetic microangiopathic complications, body mass index (BMI), osteoporosis and peripheral vascular disease were identified as risk factors of CF in both types of DM. Area under the curve in our prediction model was 0.78 in T1D and 0.71 in T2D.

Conclusion: The prevalence of CF was higher in patients with T1D than in patients with T2D with progressive increase in both types of diabetes during period of observation. This might reflect a longer survival time after CF diagnosis in patients with DM especially in T1D. Identification of risk factors for CF offer the possibility of a predictive model for the development of CF.

Supported by: Stockholm County Research Council, Bert von Kantzows Foundation, Frimurarestifelse

Disclosure: G. Tsatsaris: Grants; Stockholm County Research Council, Bert von Kantzows Foundation, Frimurarestifelse.

20

Genome wide association meta study of diabetic foot ulcers

S. Altintas1, G. Bouland2, A. Veluchamy3, M. Thangam4, E. Lindholm4, W. Meng3, J.A. Andersen1, C.S. Hansen1, L.T. Dalgaard5, C. Palmer3, E. Ahlqvist4, L.M.T. Hart6, A. Rasmussen1, P. Rossing1, T.S. Ahluwalia1;

1Steno Diabetes Center Copenhagen, Herlev, Denmark, 2Leiden University Medical Center, Leiden, Netherlands, 3University of Dundee, Dundee, Scotland, UK, 4Lund University Diabetes Center, Malmo, Sweden, 5Roskilde University, Roskilde, Denmark, 6Amsterdam University Medical Center, Amsterdam, Netherlands.

Background and aims: Diabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus. Globally, a lower limb is amputated due to diabetes every 30 seconds; foot ulceration precedes 85% of diabetes-related amputations. While several risk factors are known - including sensorimotor peripheral neuropathy (DSPN), peripheral artery disease, foot deformities, and poor glycemic control - the genetics of DFUs are poorly understood. In this study, we conducted the first genome-wide association meta-study of diabetic foot ulcers with the aim of identifying genetic loci associated with DFU risk in diabetic (type 1 and type 2) individuals with DSPN.

Materials and methods: A meta-analysis of DFUs was conducted, comprising four independent genome-wide association studies from diabetes cohorts of European ancestry (AfterEU, Denmark; SDR, Sweden; GoShare, Scotland; DCS, Netherlands). This case-control study comprised a total of 980 cases (with DFU and DSPN) and 6196 controls (no history of DFU, but with DSPN). DSPN was defined as bilateral vibration sensation threshold ≥25V or absent sensation to monofilament. Logistic regression models were applied adjusting for sex, duration of diabetes and principal components. Summary statistics from the four European cohorts were meta-analysed using fixed effects inverse-variance based meta-analysis.

Results: In the GWAS meta-analysis, we identified three common single nucleotide polymorphisms (SNPs) that were suggestive (p-value<1×10-6), from three loci; an overview of these results is given in Table 1. Two common variants - rs11069845 (intronic), and rs1534545 (missense variant) - were located in COL4A2 and ALK. The third, rs12129159, was located within 1mb from HS2ST1 and PKN2-AS1.

Conclusion: Three suggestive loci associated with DFU risk were identified in the current ongoing study. Two loci were located in COL4A2 and ALK, which have known roles in small vessel disease and neuronal development. Additional GWAS data from other participating centers will be added to the current analyses towards identification of loci associated with diabetic foot ulcers of neuropathic origin.

figure f

Disclosure: S. Altintas: None.

21

Bone morphogenetic protein-7 promotes diabetic wound healing by decreasing inflammation and matrix metalloproteinase-9 expression

E.C. Leal1,2, J. da Silva1,2, A. Figueiredo1, Y.-H. Tseng3, E. Carvalho1,2;

1Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 2Institute of Interdisciplinary Research, University of Coimbra, Coimbra, Portugal, 3Joslin Diabetes Center, Boston, USA.

Background and aims: Diabetic foot ulcers is the leading cause of prolonged hospital admission, health-related costs, and reduced quality of life for diabetic patients. Bone morphogenetic protein-7 (BMP7) is a protein of the transforming growth factor beta (TGF beta) superfamily. Some known properties of BMP7, such as the modulation of inflammation, indicate that it may promote tissue regeneration. However, the role of BMP7 in the skin is not fully understood, particularly in conditions of diabetes during wound healing. Our main aim was to evaluate the role of BMP7 in diabetic wound healing and study the underlaying mechanisms.

Materials and methods: We used male C57BL/6 mice, and diabetes was induced with intraperitoneal administration of streptozotocin (50mg/kg) for 5 consecutive days. After six weeks of diabetes, two wounds of 6 mm in diameter, were induced on the back of diabetic mice and treated topically with BMP7 (0.5 μg per wound/day) or vehicle. The wound size was measured every day up to day 10 post wounding, and the wounded skin was collected. The number of inflammatory cells present at the wound site, macrophages (CD68) and lymphocytes (CD4), was determined by immunohistochemistry. The expression of inflammatory markers (IL6, keratinocyte-derived chemokine - CXCL1/KC, TNF alpha, IL1 beta), and matrix metalloproteinase-9 (MMP9) was measured by quantitative PCR. Finally, H&E and Masson's Trichrome were used to assess histology and collagen deposition.

Results: BMP7 accelerated wound closure by 20% (p<0.05) in diabetic mice when compared to vehicle-treated wounds. The number of lymphocytes in diabetic wounds did not change with BMP7 treatment, but the number of macrophages was reduced by 30% (p<0.05) when compared to vehicle-treated wounds. In addition, BMP7 significantly decreased (p<0.05) the expression of inflammatory mediators and MMP9 expression in diabetic wounds. We also observed that in diabetic wounds treated with BMP7, the area of granulation tissue was significantly increased by 50% (p<0.05), and the collagen deposition was 35% higher (p<0.05) when compared to vehicle-treated wounds, indicating that BMP7 promotes better wound healing progression and a decrease in extracellular matrix degradation.

Conclusion: BMP7 promotes wound healing in diabetes by decreasing local inflammation and, consequently, MMP9 expression, known to be increased in inflammatory conditions. This effect of BMP7 treatment in diabetic wounds prevented the high degradation of the extracellular matrix, evident in the diabetic condition. It also led to an increase in tissue regeneration and in collagen deposition, so a better progression of wound healing. This study suggests that BMP7 is a therapeutic agent of interest for the treatment of diabetic foot ulcers.

Supported by: EFSD/Lilly EXPAND Programme, FCT, SPD

Disclosure: E.C. Leal: None.

22

Novel topical esmolol hydrochloride for diabetic foot ulcer: phase 3, randomised, double-blind, placebo-controlled, multi-centre study

A. Rastogi1, S.A. Kulkarni2, S.K. Deshpande2, S. Agarwal3, V. Vishwanathan4, A.G. Unnikrishnan5, Galnobax Study Group;

1Endocrinology, Post Graduate Institute of medical Education and Research, Chandigarh, 2Novalead Pharma, Pune, 3Ruby Hall Clinic, Pune, 4Diabetes, MV Diabetes and Resaerch center, Chennai, 5Diabetes, Chellaram Diabetes center, Pune, India.

Background and aims: Pre-clinical and phase 2 study with esmolol suggest its potential role in treatment of diabetic foot (DFU). We aimed to study the efficacy of topical esmolol for DFU healing.

Materials and methods: This is the FIRST randomized, double-blind, placebo-controlled, parallel-group, multi-centre, phase-3 study done at 27 centers across India to evaluate efficacy of topical esmolol hydrochloride gel for uninfected diabetic foot ulcers (DFU) . Participants with non-infected full thickness (UTS 1B) DFU of duration>4 week, size 2cm2-15cm2 and ABI 0.7-1.3 were randomized after a run-in phase (1 week) to receive Esmolol + standard of Care (SoC), SoC only, or vehicle + SoC (3:3:1 proportion) for 12 week (treatment phase) and followed further till 24 week. Participants visited the investigational site once a week during the 12-week treatment phase for wound measurement and at week 14, 16, 20 and 24 during follow-up phase. SoC included debridement, moist wound environment and off-loading with modified insole and shoes. The primary outcome was proportion of wound closure within 12-week in Esmolol + SoC and SoC only groups. The secondary outcomes included proportion of participants achieving target ulcer closure (24-weeks), and time to ulcer closure during treatment phase. Target ulcer closure was defined as 100% re-epithelialization, confirmed on two consecutive site visits (two weeks apart). All analyses were performed for intention-to-treat (ITT) i.e., safety evaluable population.

Results: Overall, 251 participants were screened and 176 were randomized to the three groups with baseline characteristics in Table 1. The proportion of participants who achieved target ulcer closure within 12 weeks was 41of 68 (60·3%) participants in Esmolol + SoC group compared to 30 of 72 (41·7%) participants in SoC only group (OR = 2·126, 95% CI 1·08-4·17, p =0·0276). Proportion of target ulcer closure by the end of study (week 24) was achieved in 44 of 57 (77·2%) participants in Esmolol + SoC group and 35 of 63 (55·6%) participants in SoC only group (OR = 2·708, 95% CI 1·22-5·99; p = 0·0126). The mean time for ulcer closure was similar (74·3 days for Esmolol + SoC group and 72·5 days for SoC only group). The Esmolol + SoC group showed significant advantage over SoC (p<0·05) in participants with BMI>25 kg/m2, high HbA1c, longer ulcer duration, hemoglobin<11 g/dl, low eGFR and ulcers that did not achieve 50% area reduction within initial 4 weeks of treatment. The percent ulcer area reduction from end-of-treatment to end-of study was 60·7% for Esmolol + SoC group compared to a negligible reduction of 2·7% in SoC only group (p = 0·021).

Conclusion: Topical Esmolol is a novel treatment that significantly improves healing of DFU compared to SoC.

figure g

Clinical Trial Registration Number: NCT03998436

Disclosure: A. Rastogi: None.

23

Flexor tendon tenotomy treatment of the diabetic foot: a multicentre randomised controlled trial

J. Askø Andersen1,2, A. Rasmussen3, S. Engberg3,4, J. Bencke5, M. Frimodt-Møller1, K. Kirketerp-Møller3,6, P. Rossing1;

1Complication Research, Steno Diabetes Center Copenhagen, Gentofte, 2Orthopedic Department, North Zealland Hospital, Hillerød, 3Foot Clinic, Steno Diabetes Center Copenhagen, Gentofte, 4Novo Nordisk A/S, Søborg, 5Human Movement Analysis Laboratory, Department of Orthopedic surgery,, Copenhagen University Hospital at Amager-Hvidovre, Hvidovre, 6Copenhagen Wound Healing Center, Bispebjerg Hospital, København, Denmark.

Background and aims: A fifth to a third of all individuals with diabetes will incur a diabetic foot ulcer during their life-time. There are several factors that influence the risk of incurring a diabetic foot ulcer with one being hammertoe deformities. The aim of this study was to evaluate effects on ulcer healing and prevention of needle flexor tendon tenotomy treatment of the diabetic hammertoe deformity.

Materials and methods: A multicenter randomized controlled trial of individuals with diabetes and ulcers or impending ulcers associated with hammertoes, performed between 1st of November 2019 and 31st of March 2021. Participants were stratified on the presence of ulcer, into individuals with ulcers and individuals with impending ulcers. Participants were randomized to tenotomy and standard non-surgical treatment or standard non-surgical treatment alone. Primary outcomes were time to ulcer healing and progression from impending ulcer to active ulcer.

Results: Of 224 screened individuals with diabetes, 95 (59.0% male) were included. The mean follow-up was 291 (±70) days, 28 (29.5%) had type 1 diabetes, mean diabetes duration was 20 (13-26) years, and mean age was 67.7 (±9.8) years. Of the included participants 16 had ulcers, of whom eight were randomized to intervention. Of the remaining 79 individuals with impending ulcers, 39 were randomized to intervention. For participants with ulcers, healing rates favored tenotomy (100% vs 37.5%, p=0.026) as did time to ulcer healing (p=0.04). For individuals with impending ulcers, incidence of progression to an active ulcer was lower (1 vs 7, p=0.028) and number of ulcer-free days were higher (p=0.043) in the tenotomy group. No serious adverse events were recorded.

Conclusion: This randomized study showed that the simple procedure of needle flexor tendon tenotomies was effective and safe when treating and preventing ulcers associated with the diabetic hammertoe deformity.

figure h

Clinical Trial Registration Number: NCT04154020 & NCT04154046

Supported by: the Jascha Foundation and the Aase og Ejnar Danielsen Foundation

Disclosure: J. Askø Andersen: None.

24

Saving the foot: simple orthopaedic intervention to adjust the mechanics of the ulcerated neuropathic foot improves outcomes by reducing sepsis, amputation and mortality

J. Blong1, A. Sharpe2, J. Cairney-Hill1, A. Gorman2, M. Allen2, S. Haycocks2, M. Stedman3, A. Robinson4, E. Gee1, A. Heald4;

1Orthopaedic Surgery, Salford Royal NHS Foundation Trust, Salford, 2Podiatry, Salford Royal NHS Foundation Trust, Salford, 3Res Consortium, Andover, 4Endocrinology & Diabetes, Salford Royal NHS Foundation Trust, Salford, UK.

Background and aims: Ulceration of a neuropathic foot is a poor prognostic indicator for individuals with diabetes and presents a considerable financial burden for the health economy. Orthopaedic and vascular surgeons have become embedded within most specialist diabetes foot multidisciplinary teams (DF MDT) to offer reactive interventions in the context of acute diabetic foot sepsis, such as incision and drainage of abscess, or amputation. We describe how a day-case procedure list within a DF MDT has improved outcomes by performing proactive simple surgical procedures, with a view to expediting ulcer resolution and thus minimising subsequent morbidity and mortality.

Materials and methods: Patients with ulceration (without associated abscess) were offered a percutaneous procedure performed under local anaesthetic by an orthopaedic surgeon. Purpose of surgery was to adjust the mechanics of the foot and offload the ulcerated region, expediting healing. We anticipated improved rates of ulcer resolution and reduced complication rates including diabetic foot sepsis (£11,998 per admission), amputation (£9,221 per patient) and mortality. Percutaneous tenotomies (PT) performed for toe apex ulcers. Procedure cost £501. Patient able to mobilise full weight-bearing in normal footwear immediately post-procedure. Percutaneous tendoachilles lengthening (TAL) performed for plantar metatarsal head ulcers. Procedure cost £1140. Patient partial weight-bears for four weeks in total contact cast, followed by two weeks full weight-bearing in a walker boot.

Study period April 2019 - October 2021. Primary outcome: ulcer resolution. 12 month follow-up period.

Results: Results - see table. Key findings from the intervention cohorts: successful ulcer resolution achieved for all individuals. No admissions for diabetic foot sepsis. Reduced recurrence and amputation rates. No mortality within 12 months. One complication of Achilles tendon rupture following TAL (non-concordant with immobilisation instructions). *3 of the 15 conservative cohort achieved ulcer resolution (average 20 weeks). Conservative cohort average cost £9902. Intervention cohort average cost £1211, giving average savings of £8691 per patient. This demonstrates an 88% reduction in healthcare costs.

Conclusion: These simple daycase percutaneous procedures support accelerated ulcer healing, with reduced rates of recurrence, amputation and mortality. We have demonstrated significant patient benefit and cost savings for this simple intervention, which merits full evaluation in a clinical trial.

figure i

Disclosure: J. Blong: None.

OP 05 How to become a fat cell

25

Fas (CD95) activation inhibits browning of white adipose tissue in obesity

S. Wueest1,2, P.P. van Krieken1,2, N.K. Konrad1,3, C. Koch2,3, M.S.F. Wiedemann1, M. Borsigova1,2, S. Boettcher2,3, M. Blüher4,5, D. Konrad1,2;

1Children's Hospital, Zurich, Switzerland, 2University of Zurich, Zurich, Switzerland, 3University Hospital Zurich, Zurich, Switzerland, 4University of Leipzig, Leipzig, Germany, 5University Hospital Leipzig, Leipzig, Germany.

Background and aims: We previously observed that adipocyte-specific Fas (CD95) knockout mice were protected from the development of high-fat diet (HFD)-induced adipose tissue inflammation, hepatic steatosis and insulin resistance. In these mice, Cre recombinase was under the control of the Fabp4-promotor questioning adipocyte specificity of Fas knockdown. Herein, we aimed to reassess the adipocyte-specific role of Fas on glucose metabolism in newly generated mice expressing Cre recombinase controlled by the Adipoq-promotor.

Materials and methods: 6-week-old adipocyte-specific Fas knockout (FasΔadipo) and control (FasF/F) littermates were fed a regular chow or HFD (~60% kcal fat) for 20 weeks. Glucose metabolism was assessed by intraperitoneal glucose and insulin tolerance tests. After 20 weeks, liver and fat depots were analyzed using Western blotting, qPCR and histological staining. In vitro, p53 or Fas was knocked down in subcutaneous adipocytes using CRISPR-Cas9 and siRNA, respectively, and lysates were analyzed using Western blotting. Moreover, FAS expression was determined in human subcutaneous adipose tissue and correlated to p53 and UCP1.

Results: HFD-fed FasΔadipo mice displayed improved glucose and insulin tolerance (AUC in mmol/l*min: GTT 2474±879 in FasF/F vs. 2071±646 in FasΔadipo, p<0.05; ITT 1044±94 in FasF/F vs. 739±35 in FasΔadipo, p<0.01), reduced adipose tissue inflammation as well as decreased hepatic lipid content compared to control littermates. Importantly, HFD-induced weight gain was significantly reduced in FasΔadipo mice (23.1±1.7g in FasF/F vs. 16.7±1.6g in FasΔadipo, p<0.01). The latter was associated with increased expression of browning markers such as UCP1 in inguinal adipose tissue as well as highly reduced protein levels of the UCP1-repressor p53 in isolated adipocytes. Preliminary data in cultured subcutaneous adipocytes revealed that Fas knockdown increased and Fas ligand (FasL) stimulation decreased isoproterenol-induced UCP1 protein levels, respectively. Moreover, FasL-induced downregulation of UCP1 was blunted in p53-depleted cells, indicating that Fas activation reduces browning of white adipocytes in a p53-dependent manner. In agreement, FAS (CD95) expression correlated positively with p53 but negatively with UCP1 in human subcutaneous adipose tissue.

Conclusion: Fas activation in adipocytes contributes to HFD-associated adiposity through inhibition of adipose tissue browning.

Supported by: Swiss National Science Foundation (#310030-179344 to DK)

Disclosure: S. Wueest: None.

26

Aripiprazole, but not olanzapine, directly inhibits human adipocyte differentiation and glucose metabolism

M. Vranic1, V. Ferreira2, S. Hetty1, A. Sarsenbayeva3, F. Ahmed1, G. Fanni1, Á.M. Valverde2, J. Eriksson1, M.J. Pereira1;

1Department of Medical Sciences, Uppsala University, Uppsala, Sweden, 2IIBm Alberto Sols (CSIC-UAM); CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, Spain, 3Department of Molecular and Clinical medicine, University of Gothenburg, Gothenburg, Sweden.

Background and aims: Second-generation antipsychotics (SGAs) are the cornerstone treatment of schizophrenia and other mental disorders. However, they can cause adverse metabolic effects, such as obesity and diabetes, and the exact mechanisms behind this are unknown. Different SGAs carry different propensities to induce metabolic adverse effects, e.g. olanzapine (OLA) is a high-risk drug associated with worse metabolic outcomes, while aripiprazole (ARI) is considered more neutral. We aimed to investigate the effects of SGAs on adipocyte differentiation and glucose and lipid metabolism.

Materials and methods: Human subcutaneous primary preadipocytes obtained from healthy subjects (n=6) were differentiated without or with therapeutic and supra-therapeutic concentrations of ARI (1, 2, and 10 μM), its active metabolite dehydroaripiprazole (DARI; 0.4 and 4 μM), and OLA (0.2 and 2 μM) for 14 days when differentiation rate and glucose uptake were assessed. Furthermore, the expression of genes related to cell differentiation and glucose and lipid metabolism were measured. In addition, adipose tissue samples obtained from mice treated in vivo without or with ARI or OLA for 7 months were used for gene expression analyses (n=9, control; n=9, OLA; n=8, ARI).

Results: Supra-therapeutic concentrations of ARI reduced preadipocyte differentiation rate (up to ~80%), assessed by the number of cells accumulating lipids and expression of adipogenic genes (PPARG, CEBPA, CD36; p<0.05). The expression of lipid storage genes (FASN, LPL, DGAT2, ATGL; p<0.05), and mitochondrial function genes (TFAM, PDK4, ACO1; p<0.05) were also reduced compared to control. Conversely, therapeutic levels of ARI and supra- and therapeutic levels of DARI increased the expression of mitochondrial biogenesis and lipid oxidation genes (TFAM, PDK4, CPT1B, ACO1; p<0.05), and LEP (p<0.05). Basal and insulin-stimulated glucose uptake was dose-dependently reduced (up to ~70%) in cultures exposed to ARI and DARI during differentiation (p<0.05), which corresponded to reduced expression of GLUT1 and GLUT4 (p<0.05). OLA, when present during differentiation, did not affect adipogenesis or lipid and glucose metabolism but increased LEP expression by ~2 fold (p<0.05). ARI- and OLA-treated mice had decreased expression of PPARG and GLUT4 (p<0.05), as well as increased expression of lipid oxidation genes PDK4 (p<0.05) in epididymal, but not inguinal adipose tissue. LEP expression was increased, while FASN expression was decreased in both inguinal and epididymal adipose tissues in ARI- and OLA-treated mice (p<0.05).

Conclusion: Therapeutic levels of ARI and DARI, but not OLA, negatively affected adipocyte glucose metabolism and increased the expression of lipid oxidation markers, suggesting a potential substrate switch from glucose to lipid oxidation in adipocytes. OLA, instead, increased leptin levels, which may contribute to the development of obesity in humans, potentially via CNS effects. Our results suggest that while ARI directly affects adipocyte metabolism, OLA might require long-term exposure as well as the involvement of other organs, such as the brain, in mediating its adverse metabolic effects.

Supported by: H2020 Marie Sklodowska Curie ITN TREATMENT; SSMF; SDF; EXODIAB; UU ALF

Disclosure: M. Vranic: None.

27

Senescent adipose stromal cells-derived CCL5 induces endothelial dysfunction, a key step in the onset of atheroslerosis

L. Le Pelletier1, K. Ngono Ayissi1, J. Gorwood1, F. Boccara1,2, M. Auclair1, M. Atlan1,3, B. Fève1,4, J. Capeau1, C. Lagathu1, V. Béréziat1;

1Sorbonne Université - INSERM - Centre de Recherche Saint Antoine, 2AP-HP, Saint-Antoine Hospital, Department of Cardiology, 3Tenon Hospital, Department of Plastic Surgery, 4AP-HP, Saint-Antoine Hospital, Department of Endocrinology, Paris, France.

Background and aims: Adipose tissue plays a major role in the regulation of metabolism through its storage/mobilisation and endocrine function. Aging is associated with adipose tissue redistribution, oxidative stress, and fibrosis. These alterations lead to decreased storage capacities and insulin-resistance, leading to the onset of cardiometabolic complication such as atherosclerosis. We recently demonstrated that adipose tissue aging is associated with an accumulation of senescent adipose stromal cells (ASCs) from which arise insulin-resistance and dysfunctional adipocytes. Here, we hypothesize that senescent ASCs can also contribute to the onset of endothelial dysfunction, early key step in the development of cardiometabolic disease. Our aims were firstly, to establish the proof of concept that senescent ASCs can induce endothelial dysfunction, secondly, to identify a potential candidate involved in the dialogue between ASCs and endothelial cells.

Materials and methods: ASCs were isolated from adipose tissue of young (mean ± error of the mean (SEM) age: 18.6 ± 2.6 years; mean ± SEM BMI: 24.4 ± 0.6 Kg/m2) or aged (mean ± SEM age: 60.3 ± 0.9 years; mean ± SEM BMI: 25.6 ± 0.4 Kg/m2) healhty female donors. The impact of senescent ASCs on Human Coronary Artery Endothelial Cells (HCAECs) from healthy donors was analysed trhough conditioned media experiments.

Results: We found that conditioned media from aged-donor ASCs, but not from young-donor ASCs, induced endothelial dysfunction, characterized by a decrease in Nitric Oxide (NO) production, increased senescence markers (Senescence-associated-β-galactosidase activity, and cell cycle arrest protein P16 and P21) and the acquisition of a pro-adherent phenotype (increased InterCellular Adhesion Molecule (ICAM) and Vascular Cell Adhesion Molecule (VCAM) expression). Preventing the onset of aged-donor ASCs senescence allowed to maintain the endothelial function. To identify molecules implicated in ASC - HCAEC cell communication, we compared the secretory profil of aged- versus young-donor ASCs. The secretion of CCL5 was found to be specifically increased in the conditioned media obtained from aged-donor ASCs. HCAECs exposed to CCL5 displayed endothelial dysfunction (decrease in NO production, senescence, pro-adherent phenotype, pro-migratory effect, and amplified vascular networks formation). Conversely, the addition of a CCL5 receptor antagonist was abble to prevent the deleterious effect of conditioned media from aged-donor ASCs and maintain the endothelial function of HCAECs.

Conclusion: Overall, our data demonstrate the key role of ASCs and CCL5 in age-related endothelial dysfunction. They allow to a better understanding of the contribution of adipose tissue to aged-related vascular complications, thus opening new therapeutic perspectives on the support of cardiometabolic diseases.

Disclosure: L. Le Pelletier: None.

28

Requirement of plakoglobin during the early stages of adipogenesis

F. Abou Azar1,2, S. Allali1,2, M. Abayomi1,2, S. Yuen1,2, S. Del Veliz1,2, Y. Mugabo1,2, F. Paré2, G. Lavoie3,1, P.P. Roux1,3, G.E. Lim1,2;

1Université de Montréal, 2Centre de Recherche du Centre hospitalier de l'Université de Montréal, 3Institut de recherche en immunologie et en cancérologie, Université de Montréal, Montréal, Canada.

Background and aims: Plakoglobin, also known as γ-catenin, is primarily involved in cell-cell adhesion; however, its additional roles in cellular processes have not been extensively studied. Its close homolog, β-catenin, is the main transcriptional coactivator of the Wnt signaling pathway. Previous studies have highlighted the Wnt/β-catenin signalling pathway’s anti-adipogenic properties and its ability to repress the expression of adipogenic genes to preferentially induce osteogenesis in vitro. While plakoglobin is capable of inhibiting β-catenin activity in a context-dependent manner, its role in adipogenesis remains unclear. Over-expression of the scaffold protein 14-3-3ζ in mice exacerbated high-fat diet-induced obesity. Following comparison of the 14-3-3ζ interactome in visceral adipose tissue between lean and obese mice, an enrichment of plakoglobin in the 14-3-3ζ interactome was detected, suggesting a contribution of plakoglobin to weight gain. Thus, we hypothesize that plakoglobin is an essential regulator of adipogenesis.

Materials and methods: In vitro assessment of plakoglobin’s role in adipogenesis was performed in murine 3T3-L1 pre-adipocytes and human adipose-derived stem cells, which were transfected with either overexpressing plasmids or siRNA targeting plakoglobin or β-catenin. RT-qPCR, immunoblotting, SuperTOPFlash, and Oil-Redo-O (ORO) staining were used to assess gene expression, protein abundance, Wnt/β-catenin transcriptional activity, and lipid content, respectively.

Results: Plakoglobin knockdown prior to adipocyte differentiation in 3T3-L1 pre-adipocytes reduces PPARγ2 protein abundance by 69% (p ≤ 0.05 based on two-way ANOVA and post-hoc tests), indicating an inhibition of adipogenesis. Conversely, β-catenin depletion upregulated PPARγ2 expression 180% (p ≤ 0.05 based on two-way ANOVA and post hoc tests). Rescue experiments via double knockdown of both plakoglobin and β-catenin did not affect PPARγ2 expression. In preliminary studies, plakoglobin depletion in human derived-adipose stem cells diminished PPARγ2 expression by 87% (n=2), while β-catenin knockdown led to a 270% (n=2) increase in PPARγ2 protein abundance. ORO staining showed that decreasing plakoglobin expression led to decreased lipid content in differentiated 3T3-L1 and human adipose-derived stem cells. Wnt transcriptional activity was diminished following knockdown of β-catenin by 46% (p ≤ 0.05 based on one-way ANOVA), while it was unaffected by plakoglobin depletion. Plakoglobin and β-catenin overexpression downregulated PPARγ2 abundance during adipogenesis by 63 and 34%, respectively (p ≤ 0.05 based on two-way ANOVA and post hoc tests).

Conclusion: Our study has revealed a novel and complex role for plakoglobin in adipogenesis. Comparable trends were observed in both 3T3-L1 pre-adipocytes and human adipose-derived stem cells, suggesting plakoglobin holds a species conserved effect. Additional in-depth studies are required to understand the mechanisms by which plakoglobin influences adipocyte differentiation.

Supported by: Canada Research Chairs, CIHR, Banting Research Foundation

Disclosure: F. Abou Azar: None.

29

Ampk γ2 is an essential player in adipogenesis and adipocyte function

Y. Cheng1, X. Hui2;

1Medicine, Hong Kong University, 2School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, Hong Kong.

Background and aims: Adipogenesis is the process in which the multipotent mesenchymal stem cells (MSCs) are differentiated into mature adipocytes through the pre-adipocyte stage. Adenosine monophosphate-activated protein kinase (Ampk) is a heterotrimeric serine/threonine kinase composed of one catalytic α subunit and two regulatory subunits β and γ. Ampk γ2 subunit has been overlooked and has long been believed to simply serve as a binding partner of the nucleotide. Here we uncovered the unique function of Ampk γ2 in determining adipogenesis and adipocyte function.

Materials and methods: Ampk γ2 over-expression or knockdown stromal vascular cells (SVCs) were established. We also generate adipocyte-specific Ampk γ2 knockout mice (AKO) via the Cre-LoxP strategy to elucidate the physiological effects of Ampk γ2 in adipose tissue. SVCs were isolated from the inguinal subcutaneous adipose tissue of AKO and wildtype (WT) mice. Selective deletion of the Ampk γ2 gene was induced by Tamoxifen. SVCs were differentiated to white adipocytes in vitro.

Results: Ampk γ2 was abundantly expressed in mature adipocytes. The expression of Ampk γ2 was gradually increased during adipogenesis but selectively reduced in obese adipose tissue. Knockdown or deletion of Ampk γ2 significantly compromised the adipogenic differentiation of SVCs into lipid-laden adipocytes with reduced expression of Fas, Pparγ, C/ebpα, and Fabp4. The worst adipogenesis was shown if Ampk γ2 was deleted on the first two days of differentiation, suggesting that Ampk γ2 was mainly involved in the mitotic clonal expansion phase of adipogenesis. In addition, the deletion of Ampk γ2 in mature adipocytes mitigates beige cell biogenesis. By immunofluorescence staining, we found that Ampk γ2 harbors a unique nucleus-targeting sequence. We also identified the genes that are altered by Ampk γ2 in mature adipocytes by RNA sequencing.

Conclusion: Ampk γ2 plays an essential role in adipogenesis and holds potential as a therapeutic target for obesity and metabolic diseases.

Supported by: Health and Medical Research Fund (06172346)

Disclosure: Y. Cheng: None.

30

Estrogen receptor beta knockdown reduces differentiation of preadipocytes from postmenopausal women

F. Ahmed, S. Hetty, M. Vranic, G. Fanni, M.J. Pereira, J.W. Eriksson;

Medical Science, Uppsala University, Uppsala, Sweden.

Background and aims: Estrogen deficiency in postmenopausal women is linked to redistribution of body fat, insulin resistance, and T2D. E2 signaling occurs mainly through estrogen receptors alpha (ESR1) and beta (ESR2). We have previously shown that ESR2 expression, but not ESR1, is higher in subcutaneous adipose tissue (SAT) from postmenopausal compared to premenopausal women. The functional impact of this on adipose tissue metabolism is not fully known. This study investigates the association between ESR2 expression and lipid and glucose metabolism in human subcutaneous adipose tissue (SAT) in subjects with and without T2D and in in vitro differentiated adipocytes.

Materials and methods: SAT were obtained by needle biopsies from 20 control and 20 T2D subjects with T2D matched for sex (10M/10F per group), age (58±11 vs 58±9 years) and BMI (30.8±4.6 vs 30.7±4.9 kg/m2). ESR2 gene expression in SAT was correlated to markers of obesity and glucose metabolism. ESR2 knockdown (KD) was performed in preadipocytes from postmenopausal women (age: 63±13 years, BMI 30.3±6.2 kg/m2, n=4) using CRISPR/Cas9 gene editing. In vitro differentiated KD adipocytes were characterized for differentiation rate, lipid storage, and glucose uptake.

Results: Postmenopausal females express higher levels of ESR2 in SAT compared to premenopausal women and males (1.5-fold; p<0.05) whereas ESR1 expression was not dependent on sex or menopausal status. We found that ESR2 expression was lower in subjects with obesity (BMI≥30) compared to non-obese subjects, independent of sex (p<0.05). Additionally, ESR2 expression did not differ between control and subjects with T2D. ESR2 expression in SAT from females, but not males, was negatively correlated with weight, markers of central adiposity (e.g. waist-to-hip ratio, visceral adipose tissue volume), and markers of fatty acid oxidation (e.g. CPT1A, CPTIB), and positively correlated with subcutaneous adipocyte size and markers related to lipid storage (e.g. LPL, ACACA), and glucose transport (e.g. GLUT4, AKT2) (p<0.05 all). In preadipocytes, ESR2 KD reduced preadipocyte differentiation (10-30%, p<0.05) compared to wild-type cultures on day 7 and day 14 of differentiation. This corresponded to transient reductions in the expression of differentiation markers (e.g. PPARG), markers of lipogenesis (e.g. LPL, FAS), lipolysis (e.g. HSL, PLIN1), and adipokines (e.g. ADIPOQ) (p<0.05 all). Glucose uptake was reduced in adipocytes in KD cultures (p<0.05), however, this effect was lost after normalizing for preadipocyte differentiation rate.

Conclusion: Our results indicate that ESR2 is involved in regulating human adipocyte differentiation and lipid storage, and higher ESR2 expression may promote greater energy deposition into SAT as seen after menopause. This provides insight into a potential molecular target to promote a healthy obesity phenotype.

Supported by: Swedish Diabetes Foundation, EXODIAB, the Ernfors Foundation, the Swedish Society for Medical Research

Disclosure: F. Ahmed: None.

OP 06 Intracellular regulation of insulin release

31

Glucose metabolites upstream of glyceraldehyde 3-phosphate dehydrogenase trigger activation of the mechanistic target of rapamycin complex 1 pathway in diabetic islets

E. Haythorne, M. Lloyd, J. Walsby-Tickle, J. Sandbrink, G. Cyranka, J. McCullagh, F.M. Ashcroft;

University of Oxford, Oxford, UK.

Background and aims: Hyperglycaemia leads to impaired mitochondrial respiration and hyperactivation of the nutrient-sensing mechanistic target of rapamycin complex 1 (mTORC1) pathway in pancreatic islets. Recent studies in non-islet cells suggest that glycolytic intermediates upstream of glyceraldehyde 3-phoshate dehydrogenase (GAPDH), fructose-1,6-bisphosphate (F1,6BP) and dihydroxyacetone phosphate (DHAP), signal glucose availability to mTORC1, leading to its activation. Thus, the aims of this study were to determine if this mechanism exists in beta cells/islets and if mTORC1 hyperactivation mediates mitochondrial dysfunction in diabetes.

Materials and methods: We utilised the βV59M mouse model where hyperglycaemia/diabetes is initiated via a tamoxifen-inducible KATP channel activating mutation in pancreatic beta cells. Islets were studied after 2 weeks of diabetes (>20mmol/l). Additionally, INS1 832/13 insulinoma cells were cultured at high (HG:25mM) or low (LG:5mM) glucose +/- the GAPDH inhibitor, koningic acid (5μM:KA), for 48hrs. To examine the impact of mTORC1 hyperactivation on mitochondrial efficiency, islets were incubated with/without the S6 kinase inhibitor PF-4708671 (10μM:S6Ki) for 48 hours. Metabolomics was performed using anion exchange chromatography mass spectrometry. GAPDH activity was measured biochemically. mTORC1 signalling was determined by the ratio of phosphorylated (p-S6) and total ribosomal protein S6 (tot-S6) using standard western blotting methods. Oxygen Consumption Rate (OCR) was monitored using an extracellular flux analyser.

Results: mTORC1 signalling was increased in diabetic islets (p-S6/total-S6 ratio: control = 0.36±0.02 vs diabetic = 0.70±0.082 AU, p<0.05; n=6). Compared to control islets, diabetes led to an increase in the relative abundance of F1,6BP (8.23±1.93-fold, p<0.01, n=4) and DHAP (1.43±0.04-fold, p<0.01, n=3). INS1 cells cultured at LG + KA recapitulated the effects of hyperglycaemia, with increased relative abundances of F1,6BP and DHAP, activation of mTORC1 and impaired GSIS. Diabetic islets displayed an attenuated glucose-stimulated OCR, relative to control islets, but this was significantly improved by incubation with S6Ki (diabetic = 34.45±10.72 vs diabetic+S6Ki 203.94±39.24% increase in OCR above baseline, p<0.001; n=7-8). The ATP-synthase inhibitor, oligomycin, produced significantly less inhibition of OCR in diabetic islets, compared to control islets, an effect that was prevented by mTORC1 inhibition (diabetic = 55.01±9.44 vs diabetic+S6Ki = 240.32±9.71% decrease in OCR, p<0.001; n=7-8).

Conclusion: We provide evidence that diabetes/hyperglycaemia leads to a build-up of glycolytic metabolites upstream of GAPDH which chronically activates mTORC1 in pancreatic islets. Additionally, our results suggest that hyperactivation of mTORC1 signalling is partially responsible for mitochondrial dysfunction in diabetic islets and may be involved in regulating ATP-synthase activity.

Supported by: MRC, Wellcome trust, Novo Nordisk

Disclosure: E. Haythorne: None.

32

Deletion of Carboxypeptidase E in beta cells disrupts proinsulin processing and accelerates streptozotocin-induced hyperglycaemia in mice

Y.-C. Chen1,2, A.J. Taylor2, K. Fok2, M. Komba2, X.-Q. Dai3, J.M. Fulcher4, A. Swensen4, A.E. Patterson2, R.I. Klein Geltink2, W.-J. Qian4, P.E. MacDonald3, C.B. Verchere1,2;

1Department of Surgery, University of British Columbia, Vancouver, Canada, 2BC Children's Hospital Research Institute, Vancouver, Canada, 3Department of Pharmacology, University of Alberta, Edmonton, Canada, 4Pacific Northwest National Laboratory, Richland, USA.

Background and aims: Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones, and is highly expressed in neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. In this study, we aimed to determine whether loss of Cpe in pancreatic beta cells is sufficient to disrupt proinsulin processing and accelerate development of diabetes in mice.

Materials and methods: Mice with Cpe deletion in pancreatic beta cells (βCpeKO; Cpefl/fl x Ins1Cre/+) and littermate control mice (Wt; Cpefl/fl and Cpefl/+) were treated with high fat diet (HFD) or multiple low dose streptozotocin (STZ), and body weight, plasma proinsulin, glucose tolerance, insulin sensitivity, and beta-cell area were assessed. Islets from βCpeKO mice were collected for analysis by electron microscopy, transcriptomics, peptidomics, proinsulin biosynthesis, dynamic insulin secretion, exocytosis, in vitro cell proliferation, metabolic flux, and live cell imaging. Mice with inducible beta-cell-specific Cpe deletion (iβCpeKO; Cpefl/fl x Pdx1CreER) were treated with HFD, and pancreatic beta-cell proliferation was analyzed.

Results: βCpeKO mice lack mature insulin granules in beta cells. Instead, they have 4.5-fold increased proinsulin protein in islets, and significantly elevated proinsulin immunoreactivity in plasma (37 vs. 1084 pM, Wt vs. βCpeKO, p < 0.05). However, glucose- and KCl-stimulated insulin secretion dynamics in βCpeKO islets, and insulin granule exocytosis in βCpeKO beta cells, remained largely intact. Upon HFD challenge, male and female βCpeKO mice showed comparable weight gain, glucose tolerance, and insulin sensitivity compared to Wt littermates. Interestingly, beta-cell area was increased in chow-fed βCpeKO mice (0.8 vs 1.6%, p < 0.05), and beta-cell EdU incorporation was elevated in HFD-fed iβCpeKO mice (2.6 vs 6.5%, p < 0.05). Consistent with these observations, islets from βCpeKO mice displayed increased proinsulin biosynthesis and elevated beta-cell proliferation (4.9 vs 7.4%, p < 0.05) upon high glucose treatment. In addition, transcriptomic analysis showed that βCpeKO beta cells have elevated glycolysis and HIF1α-target gene expression. Yet, high glucose treatment also led to mildly reduced mitochondrial membrane potential (1.53 vs. 1.27, p < 0.05), and increased mitochondrial (0.41 vs. 0.56, p < 0.05) and cytosolic (0.065 vs. 0.077, p < 0.05) reactive oxygen species in beta cells from βCpeKO mice. Lastly, after multiple low dose STZ treatment, βCpeKO mice had accelerated development of hyperglycemia (16.0 vs. 12.2 mM 7 days post-STZ, p < 0.05).

Conclusion: These findings confirm that Cpe mediates proinsulin processing in pancreatic beta cells. Lack of Cpe and impaired proinsulin processing is associated with beta-cell mass compensation via increased beta-cell proliferation. Under acute metabolic and oxidative stress challenges, Cpe and proper proinsulin processing are required to maintain beta-cell function and glucose homeostasis.

Supported by: This work is supported by JDRF and CIHR

Disclosure: Y. Chen: None.

33

Identification of TBL1 and TBLR1 as novel regulators of insulin transcription in beta cells

A.A. Walth1, R. Terron Exposito1, A.-C. König2, S.M. Hauck2, A. Feuchtinger3, P.E. MacDonald4, M. Rohm1;

1Institute for Diabetes and Cancer, Helmholtz Munich, Neuherberg, Germany, 2Research Unit Protein Science, Helmholtz Munich, Neuherberg, Germany, 3Core Facility Pathology & Tissue Analytics, Helmholtz Munich, Neuherberg, Germany, 4Department of Pharmacology, University of Alberta, Edmonton, Canada.

Background and aims: Insulin is a central regulator of glucose homeostasis and its synthesis by pancreatic β-cells is tightly controlled. On transcriptional level insulin synthesis is mediated by transcription factors and transcriptional cofactors such as TBL1 (transducin β-like 1) and TBLR1 (TBL-related 1). Originally identified as components of activator and repressor complexes such as the NCoR (nuclear receptor corepressor)/SMRT (silencing mediator for retinoid and thyroid receptors) repressor complex, TBL1 and TBLR1 gained interest as essential checkpoints of transcription by exchanging regulatory complexes ligand dependently. We here aimed to investigate the β-cell specific function of TBL1 and TBLR1.

Materials and methods: TBL1 and TBLR1 gene expression in islets was assessed by qPCR. To address the β-cell specific role of TBL1 and TBLR1 we generated and characterized mice with an Ins1-cre-driven double deletion of TBL1 and TBLR1 (TBL/RβKO). Islet morphology was examined through immunohistochemical analysis. TBL1 and TBLR1 interaction partners were identified in MIN6 cells using endogenous immunoprecipitation coupled to mass-spectrometry. Identified hits were verified in MIN6 and INS1 cells. Insulin promoter activity was assessed by a dual-luciferase reporter assay in INS1 cells upon siRNA mediated TBL1 and TBLR1 knock-down.

Results: We found TBL1 and TBLR1 levels to be dysregulated upon aging (n=4; p<0.05) and chronic hyperglycemia (n=6; p<0.05) in murine pancreatic islets. TBL/RβKO mice developed severe hyperglycemia (n=5; 600 mg/dl vs. 150 mg/dl at 12 weeks of age; p<0.0001) along with impaired insulin gene expression (n=6; 4.2-fold, p<0.0001). Alterations to islet architecture and reduced numbers of insulin positive cells (n=4; p<0.05) preceded the development of hyperglycemia. A transcriptome analysis revealed downregulation of β-cell identity genes, while disallowed genes - including genes from alternative islet cell types - were upregulated, suggesting dedifferentiation or impaired maturation of the β-cells. A subsequent interactome analysis identified components of the NCoR/SMRT repressor complex and PAX6 (paired box protein 6) as direct TBL1 and TBLR1 (n=3; p<0.01) interaction partners. We also found the components of the NCoR/SMRT complex to directly interact with the islet cell master regulator PAX6, suggesting TBL1 and TBLR1 as possible regulators of PAX6-mediated gene expression. Indeed, we found the insulin promoter, which is a PAX6 target, under direct TBL1 and TBLR1 control.

Conclusion: Our data support the notion that TBL1 and TBLR1 are novel regulators of insulin gene expression and β-cell functionality. Through interaction with NCoR /SMRT and PAX6, a critical regulator of β-cell maturity and identity, TBL1/TBLR1 may contribute to the maintenance of β-cell identity and its loss in pathologic conditions.

Disclosure: A.A. Walth: None.

34

Single-cell-resolution imaging of alpha/beta cell metabolic response to glucose stimulation in living human-derived Langerhans islets

F. Cardarelli1, F. Azzarello1, L. Pesce1, V. De Lorenzi1, G. Ferri1, M. Tesi2, S. Del Guerra2, P. Marchetti2;

1Scuola Normale Superiore, 2Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.

Background and aims: A cascade of highly regulated biochemical processes connects glucose stimulation to hormone secretion in specialized cells within Langerhans islets of the mammalian pancreas, the α and β cells. Given the importance of this process for systemic glucose homeostasis, non-invasive and fast strategies capable to monitor quantitatively α- and β-cells metabolic responses in living islets are highly desirable. Despite the efforts, however, no report thus far was able to probe the specific signature of α- and β-cells response to glucose stimulation in living human islets (HIs).

Materials and methods: To tackle this issue we used here a combination of label-free Fluorescence Lifetime Imaging Microscopy (FLIM) in living HIs with post-fixation immunofluorescence. More in detail, by the phasor approach to FLIM we discriminated the free and protein-bound forms of NAD(P)H molecules in optical sections of living HIs and, by means of their ratio, we defined the tissue metabolic shift upon pulsed glucose stimulation. Then, by using post-fixation immunofluorescence, we identified α and β cells, finally matching single-cell identities with their metabolic response. A cohort of 4 healthy donor patients was included in this study.

Results: A total of 15 islets were measured from the above-mentioned cohort, with 312 α cells and 654 β cells identified. We observed a neat metabolic shift towards oxidative phosphorylation in the great majority of β cells, in keeping with previous results from rat/mouse β cells. By contrast, we observed a wide spectrum of metabolic shifts in α cells, from glycolysis- to oxidative-phosphorylation-oriented ones, apparently contradicting previous reports which assessed an univocal glycolytic response to glucose in rat/mouse α cells. Interestingly, at the patient level, the heterogeneous α-cell responses reveal an inverse proportionality with respect to the amount of insulin secreted (independently probed by an ELISA assay): the higher the insulin secreted, the more glycolysis-oriented the metabolic shift measured. Noteworthy, such inverse proportionality transforms into marked linear anti-correlation (Pearson’s correlation coefficient: -0,997) if α- and β-cell responses are summed and plotted against the amount of secreted insulin (see Graph).

Conclusion: The emerging picture indicates the synergistic action of α and β cells as key signature of HI metabolic response to glucose and, in turn, as a basic constituent for the regulation of systemic glycaemia. While demonstrating the effectiveness of an optical-microscopy-based protocol to measure the specific responses of α and β cells in a living human Langerhans islet, present results pave the way to similar investigations to be conducted in diabetes and/or using drugs to restore cell functionality.

figure j

Supported by: European Research Council (grant agreement N. 866127, project CAPTUR3D)

Disclosure: F. Cardarelli: Grants; ERC Consolidator grant.

35

Mitochondrial AASS-mediated l-lysine degradation is required to maintain beta cell function

L. Cataldo1,2, Q. Gao1, K. Trost1, M. Fex2, H. Mulder2, T. Moritz1;

1The Novo Nordisk Foundation Centre for Basic Metabolic Research, University of Copenhagen., Copenhagen, Denmark, 2Lund University Diabetes Center, Lund University., Malmo, Sweden.

Background and aims: Aminoadipate-Semialdehyde Synthase (AASS) is a mitochondrial-located bifunctional enzyme with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities, involved in the first two steps in the catabolic pathway of L-lysine, an insulinotropic amino acid in humans. AASS-mediated L-lysine catabolism may contribute to fueling insulin secretion since Acetyl-CoA and L-glutamate are generated in the pathway; the former feeds the TCA cycle while L-glutamate acts as a metabolic coupling factor (MCF) for glucose-stimulated insulin secretion (GSIS). In addition, L-lysine cell accumulation is cytotoxic and a mutation in AASS was recently identified as responsible for familial hyperlysinemia, an autosomal recessive disorder. Thus, AASS-mediated degradation of L-lysine may play a dual role in β cell function by maintaining L-lysine levels at physiological levels and providing L-lysine-derived metabolites that amplify GSIS.

Materials and methods: AASS mRNA levels in human islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets and INS1 832/13 cells were transfected with scramble (control) and AASS siRNAs (AASS-KD) and GSIS was assessed by ELISA. Live cell cytosolic calcium was measured by Fluo4 dye and confocal microscopy. Glucose-stimulated mitochondrial metabolism was investigated in AASS-KD INS1 832/13 cells by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis.

Results: AASS mRNA levels were reduced in pancreatic islets from T2D vs ND donors (p=2.7e-5, n=188) and correlated negatively with hyperglycemia (HbA1c) (r=-0.158, p=0.009, n=169) but positively with GSIS stimulatory index (r=0.10, p=0.026, n=182). In vitro studies showed that AASS silencing reduced basal and GSIS in human islets. Accordingly, AASS silencing reduced insulin secretion in INS1 832/13 cells in response to basal and high glucose, as well as in response to pyruvate and cAMP-promoting agents (IBMX-FSK), but not in response to high K+. Increased cytosolic calcium responses were accompanied by reduced cytosolic ATP:ADP ratio and ATP-linked mitochondrial respiration in AASS-KD vs control cells. Metabolomics analysis indicated altered glutamate metabolism and mitochondrial TCA cycle activity, and a Warburg effect.

Conclusion: Our data suggest that AASS-mediated lysine degradation is an active metabolic pathway required to maintain normal glutamate production, mitochondrial TCA cycle and OXPHOS function, and cellular calcium homeostasis, as well as insulin secretion. Reduced AASS expression with consequent decreased AASS-mediated L-lysine degradation pathway may contribute to β cell dysfunction in T2D.

Disclosure: L. Cataldo: None.

36

Quantification of the dynamics of Ins2 gene activity

J.C.M. Chu1, H. Modi1, S. Skovsø1, C. Ellis1, N.A.J. Krentz2, Y.B. Zhao1, H. Cen1, N. Noursadeghi1, E. Panzhinskiy1, Y. Xia1, S. Xuan3, M.O. Huising4, T.J. Kieffer1, F.C. Lynn2, J.D. Johnson2;

1Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada, 2Department of Surgery, University of British Columbia, Vancouver, Canada, 3Department of Medicine Hematology and Oncology, Columbia University, New York City, USA, 4Department of Neurobiology, Physiology and Behavior, UC Davis, Davis, USA.

Background and aims: In pancreatic β-cells, the insulin gene has been shown to be expressed in a wide distribution, with examples of “extreme” β-cells exhibiting >2 fold higher insulin gene activity. Pseudo-time analyses of human single cell RNA sequencing data have suggested that cells may transition between high and low expression states of the INS gene. However, the mechanisms of these dynamics have yet to be elucidated. The the phenomenon of switching between low and high INS gene activity states has also yet to be observed in real-time.

Materials and methods: Ins2GFP knock-in mice, with the second exon of the wild-type Ins2 gene being replaced with GFP, were used for monitoring of endogenous insulin gene expression. We investigated the temporal kinetics of endogenous insulin gene activity using live-cell imaging, with complementary experiments employing FACS and single-cell RNA sequencing analyses in β-cells isolated from Ins2GFP knock-in mice. We applied inhibitors and activators of pathways related to insulin production, endoplasmic reticulum stress, and insulin secretion to determine how perturbations to β-cell function may impact Ins2 gene behavior.

Results: Live-cell imaging captured Ins2 gene activity dynamics in single β-cells over time and revealed two states in GFP expression (Ins2GFP(high), Ins2GFP(low)), consistent with bimodal expression states observed with our FACS analysis and immunofluorescence staining experiments. Autocorrelation analysis identified cells with oscillating behavior, with oscillation periods of ~17 hours. RNA velocity and hierarchical clustering analyses on single cell RNA sequencing data on cells from Ins2GFP mice revealed that β-cells with higher levels of Ins2 gene activity had a more mature β-cell profile, with genes such as Pdx1, Ucn3, and Nkx6-1 being significantly correlated with high GFP mRNA (p<0.01). Perturbation of insulin gene activity with inhibitors and activators of insulin production were shown to influence Ins2 gene activity, with some small molecules, including GLP-1, arginine, and sodium butyrate inducing transitions from Ins2GFP(low) states to Ins2GFP(high) states.

Conclusion: In conclusion, we identify a subset of pancreatic β-cells with dynamic Ins2 gene activity. Our observations define a previously uncharacterized form of β-cell plasticity in both basal and induced conditions. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.

Supported by: CIHR operating grant

Disclosure: J.C.M. Chu: None.

OP 07 Finding a phenotype for diabetic kidney disease

37

Genome-wide analysis of treatment-resistant hypertension in individuals with and without diabetes

A.A. Antikainen1,2, H. Sánez Tähtisalo2,3, S. Mutter1,2, R. Lithovius1,2, P.-H. Groop1,2, K. Kontula2,3, T. Hiltunen2,3, N. Sandholm1,2;

1Folkhälsan Research Center, 2Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, 3Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background and aims: Hypertension is a major risk factor for CVD. Almost half of individuals with hypertension fail to reach blood pressure (BP) targets despite pharmacological treatments. A subset of them have treatment-resistant hypertension (RHTN), which is defined as BP above the treatment target with at least three antihypertensive drugs, of which one is a diuretic, or controlled BP with four or more drugs. RHTN increases the risk for CVD, especially in diabetes. RHTN genetics has not been excessively studied, thus, we aimed to find genomic loci linked to RHTN in type 1 diabetes (T1D) and in the general population.

Materials and methods: We performed genome-wide association studies for RHTN in T1D in the Finnish Diabetic Nephropathy Study (606 individuals, 288 with RHTN) and in the general population in the LIFE-Fin Study (625 individuals, 297 with RHTN). Analyses were adjusted for age, sex and genomic data principal components in the minimal model, and additionally for BMI, albuminuria, and eGFR in the full model. We meta-analysed the cohorts and attempted replication in a third Finnish cohort, GENRES (N≈220), where standardized mean BP response to four different antihypertensive monotherapies was used as an indicator of drug treatment responsiveness. Finally, we performed gene and gene-set analyses with MAGMA on the FUMA platform.

Results: We discovered two variants in the minimal model with near genome-wide significance: rs1484486 (minor allele frequency (MAF)=32%, odds ratio (OR)=0.54, p=7.76×10-8) within LOC105369168, and rs11151487 (MAF=14%, OR=0.47, p=5.36×10-7) within an intron of CCDC102B. In the full model, we found also rs61009649 (MAF=38%, OR=1.81, p=4.34 ×10-7) close to a pseudogene. We were unable to replicate these in GENRES. Of note, we discovered further suggestive loci of which one from the full model (rs3138242, MAF=10%, OR=0.44, p=9.26×10-6) replicated for systolic office BP response in GENRES (β=-0.21, p=0.03, N=226). The variant is located within an intron of DCN, coding for decorin, which plays an important role in connective tissues. Interestingly, the variant is an expression QTL of DCN in fibroblasts (https://gtexportal.org/). Gene-set scoring revealed significant biological pathways (e.g., the SLRP pathway, p=5.0×10-9), and enrichment of genes with expression in heart tissues (Figure 1).

Conclusion: We studied the genetics of RHTN in individuals with and without diabetes and identified multiple candidate loci, including a variant linked to expression of decorin, and performed gene scoring revealing a significant enrichment of genes with heart tissue expression stressing the link between RHTN and CVD.

figure k

Supported by: The Finnish Foundation for Cardiovascular Research

Disclosure: A.A. Antikainen: None.

38

DNA methylation is a risk factor for kidney failure in individuals with type 1 diabetes

A. Syreeni1,2, E.H. Dahlström1, L.J. Smyth3, Y. Gupta3, C. Forsblom1,2, J. Kilner3, G.J. McKay3, A. Maxwell3, A. McKnight3,2, P.-H. Groop1, N. Sandholm1,2;

1Folkhälsan Research Center, Helsinki, Finland, 2Department of Nephrology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 3Molecular Epidemiology Research Group, Centre for Public Health, Queen’s University Belfast, Belfast, UK.

Background and aims: We previously identified DNA methylation differences at multiple CpG loci in a cross-sectional study of individuals with or without diabetic kidney disease (DKD). Here, we aimed to study DNA methylation as a risk factor for the development of kidney failure in individuals with type 1 diabetes and DKD.

Materials and methods: The study included 397 individuals with type 1 diabetes and macroalbuminuria at baseline from the Finnish Diabetic Nephropathy (FinnDiane) Study. At baseline, the mean (SD) age was 43 (±10.8) years, and 38% were women. Macroalbuminuria (>200 μg/min or >300 mg/24h) was determined from two of three overnight or 24 h urine collections. The study participants were followed up until either kidney failure developed or December 31, 2017. Data on kidney failure requiring dialysis and/or a transplant was collected from the Finnish Care Register for Health Care, study visits, or medical files. Genome-wide blood-derived DNA methylation data was generated for the Infinium HD Methylation EPIC BeadChips (Illumina) in Belfast. After quality control, we extracted M-values (M = log2(β / (1-β)) for 763,064 CpG sites using RnBeads v.2.6.0. M-values for each CpG site were analysed separately with the Cox proportional-hazards model with sex, baseline age, and six estimated white blood cell counts as covariates.

Results: During a median of 7.2 (interquartile range: 2.9-14.0) years of follow-up, 196 individuals developed kidney failure. Eleven CpGs were associated with developing kidney failure with p < 6.6×10-8 - a p-value threshold corrected for the number of studied CpGs. The top CpG cg17944885 is located on chromosome 19 between genes ZNF788P and ZNF625-ZNF20. Higher methylation at this locus was a risk factor for kidney failure (HR [95%CI] = 2.32 [1.95, 2.76], p = 1.4×10-21). Seven significant CpGs were located in or near genes; cg23597162 in JAZF1, cg12272104 in DAZAP1, cg21871803 in AHCYL2, cg12065228 in PQLC2, cg26236214 in ARHGEF7, cg19942083 in the promoter of PTPN6, and cg03262246 <1500 bp from the transcription start site of CDKN2AIPNL. In an independent cohort look-up from Belfast of kidney failure vs controls with no evidence of kidney disease (n = 519), 10 of these CpGs were significantly associated (p < 10-8). Additionally, cg17944885 was strongly associated with DKD in our cross-sectional meta-analysis of the FinnDiane and Belfast cohorts (tot n = 1,304, p = 2.0×10-44). In a cohort of 473 individuals with diabetes from the Chronic Renal Insufficiency cohort, six of eleven significant CpGs were associated with eGFR in the whole blood DNA methylation analysis (3.7×10-13p ≤ 0.05). Furthermore, all six of our eleven top CpGs that were available in the epigenome-wide meta-analysis for eGFR in 33,605 individuals from the Chronic Kidney Disease Genetics Consortium were significantly (p < 1.1×10-7; cg17944885 and CpGs in JAZF1 and PQLC2) or nominally (p < 0.05; CpGs in or near DAZAP1, AHCYL2, and PTPN6) associated with eGFR in their study.

Conclusion: DNA methylation at several CpGs show consistent associations with kidney function and the risk of developing kidney failure.

Supported by: NIH (1R01DK105154-01A1) GENIE II

Disclosure: A. Syreeni: None.

39

The genetic background predicts the type of renal lesions and the progression of fibrosis in patients with type 2 diabetes

P. Pontrelli, C. Cinefra, F. Conserva, M. Fiume, A. Gallone, F. Pesce, F. Giorgino, L. Gesualdo;

University of Bari Aldo Moro, Bari, Italy.

Background and aims: Diabetic Nephropathy (DN) is the major causes of end-stage renal failure worldwide. The occurrence of either real DN or non-diabetic renal disease (NDRD) in diabetic patients explains why kidney biopsy remains the gold standard for accurate diagnosis and treatment. Thus, there is immediate need for non-invasive biomarkers to discriminate DN and NDRD, and/or to predict DN onset. Our group has largely demonstrated that only those patients featuring specific DN lesions show an accumulation of Lys63-ubiquitinated proteins at tubular level. The aim of this project was to identify single nucleotide polymorphisms (SNPs) associated to genes involved in Lys63 ubiquitination, and asses whether these SNPs are able to predict the different type of renal damage as well as the progression of kidney disease in type 2 diabetic patients.

Materials and methods: We selected 10 HapMap SNPs within coding and regulatory sequences of both miR27b-3p, miR1228-3p and Ube2V1 gene, all involved in Lys63-ubiquitination, in order to evaluate their diagnostic and prognostic value. 201 patients were enrolled in this study, in particular: diabetic patients with a biopsy-proven diagnosis of DN (DN), diabetic patients with a biopsy-proven diagnosis of other nephropathy in the absence of DN (NDRD), diabetic patients without clinical signs of impaired renal function (T2D), diabetic patients with a biopsy-proven coexistence of both conditions (ND+NDRD), non-diabetic patients with glomerulonephritis (CKD) and non-diabetic patients without renal damage (CTRL). For each patient we analyzed: i) 10 selected HapMap SNPs using TaqMan Real-Time PCR; ii) the glomerular and tubulointerstitial fibrosis at the kidney level, quantified following Sirius Red staining of kidney biopsies using the Aperio ImageScope slide scanner; iii) relevant clinical parameters at the time of renal biopsy and at the follow-up.

Results: The analyzed SNPs showed a different genotype frequency among all the patients classes. Interestingly, SNPs rs4759275, rs4759277, rs4744422, rs3802456 showed a statistically significant difference in genotypes frequency comparing DN patients with CEU Population (p<0.04, 0.05, 0.002, 0.001 respectively) and a control cohort enclosing CTRL and T2D (p<0.02, 0.05, 0.001, 0.04 respectively). SNPs rs761214, rs10761364, rs2306692 genotypes frequency was statistically different among DN patients and the control cohort (p<0.001). The genotype frequencies of the SNPs rs10761364 (p<0.01) and rs7853195 (p<0.04) were significantly related to tubular fibrosis in DN patients, while the SNPs rs4744422 (p<0.03) and rs761214 (p<0.02) to the glomerular one. In order to evaluate the diagnostic power of the identified SNPs, we used a logistic regression model, and we observed that, when adjusted for age, sex, eGFR and glycaemic index, SNP rs10761364, discriminates DN from NDRD (p<0.05; OR=1.002-1.008; 95% CI).

Conclusion: Our data demonstrated that the allelic forms of the analyzed SNPs are linked to different renal lesions in diabetic patients. These results could provide the starting point for the creation of a new non-invasive diagnostic system based on clinical and genetic data.

Supported by: BeatDKD IMI2 Project

Disclosure: P. Pontrelli: None.

40

Loss of the transcription factor Tcf21 in adult podocytes leads to susceptibility in diabetic kidney disease

N. Teramoto1, Y. Maezawa1, T. Minamizuka1, M. Koshizaka1, Y. Endo2, Y. Akimoto3, K. Yokote1;

1Department of Endocrinology, Hematology and Gerontology, Chiba University Graduate School of Medicine, Inohana, Chuo-ku,Chiba, 2Kazusa DNA Research Institute Laboratory of Medical Omics Research, Kazusakamatari, Kisarazu, Chiba, 3Department of Microscopic Anatomy, Kyorin University School of Medicine, Mitaka city, Tokyo, Japan.

Background and aims: Diabetic kidney disease (DKD) is the most common cause of dialysis induction in Europe. Tcf21/Pod1 is a basic helix-loop-helix transcription factor that is essential for the development of the lungs, heart, and kidneys. Previously, we reported that Tcf21 regulates podocyte development. However, the precise role of Tcf21 in mature individuals and DKD remains unclear.

Materials and methods: We generated mice that lacked Tcf21 only in podocytes after birth (iKO) using a doxycycline induction system. We also constructed a diabetes mouse model using streptozotocin injections. In addition, we used cultured human podocytes to investigate the effects of Tcf21 overexpression and the factors that regulate Tcf21 expression.

Results: When the Tcf21 gene was deleted specifically in podocytes in 3-week-old mice, urinary protein was not observed. In contrast, when diabetes was induced in these mice, massive urinary protein and strong glomerulosclerosis was present in 60% of iKO mice. Ultrastructural analysis revealed glomerular basement membrane thickening and podocyte foot process effacement, suggesting that acquired podocyte-specific Tcf21 KO mice are susceptible to DKD. In addition, RNA-seq analysis of Tcf21-overexpressing podocytes showed accumulation of genes involved in the interferon pathway, extracellular matrix production, senescence, and autophagy in gene ontology analysis. Analysis of individual genes showed upregulation of interferon-related genes such as IFI6 and IFI27, increased expression of SERPINE1 (PAI-1), which is one of the senescence-associated secreted phenotypes, and decreased expression of HMGA2, whose SNIP is known to correlate with susceptibility to DKD. In addition, Tcf21 expression was suppressed by 58% and 27% after TGF-β and high glucose plus insulin stimulation, respectively.

Conclusion: Our results suggest that Tcf21 protects against DKD in mature individuals. Elucidation of the mechanism by which Tcf21 prevents DKD may lead to the identification of novel therapeutic targets.

Disclosure: N. Teramoto: None.

41

Empagliflozin attenuates obesity-related kidney dysfunction and NLRP3 inflammasome activity through the HO-1/adiponectin axis

T. Ye, J. Zhang, J. Shi, C. Kan, F. Han, N. Hou, X. Sun;

Department of Endocrinology and Metabolism, Affiliated Hospital of Weifang Medical University, Weifang, China.

Background and aims: Empagliflozin (EMPA) is a novel sodium-glucose cotransporter 2 inhibitor (SGLT2i) that produces protective cardiovascular-renal outcomes in patients with diabetes. The heme oxygenase-1 (HO-1)/adiponectin axis is an essential antioxidant system with anti-apoptotic and anti-inflammatory properties. This study explored whether EMPA improves obesity-related kidney disease by regulating the renal HO-1-mediated adiponectin axis.

Materials and methods: Four-week male C57BL/6J mice were randomly assigned to control, high-fat diet (HFD) and EMPA (10 mg/kg) groups. Mice in the control group were fed a regular diet, while mice in the other groups were fed an HFD. After receiving an HFD for 24 weeks, mice in the EMPA group were administered EMPA (10 mg/kg/day) by oral gavage for another 8 weeks. Blood biochemical and urinary albumin-to-creatinine ratios (UACR) were measured. The morphology and microstructure of the kidney were analyzed by histopathology and transmission electron microscopy. RNA-seq analysis of differential gene expression in kidneys was performed. Renal NLRP3 inflammasome with related cytokines and HO-1/adiponectin were determined.

Results: HFD mice showed significant metabolic abnormality and renal injury, including increased body weight, fat mass, urinary albumin excretion, morphologic changes, and lipid accumulation. EMPA treatment significantly decreased the final body weight (44.87 ± 1.42 g vs. 49.67 ± 1.48 g, P<0.05), fat mass (11.75 ± 0.78 g vs. 14.56 ± 0.43 g, P<0.05), and fat/weight (26.08 ± 1.09% vs. 29.37 ± 0.75%, P<0.05), compared with HFD alone. Besides, EMPA significantly improved glucose hemostasis, decreased FFA (746.30 ± 56.59 μmol/L vs. 1303.00 ± 81.14 μmol/L, P<0.05) but had no beneficial effects on triglyceride (29.31 ± 2.71 mg/dL vs. 32.87 ± 1.69 mg/dL, P>0.05). Furthermore, EMPA decreased UACR (21.01±1.99 μg/μmol vs. 45.24±4.71 μg/μmol, P<0.05), attenuated kidney injury, including reduced glomerular hypertrophy, renal fibrosis, mitochondria swell, and lipid accumulation. RNA-seq analysis showed that the differentially expressed genes shared in EMPA vs. HFD and HFD vs. control were enriched in GO and KEGG categories associated inflammation process. HFD mice showed increased renal NLRP3 activity and reduced HO-1/adiponectin axis, indicating excessive inflammation. However, EMPA significantly enhanced renal HO-1/adiponectin axis and decreased NLRP3 activity, recovering the anti-inflammation.

Conclusion: EMPA treatment improved metabolic disorders and protected against obesity-related kidney disease by activating the HO-1/adiponectin axis and reducing NLRP3 inflammasome activity. Kidney transcriptome analysis revealed that EMPA affects essential genes closely associated with inflammation. Our findings provide new knowledge concerning the mechanism for SGLT2i-mediated renal protection in obesity.

Supported by: NSFC (81870593, 82170865)

Disclosure: T. Ye: None.

42

Identification of markers for predicting the onset of chronic kidney disease in older people with type 2 diabetes by metabolomic profiling: Edinburgh Type 2 Diabetes Study

J. Krasauskaite1, B.R. Conway2, C.J. Weir1, Z. Huang1, J.F. Price1;

1Usher Institute, University Of Edinburgh, 2The Queen's Medical Research Institute, University Of Edinburgh, Edinburgh, UK.

Background and aims: Renal disease affects a large proportion of people with type 2 diabetes and it is associated with excess morbidity/ mortality. While well-established clinical biomarkers, namely estimated glomerular filtration rate (eGFR) and albuminuria are used in routine screening, these markers do not explain all of the risk. Hence, the search for new markers is a high priority. Metabolomics may reveal novel markers of chronic kidney disease (CKD) that could aid identification of patients at higher risk of renal impairment and improve risk prediction of incident CKD. We aimed to identify significant associations between metabolites and the clinically relevant outcome of incident CKD in a Scottish population of older people with type 2 diabetes and to evaluate the ability of metabolites to predict CKD onset.

Materials and methods: The Edinburgh type 2 diabetes Study (ET2DS) is a population-based cohort of 1,058 adults (49% female) with type 2 diabetes, aged 60-75 years. Nightingale metabolomic platform was used to measure 149 serum metabolite concentrations at baseline. Kidney function was determined by eGFR, calculated using the CKD-EPI equation. Incident CKD was defined as 2 of 3 eGFR records <60mL/min/1.73 m2 during follow-up. An initial multivariable-adjusted discovery screen considered the correlation between each metabolite and baseline eGFR (adjusted for age and sex). Metabolites that were significantly associated with eGFR were then related to incident CKD events in logistic regression analysis adjusted for known clinical risk factors. Risk prediction analysis involved refitting a published risk prediction model for incident CKD to evaluate the complementary value of significant metabolites.

Results: There were 823 participants in ET2DS with no CKD based on eGFR records at baseline and 217 (26%) experienced new onset CKD during follow-up (median= 6.8 years [IQR 0.9- 7.6]). Corrected for multiple testing, 68 metabolites were significantly associated with baseline eGRF (Bonferroni corrected p<0.00034). Of these, only amino acid phenylalanine (Phe) was significantly associated with incident CKD after adjustment for known clinical risk factors (OR 0.73 [95% CI 0.60- 0.89], p=0.002). Phe was added to the published risk prediction model containing the clinical variables (eGFR, age, sex, BMI, use of diabetes medications, cardiovascular disease history, smoking, hypertension, HbA1c, albumin-to-creatinine ratio). Phe remained significant in this model and higher levels of Phe reduced risk of CKD onset (HR 0.80, [95% CI 0.68-0.93] per unit of SD, p-value= 0.004). However, Phe yielded only a small improvement in risk prediction (original model concordance (c )-statistic 0.81 [95% CI 0.79- 0.84], model +Phe c-statistic 0.82 [95% CI 0.79-0.84].

Conclusion: Amino acid Phe was associated with incident CKD in people with type 2 diabetes, although, it did not improve an already well performing risk prediction model. It is possible to hypothesise that a more sophisticated multivariable analysis may reveal a combination of metabolites associated with CKD onset that together may improve the risk prediction.

Supported by: MRC

Disclosure: J. Krasauskaite: None.

OP 08 Cardiovascular disease mechanisms: something new on the table?

43

C-reactive protein, C-peptide, and risk of cardiovascual events and mortality after type 2 diabetes diagnosis: a Danish cohort study

A.D. Kjaergaard1,2, A. Gedebjerg1,3, M. Bjerre3,2, J. Nielsen4, J. Rungby5, I. Brandslund6, M. Maeng7, H. Beck-Nielsen4, A.A. Vaag8, H.T. Sørensen1,9, T.K. Hansen2,9, R.W. Thomsen1,9;

1Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, 2Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, 3Clinical Medicine, Aarhus University, Aarhus, 4Steno Diabetes Center Odense, Odense University Hospital, Odense, 5Endocrinology, Bispebjerg, Copenhagen University Hospital, Copenhagen, 6Department of Clinical Biochemistry, Lillebaelt Hospital, Vejle, 7Department of Cardiology, Aarhus University Hospital, Aarhus, 8Steno Diabetes Center Copenhagen, Copenhagen University Hospital, Copenhagen, 9Clinical Medicine, Aarhus University, Aarhus N, Denmark.

Background and aims: We investigated the relationship between high-sensitivity C-reactive protein (hsCRP, a marker of low-grade inflammation), alone or in combination with C-peptide (a marker of insulin resistance), and risk of cardiovascular events (CVEs) and mortality in patients with recent-onset type 2 diabetes (T2D) and no hospital history of CVEs.

Materials and methods: We measured serum hsCRP in 7,301 patients and C-peptide in 5,765 patients with recent-onset T2D and followed them for a first CVE, including myocardial infarction, stroke, coronary revascularization, and cardiovascular death, and death from any cause.

Results: High (>3 mg/L) versus low (<1 mg/L) hsCRP was associated with an increased CVE risk during a median follow-up of 4.8 years (adjusted hazard ratio: 1.45 [95% confidence interval: 1.08-1.96]), and with strongly increased all-cause mortality (2.49 [1.90-3.27]), mainly driven by cancer mortality. Compared to patients with low levels of both hsCRP (≤3 mg/L) and C-peptide (<1470 pmol/L), those with high levels of both biomarkers had highest risks of CVE (1.62 [1.11-2.36]) and all-cause mortality (2.42 [1.77-3.29]). The risk of CVE increased more with high C-peptide alone (1.54 [1.09-2.18]) than high hsCRP alone (1.37 [1.00-1.88]). In contrast, the risk of all-cause mortality increased much more with high hsCRP alone (1.90 [1.46-2.49]) than with high C-peptide alone (1.15 [0.82-1.61]).

Conclusion: In a contemporary cardiovascular prevention setting, elevated hsCRP is a much weaker predictor of future CVE than of all-cause mortality in patients with early T2D. C-peptide is a more accurate predictor of CVE risk than hsCRP, emphasizing the importance of targeting insulin resistance for prevention of CVE.

figure l

Supported by: Novo Nordisk Foundation

Disclosure: A.D. Kjaergaard: None.

44

Mechanistic insights into the effects of empagliflozin in patients with type 2 diabetes and heart failure

A. Elrakaybi1,2, K. Laubner1, Q. Zhou3,4, G. Päth1, H. Schmitt3, M.J. Hug5, J. Seufert1;

1Division of Endocrinology and Diabetology, Department of Medicine II, Medical Center – University of Freiburg, Freiburg, Germany, 2Department of Clinical Pharmacy, Ain Shams University, Cairo, Egypt, 3Department of Cardiology and Angiology I, Heart Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany, 4Department of Cardiology, University Hospital Basel, Basel, Switzerland, 5Pharmacy, Medical Center – University of Freiburg, Freiburg, Germany.

Background and aims: Sodium- glucose co-transporter (SGLT) 2 inhibitors were the first antidiabetic drugs to demonstrate remarkable reductions in CV mortality and hospitalisation for heart failure (HF) in patients with and without diabetes. However, the exact mechanisms are still under debate. We aimed to investigate the effect of empagliflozin on certain plasma biomarkers and potential cardiac remodelling parameters, while also determining their possible associations in diabetic patients with HF. In-vitro experiments aimed to evaluate potential anti-inflammatory effects of empagliflozin.

Materials and methods: Adult patients with type 2 diabetes (T2D) and HF with either left ventricular ejection fraction (LVEF) ≤ 45% (EFFORT-1) or LVEF > 45% (EFFORT-2) were recruited. The patients received either 25 mg empagliflozin or placebo and were followed up for 48 weeks. Endothelin-1, galectin-3, insulin-like growth factor binding protein (IGFBP)-7 and kidney injury molecule (KIM)-1 were measured using ELISA at baseline and after 2, 12, 24 and 48 weeks of randomization. Left ventricular end- diastolic diameter (LVEDD) and posterior wall diameter were evaluated at baseline, week 12, 24 and 48. Change from baseline and the corresponding differences between treatment groups were determined with a mixed effects repeated measure analysis using treatment, visit and treatment-by-visit interaction as fixed effects and baseline value as a covariate. The means and differences between log-transformed data were back transformed to original scale as ratios. Pearson or Spearman correlation coefficients were calculated. The expression of inflammatory proteins was measured in HUVECs after high glucose and TNF-α stimulation ± empagliflozin.

Results: 63 patients were recruited, 24 in EFFORT-1 and 39 in EFFORT-2. Empagliflozin significantly reduced KIM-1 levels by 38% at week 48 in EFFORT-2 compared to placebo (95% CI; -57%, -13%), with no significant differences observed in EFFORT-1. Empagliflozin improved cardiac remodelling parameters via reduction of LVEDD at all time points in EFFORT-1, reaching significance only at week 24 with difference -7.88 (95% CI; -12.2, -3.61) from placebo, whereas borderline non-significant differences were shown at week 48 [-4.40 (95% CI; -8.84, 0.042)]. In EFFORT-2, empagliflozin significantly reduced posterior wall diameter at week 48 [-2.29 (95% CI; -3.60, -0.99)]. No clinically meaningful correlations were observed between the plasma biomarkers and any of the measured parameters. In HUVECs, empagliflozin did not significantly impact the expression of inflammatory markers.

Conclusion: Empagliflozin demonstrated as shown by a decrease in the biomarker KIM-1 a renal tubular protective effect in HF patients, while it contributed to the reduction of adverse cardiac remodelling. These CV benefits of empagliflozin can most likely not be explained by anti-inflammatory actions.

Supported by: This trial was funded through an IIT Grant by Boehringer Ingelheim. A. E. was supported by DAAD

Disclosure: A. Elrakaybi: Grants; This trial was funded through an IIT Grant by Boehringer Ingelheim. A. E. was supported by the German Academic Exchange Service (DAAD) – German Egyptian Research Long-Term Scholarship (GERLS) Program.

45

Effect of semaglutide on MACE by baseline kidney function in participants with type 2 diabetes and high risk of cardiovascular disease: SUSTAIN 6 and PIONEER 6 post hoc analysis

P. Rossing1, S. Bain2, H. Bosch-Traberg3, O. Frenkel3, H.L. Heerspink4, S. Rasmussen3, L. Mellbin5;

1Steno Diabetes Center Copenhagen, Gentofte, Denmark, 2Swansea University Medical School, Swansea, UK, 3Novo Nordisk A/S, Søborg, Denmark, 4University Medical Center Groningen, Groningen, Netherlands, 5Karolinska Institutet, Stockholm, Sweden.

Background and aims: People with type 2 diabetes (T2D) are at increased risk of cardiovascular (CV) disease and chronic kidney disease (CKD). As shown in a previous post hoc analysis, semaglutide (pooled s.c. once-weekly [OW] and oral once-daily) reduces the risk of major adverse CV events (MACE) vs placebo. The current post hoc analysis investigated the association between baseline kidney function and risk of MACE (composite of CV death, nonfatal myocardial infarction and nonfatal stroke), and the effect of semaglutide on risk of MACE by baseline kidney function.

Materials and methods: Participants with T2D and at high CV risk (N=6,480) receiving semaglutide (s.c. OW 0.5 or 1.0 mg or oral 14 mg) or placebo in SUSTAIN 6 and PIONEER 6 were categorised according to baseline kidney parameters: eGFR <45 (CKD stage 3b or worse), ≥45-<60 (CKD stage 3a) and ≥60 mL/min/1.73 m2 (CKD stage 1 or 2) and urine albumin-to-creatinine ratio (UACR) <30, ≥30-≤300 and >300 mg/g. eGFR subgroup analyses used pooled SUSTAIN 6 and PIONEER 6 data; UACR subgroup analyses used SUSTAIN 6 data only (no PIONEER 6 data available). MACE risk by baseline kidney function was analysed with a Cox proportional hazards model (reference groups: eGFR ≥60 mL/min/1.73 m2 and UACR <30 mg/g). The effect of semaglutide on MACE across kidney function subgroups was assessed with unadjusted and adjusted (for important baseline predictors of CV and renal diseases) analyses.

Results: Most participants included in the SUSTAIN 6 and PIONEER 6 trials had normal or mildly decreased kidney function (eGFR <45, ≥45-<60 and ≥60 mL/min/1.73 m2; n=731, n=968 and n=4,762, respectively) and were normoalbuminuric (UACR <30, ≥30-≤300 and >300 mg/g; n=1,934, n=884 and n=420, respectively). Regardless of treatment, MACE risk was higher in participants with eGFR <45 mL/min/1.73 m2 (HR 1.52, 95% CI [1.15;1.99], p=0.0026) and ≥45-<60 mL/min/1.73 m2 (1.36, [1.04;1.76], p=0.022) vs those with ≥60 mL/min/1.73 m2 at baseline. Similarly, MACE risk was higher in participants with UACR ≥30-≤300 mg/g (HR 1.53, 95% CI [1.14;2.04], p=0.0043) and >300 mg/g (2.52, [1.84;3.42], p<0.0001) vs those with <30 mg/g at baseline. Semaglutide reduced the risk of MACE consistently across baseline kidney function subgroups in both the unadjusted and adjusted analyses vs placebo (pinteraction >0.05 for all analyses; Figure).

Conclusion: The risk of MACE was greater for participants with impaired kidney function than in those with normal kidney function. Semaglutide showed consistent reductions in MACE risk across eGFR and UACR subgroups. These findings indicate that semaglutide provides CV benefits in people with T2D and high CV risk across a broad spectrum of kidney function and damage.

figure m

Clinical Trial Registration Number: NCT01720446; NCT02692716

Supported by: Novo Nordisk A/S

Disclosure: P. Rossing: Employment/Consultancy; Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Gilead, MSD, Mundipharma, Novo Nordisk, Vifor, Sanofi Aventis. Grants; AstraZeneca, Novo Nordisk. Lecture/other fees; Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Sanofi Aventis.

46

Overexpression of miR-210 attenuates endothelial dysfunction in a mouse model of obesity and type 2 diabetes

A. Collado1, T. Jiao1, G. Zaccagnini2, J. Yang1, M. Carlström3, F. Martelli2, J. Pernow1,4, Z. Zhou1;

1Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden, 2Laboratory of Molecular Cardiology, IRCCS Policlinico San Donato, Milan, Italy, 3Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 4Karolinska University Hospital, Stockholm, Sweden.

Background and aims: MicroRNA (miR)-210 plays a protective role in many cardiometabolic diseases, and its levels are decreased in whole blood, erythrocytes, and plasma in type 2 diabetes (T2D). We recently demonstrated that miR-210 levels are lower in carotid artery plaques from patients with T2D compared to non-diabetic patients. However, the role of miR-210 in the modulation of endothelial function is not fully understood. We test the hypothesis that overexpression of miR-210 has therapeutic potential by reversing endothelial dysfunction in mice with Western diet (WD)-induced obesity and T2D.

Materials and methods: Eight weeks old miR-210 transgenic mice were treated with a WD for 12 weeks. During the last 10 days of the diet regime, doxycycline or vehicle dissolved in drinking water was given to switch on miR-210 (miR-210/on, n=7) expression or keep it off (miR-210/off, n=7), respectively. Age-matched wild-type (WT, n=7) control mice received standard chow within the same period. At the end of the treatment, body weight and glucose levels were measured. Aortic segments were isolated, and endothelial function was determined by acetylcholine-induced endothelium-dependent relaxation (EDR) using wire myographs. The expression of the miR-210 target protein tyrosine phosphatase 1B (PTP1B) and the levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85-23, revised 1996). Differences in concentration-dependent relaxations were analyzed using two-way ANOVA with repeated measures. Multiple comparisons between groups were performed with one-way ANOVA followed by the Bonferroni post hoc test. Data are shown as mean ± SD.

Results: miR-210/off mice fed with a WD had a significant increase in body weight (BW; 39.0±5.7 g miR-210/off vs. 25.5±2.2 g WT, p<0.001) and glucose levels (10.0±1.2 mM miR-210/off vs. 7.6±0.4 mM WT, p<0.01). A similar increase was found in miR-210/on mice (BW: 34.3±6.4 g, p=0.05 vs. WT; glucose: 9.8±1.9 mM, p<0.05 vs. WT). EDR in aortas from miR-210/off mice fed with WD was significantly impaired compared to vessels from WT fed with regular chow. Notably, EDR was markedly improved in aortas of miR-210/on mice fed with WD. Furthermore, the expression of PTP1B and the levels of 4-HNE were significantly elevated in the aortas from miR-210/off mice fed with WD when compared to WT (~4.3 fold, p<0.001 and 6 fold, p<0.01 increase of PTP1B and 4-HNE vs. WT, respectively). The expression was attenuated in aortas of miR-210/on mice fed with WD compared to miR-210/off mice (~2.2 fold, p<0.01 and 3 fold, p<0.05 decrease of PTP1B and 4-HNE vs. miR-210/off mice, respectively).

Conclusion: Overexpression of miR-210 ameliorates endothelial dysfunction in mice fed with WD without affecting BW or glucose levels. Increasing miR-210 levels may become a therapeutic strategy for T2D to attenuate cardiovascular dysfunction.

Supported by: the Hjärt-Lungfonden (20190341 and 20200326 to ZZ; 20190266 to JP, and 20210431 to MC).

Disclosure: A. Collado: None.

47

Inhibition of microRNA-181c rescues diabetes-impaired angiogenesis through activation of key angiogenesis mediators

E.L. Solly1,2, J. Mulangala3, B.A. Di Bartolo4, S.J. Nicholls5, P.J. Psaltis1,2, C.A. Bursill1,2, J.T.M. Tan1,2;

1Vascular Research Centre, Lifelong Health Theme, South Australian Health & Medical Research Institute, Adelaide, 2Adelaide Medical School, The University of Adelaide, Adelaide, 3Vascular Biology Program, Centenary Institute of Cancer and Inflammation, The University of Sydney, Sydney, 4Faculty of Medicine and Health, The University of Sydney, Sydney, 5Monash Cardiovascular Research Centre, Monash University, Melbourne, Australia.

Background and aims: Diabetic vascular complications are characterized by impaired angiogenic responses to ischemia. Many patients remain refractory to current therapies, highlighting the need to identify novel therapeutic targets. We recently identified an anti-angiogenic role for miRNA-181c. However, it’s role in diabetes-impaired angiogenesis was unknown. This study aimed to elucidate the role of miRNA-181c in diabetes-impaired angiogenesis.

Materials and methods: Human coronary artery endothelial cells were transfected with a miRNA-181c inhibitor (antimiR-181c) or negative control (antimiR-Neg), exposed to glucose (5mM or 25mM, 48h) then subjected to Matrigel tubulogenesis assay or Boyden Chamber migration assay. Protein levels of angiogenic mediators (VEGFA) and activation of angiogenesis pathways (p-ERK2, p-eNOS, p-p38 MAPK) was determined by Western Blot. In vivo, we assessed the effect of miR-181c inhibition on diabetes-impaired angiogenesis using murine models of hindlimb ischemia and wound healing. Hindlimb blood-flow reperfusion was measured longitudinally by Laser Doppler imaging and gene expression was assessed 3-days post-ischemic induction when angiogenesis is important. Wound area was calculated daily. Neovascularization was assessed by CD31 (capillaries) and smooth muscle α-actin (arterioles) immunostaining in hindlimbs and wounds.

Results: Inhibition of miRNA-181c increased tubule formation (antimiR-181c: 99.9±7.1 vs. antimiR-Neg: 74.7±3.9, n=11, P<0.01) and cellular migration (antimiR-181c: 115.7±23.4 vs. antimiR-Neg: 37.9±7.1, n=6, P<0.05) in high glucose. Mechanistically, this was associated with increased VEGFA levels (antimiR-181c: 112.8±7.9 vs. antimiR-Neg: 90.8±5.8, n=17, P<0.05) and activated ERK2 signalling (antimiR-181c: 113.9±11.9 vs. antimiR-Neg: 79.9±8.9, n=12, P<0.05) in high glucose. In diabetic mice, inhibition of miRNA-181c increased blood flow reperfusion to the ischemic hindlimb, compared to diabetic control mice (Diabetic antimiR-181c: 0.432±0.04 vs. Diabetic antimiR-Neg: 0.282±0.03, n=11, P<0.001), returning it back to non-diabetic levels (0.373±0.04). This was associated with improved early induction of the pro-angiogenic mediator Erk2 (Diabetic antimiR-181c: 93.8±10.1 vs. Diabetic antimiR-Neg: 60.9±9.3, n=8, P<0.05) and an increase in hindlimb arteriolar density (Diabetic antimiR-181c: 133.6±23.8% vs. Diabetic antimiR-Neg: 72.8±10.3%, n=11, P<0.05). Inhibition of miRNA-181c significantly increased the rate of wound closure (Diabetic antimiR-181c: 76.8±3.6% vs. Diabetic antimiR-Neg: 59.7±4.3%, n=11, P<0.01) and increased the number of CD31 wound neovessels (Diabetic antimiR-181c: 142.5±39.2% vs. Diabetic antimiR-Neg: 54.3±13.3%, n=11, P<0.05) in diabetic mice.

Conclusion: Inhibition of miRNA-181c rescues diabetes-impaired angiogenesis. This presents miRNA-181c as a novel therapeutic target for the prevention of diabetic vascular complications.I

Supported by: THRF, Diabetes Australia

Disclosure: E.L. Solly: None.

48

Association between microangiopathic complications and cardiac structure and function in asymptomatic patients with type 2 diabetes

M. Nguyen, S. Pinto, P. Poignard, P. Valensi;

Jean Verdier hospital - APHP, Bondy, France.

Background and aims: Diabetic retinopathy has been associated with an increased risk of cardiac events including heart failure and with echocardiographic alterations. The pathophysiology of diabetic cardiomyopathy is complex and plurifactorial. The present study aimed to examine the relationship between microangiopathic complications and these alterations in patients with type 2 diabetes (T2D).

Materials and methods: We included 699 patients (male/female 54%/46%) with T2D, free of cardiac history and symptom but with other cardio-vascular risk factors. They were separated in 4 groups according to the number (from 0 to 3) of microangiopathic complications among retinopathy, nephropathy and peripheral neuropathy (G0 to G3). An echocardiography was performed, with measurement of structural and functional parameters. Silent coronary disease was assessed by performing a stress myocardial scintigraphy to detect silent myocardial ischemia (SMI), and a coronary angiography in the patients with SMI. NT-proBNP was measured in 243 of them.

Results: A higher number of microangiopathic complications was associated with male gender, age, diabetes duration, hypertension and SMI (p<0.005 for all comparisons). Left ventricle systolic dysfunction (ejection fraction <50%), dilatation, hypokinesia and hypertrophy were detected in 3.9%, 8.4%, 7.6% and 34.1% of the population, respectively. The prevalence of hypokinesia and hypertrophy increased from G0 to G3 (p=0.02 and 0.03), as well as interventricular septal and posterior wall thickness and NT-proBNP levels (p<0.0001 for all comparisons). Multivariate analyses showed that hypertrophy, septal thickness and NT-proBNP were significantly associated with the number of microangiopathic complications, independently from gender, diabetes duration, hypertension and SMI.

Conclusion: The association between the number of microangiopathic complications and these left ventricle structural and functional alterations, independent from major potential confounding factors, particularly silent coronary disease, stands for a microvascular contribution to the development of diabetic cardiomyopathy.

Disclosure: M. Nguyen: None.

OP 09 Fighting diabetes with tubes, scanners, and catheters

49

Advances in diabetes management: has pregnancy glycaemic control in women with type 1 diabetes changed in the last decades?

F. Citro1, F. Nicolì1, M. Aragona2, L. Battini3, C. Bianchi2, S. Del Prato1, A. Bertolotto2;

1Department of Clinical and Experimental Medicine, University of Pisa, 2Department of Medicine, University Hospital of Pisa, 3Maternal-Infant Department, University Hospital of Pisa, Pisa, Italy.

Background and aims: Over the recent years, multiple therapeutic and management opportunities have been made available to treat pregnant women with type 1 diabetes (T1DM). However, analysis assessing whether these different approaches may have any specific advantage/disadvantage in metabolic control and outcomes is still limited.

Materials and methods: We performed a retrospective analysis on pregnant women with T1DM, managed between 2008 and 2020 at the Pisa University Hospital, to analyze metabolic data according to types of basal insulin (NPH, detemir or glargine), insulin administration ways [Multiple Daily Injections (MDI) or Continuous Subcutaneous Insulin Infusion (CSII)] and glucose monitoring systems [Self-Monitoring of Blood Glucose (SMBG) or Continuous/Flash Glucose Monitoring (CGM/FGM)] that were adopted during pregnancy.

Results: We identified 136 T1DM women (age: 32 [IQR 30-35] years old; preconception HbA1c: 58.1±11.6 mmol/mol). Of them, 103 (76%) were on MDI based on NPH (n: 53, 51%), detemir (n: 35, 34%) or glargine (n: 15, 15 %). The remaining 33 women (24%) were on CSII. A CGM/FGM system was used in 33 (24%) women (20 (19%) on MDI and 13 (39%) on CSII). HbA1c, fasting plasma glucose, lipid profile and weight gain at baseline and during pregnancy were comparable among women treated with different basal insulins. Pregnancy planning was more common in women on CSII (94% vs. 60%, p=0.001) and, as compared to women on MDI treatment, they had better pregestational HbA1c (54±5.4 vs. 60±13 mmol/mol; p=0.044), first trimester fasting plasma glucose (103±38 vs. 140 ± 59 mg/dL, p=0.004), lower pregnancy weight gain (10.7±4.0 vs. 13.8±6.2 kg, p=0.018) and lower pre-prandial insulin dose at first (0.25±0.09 vs. 0.38 ± 0.18 UI/kg, p= 0.002), second (0.30±0.11 vs. 0.43±0.20 UI/kg, p=0.003) and third (0.42±0.20 vs. 0.54±0.24 UI/kg; p=0.017) trimester. Women using CGM/FGM had significantly lower pregestational (54.1±8.0 vs. 59.9±13.1 mmol/mol; p=0.041) and first trimester (46.5±5.5 vs. 51.2±7.1 mmol/mol, p=0.034) HbA1c levels than those on SMBG. Mode of delivery (vaginal or caesarean section) and neonatal outcomes (birth weight, macrosomia, Large for Gestational Age (LGA), Small for Gestational Age (SGA), preterm birth, Apgar score at 5’, congenital malformations) were comparable in all groups. At logistic regression analysis, in the whole group, third trimester HbA1c level was associated with LGA risk [OR=2.596 (1.408-4.787)].

Conclusion: Treatment with NPH or long-acting insulin analogs didn’t significantly change pregnancy metabolic data of women with T1DM, although CSII and CGM/FGM can optimize preconception and first trimester pregnancy glycemic control. Nonetheless, irrespective of the therapeutic management, third trimester HbA1c remains the strongest risk factor for LGA.

Disclosure: F. Citro: None.

50

Fully automated closed-loop insulin delivery vs standard insulin therapy in adults with type 2 diabetes: an open-label, single-centre randomised crossover trial

C.K. Boughton1, A.B. Daly1, M. Nwokolo1, S. Hartnell2, M.E. Wilinska1, A. Cezar1, M.L. Evans1, R. Hovorka1;

1Box 289, University of Cambridge, 2Wolfson Diabetes and Endocrine Clinic, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

Background and aims: Despite advances in oral and injectable therapies for type 2 diabetes (T2D), many adults with T2D requiring insulin do not reach the recommended target HbA1c levels and there is a clear need for novel therapeutic approaches. We evaluated the safety and efficacy of fully closed-loop insulin delivery, not requiring meal bolusing, compared with standard insulin therapy in adults with T2D.

Materials and methods: In an open-label, single-centre, randomised, crossover study, 26 adults with T2D (7 females, mean±SD age 59±11y, diabetes duration 18±8y, baseline HbA1c 75±15 mmol/mol [9.0±1.4%], BMI 35.3±8.6kg/m2) underwent two 8-week periods of unrestricted living comparing CamAPS HX fully closed-loop using Fiasp (CL), with standard insulin therapy and a masked continuous glucose monitor (control) in random order. There was a 2-4 week washout between intervention periods. Primary endpoint was time spent in target glucose range (3.9 to 10.0 mmol/L).

Results: The proportion of time spent in target glucose range was mean±SD 66.3±14.9% with CL vs. 32.3±24.7% with control (mean difference 35.3 percentage points [95%CI 28.0, 42.6]; P<0.001). The proportion of time spent above target glucose range (>10.0 mmol/L) was 33.2±14.8% with CL vs. 67.0±25.2% with control (mean difference 35.2 percentage points [95%CI -42.8, -27.5]; P<0.001). Mean glucose was lower during CL use (9.2±1.2 mmol/L vs. 12.6±3.0 mmol/L; mean difference 3.6 mmol/L [95%CI 2.5, 4.6]; P<0.001). There was a reduction in HbA1c at the end of the CL period at 57±9 mmol/mol [7.3±0.8%] compared to 72±13 mmol/mol [8.7±1.2%] at the end of the control period (mean-adjusted difference 15mmol/mol [95%CI 11, 20]; 1.4% [95%CI 1.0, 1.8]; P<0.001). The proportion of time in hypoglycaemia (<3.9 mmol/L) was low and similar between treatment periods (median [IQR] 0.43% [0.20, 0.77] in the CL period vs. 0.08% [0.00, 1.05] in the control period; P=0.751). There were no episodes of severe hypoglycaemia in either group.

Conclusion: Fully closed-loop insulin delivery improved glucose control without increasing the risk of hypoglycaemia compared to standard insulin therapy in adults with T2D. The treatment may represent a safe and effective method of achieving glycaemic target in this group.

figure n

Clinical Trial Registration Nubmer: NCT04701424

Supported by: The study was supported by the National Institute for Health Research Cambridge Biomedical Research

Disclosure: C.K. Boughton: None.

51

Important decrease of hospitalisations for acute diabetes events before and after FreeStyle Libre ® system initiation in type 2 diabetes with basal insulin therapy in France

J.-P. Riveline1, F. Levrat-Guillen2, B. Detournay3, E. Vicaut4, G. De Pouvourville5, C. Emery3, B. Guerci6;

1Department of Endocrinology and Diabetology, Lariboisière Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France, 2Abbott Diabetes Care, Maidenhead, UK, 3CEMKA, Bourg-La-Reine, France, 4Clinical Research Unit, Fernand Vidal Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France, 5Department of Economics, ESSEC Business School, Cergy-Pontoise, France, 6Department of Endocrinology, Diabetology and Nutrition, Brabois Adult Hospital, Vandoeuvre-lès-Nancy, France.

Background and aims: Glycemic management in people with T2D (PwT2D) who are not on intensive insulin regimens is of considerable importance. Due to the progressive nature of T2D, initiation of insulin treatment may be necessary to achieve glycemic target. Initiation of basal-insulin therapy in PwT2DM is associated with serious diabetes-related acute events, such as severe hypoglycemia or diabetes ketoacidosis (DKA) that can be life-threatening. We assessed the impact of initiating FreeStyle Libre® glucose monitoring system (FSL) as compared to the usual self-glucose monitoring of glycemia on hospitalizations for acute diabetes events (ADEs) in PwT2D on basal insulin scheme +/- other hypoglycemiant agents.

Materials and methods: A retrospective study on the overall French national SNDS reimbursement claims database (≈66 million people) was conducted on all French PwT2D on basal insulin therapy receiving a first reimbursement of FreeStyle Libre between 01/08/2017 to 31/12/2018. The analysis looked at claims data for the 12 months before, and up to 24 months after FSL initiation. Hospitalizations for diabetes-related acute events were identified, using ICD-10 codes as main or related diagnosis: severe hypoglycemia (SH) events (E160, E161, E162 and T383); DKA events (ICD-10 codes E101 and E111), comas (ICD-10 codes E100, E110 and E140) and hyperglycemia related stays (ICD 10 code R739).

Results: We identified 5,933 PwT2D on a basal insulin therapy who initiated FSL during the selection period. Only 78.9% of patients were on both basal insulin and other hypoglycemiant agents, of which 40% were documented as receiving sulphonylurea. Amongst PwT2D on a basal insulin therapy, 2.01% experienced at least one hospitalization for any ADE in the year before FSL initiation compared to 0.75% (1 year after) and 0.60% (2 years after) (Fig. 1), and these results are similar independently of the sulfonylurea use or not. This reduction in ADEs was mainly driven by 75% fewer DKA admissions, with a 44% reduction in admissions for SH. These patterns of reduction in ADEs persisted after 2 years, with a further 43% reduction in DKA rates.

Conclusion: This sub-group analysis of our previous RELIEF study strongly suggests the value of the FSL system in a PwT2D population initially treated with basal insulin therapy in reducing diabetes related acute events and their long-term consequences.

figure o

Supported by: Funded by Abbott Diabetes Care

Disclosure: J. Riveline: Honorarium; Abbott.

52

Accuracy of a real-time continuous glucose monitoring system during cardiac surgery with hypothermic extracorporeal circulation

D. Herzig1, M. Vettoretti2, D.P. Guensch3, A. Melmer1, A.C.K. Goerg1, A. Kadner4, A. Facchinetti2, A. Vogt3, L. Bally1;

1Department of Diabetes, Endocrinology, Nutritional Medicine and Metabolism, Bern University Hospital, University of Bern, Bern, Switzerland, 2Department of Information Engineering, University of Padova, Padova, Italy, 3Department of Anaesthesiology and Pain Medicine, Bern University Hospital, University of Bern, Bern, Switzerland, 4Department of Cardiac and Vascular Surgery, Bern University Hospital, University of Bern, Bern, Switzerland.

Background and aims: Frequent blood glucose testing is a key element of perioperative care. Continuous glucose monitoring (CGM) allows monitoring glucose levels in real-time and guide clinical decision making. Several studies have shown the feasibility of CGM in the inpatient setting with satisfactory performance. The accuracy of sensors, however, may be affected in extreme conditions that have not been well studied. Here we evaluated the accuracy of the Dexcom G6 CGM system during cardiac surgery using hypothermic extracorporeal circulatory circulation (ECC).

Materials and methods: Sixteen adults wore the DexcomG6 CGM sensor 22.8 [11.2, 79.4 hours] (median [min, max]) prior to cardiac surgery with hypothermic ECC until hospital discharge or a maximum of 10 days. A subset of 11 patient also underwent deep hypothermic cardiac arrest (DHCA). A calibration was performed with arterial blood at the time of anaesthesia induction using the Accu-Chek® Inform II meter. During surgery, blood for reference values was sampled every 20 minutes from the arterial line and the heart-lung machine during ECC/DHCA. Post-surgery reference values were obtained from capillary blood measurements using the Accu-Chek® Inform II meter. Core body temperature was monitored using an esophageal probe. The primary endpoint was the mean absolute relative difference (MARD) between sensor and reference method during surgery.

Results: Mean±SD surgery duration was 5.4±1.7 hours with body temperature decreasing to 25.2±4.1Co (range 20.9 to 32.0Co). Individual intraoperative reference glucose levels ranged between 5.5±1.7 and 16.1±2.6 mmol/l. MARD of 256 paired CGM/reference values was 23.8% during surgery. MARD was 29.1% during ECC (154 pairs) and 41.6% during DHCA (10 pairs), with a negative bias in all three periods (signed relative difference was -13.7%, -26.6% and -41.6, respectively). During surgery, a total of 86.3% pairs were in Clarke error grid Zones A or B (A, 51.6%) and 41.0% of intraoperative sensor readings were within the limits specified by the ISO 15197:2013 norm. Sensor readings were available for 91.8% [56.8%, 100.0%] of the intraoperative time. In the postoperative period (from end of surgery until hospital discharge), MARD was 15.0% (144 pairs).

Conclusion: Extreme conditions such as deep hypothermia in cardiac surgery challenge the performance of Dexcom G6 system. However, the accuracy of sensor recovered in most cases with adequate performance in the post-surgery period.

Supported by: Swiss Helmut Horten Foundation, Swiss Foundation of Anaesthesiology and Intensive Care, Dexcom

Disclosure: D. Herzig: None.

53

Evidence of significant reduction in pain and sensory symptoms of diabetic neuropathy with 10kHz spinal cord stimulation: 24-month RCT outcomes

E. Petersen1, T.G. Stauss2, J.A. Scowcroft3, J.L. White4, S.M. Sills5, K. Amirdelfan6, M.N. Guirguis7, J. Xu8, C. Yu9, A. Nairizi10, D. Patterson11, V. Galan12, R.S. Taylor13, D. Caraway14, N.A. Mekhail8;

1Neurosurgery, University of Arkansas for Medical Sciences, Little Rock, USA, 2Advanced Pain Management, Greenfield, USA, 3Pain Management Associates, Lee's Summit, USA, 4Accelerated Enrolment Solutions, Orlando, USA, 5Touchstone Interventional Pain Center, Medford, USA, 6IPM Medical Group, Walnut Creek, USA, 7Ochsner Clinic Foundation, New Orleans, USA, 8Cleveland Clinic Foundation, Cleveland, USA, 9Swedish Pain & Headache Center, Seattle, USA, 10Reno Tahoe Pain Associates, Reno, USA, 11Nevada Advanced Pain Specialists, Reno, USA, 12Pain Care, Stockbridge, USA, 13University of Glasgow, Glasgow, UK, 14Nevro, Redwood City, USA.

Background and aims: Painful diabetic neuropathy (PDN) can lead to severe deterioration in quality of life, loss of function, and increased health care costs. A large randomized controlled study was undertaken to provide high level clinical evidence for the use of 10kHz spinal cord stimulation (SCS), which does not cause paresthesias like traditional SCS, for the treatment of PDN.

Materials and methods: Prospective, multicenter, RCT to document the impact of 10kHz SCS on PDN. Participants had PDN symptoms ≥12 months (M), refractory to medications, lower limb pain intensity ≥5cm (0-10cm visual analog scale [VAS]), and hemoglobin A1c ≤10%. Patients (N=216) were allocated 1:1 to 10kHz SCS (Nevro Corp.) plus conventional medical management (CMM) or CMM alone with optional crossover at 6 M.

Results: There were 90 patients implanted in the 10kHz arm after meeting 50% pain relief requirement in a temporary trial, and 84 (93%) remained in follow-up at 24 months. The 10kHz SCS patients maintained substantial pain relief from 3 M, averaging an 81.9% (95%CI 77.3 - 86.5) decrease at 24 M. At 6 M follow up, 0% of 10kHz SCS participants but 93% of eligible CMM patients elected to crossover. At 18 M follow-up after SCS, both groups (n=138) reported similar significant improvements in pain, sleep disturbance, and in pain interference with mood and daily activities (see Fig 1). In addition, the neurological assessment found a majority of patients treated with SCS experienced sensory improvements. In terms of health care utilization, hospitalizations were 38% less in the 10kHz group at 6 M. There were no stimulation-related neurological deficits and 6 total explants (3.9%), 5 due to procedure-related infections and 1 as a precaution for endocarditis.

Conclusion: The largest RCT to date of SCS management of PDN demonstrates safety, durable pain relief, and clinically meaningful improvement on neurological examination over 24 M with 10kHz SCS. These PDN patients with moderate to severe pain that was refractory to CMM achieved clinically important improvements in quality of life, and reduced health care utilization with a safe, reversible, minimally invasive procedure.

figure p

Clinical Trial Registration Number: NCT03228420

Supported by: Nevro

Disclosure: E. Petersen: Employment/Consultancy; Nevro, Medtronic Neuromodulation, Abbott Neuromodulation, Saluda.

54

Duodenal jejunal bypass liner (DJBL) treatment for type 2 diabetes and obesity: glycaemic and CVD risk factor improvements vs risks in patients treated worldwide

R.E.J. Ryder1, P. Sen Gupta2, T. Battelino3, P. Kotnik3, J. Teare4, A. Ruban4, H. Frydenberg5, L. Munro5, S. Fishman6, R. Cohen7, C. de Jonge8, J.-W. Greve9, H. Sourij10, K. Laubner11, J. Seufert11;

1City Hospital, Birmingham, UK, 2Guy's and St Thomas' Hospitals,, London, UK, 3University Medical Center, Ljubljana, Slovenia, 4Imperial College, London, UK, 5Epworth Hospital, Richmond, Australia, 6Sourasky Medical, Tel Aviv, Israel, 7Oswaldo Cruz Hospital, Sao Paulo, Brazil, 8Catharina Hospital, Eindhoven, Netherlands, 9Zuyderland MC, Heerlen, Netherlands, 10Medical University, Graz, Austria, 11Freiburg University, Freiburg, Germany.

Background and aims: There is uncertainty over the balance of benefits and risks of proximal intestinal exclusion with a temporarily inserted DJBL for treatment of type 2 diabetes and obesity. An online registry was established under the auspices of the Association of British Clinical Diabetologists (ABCD) in 2017.

Materials and methods: DJBL safety and efficacy data were entered into the ABCD registry from patients worldwide. To evaluate the glycaemic effectiveness of DJBL, we categorised patients into groups according to baseline HbA1c.

Results: As of March 2022, data had been submitted on 1022 patients (mean ± SD age 51.3 ± 11.4 years, 52.5% male, 84.9% type 2 diabetes, BMI 41.1 ± 8.7 kg/m2) from 34 centres in 10 countries (table). In those with both baseline and time of removal data, mean ± SD weight fell by 13.3 ± 9.7 kg from 120.2 ± 25.3 to 106.9 ± 23.8 kg (n=811, p<0.001), HbA1c by 13.7 ± 15.9 from 67.6 ± 19.8 to 54.0 ± 13.9 mmol/mol (by 1.3 ± 1.5, from 8.3 ± 1.8 to 7.1 ± 1.3 %) (n=646, p<0.001), systolic BP fell from 135.7 ± 18.0 to 129.5 ± 17.0 mmHg (n=448, <0.001) and cholesterol fell from 4.8 ± 1.3 to 4.2 ± 1.0 mmol/L (n=467, p<0.001). In those with baseline HbA1c ≥ 53 mmol/mol, HbA1c fell by 17.0 ± 16.3 from 74.6 ± 16.3 to 57.6 ± 12.9 mmol/mol (n = 506, p<0.001); with baseline HbA1c ≥ 64 mmol/mol, HbA1c fell by 20.7 ± 16.9 from 80.8 ± 15.0 to 60.1 ± 13.4 mmol/mol (n=365, p<0.001); with baseline HbA1c ≥ 75 mmol/mol , HbA1c fell by 27.0 ± 18.0 from 90.0 ± 13.9 to 63.0 ± 14.4 mmol/mol (n=207, p<0.001); with baseline HbA1c ≥ 86 mmol/mol , HbA1c fell by 34.9 ± 18.1 from 99.1 ± 13.2 to 64.1 ± 15.9 mmol/mol (n=111, p<0.001). There were 43 (4.2%) serious adverse events (SAE) and 139 (13.6%) less serious SAEs (table). All SAE patients made a full recovery.

Conclusion: The data demonstrate that, in response to DJBL, the higher the initial HbA1c, the greater its fall, with HbA1c fall 27.0 mmol/mol (2.5 %) when initial HbA1c ≥ 75 mmol/mol (≥ 9.0 %) and 34.9 mmol/mol (3.2 %) when initial HbA1c ≥ 86 mmol/mol (≥ 10.0 %). In view of impact on microvascular and macrovascular risk factors, the benefits of DJBL therapy could potentially reduce complications of diabetes. The SAE rate was not insignificant but acceptable. As all patients made a full recovery and many experienced significant benefit despite the SAE, this registry data from a large patient number suggests that the benefits of DJBL outweigh the risks. With monitoring during the time of DJBL and prompt removal if indicated, this treatment is a useful option.

figure q

Supported by: ABCD

Disclosure: R.E.J. Ryder: None.

OP 10 Beta cells: protecting what is precious

55

Early effects of treatment with intralymphatic administration of rhGAD65 in LADA appear similar to those observed in type 1 diabetes

I. Hals1,2, A. Björklund3, C.N.D. Balasuriya2, R. Casas4, J. Ludvigsson4,5, V. Grill1;

1Dept of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway, 2Clinic of Medicine, St Olavs hospital, Trondheim University Hospital, Trondheim, Norway, 3Dept. of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden, 4Dept of Biomedical and Clinical Sciences, Linköping university, Linköping, Sweden, 5Crown Princess Victoria Children's Hospital, University Hospital, Linköping, Sweden.

Background and aims: We perform an investigator-initiated open-labelled 12 months pilot study in which treatment with intralymphatic administration of the antigen specific immunotherapy Diamyd® (rhGAD65) is tested for the first time in patients with LADA. We aim to evaluate safety, immune response and insulin secretion after intralymphatic treatment with rhGAD65 in LADA.

Materials and methods: Fourteen LADA individuals, women/men: 7/7, with high anti-GAD titers (>190 RU/ml) and not on insulin therapy received 3 intralymphatic injections of 4 μg rhGAD65, one month apart. Safety parameters, immune responses and insulin secretion capacity were summarized in an interim analysis 5 months after baseline.

Results: Baseline values (mean ± SD) were: fasting C-peptide 0.65 ± 0.36 nmol/L, HbA1c 43 ± 7 mmol/mol and BMI 26.5 ± 5.2 kg/m2, diabetes duration 5.9 ± 3.8 months, age 47 ± 8 years. Half the study population was HLA-DR3DQ2 positive. Interim analysis at 5 months from baseline showed no treatment-related serious adverse events. There was a clear GAD-specific immune response both in terms of anti-GAD and T-cell reactions. At 2 and 5 months from baseline, median (Q1, Q3) GAD65 stimulation index of PBMC proliferation was 2.16 (1.25, 3.06) and 2.34 (1.81, 3.58) respectively, p< 0.01 for increase from baseline (corresponding findings in type 1 diabetes (n=56): 2.77 (1.96, 6.05) and 3.19 (2.02, 6.19), p< 0.0001). At 5 months from baseline, supernatant concentrations of cytokines from GAD65-stimulated secretion by PBMCs were increased for interleukin (IL) IL-1 beta, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17, tumor necrosis factor (TNF) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1 and interferon (IFN) gamma, p< 0.05 or less for difference. Insulin secretion assessed from meal stimulation tests showed a modest decline in terms of postprandial 2 hour levels of C-peptide: at baseline median (Q1, Q3) levels were 1.95 (1.35, 2.30) nmol/L and after 5 months 1.45 (1.25, 1.85) nmol/L, p< 0.025 for difference. Parameters of glucagon stimulation tests were not affected.

Conclusion: Intralymphatic treatment with rhGAD65 in LADA appears safe. It induces important immune responses that are similar to those observed after intralymphatic rhGAD65 in type 1 diabetes.

Clinical Trial Registration Number: NCT04262479

Supported by: Liaison committee between Central Norway RHA and NTNU

Disclosure: I. Hals: None.

56

Loss of beta cell Scn9a Na + channel activity is protective in the context of type 1 diabetes while suppressing glucose stimulated insulin secretion

P. Overby, S. Provenzano, G. Sun, N. Nahirney, J. Kolic, S. Skovsø, J.D. Johnson;

Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, Canada.

Background and aims: Type 1 diabetes is caused by β-cell death resulting in insulin insufficiency. Somewhat paradoxically, the recent clinical success of verapamil provides rationale for identifying protective therapeutic targets that suppress insulin secretion by limiting excitotoxicity. We previously used high-throughput screening to identify the FDA-approved Na+ channel inhibitor carbamazepine as a drug that protects mouse β-cells from multiple stressors. Follow-up studies demonstrated a decrease in spontaneous diabetes incidence in non-obese diabetic (NOD) mice treated with carbamazepine. Here, we aimed to characterize the effects of carbamazepine and related drugs on human islet survival and function, and to investigate the specific role of Scn9a (Nav 1.7), which is the most highly expressed voltage-depended Na+ channel gene in β-cells and the likely cellular target of carbamazepine.

Materials and methods: Human islet cell death in the presence of carbamazepine and its analogues (e.g. oxcarbazepine), structurally unrelated Nav1.7 inhibitors (e.g. ProTx-II), and a Nav1.7 agonist (e.g. veratridine) were investigated using high-content imaging. The dynamics of glucose-stimulated insulin secretion from human and mouse islets were assessed by perifusion. The specific role of Scn9a in β-cell function and type 1 diabetes was assessed in ‘floxed' NOD mice crossed with the Ins1Cre knockin allele or injected at 7 weeks with adeno-associated virus (AAV8) containing Cre under the control of an Ins1 promoter.

Results: Carbamazepine and oxcarbazepine protected human β-cells from cytokine- and ER stress-induced death, while veratridine increased cell death under these conditions. Glucose-stimulated insulin secretion was moderately reduced in perifused human islets in combination with carbamazepine (p=0.0044), while veratridine increased insulin secretion (p=0.035). We observed a significant reduction in glucose stimulated insulin secretion between NOD:Scn9afl/fl;Ins1Cre knockout mice and their wildtype littermate NOD:Scn9awt/wt;Ins1Cre controls (p<0.0001). NOD:Scn9afl/fl injected with AAV8-Ins1Cre showed reduced type 1 diabetes incidence relative to injected NOD:Scn9awt/wt controls.

Conclusion: Collectively, our studies to date point to Scn9a as a potential pre-clinical drug target to protect β-cells from excitotoxicity in type 1 diabetes.

Supported by: JDRF

Disclosure: P. Overby: None.

57

Tirzepatide improves multiple aspects of beta cell function

K. Mather1, A. Mari2, J. Li1, S. Urva1, T. Heise3, J. DeVries4, E. Pratt1, R. Heine1, M. Thomas1, Z. Milicevic1;

1Eli Lilly and Company, Indianapolis, USA, 2National Research Council Institute, Rome, Italy, 3Profil Institute for Clinical Research, Neuss, Germany, 4Profil Institut für Stoffwechselforschung GmbH, Neuss, Germany.

Background and aims: In this study we investigated the effect of tirzepatide (TZP) 15 mg (novel GIP-GLP-1 dual receptor agonist), placebo and semaglutide 1 mg (SEMA) on beta-cell function.

Materials and methods: In a 28-week double-blind randomised controlled trial in people with type 2 diabetes the effect of TZP 15 mg (GIP-GLP-1 dual receptor agonist), placebo and SEMA 1 mg on beta-cell function was analysed using euglycaemic and hyperglycaemic glucose clamps (EGC, HGC) and Mixed Meal Tolerance Tests (MMTT).

Results: (Table): During EGC and HGC, TZP improved insulin sensitivity (M-value), first and second phase insulin secretion (ISR) and beta-cell glucose sensitivity (ratio of ISR and glucose increments) more than SEMA and placebo. During MMTT, both fasting and AUC glucose were reduced more with TZP than with SEMA or placebo, while incremental glucose AUC was reduced similarly with TZP and SEMA vs placebo. Despite a similar glucose response the total and incremental insulin AUC were reduced by TZP but not by SEMA. Model-derived beta-cell glucose sensitivity was similarly increased with TZP and SEMA from baseline. In contrast, ISR at 7.2 mmol/L glucose increased more with TZP than with placebo and SEMA.

Conclusion: The glucose-lowering effect of TZP is mediated through improvements in multiple aspects of beta-cell function, including beta-cell glucose sensitivity and ISR, and in insulin sensitivity, resulting in pronounced lowering of fasting and postprandial glucose. These effects help explain the superior improvement in glucose control seen with TZP vs comparators in clinical trials.

figure r

Clinical Trial Registration Number: NCT03882970

Disclosure: K. Mather: Employment/Consultancy; Eli Lilly and Company. Stock/Shareholding; Eli Lilly and Company.

58

Shotgun proteomics unveils mechanisms behind metformin-induced protection against pro-inflammatory cytokine-induced human islet cell damage

M. Tesi1, L. Giusti2, F. Ciregia1, L. Marselli1, L. Zallocco1, M. Suleiman1, C. De Luca1, S. Del Guerra1, D.L. Eizirik3, M. Cnop3, M.R. Mazzoni1, P. Marchetti1, A. Lucacchini1, M. Ronci4;

1University of Pisa, Pisa, Italy, 2University of Camerino, Camerino, Italy, 3Université Libre de Bruxelles, Brussels, Belgium, 4University of Chieti-Pescara, Chieti, Italy.

Background and aims: Metformin (Met), a drug commonly used for the treatment of type 2 diabetes (T2D), has been shown to have direct protective actions on human β-cells exposed to gluco- and/or lipotoxic conditions and on islets from T2D donors. We presently assessed whether Met could relieve stress induced by pro-inflammatory cytokines in human β-cells and investigated the underlying mechanisms by shotgun proteomic analysis.

Materials and methods: Altogether, 14 human islet preparations were used. Islets were exposed to 50 U/ml interleukin-1β plus 1000 U/ml interferon-γ for 48 h, with or without 2.4 μg/ml Met, a therapeutical concentration of the drug. Glucose-stimulated insulin secretion studies, caspase 3/7 activity assay, shotgun label free proteomics and Western blots were performed.

Results: Met prevented the reduction of the insulin stimulation index (control islets, Ctrl: 5.4±1.7; cytokines: 3.4±1.1, p<0.05 vs Ctrl; cytokines plus Met: 4.7±1.6; mean±SEM) and the increase of caspase 3/7 activity (Ctrl: 1±1.1; cytokines: 1.5±1.1, p<0.001 vs Ctrl; cytokines plus Met: 1.2±0.1, p<0.01 vs cytokines and Ctrl) induced by cytokine exposure. Proteomics analysis identified 2,304±392 proteins. Cytokines significantly altered 244 proteins (145 up- and 99 downregulated), while in the presence of Met cytokines modified 231 proteins (128 up- and 103 downregulated). There were 212 differentially expressed proteins in common. Among the proteins regulated in opposite direction in the two conditions, were proteins involved in vesicle motility (e.g. transgelin, Ras-related protein Rab-14), defence from oxidative stress (e.g. peroxiredoxins, PRDX2 and PRDX5), metabolism (e.g. flavin reductase, mitochondrial ATP synthase subunit O), protein synthesis (e.g. 40S ribosomal proteins and eukaryotic translation initiation factor 4E), glycolysis and its regulation (e.g. triosephosphate isomerase and pyruvate kinase), cytoskeletal proteins and proteins interacting with the cytoskeleton (e.g. myosin light polypeptide 6, Ras-related protein Ral-A and coactosin-like protein). Met inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling and cell senescence. Some of the key changes were confirmed by Western blot.

Conclusion: Met prevents part of the deleterious actions of pro-inflammatory cytokines on human β-cells, which is accompanied by islet proteome modifications. These results suggest that Met, a widely used drug for the treatment of T2D, might be repurposed for other types of diabetes.

Supported by: INNODIA, INNODIA HARVEST, PRIN2017, CAPTUR3D

Disclosure: M. Tesi: None.

59

Irisin administration restores beta cell functional mass in a mouse model of type 2 diabetes

N. Marrano1, G. Biondi1, A. Borrelli1, M. Rella1, L. Roberto2, A. Cignarelli1, S. Perrini1, L. Laviola1, F. Giorgino1, A. Natalicchio1;

1Department of Emergency and Organ Transplantation, University of Bari Aldo Moro, Bari, 2Transgenic Mice Facility, Biogem S.c.a.r.l., Ariano Irpino (AV), Italy.

Background and aims: Irisin is a hormone secreted by skeletal muscle following physical activity or excess of saturated fatty acids (SFAs), able to improve metabolic homeostasis and promote energy expenditure. Serum irisin levels are reduced in type 2 diabetes (T2D), while exogenous irisin administration improves glycemic control in diabetic mice. We have previously demonstrated that irisin protects human and rodent beta-cells and pancreatic islets from SFAs-induced apoptosis, increases insulin byosinthesis and glucose-stimulated insulin secretion (GSIS), and promotes beta-cell proliferation, both in vitro and in vivo in healthy wild type mice. We have also demonstrated that irisin restores the defective GSIS and reduces the high rate of apoptosis characteristic of islets from T2D patients. The beta-cellular effects of irisin administration to T2D mice are still unknown.

Materials and methods: 6 weeks-old C57Bl/6 mice (n = 8) were fed a high-fat diet (HFD, 60% of energy deriving from fat) for 10 weeks and then intraperitoneally injected with streptozotocin (STZ, 100 mg/Kg) to induce diabetes. 4 standard diet (SD)-fed mice were used as control. HFD/STZ mice were treated with 0.5 μg/g irisin (n = 4) or vehicle (n = 4), for 14 days. Fasting glycemia, insulinemia, and body weight have been measured throughout the study. Glucose tolerance and pancreatic islets function have been assessed on the last day of the study through an intraperitoneal glucose tolerance test. Pancreatic islets architecture has been evaluated through immunofluorescence analyses.

Results: As expected, compared to SD mice, HFD/STZ mice showed higher glycemia and body weight, glucose intolerance, and reduced GSIS; in addition, HFD/STZ mice showed reduced islets volume (-78 %; p<0.001 vs SD), beta-cell area (-35 %; p<0.001 vs SD), and insulin content (-60 %; p<0.001 vs SD), while increased alpha-cell area (+54 %; p<0.01 vs SD). Irisin administration significantly restored glycemia (-31 %; p<0.05 vs HFD/STZ), body weight (-13 %; p<0.001 vs HFD/STZ), glucose tolerance (-27 % AUC; p<0.001 vs HFD/STZ), GSIS (+23 % AUC; p<0.005 vs HFD/STZ), islets volume (+61 %; p<0.01 vs HFD/STZ), beta-cell (+34 %; p<0.001 vs HFD/STZ) and alpha-cell (-49 %; p<0.01 vs HFD/STZ) area, and insulin content (+36 %; p<0.001 vs HFD/STZ). Of note, irisin induced a 9-fold increase in beta-cell proliferation rate (p<0.001 vs SD and HFD/STZ).

Conclusion: These results show for the first time that irisin restores beta-cell functional mass also when administered in vivo to diabetic mice, likely by promoting beta-cell proliferation. In conclusion, irisin could be considered a valid new anti-diabetes therapeutic strategy.

Supported by: “EU–ESF PON R&I 2014-20, AIM” and “Tecnopolo per la Medicina di Precisione CUP B84I18000540002”

Disclosure: N. Marrano: None.

60

Non-invasive evaluation of the preservation effect of imeglimin on pancreatic beta cell mass using 111 In-labeled exendin-4 SPECT/CT imaging

M. Fauzi, T. Murakami, H. Fujimoto, A. Botagarova, K. Sakaki, S. Kiyobayashi, N. Inagaki;

Department of Diabetes, Endocrinology, and Nutrition, Kyoto University, Graduate School of Medicine, Kyoto, Japan.

Background and aims: Progressive loss of β-cell mass (BCM) is one of the hallmarks of type 2 diabetes. However, observing BCM requires an invasive method and can only provide cross-sectional data. Recently, a noninvasive approach for observing BCM on living subjects using indium 111-labeled exendin-4 derivative ([Lys12(111In-BnDTPA-Ahx)]exendin-4) (111In-Ex4) has been developed. Imeglimin, a novel antidiabetic agent, has been reported to increase insulin secretion by optimizing mitochondrial function, which lowers the blood glucose levels. However, the effect of imeglimin on BCM preservation has not been fully understood. The Leprdb/db (db/db) mouse is a diabetic mouse model with obesity, in which diminished mitochondrial number in pancreatic β-cells and rapidly decreased BCM were observed. Therefore, we investigated the effect of imeglimin on in-vivo BCM in db/db mice using 111In-Ex4 single-photon emission computed tomography/computed tomography (SPECT/CT) technique.

Materials and methods: Five-week-old male db/db mice were assigned to imeglimin- or vehicle-treated group. The drug was administered orally at a dose of 150 mg/kg B.I.D. for five weeks. 111In-Ex4 SPECT/CT scans were conducted to evaluate BCM changes longitudinally at 5 (baseline), 7, and 10 weeks of age. Oral glucose tolerance tests (OGTTs) were conducted with the same interval as the SPECT/CT scans. Following the longitudinal SPECT/CT scans, immunohistochemical analysis of BCM was conducted. Electron microscopy was also used for evaluating the mitochondrial structure of pancreatic β-cells. On islets harvested from 8-week-old db/db mice after 1-week imeglimin or vehicle treatment, mitochondrial membrane potentials were evaluated with JC-1 dye, a fluorescent lipophilic carbocyanine dye.

Results: Imeglimin-treated group demonstrated significantly lower random blood glucose levels compared to the vehicle-treated group during the age of 6-10 weeks (p < 0.05). Furthermore, OGTT results showed significantly better glucose tolerance at 10 weeks old (p < 0.05). Although the db/db mice in the vehicle-treated group showed progressive loss of RI intensities in the pancreas, based on SPECT/CT during 5-week treatment, the intensity loss was significantly attenuated in the imeglimin-treated group at 10 weeks (p < 0.01). This result was confirmed using the BCM calculation with immunohistochemistry (p < 0.05). In electron microscopy analysis, imeglimin-treated group showed significantly smaller numbers of mitochondria with abnormal structures, such as loss of proper cristae formation, compared with the vehicle-treated group (p < 0.01). This observation was in line with ex-vivo analysis of islet mitochondrial membrane potentials of db/db mice; islets of imeglimin-treated group displayed significantly higher red/green fluorescence intensity ratios (p < 0.05), which indicated functionally healthier mitochondria with preserved membrane potentials in the islet cells.

Conclusion: Imeglimin treatment on db/db mice demonstrated the preservation effect of BCM according to longitudinal observation with 111In-Ex4 SPECT/CT. This effect may be caused partly by preserved mitochondrial structures and mitochondrial membrane potentials of pancreatic β cells treated with imeglimin.

Disclosure: M. Fauzi: None.

OP 11 Adipose tissue profiling and cardio-metabolic risk

61

The effect of ovariectomy and estradiol substitution on metabolic parameters and transcriptomic profile of adipose tissue in a prediabetic model

I. Markova1, M. Huttl1, D. Miklankova1, L. Sedova2, O. Seda2, H. Malinska1;

1Centre for Experimental Medicine, Institute for Clinical & Experimental Medicine, 2Institute of Biology and Medical Genetics, the First Faculty of Medicine, Prague, Czech Republic.

Background and aims: The postmenopausal period leads to the development of abdominal obesity, insulin resistance, hepatic steatosis, and other lipid and carbohydrate metabolism disorders. Adipose tissue, its metabolic and endocrine activity, partially regulated by sex hormones, may play a significant role in the development of these metabolic changes. The pathogenesis of these disorders and the possible effect of hormonal substitution are not precisely known. The study aimed to analyse the effect of ovariectomy and estradiol substitution in a prediabetic model with insulin resistance - females of the hereditary hypertriglyceridemic rat strain (HHTg).

Materials and methods: HHTg females underwent bilateral ovariectomy (HHTg-OVX) or sham surgery (SHAM) at 8 weeks. Two weeks after surgery half of the HHTg-OVX rats were substituted with 17-β estradiol (OVX-E) at a dose of 12.5 μg/kg b.wt./day for 12 weeks subcutaneously.

Results: Ovariectomy in HHTg females was accompanied by weight gain, increased serum leptin levels, impaired glucose tolerance, and decreased adipose tissue insulin sensitivity (p<0.05). Serum concentrations of glucose, insulin, cholesterol, NEFA, adiponectin and inflammatory cytokines MCP-1 and hsCRP did not differ between groups. In contrast, ovariectomy resulted in significant ectopic deposition of triacylglycerols (TAG) in the liver (p<0.001) and kidneys (p<0.001), although serum TAG levels were reduced after ovariectomy. Estradiol substitution alleviated the development of only some of the metabolic disorders associated with ovariectomy; in particular, it improved insulin sensitivity and reduced ectopic TAG deposition (liver: -16%, p<0.01; kidneys: -23%, p<0.001). Transcriptomic analysis of visceral adipose tissue revealed 813 differentially expressed transcripts in HHTg-OVX vs. SHAM, mostly pertaining to the regulation of lipid and glucose metabolism, cell cycle and oxidative stress (AMPK, FoxO, PI3-Akt signaling pathways). Estradiol substitution affected 1059 transcripts with significant overrepresentation in the signaling pathways of lipid metabolism, especially steroid biosynthesis, regulation of lipolysis in adipocytes and glycerol lipid metabolism. The principal component and hierarchical clustering analyses of transcriptome shifts corroborated the metabolic data, showing higher similarity of transcript clusters between OVX-E and SHAM groups, both contrasting with HHTg-OVX. Still, there was a clear distinction between OVX-E and SHAM with regard to transcript clusters related to steroid biosynthesis, glycerolipid or glutathione metabolism.

Conclusion: Ovariectomy in HHTg females worsens adipose tissue insulin resistance and significantly potentiates ectopic lipid deposition. Changes in the visceral adipose tissue transcriptome, especially in pathways relevant to lipid metabolism, oxidative stress, and insulin signaling, may contribute to metabolic abnormalities. Estradiol substitution may partially alleviate some of these disorders.

Supported by: MH CZ No. NU20-01-00083 and IKEM, IN00023001

Disclosure: I. Markova: None.

62

Mir-15b mediates the obesity-induced adipocyte insulin resistance by targeting insulin receptor

L. Xingjing;

Department of Endocrinology, Zhongda Hospital, Institute of Diabetes, Medical School, Southeast Univ, Nanjing, China.

Background and aims: In recent years, the morbidity of obesity has been increasing rapidly worldwide, which is a major risk factor for type 2 diabetes mellitus (T2DM). Obesity, mainly characterized by abnormal and excessive white adipose tissue accumulation, is the most common cause of insulin resistance (IR), where the insulin target tissues fail to respond normally to circulating insulin. However, the precise mechanism by which obesity affects insulin resistance in the major insulin sensitive tissues remains unclear. Adipose glucose uptake plays a significant role in systemic insulin sensitivity, therefore clarifying the regulatory factors of adipose insulin sensitivity is of great significance to find effective therapeutic targets of obesity. Obesity causes the increase of hepatic miR-15b, which provokes hepatocyte insulin resistance, but has no effect in skeletal muscle. The upregulation of miR-15b induced by obesity causally resulted in an impairment of hepatocyte insulin signaling and the decrease of the insulin receptor (INSR) expression. However, no studies have explored whether miR-15b is involved in adipose tissue insulin resistance induced by obesity so far. Aim: To study the role of miR-15b in the adipose tissue of DIO mice and IR 3T3-L1 adipocytes

Materials and methods: We fed mice with high-fat diet (HFD) for 10 weeks to construct obese and insulin-resistant (IR) mice models, and treated3T3-L1 adipocytes with chronic hyperinsulinemia to establish IR adipocyte models. Cell transfection was performed using riboFECTTMCP or Lipofectamine2000. Insulin stimulated fluorescence labeled 2-NBDG uptake assay was used to detect the capacity of glucose uptake in adipocytes. The expression levels of miR-15b, the insulin receptor (INSR) and its downstream insulin signaling molecules were detected by real-time PCR and Western blot respectively.

Results: We found that expression of miR-15b was increased, while INSR expression was downregulated in adipose tissue of diet-induced-obese (DIO) mice. In IR 3T3-L1 adipocytes, the expression of miR-15b also ascended, accompanied by the decrease of INSR expression. Bioinformatics analysis and luciferase reporter analysis suggested that INSR was a potential target of miR-15b. Overexpression of miR-15b led to decreased INSR expression and impaired insulin signal transduction in adipocytes, and inhibition of endogenous miR-15b can reverse the downregulation of INSR and insulin resistance induced by high insulin. In addition, when miR-15b was overexpressed, the simultaneous overexpression of INSR partially alleviated the insulin resistance in adipocytes.

Conclusion: These results suggested that the impaired insulin signaling in adipocytes caused by obesity was at least partially mediated by the downregulation of INSR induced by elevated miR-15b.

Supported by: No. 81570734

Disclosure: L. Xingjing: None.

63

Impact of rare heterozygous mutations of PCSK1 on obesity: implication for treatment with MC4R agonists

L. Folon1, M. Baron1, M. Derhourhi1, B. Balkau2, G. Charpentier3, S. Franc3, R. Roussel4, M. Canouil1, P. Froguel1,5, A. Bonnefond1,5;

1Inserm UMR1283, CNRS UMR8199, Lille, France, 2Inserm U1018, Villejuif, France, 3CERITD, Evry, France, 4Inserm U1138, Paris, France, 5Imperial College London, London, UK.

Background and aims: Pathogenic mutations in key genes involved in the leptin-melanocortin pathway (critical for the control of food intake) are well-established causes of monogenic forms of obesity. Recently, the melanocortin-4 receptor (MC4R) agonist setmelanotide has been developed to efficiently treat obese patients with homozygous mutations in POMC and LEPR. Its relevance to other forms of monogenic obesity is currently discussed. PCSK1 encodes the PC1/3 enzyme that is involved in this pathway. Bi-allelic pathogenic mutations in PCSK1 lead to early-onset obesity associated with severe endocrinopathy, but the clinical impact of heterozygous pathogenic PCSK1 mutations on obesity is still elusive. We performed large-scale functional genetic studies to clarify the link between heterozygous PCSK1 mutations and obesity.

Materials and methods: All 14 coding exons of PCSK1 were sequenced in 10,000 individuals (including obese and/or diabetic patients and controls) by next-generation sequencing. The detected heterozygous variants were created by mutagenesis and inserted into plasmids. Functional assays of each variant were performed in HEK293 cells: enzymatic activity was analysed using a fluorescent PC1/3 substrate and protein expression in cell lysates was studied by western blotting. Then, we determined different pathogenicity groups of these rare variants by clustering them through machine learning (k=5). Finally, we compared the pathogenicity of PCSK1 variants predicted by in silico REVEL (rare exome variant ensemble learner) test with results of in vitro analyses.

Results: We identified 66 rare heterozygous variants of PCSK1 (minor allele frequency < 1%). The variants were classified into 5 groups (A-E) based on their enzymatic activity, compared to the activity of the wild-type protein. The 17 variants in group A had complete loss of enzymatic activity whereas group B included 11 variants with partial loss of enzymatic activity. These loss of function variants were located mostly in catalytic domain but also in the prodomain and P domain of PC1/3. They strongly affect the structure of the protein preventing the expression of the mature active form of PC1/3. We found that class A variants increased the risk of developing obesity by 6-fold in both children (P=0.097; OR=6.0) and adults (P=0.038; OR=5.6). In contrast, class B variants were not associated with obesity risk (P=0.86; OR=1.1). Importantly, we observed a lack of sensibility and specificity of the largely used in silico tools predicting putative heterozygous pathogenic PCSK1 mutations. Indeed, the pathogenic variants predicted by REVEL showed 4 false negatives and 11 false positives compared to the results of in vitro functional assays.

Conclusion: Our results strongly suggest that only obese individuals carrying PCSK1 heterozygous mutations proven pathogenic through in vitro functional testing (i.e. category A) may be eligible for setmelanotide treatment, but not the carriers of mutations with only intermediate in vitro deleterious effect. Therefore, in vitro tests are required following the molecular diagnosis of rare heterozygous PCSK1 mutations before initiating expensive medication.

Disclosure: L. Folon: None.

64

Obesity specific N6-methyladenosine (m6A) RNA modification in human adipose tissue

T. Rønningen1, Y. Zeng2, M. Visnovska1, M.B. Dahl3, J. Wang3, L. la Cour Poulsen1, J.A. Kristinsson4, T. Mala4, J.K. Hertel5, J. Hjelmesæth5, T.G. Valderhaug6, H.H. He2, Y. Böttcher3;

1Department of Clinical Molecular Biology (EpiGen), Akershus University Hospital, Lørenskog, Norway, 2Princess Margaret Cancer Centre, Toronto, Canada, 3Department of Clinical Molecular Biology (EpiGen), University of Oslo, Oslo, Norway, 4Department of Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital, Oslo, Norway, 5Morbid Obesity Centre, Vestfold Hospital Trust, Tønsberg, Norway, 6Department of Endocrinology, Akershus University Hospital, Lørenskog, Norway.

Background and aims: Omental visceral adipose tissue (OVAT) has distinct gene expression and epigenetic profiles compared to subcutaneous adipose tissue (SAT), contributing to metabolic dysfunction. Post-transcriptional RNA modifications such as N6-methyladenosine (m6A) play important roles in regulating RNA metabolism. The FTO gene, whose genetic variants associate with obesity, encodes one of two known m6A demethylases, suggesting a mechanistic role for m6A in obesity. The aim of this study was (i) to map m6A signatures genome wide in paired samples of human adipose tissue and primary adipocytes, (ii) to identify obesity specific signatures and (iii) to decipher whether m6A discriminates for its depot of origin.

Materials and methods: RNA was extracted from intra-individually paired biopsies of SAT and OVAT from patients with obesity (BMI >35, n=10) and normal-weight controls (BMI <25, n=3). RNA was also obtained from primary adipocytes from 5 patients with obesity. M6A signatures were mapped using methylated RNA immunoprecipitation combined with next generation sequencing (meRIP-seq). meRIP-seq data were analyzed using established bioinformatics pipelines; peak calling was performed using MeTPeak and differential methylation analysis was performed using RADAR.

Results: We identified an average of ~6000 mRNA transcripts containing m6A in OVAT and SAT. Of those, we identified a number of genes (n~100) with differential m6A methylation in patients with obesity compared to normal weight controls. Further, by intersecting gene lists with m6A data from primary adipocytes, we also identified whether the specific m6A signature originates from adipocytes or cells from the stromal vascular fraction. Whilst most of the ~6000 methylated transcripts were conserved between SAT and OVAT, we identified a catalogue of genes (n=338) showing depot specific methylation.

Conclusion: We here present the first genome wide mapping of m6A in paired samples of human adipose tissue, identifying several novel targets linking m6A to obesity. Functional analyses of target genes will further elaborate on their role in obesity and metabolic dysfunction.

Supported by: South-Eastern Norway Regional Health Authority

Disclosure: T. Rønningen: None.

65

Waist-to-hip ratio is a stronger, more consistent predictor of all-cause mortality than BMI

I. Khan1, M. Chong2, A. Le2, P. Mohammadi-Shemirani2, R. Morton2, C. Brinza3, M. Kiflen4, S. Narula2, L. Akhabir2, S. Mao2, K. Morrison2, M. Pigeyre2, G. Paré2;

1College of Medicine and Health, University College Cork, Cork, Ireland, 2Pathology and Molecular Medicine, McMaster University, Hamilton, Canada, 3School of Medicine, Queen's University, Kingston, Canada, 4Temerty Faculty of Medicine, University of Toronto, Toronto, Canada.

Background and aims: Current clinical guidelines recommend a body mass index (BMI) between 18.5-24.9 kg/m2. However, BMI does not consider inter-individual variation in body composition and is thus not consistent in predicting risk of disease or mortality. We sought to determine whether current BMI recommendations are valid when accounting for body composition, and which of BMI, waist-to-hip ratio (WHR), and fat mass index (FMI) is the strongest and most consistent predictor of all-cause mortality across the range of body composition.

Materials and methods: We partitioned the British Caucasian UK Biobank (UKB) population (N= 387,672) into two subsets: a discovery cohort (N = 337,078) and validation cohort (N = 50,594). The discovery cohort was used to derive genetically-determined adiposity measures while the validation cohort was used for all subsequent analyses. Observational relationships between BMI, WHR, and FMI with mortality from all-cause, cancer, cardiovascular disease (CVD), respiratory disease, or non-CVD, - were analysed. Mendelian randomization (MR) was then used to assess causality of observed associations and to investigate effects across percentiles of BMI, WHR, and FMI.

Results: The study population consisted of 25,297 deaths and 25,297 randomly selected age- and sex-matched controls from the UKB (mean (SD) age: 61.6 (6.2), 59.3% male). Observational relationships between BMI and FMI with all-cause mortality were J-shaped, whereas the relationship between WHR with all-cause mortality increased linearly. Genetically-determined WHR had a stronger association with all-cause mortality compared to BMI or FMI, and exhibited a stronger effect in males compared to females (OR per SD of genetically-predicted WHR (95% CI): 1.51 (1.32 - 1.72); genetically-predicted BMI (95% CI): 1.29 (1.20 - 1.38); genetically-predicted FMI (95% CI): 1.26 (1.20 - 1.32), P for all analyses and comparisons < 0.05). Unlike BMI or FMI, the relationship between genetically-determined WHR and all-cause mortality was consistent across all percentiles of BMI, FMI, or WHR (P > 0.05).

Conclusion: In contrast to BMI, WHR has the strongest causal effect on risk of mortality regardless of the levels of adiposity and body composition, but show evidence of differential effects according to sex. Clinical recommendations and interventions should prioritize setting healthy WHR targets for males and females rather than general BMI targets. More precise recommendations for body shape may make a significant difference in disease burden and deaths due to excess of adiposity.

Supported by: OGS-M

Disclosure: I. Khan: None.

66

Single-cell RNA sequencing of human visceral adipose tissue identifies new macrophage clusters in health and obese type 2 diabetes

Y. Yuan, H. Wang, H. Sun, Y. Bi;

Department of Endocrinology, Affiliated Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China.

Background and aims: Adipose tissue macrophages are closely associated with type 2 diabetes mellitus (T2DM). However, in the development of obese T2DM, the atlas of human visceral adipose tissue macrophages and its evolvement remain unrevealed. This study aims to decipher the composition of human visceral adipose tissue macrophages and investigate their roles in T2DM.

Materials and methods: Non-obese non-T2DM, obese non-T2DM and obese T2DM subjects were enrolled for single-cell RNA sequencing of the stromal vascular fraction (SVF) of visceral adipose tissue. The sequencing data was analyzed to identify and annotate macrophage clusters and functional analysis was performed. Weighted gene correlation network analysis (WGCNA) was performed to reveal the distinct genetic and functional changes of macrophage clusters in obesity and T2DM. Flow cytometry was employed to validate the macrophage clusters, and to evaluate their abilities of lipid-buffering and tumor necrosis factor-α (TNF-α) production.

Results: Four macrophage clusters were identified in human visceral adipose tissue, including 1) perivascular macrophages (PVMs) with potential angiogenic and anti-inflammatory capacities. 2) Phagocytic macrophages (PMs) with endocytic potentials. 3) Monocyte-derived macrophages (MMs) that could regulate inflammatory response and the activation of T cells; 4) Lipid-associated macrophages (LAMs) that harbored lipid metabolic potentials. PVMs were located adjacent to blood vessels, while the other three clusters infiltrated between adipocytes, with PMs forming crown-like structures (CLSs). Among the four macrophage clusters, PVMs, MMs and LAMs have been reported previously, while PMs were discovered and defined for the first time. Flow cytometric analysis revealed that the levels of PMs raised significantly in T2DM patients, and their levels positively correlated with fasting blood glucose and HbA1c. Further functional experiments suggested that PMs had the highest lipid-uptake capacities and intracellular lipid content among the four macrophage clusters, which increased significantly in obese non-T2DM subjects and dropped in obese T2DM subjects. In addition, PMs had the highest levels of TNF-α+ cells, which were significantly higher in obese non-T2DM and obese T2DM subjects as compared to non-obese non-T2DM subjects.

Conclusion: The study analyzed the atlas of human visceral adipose tissue macrophages in non-obese non-T2DM, obese non-T2DM and obese T2DM states for the first time. Four distinct macrophage clusters were identified, among which PMs were newly discovered and defined. Flow cytometric analysis revealed that PMs may be closely involved in the pathogenesis of T2DM. The results deepened the understanding of human visceral adipose tissue macrophages and laid foundations for the understanding of the role of adipose tissue macrophages in obesity and T2DM, as well as the development of novel therapeutical strategies targeting obese T2DM.

Supported by: NSFC, China

Disclosure: Y. Yuan: None.

OP 12 GWAS and more

67

Genetics of serum C-peptide in type 1 diabetes

A.D. Paterson1, D. Roshandel1, A. Spiliopoulou2, S. McGurnaghan2, S.B. Bull3, P.M. McKeigue2, H.M. Colhoun2;

1The Hospital for Sick Children, Toronto, Canada, 2University of Edinburgh, Edinburgh, UK, 3Lunenfeld-Tanenbaum Research Institute, Toronto, Canada.

Background and aims: Heritability of serum C-peptide (CP) in type 1 diabetes (T1D) is 26%. Two independent genome-wide association studies (GWAS) of CP in T1D have identified multiple loci in the HLA region, insulin gene (INS), and a locus on chromosome 1. Although some of these loci overlap with those for T1D, some are independent. However, the identified loci together account for only a small proportion of CP variation. Here, we performed a large meta-GWAS of CP, including studies from the two prior GWAS, to improve the statistical power and detect new loci. We also investigated the association of T1D genetic risk score (GRS) at the HLA region with CP.

Materials and methods: 7,248 unrelated European subjects with T1D from four studies were included: the Scottish Diabetes Research Network Type 1 Bioresource (SDRNT1BIO) (n = 4,824, random CP), Diabetes Control and Complications Trial (DCCT) (n = 1,304, fasting CP), Coronary Artery Calcification in Type 1 Diabetes (CACTI) (n = 529, fasting CP) and Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) (n = 591, random CP). GWAS genotyping was performed with Illumina arrays. Ungenotyped variants were imputed using TOPMed, the largest imputation reference panel to date, which imputes lower frequency variants with better quality than previous panels. SNPs with minor allele frequency >0.01 and high imputation quality (INFO >0.80) present in both SDRNT1BIO and DCCT (the two large cohorts) were included in the meta-GWAS (n = 8,150,645). Classical HLA alleles, amino acid sequences and additional SNPs in the HLA region were imputed using multi-ethnic HLA reference panel on Michigan imputation server. Study-specific effect sizes and standard errors for scores and/or loci were estimated by multiple linear regressions of log (CP) with adjustment for covariates (sex, age at diagnosis, T1D duration). Meta-GWAS analysis was performed using METAL v1.5 weighting effect sizes using the inverse of the standard errors. We also tested association of T1D GRS at the HLA region with CP. T1D GRS at the HLA region was explored in total and separately for DR-DQ and non-DR-DQ haplotypes. Non-DR-DQ haplotypes were divided into class I, DPB1, and intergenic regions between DRB1 and DQA1 which do not track classic HLA alleles but regulate class II gene expression.

Results: We identified association at the HLA region (rs9271349, A>G, Chr6:32616053, β (SE) = 0.57 (0.08), p = 1.62E-12). In the HLA imputation, DRB1*06:02 (β (SE) = 0.90 (0.11), p = 1.18E-16) and DRB1*15:01 (β (SE) = 0.70 (0.10), p = 1.68E-12) haplotypes were associated. Five amino acid changes within HLA-DRB1, HLA-DQB1 and HLA-A reached the genome-wide significance threshold. DR-DQ GRS was not associated with CP whereas non-DR-DQ GRS was associated with lower CP levels in all four studies: SDRNT1BIO (β = -0.17, p = 2.00E-8), DCCT (β = -0.06, p = 0.045), CACTI (β = -0.08, p = 0.015) and WESDR (β = -0.10, p = 6.44E-4). The signal mostly came from class I haplotypes (A*2402, B*3906 and A*2902); and intergenic regions (rs9271346, rs116522341 and rs1281934). HLA-A*2402, HLA-B*3906 and intergenic GRSs were associated with lower CP whereas HLA-A*2902 GRS was associated with higher CP.

Conclusion: There is heterogeneity of T1D HLA variants in terms of their associations with CP. T1D DR-DQ GRS is not associated with CP. This could eventually provide insight into mechanisms and opportunities to preserve beta cell function.

Supported by: MRC: MR/T032340/1; CIHR: UCD-170583

Disclosure: A.D. Paterson: None.

68

Genome-wide association study on stroke in type 1 diabetes

E.H. Dahlstrom1,2, A. Antikainen1,2, L. Thorn1,2, A. Syreeni1,2, S. Hägg-Holmberg1,2, J. Putaala3, V. Harjutsalo1,2, P.-H. Groop1,4, N. Sandholm1,2;

1Folkhalsan Research Center, 2Research Program for Clinical and Molecular Metabolism, University of Helsinki and Helsinki University Hospital, 3Neurology, Helsinki University Hospital and University of Helsinki, 4Department of Nephrology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background and aims: The risk of stroke is markedly increased in type 1 diabetes. In addition, individuals with type 1 diabetes experience stroke at a considerably younger age. Despite the elevated risk of stroke, few studies have assessed genetic factors influencing stroke risk in type 1 diabetes. Therefore, our aim was to identify genetic risk factors for stroke in a genome-wide association study (GWAS).

Materials and methods: This study included 4,916 individuals with type 1 diabetes from the Finnish Diabetic Nephropathy (FinnDiane) Study with data available on stroke. Cases had experienced a stroke based on Finnish nationwide registry data (ICD: I60, I61, I62, I63, I64), with majority of the events verified from medical records by a neurologist. Controls were required to be older than 35 years and have a diabetes duration of more than 20 years. Genotyping was performed with Illumina HumanCoreExome chips at the University of Virginia, US. Imputation was done with Beagle at the Finnish Institute for Molecular Medicine (FIMM), using a Finnish reference panel (SISu v.3). We performed GWAS with the score test adjusted for the calendar year of diabetes onset, sex, and genotyping batches using the RVTESTS software. We included 8,812,836 variants with minor allele frequency (MAF) >1% and good imputation quality (r2 >0.60).

Results: One locus, containing 8 variants, was significant at a genome-wide threshold (p<5×10-8) and associated with stroke in type 1 diabetes (lead variant: rs146444827, MAF=1.2%, OR=4.25, p=2.2×10-8). The variants were located on chromosome 16 near or in the intronic region of the ADAMTS18 gene—a member of the ADAMTS gene family containing genes important in angiogenesis and coagulation. Variants within or near this gene have previously been associated with human cortical structure in brain MRI, brain sulcal depth, and body mass index (BMI) in GWAS. Furthermore, two other variants were suggestively associated with stroke (p<5×10-7). These were located on chromosome 14 within the NRXN3 gene, which has also been associated with BMI and specifically expressed in the brain (rs77936402, MAF=1.8%, OR=2.91, p<3.7×10-7), and on chromosome 1 in the PTGFR gene (rs145968355, MAF=1.2%, OR=3.77, p<1.7×10-7).

Conclusion: We have performed the largest GWAS to date on stroke in type 1 diabetes, and identified one new locus near ADAMTS18 gene, and two additional loci suggestively associated with stroke.

Supported by: EFSD/Sanofi European Diabetes Research Programme in Macrovascular Complications

Disclosure: E.H. Dahlstrom: None.

69

Identification of novel type 1 and type 2 diabetes genes by colocalisation of human islet eQTL and GWAS variants

A. Piron1, M.L. Colli1, M. Defrance2, D.L. Eizirik1, J.M. Mercader3, M. Cnop1;

1ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium, 2Interuniversity Institute of Bioinformatics in Brussels (IB2), Université Libre de Bruxelles, Brussels, Belgium, 3Programs in Metabolism and Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, USA.

Background and aims: Type 2 diabetes (T2D) results from progressive pancreatic beta cell failure. It is tightly related with glycemic traits, e.g. HbA1c, fasting and 2h post-challenge glucose and fasting insulin. Type 1 diabetes (T1D) results from an autoimmune destruction of beta cells. In both diseases genetic factors play a key role, but how genetic variants lead to beta cell failure remains poorly understood. Here, we analyzed the relationship between genetic variants associated with T2D, T1D, and glycemic traits (from genome-wide association studies, GWAS) and cis-expression quantitative trait loci (eQTL) in human islets. We developed a novel colocalization approach to assess whether genetic variants associated with traits share association signals with gene expression, leveraging a dataset of >400 samples.

Materials and methods: Colocalization analyses were performed between human islet eQTL from TIGER (http://tiger.bsc.es/) and GWAS from DIAMANTE (T2D), Type 1 Diabetes Genetics Consortium and MAGIC (glycemic traits). A novel colocalization method was developed that prioritizes candidate variants prior to testing with coloc R package. It shortlists candidate variants with specifically tailored rank-rank hypergeometric overlap analysis detecting linkage disequilibrium-induced overlap of variants. It identifies overlapping signals when multiple GWAS SNPs exist in the region of interest, a scenario in which coloc performs suboptimally. As a further improvement, the shortlisting method uses eQTL effect (up- or downregulation) and GWAS directions (increasing or decreasing risk) to further reduce the analyzed set of variants. The function of some of the novel genes was investigated in human islets and EndoC-bH1 cells.

Results: In total, 158 colocalizations were identified: 27 for T1D, 84 for T2D, 21 for HbA1c, 21 for fasting glucose, 4 for 2h post-challenge glucose, 1 for fasting insulin. The novel shortlisting method followed by coloc identified 58 new colocalizations of GWAS and eQTL SNPs that were not detected by coloc alone. Out of the 84 T2D eGenes, 16 also show colocalization with at least one additional glycemic trait. Five variants with low minor allele frequency (MAF<0.1) colocalized for T2D. Among them we discovered colocalization between MYO5C expression and T2D risk (MAF=0.05), with the protective allele being associated with increased MYO5C expression. A well-known eQTL for ADCY5 colocalized with T2D and all glycemic traits. T1D and T2D co-localizations were detected in the intronic region of GLIS3 that has been reported to modulate apoptosis in beta cells in both diseases. A FUT2 eQTL colocalized with a T1D GWAS signal; with the T1D protective allele being associated with increased FUT2 expression. Interestingly, infection of human beta cells by coxsackievirus B1 decreased FUT2 expression.

Conclusion: We identified 158 eQTLs colocalizing with T1D, T2D and glycemic traits, including 27 T1D, 37 glycemic and 30 T2D eGenes that are novel. This human islet expression regulatory variation sheds light on genes mediating genetic variant effects, representing an invaluable asset to understand glycemic traits and the pathophysiology of T2D and T1D.

Supported by: J.M.M. is supported by American Diabetes Association (1-19-ICTS-068) and by NHGRI, grant U01HG011723

Disclosure: A. Piron: None.

70

The impact of rare pathogenic variants of GLIS3 on type 2 diabetes

S. Meulebrouck1, M. Canouil1, M. Derhourhi1, B. Balkau2, G. Charpentier3, S. Franc3, M. Michel4, R. Roussel4, M. Vaxillaire1, P. Froguel1,5, A. Bonnefond1,5;

1Inserm UMR1283, CNRS UMR8199 - EGID, Lille, France, 2Inserm U1018, Villejuif, France, 3CERITD, Evry, France, 4Inserm U1138, Paris, France, 5Imperial College London, London, UK.

Background and aims: Type 2 diabetes (T2D) is due to a combination of environmental and genetic factors. There are monogenic forms, caused by a mutation in a single gene, that are usually (but not always) more severe and appear earlier than polygenic forms. Among the more than 30 genes responsible for monogenic diabetes, the biallelic pathogenic mutations of GLIS3, which encodes a key transcription factor for β cell lineage, are known to cause a rare autosomal recessive syndromic form of neonatal diabetes. We hypothesized that pathogenic heterozygous GLIS3 variants could also be associated with a dominantly transmitted form of monogenic diabetes or even contribute to common T2D.

Materials and methods: We sequenced GLIS3 in 5,471 individuals. The identified rare heterozygous variants were then functionally investigated through luciferase assays based on their ability to bind to the promoter region of the INS gene and activate its transcription. The pathogenicity of each variant was then assessed by combining our luciferase results with the other criteria from the American College of Medical Genetics and Genomics (ACMG). Finally, we performed adjusted association studies between the cluster of pathogenic or likely pathogenic (P/LP) variants and T2D risk, and we analyzed the phenotype of carriers and non-carriers.

Results: We identified 105 rare heterozygous variants of GLIS3. Through luciferase assays, we subsequently found 49 loss-of-function variants, presenting a decrease in luciferase activity compared to the wild-type GLIS3. By combining our functional results with the other ACMG criteria, we identified 18 P/LP variants. These variants were enriched at the C-terminus of the protein, containing its transactivation domain. Finally, the association studies showed that these P/LP variants were strongly associated with T2D risk (P = 0.003; OR = 3.91). Strikingly, all the T2D patients carrying a rare P/LP GLIS3 variant were treated with sulfonylureas, suggesting an early alteration of insulin secretion that responded to this class of medication. Importantly, both age of onset of diabetes and body mass index were similar between carriers and non-carriers.

Conclusion: We conclude that heterozygous P/LP variants of GLIS3 strongly increase the risk to develop T2D, and can not be clinically distinguished from the other common forms of T2D using classic monogenic diabetes clinical criteria. Genetic screening may be useful for precision medicine, as GLIS3 deficiency may severely impair insulin secretion and seem to respond to sulfonylureas as MODY1 and 3 do.

Disclosure: S. Meulebrouck: None.

71

Sequencing of 448 Greenlandic individuals uncovers a novel splice-affecting HNF1A variant with large population impact on diabetes

M.E. Jørgensen1,2, A.C.B. Thuesen3,4, F.F. Stæger5, A. Kaci6, M.H. Solheim6, I. Aukrust6, E. Jørsboe3, C.G. Santander7, A. Gilly8, M.L. Pedersen1,9, E. Zeggini8, L. Bjørkhaug10, A. Albrechtsen7, I. Moltke7, T. Hansen3;

1Steno Diabetes Center Greenland, Nuuk, Greenland, 2Clinical Epidemiology, Steno Diabetes Center Copenhagen, Herlev, Denmark, 3Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark, 4Clinical Research, Steno Diabetes Center Copenhagen, Herlev, Denmark, 5Section for Computational and RNA Biology, Department of Biology,, University of Copenhagen, Copenhagen, Denmark, 6Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway, 7Section for Computational and RNA Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark, 8German Research Center for Environmental Health, Neuherberg, Germany, 9Greenland Center for Health Research, Institute of Health and Nature, University of Greenland, Nuuk, Greenland, 10Department of Safety, Chemistry and Biomedical laboratory science, Western Norway University of Applied Sciences, Bergen, Norway.

Background and aims: The genetic disease architecture of Inuit includes a large number of common high-impact variants. Identification of such variants contributes to our understanding of the genetic aetiology of diseases and improves global equity in genomic personalised medicine. We aimed to identify and characterise novel variants in genes associated with Maturity Onset Diabetes of the Young (MODY) in the Greenlandic population.

Materials and methods: Using combined data from Greenlandic population cohorts of 4497 individuals, including 448 whole genome sequenced individuals, we screened 14 MODY genes for known and novel variants. We functionally characterised an identified novel variant and assessed its association with diabetes prevalence and cardiometabolic traits and population impact.

Results: We identified a novel HNF1A variant with an allele frequency of 1·9% in the Greenlandic Inuit and absent elsewhere. Functional assays indicate that it prevents normal splicing of the gene. The variant caused lower 30-min insulin (β=-232 pmol/L, βSD=-0·695, P=4·43×10-4) and higher 30-min glucose (β=1·20 mmol/L, βSD=0·441, P=0·0271) during an oral glucose tolerance test. Furthermore, the variant was associated with type 2 diabetes (OR 4·4, P= 7·24×10-6) and HbA1c (β=0·11 HbA1c%, βSD=0·21, P=0·0052). The variant explained 2·5% of diabetes variance in Greenland.

Conclusion: The reported variant has the largest population impact of any previously reported variant within a MODY gene. Together with the recessive TBC1D4 variant, we show that close to 1 in 5 cases of diabetes (18%) in Greenland has a monogenic diabetes aetiology compared to 1-3% in large populations.

Supported by: Novo Nordisk Foundation, Independent Research Fund Denmark, and Karen Elise Jensen’s Foundation.

Disclosure: M.E. Jørgensen: None.

72

A common variant in the ketohexokinase gene is associated with fructosuria and cardiometabolic outcomes

A.M. Buziau1, C.G. Schalkwijk1, J.L.J. Scheijen1, P.I.H. Simons1, C.J.H. van der Kallen1, S.J.P. Eussen1, N.C. Schaper2, P.C. Dagnelie1, M.M.J. van Greevenbroek1, A. Wesselius2, C.D.A. Stehouwer2, M.C.G. Brouwers2;

1Internal Medicine, Maastricht University, 2Maastricht University Medical Center+, Maastricht, Netherlands.

Background and aims: There is an ongoing discussion on whether fructose per se is disadvantageous for cardiometabolic health. Recently, pharmacological inhibition of ketohexokinase (KHK), the first enzymatic step of fructolysis, was shown to reduce intrahepatic lipid content in humans. The study of individuals carrying functional variants in the KHK gene will provide more insight into the lifelong effects of inhibition of KHK, as these individuals have been exposed from birth to a KHK protein with less enzymatic activity. The aims of the present study were: 1) to study whether a common missense variant in KHK (rs2304681) is a functional variant that affects fructosuria (similar to pharmacological inhibition of KHK); and 2) to study the association between rs2304681 and cardiometabolic outcomes.

Materials and methods: First, linear regression analyses were performed to study the association between rs2304681 and 24h urinary fructose levels (quantified by tandem mass spectrometry), with adjustment for age, sex, and type 2 diabetes (T2D), in the Maastricht Study, a population-based cohort. Second, we used summary-level data on the association of rs2304681 with non-alcoholic fatty liver disease, T2D, hypertension, and myocardial infarction, obtained from publicly available databases.

Results: First, the rs2304681 minor A allele (frequency: 0.36) was associated with higher 24h urinary fructose levels (unstandardized beta: 0.064; 95% CI: 0.027-0.100; n=1,471). Second, the rs2304681 minor A allele protected from hepatic steatosis (OR: 0.972; 95% CI: 0.957-0.988; n=36,703; UK Biobank), T2D (OR: 0.985; 95% CI: 0.975-0.99; n=1,331,670; fixed-effects meta-analysis in the AGEN and the European DIAMANTE cohorts) and myocardial infarction (OR: 0.976; 95% CI: 0.961-0.992; n=583,191; fixed-effects meta-analysis in the CARIoGRAMplusC4D and the UK Biobank cohorts). Two studies both showed a protective effect on the risk of hypertension (OR: 0.988; 95% CI: 0.976-0.999; n=440,285; UK Biobank; and Z-score: -2.59; p = 0.01; n=192,763; the combined CHD Exome+, ExomeBP, and GoT2D cohorts).

Conclusion: Lifelong impairment of KHK activity (reflected by rs2304681) is associated with fructosuria and protection from cardiometabolic disease. These findings suggest that fructose per se has harmful cardiometabolic effects, which may be mitigated by pharmacological inhibition of KHK.

Supported by: Dutch Diabetes Research Foundation

Disclosure: A.M. Buziau: None.

OP 13 Beyond type 1 and type 2

73

Clusters of prediabetes and type 2 diabetes stratify all-case mortality in a cohort of participants undergoing invasive coronary diagnostics

K. Prystupa1,2, G.E. Delgado3,4, A.P. Moissl5,3, M.E. Kleber3,6, M. Heni1,2, A.L. Birkenfeld1,2, A. Fritsche1,2, W. März3,7, R. Wagner1,2;

1Department of Internal Medicine IV, Division of Endocrinology, Diabetology and Nephrology, University of Tuebingen, Tübingen, 2Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tübingen, Tübingen, 3Vth Department of Medicine (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Mannheim, 4Center for Preventive Medicine and Digital Health Baden-Württemberg (CPDBW), Medical Faculty Mannheim, Heidelberg University, Mannheim, 5Institute of Nutritional Sciences, Friedrich Schiller University Jena, Jena, 6SYNLAB MVZ für Humangenetik Mannheim GmbH, Mannheim, 7SYNLAB Academy, SYNLAB Holding Deutschland GmbH, Mannheim, Germany.

Background and aims: The risk for complications and mortality among patients with type 2 diabetes (T2D) is heterogeneous. Different trajectories can be identified in the prediabetic state, which comprises heterogeneous metabolic clusters. It is not known whether such pathophysiologic clusters of prediabetes and diabetes affect survival in at-risk persons being evaluated for coronary heart disease.

Materials and methods: The LURIC Study recruited patients referred for coronary angiography at a median age of 63(IQR 56-70) who have since been followed up for an overage of 20 years. Clustering of 1269 subjects without diabetes was performed with oGTT-derived glucose and insulin; fasting triglyceride, high-density lipoprotein, BMI, waist, and hip circumference. Patients with T2D (n=794) were clustered using age, BMI, glycemia, homeostasis model assessment, and islet autoantibodies. Associations of clusters with mortality were analyzed using Cox regression.

Results: Individuals without diabetes were classified into six subphenotypes, with 884 assigned to subjects at low-risk (cluster 1,2,4) and 385 at high-risk (cluster 3,5,6) for diabetes. We found significantly increased mortality in clusters 3 (hazard ratio (HR) 1.42), 5 (HR 1.43), and 6 (HR 1.46) age-, BMI-, HbA1c- and sex-adjusted. In the T2D group, 508 were assigned to mild age-related diabetes (MARD), 183 to severe insulin-resistant diabetes (SIRD), 84 to mild obesity-related diabetes (MOD), 19 to severe insulin-deficient diabetes (SIDD). Compared to the low-risk non-diabetes group, crude mortality was not different in MOD. Increased mortality was found for MARD (HR 2.2), SIRD (HR 2.2), and SIDD (HR 2.5).

Conclusion: Metabolic clustering successfully stratifies survival even among persons already undergoing invasive coronary diagnostics. Novel clustering approaches based on glucose metabolism can identify persons who require specific attention as they have an increased mortality risk.

Supported by: BMBF

Disclosure: K. Prystupa: None.

74

Novel clusters of prediabetes and their association with progression and regression: a 3-year follow-up study

Y. Liu, M. Sang, S. Qiu, Z. Sun;

Southeast University, Nanjing, China.

Background and aims: Cluster analysis may assist in stratifying heterogeneous clinical presentations of prediabetes. However, the association of cluster-based subgroups of prediabetic with its progression to diabetes and regression to normoglycemia remains unclear. This study was aimed to address this issue with novel clusters of prediabetes derived from four parameters.

Materials and methods: We developed a k-means clustering model in participants with prediabetes (N=4,138) from the SENSIBLE and SENSIBLE-Addition studies, based on body mass index (BMI), age, triglyceride-and-glucose (TyG) index, and hemoglobin A1c (HbA1c). TyG index was used to assess insulin resistance. Of the included participants with prediabetes at baseline, 1,629 were followed-up for 3 years. Prediabetes was defined as impaired fasting glucose and/or impaired glucose tolerance based on World Health Organization 1999 criteria. Logistic regression analyses were performed to obtain the odds ratios (ORs) and 95% confidence intervals (CIs).

Results: Three clusters were identified, with cluster 0, 1 and 2 accounting for 28.7%, 30.6% and 40.7%, respectively. Participants with prediabetes were featured by the youngest and the lowest HbA1c in cluster 0, the highest BMI and TyG index in cluster 1, and the oldest and the lowest BMI in cluster 2. After the adjustment for gender, ethnic groups, serum uric acid, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and estimated glomerular filtration rate, cluster 1 (OR 3.61, 95% CI: 2.21-5.90) and cluster 2 (OR 3.01, 95% CI: 1.87-4.84) were both associated with increased risk of progression to diabetes when compared with cluster 0. Moreover, cluster 1 and cluster 2 were both associated with decreased chance of regression to normoglycemia (OR 0.42, 95% CI: 0.31-0.57; and OR 0.48, 95% CI: 0.37-0.63, respectively).

Conclusion: Our cluster-based analysis showed that participants featured by older age and higher degree of insulin resistance, obesity, or insulin resistance had higher risk of progression to diabetes and lower risk of regression to normoglycemia. While young participants with sufficient attention to BMI and blood glucose management were more likely to regress to normoglycemia.

figure s

Supported by: the National Key R&D Program of China (2016YFC1305700)

Disclosure: Y. Liu: None.

75

Incidence of HbA 1c -defined prediabetes and progression to type 2 diabetes: a nationwide study with routine care laboratory data

S.K. Nicolaisen1, R.W. Thomsen1, D. Witte2, H.T. Sørensen1, L. Pedersen1;

1Department of Clinical Epidemiology, Aarhus University Hospital and Aarhus University, 2Steno Diabetes Center Aarhus and Department of Public Health, Aarhus University, Aarhus, Denmark.

Background and aims: Since glycated hemoglobin (HbA1c) became a diagnostic tool for diabetes in 2011, HbA1c testing has increasingly been used. Population-based routine clinical care laboratory databases may therefore be a valuable research tool in prediabetes research. We aimed to describe HbA1c measurement patterns in the entire Danish population, to identify and characterize individuals with incident HbA1c-defined prediabetes, and to examine their risk of progression to diabetes.

Materials and methods: All HbA1c measurements in the Danish nationwide laboratory databases during 2012-2018 were characterized. The general population incidence rate of prediabetes (HbA1c 42-47 mmol/mol) was calculated. Among individuals with prediabetes, the 5-year cumulative incidence of diabetes (HbA1c >=48 mmol/mol) was assessed.

Results: Among 5,483,467 Danish residents, 13,107,797 HbA1c measurements from 3,183,880 (58%) individuals were available in the database for the 2012-2018 period. A total of 373,628 individuals were identified with incident HbA1c-defined prediabetes, corresponding to an incidence rate of 15.5 (95% CI 15.5-15.6) per 1,000 person-years. The median HbA1c at prediabetes diagnosis was 43 mmol/mol (IQR 42-44), median age was 67.8 years (IQR 57.9-76.4), and 52.0% were women. During a median follow-up of 2.7 years, 46,821 (12.5%) developed HbA1c-defined diabetes, yielding an estimated cumulative 5-year diabetes incidence of 20.1% (95% CI 19.9-20.3).

Conclusion: More than half of the Danish entire nation had at least one HbA1c measurement registered during 2012-2018. The incidence rate of prediabetes was 15.5 per 1000 person-years, and likely due to extensive testing, persons were identified at an early stage of prediabetes. Within 5 years, one in five individuals with prediabetes will progress to diabetes.

Disclosure: S.K. Nicolaisen: None.

76

Antibody positive patients with type 2 diabetes who rapidly progress to insulin have similar characteristics and type 1 genetic risk scores as patients with type 1 diabetes

V. Simpson1,2, N. Thomas1,2, A.V. Hill1,2, B.M. Shields1,2, S. Deshmukh1, T.J. McDonald1,2, A.G. Jones1,2, StartRight Study Group;

1University of Exeter, 2Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.

Background and aims: New EASD/ADA guideline recommend testing islet autoantibodies in all clinically suspected adult-onset type 1 diabetes (T1D), including those treated as type 2 diabetes (T2D) with early progression to insulin, and advise that a positive islet antibody in this setting confirms type 1 diabetes. We assessed whether a positive islet autoantibody in the setting of initial treatment as type 2 diabetes and early progression to insulin confirmed the genetic and C-peptide progression characteristics of type 1 diabetes.

Materials and methods: In 1793 participants with recently diagnosed adult-onset diabetes (duration at recruitment <12 months, median 5 months) we compared the baseline characteristics, T1D genetic risk score (T1DGRS) and rate of loss of beta-cell function (annual urine c-peptide creatinine ratio (UPCR), over median 2 years) between definite T1D, defined as a clinical diagnosis of T1D, treated with insulin within 14 days of diagnosis and ≥2 positive islet-autoantibodies (of GAD, IA2, Znt8, n=305) and patients treated as T2D (diagnosed or suspected T2D without insulin for >2 weeks from diabetes diagnosis) with positive islet autoantibodies and insulin treatment within 3 years of diagnosis (n=94). We also compared these characteristics in antibody positive T2D not progressing to insulin within 3 years (n=79) and autoantibody negative tT2D (n=687).

Results: T1DGRS and C-peptide progression are shown in figure 1. Participants with positive autoantibodies and rapid progression to insulin treatment had genetic susceptibility consistent with confirmed T1D: T1DGRS 13.0 (95%CI 12.7-13.4), compared to 12.9 (95%CI 12.7-13.1) in multi-antibody positive T1D (p=0.63). These participants also had rapid loss of C-peptide, again consistent with confirmed T1D: annual change in UPCR -37% (-25,-47) in comparison to -36% (-30,-41) in multiple antibody positive T1D (p=0.8). In contrast, positive islet autoantibodies in those without early progression to insulin had T1D genetic susceptibility and C-peptide loss more similar to T2D than T1D: in antibody positive participants without progression to insulin and antibody negative T2D T1DGRS was 10.8 (95%CI 10.2-11.5) and 10.0 (95%CI 9.8-10.2) (p <0.001) respectively, annual change in UCPCR -10% (0,-25%) and -5% (0,-15%) (p<0.001) respectively. In a sub-group of those autoantibody positive (n = 40) and early insulin treatment, but without insulin within 6 months of diagnosis, T1DGRS and UCPCR remained consistent with classical type 1 diabetes (T1DGRS 12.9 (95% CI 12.5-13.4), UCPCR annual change -37% (95% CI -19%,-51%).

Conclusion: In people initially treated as T2D with early progression to insulin a positive islet autoantibody test confirms the genetic characteristics and rate of C-peptide loss of classical T1D. These patients should therefore be considered to have, and be treated as, T1D.

figure t

Clinical Trial Registration Number: NCT03737799

Supported by: Diabetes UK

Disclosure: V. Simpson: None.

77

Children with newly-diagnosed diabetes but no autoantibodies shoud be genetically tested

M. Harsunen1,2, J.L.T. Kettunen2,3, T. Härkönen1,2, P. Vähäsalo4,5, R. Veijola4,5, J. Ilonen6, P.J. Miettinen1,7, M. Knip1,2, T. Tuomi2,3;

1New Children's Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, 2Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, 3Abdominal Centre, Endocrinology, University of Helsinki and Helsinki University Hospital, Helsinki, 4Department of Pediatrics, PEDEGO Research Unit, Medical Research Center, University of Oulu, Oulu, 5Department of Children and Adolescents, Oulu University Hospital, Oulu, 6Immunogenetics Laboratory, Institute of Biomedicine, University of Turku, Turku, 7Translational Stem Cell Biology and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Background and aims: Patients with monogenic forms of diabetes may be misclassified and treated suboptimally. While the prevalence of type 1 diabetes (T1D) is high in children in Finland, that of monogenic diabetes is not known. We assessed the prevalence and clinical manifestations of monogenic diabetes in children, who were negative for T1D related autoantibodies (AAB) or positive only for low titer islet cell antibodies (ICA) at diagnosis.

Materials and methods: A next generation sequencing gene panel including 42 genes was used to identify monogenic diabetes in participants of the Finnish Pediatric Diabetes Register, covering approximately 90% of newly-diagnosed diabetes in Finland (<16 years). Five AAB (ICA, IAA, GADA, IA-2A, ZnT8A) and HLA class II genotypes were analysed at diagnosis.

Results: Out of 6482 participants initially registered with T1D, we sequenced DNA for 152 (2.3%) testing negative for all AAB and 49 (0.8%) positive only for ICA ≤10 JDFU (ICAlow). Monogenic diabetes was revealed in 19 (12,5%) of the AAB-negative and 2 (4%) of the ICAlow individuals. The genes involved were GCK, HNF1A, HNF4A, HNF1B, INS, KCNJ11, RFX6, LMNA and WFS1. None of these patients had ketoacidosis at diagnosis or carried an HLA genotype conferring high risk for T1D. The diagnosis of monogenic diabetes led to a change in the treatment of many patients. A switch from insulin treatment to oral medication has been successful in patients with variants in HNF1A, HNF4A or KCNJ11, significantly improving their glycemic control and quality of life. The figure illustrates continuous glucose monitoring before and after a transfer from insulin pump to sulfonylurea in a patient with KCNJ11 variant. Next, we will proceed to exome sequencing of the AAB-negative children without genetic diagnosis.

Conclusion: More than 10% of AAB-negative children had a genetic variant associated with monogenic diabetes. Because diagnosis of monogenic diabetes can lead to major changes in the treatment, we recommend referring all children with newly-diagnosed diabetes who are AAB-negative to genetic testing. Low-titer ICA in the absence of other AAB does not always indicate a diagnosis of T1D.

figure u

Disclosure: M. Harsunen: None.

78

Penetrance of MODY is substantially lower in clinically unselected cohort: important implications for opportunistic genomic testing

K. Patel1, U. Mirshahi2, K. Colclough3, C. Wright1, A. Wood1, R. Beaumont1, T. Laver1, R. Stahl2, A. Golden2, J. Goehringer2, Geisinger-Regeneron DiscovEHR Collaboration, T. Frayling1, A. Hattersley1, D. Carey2, M. Weedon1;

1University of Exeter, Exeter, UK, 2Geisinger Health System, Danville, USA, 3Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.

Background and aims: Accurate estimates of penetrance are needed for counselling of individuals with a mutation in common MODY (Maturity Onset Diabetes of the Young) genes. We aimed to comprehensively assess the penetrance of the three most common causes of MODY (HNF1A, HNF4A and GCK) in large genotype-first (clinically unselected) and phenotype-first (clinically selected) cohorts.

Materials and methods: We analysed clinical and genetic sequencing data from four different cohorts: 1742 probands referred for clinical MODY testing; 2194 family members of the MODY probands; clinically unselected 132,194 individuals from an American hospital-based cohort; and 198,748 individuals from a UK population-based cohort.

Results: Age-related penetrance of diabetes for pathogenic variants in HNF1A and HNF4A was substantially lower in the clinically unselected cohorts compared to clinically referred probands (ranging from 32% to 98% at age 40yrs for HNF1A, and 21% to 99% for HNF4A). The background rate of diabetes, but not clinical features or variant type, explained the reduced penetrance in the unselected cohorts. In contrast, the penetrance of mild hyperglycaemia for pathogenic GCK variants was similarly high across cohorts (ranging from 89 to 97%) despite substantial variation in the background rates of diabetes.

Conclusion: Penetrance of HNF1A/HNF4A-MODY but not GCK-MODY is substantially lower in the genotype-first approach and has important implications for opportunistic screening during genomic testing. Our finding suggests the need to tailor genetic interpretation and counselling for individuals based on the context in which a pathogenic variant was identified.

Supported by: Wellcome Trust, MRC

Disclosure: K. Patel: None.

OP 14 Exercising your tissues in shape

79

Does exercise matter? The effects of different volumes of exercise on beta cell function in patients with newly diagnosed type 2 diabetes

M. Lyngbaek1, G. Legaard1, N. Nielsen1, T. Almdal2, M. Ried-Larsen1, K. Karstoft2;

1Centre for Physical Activity Research, Copenhagen University Hospital - Rigshospitalet, 2Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark.

Background and aims: Dietary weight loss may lead to improved beta-cell function (disposition index: DI = insulin sensitivity x insulin secretion) in persons with type 2 diabetes (T2D). However, the role of exercise in adjunct to diet therapy remains unclear. Thus, we performed a study assessing beta-cell function both at supraphysiological conditions using the gold standard hyperglycemic clamp, and at physiological conditions using a mixed meal tolerance test. We expected to observe a dose-dependent improvement in beta-cell function when adding exercise to dietary weight loss.

Materials and methods: Persons with T2D duration < 7 years and obesity/overweight were randomized 1:1:1:1 (stratified by sex) to either 16 weeks of 1) Standard care 2) Dietary intervention 3) Dietary intervention + exercise 3 times/week 4) Dietary intervention + exercise 6 times/week. The exercise interventions consisted of both aerobic and resistance training. The dietary intervention aimed at a 25% caloric deficit and weight loss. The primary outcome was the change in beta-cell function assessed with clamp-derived late-phase disposition index. Major secondary outcomes included clamp- and mixed meal-derived changes in beta-cell function, insulin secretion, and insulin sensitivity from baseline to 16-weeks follow-up. The full protocol has been published.

Results: We randomized 82 participants (35% females, mean age (SD) of 58.2 (9.8) years, BMI 33.1 (3.7) kg/m2, median T2D duration (IQR) of 4.0 (1.9 to 5.5) years. Adherence to the diet (25-30% energy reduction) and exercise (>85%) was similar across intervention groups. Five persons (6%) were lost to follow-up. From the hyperglycemic clamp we observed improvement in late-phase disposition index in all three intervention groups. The mean difference (MD) from standard care (95% confidence intervals (CI)) was 58 (16 to 116)%, 105 (49 to 182)% and 137 (73 to 225)% for diet only, moderate volume exercise, and the high volume exercise, respectively. While the improvement was larger in the high volume exercise group compared to diet group (MD (95% CI): 50 (10 to 104)%), no difference where observed between the exercise groups (high volume exercise vs. moderate volume exercise MD (95% CI): 16 ( -16 to 59)%) or between moderate volume exercise and the diet group (MD (95% CI): 29 (-5 to 77)%). No differences in glucose-stimulated insulin secretion were observed between the groups. Improvements in insulin sensitivity followed a similar pattern as observed for the disposition index. The observations during the meal test confirmed the findings from the hyperglycemic clamp.

Conclusion: Sixteen weeks of high volume of exercise in combination with dietary weight loss improves beta-cell function more than standard care or diet alone in persons with T2D. However, there was no significant difference between moderate and high volume of exercise in beta-cell function, suggesting that increasing exercise volume from 3 to 6 times/week, may not amplify the beta-cell function. The improvement in beta-cell function was mainly mediated by improved insulin sensitivity, whereas adding exercise to the dietary intervention did not seem to increase glucose- or meal-stimulated insulin secretion beyond dietary weight loss alone.

Clinical Trial Registration Number: NCT03769883

Supported by: TrygFonden ID 124708 and Svend Andersen Fonden

Disclosure: M. Lyngbaek: Grants; Danish Diabetes Academy NNF17SA0031406.

80

Improved beta cell function following eight weeks high-intensity interval training combining rowing and cycling in type 2 diabetes

M.H. Petersen1, M.E. de Almeida2, E.K. Wentorf2, J.V. Stidsen1, N. Ørtenblad2, K. Højlund1;

1Steno Diabetes Center Odense, 2Department of Sports Science and Clinical Biomechanics, Odense, Denmark.

Background and aims: High-intensity interval training (HIIT) and recruitment of several muscle groups seem to enhance the effect of exercise training on glucose homeostasis in type 2 diabetes. However, the effect of these factors on beta-cell function in type 2 diabetes remains to be established. Here, we investigated the effect of a novel HIIT-protocol engaging upper and lower body muscles on the beta-cell function in individuals with type 2 diabetes compared with glucose-tolerant obese and lean controls.

Materials and methods: Fifteen obese men with type 2 diabetes, and age-matched, glucose-tolerant obese (n=15) and lean (n=18) men underwent an 8-week, supervised, HIIT-protocol combining rowing and cycling. Before and after the training period, the participants were evaluated by the Botnia clamp combining an IVGTT with a hyperinsulinemic-euglycemic clamp to measure first- and second-phase insulin secretion, insulin sensitivity and estimate beta-cell function adjusted for insulin sensitivity (disposition index).

Results: At baseline, insulin sensitivity was ~40% lower, and first-phase insulin secretion and the disposition index were >90% lower in men with type 2 diabetes compared with both lean and obese controls (all p<0.05). The HIIT-protocol induced a marked increase (~30-40%) in insulin sensitivity in all groups (all p<0.05). In patients with type 2 diabetes, this was accompanied by a large (>200%) but highly variable improvement in the disposition index (p<0.05), and a clinically relevant reduction in HbA1c (~4 mmol/mol). However, first-phase insulin secretion and the disposition index were still markedly reduced in men with type 2 diabetes (all p<0.05). In lean and obese controls, HIIT also improved (~30%) the disposition index (all p<0.05) and tended to reduce the second-phase insulin response (p<0.10). No group-differences were seen in the HIIT-induced responses on insulin secretion and insulin sensitivity.

Conclusion: HIIT combining rowing and cycling induces a large, but highly variable increase in beta-cell function adjusted for insulin sensitivity in men with type 2 diabetes, and to a numerically smaller extent in obese and lean glucose-tolerant men. However, after the HIIT-protocol, the beta-cell function was still severely impaired in men with type 2 diabetes, even when adjusted for the improvement in insulin sensitivity, suggesting that this component of type 2 diabetes is less reversible than insulin sensitivity.

Clinical Trial Registration Number: 17/31977

Supported by: Novo Nordisk Foundation and Sawmill owner Jeppe Juhl and wife Ovita Juhls Memorial Bursary

Disclosure: M.H. Petersen: None.

81

Exercise-responsive non-coding RNAs in the regulation of skeletal muscle metabolism

E. Caria1, I. Sen1, M. Katayama1, M. Savikj2, A.M. Abdelmoez2, J.A.B. Smith2, N.J. Pillon1, J.R. Zierath2, A. Krook1;

1Department of Physiology and Pharmacology, Karolinska Institutet, 2Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Exercise triggers profound structural and metabolic adaptations in skeletal muscle. While this largely accounts for the benefits of exercise in the treatment or prevention of type 2 diabetes (T2D), a detailed understanding of the molecular mechanisms involved remains unclear and warrants further research. Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), have recently emerged as important regulators of skeletal muscle physiology, and their expression in skeletal muscle is modulated by different exercise training programs. miRNAs are also incorporated into exosomes and secreted upon exercise to exert autocrine, paracrine and/or endocrine effects on tissue-specific or whole-body glucose homeostasis. Here, we aimed to identify exercise-responsive serum exosomal miRNAs (study A) or skeletal muscle-derived lncRNAs (study B) that mediate skeletal muscle adaptations to exercise training or acute exercise in individuals with or without T2D.

Materials and methods: In study A, young healthy individuals (n=12) underwent three weeks of aerobic exercise training, and serum was collected before and after for exosome isolation. Differentially expressed exosomal miRNAs were identified using a miRNA focus PCR panel. Functional characterization of the identified candidates was performed by transfecting primary human skeletal muscle cells with miRNA mimics, and effects on glucose metabolism were assessed using radiolabelled substrate assays. In study B, a separate cohort of individuals with T2D (n=19) or normal glucose tolerance (NGT, n=17) underwent 30 minutes of cycling exercise at 85% of their maximum heart rate. Vastus lateralis muscle biopsies were taken at baseline, immediately after, and three hours after exercise, and RNA sequencing was performed to identify differentially expressed, skeletal muscle-derived lncRNAs.

Results: In study A, exosomal miR-136-3p and miR-139-5p were increased in the circulation following three weeks of aerobic exercise training. Overexpression of miR-136-3p or miR-139-5p in human primary skeletal muscle cells elicited exercise-like metabolic effects, in terms of increased glucose uptake (miR-136-3p) and glycogen synthesis (miR-139-5p). In study B, a single bout of exercise changed the expression of more than 200 lncRNAs in the T2D group, whereas this number was much lower in the NGT group (24). We identified exercise-responsive lncRNAs that were co-expressed with metabolic genes and shortened the candidate list to 41 lncRNAs for screening, according to their fold-changes versus baseline and expression level in human myotubes.

Conclusion: Exosomal miR-136-3p and miR-139-5p are increased in serum following exercise training. Increased miR-136-3p or miR-139-5p lead to metabolic adaptations in skeletal muscle. An acute exercise bout alters the expression of several lncRNAs specifically in skeletal muscle from subjects with T2D. Further functional characterization of these candidates could provide mechanistic insights into the regulation of skeletal muscle metabolism by exercise, particularly in the context of T2D.

Disclosure: E. Caria: None.

82

Exercise-induced crosstalk between immune cells and adipocytes in humans: role of oncostatin-M

L. Dollet1,2, L.L. Lundell2, A.V. Chibalin3, L.A. Pendergrast3, N.J. Pillon1, K. Caidahl3, A.S. Deshmukh2, T. De Castro Barbosa4, M. Rydén4, R. Barrès2, H. Wallberg-Henriksson1, J.R. Zierath2,3, A. Krook1;

1Department of Physiology and Pharmacology, Karolinska Institutet, Solna, Sweden, 2Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark, 3Department of Molecular Medicine and Surgery, Karolinska Institute, Solna, Sweden, 4Department of Medicine (H7), Karolinska Institutet, Stockholm, Sweden.

Background and aims: The discovery of novel exercise-regulated circulatory factors has fueled interest in the role of organ-crosstalk, especially between skeletal muscle and adipose tissue in conferring the beneficial effects of exercise on insulin sensitivity and metabolism. We aimed to study the adipose tissue secretome in the context of exercise, and its potential role in inter- and intra-organ crosstalk in the response to exercise in individuals with normal glucose tolerance or type 2 diabetes.

Materials and methods: Men and women with normal glucose tolerance (NGT, n=20) or type 2 diabetes (T2D; n=28) performed an acute exercise bout (30 min cyclometer). Leg adipose tissue biopsies were collected from the participants at rest, immediately after exercise, and after 3h recovery for RNA sequencing analysis. Putative exercise-induced secreted proteins were selected, and cultured human adipocytes were exposed in vitro to recombinant candidate proteins. Phosphorylation arrays and immunoblot analysis were performed to track signalling events, and lipolytic responses were measured. Plasma measurements were performed using ELISA. THP1 human monocytes were differentiated in vitro, and gene expression of candidates were measured after acute exposure to an adrenergic agonist and conditioned media from contracted cultured myotubes.

Results: Transcriptomic analysis revealed time-dependent changes, with 712 and 893 genes altered post exercise, and 1353 and 4316 genes altered after recovery in adipose tissue from individuals with NGT or T2D respectively (FDR<0.05). The differential response in T2D adipose tissue was associated with a sustained activation of inflammatory pathways as shown by gene ontology analysis. Oncostatin-M was identified as one of the most upregulated putative secreted factors post exercise (logFC=3.35 and 2.95 for NGT and T2D). Oncostatin-M plasma level was increased after exercise in both groups (2-way ANOVA p<0.001). Exposure of isolated human adipocytes to recombinant oncostatin-M activated phospho-ERK and MAPK signalling and stimulated basal lipolysis (+47%, p<0.001). RNA sequencing in adipose tissue cell populations showed that oncostatin-M expression mainly arises from the macrophage fraction, while the corresponding receptors are expressed in adipocytes. Accordingly, oncostatin-M expression was induced in Thp1 macrophages in response to adrenergic stimuli (+620%, p<0.001), as well as in response to conditioned media from exercised myotubes (+ 79% at 3h, 2-way ANOVA p<0.001).

Conclusion: Collectively, our findings reveal that the adipose tissue transcriptome profile is robustly altered by exercise in a time-dependent manner, with a sustained increase in an inflammatory signature unique to Type 2 diabetes. Our results suggests that immune cells may be an important player in the crosstalk between skeletal muscle and adipose tissue during exercise, through the secretion of factors such as oncostatin-M to regulate adipocyte metabolism.

Supported by: NNF

Disclosure: L. Dollet: Grants; NNF20OC0060969.

83

High intensity interval training improves whole-body insulin sensitivity and skeletal muscle oxidative capacity and favourably affects hepatic fat storage

M. Bergman1, R. Mancilla1, P. Veeraiah2, V.H.W. de Wit-Verheggen1, Y.M.H. Bruls2, J. Hoeks1, V.B. Schrauwen-Hinderling1,2, M.K.C. Hesselink1;

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

Background and aims: High intensity interval training (HIIT) is a time-efficient alternative for conventional exercise training and considered a promising exercise modality to counteract obesity-related metabolic impairments. We studied if HIIT prompts beneficial effects on insulin sensitivity, muscle oxidative capacity and intrahepatic lipid (IHL) content in overweight/obese individuals. Since carbohydrate-rich and insulinogenic sports drinks are frequently consumed after training and may impact the outcome of the training, we also explored if co-ingestion of a standardized glucose and casein hydrolysate post-exercise affects the anticipated HIIT-mediated metabolic improvements.

Materials and methods: Twenty-three overweight and obese adults (10 males and 13 females, mean age: 64 ± 7.5 years, BMI: 31.8 ± 3.3 kg/m2) completed 12 weeks of progressive HIIT on a stationary bike while consuming either water during/post exercise (HIIT+WAT) or with co-ingestion of a glucose and casein hydrolysate immediately post-exercise (HIIT+CHO/PRO). Before and after the HIIT program, muscle oxidative capacity (expressed as the PCr recovery rate constant) was assessed via phosphorus magnetic resonance spectroscopy (31P-MRS) and IHL content and composition were determined by proton magnetic resonance spectroscopy (1H-MRS). Whole-body insulin sensitivity was assessed by a 2-step hyperinsulinemic-euglycemic clamp (10 - 40 mU), along with maximal aerobic capacity (VO2max) on a cycle ergometer and body composition by BodPod.

Results: VO2max improved significantly from 41.0 ± 1.7 to 44.8 ± 1.8 ml/FFM/min in the HIIT+WAT group and from 42.8 ± 1.9 to 47.5 ± 2.0 ml/FFM/min in HIIT+CHO/PRO, whereas the PCr recovery rate constant improved in the HIIT+WAT group from 0.030 ± 0.002 to 0.041 ± 0.003 s-1 and in the HIIT+CHO/PRO group from 0.031 ± 0.002 to 0.038 ± 0.003 s-1. In the combined group, HIIT tended (P=0.06) to decrease IHL content significantly (P=0.048), increased the polyunsaturated fatty acid (PUFA) fraction and tended (P=0.08) to decrease the monounsaturated fatty acid (MUFA) hepatic fat content, whereas the saturated fatty acid fraction did not change. Whole-body insulin sensitivity, as reflected by the M-value, improved significantly upon HIIT, to a similar extent in both groups (HIIT+WAT: Δ 8.2 ± 3.7 and HIIT+CHO/PRO: Δ 6.9 ± 4.1 μmol/FFM/min, P = 0.01). No changes in body weight and body composition were observed upon HIIT in either of the groups.

Conclusion: HIIT promotes both insulin sensitivity and muscle oxidative capacity in overweight/obese individuals. Moreover, IHL composition changes slightly (a shift towards more PUFA and less MUFA) in overweight/obese individuals. These benefits can be attained regardless of co-ingesting glucose and proteins after exercise and occurred in the absence of changes in body weight or body composition.

Clinical Trial Registration Number: NCT03405545

Disclosure: M. Bergman: None.

84

The plasma and tissue metabolomics responses to high intensity interval training and its importance in insulin resistance in obesity and type 2 diabetes

P.M. Møller1, M.H. Petersen1, M.E. De Almeida2, N. Ørtenblad2, J. Havelund3, N.J.K. Færgeman3, K. Højlund1;

1Steno Diabetes Center Odense, Odense University Hospital, Odense C, 2Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense M, 3Biochemistry and Molecular Biology, Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.

Background and aims: Insulin resistance (IR) links obesity with type 2 diabetes (T2D). Evidence suggests that IR is associated with a range of specific changes in circulating and inter-tissue metabolites. Physical activity improves insulin sensitivity in obesity and T2D. Recent data suggests that high-intensity interval training (HIIT) and recruitment of several muscle groups enhance the beneficial effects of exercise on glucose homeostasis. Here, we aimed to uncover the nearly undiscovered plasma and tissue metabolomics signatures of a novel HIIT protocol engaging upper and lower body muscles in obesity and T2D.

Materials and methods: Plasma samples and skeletal muscle and subcutaneous adipose tissue (SAT) biopsies were obtained from obese patients with T2D (n=15) and matched glucose-tolerant obese (n=15) and lean (n=18) individuals before and after their participation in an 8-week HIIT protocol combining rowing and cycling 3 times/week. Insulin sensitivity, VO2max, and body composition were assessed before and after the HIIT protocol. Plasma and tissue samples were investigated by untargeted metabolomics analyses using LC-MS. The metabolomics data will be used for biocomputational analyses, including clustering and correlation analyses.

Results: The HIIT protocol markedly increased insulin sensitivity (~30-40%) in all groups (p<0.05), and also improved VO2max (p<0.05), and reduced total body fat (p<0.001). These physiological changes in response to HIIT were accompanied by changes in the plasma metabolome, predominantly in T2D patients. The changes included the induction of lactic acid and amino acid derivatives (p<0.05), including BCAA degradation products, previously observed to increase glucose and fatty acid uptake in primary human adipocytes. The largest baseline differences were observed in the lipidome of T2D patients compared with the obese and lean groups (p<0.05). This included increased levels of sphingolipids, and diacylglycerols, previously coupled to tissue IR. The plasma data will be supplemented and compared to metabolomics and lipidomic analyses of fat and muscle biopsies, and further enrichment and correlation analyses with clinical data will be performed.

Conclusion: T2D patients display an altered lipidomic profile in plasma, whereas HIIT induces changes in the plasma metabolome, which could suggest a switch in energy metabolism and organ crosstalk.

Supported by: SDCO PhD grant, Region Syddanmark PhD grant

Disclosure: P.M. Møller: None.

OP 15 Preserving kidney function

85

Effects of treatment with semaglutide, empagliflozin and the combination on kidney function in type 2 diabetes: impact of oxygenation and perfusion

S. Gullaksen1,2, L. Vernstrøm1,2, S.S. Sørensen1,2, K.L. Funck2, P.L. Poulsen2, E.G. Laugesen2;

1Dept. of Clinical Medicine, Aarhus University, Aarhus, 2Aarhus University Hospital, Dept. of Endocrinology and Internal Medicine, Denmark.

Background and aims: Glucagon-like peptide-1 receptor agonists (GLP-1ra) and sodium-glucose cotransporter-2 inhibitors (SGLT-2i) have each shown kidney protective effects in large clinical outcome-studies. The mechanisms behind these effects are sparsely elucidated and the effects of combination therapy remains obscure. In this randomized clinical trial, we compared kidney function in persons with type 2 diabetes receiving either the GLP1-ra semaglutide (SEMA), the SGLT2-i empagliflozin (EMPA) or the combination (COMBI). EMPA treatment was double blinded. Kidney oxygenation was primary outcome and regional (cortex and medulla) assessments were included.

Materials and methods: 80 participants with type 2 diabetes (age 68.1 ± 6.5 years, male 76.25 %, diabetes duration 8[4;12] years), were randomized to either placebo, EMPA, SEMA or COMBI for 32 weeks. Kidney excretory function was assessed by urinary albumine/creatinine ratio (UACR) and glomerular filtration rate (GFR). Magnetic resonance imaging was used to assess kidney oxygenation with R2*(Hz) (higher values indicating lower oxygenation) by a blood oxygen level depedent sequence (BOLD) and renal perfusion (arterial spin labelling (ASL)).

Results: UACR was reduced significantly for the COMBI group (-35% 95% CI [-46;-92]%, p=0.01), with the same tendency for each of the separate treatment arms. GFR decreased significantly from baseline and compared to placebo in all three treatment groups (SEMA -19 mL/min 95% CI [-7;-31]mL/min, p=0.001; EMPA -18 mL/min 95% CI [-6;-30]mL/min, p<0.001 and COMBI -26mL/min 95% CI [-14;-38]mL/min, p<0.001). Medullary renal oxygenation was reduced compared to placebo in both EMPA and the COMBI groups (figure 1A) (0.91s-1 95% CI [0.16;1.65]s-1, p=0.01 and 0.91s-1 95% CI [0.26;1.57]s-1, p< 0.01, respectively). EMPA reduced medullary oxygenation significantly compared to SEMA (1.03s-1 95% CI [0.22;1.84]s-1, p=0.01). No significant changes were observed in cortical oxygenation (figure 1A). Compared to placebo, cortical perfusion was reduced in SEMA and COMBI, whereas medullary perfusion was reduced across treatments, figure 1B.

Conclusion: Our data demonstrate joined effects of EMPA and SEMA on UACR and GFR and reveal important differential effects of EMPA and SEMA on renal physiology. As hypoxia is considered a main driver in kidney disease, we hypothesized that EMPA and/or SEMA would exert their beneficial effects through improved kidney oxygenation. Contrary to our hypothesis, EMPA treatment for 32 weeks was not associated with improved medullary oxygenation but even slightly reduced it. This is in line with recent rodent studies. SEMA treatment is associated with a substantial reduction in kidney perfusion, probably leading to reduced hyperfiltration. To our knowledge, these effects have not previously been demonstrated in humans.

figure v

Clinical Trial Registration Number: EUDRACT 2019-000781-38

Supported by: NNF, DMA Research Foundation, Health Research Foundation of Central Denmark Region

Disclosure: S. Gullaksen: None.

86

Low blood oxygen saturation is associated with microvascular complications in individuals with type 1 diabetes

J.-C. Laursen, H.I. Mizrak, S.K. Heckquet, H. Kufaishi, M. Frimodt-Møller, C.S. Hansen, P. Rossing;

Steno Diabetes Center Copenhagen, København, Denmark.

Background and aims: Blood oxygen saturation (SpO2) is lower in both type 1 diabetes (T2D) and type 2 diabetes (T2D) as compared with non-diabetes (CON). Hypoxia is thought to play a role in the progression of diabetic complications, but it is unknown whether low SpO2 is associated with diabetic complications.

Materials and methods: Cross-sectional study in T1D and T2D separately. SpO2 was measured in the supine body position with pulse oximetry and patients were divided in low SpO2 (< 96%) and high SpO2 (≥ 96%) which has been proposed as a threshold. Outcomes were albuminuria (two out of three consecutive measurements ≥ 30 mg/g), any retinopathy (fundus photography), neuropathy (symmetric vibration perception threshold ≥ 25 V), and cardiovascular disease (CVD) (history). Odds ratios (OR) were adjusted for diabetes duration, gender, smoking, exercise, body mass index, and blood hemoglobin.

Results: We included 663 patients with T1D (23 with low vs. 640 with high SpO2) and 425 with T2D (43 with low vs. 382 with high SpO2). In T1D, the OR (95% CI, p-value) with low vs. high SpO2 was for albuminuria 3.3 (1.3 to 8.3, p < 0.01), for retinopathy 2.9 (1.1 to 7.8, p = 0.03), for neuropathy 5.3 (1.9 to 14.9, p < 0.01), and non-significant for CVD (1.0 (0.3 to 3.5, p = 0.95)). In T2D, there were no significant differences between low vs. high SpO2 in the odds of having albuminuria, retinopathy, neuropathy, or CVD (Figure 1).

Conclusion: SpO2 below 96% was associated with the microvascular complications, albuminuria, retinopathy, and neuropathy in T1D, but not with the macrovascular complication CVD. This could reflect that microvascular lung damage is related to hypoxia. No associations were observed in T2D.

figure w

Supported by: NNF14OC0013659

Disclosure: J. Laursen: Lecture/other fees; For Boehringer Ingelheim, all fees donated to Steno Diabetes Center Copenhagen.

87

Development in eGFR trajectories in people with diabetic nephropathy

C.G. Poulsen1, K. Jesse1, B. Carstensen1, M. Frimodt-Moeller1, T.W. Hansen1, F. Persson1, D. Vistisen1,2, P. Rossing1,2;

1Steno Diabetes Center Copenhagen, 2University of Copenhagen, Copenhagen, Denmark.

Background and aims: Diabetic nephropathy (DN) is a frequent and serious complication to both type 1 diabetes (T1D) and type 2 diabetes (T2D) and is characterized by a progressive loss of kidney function, elevated blood pressure and increased risk of cardiovascular disease and death. The effect of advancing diabetes care over the past decades on progression and prognosis for persons diagnosed with DN requires updating. In this study we analyzed the development over calendar time in eGFR trajectories from the time of DN diagnosis.

Materials and methods: In a retrospective cohort study, data was collected from electronic health records from persons attending the outpatient clinic at Steno Diabetes Center Copenhagen, Denmark between 2001-2020. Inclusion criteria were: T1D/T2D and DN, defined as urine albumin to creatinine ratio (UACR) > 300mg/g or urine albumin excretion rate (UAER) > 300 mg/24hours in two separate measurements > 60 days apart. Individual eGFR trajectories were calculated separately for T1D and T2D, using mixed-effects models with fixed effects of age, sex, date of DN diagnosis, duration of DN, and random effects of person and duration of DN.

Results: The T1D cohort included 891 persons, 59.7% were male and median (IQR) age at DN diagnosis was 50 (38-62) years. Figure 1A shows the estimated trajectories for eGFR for a person diagnosed with T1D and DN at age 50 in 2000, 2005, 2010 and 2015. eGFR at the time of DN diagnosis increased over time with 1.8 ml/min/1.73m2 per calendar year. The T2D cohort included 1447 persons, 71.9% were male and median (IQR) age at DN diagnosis was 65 (58-72) years. Figure 1B shows the estimated trajectories for eGFR for a person diagnosed with DN at age 65 in 2000, 2005, 2010 and 2015. eGFR at the time of DN diagnosis increased with 0.7 ml/min/1.73m2 per calendar year. For both T1D and T2D, the trajectories depict a tendency towards an attenuating decline in eGFR over time, when diagnosed with DN in more recent years, but with a faster decline in the first five years after diagnosis of DN.

Conclusion: Kidney function at time of DN diagnoses has increased over the past 20 years, especially in T1D. Moreover, the eGFR decline after diagnosis seems attenuating when diagnosed in more recent years. This may be explained by improved awareness and treatment.

figure x

Supported by: Skibsreder Per Henriksen, R. og Hustrus Fond

Disclosure: C.G. Poulsen: None.

88

Shifts in KDIGO CKD risk groups with empagliflozin: Reno-protection from SGLT2 inhibition across the spectrum of risk

S.E. Inzucchi1, B. Zinman2, M. Mattheus3, D. Steubl3, A.P. Ofstad4, C. Wanner5;

1Yale School of Medicine, New Haven, USA, 2Mount Sinai Hospital, University of Toronto, Toronto, Canada, 3Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim, Germany, 4Boehringer Ingelheim Norway KS, Asker, Norway, 5Würzburg University Clinic, Würzburg, Germany.

Background and aims: Chronic kidney disease (CKD) is a common complication of type 2 diabetes (T2D), manifested by progressive decline in glomerular filtration rate (GFR) that can lead to end-stage kidney disease (ESKD) and need for renal replacement therapy. The impacts of CKD are increased cardiovascular (CV) and mortality risk, and healthcare costs. SGLT2 inhibitors reduce the progression of established CKD. The KDIGO ‘heat map’ has 18 categories of kidney function based on estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio. These categories are ordered into 4 groups with low, moderate, high and very high risk for progression to ESKD. The heat map is used clinically to track patients with or at risk for CKD and to encourage prompt subspecialty referral as needed. We sought to determine the effect of the SGLT2 inhibitor empagliflozin (empa) on changes in CKD risk by assessing worsening and improvement in KDIGO risk group in EMPA-REG OUTCOME.

Materials and methods: 7020 patients with T2D and established CVD were randomised and treated with empa 10, 25 mg (pooled for subsequent analyses) or placebo and followed for a median of 3.1 yrs. In this post hoc analysis, we categorised trial patients based on their KDIGO CKD risk group at baseline and their last value on-treatment. Worsening risk was defined as a shift to a more advanced group (e.g., low to moderate). Improvement in risk was defined as a shift to a less advanced group (e.g., high to moderate). We then compared the proportion (%) of patients who experienced worsening or improvement in risk status between empa and placebo by baseline KDIGO CKD risk groups. Odds ratios (OR) with 95% confidence intervals (CI) were calculated using logistic regression models, adjusting for study treatment, sex, region, baseline age, BMI, HbA1c and KDIGO risk.

Results: In the placebo group the % of patients with worsening in their KDIGO CKD risk were 32.1%, 31.5% and 36.3% in the low, moderate and high risk baseline groups, respectively. The corresponding values in the empagliflozin groups were lower: 26.0%, 24.4% and 23.3%, respectively. The OR (95% CI) for worsening risk across all baseline groups was 0.70 (0.62, 0.78) in favour of empa (Figure). Improvement in KDIGO CKD risk group was experienced by 18.7%, 16.8% and 18.5% of patients assigned to placebo in the moderate, high and very high baseline risk groups. The corresponding values in empagliflozin were higher: 24.6%, 27.1% and 26.2%, respectively. The OR (95% CI) for improved risk across all baseline groups was 1.56 (1.30, 1.86) in favour of empa (Figure).

Conclusion: The use of empa was associated with a 30% lower odds of worsening and >50% higher odds of improvement in KDIGO CKD risk groups. These data support greater use of SGLT2 inhibitors across a broad spectrum of CKD risk.

figure y

Clinical Trial Registration Number: EMPA-REG OUTCOME (NCT01131676)

Supported by: Boehringer Ingelheim & Eli Lilly and Company Diabetes Alliance

Disclosure: S.E. Inzucchi: Honorarium; AstraZeneca, Boehringer Ingelheim, Novo Nordisk, Merck, Lexicon, Esperion, vTv Therapeutics, Abbott, Pfizer.

89

Sotagliflozin, a dual SGLT1 and SGLT2 inhibitor, reduces the risk of cardiovascular and renal disease as assessed by Steno Risk Engines in adults with type 1 diabetes

E.B. Stougaard1, P. Rossing1,2, D. Vistisen1,3, P. Banks4, M. Girard4, M.J. Davies4, F. Persson1;

1Steno Diabetes Center Copenhagen, Herlev, Denmark, 2Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark, 3Department of Public Health, University of Copenhagen, Copenhagen, Denmark, 4Lexicon Pharmaceuticals, Inc., The Woodlands, USA.

Background and aims: Sotagliflozin (SOTA) as an adjunct to insulin therapy improves glycemic control, without an increased risk of hypoglycaemia, reduces body weight and blood pressure (BP), and increases time in range in adults with type 1 diabetes (T1D). Treatment with SOTA is associated with an increased risk of diabetic ketoacidosis (DKA). SOTA demonstrated CV and renal benefits in high-risk adults with type 2 diabetes. The cardiorenal benefits along with the cardiometabolic effects may collectively outweigh the risk of DKA when evaluating SOTA for adults with T1D. The present analysis estimated the risk of CVD and end-stage kidney disease (ESKD) in adults with T1D treated with SOTA.

Materials and methods: Patient-level data were used from the three Phase 3 inTandem trials evaluating 2977 adults with T1D randomized to once-daily placebo, SOTA 200 mg, or SOTA 400 mg for 24 weeks. Analyses focused on 3 cohorts (1. SOTA 200 mg; 2. SOTA 400 mg; 3. SOTA pooled). A subgroup analysis was performed in patients with a BMI ≥27 kg/m2. For each patient, the cumulative risks of developing CVD and ESKD were estimated using the Steno T1 Risk Engines, which are validated prediction models for predicting 5- and 10-year risk of CVD and 5-year risk of ESKD. For CVD risk estimation, only patients without previous CVD (98% of overall cohort) at baseline were included. Baseline values for age and duration of diabetes were used in the model at Week 24. Missing albuminuria values were set to normal. Smoking and exercise history were not collected, and these variables were set to No. The estimated risk was calculated at baseline and Week 24 in both treatment groups. If a participant did not have a Week 24 assessment, a baseline observation carried forward approach was used. The difference in least-square mean percent change in estimated risk from baseline (95% CI and p-value) was compared between groups using a mixed model with percent change from baseline as dependent and including the treatment group as fixed effect, and the baseline value as covariate.

Results: SOTA significantly reduced 5- and 10-year CVD risk scores by approximately 4 to 7% compared to placebo at 24 weeks (Table). ESKD risk score was numerically reduced with SOTA 200 mg and significantly reduced with SOTA 400 mg relative to placebo. Similar results were observed with SOTA pooled and in patients with baseline BMI ≥27 kg/m2.

Conclusion: Using the Steno T1 Risk Engines, the estimated risk of CVD and ESKD was significantly reduced with SOTA compared to placebo in adults with T1D. This analysis provides additional clinical results that may positively enhance the benefit/risk assessment of SOTA use in T1D.

figure z

Disclosure: E.B. Stougaard: None.

90

Morphological and functional ultrasound features of diabetic kidney disease phenotypes in people with type 2 diabetes

M. Garofolo1, V. Napoli2, D. Lucchesi1, S. Accogli2, M. Mazzeo2, P. Rossi2, P. Falcetta1, M. Giambalvo1, E. Neri2, G. Penno1, S. Del Prato1;

1Department of Clinical and Experimental Medicine, University of Pisa, 2Department of Translational Research, Academic Radiology, University of Pisa, Pisa, Italy.

Background and aims: Diabetic kidney disease (DKD) develops in 40% of people with type 2 diabetes (T2D) and is the leading cause of end stage kidney disease. Non-albuminuric DKD has become the prevailing DKD phenotype (PH) and exhibits distinct clinico-pathological characteristics from those with albuminuria, including a higher proportion of females, better risk factors profile, and possibly distinct renal structural lesions on biopsy. Whether DKD PHs may also recognize differences in kidney morphological and vascular features has not been yet explored. We evaluated to which extent kidney ultrasonography (US) may differentiate DKD PHs in T2D subjects, in a cross-sectional, single-center study.

Materials and methods: DKD PHs were defined by KDIGO baseline eGFR strata and albuminuria categories: no-DKD (preserved eGFR and normoalbuminuria), albuminuria and preserved eGFR (DKD stages 1-2), decreased eGFR and normoalbuminuria (NA-DKD), and albuminuria with decreased eGFR (MA-DKD). Total and parenchymal renal volumes were calculated applying the ellipsoid formula for conventional (2D) US and with manual segmentation for 3D US (by X-matrix array technology); total and parenchymal renal volumes were adjusted for body surface area (aTRV, aPRV). Renal resistive index (RI) was also determined at the renal interlobar arteries. All US procedures were made by a radiologist blinded to patients’ characteristics.

Results: Out of 256 subjects, 26.2% had no-DKD, 24.6% DKD stages 1-2, 24.2% NA-DKD and 25.0% MA-DKD. No difference in albuminuria were observed between DKD stages 1-2 and MA-DKD (or between no-DKD and NA-DKD). Consistently, eGFRcreat levels did not differ in MA-DKD as compared to NA-DKD (or between no-DKD and DKD stages 1-2). Compared to no-DKD, RI was higher in all DKD PHs, being the highest in MA-DKD and with a significant trend of RI >0.70 prevalence to increase across PHs (p=0.022). aTRV3D and aPRV3D significantly differed across PHs (p<0.0001 for both). Compared to no-DKD (aTRV3D 180 ml/m2, [IQR 162-205], and aPRV3D 150 ml/m2, [132-162]), volumes were higher in DKD stages 1-2 (aTRV3D 198 ml/m2, [170-224] and aPRV3D 163 ml/m2, [140-184]) and reduced in both NA-DKD and MA-DKD, with significantly lower volumes in NA-DKD (aTRV3D 153 ml/m2, [136-168], and aPRV3D 127 ml/m2, [106-141]) as compared to MA-DKD (aTRV3D 164 ml/m2, [143-186], and aPRV3D 138 ml/m2, [122-150]; p=0.017 and 0.011 for aTRV3D and aPRV3D, respectively). These differences were confirmed for 2D volumes, and for DKD PHs established by GFR estimation based on creatinine and cystatin C CKD-EPI equation. In adjusted logistic regression models, compared to no-DKD, RI and aPRV3D were associated with DKD stages 1-2 and MA-DKD; only aPRV3D with NA-DKD. Compared to no-DKD, ROC curves, traced on top of conventional risk factors, showed that US parameters did not improve characterization of DKD stages 1-2 and MA-DKD, while aPRV3D significantly improved phenotyping of NA-DKD.

Conclusion: In type 2 diabetes, the emerging NA-DKD phenotype showed reduced TRVs and PRVs even when compared with MA-DKD with similar eGFR reduction. These findings support the hypothesis that different damage pathways underly the progression of different DKD phenotypes.

Disclosure: M. Garofolo: None.

OP 16 Flames and scars in the liver

91

Role of the constitutive androstane receptor CAR in sex- and gut microbiota-dependent non-alcoholic fatty liver disease

M. Huillet1, F. Lasserre1, A. Polizzi1, V. Alquier-Bacquie1, A. Fougerat1, C. Rives1, C. Martin1, J. Bruse1, J.H. Wan2, B. Chassaing3, S. Lotersztajn2, L. Gamet-Payrastre1, H. Guillou1, N. Loiseau1, S. Ellero-Simatos1;

1Toxalim, UMR 1331, INRAE, Université de Toulouse, Toulouse, 2INSERM-UMR 1149, Centre de Recherche sur l'Inflammation, Paris, 3INSERM U1016, CNRS UMR 8104, Université de Paris, Paris, France.

Background and aims: Non-alcoholic fatty liver diseases (NAFLD), including steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocarcinoma, are sexually-dimorphic diseases, in which the gut microbiota seems to play a causal role. Mechanisms involved in disease progression remain incompletely understood. The constitutive androstane receptor (CAR, official name NR1I3) is a liver enriched-nuclear receptor, that regulates the transcription of genes involved in both xenobiotic detoxification and energy metabolism. In addition, CAR represents a potential candidate for the recognition of microbial signals by its ability to bind a large number of exogenous molecules. In this context, we aimed to investigate the potential role of CAR in the progression of NAFLD.

Materials and methods: First, we analyzed hepatic CAR expression using publicly available transcriptomic data from NAFLD human cohorts of the Gene Expression Omnibus (GEO) repository. Then, we used adult C57Bl6/J CARWT (WT) or CARKO (KO) female and male mice fed a chow diet (CD) or a sucrose and lipid enriched diet (western-diet, WD) for 14 weeks (n= 8-12/group). Gut microbiota composition, glucose and lipid metabolism and hepatic phenotype were studied using 16S-sequencing, glucose tolerance test, hepatic histology (H&E and sirus red staining), hepatic qPCR and plasma biochemical analysis.

Results: In clinical studies, CAR expression was lower in NASH patients with advanced fibrosis than in those with mild fibrosis (adjusted p-value (padj)=0.04 GSE163211, padj=0.01 GSE49541), and in patients with cirrhosis or hepatocarcinoma compared to healthy patients (padj =3x10-19 GSE10143, padj =10-40 GSE14520). Thus, hepatic expression of CAR seems to decrease during NAFLD progression in humans. In preclinical mouse studies, WD feeding increased body weight and induced liver triglyceride deposition (steatosis) in both males and females, with no significant effect of CAR deletion. KO WD fed males had significantly higher circulating levels of alanine aminotransferase (584±175 vs. 202±113 U.L-1, p=10-5) and alkaline phosphatase (242±48 vs. 113±37 U.L-1, p=10-5), indicative of liver damages, as well as increased liver fibrosis (% sirus red staining: 0.3±0.15 vs. 0.7±0.3, p=0.005; Col1a1 mRNA: 2.5 fold increase, p=10-5) , compared with WT males. In females, we observe no influence of genotype on WD-induced liver damages or fibrosis. Caecal microbiota composition of KO WD-fed males was significantly different from WT WD-fed males. To assess the role of gut microbiota in CAR-dependent liver dysfunctions, KO and WT male mice were co-housed in same cages and fed a WD. There were no differences in liver damages between WT and KO co-housed males, indicating that gut microbiota transfer by coprophagy attenuated WD-induced CAR-dependent liver damages.

Conclusion: CAR plays a sex- and gut microbiota-dependent role in WD-induced NAFLD in mice. We hypothesize that hepatic CAR is involved in the detoxification of gut microbial metabolites and we will further investigate this using portal vein metabolomics to unravel potential deleterious metabolites involved in sexually-dimorphic NAFLD.

Supported by: JPI HDHL ANR FATMAL

Disclosure: M. Huillet: None.

92

Hepatic fat and macrophages are increased in livers of diabetic patients without NAFLD

A. Korn1,2, C. Nadeem1, E. Bos1, H.W.M. Niessen1,2, S. Simsek3,4, P.A.J. Krijnen1,2;

1Pathology, Amsterdam UMC, Location VUmc, Amsterdam, 2Amsterdam Cardiovascular Sciences, Amsterdam, 3Internal Medicine, Northwest Clinics, Alkmaar, 4Internal Medicine, Amsterdam UMC, Location VUmc, Amsterdam, Netherlands.

Background and aims: Diabetes mellitus (DM) is strongly associated with non-alcoholic fatty liver disease (NAFLD), which increases risk of severe liver disease as well as extra-hepatic microvascular disorders. NAFLD is diagnosed in liver biopsies when fat accumulation, lymphocytes, ballooning of hepatocytes and/or fibrosis are found, but it is often diagnosed relatively late due to limitations of current diagnostic techniques and minor clinical manifestations in the early stages of development. This study aimed to analyse putative early changes in the liver of deceased DM patients that were without clinical diagnosis of NAFLD and did not have histopathological characteristics of NAFLD at autopsy, and analysed age/sex effects hereon.

Materials and methods: Liver tissue was obtained at autopsy from 24 DM patients and 66 non-diabetic control patients. All patients did not have histopathological characteristics of NAFLD. Hepatic fat (percentage and number of cells) and inflammatory cells (CD45-positive lymphocytes and CD68-positive macrophages) were studied through (immuno)histochemical analysis.

Results: We observed a 5-fold increase in percentage of fat (0.7±1.1, p=0.0007) and an 8-fold increase in amount of fat cells in DM patients (94.6±133.5, p<0.0001) compared with the non-diabetic controls (% fat: 0.4±0.9; fat cells: 19.9±47.8). The number of CD45-positive lymphocytes was not affected in the diabetic livers (p=0.06), whereas the number of CD68-positive cells was increased in DM patients (p=0.0002) compared to controls. Fat percentage and fat cells were significantly higher in DM2 patients compared to both DM1 (p=0.02 and p=0.04 respectively) and non-diabetic controls (p=0.0007 and p<0.0001 respectively), while the number of CD68-positive cells was significantly elevated in both DM groups, regardless of type (DM1: p=0.0008; DM2: p=0.007). We found no significant differences between men and women in the non-diabetic control groups nor in the diabetic patient group, regardless of age. No correlation was found between hepatic fat and inflammation.

Conclusion: Hepatic fat and the number of macrophages is increased in the liver in DM patients that are not yet diagnosed with NAFLD, warranting early clinical investigation for signs of NAFLD in patients with DM.

figure aa

Supported by: EFSD/Sanofi European Diabetes Research Programme in Macrovascular Complications

Disclosure: A. Korn: None.

93

Ubiquitin-proteasome system dysfunction in the liver of severely obese men with and without type 2 diabetes

B. Stocks1, E. Näslund2, J.R. Zierath3, A.S. Deshmukh1;

1Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen N, Denmark, 2Department of Clinical Sciences, Karolinska Institutet, Stockholm, Sweden, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Dysregulated liver metabolism, in particular insulin resistance, is a predominant factor in the pathogenesis of type 2 diabetes. Protein degradation via the ubiquitin-proteasome system has been implicated in the development of type 2 diabetes. Through a diversity of signals, ubiquitination regulates a range of cellular processes such as protein degradation, enzyme activation and sub-cellular localisation. Nonetheless, a global understanding of ubiquitin-proteasome system dysfunction in obesity and type 2 diabetes has yet to be established. As the human genome encodes more than 600 E3 ligases (which catalyse the transfer of ubiquitin to protein substrates) and over 100 deubiquitinases, a systems approach is required to truly understand ubiquitin-proteasome system dysfunction. We aimed to characterise the regulation of the ubiquitin-proteasome system in the liver of severely obese men with and without type 2 diabetes.

Materials and methods: Liver samples from 9 non-obese men, 8 severely obese men (Ob) and 10 severely obese men with type 2 diabetes (Ob-T2D) were collected during cholecystectomy (non-obese) or Roux-en-Y gastric bypass surgery (Ob and Ob-T2D). Samples were lysed in 4% SDS and digested using Lys-C and trypsin. Peptides were measured via liquid chromatography tandem mass spectrometry (LC-MSMS) using an EASY nanoLC coupled to an Exploris 480 Orbitrap on a 100-min gradient using data independent acquisition. Protein ubiquitination was assessed via immunoblotting in human and rodent liver.

Results: We identified the regulation of 4319 proteins in the livers of 9 non-obese, 8 severely obese non-diabetic men (Ob) and 10 severely obese men with type 2 diabetes (Ob-T2D), collected during cholecystectomy (non-obese) or Roux-en-Y gastric bypass surgery (Ob and Ob-T2D). We found 162 proteins that were differentially regulated in Ob and/or Ob-T2D compared to non-obese men. Of these, 11 ubiquitin-proteasome system proteins were regulated, including the E3 ligases COPB2, DCAF11, DCUN1D1, EML2, LRSAM1, SEC13, SEC31A, STRN and TBL2, the deubiquitinase UCHL1, and the 26S proteasome protein PSMD7. Furthermore, protein ubiquitination is dysregulated in the liver of Ob and Ob-T2D patients.

Conclusion: These data identify ubiquitin-proteasome system dysfunction in the liver of severely obese men with and without type 2 diabetes, providing protein targets for future mechanistic and therapeutic research.

Supported by: EFSD Rising Star Fellowship Programme

Disclosure: B. Stocks: Grants; EFSD Rising Star Fellowship Programme.

94

Liver inflammation increases the proliferation of oval cells during non-alcoholic fatty liver disease

S. Calero Pérez1,2, I. Barahona1,2, P. Valdecantos1,2, Á.M. Valverde1,2;

1IIBm Alberto Sols (CSIC-UAM), 2CIBERdem (ISCIII), Madrid, Spain.

Background and aims: Oval cells (OCs) are hepatic progenitor cells with an emerging role in repopulation capacity and differentiation towards hepatocytes or colangiocytes under conditions of liver damage. However, their susceptibility to an inflammatory environment in the context of obesity-associated insulin resistance and non-alcoholic liver disease (NAFLD) is unknown. On that basis, the aim of this study was, first, to identify the impact of an environment mimicking metainflammation in obesity and NAFLD in OC expansion in mice and, second, to study the interactome between OCs and immune cells of the liver at the molecular and cellular levels.

Materials and methods: To induce NAFLD, eight week-old male mice (C57Bl6j x 129 sv) were fed a Western Diet (WD) containing 21.1% fat, 41% sucrose, and 1.25% cholesterol and a high sugar solution (23.1 g/L d-fructose and 18.9 g/L d-glucose) for 12 weeeks. Additionally, CCl4 (0.32 μg/g body weight) was injected intra-peritoneally (i.p.) once per week, starting one week after the WD. OCs were isolated from mouse livers and established in culture. To generate a NAFLD-like proinflammatory environment, peritoneal macrophages or bone marrow-derived macrophages (BMDM) were treated with a mixture of palmitate (400 μM) and LPS (150 ng/ml) (PA/LPS) for 8 h, after which the culture medium was replaced with fresh medium for a further 16 h. The conditioned medium (CM) was used to stimulate OCs for different time-periods.

Results: NAFLD was evaluated in mice upon 12 weeks of intervention and histopathological analysis of liver sections showed features of non-alcoholic steatohepatitis (NASH) including steatosis, ballooning, inflammation and fibrosis, as revealed by the NAS score (p<0.001, n=6-11), serum transaminases (p<0.001, n=6-11), Col1a1 mRNA levels (p<0.001, n=6-11) and hydroxyproline (p<0.001, n=6-11). Importantly, a marked increase in A6/SOX9 positive cells (p<0.001, n=6-11), surrounded by inflammatory monocytes (Ly6c+) was found in all mice with NASH (p<0.01, n=3), together elevated mRNA levels of the progenitor markers Epcam and Krt19 (p<0.001, n=6-11), pointing to a major presence of OCs in livers with NASH. Stimulation of OCs with CM collected from PA/LPS-stimulated macrophages for 5-30 min triggered a rapid activation of proinflammatory signaling cascades including phosphorylation of STAT3 (p<0.001, n=3), p65-NFκB (p<0.05, n=3), degradation of IκBα (p<0.05, n=3) and nuclear translocation of p65-NFκB (p<0.001, n=3). Treatment for 24 h with PA/LPS-CM increased the proliferation of OCs as reflected by increased PCNA levels (p<0.05, n=3). Gene expression levels related to OCs were obtained from the NCBI Gene Expression Omnibus database based on the gene chips of fibrotic livers associated to NAFLD (GSE130970, GSE49541, GSE162694 and GSE48452). In line with data in mice, expression of the progenitor markers SOX9, EPCAM and KRT7, was significantly increased in NAFLD patients with advanced fibrosis (p<0.001 n=8-103).

Conclusion: Our results have demonstrated that OC expansion concurs with inflammatory features during NAFLD progression in mice. The molecular studies revealed that the interactome between liver inflammatory cells and OCs favors OCs proliferation, an effect that opens therapeutic perspectives to preserve OCs proliferation under liver damage associated to NAFLD, but favoring their plasticity.

Supported by: RTI2018-094052-B-100 (MICINN/AEI/FEDER, EU), S2017/BMD-3684 (Comunidad de Madrid, Spain)

Disclosure: S. Calero Pérez: None.

95

Phthalate exposure is associated with NAFLD, but not with liver fibrosis in the United States

S. Ciardullo1, E. Muraca2, R. Cannistraci1, G. Lattuada2, G. Perseghin1;

1University of Milano Bicocca, 2Policlinico di Monza, Monza, Italy.

Background and aims: Recent epidemiological observations have reported an association between phthalates exposure and insulin resistance and obesity while data are scarce on the association between these pollutants and nonalcoholic fatty liver disease (NAFLD) and liver fibrosis.

Materials and methods: To address this gap in knowledge we performed a population-based cross-sectional study using data from the 2017-2018 cycle of the National Health and Nutrition Examination Survey, a complex survey conducted to gain information on the health status of the general population of the United States. The target population consisted in all adult participants with available data on vibration controlled transient elastography (VCTE) and on urinary phthalate concentrations, with the exclusion of individuals with chronic viral hepatitis and/or significant alcohol consumption (>1 standard drink/day in women and >2 standard drink/day in men). Concentrations of mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) were measured in a spot morning urine sample using liquid chromatography-electrospray ionization-tandem mass spectrometry. Multivariable logistic regression analysis was used to evaluate the association between phthalate concentrations and liver steatosis (controlled attenuation parameter > 274 dB/m) and significant liver fibrosis (liver stiffness measurement > 8 kPa) after adjustment for confounders.

Results: A total of 1793 participants were included. Weighted prevalence of NAFLD and significant fibrosis were 40.3% and 6.8%, respectively. Patients with NAFLD showed higher levels of both MEOHP and MEHHP, while no difference was found in MEHP. Concentrations of all three metabolites did not differ significantly between participants with and without significant liver fibrosis. After adjustment for potential confounders including age, race-ethnicity, body mass index, glomerular filtration rate and diabetes, we found a significant positive association between both MEOHP (OR 1.01, 95% CI 1.00-1.02 per unit increase, p=0.014) and MEHHP (OR 1.01, 95% CI 1.00-1.03 per unit increase, p=0.031) and NAFLD, while no significant association was found for MEHP. None of the three molecules was associated with fibrosis in the multivariable model.

Conclusion: Our findings have revealed an association between phthalate exposure and NAFLD, but not liver fibrosis independently of diabetes and obesity. Further studies elucidating potential pathophysiological mechanisms are needed.

Disclosure: S. Ciardullo: None.

96

The fibrotic NASH index: a simple non-invasive score to screen for liver disease in individuals with metabolic risk factors

F. Tavaglione1,2, O. Jamialahmadi2, A. De Vincentis3, S. Qadri4, V. Bruni5, S. Carotti6, G. Perrone6, D. Tuccinardi7, S. Manfrini7, P. Pozzilli7, A. Picardi1, H. Yki-Järvinen4, L. Valenti8, U. Vespasiani-Gentilucci1, S. Romeo2,9;

1Clinical Medicine and Hepatology Unit, Campus Bio-Medico University, Rome, Italy, 2Department of Molecular and Clinical Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 3Internal Medicine Unit, Campus Bio-Medico University, Rome, Italy, 4Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 5Bariatric Surgery Unit, Campus Bio-Medico University, Rome, Italy, 6Department of Pathology, Campus Bio-Medico University, Rome, Italy, 7Department of Endocrinology and Diabetes, Campus Bio-Medico University, Rome, Italy, 8Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milano, Italy, 9Clinical Nutrition Unit, Department of Medical and Surgical Sciences, University Magna Graecia, Catanzaro, Italy.

Background and aims: Type 2 diabetes is a key risk factor for non-alcoholic fatty liver disease (NAFLD), which is the major cause of chronic liver disease worldwide. Indeed, up to 65% of individuals with type 2 diabetes have NAFLD. Given the huge number of individuals at high risk of NAFLD, accurate and affordable non-invasive screening strategies for liver disease are urgently needed. Herein, we aimed to develop a simple non-invasive score using routine laboratory tests to identify, among individuals at high risk of NAFLD, those with fibrotic non-alcoholic steatohepatitis (NASH) defined as NASH, NAFLD activity score (NAS) ≥4, and fibrosis stage ≥2.

Materials and methods: The derivation cohort included 264 morbidly obese individuals undergoing intraoperative liver biopsy in Rome, Italy. The best predictive model was developed and internally validated using a bootstrapping stepwise logistic regression analysis (2000 bootstrap samples). Performance was estimated by the area under the receiver operating characteristic curve (AUROC). External validation was assessed in three independent European cohorts (Finland, n=370; Italy n=947; England n=5,368) of individuals at high risk of NAFLD, namely with type 2 diabetes, overweight/obesity, and/or metabolic syndrome. All cohorts received Local Research Ethics approval and all participants gave written informed consent to the study.

Results: The final predictive model, designated as Fibrotic NASH Index (FNI), combined aspartate aminotransferase (AST), high-density lipoprotein (HDL) cholesterol, and hemoglobin A1c (HbA1c). Performance for fibrotic NASH was satisfactory in both derivation and external validation cohorts (AUROCs 0.78 and 0.80-0.95, respectively). In the derivation cohort, rule-out and rule-in cut-offs were 0.10 for sensitivity ≥0.89 (negative predictive value [NPV] 0.93) and 0.33 for specificity ≥0.90 (positive predictive value [PPV] 0.57), respectively. In external validation cohorts, sensitivity ranged from 0.87 to 1 (NPV 0.99-1) and specificity from 0.73 to 0.94 (PPV 0.12-0.49) for rule-out and rule-in cut-off, respectively.

Conclusion: FNI is the first score for fibrotic NASH based on simple blood tests, namely AST, HDL cholesterol, and HbA1c. FNI may represent an accurate and affordable non-invasive tool to screen for liver disease individuals with metabolic risk factors in primary healthcare and diabetology clinics.

Supported by: VR; VINNOVA; Svenska Diabetesstiftelsen; Hjärt-Lungfonden; KAW; NNF; SSF; AZN

Disclosure: F. Tavaglione: None.

OP 17 Toying with monitoring: from Present Continuous to Future Perfect

97

Gaps remain in achieving target type 1 diabetes glycaemic goals despite advanced technologies

L. Laffel1, J. Liu2, L. Titievsky3, K. Hagan3, T. Liu3, K. Chandarana3, J. Gaglia3, W. Wolf2, J. Bispham4, K. Chapman2, D. Finan2, R. Bergenstal5;

1Dept of Pediatric & Adoles. Med., Joslin Diabetes Centre, Boston, 2T1D Exchange, Boston, 3Vertex Pharmaceuticals, Boston, 4Evidera PPD, Waltham, 5International Diabetes Center, HealthPartners Institute, Minneapolis, USA.

Background and aims: Continuous glucose monitoring (CGM) metrics and self-reported disease characteristics (severe hypoglycaemic events [SHEs], HbA1c) warrant further description in people with T1D using CGM and pumps, including hybrid closed-loop systems (HCLS).

Materials and methods: We conducted a one-time online survey of adults with T1D in the T1D Exchange registry in the USA or online communities, where ~50% of participants contributed up to 1 year of CGM data. Patients were asked about their medical history (SHEs, HbA1c) while glucose management indicator (GMI), prolonged hypoglycaemic events (<54 mg/dL), time in and below range (TIR/TBR), and coefficient of variation (CV) were derived from CGM data.

Results: Patients who completed the survey and contributed CGM data (N=926) had a mean age of 42y and T1D duration of 25y; 73% were female, 96% white; 94% had ≥1 year of CGM use. Mean HbA1c was 6.6% (69.0% had HbA1C <7%). While most patients met consensus glycaemic targets (HbA1c, GMI, TIR, TBR, and CV), with higher proportions observed in those using HCLS versus those using pump + CGM (not HCLS) and MDI + CGM (Table), patients continued to have significant hypoglycaemia based on CGM data and an average of 1.1 SHEs in the prior year.

Conclusion: Despite improvements in glycaemic control (TIR, TBR, and self-reported HbA1c) with advanced technologies, many patients are still unable to achieve clinical targets and experience significant hypoglycaemia, highlighting the unmet need for novel T1D treatments.

figure ab

Supported by: Vertex Pharmaceuticals

Disclosure: L. Laffel: Other; Advisory boards/consulting for Medtronic, Dexcom, Janssen, Boehringer Ingelheim, Provention, Dompe, Lilly, Roche, and Insulet.

98

Intermittently scanned continuous glucose monitoring is associated with a long-term glucose-lowering effect for type 1 diabetes patients in poor glycaemic control

M.H. Jensen1,2, S.L. Cichosz2, P. Gustenhoff3, A. Nikontovic1, O. Hejlesen2, P. Vestergaard3,1;

1Steno Diabetes Center North Denmark, Aalborg University Hospital, 2Department of Health Science and Technology, Aalborg University, 3Department of Endocrinology, Aalborg University Hospital, Aalborg, Denmark.

Background and aims: Lowering glucose levels is a complex task for patients with type 1 diabetes and they often lack close contact to health care professionals. Several studies have shown that reduced HbA1c can be obtained from use of intermittently scanned continuous glucose monitoring (isCGM), but the studies are typically of a shorter duration of 3-6 months with strict inclusion/exclusion criteria or with patient populations with mixed indications. The aim of this study was to investigate the long-term effect of isCGM on HbA1c in type 1 diabetes patients with poor glycaemic control in a region-wide real-world setting.

Materials and methods: All type 1 diabetes patients receiving an isCGM device due to poor glycaemic control (≥70 mmol/mol) in the period of 2020-21 in Region North Denmark (“T1D-CGM”) were compared with all T1D patients without isCGM (“T1D-NOCGM”) in the same period. Patients in the T1D-NOCGM group inherited the date of initiation of isCGM as an index date from the T1D-CGM patient with the closest year of birth. A multiple linear regression model adjusted for age, sex, diabetes duration and use of continuous subcutaneous insulin infusion was constructed to estimate the difference in change from baseline HbA1c between the two groups. Change from baseline HbA1c was for each person calculated as the difference between the average of all values one year before initiation of isCGM (baseline HbA1c) and the average of all values two years after.

Results: The cohort consisted of 897 patients receiving isCGM and 1,630 without isCGM. Included T1D-CGM patients were 47% female, had an average age of 46 years (SD: 16 years), a diabetes duration of 12 years (SD: 8 years) and a baseline HbA1c of 80 mmol/mol (SD: 12 mmol/mol). T1D-NOCGM patients were 41% female, had an average age of 54 years (SD: 17 years), a diabetes duration of 12 years (SD: 10 years) and a baseline HbA1c of 64 mmol/mol (SD: 16 mmol/mol). The estimated adjusted difference in change from baseline HbA1cbetween T1D-CGM vs T1D-NOCGM was -5.9 mmol/mol (95% CI: -6.7 to -4.7 mmol/mol; p<0.0001) and the development in average HbA1c levels can be seen in the figure below.

Conclusion: Our results indicate that type 1 diabetes patients with poor glycaemic control from Region North Denmark benefit from using isCGM with a sustained 24-month improvement in HbA1c. However, results should be interpreted with care, as the study was not randomized. Results need to be further investigated, for example, with subgroup analysis to clarify if all subgroups benefit equally from CGM.

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Disclosure: M.H. Jensen: Employment/Consultancy; Abbott Laboratories A/S. Stock/Shareholding; Novo Nordisk A/S.

99

Conversion from FreeStyle Libre to Freestyle Libre 2 is associated with improvements in continuous glucose monitoring metrics

F.W. Gibb, R.H. Stimson, R.J. Wright, S. Forbes, M.W.J. Strachan, A. Dover;

Edinburgh Centre for Endocrinology & Diabetes, Edinburgh, UK.

Background and aims: Real-time continuous glucose monitoring (rtCGM) is associated with improvements in glycaemic control in comparison to Freestyle Libre. The ALERTT1 study indicated improvements in both high and low glucose metrics favouring rtCGM over the original Freestyle Libre system. We sought to assess whether Freestyle Libre 2, with alarm functions, was associated with similar improvements.

Materials and methods: An observational study to assess changes in CGM metrics, at six months, in 415 adults with type 1 diabetes (≥ 1 year's duration) after converting from Freestyle Libre to Freestyle Libre 2 . Secondary outcomes included predictors of reduction in time below range (TBR) ≥ 0.5% (<3.9mmol/l) and increase in time in range (TIR) ≥5.0% (3.9 - 10 mmol/l). Participants were included if ≥70% CGM data (2 week block) was available both at baseline and six months.

Results: Low and high glucose alarms were used by 74.5% and 58.6%, respectively. In all users, TBR fell by a median of 1.0% (IQR -2.7 - 0.3, p <0.001) after 6 months but TIR was unchanged (p = 0.920) (table). Average duration of low glucose events (<3.9 mmol/l) fell by 14 minutes (-41 - 13, p <0.001) and the number of low glucose events per 2 weeks fell from 7 to 4 (p <0.001) . Low alarm thresholds were significantly correlated with baseline TBR (R -0.232, p < 0.001) and high alarm thresholds were significantly correlated with baseline time above range (R 0.168, p = 0.009). Alarm thresholds were not independently associated with glycaemic response, however low alarm use was independently associated with a fall in TBR of ≥0.5% (OR 2.9 [95% CI 1.5 - 5.6], p = 0.002) and high alarm use was associated with a rise in TIR of ≥5% (OR 2.17 [1.2 - 4.2], p = 0.013).

Conclusion: Freestyle Libre 2 was associated with modest but clinically relevant improvements in hypoglyaemia metrics but did not improve TIR or average glucose. Low and high glucose alarms were independently associated with improvements in TBR and TIR, respectively, and users of this system should be encouraged to use alarms at thresholds appropriate for the individual's circumstances.

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Disclosure: F.W. Gibb: Honorarium; Abbott Diabetes, Insulet, Novo Nordisk, Lilly.

100

Efficacy and safety of real-time continuous glucose monitoring guiding insulin administration in the hospital: a randomised clinical trial

G.E. Umpierrez1, R.J. Galindo1, M.A. Urrutia1, P. Vellanki1, A.L. Migdal1, G.M. Davis1, F.J. Pasquel1, M. Fayfman1, T. Idrees1, L. Peng1, L.G. Singh2, E.K. Spanakis2;

1Div. of Endocrinology, Emory University School of Medicine, Atlanta, 2Div. of Endocrinology, University of Maryland Medical Center, Baltimore, USA.

Background and aims: Inpatient studies in insulin-treated patients have reported high accuracy and higher detection of hypo- and hyperglycaemic events with the use of real-time continuous glucose monitoring (RT-CGM) compared to capillary point-of-care (POC) glucose testing. In a randomised controlled study, we tested the efficacy and safety of RT-CGM in adjusting inpatient insulin therapy.

Materials and methods: A total of 173 general medicine and surgery patients with type 1 and type 2 diabetes treated with a basal bolus insulin regimen were randomised to a standard of care (POC group, n=85) wearing a blinded Dexcom G6 CGM for up to 10 days with insulin dose adjusted based on POC results, or to a RT-CGM group (n=88) who had their insulin adjustment based on daily Dexcom G6 RT-CGM profile. Primary endpoints were differences in time in range (TIR: 3.9-10 mmol/L) and hypoglycemia (<3.9 and <3.0 mmol/L).

Results: There were no significant differences in TIR (54.51%±27.72 vs 48.64%±24.25, p=0.14), mean daily glucose (10.2±2.2 mmol/L vs 10.4±2.2 mmol/L, p=0.36), percent of patients with CGM values <3.9 mmol/L (36% vs 39%, p=0.68) or <3 mmol/L (14% vs 24%, p=0.12) between RT-CGM-guided and POC group. Among patients with ≥ 1 hypoglycaemic event, compared to POC, the RT-CGM group experienced a significant reduction in hypoglycaemia reoccurrence (1.80±1.54 vs 2.94±2.76 events/patient, p=0.037), lower percentage of time below range (TBR) <3.9 mmol/L (1.89%±3.27 vs 5.47%±8.49, p=0.024), lower recurrent nocturnal hypoglycaemic events (1.21±0.43 vs 1.93±0.92, p=0.02) and lower incidence-rate ratio <3.9 mmol/L (0.53, 95% CI:0.31-0.92) and <3.0 mmol/L (0.37, 95% CI:0.17-0.83).

Conclusion: Our results indicate that the inpatient use of Dexcom G6 RT-CGM is safe and effective in guiding insulin adjustment resulting in a similar improvement in glycaemic control and in a significant reduction of recurrent hypoglycaemic events compared to POC-guided insulin adjustment.

figure ae

Clinical Trial Registration Number: NCT03877068

Supported by: Dexcom

Disclosure: G.E. Umpierrez: Grants; Dexcom, Baxter.

101

Cutaneous reactions associated with diabetes adhesives devices: results of the CutaDiab study

M. Diedisheim1, A. Sola-Gazagnes1, A. Carlier2, L. Potier2, A. Hartemann3, S. Jacqueminet3, C. Pecquet4, J.-B. Julla5, T. Vidal-Trecan5, J.-F. Gautier5, E. Larger1, D. Dubois Laforgue1, R. Roussel2, J.-P. Riveline5;

1Diabetology, Cochin Hospital, APHP, 2Diabetology, Bichat Hospital, APHP, 3Diabetology, Pitié-Salpêtrière Hospital, APHP, 4Dermatology, Tenon Hospital, APHP, 5Diabetology, Lariboisière Hospital, APHP, Paris, France.

Background and aims: The use of devices adhering to the skin for between 2 and 14 days is constantly increasing, as continuous glucose measurement systems (CGM) and insulin pumps. Skin reactions have been observed in patients with diabetes using these new technologies, sometimes leading to discontinuation. Prevalence and consequences of these skin intolerances are unknown. The objective of this study is to determine the prevalence and consequences of cutaneous reactions to CGM or pumps.

Materials and methods: This is an observational, cross-sectional, multicentre study involving four university hospital diabetology departments (Paris, France). A form with about fifty questions concerning diabetes evolution, use of pump and CGM, and description and consequences of possible cutaneous reaction was drawn up on touchpad. All adult patients with diabetes seen in consultation over a period of 6 months and using or having used in the last 10 years a system with skin adhesives were included. Included devices were insulin patch pump (e.g. OMNIPOD®, cell Novo®), pump with externalized catheter (e.g. PARADIGM®, ANIMAS®, MINIMED 640G®, YpsoPump®) and CGM system (Free Style®, DexCom® sensors, Enlite® sensors). Non-parametric tests were used for quantitatives values (Mann-Withney or Kruskal-Wallis tests), and chi-squared test for qualitative values.

Results: 851 patients were included. At least one cutaneous reaction was reported in 28% of CGM users (231/833) and in 29% of pump users (108/374). Patients with cutaneous reaction were more often women for CGM (63 vs 50% for patients with and without cutaneous reaction respectively, p<0.001) and for pump (73 vs 58%, p=0.006), and had more often type 1 diabetes than type 2 diabetes for CGM (84 vs 73%, p<0.001), without difference of type of diabetes for pump (91 vs 90% of type 1 diabetes). Symptoms were similar for CGM and pump reactions: erythema and pruritus in 70-75% of patients, pain in 20-25%, vesicle and peeling in 12-15%. The first symptom appeared within 24 hours of the first use of the device for 24% of CGM reactions, and 22% of pump reactions, or more than 6 months after the first use for 38% of CGM reactions, and 47% of pump reactions. Among patients with cutaneous reaction, this reaction does not modify the use of the device for 82% of reactions to CGM, and 80% for pump. Device use was definitely stopped for 12% of CGM reaction (3.2% of all users, 27/833), and for 7% of pump reaction (2.1% of all users, 8/374). The other reactions led to a decrease in use or to a change of device (6% for CGM, 13% for pump). Cutaneous reaction was more common for tubeless pumps than tubed pumps (31 vs 23%, p=0.03). These cutaneous reactions were no larger than the adhesive in 89% of cases for CGM and 93% for pump, suggesting skin irritation rather than an allergy.

Conclusion: Cutaneous reaction is a common reaction in pump or CGM users, but without consequences for the use of the device in the vast majority of cases, with very similar symptoms between CGMs and pump reactions. However, this reaction, which can appear more than 6 months after initiation of the device, leads to the interruption of its use in 2-3% of patients.

Clinical Trial Registration Number: NCT04853810

Disclosure: M. Diedisheim: None.

102

Impact of treatment costs on the cost-effectiveness of real-time continuous glucose monitoring vs self-monitoring of blood glucose in type 1 diabetes patients in the United Kingdom

H. Alshannaq1,2, G. Cogswell1, G.J. Norman1, P.M. Lynch1, S. Roze3;

1Dexcom, San Diego, USA, 2University of Cincinnati, Cincinnati, USA, 3Vyoo Agency, Lyon, France.

Background and aims: Previous studies have shown that real-time continuous glucose monitoring (rt-CGM) is cost-effective for patients with Type 1 diabetes (T1D) in multiple countries including the UK. However, rt-CGM technology is a rapidly evolving field with continued advancements in clinical efficacy and production efficiency. New generations of the technology are expected to be introduced into clinical practice, which will vary in price and budget impacts to global healthcare systems. The aim of this analysis was to examine the sensitivity of existing cost-effectiveness evidence to changes in treatment costs of rt-CGM technology from a UK health care system perspective.

Materials and methods: The IQVIA CORE diabetes model was used to conduct the treatment costs impact analysis. We used base case baseline data from a published cost-effectiveness analysis for rtCGM in patients with T1D in the UK compared to self-monitoring of blood glucose (SMBG). Clinical data were sourced from the DIAMOND trial of adults with T1D on multiple daily injections of insulin and adapted to the UK. The baseline mean age (SD) of the cohort was 47.6 years (12.7) and proportion of female 56%. Mean baseline HbA1c for the cohort was 8.6% (70 mmol/mol). Reduction in HbA1c was -1.0% and -0.4% for rt-CGM and SMBG, respectively. For treatment costs impact, we conducted sensitivity analyses for -20%, -30%, and -50% reduction from the published treatment cost of GBP 1850. The analysis was conducted from the UK National Health Service payer perspective over a lifetime horizon.

Results: Rt-CGM was associated with a 1.489 incremental gain in quality-adjusted life years (QALYs) compared with SMBG (mean [SD] 11.47 [2.04] versus 9.985 [1.84] QALYs). Variation in treatment costs had a significant impact on the incremental cost-effectiveness ratio (ICER). The ICER was reduced by 46% when treatment costs were reduced by 20% (ICER 9,558 to 5,140 GBP/QALY gained) and by 69% when treatment costs decreased by 30% (ICER 9,558 to 2,933 GBP/QALY gained). Rt-CGM became dominant compared to SMBG when treatment costs decreased by 50% (ICER -1490).

Conclusion: Our analysis shows that treatment cost for rt-CGM is a significant driver of cost-effectiveness against SMBG. Reductions in treatment cost will position rtCGM as a long-term economic strategy to manage diabetes for patients with T1D in the UK.

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Disclosure: H. Alshannaq: Employment/Consultancy; Dexcom.

OP 18 Cross-talk communication in the pancreas

103

Chronic hyperglycaemia leads to dysregulated δ-cell metabolism and reduced glucose-stimulated somatostatin secretion from the pancreatic islets

T.G. Hill1, A.I. Tarasov2, M. Wallace3, E. Haythorne4, L.J.B. Briant1, G. Cyranka4, F.M. Ashcroft4;

1Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford, Oxford, 2School of Biomedical Sciences, Ulster University, Coleraine, 3Wellcome Centre for Human Genetics, University of Oxford, Oxford, 4Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, UK.

Background and aims: Reduced islet β-cell glucose metabolism-insulin secretion coupling and elevated postprandial α-cell glucagon secretion represent hallmark features of type-2 diabetes (T2D). Glucotoxicity has been extensively shown to trigger β-cell metabolic dysregulation, causing reduced glucose-stimulated insulin release. Chronic hyperglycaemia also leads to dysregulated glucagon secretion although the underlying causes remain largely obscure. We explored whether chronic hyperglycaemia alone also interferes with islet δ-cell function and glucose-stimulated somatostatin (Sst) secretion.

Materials and methods: Isolated WT (C57BL/6J), transgenic δ-cell-specific (δGCaMP6 and δRFP) mice, and non-diabetic cadaver donor human islets were chronically cultured under either normo-(Cont, control) or hyperglycaemic (Hyp, 25 mmol/l) glucose conditions in vitro for 14 days. Diabetic mouse islets were isolated from tamoxifen-inducible β-cell-specific Kir6.2-V59M (βV59M) mice subjected to 14 days of chronic in vivo hyperglycaemia (plasma glucose ≥ 25 mmol/l). δ-cell intracellular Ca2+ ([Ca2+]i) and ATP ([ATP]i) were imaged using GCaMP6 and Perceval sensors respectively and a wide-field Zeiss Axio Zoom V16. Sst-14 secretion from islets isolated from WT or βV59M mice, and non-diabetic human donors, was quantified by radioimmunoassay.

Results: (i) Mouse islet δ-cells exposed to chronic hyperglycaemia in vitro demonstrated a smaller increase in [Ca2+]i in response to stimulation with 20 mmol/l glucose (δGCaMP6 F/F0 AUC at 20 mmol/l glucose; Cont, 5.89 ± 0.20 vs Hyp, 1.74 ± 0.72, p < 0.05), when compared to normoglycaemia-exposed controls. (ii) Mouse islet δ-cells subjected to either chronic in vitro or in vivo hyperglycaemia also revealed impaired [ATP]i responses when subjected to acute glucose stimulation (δRFP islets transfected with Perceval: F/F0 AUC at 20 mmol/l glucose: Cont, 6.28 ± 0.21 vs Hyp, 2.06 ± 0.52, p < 0.01. βV59M islets transfected with Perceval F/F0 AUC at 20 mmol/l glucose: Cont, 2.87 ± 0.21 vs Hyp, 1.30 ± 0.17, p < 0.05); and (iii) lower glucose-stimulated Sst secretion (fold-change 20 mmmol/l glucose vs 1 mmol/l glucose; WT islets: Cont, 4.30 ± 0.13 vs Hyp, 2.36 ± 0.84; p < 0.05; βV59M islets: Cont, 24.75 ± 4.67 vs Hyp, 9.73 ± 3.49, p < 0.01), when compared to normoglycaemia-exposed controls. (iv) Similarly, human islets exposed to chronic in vitro hyperglycaemia also showed decreased glucose-stimulated Sst secretion when compared to normoglycaemic control islets.

Conclusion: These data demonstrate that chronic hyperglycaemia alone results in dysregulation of glucose-stimulated δ-cell function and reduced Sst secretion. This may, via loss of normal paracrine regulation of the α-cells, contribute to the abnormal increase in postprandial α-cell glucagon secretion seen in T2D patients.

Supported by: Novo Nordisk-Oxford Fellowship

Disclosure: T.G. Hill: None.

104

Paracrine signalling in delta cells is disturbed in human type 2 diabetes

L. Matuseviciene1, O.M. Hmeadi2, P.-E. Lund2, S. Barg2;

1Uppsala University, 2Uppsala Universitet, Uppsala, Sweden.

Background and aims: Pancreatic delta cells secrete somatostatin in response to elevated glucose, which in turn suppresses insulin and glucagon secretion from beta and alfa cells, respectively. In type 2 diabetes, this paracrine regulation is disturbed, but the underlying mechanisms remain unclear. Research on isolated delta cells is rare and relatively little is known about the cellular control of somatostatin release. The aim of this study was to quantify the effects of intra-islet signalling pathways on exocytosis and membrane potential in delta cells from non-diabetic and type 2 diabetic islets.

Materials and methods: Live cell imaging and electrophysiology were performed in dispersed pancreatic islets of non-diabetic (ND) or type 2 diabetic (T2D) cadaveric human donor islets. To identify delta cells and for imaging exocytosis by TIRF-microscopy, dispersed islet cells were transduced with adenovirus coding for fluorescent granular marker under control of somatostatin promoter. Exocytosis was evoked by elevated K+ in presence of 10mM glucose/200μM diazoxide (control) and either of the following: 100nM insulin, 10nM glucagon, 400nM somatostatin, 400nM GABA and 5μM adrenaline, 2μM forskolin, or 10nM exendin4. In addition, standard whole-cell voltage clamp and capacitance recordings were performed on single delta cells to study effects on exocytosis, ion channels. Somatostatin receptor 2 (SSTR2) expression was analysed by confocal microscopy of immunostained human pancreatic tissue sections.

Results: K+-stimulated exocytosis of somatostatin granules followed a biphasic timecourse, with a tendency towards reduced exocytosis in delta cells of T2D donors (-28%, p=0.08, 6 ND donors, n=50 cells; 4 T2DM donors, n=25 cell); this was strengthened by capacitance recordings (-40%, p=0.018; 10 ND donors, n=68 cells; 4 T2D donors, n=42 cells). There were no changes in Ca2+-currents that could explain the decrease in exocytosis, but Na+-currents were reduced in T2D cells, compared with ND (-31%, p=0.02; 10 ND donors, n=66 cells; 4 T2D donors, n=34 cells). K+-stimulated exocytosis decreased in presence of somatostatin when compared with diazoxide only (-39%, p=0.03, 4 ND donors, n=18 cells) and robustly increased by glucagon (+50%, p<0.0001; 5 ND donors, n=28 cells), exendin-4 (+61%, p<0.0001; 4 ND donors, n=17 cells), forskolin (+68%, p<0.0001, 5 ND donors, n=23 cells), and GABA (+66%, p<0.0001, 5 ND donors, n=22 cells). No significant difference in the compounds’ effects were detected between ND and T2D, or in 1 mM vs 10 mM glucose. In contrast, adrenaline inhibited exocytosis in ND delta cells (-59%, p<0.0001; 8 ND donors, n=41 cells), but significantly increased in T2D delta cells (+56%, p<0.0001; 2 T2D donors, n=13 cells; 6 ND donors, n=50 cells). In addition, short term exposure to GABA depolarized the membrane potential of delta cells, while somatostatin repolarized and inhibited electrical activity. Finally, immunostaining of human pancreas sections revealed internalization and reduced surface expression of SSTR2 in beta cells of T2D donors.

Conclusion: Human delta cells are responsive to autocrine inhibition by somatostatin, as well as a variety of paracrine signalling pathways. Type 2 diabetes is associated with decreased delta cell secretory capacity and a prominent reversal of adrenalin’s effect from inhibitory to stimulatory. The latter may indirectly contribute to hyperglycemia in type 2 diabetes by strengthening somatostatin-dependent inhibition of insulin secretion.

Supported by: NNF, DF, BDF, VR, MF-UU, EXODIAB, Ernfors, Rudbergs

Disclosure: L. Matuseviciene: None.

105

The requirement of beta cells to the glucose responsiveness of alpha cells depends on the depolarisation status of alpha cells

F. Khattab, P. Gilon;

IREC EDIN, UCLouvain, Brussels, Belgium.

Background and aims: The mechanisms by which glucose controls glucagon release are largely unknown. In particular, it is unclear whether glucose controls α-cell activity directly or indirectly via β- or δ-cells. In this study, we used mouse pseudo-islets (PI) of various compositions to investigate the role of β- and δ-cells in the control of glucagon secretion by glucose.

Materials and methods: Several mouse models expressing a fluorescent protein specifically in α-, β-, or δ-cells were used. Islets were dispersed and cells were FACS-sorted. Highly pure populations of α-, β-, and δ-cells were then reaggregated into various types of pseudo-islets by culturing them during 5-6 days. The effect of a change of the glucose (G) concentration between 1 (G1) and 10 mmol/l (G10) was tested on glucagon and insulin secretions from islets or PI in perifusion experiments. Perifusion media contained a 2.1 mmol/l mixture of amino acids present at physiological concentrations and containing (in mmol/l) 0.4 alanine, 0.5 glutamine, 0.2 lysine, 0.25 glycine, 0.15 leucine, 0.25 valine, 0.15 threonine, 0.1 serine, and 0.1 arginine.

Results: Dispersed islets cells, including pure α-cells, spontaneously reaggregated into pseudo-islets within 5-6 days. As expected, control islets showed an increase in glucagon secretion in response to a drop of glucose concentration from G10 to G1. The response was similar in pseudoislets obtained after reaggregation of all cell types. However, glucagon release from pseudo-islets made of pure α-cells (α-PI) was impaired in response to glucose. The addition of β-cells (α/β-PI), but not δ-cells (α/δ-PI) to α-cells restored normal glucagon secretion in response to G1. To determine whether this restoration was specifically due to the presence of β-cells or simply to the reestablishment of cell-to-cell contacts with another cell type, α-cells were reaggregated with mouse embryonic fibroblasts (α/MEF-PI). The presence of MEF did not restore a normal glucagon response to G1. We also generated α/β/MEF-PI to verify that MEF did not interfere with glucagon secretion. α/β/MEF-PI showed a normal simulation of glucagon secretion in response to G1, further confirming the importance of β-cells. Interestingly, in the presence of diazoxide (a KATP channel opener) and high potassium (30 mmol/l K+) which, together, clamp the membrane potential at a depolarized level, a drop of glucose concentration from G10 to G1 stimulated glucagon release from α-PI, suggesting that glucose modulates the efficacy of Ca2+ on exocytosis in α-cells independently of β and δ-cells.

Conclusion: We show, for the first time, that mouse α-cells can reaggregate into pseudo-islets. We demonstrate that, in conditions where the plasma membrane is not clamped, glucagon secretion in response to glucose from pure α-PI is impaired, and that the addition of β-cells, but not δ-cells, restores a normal control of glucagon secretion by α-cells. However, in conditions where the plasma membrane is clamped at a depolarized level, low glucose efficiently stimulates glucagon release from pure α-PI, i.e. independently of β- and δ-cells.

Supported by: FRIA, FNRS, Helmsley, ARC, SFD, EFSD/JDRF/Lilly Programme

Disclosure: F. Khattab: None.

106

Antecedent hypoglycaemia enhances intra-islet α-δ-cell paracrine feedback loop to impair α-cell glucagon secretion

R. Gao, S. Acreman, P. Rorsman, Q. Zhang;

OCDEM, University of Oxford, Oxford, UK.

Background and aims: Glucagon released by pancreatic α-cells is a principal glucose-elevating hormone that counter-regulates hypoglycaemia. Glucagon secretion can be regulated by inhibitory somatostatin released from neighbouring δ-cells. Appropriate somatostatin secretion maintains a balanced islet glucagon output but abnormal islet paracrine tone due to somatostatin hypersecretion often leads to defective secretion of glucagon and impairs glucose counter-regulation. However, how δ-cell releases somatostatin and how it becomes dysregulated remain obscure. Here we studied the crosstalk between α- and δ-cells, and how this cellular communication is affected by antecedent hypoglycaemia.

Materials and methods: Glucagon and somatostatin secretion was assessed using static hormone secretion assays. α- and δ-cell intracellular Ca2+ dynamics were monitored using live cell imaging with islets from reporter-expressing mice (GCaMP6f specifically expressed in α- or δ-cells, GCG-GCaMP6f or SST-GCaMP6f, respectively). Direct interaction between α-cell exocytosis and its adjacent δ-cell response was characterised using a combination of electrophysiology and high-speed Ca2+ imaging.

Results: To study α-δ-cell crosstalk, single α-cells within intact SST-GCaMP6f islets were stimulated with depolarisations from -70 to 0 mV using voltage-clamp technique. Stimulation of α-cell led to rapid increase of Ca2+ in neighbouring δ-cells (~900 ms delay). The magnitude of depolarisation-triggered α-cell exocytosis was positively correlated with the AUCs of δ-cell Ca2+ transients (R=0.56, P<0.001), suggesting δ-cells respond to paracrine factors released by α-cells. Blockade of glucagon receptor or glutamate receptor strongly reduced δ-cell Ca2+ activity, exocytosis and somatostatin secretion at low glucose (where α-cells’ activity is high). Glucagon-dependent δ-cell Ca2+ activity is originated from ER and mediated through both Gαs and Gαq. Antagonising Gαs or Gαq pathway reduced basal δ-cell activity by 48.7% (P=0.002) or 47.5% (P=0.004), respectively. This paracrine interaction is sensitive to metabolic cues and is strengthened following exposure to hypoglycaemia. In islets pretreated with hypoglycaemia (2 mM glucose, 2-hr, followed by overnight recovery in 5 mM glucose), basal somatostatin hypersecretion (P=0.026) and δ-cell Ca2+ hyperactivity (P=0.047) were observed. These were correlated with reduced glucagon secretion (P=0.006) and α-cell activity (P<0.001) in response to 1 mM glucose. At single cell level, δ-cells became more sensitive to α-cell exocytosis (R=0.69, P<0.001). Hypoglycaemic exposure also induced persistent δ-cell structural changes with protrusion extension by ~1.5 μm (P=0.007). These effects were reversed by including a glucagon receptor blocker during the hypoglycaemic pretreatments, indicating a key role of intensive glucagon release during antecedent hypoglycaemia in δ-cell plasticity.

Conclusion: Pancreatic α- and δ-cells are tightly coupled to form a reciprocal paracrine network, a mechanism that fine-tunes glucagon secretion to prevent glucagon overshoot. This coupling is plastic and sensitive to metabolic cues: δ-cell sensitivity to adjacent α-cell activity is enhanced by antecedent hypoglycaemia, leading to defective glucagon response to later hypoglycaemia. These hypoglycaemia-induced dysregulated α-δ-cell interaction may underlie the defective glucagon secretion in recurrent hypoglycaemia in diabetes.

Supported by: The Royal Society, Medical Research Council, Diabetes UK

Disclosure: R. Gao: None.

107

Insulin-like growth factor binding protein 7 reduces glucose stimulated insulin secretion and downregulates key beta cell transcription factors

E. Westholm, A. Karagiannopoulos, A. Wendt, L. Eliasson;

Islet Cell Exocytosis Group, Faculty of Medicine, Malmö, Sweden.

Background and aims: Cystic fibrosis (CF) is one of the most prevalent genetic diseases in the world and is caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common non-pulmonary complication of CF is cystic fibrosis-related diabetes mellitus (CFRD), which is characterised by impaired insulin secretion. In a ferret CFTR-KO model, the secretome from pancreatic ductal epithelium showed an upregulation of transforming growth factor β1 (TGFβ1). In the same study TGFβ1 stimulated an increased expression of insulin-like growth factor binding protein 7 (IGFBP7) in the surrounding pancreatic stromal cells. IGFBP7 can bind insulin with high affinity and interact with the insulin receptor and IGF receptors. The aim of this project is to investigate if IGFBP7 affects β-cell physiology, especially insulin secretion.

Materials and methods: EndoC-βH1 cells were used in glucose stimulated insulin secretion (GSIS) experiments. In acute IGFBP7 treatment, the GSIS assay buffer was supplemented with 100 nM IGFBP7. C-peptide was measured as a proxy for secreted insulin using ELISA and the results were normalised to total protein (ng/mg prot/h). In long-term treatment, cells were cultured 72 h with 100 nM IGFBP7 prior to GSIS, and secreted insulin was normalised to total insulin (%/h). Gene expression after long-term treatment was assessed with RT-qPCR. RNA from EndoC-βH1 cells, 4 passages, and primary human islets from 4 donors was prepared using the TruSeq library kit and sequenced on an Illumina platform. Transcripts were quantified with Salmon v. 0.14 and results were processed using the DESeq2 tool and are reported as normalised counts. For GSIS and RT-qPCR data, statistical analysis was done using paired T-test.

Results: In acute treatment experiments, IGFBP7 reduced the secretion of C-peptide at 20 mM glucose stimulation (CTRL 111 ng/mg prot/h vs. IGFBP7 90 ng/mg prot/h, n=5, P=0.018). A similar effect was observed with long-term treatment of IGFBP7 (CTRL 5.2 %/h vs. IGFBP7 4.0 %/h, n=4, P=0.0085). Long-term treatment with IGFBP7 did not significantly change total insulin content of EndoC-βH1 cells. With long-term treatment, there was a decreased expression of genes encoding for important β-cell transcription factors such as PDX1, NKX6-1, PAX4, FOXO1 (n=4, P<0.05). We also saw a reduction of VAMP2 (n=4, P<0.05). In RNA sequencing data, there was similar expression of potential IGFBP7 binding receptor genes INSR, IGF1R, IGF2R as with TGFBR1 and KCNJ11, in EndoC-βH1 cells and primary human islets. However, the expression of IGFBP7 was almost 50 times higher in primary human islets in comparison to EndoC-βH1 cells.

Conclusion: Combined, these experiments show that IGFBP7 cause impaired β-cell function with reduced insulin secretion and decreased gene expression levels of important β-cell genes. In the context of cystic fibrosis and CFRD, we hypothesize that elevated IGFBP7 functions as a mediator of pathophysiological intra-pancreatic crosstalk between defect ductal cells and islet cells. Such cross talk likely plays a role in disease development.

Supported by: CF Trust International consortium SRC019 and RfCF

Disclosure: E. Westholm: None.

108

Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis

F. Brozzi1, C. Cosentino1, C. Jacovetti1, K. Wu1, V. Menoud1, M.B. Bayazit1, C. Guay1, R. Regazzi1,2;

1Department of Fundamental Neurosciences, University of Lausanne, 2Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland.

Background and aims: Type 1 diabetes (T1D) is an autoimmune disease characterized by immune cell infiltration in the islets of Langerhans (insulitis). Immune cells release pro-inflammatory cytokines and extracellular vesicles (EVs) in the site of infiltration, causing β-cell death. This study focuses on decoding the dialogue between T-cells and β-cells during T1D pathogenesis, on the analysis of T-cell EV content and of their functional impact on β-cells. We have previously demonstrated that specific miRNAs are transferred from T-cells to β-cells via EVs during insulitis, causing β-cell inflammation and apoptosis. tRNA-derived fragments (tRFs) are also emerging as major components of T-cell EVs and may potentially be delivered to β-cells during the autoimmune attack. tRFs are fragments originating from transfer RNAs (tRNAs) and have several functions in the control of cellular processes, including apoptosis. Indeed, our preliminary results show an increase in tRF expression in islet cells of pre-diabetic NOD mice. The aim of the present study is to identify the tRFs that are transferred from T-cells to islet cells during T1D pathogenesis and affect β-cell function, inflammation, and survival.

Materials and methods: Small RNAseq analysis was performed in EVs isolated from NOD CD4+/CD25- T-cells and in pancreatic islet cells treated for 24h with NOD T-cell EVs. To prove the direct transfer of selected tRFs, the RNA in the CD4+/CD25- T-cells was tagged with a uridine analogue (EU), and then isolated form the β-cells treated with the EVs produced from EU-tagged T-cells. The tagged RNA was analyzed by qPCR to identify selected tRFs transferred to β-cells. RNA mimics were used to overexpress the candidate tRFs in β-cells, with the aim of reproducing the EV-dependent rise of the selected RNA and study its effect. Apoptosis was assessed with Propidium Iodide (PI)/ Hoechst staining and cleaved caspase-3 assay.

Results: The small RNA data sets from T-cell EVs and from islet cells treated with EVs were compared with the tRFs that are significantly up regulated in the islets of pre-diabetic NOD mice (265 tRFs in 8 weeks-old vs 4 weeks-old mouse islets, adjusted p-value=0.05, n=4). From this comparison we identified a subset of 7 tRFs that are transferred from T-cells to β-cells in the initial phases of T1D. Their expression increased in islets during insulitis (2.0 to 6.2 Fold Change (FC), in 8 weeks-old vs 4 weeks-old mouse islets, p-value <0.05, n=4, t-Test ) and after in-vitro incubation with T-cell EVs (2.0 to 4.8 FC in EV treated vs control islets, p-value<0.05, n=4, t-Test). Moreover, with an RNA-tagging strategy, we were able to recover in EV-treated β-cells some of the miRNAs and tRFs that were increased in NOD islet cells during insulitis. The overexpression of three tRFs candidates increased β-cell apoptosis in primary islet cells (1.6 to 1.8 FC, tRFs vs Ctrl, p-value <0.05, ANOVA).

Conclusion: We provide a direct “proof of concept” that tRFs can be shuttled between T-cells and β-cells via EVs, and identify a new mechanisms through which T-cells may trigger β-cell dysfunction and apoptosis in T1D pathogenesis. Mechanistic studies will be done to evaluate the mode of action of selected tRFs and to evaluate the potential design of new strategies for the treatment of diabetic patients.

Disclosure: F. Brozzi: None.

OP 19 GLP1 agonists: from here to eternity

109

Dulaglutide in youth with type 2 diabetes: results of the AWARD-PEDS randomised, placebo-controlled trial

S. Arslanian1, T. Hannon2, P. Zeitler3, L. Chao4, C. Boucher-Berry5, M. Barrientos-Pérez6, E. Bismuth7, S. Dib8, J. Cho9, D. Cox9;

1UPMC Children's Hospital of Pittsburgh, Pittsburgh, USA, 2Riley Hospital for Children, Indianapolis, USA, 3Children's Hospital Colorado, Aurora, USA, 4Children's Hospital of Los Angeles, Los Angeles, USA, 5Children's Hospital of the University of Illinois, Chicago, USA, 6Angeles Hospital of Puebla, Puebla, Mexico, 7University Robert Debré Hospital, Paris, France, 8Federal University of São Paulo State, São Paulo State, Brazil, 9Eli Lilly and Company, Indianapolis, USA.

Background and aims: AWARD-PEDS was a Phase 3 trial to assess the efficacy and safety of dulaglutide (DU), a once-weekly GLP-1 receptor agonist, in youth (10 to <18 years old) with type 2 diabetes (T2D) treated with lifestyle alone or on stable metformin with or without basal insulin. The primary aim was to demonstrate superiority of DU (pooled doses) vs placebo for change in HbA1c at 26 weeks.

Materials and methods: Participants (mean age, 14.5 yrs; mean BMI, 34.1 kg/m2) were randomised to placebo (N=51), DU 0.75 mg (N=51), or DU 1.5 mg (N=52). Analyses included all patients with ≥1 dose of study drug, excluding data after initiation of rescue therapy.

Results: DU was superior to placebo (figure) in improving glycaemic control measured by change in HbA1c, percent of patients with HbA1c <53 mmol/mol (7%), and change in fasting glucose at Week 26. No effect of DU was observed on BMI change (p=0.776). Fewer patients assigned to DU compared to placebo required rescue therapy (2.9% vs 17.6% respectively, p=0.003). Incidence of common GI adverse events was higher in DU group vs placebo [nausea (14.6% vs 7.8%), vomiting (15.5% vs 3.9%), diarrhoea (18.4% vs 13.7%)] but comparable to that observed in adults.

Conclusion: In conclusion, in youth with inadequately controlled T2D treated with or without metformin and/or basal insulin, once weekly DU 0.75 mg or 1.5 mg was superior to placebo in improving glycaemic control without an effect on BMI through 26 weeks, with a safety profile consistent with that established in adults.

figure ag

Clinical Trial Registration Number: NCT02963766

Supported by: Eli Lilly and Company

Disclosure: S. Arslanian: Grants; Eli Lilly and Company, Novo Nordisk. Other; Advisory Board: Eli Lilly and Company, Novo Nordisk; DMC member: AstraZeneca, Eli Lilly and Company.

110

Beta cell function and sensitivity to incretins before and after Roux-en-Y gastric bypass in patients with type 2 diabetes

M.S. Svane1, M. Hindsø1, C. Martinussen1, C. Dirksen1, N.B. Jørgensen1, B. Hartmann2, V.B. Kristiansen1, J.J. Holst2, K.N. Bojsen-Møller1, S. Madsbad1;

1Copenhagen University Hospital Hvidovre, Hvidovre, 2University of Copenhagen, Copenhagen, Denmark.

Background and aims: Roux-en-Y gastric bypass (RYGB) improves glucose tolerance in patients with type 2 diabetes (T2D), but whether RYGB affects beta-cells independently of signals from the gut is unclear. Also, the sensitivity of the beta-cell to glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) remains to be clarified. We aimed to investigate beta-cell function before and after RYGB, and particularly, the sensitivity of the beta-cell to the incretin hormones. We hypothesized that sensitivity to GIP would be increased after RYGB because of relieve of glucotoxicity.

Materials and methods: Nine patients with obesity and T2D (5 women, age: 45±3 years, weight: 127.4±8.1 kg, BMI: 42.7±2.3 kg/m2) were examined with a 75 g OGTT and three 90-min hyperglycemic clamps at ~15 mM plasma glucose (CV 2%) with primed continuous co-infusions of either GIP (1.5 pmol/kg/min), GLP-1 (1 pmol/kg/min) or saline before (pre) and 3 months (3mo) after RYGB. Beta-cell function was calculated separately for the OGTT (as beta-cell glucose sensitivity (β-GS): slope between insulin secretion rates and plasma glucose concentrations) and the intraveneous stimulation (first phase as the acute insulin response to glucose (AIR) (positive incremental (pi)AUC0-10 min) and second phase (piAUC 20-90 min) during clamps).

Results: After RYGB, fasting plasma glucose decreased (pre: 7.9±0.3 mmol/L, 3mo: 5.4±0.3, p<0.01) and insulin sensitivity improved (HOMA2 IR: pre 4.1±0.3, 3mo: 2.4±0.2, p<0.01). Weight loss was 19±0.7 kg (14.9%). During the OGTT, 2-hour plasma glucose decreased (pre: 13.6±0.7 mmol/L, 3mo: 7.3±0.8, p<0.01) and insulin secretion increased resulting in a two-fold increase in β-GS (pre: 0.9±0.1 (mmol/kg/min)/mmol/L, 3 mo: 1.8 ±0.1, p<0.01). GIP secretion decreased slightly after RYGB (piAUC pre: 5270±913 pmol/L· min, 3mo: 4310±339, p=0.01), whereas GLP-1 secretion increased markedly (pre: 1156±583 pmol/L· min, 3mo: 6007±691, p<0.01). During the saline clamp both first and second phase of insulin secretion increased after RYGB (AIR pre: 15±5 pmol/kg, 3mo: 58±15, p<0.01; piAUC 20-90 min pre: 296±54, 3mo: 445±54, p<0.05). GIP-infusion augmented second phase of insulin secretion equally before and after RYGB (pre: +96±16% vs saline-clamp, 3mo: +90±14%, p=0.57). GLP-1 infusions increased insulin secretion even more, with a small decline in the potentiating effect after RYGB (Pre: +399±84%, 3mo: +233±32%, p=0.03).

Conclusion: In patients with obesity and T2D, beta-cell function increases after RYGB both in response to oral and iv glucose. An insulinotropic effect of GIP was demonstrated before surgery and this effect was maintained postoperatively. The potentiating effect of GLP-1 on insulin secretion was pronounced both before and after surgery with a slight decline observed after RYGB. Thus, the improved beta cell function in response to an oral glucose stimulus after RYGB is the result of GLP-1 hypersecretion, rather than an increased insulinotropic action of the incretins.

Clinical Trial Registration Number: NCT04782999

Supported by: European Research Council, Horizon 2020, Grant: BYPASSWITHOUTSURGERY

Disclosure: M.S. Svane: Grants; European Research Council, Novo Nordisk Foundation, Danish Diabetes Association.

111

Ly3437943 (LY), a novel triple GIP/GLP-1/glucagon receptor agonist, provides glucose lowering and weight loss in patients with type 2 diabetes after 12 weeks of treatment

Z. Milicevic, S. Urva, M. Loh, T. Coskun, Y. Du, C.T. Benson, C. Loghin, A. Haupt;

Eli Lilly and Company, Indianapolis, USA.

Background and aims: Multi-receptor incretin agonists are being developed for several metabolic disorders. LY is an investigational triple agonist with potent activity on glucose-dependent insulinotropic polypeptide (GIP), glucagon-like polypeptide-1 (GLP-1), and glucagon receptors. LY was safely studied in a prior first-in-human study, and pharmacokinetic properties supported once weekly dosing. The primary objective of this randomized, double-blind, placebo-controlled, Phase 1 proof-of-concept study was to assess the safety and tolerability of multiple ascending doses of LY in patients with type 2 diabetes (T2D).

Materials and methods: Seventy-two patients were randomized (9:3:1) to 5 rising dose cohorts of subcutaneous LY, placebo, and dulaglutide 1.5mg, respectively. Within cohort, dose-escalation was implemented at highest 2 cohorts. Vital signs, laboratory data and adverse events (AEs) were monitored to assess safety and tolerability. Efficacy was assessed by monitoring change in glycated hemoglobin (HbA1c) and body weight at week 12.

Results: The most common treatment-emergent AEs were gastrointestinal (nausea and diarrhea), which were mostly mild in severity. By week 12, mean systolic and diastolic blood pressure decreased from baseline in LY compared to placebo group, while pulse and heart rate increased from baseline within most of the LY cohorts and the dulaglutide cohort, but not with placebo. By week 12, mean HbA1c decreased from baseline in all groups, with higher doses of LY showing statistically significant, placebo-adjusted decreases of up to 17.1 mmol/mol. Except at the initial cohort, dose-dependent decreases in mean placebo-adjusted body weight of up to 8.96 kg were observed with LY.

Conclusion: LY3437943 exhibits a safety and tolerability profile similar to other incretins. The promising glycemic and body weight loss efficacy within this study highlights the potential for LY to provide additional benefit versus existing therapies in treatment of T2D and obesity.

Clinical Trial Registration Number: NCT04143802

Disclosure: Z. Milicevic: Employment/Consultancy; Eli Lilly and Company. Stock/Shareholding; Eli Lilly and Company.

112

Effect of liraglutide on muscle fat infiltration in adults with overweight or obesity: a randomised clinical trial

K.V. Patel1, A. Pandey2, C. Ayers2, J. Linge3, O.D. Leinhard3, P.H. Joshi2, I.J. Neeland4;

1Houston Methodist Hospital, Houston, USA, 2University of Texas Southwestern Medical Center, Dallas, USA, 3AMRA Medical and Linköping University, Linköping, Sweden, 4University Hospitals Harrington Heart and Vascular Institute and Case Western Reserve University School of Medicine, Cleveland, USA.

Background and aims: Excess muscle fat is observed in obesity and associated with greater burden of cardiovascular risk factors and higher risk of mortality. Liraglutide reduces total body weight and visceral fat but its effect on muscle fat infiltration (MFI) is unknown.

Materials and methods: This study is an analysis of a randomized, double-blind, placebo-controlled trial that examined the effects of liraglutide plus a lifestyle intervention on visceral adipose tissue among adults with body mass index ≥30 kg/m2 or ≥27 kg/m2 and metabolic syndrome without diabetes. Participants were randomly assigned to a once-daily subcutaneous injection of liraglutide (target dose 3.0 mg) or matching placebo for 40 weeks. Body composition was assessed by magnetic resonance imaging at baseline and follow-up. MFI was measured as the average proportion of fat in viable muscle tissue (muscle tissue with fat fraction <50%) of the bilateral anterior thighs. Treatment effects and 95% confidence intervals of liraglutide and placebo on MFI were calculated by means of generalised linear mixed models with random effects for participants. The associations of changes in MFI with changes in measures of body composition and cardiometabolic biomarkers were assessed using Spearman correlation coefficients.

Results: Among the 128 participants with follow-up imaging (92.2% women, 36.7% Black), median MFI at baseline was 7.8%. The percent change in MFI over follow-up was greater among participants randomized to liraglutide (n = 73) compared with placebo (n = 55) (-2.81% vs. 0.29%, p-value = 0.001) (Figure). Longitudinal change in MFI was significantly correlated with change in body weight (r = 0.30), visceral adipose tissue (r = 0.41), and abdominal subcutaneous adipose tissue (r = 0.32) but not with change in total body lean tissue or total thigh muscle. Change in MFI was significantly associated with change in N-terminal pro-B-type natriuretic peptide (r = 0.28) and high-sensitivity C-reactive protein (r = 0.29) but not change in fasting plasma glucose or insulin.

Conclusion: Among adults with overweight or obesity free of diabetes, once daily subcutaneous liraglutide reduced mean anterior thigh MFI compared with placebo independent of changes in thigh muscle volume. The contribution of MFI improvement to the cardiometabolic benefits of liraglutide require further study.

figure ah

Clinical Trial Registration Number: ClinicalTrials.gov Identifier: NCT03038620

Supported by: Funding: Novo Nordisk

Disclosure: K.V. Patel: None.

113

Glucagon-like peptide 1 receptor agonists and gallbladder or biliary diseases: data from the FDA Adverse Event Reporting System

H. Zhang, L. He, J. Wang, N. Yang, W. Li, L. Xu, Y. Li, F. Ping;

Department of Endocrinology, Key Laboratory of Endocrinology of National Health Commission, Peking U, Beijing, China.

Background and aims: Associations between glucagon-like peptide 1 receptor agonists (GLP-1RAs) and gallbladder or biliary diseases remains controversial in real-world settings. We aimed to make a detailed analysis of the gallbladder or biliary diseases reports for GLP-1RAs versus sodium-glucose cotransporter-2 inhibitors (SGLT-2i) or non-GLP-1RAs in the United States Food and Drug Administration Adverse Event Reporting System (FAERS).

Materials and methods: The FAERS database was mined from the first quarter of 2013 to the fourth quarter of 2020. Disproportional analyses with proportional reporting ratios (PRR) and 95% confidence intervals (CI) and multiple logistic analyses with odds ratios (OR) and 95% CI were performed.

Results: 1,109 cases of gallbladder or biliary diseases were extracted from the FAERS database. Significant associations between GLP-1RAs and increased reporting of gallbladder or biliary diseases were observed in the disproportional analyses (PRR: 2.366 [95% CI: 2.057-2.721]) and multiple-adjusted analyses (OR: 2.992 [95% CI: 2.533-3.534], P<0.0001), which remained consistent across the subcategories. Liraglutide (PRR: 5.001 [95% CI: 4.277-5.847]) and semaglutide (PRR: 6.267 [95% CI: 5.143-7.635]) showed significant signals of gallbladder or biliary diseases; exenatide and dulaglutide showed significant signals of only cholelithiasis and biliary obstruction, respectively. More gallbladder or biliary diseases reports with GLP-1RAs were observed in males than females (P=0.0134) and groups with indications of weight reduction than glycemic control (P<0.0001).

Conclusion: GLP-1RAs was associated with increased reporting of not only gallbladder disorders but also biliary diseases in a large pharmacovigilance database, particularly semaglutide and liraglutide, adding to available evidence from trials through real-word data.

figure ai

Supported by: Natural Science Foundation of Beijing Municipality Beijing (M22014)

Disclosure: H. Zhang: None.

114

Once-daily oral small molecule GLP-1R agonist PF-07081532 robustly reduces glucose and body weight within 4-6 weeks in adults with type 2 diabetes and non-diabetic adults with obesity

C. Buckeridge1, N. Tsamandouras1, S. Carvajal-Gonzalez1, L.S. Brown2, K.L. Chidsey1, A.R. Saxena1;

1Pfizer Worldwide Research and Development, Cambridge, 2Pfizer Worldwide Research and Development, Collegeville, USA.

Background and aims: PF-07081532 is a small molecule glucagon-like peptide-1 receptor (GLP-1R) agonist for once-daily oral administration. This Phase 1, randomised, double-blind, placebo-controlled, multiple ascending dose study investigated the safety, tolerability, pharmacokinetics and pharmacodynamics of PF-07081532 administered for 28 or 42 days in adults with type 2 diabetes mellitus (T2DM) inadequately controlled on metformin, and in non-diabetic adults with obesity.

Materials and methods: The study enrolled 51 participants with T2DM who received study drug once-daily for 28 or 42 days, and 15 participants with obesity who received study drug once-daily for 42 days. Participants were randomised to PF-07081532 or placebo in a 4:1 ratio. The daily dose was titrated over the dosing period up to a target dose; 61 participants completed inpatient dosing.

Results: In participants with T2DM, the mean baseline in fasting plasma glucose (FPG) and mean daily glucose (MDG) were 192 mg/dL and 212 mg/dL, respectively, and were similar across treatment groups. Mean baseline body weight for participants with T2DM and participants with non-diabetic obesity were 90 kg and 98 kg, respectively. PF-07081532 administered orally once-daily resulted in robust reductions in glucose and weight. Modelled mean reductions in MDG in participants with T2DM ranged up to 91 mg/dL over 28 days and 99 mg/dL over 42 days, compared with 29 mg/dL with placebo. Decreases in FPG of up to 79 mg/dL were observed over 28 days and 102 mg/dL over 42 days. Observed body weight reduction in participants with T2DM ranged up to 5 kg over 28 to 42 days, compared with 2 kg for placebo, with a similar magnitude of decrease in participants with non-diabetic obesity. Most adverse events were mild and consistent with the mechanism of action. No clinically significant, adverse trends in laboratory measures, electrocardiogram or vital sign abnormalities were apparent.

Conclusion: In adults with T2DM, once-daily oral administration of the GLP-1R agonist, PF-07081532 robustly reduced plasma glucose and body weight with a safety and tolerability profile consistent with the GLP-1R agonist class. Similar changes in body weight were observed in participants with non-diabetic obesity.

Clinical Trial Registration Number: NCT04305587

Supported by: Sponsored by Pfizer Inc.

Disclosure: C. Buckeridge: Employment/Consultancy; Pfizer Inc. Stock/Shareholding; Pfizer Inc.

OP 20 NAFLD and treatment

115

One night of prolonged fasting improves nocturnal substrate oxidation without modulating hepatic glycogen in individuals with NAFL and healthy age-matched individuals

K.H.M. Roumans1, A. Veelen1, C. Andriessen1, J. Mevenkamp1,2, P. Veeraiah1,2, B. Havekes3, H.P.F. Peters4, L. Lindeboom1,2, P. Schrauwen1, V.B. Schrauwen-Hinderling1,2;

1Department of Nutrition and Movement Sciences, Maastricht University, Maastricht, 2Department of Radiology and Nuclear Medicine, Maastricht University Medical Center, Maastricht, 3Department of Internal Medicine, Maastricht University Medical Center, Maastricht, 4Unilever Food Innovation Center, Wageningen, Netherlands.

Background and aims: Increasing overnight fasting time, as with time-restricted eating, has been shown to improve metabolic health. The mechanisms underlying these beneficial metabolic effects remain inconclusive, but may be related to larger fluctuations in hepatic glycogen which in turn could induce a higher fat oxidation during the night. Increasing fat oxidation may especially be an interesting therapeutic strategy for people with a high amount of ectopic fat accumulation, as with non-alcoholic fatty liver (NAFL). Here, we investigated whether acutely prolonging an overnight fast from 9.5-hours to 16-hours, without changing total daily energy intake, reduces overnight hepatic glycogen and improves substrate metabolism in individuals with NAFL compared to age-matched healthy lean individuals.

Materials and methods: Eleven participants with NAFL and ten control participants participated in a randomized cross-over trial, restricting food intake to either a 14.5- or 8-hour time period, thereby creating an overnight period of 9.5-hours of fasting or 16-hours of fasting. Hepatic glycogen was measured with 13C-MRS after a standardized lunch at 2 pm and the next morning at 6.30 am in both arms. Nocturnal substrate oxidation was measured with whole-room indirect calorimetry (respiration chamber) and a meal test was performed in the morning after the overnight fast to assess the metabolic response to a meal using indirect calorimetry (ventilated hood) and assessment of plasma metabolites from blood draws.

Results: Hepatic glycogen levels were not affected by extending overnight fasting time in the NAFL or control group. Extending fasting time led to a lower nocturnal carbohydrate oxidation and higher fat oxidation in both groups (intervention * time, p < 0.005; for carbohydrate and fat oxidation). However, in both arms, the respiratory exchange ratio measured during the night remained higher in the NAFL group compared with the control group (population p < 0.001, figure 1). During the meal test, the AUC of triglycerides appeared higher with a 16-hour versus 9.5-hour fast in both the NAFL and control group (intervention, p = 0.014), but no other postprandial differences between short and extended fasting were observed.

Conclusion: These results suggest that a prolonged overnight fast can improve nocturnal substrate oxidation, and that these improvements are not mediated by changes in hepatic glycogen depletion.

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Clinical Trial Registration Number: NCT03593343 and NCT04510155

Supported by: PPP Allowance by top Sector Life Sciences & Health and by Unilever R&D Wageningen

Disclosure: K.H.M. Roumans: None.

116

Therapeutic benefits of 17a-estradiol in hepatic fibrosis

S. Ali Mondal, R. Sathiaseelan, B. Miller, M. Stout;

Aging & Metabolism, Oklahoma Medical Research Foundation, Oklahoma City, USA.

Background and aims: Diabetes is an independent risk factors for the development of non-alcoholic steatohepatitis (NASH), the leading cause of liver transplant in the world. Moreover, liver fibrosis is the best determinant of mortality in NASH, therefore the reversal of liver fibrosis is a major focus in the field. There is currently no approved therapy for treating liver fibrosis. Men and postmenopausal women are at a higher risk of developing liver fibrosis compared to premenopausal women, thereby indicating a role of endogenous estrogens in controlling the progression of liver fibrosis. In this study, we sought to determine if 17α-E2 can prevent and/or reverse collagen deposition and/or increase collagen degradation in a CCl4-induced liver fibrosis mice model.

Materials and methods: 12-14 weeks old C57BL/6Jmale mice were used and randomized into five groups: (a) Vehicle group (n=15): mice were injected with olive oil intraperitoneally(i.p.) twice weekly for 8 weeks. (b) CCl4 group (n=15): mice were injected with 40% CCl4 (at a dose of 1ul/g bodymass)i.p. weekly twice for 8 weeks. Mice were maintained on Chow TestDiet 58YP (c) CCl4-17α-E2-Preventive group: Chow+17α-E2 (TestDiet 58YP + 17α-E2,14.4ppm) treatment started concomitantly with 40% CCl4 injection (1ul/g bodymass) i.p., weekly twice for 8 weeks. (d) CCl4-17α-E2-Therapeutic group: Chow+17α-E2 (TestDiet 58YP + 17α-E2,14.4ppm) treatment started on day 29 (after week 4) of CCl4(1ul/g bodymass, i.p.) injections to find out the effect of 17α-E2 in treating already developed fibrotic liver. (e) A short-term liver injury group (CCl4 -Baseline; n=18) was introduced using 40% CCl4 injected (i.p.), weekly twice at a dose of 1μl/g bodymass for 4 weeks to check the baseline development of liver fibrosis. The preventive and therapeutic effects of 17α-E2 treatment on collagen turnover rates were evaluated using stable isotope (D2O) labeling techniques.

Results: Compared with control mice, mice receiving CCl4 displayed a robust upregulation of hepatic collagen synthesis rates (P<0.0001)and declines in collagen degradation(P<0.0001) in parallel with significant elevations in TGFꞵ1(P<0.0001) and lysyl oxidase like-2 protein (LOXL2)(P<0.0001), which are responsible for hepatic stellate cell activation and collagen crosslinking, respectively. Conversely, mice receiving 17α-E2 demonstrated significantly reduced collagen synthesis rates(P<0.0001) and greater collagen degradation rates(P<0.001), which was mirrored by declines in TGFꞵ1(P<0.05) and LOXL2 protein(P<0.05), especially in the therapeutic group. These improvements were associated with increased matrix metalloproteinases-2 activity(P<0.05) and elevated levels of PPARγ(P<0.05) in both the preventive and therapeutic groups, which are established mechanisms related to the regression of liver fibrosis.

Conclusion: These findings indicate that 17α-E2 acts in a multimodal fashion to reduce fibrotic burden in the liver. Future studies will be needed to determine the cell-type-specific mechanisms by which 17α-E2 affects collagen deposition and degradation in the liver.

Supported by: NIH [R00 AG051661; R01 AG070035]

Disclosure: S. Ali Mondal: None.

117

The effect of a 16-week diet intervention with or without differing amounts of exercise volume on hepatic fat content in people with type 2 diabetes

C. Durrer1, M. Lyngbæk1, B. Liebetrau1, G.E. Legård1, T.P. Almdal2, M.A.V. Lund3, M. Ried-Larsen1;

1Centre for Physical Activity Research, Rigshospitalet, 2Hormone and Metabolic Diseases, Rigshospitalet, 3Department of Biomedical Sciences, Copenhagen University, Copenhagen, Denmark.

Background and aims: Diet and exercise with the goal of weight loss continue to be the first line treatment for non-alcoholic fatty liver disease (NAFLD) and represent a means to treat the disease which otherwise has no approved pharmacological treatment. Exercise, even independent of weight loss, can relieve hepatic insulin resistance and decrease fat liver content but its effectiveness in addition to dietary changes is unclear. The aim of this project was to test the hypothesis that an energy-reduced diet, with or without concomitant exercise training (at two different levels of exercise volume) in people with type 2 diabetes (T2D) will reduce liver fat in a dose-dependent manner.

Materials and methods: The study was a secondary analysis of a 16-week parallel group, 4-arm assessor blinded randomized controlled trial. People with T2D aged 18-80 years, diabetes duration <7 years, absence of severe comorbidities were eligible for the study. Participants were randomly allocated (1:1:1:1, stratified by sex) to either 1) No intervention control (CON), 2) Dietary intervention (DIET), 3) DIET + moderate volume exercise (MVE), or 4) DIET + high volume exercise (HVE). Medications were managed using a pre-defined algorithm by a study endocrinologist blinded to participant allocation. Hepatic fat content was quantified via magnetic resonance imaging T1-minimized signal fat fraction. Briefly, the liver was segmented based on vascular anatomy and the fat fraction was assessed in each segment. The values were then averaged to give a mean fat fraction for the entire liver. Statistical analysis was performed using a constrained baseline approach via a mixed effects linear model. Data were log-transformed to meet model assumptions and estimated marginal means were back-transformed to their original scale. Mean differences are presented as ratio (percentage) differences with associated 95% confidence intervals.

Results: Following the 16-week intervention there were reductions in liver fat by -53% [CI95: -75 to -10]; P=0.022 in the DIET group, -72% [CI95: -85 to -47]; P<0.001 in the MVE group, and -83% [CI95: -91 to -68]; P<0.001 in the HVE group compared to CON. There were no significant differences in liver fat reduction between the DIET and MVE group or the MVE and HVE group, but there was a larger reduction in liver fat in the HVE group compared to the DIET group (-63% [CI95: -79 to -35]; P=0.001). Marginal means and associated 95% confidence intervals at follow-up are presented in Figure 1, the dashed red line represents the constrained baseline.

Conclusion: A 16-week intervention with a 25% caloric deficit is able to reduce liver fat in people with T2D. There is an additional reduction in liver fat only when a high-volume of exercise training is added to the diet intervention.

figure ak

Clinical Trial Registration Number: NCT03769883

Supported by: TrygFonden, Svend Andersen fonden, CIHR

Disclosure: C. Durrer: None.

118

Ezetimibe combination therapy with statin for non-alcoholic fatty liver disease: a randomised controlled trial (ESSENTIAL study)

Y. Kim;

Department of Internal Medicine, Yonsei University College of Medicine, Severance Hospital, Seoul, Republic of Korea.

Background and aims: The effect of ezetimibe, Niemann-Pick C1-Like 1 inhibitor, on liver fat is not clearly elucidated. Our primary objective was to evaluate the efficacy of ezetimibe plus rosuvastatin versus rosuvastatin monotherapy to reduce liver fat using magnetic resonance imaging-derived proton density-fat fraction (MRI-PDFF) in patients with nonalcoholic fatty liver disease (NAFLD).

Materials and methods: A randomized controlled, open-label trial of 70 participants with NAFLD confirmed by ultrasound were assigned to receive either ezetimibe 10 mg plus rosuvastatin 5 mg daily or rosuvastatin 5 mg for up to 24 weeks. Liver fat change was measured as average values in each of nine liver segments by MRI-PDFF. Magnetic resonance elastography (MRE) was used to measure liver fibrosis change.

Results: Combination therapy significantly reduced liver fat compared with monotherapy by MRI-PDFF (mean difference: 3.2%; p=0.020). There were significant reductions from baseline to study completion by MRI-PDFF for both the combination and monotherapy groups, respectively (18.1% to 12.3%; p<0.001 and 15.0% to 12.4%; p=0.003). Individuals with higher body mass index, type 2 diabetes, insulin resistance, and severe liver fibrosis were likely to be good responders to treatment with ezetimibe. MRE-derived change in liver fibrosis was not significantly different (both groups, p>0.05). CAP (controlled attenuation parameter) by transient elastography was significantly reduced in the combination group (321dB/m to 287 dB/m; p=0.018), but not in the monotherapy group (323 dB/m to 311 dB/m; p=0.104).

Conclusion: Ezetimibe and rosuvastatin were found to be safe to treat participants with NAFLD. Further, ezetimibe combined with rosuvastatin significantly reduced liver fat in this population.

Clinical Trial Registration Number: NCT03434613

Supported by: Yuhan Corporation

Disclosure: Y. Kim: None.

119

Time-restricted feeding and obeticholic acid/semaglutide drug combination have a different impact on non-alcoholic steatohepatitis in diet-induced obese mice

F. Briand, N. Breyner, E. Grasset, T. Sulpice;

PHYSIOGENEX, Escalquens, France.

Background and aims: Combination of therapies is a promising strategy for the treatment of NASH and liver fibrosis. Here we evaluated whether the Farnesoid X Receptor agonist obeticholic acid (OCA) combined with the glucagon-like peptide-1 (GLP-1) receptor agonist semaglutide (SEMA) would show superior effects than weight loss induction with time-restricted feeding (TRF) in mice.

Materials and methods: Mice were fed a 60% high fat/2% cholesterol diet with 10% fructose supplemented drinking water (HFCF diet) for 25 weeks to induce obesity and NASH/liver fibrosis. After the diet-induction period, mice were kept on HFCF diet and treated with vehicle (control) or OCA 30mg/kg p.o. QD + SEMA 0.06mg/kg s.c. QD, or placed on TRF from the last 3 hours of the dark cycle till the end of the light cycle, without access to food, but free access to normal drinking water (i.e. without fructose) every day, for 6 weeks.

Results: OCA+SEMA induced a 20% lower caloric intake, which led to a 27% lower body weight (p<0.001 vs. control). TRF had a weaker effect on caloric intake (-9%) but also reduced body weight (-11%, p<0.05 vs. control). Both OCA+SEMA and TRF reduced the HOMA-IR index of insulin resistance and significantly reduced plasma ALT and AST levels, but these effects were more pronounced with OCA+SEMA. TRF, and to a greater extent OCA+SEMA, significantly reduced liver weight, hepatic fatty acids, triglycerides, and total cholesterol levels. Both OCA+SEMA and TRF led to significantly lower NAFLD activity score. However, OCA+SEMA did not alter liver fibrosis, while TRF showed a clear anti-fibrotic effect in the liver, with lower % Sirius Red labelling (p<0.05 vs. control).

Conclusion: OCA+SEMA combination reduced body weight and NAFLD activity score but did not improve hepatic fibrosis, while TRF improved both NASH and liver fibrosis. These TRF benefits should be further investigated in obese NASH patients.

Disclosure: F. Briand: Employment/Consultancy; PHYSIOGENEX. Stock/Shareholding; PHYSIOGENEX.

120

Cellular resistance to reactive metabolites is reversely affected by acute glucose stress and caloric restriction in type 2 diabetes patients with complications

A. Sulaj1,2, E. von Rauchhaupt1,2, C. Rodemer1, R. Bulkescher1, E. Kliemank1, S. Kopf1,2, P.P. Nawroth1,2, J. Szendroedi1,2, J. Zemva1;

1University Hospital of Heidelberg, Heidelberg, 2German Center for Diabetes Research, Neuherberg, Germany.

Background and aims: Reactive oxygen (ROS) and reactive dicarbonyl species (RCS) such as methylglyoxal (MG), are reactive metabolites (RM) known to contribute to the progression of diabetic complications. Depending on the dose, RM can also act protective by inducing hormetic reactions rendering cells more resistant to potentially harmful RM. This study investigated the cellular resistance to ROS and RCS in patients with type 2 diabetes with (T2D+) and without (T2D-) diabetic complications compared to healthy subjects (Ctrl) under acute glucose stress as well as under caloric restriction, to test whether these conditions have an impact on hormesis.

Materials and methods: An oral glucose tolerance test (oGTT) was performed before and after 5 days of fasting-mimicking diet (FMD) in T2D+ and T2D- patients and in healthy subjects (n=10: 6 males, 4 females per group). Study participants were matched for age, BMI, diabetes duration, HbA1c and antihyperglycemic medication. Peripheral blood mononuclear cells (PBMCs) were isolated before and 2h after the oGTT. PBMCs were immediately plated on a 96-well plate. To test resistance to ROS and RCS, cells were incubated with increasing levels of H2O2 and MG respectively for 24hrs. Afterwards, cell viability was measured with a CellTiter-Glo assay to calculate the corresponding EC50-value. Differences in EC50-value before and after oGTT were expressed as percentage changes (ΔEC50).

Results: Age and BMI were as expected comparable between study groups (T2D+: 66.3 [61.6, 71.0] yrs, 29.0 [26.5, 31.5] kg/m2; T2D-: 63.8 [60.2, 67.4] yrs, 27.9 kg/m2 [25.7, 30.2]; Ctrl: 63.8 [58.6, 69.0] yrs, 28.8 [25.5, 32.2] kg/m2. HbA1c was comparable between T2D+ and T2D- patients, but significantly higher in T2D+ (7.4 [6.9, 7.9] %, p < 0.0001) and T2D- (7.0 [6.2, 7.7] %, p < 0.001) compared to Ctrl (5.5 [5.1, 5.8] %). ΔEC50 of MG increased in healthy controls before (+12.9 %) and after (+12.6 %) FMD, whereas ΔEC50 of MG was unaffected in T2D- (before FMD: +1.8 %; after FMD: -1.7 %). In T2D+ ΔEC50 of MG significantly decreased after FMD (before FMD: -3.9 %; after FMD: -19.0 %, p = 0.005) and was reduced compared to T2D- (-1.7 % vs. -19.0 %, p = 0.027) and healthy controls (+12.6 % vs. -19.0 %, p = 0.035). ΔEC50 of H2O2 showed the same trend in all groups as for MG, but did not reach significance. However, EC50 of H2O2 before oGTT was significantly higher in T2D+ as compared to T2D- (before FMD: 134.3 μM vs. 91.8 μM, p = 0.028; after FMD: 134.0 μM vs. 98.6 μM, p= 0.032) and Ctrl (before FMD: 134.3 μM vs. 116.5 μM, p = 0.030; after FMD: 134.0 μM vs. 125.8 μM, p = 0.038). These results could mainly be attributed to higher EC50 values in female study participants.

Conclusion: Acute glucose stress induces cellular resistance to ROS and RCS in a hormetic manner in healthy subjects, a response which in diabetic patients seems to be lost during progression into diabetic complications. This response could not be restored after 5 days of caloric restriction. Furthermore, our data suggest for different capacities in handling of RM between female and male diabetic patients.

Supported by: German Research Foundation DFG (SFB 1118), German Center for Diabetes Research DZD (82DZD07C2G)

Disclosure: A. Sulaj: None.

OP 21 Retinopathy future vision

121

An early window of opportunity: risk factors for diabetic retinopathy are associated with early retinal neurodegenerative changes: The Maastricht Study

F. van der Heide1, S. Mokhtar1, R. Henry1, A. Kroon1, P. Dagnelie1, T. Berendschot1, S. Eussen1, J. Schouten2, M. Schram1, C. van der Kallen1, M. van Greevenbroek1, H. Savelberg1, N. Schaper1, C. Webers1, C. Stehouwer1;

1University of Maastricht, Maastricht, 2Canisius ziekenhuis, Nijmegen, Netherlands.

Background and aims: If determinants of retinal neurodegeneration, which precedes diabetic retinopathy, can be identified, there may already be an opportunity for the early prevention of diabetic retinopathy in individuals who are at risk for type 2 diabetes (e.g. individuals with prediabetes or obesity). We investigated the associations of risk factors for diabetic retinopathy with retinal neurodegenerative changes, using population-based data from an observational cohort study.

Materials and methods: We used cross-sectional data from The Maastricht Study (up to 5,666 participants, 50.5% men, mean ± SD age 59.7±8.7 years, and 22.6% with type 2 diabetes [the latter oversampled by design]). We investigated the associations of risk factors for diabetic retinopathy with retinal sensitivity, an index of retinal function, and retinal nerve fibre layer (RNFL) thickness, an index of retinal neural structure. We used linear regression analyses (results expressed as standardized betas) and adjusted for potential confounders (age, sex, educational level and key cardiovascular risk factors). We tested for interaction by sex and type 2 diabetes status.

Results: After full adjustment, greater HbA1c and lower healthy diet score (quantified with the Dutch healthy Diet index) were associated with lower retinal sensitivity (standardized betas [95%CI] -0.05 [-0.08; -0.02] and -0.06 [-0.09; -0.03], respectively) and lower RNFL thickness (-0.05 [-0.08; -0.02] and -0.03 [-0.06; -0.00]); high versus light alcohol consumption was associated with lower RNFL thickness (-0.08 [-0.16; -0.01],respectively), but not with retinal sensitivity (0.04 [-0.03; 0.10]); current versus never smoking was associated with lower retinal sensitivity (-0.14 [-0.22; -0.06]), but not with RNFL thickness (0.09 [-0.00; 0.18]); in individuals with, but not in individuals without, type 2 diabetes greater 24-hour ambulatory systolic blood pressure was associated with lower retinal sensitivity and lower RNFL thickness (in individuals with type 2 diabetes, -0.06 [-0.12; -0.04] and -0.06 [-0.13; 0.00], respectively); and, higher total cholesterol level was associated with greater retinal sensitivity (0.05 [0.02; 0.08]) and greater RNFL thickness (the latter only in individuals with type 2 diabetes; in individuals with type 2 diabetes, 0.09 [0.03; 0.16]). Sex did not modify any of the associations under study.

Conclusion: This population-based study found that most risk factors for diabetic retinopathy were independently associated with retinal neurodegenerative changes. Hence, early prevention of risk factors for diabetic retinopathy, already in individuals with prediabetes or obesity, may contribute to the prevention of diabetic retinopathy.

Supported by: ERDF, PL, DMEA, SW, PSID, CVCM, CARIM, CAPHRI, NUTRIM, SA, Perimed, DF, OF, HE, JC, NNF, SAN

Disclosure: F. van der Heide: None.

122

Transcriptome analysis reveals that retinal neuromodulation is the main underlying mechanism of the neuroprotective effect of sitagliptin in diabetic retina

H. Ramos, P. Bogdanov, J. Huerta, A. Deàs-Just, C. Hernández, R. Simó;

Diabetes and Metabolism Research Unit, Vall d'Hebron Research Institute (VHIR), Barcelona, Spain.

Background and aims: The neurovascular unit (NVU) is a functional coupling between neurons, glial cells and blood vessels that integrates vascular flow with metabolic activity. Its impairment is an early event in the pathogenesis of diabetic retinopathy (DR), which participates in the neurodegeneration and the early microvascular impairment of the diabetic retina. Consequently, NVU becomes an emergent therapeutic target of DR. The reduction of synaptic protein expression, the impairment of neurotransmission and alterations in neuronal morphology have been described as underlying mechanisms of NVU impairment. We previously reported that topical administration (eye drops) of sitagliptin, a dipeptidyl peptidase-4 inhibitor (DPP-4i), prevented retinal neurodegeneration induced by diabetes in db/db mice. To further explore the mechanisms involved in the beneficial effects of DPP-4i on diabetes-induced retinal neurodegeneration, we have compared by transcriptomic analysis the retinal expression patterns of mRNA in vehicle-treated db/db mice (an experimental model of DR) vs. db/db mice treated with sitagliptin.

Materials and methods: Ten db/db mice, aged 10 weeks, were topically treated with sitagliptin eye drops (5 μL/eye; concentration: 10 mg/mL) for 2 weeks twice per day, while other ten db/db received a topical administration of vehicle (5 μL/eye). Ten db/+ mice (non-diabetic mice) were assigned as control group. Before euthanasia, full-field electroretinogram recordings were used to address retinal functionality. At 12 weeks, after euthanasia, one eye was used for a transcriptome analysis and the other for its validation through RT-PCR and for protein assays through Western Blotting (WB) and Immunohistochemistry (IHC).Biological relevance of the transcriptome analysis was assessed through gen set enrichment analyses (GSEA) on two different annotation databases [Gene Ontology and Reactome Pathway Knowledge base].

Results: Diabetic mice topically treated with sitagliptin presented different expression patterns of mRNA in the retina in comparison to those treated with vehicle. GSEA revealed a positive enrichment, after sitagliptin treatment, of multiple candidates linked to synaptic transmission. Additionally, we observed in both, RT-PCR and WB/IHC assays, that presynaptic proteins involved in vesicle biogenesis, mobilization, docking, fusion and recycling, were down-regulated in db/db mice retinas in comparison with non-diabetic controls. Topical administration of sitagliptin inhibits this diabetes-induced down-regulation and improves the functionality of diabetic retinas without any effect on blood glucose levels.

Conclusion: Sitagliptin exerts neuroprotective effects in db/db mice retinas by inhibiting the down-regulation of key presynaptic proteins induced by diabetes. Notably, this effect is unrelated to the improvement of metabolic control. This finding opens up a new strategy for treating not only DR but also other retinal diseases in which synaptic abnormalities/neurodegeneration play a crucial role.

Supported by: UEB-VHIR, UAT-VHIR, VHIR, MICINN, CIBERDEM, UAB

Disclosure: H. Ramos: None.

123

Presence of retinopathy and kidney and cardiovascular events in type 2 diabetes and normoalbuminria: a post-hoc analysis of The PRIORITY study

T. Hansen1, V. Rotbain Curovic1, N. Tofte1, M.K. Lindhardt1,2, C. Delles3, M. Frimodt-Møller1, H. Mischak4, F. Persson1, H. von der Leyen5, P. Rossing1, on the behalf of the PRIORITY Study Group;

1Steno Diabetes Center Copenhagen, Herlev, Denmark, 2Department of Medicine, Holbæk Hospital, Holbæk, Denmark, 3Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK, 4Mosaiques Diagnostics, Hannover, Germany, 5Hannover Clinical Trial Center, Hannover Medical School, Hannover, Germany.

Background and aims: To evaluate the association between diabetic retinopathy and development of albuminuria, impaired kidney function and cardiovascular events in persons with type 2 diabetes and normoalbuminuria.

Materials and methods: Post-hoc analysis of the prospective observational PRIORITY study including 1756 persons with type 2 diabetes and normoalbuminuria followed for three years. The study was originally designed to investigate the prediction of a urinary proteomic risk classifier (CKD273) for development of albuminuria. Diabetic retinopathy included information from medical records on non-proliferative and proliferative changes, presence of macular oedema and history of laser treatment. Cox proportional hazard models were fitted to investigate baseline retinopathy status to development of 1) microalbuminuria (urinary albumin-creatinine ratio >30mg/g on ≥ 2 out of 3 urine samples); 2) chronic kidney disease (eGFR <60 ml/min/1.73m2); and 3) cardiovascular events (myocardial infarction, stroke, coronary intervention, and hospitalization for heart failure). Adjustment included sex, baseline age, diabetes duration, HbA1c, systolic blood pressure, eGFR, urinary albumin-creatinine rate and urinary proteomic risk classifier status. Baseline LDL cholesterol, body mass index and history of cardiovascular disease were also included in the adjustment for cardiovascular events.

Results: At baseline, 287 (16.3%) had retinopathy. Compared to persons without retinopathy, they were older (mean ±SD: 62.7±7.7 vs 61.4±8.3 years, p=0.019), had longer diabetes duration (17.9±8.4 vs. 10.6±7.0 years, p<0.001) and higher HbA1c (62±13 vs. 56±12 mmol/mol, p<0.001). The adjusted hazard ratios of retinopathy at baseline for development of albuminuria (n=197), chronic kidney disease (n=166) and cardiovascular events (n=64) were: 1.54 (95%CI: 1.06, 1.73), 0.89 (95%CI: 0.57, 1.38), and 2.56 (95%CI: 1.40, 4.66), compared to persons without retinopathy.

Conclusion: Individuals with normoalbuminuric type 2 diabetes and retinopathy had higher risk of developing albuminuria, but not impaired kidney function, and had a markedly higher risk of cardiovascular disease during the 3-year follow-up, compared to individuals without retinopathy.

Clinical Trial Registration Number: NCT02040441

Supported by: European Union Seventh Framework Programme (FP7/20072-013)

Disclosure: T. Hansen: None.

124

Stroke incidence increases with severity of diabetic retinopathy and maculopathy in people with type 1 diabetes

M.I. Eriksson1,2, K. Hietala1,3, P. Summanen4,1, C. Forsblom1,5, J. Putaala6, A. Ylinen1,2, S. Hägg-Holmberg1,2, P.-H. Groop1,5, L.M. Thorn1,7, on behalf of the FinnDiane Study;

1Folkhälsan Institute of Genetics, Folkhälsan Research Center, Helsinki, 2Research Program in Clinical and Molecular Metabolism, University of Helsinki, Helsinki, 3Department of Ophthalmology, Central Finland Hospital, Jyväskylä, 4Department of Ophthalmology, University of Helsinki and Helsinki University Hospital, Helsinki, 5Department of Nephrology, University of Helsinki and Helsinki University Hospital, Helsinki, 6Department of Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, 7Department of General Practice and Primary Health Care, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background and aims: Although the retinal vessels are suggested to mirror the brain’s vasculature, there are limited data on this association in type 1 diabetes. We therefore aimed to study the link between severity of diabetic retinopathy (DR) and stroke in type 1 diabetes.

Materials and methods: We included 1,327 participants with type 1 diabetes from the Finnish Diabetic Nephropathy Study (mean age 38.9±10.9 years, men 51.9%, kidney disease 35.9%). Exclusion criteria were stroke before baseline or missing DR data at baseline. Baseline visits were conducted in 1994-2006. Strokes were identified from registers, until the end of 2017, and verified from medical files. DR was graded according to the Early Treatment Diabetic Retinopathy Study (ETDRS) scale. Data on macular oedema were available for 637 (48.0%) participants, and any clinically relevant macular oedema was considered as maculopathy.

Results: During a median of 17.7 (14.0-19.2) follow-up-years, 133 (10.4%) had a stroke (97 ischemic, 27 haemorrhagic, 9 unclassified). At baseline, 45.1% had no or mild DR (ETDRS 10-30), 16.0% moderate to severe DR (ETDRS 40-55), and 39.0% proliferative DR (ETDRS 60-80). Of the participants with available macular oedema data, 74.9% had maculopathy. Table 1 presents incidence rates and HR for stroke by severity of DR and maculopathy. In Cox regression analysis, adjusted for diabetes duration, sex, systolic and diastolic blood pressure, BMI, LDL cholesterol, triglycerides, and smoking, ETDRS 40-55 and ETDRS 60-80 were predictors of stroke (Table 1). Additionally, maculopathy was a predictor of stroke when analysed separately in a similar model (Table 1). After further adjustment for kidney disease, retinopathy severity (p=0.081 and p=0.171), and maculopathy (p=0.063) were no longer significant for any stroke. ETDRS 60-80 remained a significant predictor of haemorrhagic stroke (HR 10.2, 95% CI 1.2-86.2, p=0.033) in the final Cox model.

Conclusion: In our study, the incidence of stroke increased by the severity of DR and prevalence of maculopathy. Proliferative DR was a predictor of haemorrhagic stroke even in the presence of kidney disease.

figure al

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

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

125

Fenofibrate use and diabetic retinopathy progression in patients with type 2 diabetes: a propensity-matched cohort study

N. Kim, J. Kim, K. Kim, J. Bae, K. Kim, S. Kim;

Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea.

Background and aims: To determine the association between fenofibrate use and diabetic retinopathy progression in patients with type 2 diabetes (T2D) and metabolic syndrome undergoing statin therapy in a real-world database.

Materials and methods: In this propensity-matched cohort study, patients with T2D and metabolic syndrome (≥ 30 years) receiving statin therapy were matched 1:2 by propensity score into the statin plus fenofibrate group (n=23,692) and statin-only group (n=46,223). The primary outcome was a composite of diabetic retinopathy progression including vitreous hemorrhage, vitrectomy, laser photocoagulation, intravitreous injection therapy and retinal detachment.

Results: For the primary outcome, the incidence rate per 1,000 person year was 14.25 in the statin-only group and 12.65 in the statin plus fenofibrate group. The risk of the primary outcome was significantly lower (hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.83 to 0.96; p=0.001) in the statin plus fenofibrate group than in the statin-only group. Patients with retinopathy at baseline showed marked benefits of fenofibrate treatment (HR, 0.86; 95% CI, 0.78 to 0.94; p=0.001). In addition, the statin plus fenofibrate group exhibited significantly lower risks of vitreous hemorrhage (HR, 0.87; 95% CI, 0.78 to 0.96; p=0.008), laser photocoagulation (HR, 0.89; 95% CI, 0.81 to 0.98, p=0.022) and intravitreous injection therapy (HR, 0.78; 95% CI, 0.66 to 0.92; p=0.003) than those in the statin-only group. There was no significant interaction between the different characteristics at baseline and the treatment effect.

Conclusion: In this propensity-weighted cohort study, the addition of fenofibrate to statins was associated with significantly lower risk of diabetic retinopathy progression than statin therapy alone in patients with T2D and metabolic syndrome.

Supported by: Abbott Laboratories, Korea

Disclosure: N. Kim: None.

126

Blocking haemopexin with specific antibodies: a new experimental strategy for treating diabetic retinopathy

R. Simó1, P. Bogdanov1, A. Duarri2, A. Salas2, D. Sabater1, H. Ramos1, H. Isla2, C. Hernández1;

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

Background and aims: By means of a proteomic analysis we identified hemopexin (Hpx) as a genuine and abundant protein in the vitreous fluid of patients with diabetic macular edema (DME). In addition, we provided first evidence that Hpx is overexpressed in the retina of diabetic patients, and induces hyperpermeability due to the breakdown of the blood-retinal barrier (BRB), an essential pathogenic mechanism for the development of DME. The aims of the present study are: 1) To evaluate whether the inhibition of Hpx by specific antibodies (intravitreally administered) is able to arrest vascular leakage in two experimental models of diabetes 2) To explore in in vitro models whether Hpx antibodies are able to inhibit microvascular angiogenesis.

Materials and methods: In vivo studies: An intravitreal (IVT) injection of 1.5 μl of anti-Hpx antibody (1 mg/ml), (n=10) or saline (n=10) in each eye was administered to db/db mice at age of 12 weeks. Ten non-diabetic mice (db/+) matched by age served as a control group. Retinal vascular permeability was examined 5 days after intravitreal injection using the Evans Blue albumin method. HPX expression (mRNA) was determined by RT-PCR. The same protocol and assessments were performed using Long-Evans rats without diabetes (n=9) and with STZ-induced diabetes (n=9). In vitro studies: The microvascular angiogenesis was tested by using the scratch wound healing technique and sprouting assay, both stimulated by VEGF treatment (25 ng/mL). For the former we used human retinal endothelial cells (HRECs) and the inhibitory effect of anti-Hpx antibodies (0.36 mcg/mL) was tested. To examine sprouting we measured the area (mm2) of sprouted cells using choroidal explants from Long-Evans rats which were cultured under different conditions: 5.5 mM glucose, 30 mM glucose, 30 mM + bevacizumab (125 and 250 mcg/mL), 30 mM glucose + 0.1 microM dexamethasone, 30 mM glucose + anti-Hpx antibodies (0.36 mcg/mL). In addition, explants from Long-Evans rats with STZ-induced diabetes treated with IVT injection of anti-Hpx antibodies (1mg/ml) vs. treated with vehicle were compared.

Results: A higher expression of Hpx was detected in retina from diabetic animals in comparison with non-diabetic animals (p<0.05). IVT injections of anti-Hpx antibodies significantly reduced vascular leakage in the two diabetic models in comparison with vehicle (p<0.01). Treatment with Hpx antibodies was able to significantly reduce the HRECs migration induced by high glucose concentration with and without VEGF after scratch (p<0.01). In addition, Hpx neutralization inhibited the vessel outgrowth from choroidal explants in non-diabetic rats (60% of reduction of the sprouting area; p<0.01) and STZ-induced diabetic rats (75% reduction; p<0.01 of the sprouting area). Notably, this effect was similar to obtained with bevacizumab or dexamethasone.

Conclusion: The blockade of Hpx-induced permeability by using IVT injections of anti-Hpx antibodies can be envisaged as a new strategy for treating DME. The in vitro and ex-vivo antiangiogenic effect also suggest proliferative retinopathy as a potential target, but in vivo experiments are lacking. Further research is needed to confirm Hpx not only as a therapeutic target of advanced diabetic retinopathy but also to explore its role as an alternative to anti-VEGF agents.

Supported by: ISCiii (DTS19/00171)

Disclosure: R. Simó: None.

OP 22 Giving birth with diabetes

127

Glycaemic control and complication rate through pregnancy and thirteen-years post-partum in women with microalbuminuria (type 1 diabetes)

N. Asatiani, R. Kurashvili, E. Inashvili, E. Shelestova, T. Akhobadze;

Diabetes in Pregnancy, National Center for Diabetes Research, Tbilisi, Georgia.

Background and aims: The aim of the present work was to assess the degree of glycemic control and complications rate through pregnancy and thirteen-years post-partum in women with microalbuminuria (T1DM).

Materials and methods: In total 191 patients with T1DM were enrolled in the study. Based on albuminuria levels in the 1st trimester women were separated into 2 groups (Gr.). Gr.1 - 116 women with normoalbuminuria, Gr.2 - 75 women with microalbuminuria. Preconception care was performed in 46.5%- Gr.1 and 57.3% -Gr.2. Strict metabolic control was maintained and fetal surveillance was performed throughout the pregnancy. Repeated examinations were performed 13 years’ post-partum.

Results: At entry HbA1c(%) levels for Gr.1 and 2 were: 7.42 (0.15) and 7.25 (0.14); by the end of the pregnancies they statistically decreased in both groups (Gr.1- P=0.000, Gr.2 - P=0.000). At entry percent (%) of retinopathy for Gr.1- 8.6 and Gr.2 - 20.0; by term the percent has not increased. The percent of women with macroalbuminuria increased, together with the growth of gestational age. By term macroalbuminuria was observed in 2.5% (Gr.1) and in 14.6% (Gr.2) of patients (P=0.0094, OR-5.67). In Gr.1 percent of pre-eclampsia and preterm deliveries before 37 weeks of gestation was lower, than in Gr.2 (pre-eclampsia - P = 0.0064, OR -4.64; preterm deliveries P = 0.048; OR -2.8). Perinatal mortality was observed in Gr.1 - 0.8% and in Gr.2 - 6.6% of women (P - 0.006, OR - 7.73). Repeated examinations 13 years post-partum showed that HbA1c levels were statistically higher, than at the end of pregnancy: Gr.1-7.7 (0.41) (P=0.0002), Gr.2 - 8.05 (0.26) (P=0.000). Repeated examinations showed that percent of retinopathy, neuropathy, micro and macroalbuminuria, increased in both groups. Besides, patients from Gr.2 had statistically higher complications rate, than patients from Gr.1 (retinopathy - P=0.0002, OR - 3.45; microalbuminuria - P<0.0001, OR - 10.2; macroalbuminuria -P=0.0032, OR- 3.03). Chronic kidney disease were observed in 36 patents. Nine patients from Gr.2 are on regular hemodialysis, and in two patient kidney transplantation was performed.

Conclusion: If microalbuminuria was detected in the 1st trimester, the risk of preeclampsia increased 4.6 times, the risk of preterm delivery increased 2.8 times and risk of perinatal mortality increased 7.3 times, compared to patients with normoalbuminuria. In both groups glycemia control deterioration was observed thirteen years post-partum. The higher complication percent was found if pregnancy proceeded with microalbuminuria, the risk of retinopathy increased 3.4 times and the risk of CKD increased 3 times compared to patients with normoalbuminuria.

Disclosure: N. Asatiani: None.

128

Glucose response patterns based on 75g OGTTs during pregnancy and their association with the risk of macrosomia: a latent class analysis of three cohort studies

L. Fritsche1, A. Hulman2, K. Prystupa3, M. Heni4, S.L. White5, A.L. Birkenfeld6, A. Peter4, A. Fritsche3, A. Kun7, L. Poston5, A. Tabak8, R. Wagner9;

1Institute for Diabetes Research and Metabolic Diseases, Helmholtz Center Munich, Tuebingen, Germany, 2Steno Diabetes Center, Aarhus, Denmark, 3Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tuebingen, Tuebingen, Germany, 4Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicin, University Hospital Tuebingen, Tuebingen, Germany, 5Department of Women and Children’s Health, King's College London, London, UK, 6. Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tuebingen, Tuebingen, Germany, 7Department of Obstetrics & Gynaecology, Tolna County Balassa János Hospital, Szekszárd, Hungary, 8Department of Public Health, Faculty of Medicine, Semmelweis University, Budapest, Hungary, 9German Center for Diabetes Research, Neuherberg, Germany.

Background and aims: Studies in the general population show that different glucose response patterns based on oral glucose tolerance tests (OGTT) are associated with different clinical characteristics and long-term outcomes. We aimed to identify comparable glucose response patterns in pregnancy and their association with maternal and fetal outcomes.

Materials and methods: We used latent class trajectory modelling to identify glucose response patterns using a 5-point 75g OGTT in 470 pregnant women at 27.3±2.2 weeks of gestation. We assessed these classes and examined pregnancy outcomes in two independent European cohorts with 3-point OGTTs: a population based Hungarian cohort with 7073 pregnant women and the control group of the UK Pregnancies Better Eating and Activity Trial comprising of 610 obese women. Pregnancy outcomes in the different classes were analysed using generalized estimating equations (GEE).

Results: We identified five different glucose response patterns (classes, Fig.1). Rate of gestational diabetes (GDM) was lowest in class 1 (< 7%) and highest in class 5 (100%) for all three cohorts. Class 3 was characterized by transient hyperinsulinemia at 30 minutes (Fig.1) but the prevalence of GDM was only 25-36%. Compared to class 1, women in class 3 had higher gestational weight gain (GWG, β=0.76 SE: 0.28kg, p=0.0076, adjusted for study, age, gestational age, pre-gestational BMI, GDM treatment and AUCGlucose). New-borns in class 3 had the highest risk of macrosomia (OR 1.49 95%CI: [1.17, 1.90] vs. class 1) after adjustment for study, maternal age, parity and smoking, but this was attenuated by additional adjustment for pre-pregnancy BMI and GWG.

Conclusion: We found an easily identifiable group of pregnant women who have an increased risk of macrosomia often without formally meeting GDM criteria. These women would most likely benefit from a therapy (e.g. nutritional counselling) targeting excessive glucose excursions and GWG.

figure am

Clinical Trial Registration Number: NCT04270578, ISRCTN89971375

Supported by: BMBF (01GI0925) to DZD

Disclosure: L. Fritsche: None.

129

Changes in body fat partitioning and insulin resistance between preconception and postpartum in Singaporean women: the SPRESTO study

S. Sadananthan1, N. Michael1, Y. Manuel1,2, K. Thirumurugan1, J. Yaligar1, M. Tint1,2, K. Tan3, K.M. Godfrey4, P.D. Gluckman1, Y. Chong1,2, Y. Lee1,2, J.K.Y. Chan3, S.-Y. Chan1,2, J.G. Eriksson1,2, S. Velan1;

1Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*STAR), Singapore, Singapore, 2National University of Singapore, Singapore, Singapore, 3KK Women’s and Children’s Hospital, Singapore, Singapore, 4MRC Lifecourse Epidemiology Unit & NIHR Southampton Biomedical Research Centre, University of Southampton & University Hospital Southampton NHS Foundation Trust, Southampton, UK.

Background and aims: Pregnancy is a period when the body experiences dynamic changes in body composition, hormone levels, insulin resistance, and a build-up of energy reserves in the form of fat to support lactation. While postpartum weight retention has been linked to increased metabolic risk, the role of changes in body fat partitioning has not been well investigated. In the Singapore Preconception Study of Long-Term Maternal and Child Outcomes (S-PRESTO), we characterized the changes in body weight, body fat partitioning, and insulin resistance between preconception and 3 months postpartum.

Materials and methods: 1039 women (aged 18-45 years, Chinese, Malay, and Indians) who intended to get pregnant within the next 12 months were recruited; those who conceived were followed through their pregnancy and at 3 months postpartum. 68 participants underwent MRI at both preconception and postpartum time points; volumetric abdominal MRI was used for the segmentation and quantification of deep subcutaneous (DSAT), superficial subcutaneous (SSAT), and visceral adipose tissue (VAT) volumes. Ectopic fat accumulation within the liver and muscle (intramyocellular lipids (IMCL)) was determined using magnetic resonance spectroscopy. Insulin resistance was assessed using homeostatic model assessment for insulin resistance 2 (HOMA2-IR) preconception, at week 24-28 of gestation, and at 3 months postpartum.

Results: As expected, both body weight and HOMA2-IR significantly increased during pregnancy. At 3 months postpartum, HOMA2-IR returned to preconception levels, while body weight was higher than preconception (Table 1); subcutaneous fat depots (DSAT and SSAT) and IMCL at postpartum were similar to the preconception levels, but VAT (p = 0.001) and liver fat (p = 0.027) were increased 3 months after delivery. There were no associations between the changes in fat depots and the change in HOMA2-IR.

Conclusion: Our findings indicate that subcutaneous fat depots may be preferentially mobilized in the early postpartum period, relative to VAT and ectopic fat in the liver. While elevated VAT and liver fat have been generally linked to elevated insulin resistance, increases in both these depots were not accompanied by an increase in HOMA2-IR in the early postpartum period. Further assessments are required to evaluate whether the dissociation between these pathogenic fat depots and HOMA2-IR is a transient phenomenon.

figure an

Clinical Trial Registration Number: NCT03531658

Supported by: NMRC/TCR/004-NUS/2008; NMRC/TCR/012-NUHS/2014

Disclosure: S. Sadananthan: Grants; National Medical Research Council, Singapore National Research Foundation (NMRC/TCR/004-NUS/2008; NMRC/TCR/012-NUHS/2014), Singapore Institute for Clinical Sciences.

130

Maternal diabetes and attention-deficit/hyperactivity disorder in childhood

C.E. Cesta1, K.K.C. Man2,3, L.J. Kjerpeseth4, L. Gao2, Nordic Pregnancy Drug Safety Studies (NorPreSS) consortium, A.Y.L. Chan2,5, M.H.C. Hsieh6, E.C.C. Lai6, H. Zoega7,8, I.C.K. Wong3,2;

1Karolinska Institutet, Stockholm, Sweden, 2The University of Hong Kong, Hong Kong, Hong Kong, 3UCL School of Pharmacy, London, UK, 4Norwegian Institute of Public Health, Oslo, Norway, 5University of Groningen, Groningen, Netherlands, 6National Cheng Kung University, Tainan, Taiwan, 7University of Iceland, Reykjavik, Iceland, 8UNSW Sydney, Sydney, Australia.

Background and aims: Recent studies suggest an increased risk of attention-deficit/hyperactivity disorder (ADHD) in the children born to mothers with diabetes mellitus during pregnancy, including pregestational diabetes (PGDM) and gestational diabetes mellitus (GDM). However, current evidence remains inconclusive. This study aims to assess the association between prenatal exposure to maternal diabetes mellitus (MDM) and the risk of ADHD in childhood.

Materials and methods: This is a multinational cohort study with linked mother-child pairs using healthcare databases from Asia (Hong Kong (HK), Taiwan) and Northern Europe (NorPreSS: Finland, Iceland, Norway, Sweden) including children born between 2001-2018 with follow-up through 2020 (subject to data availability). Cox proportional hazard regression models and propensity score fine stratification, including demographic, comorbidity, and comedication covariates, were used to calculate hazard ratios (HR) with a 95% confidence interval (CI) for each comparison.

Results: We included 4,554,325 million mother-child pairs (HK: 535,924; Taiwan: 887,120; NorPreSS: 3,131,281) in the analyses, of which 6.1% of children were prenatally exposed to MDM (71,232 to PGDM; 207,171 to GDM). A total of 158,154 children had ADHD (HK: 16,453; Taiwan: 85,471; NorPreSS: 56,230). Children born to mothers with MDM were at a higher risk of developing ADHD (HK: PS-weighted HR 1.18, 95%CI 1.11-1.26; Taiwan: 1.13, 1.09-1.18; NorPreSS: 1.22; 1.18-1.26). The elevated risk was present separately for PGDM (HK=1.20, 0.98-1.46; Taiwan=1.63, 1.45-1.83; NorPreSS=1.31, 1.24-1.38) and for GDM (HK=1.18, 1.10-1.25; Taiwan=1.11, 1.07-1.15; NorPreSS=1.17, 1.12-1.22). Study sized allowed for a sibling-matched GDM analysis, which showed no association between prenatal exposure to GDM and the risk of ADHD (HK: 1.00, 0.86-1.16; Taiwan: 0.96, 0.85-1.07).

Conclusion: Our findings suggest that children prenatally exposed to maternal diabetes in general, and specifically to PGDM and GDM, have an increased risk of ADHD in childhood. However, for at least GDM exposure the risk is largely due to unmeasured familial confounding. Further investigation will assess the role of pharmacological treatment of MDM during pregnancy and glycaemic control on the association between maternal diabetes and child ADHD.

Supported by: Hong Kong Research Council (no.1711202), NordForsk (no.83539), MSCA (no.844728)

Disclosure: C.E. Cesta: Grants; EU Horizon 2020 MSCA global fellowship (no. 844728).

131

Pregestational diabetes and risk of congenital heart defects in the offspring: French nationwide study using the French PMSI-MCO database

M. Lemaitre, G. Bourdon, A. Bruandet, X. Lenne, D. Subtil, T. Rakza, A. Vambergue;

CHRU Lille, Lille, France.

Background and aims: Congenital Heart defects (CHD) are the most common type of congenital malformations in offspring of women with pregestational diabetes. Our aim was first to estimate the incidence of CHDs in the offspring of mothers with type 1 diabetes and those with type 2 diabetes using data from the national French Medical Information System Program in Medicine, Surgery and Obstetrics database (PMSI-MCO), compared to the general population. Secondly, we investigated if the association between maternal diabetes and CHD varied with type of diabetes (Type 1 diabetes (T1D) or type 2 diabetes (T2D) in a large national cohort in France.

Materials and methods: The presence of CHDs and maternal diabetes were screened for according to the International Classification of Diseases, 10th [Revision], in the PMSI-MCO database, from 2012 to 2020. A logistic model was used to estimate risk factors for maternal-fetal prognostic indicators in women with T1D, in women with T2D, and in the control population by adjusting for maternal age, gender of the newborn, prematurity, Small for gestational Age (SGA), Large for gestational Age (LGA) and, mode of delivery.

Results: 6,076,251 mother-infant pairs were included. The rate of congenital malformations was 6.2% in the control group, 8.0% in women with T1D and 8.4% in women with T2D (p<0.001). The incidence of CHD was 8.0 per 1000 births in the control group, 29.6/1000 and 27.4/1000 in women with T1D and those with T2D, respectively (p<0.001). The risk of CHD was 2.07 times higher in women with type 1 diabetes [CI 95% [1.91-2.24], p<0.001] and 2.20 times higher in women with type 2 diabetes [CI 95% [1.99-2.44]], p<0.001] with no difference found between T1D and T2D (p=0.336). Cesarean section, SGA, LGA, and prematurity were also associated with an excess risk of CHD.

Conclusion: Pregestational diabetes is a risk factor for the development of CHD in the offspring without a significant difference between the two types of diabetes. Metabolic control is a modifiable risk factor that can be addressed to reduce the risk of CHD.

Disclosure: M. Lemaitre: None.

132

Beta cell function, hepatic insulin clearance and insulin sensitivity in South Asian and Nordic women after gestational diabetes

A. Sharma1,2, I. Nermoen1,2, E. Qvigstad3,2, C. Sommer2, N. Sattar4, J. Gill4, H. Gulseth5, S. Sollid6, K. Birkeland2,3, S. Lee-Ødegård2,3;

1Ahus, Lorenskog, Norway, 2University of Oslo, Oslo, Norway, 3Oslo University Hospital, Oslo, Norway, 4University of Glasgow, Glasgow, UK, 5Norwegian Institute of Public Health, Oslo, Norway, 6Vestre Viken Drammen Hospital, Drammen, Norway.

Background and aims: The risk of developing type 2 diabetes (T2D) after gestational diabetes mellitus (GDM) is twice as high in South Asian compared to Nordic women. We aimed to assess and compare ethnic differences in β-cell function and insulin sensitivity (IS) in women with normal glucose tolerance (NGT), prediabetes and T2D, 1-3 years after a GDM pregnancy.

Materials and methods: We performed an OGTT in South Asian (n=179) and Nordic (n=108) women living in Norway. Based on the OGTT, we calculated the pre-hepatic insulin secretion rate (ISR) by deconvolution of C-peptide kinetics, and hepatic insulin clearance (HIC) as the ratio of ISR to peripheral insulin levels. First phase insulin secretion was assessed by the insulinogenic index (IGI). Fasting insulin secretion was represented by the homeostasis model assessment (HOMA)2-β. β-cell glucose sensitivity (βC-GS) was approximated from cross-correlation of ascending glucose and ISR levels during the OGTT. Hepatic insulin sensitivity (IS) was estimated by HOMA2-S, peripheral IS by the muscle IS index (muscle-ISI) and whole-body IS by the Matsuda index (Matsuda-ISI). β-cell function adjusted for insulin resistance was estimated by the disposition index (DI); estimated by IGI and either HOMA2-IR (DI-HOMA2-IR) or Matsuda-ISI (DI-Matsuda).

Results: Compared to Nordic women, South Asians had higher levels of glucose, insulin, C-peptide and ISR during the OGTT. HOMA2-β and βC-GS (p<0.05) were higher in South Asian vs. Nordic women. HIC was lower, and so were HOMA2-S, muscle-ISI, Matsuda-ISI and both DI-HOMA2-IR and DI-Matsuda. In NGT women, our results were similar, expect for a higher IGI, but similar DI-HOMA2-IR and DI-Matsuda between South Asian and Nordic women. In women with prediabetes or T2D, both ethnicities displayed similar OGTT glucose levels, but South Asian exhibited higher OGTT insulin and ISR levels than Nordic women (Table 1).

Conclusion: In women examined 1-3 years after a GDM pregnancy, South Asians displayed a ‘stressed’ β-cell function compared to Nordic women. South Asians also displayed lower IS and HIC across all glucose tolerance categories. In spite of NGT, South Asian women exhibited higher fasting and first-phase insulin secretion. Hence, a more ‘stressed’ β-cell function in addition to lower IS and HIC in South Asian women may increase their propensity to develop T2D after GDM.

figure ao

Supported by: The Research Council of Norway, grant number 273252.

Disclosure: A. Sharma: None.

OP 23 Are we too slow to outlaw the low?

133

Severe hypoglycaemia presenting to a hospital emergency department: clinical characteristics and mortality outcomes

S.H. Song1, B.M. Frier2;

1Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, 2The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK.

Background and aims: The prevalence of young-onset type 2 diabetes (YOT2D), diagnosed below age 40, is increasing with many requiring insulin. Severe hypoglycaemia (SH) is common in insulin-treated diabetes and may be a marker of underlying illness. The frequency of SH in YOT2D was ascertained in all people requiring treatment in a hospital emergency department (ED), and clinical characteristics and mortality outcomes were examined.

Materials and methods: Patients with SH who attended ED at Northern General Hospital, Sheffield, UK between January 2019 and March 2022 were identified. Complete collection of SH events was ensured by interrogating several sources: ambulance service, hospital ED, inpatient and general practice records. Data included diabetes type, clinical characteristics and comorbidities, and were analysed by age of diabetes onset, below and above age 40. Charlson score quantified the comorbidity burden.

Results: A total of 537 episodes of SH occurred in 442 patients, of whom 155 (35.1%) had type 1 diabetes (T1D), 184 (41.6%) had type 2 diabetes (T2D), 7 (1.6%) had insulin-treated secondary pancreatic diabetes and 96 (21.7%) were non-diabetic, with mean blood glucose concentrations at presentation of 1.8±0.7, 2.0±0.7, 1.8±0.8 and 2.0±0.7 mmol/L respectively. SH events recurred in 61 (13.8%) patients (2-10 ED visits per person), totalling 153 (28.5%) attendances. Forty-five (24.5%) patients with T2D were taking neither sulfonylureas nor insulin. T1D had more impaired hypoglycaemia awareness (51.0% vs 14.6%, p<0.005) and history of SH (56.1% vs 15.8%, p<0.005) than T2D, while cognitive impairment was less prevalent (7.1% vs 19.6%, p=0.001). T2D had higher Charlson score (8.5±3.1 vs 4.3±3.1, p<0.005) and greater socioeconomic deprivation than T1D (20% most deprived decile; 53.3% vs 38.1%, p=0.006). More T2D required hospital admission than T1D (74.4% vs 60.3%, p=0.002). Of those diagnosed age <40, 131 (84.5%) had T1D (YOT1D) and 24 (13.0%) had T2D (YOT2D) with median (range) age of diabetes onset 18 (1-39) and 35 (18-39) years respectively. YOT2D had more cardiorenal disease (66.7% vs 42.7%, p=0.04) and heart failure (41.7% vs 13.7%, p=0.003) than YOT1D. Mental illness was equally common in both types of diabetes (YOT1D vs YOT2D; 50.4% vs 45.8%, p=NS). Of those diagnosed age >40, T2D had more cardiorenal disease (84.0% vs 47.8%, p<0.005), heart failure (30.8% vs 8.7%, p=0.026) and liver disease (20.5% vs 4.3%, p=0.046) than T1D. Most SH occurred in YOT1D (188 episodes) and older-onset T2D (179 episodes). Only 28 episodes occurred in YOT2D. Mortality after index presentation to ED was higher in T2D than T1D (38.0% vs 14.2%, p<0.005) with shorter mean time to death (239.8±290.6 vs 470.0±318.2 days, p=0.002). Compared to T1D, the T2D cohort had more cancer (34.3% vs 9.1%, p=0.029), respiratory disease (40.0% vs 9.1%, p=0.008) and cardiorenal disease (87.1% vs 63.6%, p=0.024). The mortality was similar in non-diabetics to T2D (37.5% vs 38.0%, p=NS) but death occurred significantly earlier (113.7±218.4 vs 239.8±290.6 days, p=0.014). Charlson score was elevated (non-diabetic vs T2D; 7.7±2.8 vs 9.9±2.7, p<0.005) and cardiorenal disease was prevalent in both groups (non-diabetic vs T2D; 94.4% vs 87.1%, p=NS).

Conclusion: SH is uncommon in YOT2D except when certain comorbid conditions co-exist. SH may be a biochemical marker of severe underlying medical disorders that predicts a poor prognosis.

Disclosure: S.H. Song: None.

134

Determining the incidence and clinical predictors of severe hypoglycaemia in patients receiving insulin and sulphonylureas for type 2 diabetes

R.L.M. Cordiner, K. Bedair, L. Donnelly, G. Leese, E.R. Pearson;

Level 5, Mailbox 12, University of Dundee, Dundee, UK.

Background and aims: The most serious adverse effect of sulphonylureas (SU) and insulin treatment of patients with type 2 diabetes (T2DM) is hypoglycaemia, which may result in significant morbidity and mortality. We aimed to investigate the incidence and clinical predictors of severe hypoglycaemia (SH) requiring paramedic intervention in patients treated with SU or insulin for T2DM in real-world data from patients in Tayside and Fife regions of Scotland

Materials and methods: We undertook a retrospective cohort study of n=23016 patients with T2DM treated with insulin or SU in NHS Tayside and Fife between 2008 and 2016. Data were from a SCI-diabetes extract linked to unique Scottish Ambulance Service data in the region. We calculated the incidence rates of severe hypoglycaemia (SH) per 1000 person years. Chi-Squared tests were conducted to evaluate clinical and biochemical characteristics of patients who had experienced SH episodes within the region. A fixed effect Poisson regression model was developed to identify predictors of SH. We then developed and validated a clinical prediction tool to predict individual risk for SH.

Results: The incidence of SH in SU treated T2DM was 0.5% per year, and 1.8% in those treated with insulin. SH was less common in males treated with SU (0.47%-year male; 0.66% female, Chi-Squared Test p<0.0001), but more common in males treated with insulin (1.9%/year male, 1.7 %/year female, Chi-Squared Test p<0.0001). The incidence of SH in females treated with gliclazide was almost double that of males (0.41% male, 0.75% female, Chi-Squared Test p<0.0001). There was increased risk of SH with increasing age, duration of diabetes and creatinine, and lower HBA1c and a BMI <30 kg/m2. Modified release gliclazide was the SU with lowest risk of SH, with incidence of 0.02% per year versus 1.3% with glibenclamide. The model was trained on the observation period data from the Tayside population (n=8903) with replication in Fife (n=8737), the mean model error was 0.037 per 1000 person years, suggesting that the model performed well with little error and could have potential clinical utility.

Conclusion: The incidence of SH associated with SU is low, more than a third lower than that of insulin. However, some individuals are at high risk even when treated with SU - e.g. older women with long duration of diabetes and low HbA1c. Our validated prediction tool has the clinical potential to identify those at high risk of SH and optimise their treatment to minimise risk (by switching of insulin or SU, reducing dose, or changing to Gliclazide MR).

Supported by: Wellcome Trust New Investigator Award

Disclosure: R.L.M. Cordiner: None.

135

Dishabituation with high intensity exercise improves hormonal and symptom responses to hypoglycaemia in people with type 1 diabetes and impaired awareness of hypoglycaemia

C.M. Farrell1, A.D. McNeilly1, S.M. Hapca2, T.W. Jones3, P.A. Fournier3, D. West4, R.J. McCrimmon1;

1Clinical Research Centre, Ninewells Hospital, Dundee, UK, 2Computing Science, University of Stirling, Stirling, UK, 3University of Western Australia, Perth, Australia, 4Faculty of Medical Sciences, Newcastle University, Newcastle, UK.

Background and aims: Impaired awareness of hypoglycaemia (IAH) in Type 1 diabetes (T1D) is a major risk factor for severe hypoglycaemia. We recently proposed that IAH develops as a form of adaptive memory (Habituation) to repeated hypoglycaemia. Consistent with this, we found that introduction of a single novel stress stimulus [high intensity exercise (HIE)] partially restored counterregulatory (CRR) hormonal and symptom responses to hypoglycaemia in people with T1D and IAH; a process referred to as Dishabituation. For HIE to prove to be an effective therapeutic intervention, it is important to demonstrate sustained efficacy in individuals with TID who suffer from IAH.

Materials and methods: To address this question, we conducted a 4-week pilot, single centre, randomised parallel-group study using HIE in people with T1D and IAH: HIT4HYPOS. Individuals (n=18) underwent a 4-week run-in period of insulin optimisation, before randomisation to HIE (3 session per week [4 x 30 s cycle sprints separated by 2 min of recovery] to reach an intensity corresponding to ≥ 90% peak heart rate achieved during VO2peak assessment) for 4 weeks plus use of real-time continuous glucose monitoring (rtCGM) or Control (rtCGM alone). At baseline and following the intervention they underwent a 90-minute hyperinsulinaemic hypoglycaemic clamp at 2.5 mmol/l with measurement of hormonal CRR and symptom scores.

Results: Change from baseline was compared between groups using a generalised estimated equation, adjusted for baseline and euglycaemia. In comparison to Control, HIT significantly increased glucagon (Δ mean [SEM] 0.9 [3.89] vs 16.2 [6.21] ng/l, p < 0.01) and maintained noradrenaline (-987.71 [447.37] vs 514.03 [731.73] pmol/l, p < 0.01), but not adrenaline responses (298.1 [687.42] vs 1129.7 [747.40] pmol/l; p = 0.11) during equivalent hypoglycaemia. Post intervention total symptom scores during hypoglycaemia were greater in the HIT group than the Control group (Edinburgh Hypoglycaemia scale; 25.0 +/- 2.0 vs 20.4 +/- 2.4 vs, p <0.05).

Conclusion: These findings suggest that 4 weeks of HIE provide sustained benefits on the hormonal and symptomatic CRR to hypoglycaemia in people with T1D and IAH. Of note there was a small but significant increase in hypoglycaemia-induced glucagon. Therefore, HIT may represent a novel therapeutic intervention for people with T1D and IAH.

Clinical Trial Registration Number: ISRCTN15373978

Supported by: Diabetes UK and JDRF

Disclosure: C.M. Farrell: Grants; Diabetes UK 17/0005591, JDRF 3-SRA-2017-485-S-B.

136

Decreased branched-chain amino acids and elevated fatty acids during antecedent hypoglycaemia in type 1 diabetes

R. She1,2, N. Al-Sari3, I. Mattila3, P. Henriksen3, J. Pedersen4, A.-S. Sejling1,5, C. Legido-Quigley3,6, U. Pedersen-Bjergaard1,2;

1Department of Endocrinology and Nephrology, Nordsjællands Hospital, Hillerød, Denmark, 2Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark, 3Steno Diabetes Center Copenhagen, Herlev, Denmark, 4Department of Internal Medicine, Herlev and Gentofte Hospital, Herlev, Denmark, 5Novo Nordisk A/S, Bagsværd, Denmark, 6King's College London, Institute of Pharmaceutical Science, UK.

Background and aims: Hypoglycaemia is a major limiting factor in achieving recommended glycaemic targets for individuals in insulin treatment and can lead to late diabetes complications, incremental morbidity, and mortality. While counterregulatory hormonal responses have been studied extensively in patients with type 1 diabetes, a more comprehensive assessment of the metabolic responses has not been done previously. This post hoc study explored potential responses of the metabolome to hypoglycemia.

Materials and methods: A post hoc study examining the metabolome from participants with euglycemia and hypoglycemia during hyperinsulinemic clamps. Setting and Participants: Twenty-one outpatients with type 1 diabetes were recruited in the outpatient clinic. Intervention: On two consecutive days participants underwent a period of euglycemia (5.0-5.5 mmol/l) and a period of hypoglycemia (2.0-2.5 mmol/l) during the hyperinsulinemic glucose clamp. Primary Outcome Measure: Plasma samples were taken 40 min after reaching euglycaemia and 20 min and 40 min after reaching hypoglycaemia. Non-targeted plasma metabolomic analyses were conducted using two-dimensional gas chromatography/time-of-flight mass spectroscopy. Metabolites were analyzed by a linear mixed effect model, adjusting for age and sex. P-values were adjusted for multiple testing using the Benjamini-Hochberg method.

Results: In total, 79 metabolites were identified. Concentrations of the branched-chain amino acids, leucine, and isoleucine, decreased during hypoglycaemia at day 1: isoleucine (β±SE: -0.72±0.16, p = 2.2x10-3), leucine (β±SE: -0.78±0.18, p = 3.8x10-3). At day 2, five amino acids including all three branched-chained amino acids decreased: Leucine (β±SE: -0.62±0.13, p = 0.002), isoleucine (β±SE: -0.59±0.19, p = 0.026), valine (β±SE: -0.52±0.12, p =0.002), methionine (β±SE: -0.62±0.19, p = 0.026) and phenylalanine (β±SE: -0.65±0.20, p = 0.026). Two fatty acids were increased, oleic acid (β±SE: 0.50±0.14, p = 0.016) and tetradecanoic acid (β±SE: 0.63±0.17, p = 0.013). More metabolites responded to hypoglycemia on day 2; however, metabolic responses were not significantly different between the two days. Hormonal counter-regulatory responses to hypoglycemia were not different between the two days.

Conclusion: In conclusion, this study found that the metabolome alternates in response to insulin-induced hypoglycemia, resulting in decrement of several amino acids and increment of two fatty acids. The hormonal counterregulatory response was indifferent between the first and subsequent hypoglycemic episode, and while we also could not find a significant change in metabolic responses, the additional significant changes in metabolites during day 2 suggest an altered metabolic response to a subsequent hypoglycemic episode, which is not a result of an attenuation of the hormonal counterregulatory response.

Clinical Trial Registration Number: NCT01337362

Supported by: Steno Collaborative Grants research programme

Disclosure: R. She: None.

137

Recurrent low glucose exposure (RLG) induces intrinsic metabolic adaptations in pancreatic alphaTC1.9 cells

K.M. Partridge, N.G. Morgan, K.L.J. Ellacott, C. Beall;

RILD Building, Institute of Biomedical and Clinical Sciences, Exeter, UK.

Background and aims: Recurrent hypoglycaemia is a severe complication associated with insulin-treated diabetes. Over time, pancreatic alpha cells fail to release glucagon in response to recurrent bouts of hypoglycaemia for poorly defined reasons. Previous studies have focused on potential changes in the brain-pancreas axis as a mechanism to explain this diminished alpha cell hypoglycaemia counter-regulation. However, little is known about whether or how alpha cell metabolism/function is altered following recurrent hypoglycaemia. We therefore sought to determine the intrinsic alpha cell metabolic changes after recurrent bouts of low glucose (RLG) in a murine alpha cell line, alphaTC1.9.

Materials and methods: To mimic recurrent hypoglycaemia seen in diabetes, alphaTC1.9 cells were treated with between zero - four bouts of RLG (0.5 mmol/l) then allowed to recover overnight in euglycaemic-like levels of glucose (5.5 mmol/l). Cell glycolytic rate was measured using a Seahorse XFe96 flux analyser by calculating proton efflux rates derived from glycolysis (glycoPER), the extracellular acidification rate (ECAR) and oxygen consumption rates (OCR) - an indicator of mitochondrial metabolism. Acute glucose utilisation (ΔECAR) was measured after injection of between 0.1 - 11.7 mmol/l glucose. Data were analysed using unpaired two tailed t-test or Two-Way ANOVA with multiple comparisons test.

Results: RLG augmented basal glycolysis compared to acute low glucose (ALG) (pmol/min/μg) (ALG; 5.095 ± 0.539, RLG; 10.801 ± 0.687, p < 0.0001; n = 33 - 35). After mitochondrial inhibition, RLG increased compensatory glycolysis (pmol/min/μg) (ALG; 12.494 ± 0.701; RLG; 22.088 ± 1.038; p < 0.0001, n = 33 - 35). RLG enhanced baseline OCR (ALG; 32.543 ± 0.717, RLG; 39.248 ± 1.278, p < 0.0001, n = 33 - 35). After four prior bouts of low glucose, RLG enhanced acute glucose utilisation (mpH/min/μg) in a concentration-dependent manner (0.1 mmol/l; ALG 0.089 ± 0.025, RLG 0.159 ± 0.039; 0.5 mmol/l; ALG 0.566 ± 0.051, RLG 0.703 ± 0.065, 5.5 mmol/l; ALG 1.148 ± 0.077, RLG 1.383 ± 0.059, p < 0.05, 11.7 mmol/l; ALG 1.204 ± 0.104, RLG 1.476 ± 0.077, p < 0.05, n = 20 - 24).

Conclusion: We demonstrate for the first time that intrinsic metabolic adaptations in an alpha cell line are induced by prior bouts of antecedent low glucose. RLG increased alpha cell basal glycolytic flux and glucose utilisation, even at low/hypoglycaemic levels of glucose. RLG also enhanced basal oxidative metabolism suggesting that mitochondria are working harder at baseline. These data suggest that enhanced glucose utilisation may play a role in driving the alpha cell dysfunction associated with defective counterregulatory responses to hypoglycaemia.

Supported by: DUK PhD studentship

Disclosure: K.M. Partridge: None.

138

The impact of CGM with a predictive hypoglycaemia alert function on hypoglycaemia in physical activity for people with type 1 diabetes: PACE study

S. Rilstone1, N. Oliver1, B. Tanushi1, N. Hill2;

1Imperial College London, 2Imperial College Healthcare NHS Trust, London, UK.

Background and aims: The benefits of exercise for people Type 1 Diabetes (T1D) are significant, and include improvement in cardiovascular risk factors. Uptake of exercise is low in people with T1D, with fear of hypoglycaemia cited as the main barrier to exercise. Insulin and fuelling strategies are complex, and varying insulin sensitivity associated with different exercises regimens can make diabetes management challenging, resulting in hypoglycaemia. Technology has great potential to improve self-management of diabetes during exercise, offering real time glucose feedback with trend arrows, and alarms to alert the user of impending hypoglycaemia during exercise when close observation of glycaemia may be challenging. The aim of the study is to assess the impact of Dexcom G6 real-time CGM (RT-CGM) with a predictive hypoglycaemia alert function on the frequency, duration and severity of hypoglycaemia occurring before, during and after regular physical activity in people with type 1 diabetes.

Materials and methods: People with type 1 diabetes on multiple daily injections or insulin pump therapy who exercised regularly (150min/week or more) have been recruited to this observational, within subject crossover study. Participants undertook 10 days of blinded CGM, whilst living their normal lives including their usual activity which was recorded using a sports watch. At the end of the 10 days, the RT-CGM was unblinded so that participants were able to see their glucose data. Participants were randomised 1:1 to use the “urgent low soon” alert switched on or off. The urgent low soon alert is an alarm that sounds when the algorithm predicts that the wearer will be 3.1mmol/l or less in the next 20 minutes. Participants continued to undertake and record their usual exercise. After 40 days the participants crossed over, so the participants that had the alarm off now switched it on, and vice versa for a further 40 days. Participants recorded their carbohydrate and insulin doses throughout the study.

Results: 24 participants were randomised. The participants (8 men, 16 women) had a mean (SD) age of 35 (11) years, duration of diabetes of 14 (7) years, BMI 26 (4.7)kg/m2 and HbA1c of 58 (12) mmol/mol]. 1 person was excluded from the analysis due to incomplete data. The baseline median percentage time < 3.0mmol/l was 0.25 (0.0.5-0.89)%. At the end-point the percentage time <3.0mmol/l was significantly lower with urgent low soon alerts on (median treatment difference 0.12% (95% CI 0.04-0.2, p=0.0036), compared to the urgent low soon off period. Time spent in hypoglycaemia below 2.8mmol/L was significantly different in the 24 hours after activity favouring the urgent low soon (p=0.035).

Conclusion: A CGM device with a an urgent low soon alert reduces exposure to level 2 hypoglycaemia compared to real time continuous glucose monitoring with a threshold alarm. Additionally an urgent low soon alert reduces exposure to disabling hypoglycaemia below 2.8mmol/l in the 24 hours after exercise compared to a threshold alert. These data suggest people with T1D undertaking regular exercise benefit most from a predictive alert monitoring system.

Clinical Trial Registration Number: NCT04142944

Supported by: Dexcom

Disclosure: S. Rilstone: Grants; Dexcom.

OP 24 How to burn energy

139

The oxidative phenotype of abdominal and femoral adipose tissue in women with normal weight or obesity

I.G. Lempesis1,2, N. Hoebers2, Y. Essers2, J.W.E. Jocken2, L.J. Dubois3, E.E. Blaak2, K.N. Manolopoulos1,4, G.H. Goossens2;

1Institute of Metabolism and Systems Research (IMSR), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, 2Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, Netherlands, 3The M-Lab, Department of Precision Medicine, GROW School for Oncology and Reproduction, Maastricht University Medical Centre+, Maastricht, Netherlands, 4Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK.

Background and aims: Body fat distribution is closely associated with the risk for developing obesity-related cardiometabolic diseases. Rodent and human studies suggest that mitochondrial dysfunction in adipose tissue (AT) contributes to obesity-related metabolic complications. However, whether mitochondrial function differs between AT depots remains unknown. Here, we aimed to explore whether the oxidative phenotype differs between upper and lower body subcutaneous AT in humans.

Materials and methods: We investigated the abdominal and femoral AT oxidative machinery in 21 postmenopausal women (age 50-65 yrs) with normal weight (PwNW; BMI 18-25 kg/m2) versus obesity (PwOB; BMI 30-40 kg/m2). In vivo fractional extraction (FE) and release (FR) of oxygen, carbon dioxide, and several metabolites across abdominal and femoral AT were determined using the arterio-venous balance technique. Oxidative phosphorylation (OXPHOS) protein levels were determined in abdominal and femoral AT biopsies and differentiated adipocytes derived from the same donors. Finally, we assessed ex vivo mitochondrial respiration in abdominal and femoral adipocytes (Seahorse XF96 analyzer).

Results: The FE of oxygen (22.6 ± 2.1 vs. 35.8 ± 3.1%, p=0.009) and the FR of carbon dioxide (4.1 ± 0.8 vs. 7.0% ± 1.2, p=0.076) across abdominal AT were lower compared to femoral AT. Adipocyte size (68.5 ± 1.9 vs. 68.4 ± 1.6 μm, p= 0.791) and blood flow (9.3 ± 2.1 vs. 5.8 ± 1.8 mL/min, p= 0.296) were not different between abdominal and femoral AT. OXPHOS protein expression was lower both in AT (complexes I and III) and in adipocytes (complexes III and V) in PwOB than PwNW but did not differ between depots. Basal respiration (p = 0.020), maximal respiration (p= 0.001) and spare respiratory capacity (p=0.001) were lower in abdominal compared to femoral adipocytes derived from both PwNW and PwOB.

Conclusion: These findings demonstrate for the first time that AT oxygen extraction and adipocyte oxygen consumption are lower in ABD than FEM AT in postmenopausal women with normal weight or obesity, already at the preclinical stage, independent of adipocyte size.

Supported by: EFSD/Lilly Research Programme, EFSD Anniversary Programme

Disclosure: I.G. Lempesis: None.

140

Pdgfrα-specific deletion of Alms1 in mice recapitulates the obesity and insulin resistance of global Alms1 KO and this cannot be explained by discoordinated ciliary dynamics

E.J. McKay1, I.H. Luijten1, D. McCormick1, P. Mill2, R.K. Semple1;

1Centre for Cardiovascular Science, University of Edinburgh, 2Institute for Genetics and Cancer, University of Edinburgh, Edinburgh, UK.

Background and aims: Alström Syndrome (AS) is a rare autosomal recessive disease featuring early onset, severely insulin resistant diabetes, fatty liver and heart failure among other features. These cardiometabolic complications occur in the face of only moderate obesity in many patients. AS is caused by biallelic loss-of-function mutations in the ALMS1 gene, encoding a large centrosomal protein. The precise derangement of centrosomal and/or primary ciliary function caused by loss of ALMS1 is unknown, as most studies have reported primary cilia to be morphologically normal. Abnormal cell cycle kinetics have been suggested to play a role in some facets of the syndrome. Several global knockout (KO) mouse models have been described to recapitulate key metabolic components of AS, but none have used tissue-specific KO to tease out contributions of different cell types to pathology. Hypotheses: 1) 1) The metabolic profile of AS closely resembles that of lipodystrophy. We thus hypothesised that loss of Alms1 function in mesenchymal stem cell populations, such as adipose precursor cells, would recapitulate the metabolic derangement in AS. 2) Alms1 loss leads to disco-ordination of ciliary and cell cycles, and it is such discoordination rather than abnormal ciliary structure, that compromises tissue homeostasis.

Materials and methods: 1) A novel global KO mouse was generated by crossing the EUCOMM Tm1c Alms1 line with global CAG-Cre mice. A Pdgfrα-Cre driver was used to abrogate Alms1 function only in mesenchymal progenitor cells and their descendants including preadipocytes and adipocytes. We undertook metabolic phenotyping of global and Pdgfrα+ Alms1-KO mice on a 45% high-fat diet. 2) 2) NIH-3T3 flp-in cells were edited using CRISPR-Cas9 to cause biallelic loss-of-function mutations of Alms1. The cycle and ciliary biosensor, Arl13bCerulean-Fucci2a, was stably expressed in Alms1-/- and WT control cells using the flp-in system. Live cell imaging of these cells was then performed, followed by analysis of cell cycle and ciliary coordination dynamics.

Results: 1) Consistent with previous models and the human disease, global Alms1 KO mice were hyperphagic, obese, insulin resistant, and had severe hepatosteatosis. KO of Alms1 only in MSCs and their descendants recapitulated key metabolic phenotypes of global KO animals including obesity and insulin resistance. Interestingly hyperphagia was also seen despite preservation of neuronal Alms1. 2) No significant difference in the time between deciliation and cytokinesis, or the time between cytokinesis and ciliation was observed.

Conclusion: 1. MSC-derived lineages are critical in driving the severe metabolic syndrome in AS 2. Hyperphagia in AS does not depend on neuronal Alms1 deficiency 3. Discoordinated ciliary and cell cycles do not appear to explain the critical role of MSCs in the metabolic syndrome of AS.

Supported by: BHF, Wellcome Trust

Disclosure: E.J. McKay: None.

141

A larger brown fat volume and lower mean radiodensity are related to a greater cardiometabolic risk, especially in young men

F. Acosta1,2, G. Sanchez-Delgado2,3, B. Martinez-Tellez2,4, F. Osuna-Prieto2,5, A. Mendez-Gutierrez6,7, C. M. Aguilera6,8, A. Gil6,8, J. Llamas Elvira9, J. Ruiz Ruiz2,8;

1Turku PET Centre, University of Turku, Turku, Finland, 2PROFITH research group, University of Granada, Granada, Spain, 3Pennington Biomedical Research Center, Baton Rouge, USA, 4Department of Medicine, Division of Endocrinology, Einthoven Lab for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, Netherlands, 5Department of Analytical Chemistry, University of Granada, Granada, Spain, 6Department of Biochemistry and Molecular Biology II, Biomedical Research Centre, Granada, Spain, 7Biohealth Research Institute in Granada, Granada, Spain, 8Biohealth Research Institute, Granada, Spain, 9University Hospital Virgen de las Nieves, Granada, Spain.

Background and aims: Brown adipose tissue (BAT) is important in the maintenance of cardiometabolic health in rodents. Recent reports appear to suggest the same in humans, although if this is true remains elusive, partly because of the methodological bias that affected previous research. The present cross-sectional work reports the relationships of cold-induced BAT volume, activity (peak standardized uptake, SUVpeak) and mean radiodensity (an inverse proxy of the triacylglycerols content) with the cardiometabolic and inflammatory profile of 131 young adults, and how these relationships are influenced by sex and body weight.

Materials and methods: This was a cross-sectional study carried out in Granada (south of Spain) during October-December 2015 and 2016. Subjects underwent personalized cold exposure for 2 h to activate BAT, followed by static 18F-fluorodeoxyglucose positron emission tomography-computed tomography scanning to determine BAT variables. Information on cardiometabolic risk (CMR) and inflammatory markers was gathered, and a CMR score and fatty liver index (FLI) calculated. Anthropometry, body composition and lifestyle behaviors (potential confounders) were also measured.

Results: In men, BAT volume was positively related to homocysteine and liver damage markers concentrations (independently of BMI and seasonality) and the FLI (all P≤0.05). In men too, BAT mean radiodensity was negatively related to the glucose and insulin concentrations, alanine aminotransferase activity, insulin resistance, total cholesterol/HDL-C, LDL-C/HDL-C, the CMR score, and the FLI (all P≤0.02). In women it was only negatively related to the FLI (P<0.001). These associations were driven by the results for the overweight and obese subjects (see figure below). No relationship was seen between BAT and inflammatory markers (P>0.05).

Conclusion: These findings show that a larger BAT volume and a lower BAT mean radiodensity are related to a higher CMR, especially in young men, which may support that BAT acts as a compensatory organ in states of metabolic disruption.

figure ap

Clinical Trial Registration Number: NCT02365129

Supported by: Potential support (not decided yet) - Turku Foundation, University of Turku

Disclosure: F. Acosta: None.

142

Brown adipose tissue content of triglycerides, but not SUV, is associated with cardiovascular risk markers in volunteers from a tropical region

M. Monfort-Pires1, G.R. Silva2, P. Dadson1, G.A. Nogueira3, M. U-Din1, K.A. Virtanen1, L.A. Velloso4;

1Turku PET Centre, Turku PET Centre, Turku University Hospital, Turku, Finland, 2Nutrition, School of Public Health, University of Sao Paulo, Sao Paulo, Brazil, 3Clinical Medicine, University of Campinas, Campinas, Brazil, 4Medicine, University of Campinas, Campinas, Brazil.

Background and aims: Since the discovery of active brown adipose tissue (BAT) in human adults, the tissue has been considered a potential target for the treatment of obesity, diabetes, and cardiovascular diseases. A detailed characterization of BAT structure and function could help understand its role in metabolism, but most studies evaluating BAT structure and function were performed in temperate climate regions, while 40% of the world’s population are living in tropical areas.

Materials and methods: We used 18F-fluorodeoxyglucose positron emission tomography - combined with magnetic resonance imaging to evaluate BAT activity (FDG standardized uptake value - SUV), BAT volume and BAT content of triglycerides (TG) in 30 lean and 15 subjects with overweight or obesity living in a tropical area in Southeast Brazil. Body composition was analyzed using DXA and magnetic resonance imaging and fasting blood samples were withdrawn for several determinations. The study was approved by the Local Ethics Committee, and it has been performed in accordance with the ethical standards laid down in the Helsinki Declaration.

Results: We observed that BAT activity (FDG SUV) and BAT volume are not correlated with leanness (p>0.05 for all); instead, BAT triglyceride content is correlated with waist circumference (correlation coefficient r=0.71; p<0.01) and markers of cardiovascular risk, such as Castelli Index I (correlation coefficient r=0.51; p<0.01) and II (correlation coefficient r=0.47; p<0.01). Also, it was inversely associated with HDL (correlation coefficient r= -0.48; p<0.05). To further investigate this association, we performed an analysis of variance (ANOVA) comparing BMI, waist circumference, total fat mass, visceral adipose tissue, HDL, Castelli I and Castelli II indexes against BAT activity, volume, and TG content. BAT mean SUV and volume did not differ across categories of BMI or waist circumference, or across tertiles of total fat mass and visceral adipose tissue, HDL, Castelli I and II indexes (p>0.05 for all). However, we detected a difference between low and high BMI (68.8 ± 6.1 versus 77.8 ± 5.1 % of TG) and waist circumference (68.7 ± 4.9 versus 77.6 ± 5.3 % of TG) and between the first and 3rd tertile of visceral fat mass (68.3 ± 4.2 versus 78.4 ± 4.3 % of TG) for the BAT TG content (p<0.05 for all). In addition, differences between tertiles were observed for HDL cholesterol, Castelli I and Castelli II indexes (p<0.05 for all).

Conclusion: This study expands knowledge regarding the structure and function of BAT in people living in tropical areas. In addition, we provide evidence that BAT triglyceride content could be a marker of cardiovascular risk.

Supported by: FAPESP: #2016/10616-7/#2017/22586-8/2019/02055-3

Disclosure: M. Monfort-Pires: None.

143

Humans with metabolically active brown fat demonstrate higher capacity to catabolise branched chain amino acids

M. U-Din1, V. de Mello Laaksonen2, K. Hanhineva2, J. Newman3, K. Kristiansen4, M. Klingenspor5, T. Fromme5, T. Niemi6, M. Taittonen6, T. Saari1, J. Raiko1, P. Nuutila1, K.A. Virtanen1;

1Turku PET Centre, Turku, Finland, 2University of Eastern Finland, Kuopio, Finland, 3University of California, Davis, USA, 4University of Copenhagen, Copehagen, Denmark, 5Technical University Munich, Freising, Germany, 6Turku University Hospital, Turku, Finland.

Background and aims: Branched chain amino acids (BCAA: valine, leucine, isoleucine) may synergize with circulatory lipids to induce insulin resistance. Metabolically active brown adipose tissue (BAT) may be a potent site for BCAA catabolism that participates in systemic clearance of BCAA. The study aimed to investigate the systemic and BAT-specific metabolism of BCAA in healthy human adults.

Materials and methods: Seventy-nine (25M/54F) humans with BMI 27 ± 6 kg/m2 and whole-body insulin sensitivity (M-value) of 39 ± 20 μmol/kg/min participated. M-value was determined with hyperinsulinemic-euglycemic clamp. Circulatory BCAA were determined with non-targeted metabolomics on serum samples. Tissue BCAA were determined with non-targeted metabolomics, and tissue transcriptomics were determined with RNA-sequencing on human supraclavicular BAT samples. BAT metabolism was determined with PET imaging with either [18F]-FDG or [18F]-FTHA radiotracers to measure glucose or NEFA uptake, respectively, under cold stimulation. The subjects manifesting BAT glucose uptake ≥ 3.0 μmol/100g/min or NEFA uptake ≥ 0.7 μmol/100g/min were classified as subjects possessing a high metabolically active BAT (High-BAT).

Results: Circulatory levels of BCAA were directly related to obesity markers and inversely related to M-value. The subjects with high BAT metabolism (High-BAT) demonstrated lower levels of circulatory BCAA compared to Low-BAT subjects (valine p = 0.009, leucine p = 0.056, isoleucine p = 0.09); the differences persisted whilst adjusting for whole-body fat percentage (ANCOVA, valine p = 0.01, leucine p = 0.06, isoleucine p = 0.095). In BAT, there was a trend of lower levels of valine and isoleucine in High-BAT compared to Low-BAT (p=0.07 and p=0.07, respectively) while there was no difference in the mRNA expression of the cellular transporters of BCAA uptake (SLC7A5, SLC38A7, SLC38A9, SLC6A15, SLC3A2) between High-BAT and Low-BAT. There was no relationship between the circulatory and BAT levels of BCAA (all p > 0.19) indicating that the differences in tissue levels of BCAA (High-BAT vs Low-BAT) are likely due to the differences in catabolic capacity. In line with this, the mRNA expression of the genes in BAT regulating BCAA catabolism BCKDHA, ACADSB, and HIBADH, was higher in High-BAT in comparison to Low-BAT (all p < 0.05). The expression of the gene encoding BCAA mitochondrial transport-mediator (SLC25A44), BCKD complex (BCKDHA, BCKDHB, DBT, DLT), and several others involved in the catabolism of BCAA (ACAD8, AUH, MCCC1, ACAT1, MCEE) was in direct relationship with the expression of the thermogenic gene, UCP1 (all p < 0.05).

Conclusion: These results suggest that humans with metabolically active BAT have a greater capacity for systemic clearance of BCAA in comparison to those with lower metabolically active BAT. In BAT, the genes encoding proteins responsible for the catabolism of BCAA are linked with thermogenic capacity indicating BCAA catabolism may participate to fuel BAT thermogenesis. An enhancement of BAT metabolism may prove to be a useful target for combating systemic BCAA induced metabolic disruptions in insulin resistance.

Supported by: AoF

Disclosure: M. U-Din: None.

144

Hepatocyte PPARα is required for the sensing of adipose-derived fatty acids and for full brown adipose tissue activation during lipolysis

A. Fougerat1, G. Schoiswohl2, A. Polizzi1, M. Régnier1, C. Wagner2, B. Tramunt3, S. Ellero-Simatos1, L. Payrastre1, C. Postic4, W. Wahli5, N. Loiseau1, A. Montagner3, D. Langin3, A. Lass2, H. Guillou1;

1Toxalim, INRAE, Toulouse, France, 2Institute of Molecular Biosciences, Graz, Austria, 3Institute of Metabolic and Cardiovascular Diseases, Toulouse, France, 4INSERM U1151/CNRS UMR 8253, Institut Necker-Enfants Malades (INEM), Paris, France, 5Center for Integrative Genomics, Lausanne, Switzerland.

Background and aims: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor and a member of the PPAR family, which also includes PPARβ/δ and PPARγ. In hepatocytes, PPARα acts as a lipid sensor that controls the expression of genes involved in whole-body energy homeostasis during fasting, including fatty acid oxidation and ketogenesis. During fasting, PPARα is also required for the expression of the hormone fibroblast growth factor 21 (FGF21), a hepatokine with systemic metabolic effects. In this work, we investigated the role of hepatocyte PPARα activity in the dialogue between the liver and adipose tissues.

Materials and methods: We used mouse models of selective deletion of PPARα in hepatocytes and of adipose triglyceride lipase (ATGL) in adipocytes as a model of defective lipolysis. First, we performed liver whole genome expression analysis in fasted mice upon cell-specific deletion of adipocyte ATGL or hepatocyte PPARα. Second, we tested the consequences of hepato-specific PPARα deficiency during pharmacological induction of adipocyte lipolysis with a β3-adrenergic receptor agonist.

Results: In the absence of ATGL in adipocytes, ketone body and FGF21 production is impaired in response to starvation. Liver transcriptome analysis reveals that adipocyte ATGL is critical for regulation of hepatic gene expression during fasting, with a strong induction of PPARα target gene expression. Adipose tissue lipolysis, induced by acute activation of the β3-adrenergic receptor, also triggers PPARα-dependent responses in the liver, including gene expression, FGF21 production and ketogenesis. Hepatic PPARβ/δ is dispensable for these β3-adrenergic responses. In addition, the absence of hepatocyte PPARα alters brown adipose tissue (BAT) morphology and reduces the expression of BAT markers upon stimulation of β3-adrenergic signaling while the deletion of FGF21 in hepatocytes does not affect BAT activation under this condition.

Conclusion: Altogether, our results support a dominant role for adipose ATGL in generating fatty acids that trigger hepatocyte PPARα activity and underscore the critical role of hepatic PPARα not only in the sensing of lipolysis-derived lipids but also in triggering BAT activation independent of hepatocyte FGF21 production. Intact PPARα activity in hepatocytes is required for cross-talk between adipose tissues and the liver during fat mobilization.

Supported by: ANR Hépadialogue/Région Occitanie/Agreenskills

Disclosure: A. Fougerat: None.

OP 25 Lipid in and out of the liver

145

Skeletal muscle derived myokine affects insulin sensitivity and lipogenesis in a human hepatocyte spheroid model

J.-B. Potier1,2, A. Dumond2, A. Fardellas3, M. Pinget2, M. Aouadi3, K. Bouzakri2,1;

1Ilonov, Strasbourg, France, 2Université de Strasbourg, Centre européen d'étude du diabète, Strasbourg, France, 3Karolinska Institutet, Center for Infectious Medicine, Huddinge, Sweden.

Background and aims: Inter-organ crosstalk has recently emerged as a crucial aspect of body homeostasis, especially in metabolic diseases such as type 2 diabetes (T2D) and NAFLD. Skeletal muscle is highly implied in this crosstalk, through the secretion of specifics cytokines called myokines. Among those, our laboratory has focused on the myokine X (MyoX). Due to our previous results on the prevention of diabetes and to the pathophysiological link between this disease and NAFLD, we chose to investigate the effect of the MyoX on the liver. Primary human hepatocytes (PHH) spheroids, the gold standard model for the in vitro study of liver diseases, were used for our experiments.

Materials and methods: PHH spheroids were cultured in lean and metabolic syndrome (MetS) conditions treated, or not, with different concentrations of MyoX: 0.1, 1 and 10 μg/ml. Spheroids were then stimulated with insulin and protein lysates were collected. Effects of MyoX on the expression of proteins related to glucose (Akt, ERK1/2, GLUT2) and lipid (SREBP1c, FAS, CPT1A) metabolism, two pathways implied in NAFLD development, were assayed by western blot. Furthermore, Nile Red staining was performed for the measurement of triglyceride accumulation.

Results: Concerning glucose metabolism, noticeable effects were observed for the phosphorylation of Akt, which was stronger at a dose of 10 μg/ml in lean condition, suggesting that the MyoX might enhance the intensity of the insulin downstream. Likewise, in the same condition, the slight increase in triglyceride accumulation, observed with the Nile Red staining, could be explained by an insulindependent de novo lipogenesis mechanism confirmed by the increase of SREBP1c phosphorylation (an insulin-dependent transcription factor responsible for the storage of fatty acid as triglycerides). Concerning MetS conditions, beneficial effects were observed at the lower dose of MyoX, with a decrease in triglyceride amount related to the reduction of SREBP1c phosphorylation, associated with no detrimental effect on p-Akt.

Conclusion: Taken together, our analysis suggest that high doses of MyoX might act as a sensitizer and exacerbates the effects of insulin in PHH in lean condition, enhancing the de novo lipogenesis. In MetS condition, potential beneficial effects were noticed for the lower dose of MyoX, suggesting that our protein might be of an interest for the prevention of NAFLD. Further studies should beperformed to determine the optimal dose of the myokine.

Disclosure: J. Potier: None.

146

The PNPLA3 I148M variant increases intrahepatic lipolysis and beta oxidation and decreases de novo lipogenesis and hepatic mitochondrial function in vivo in humans

P.K. Luukkonen1,2, K. Porthan2, N. Ahlholm2, F. Rosqvist3,4, S. Dufour1, X.-M. Zhang1, J. Dabek2, T.E. Lehtimäki5, W. Seppänen5, M. Orho-Melander6, L. Hodson4, K.F. Petersen1, G.I. Shulman1, H. Yki-Järvinen2;

1Yale University, New Haven, USA, 2Minerva Foundation Institute for Medical Research, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 3Uppsala University, Uppsala, Sweden, 4University of Oxford, Oxford, UK, 5University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 6Lund University, Malmö, Sweden.

Background and aims: The PNPLA3 I148M variant is the strongest genetic risk factor of non-alcoholic fatty liver disease (NAFLD) but the underlying mechanisms remain unknown. We studied the effect of this variant on hepatic metabolism in vivo under multiple physiological conditions by combining a recruit-by-genotype approach with state-of-the-art stable isotope techniques.

Materials and methods: We recruited 93 healthy participants (mean age 53±1 yrs, BMI 30±1 kg/m2, 19% men; 37 homozygous carriers [148MM] and 56 non-carriers [148II]). Hepatic de novo lipogenesis (DNL) was assessed after an overnight fast using D2O (148MM, n=19; 148II, n=36). Hepatic fate of exogenous fatty acids (FA) was determined using a mixed meal enriched in 13C-labeled FA (148MM, n=12; 148II, n=14). Endogenous glucose production, ketogenesis and hepatic mitochondrial citrate synthase flux (VCS) were assessed before and after a 6-day ketogenic diet by Positional Isotopomer NMR Tracer Analysis (PINTA) by infusing [3-13C]-lactate, [13C4]-β-hydroxybutyrate (β-OHB) and [2H7]-glucose (148MM, n=6; 148II, n=6). Intrahepatic triglycerides (IHTG) were assessed by NMR spectroscopy and hepatic mitochondrial redox state by plasma [β-OHB]/[acetoacetate].

Results: After an overnight fast, the 148MM group had higher plasma [β-OHB] (+104%, p<0.05) and lower DNL (-47%, p<0.01) than the 148II group, independent of plasma [NEFA] and [insulin]. After a mixed meal, exogenous 13C-labeled FAs in the 148MM group were channeled more to ketogenesis (+265%, p<0.001), which associated with an increased hepatic mitochondrial redox state (+36%, p<0.05). During a ketogenic diet, the 148MM group had greater intrahepatic lipolysis as reflected by a larger reduction in IHTG (77%, p<0.05) than the 148II group, which associated with: 1) increased plasma [β-OHB] (+90%, p<0.05), 2) increased mitochondrial redox state and 3) decreased VCS (-31%, p<0.01). Plasma [GDF-15], a mitochondrial stress marker, was increased by 40% (p<0.01) in the 148MM group compared to the 148II group.

Conclusion: The PNPLA3 I148M carriers have alterations in both intrahepatic anabolic/catabolic processes and mitochondrial function as reflected by increased intrahepatic lipolysis, decreased DNL and increased hepatic mitochondrial β-oxidation/ketogenesis. These changes associated with an increased mitochondrial redox state and reductions in hepatic mitochondrial VCS. These results provide new insights in the mechanisms by which the PNPLA3 I148M promotes NAFLD (Figure).

figure aq

Clinical Trial Registration Number: NCT03737071

Supported by: Novo Nordisk Foundation, Sigrid Jusélius Foundation

Disclosure: P.K. Luukkonen: Grants; Novo Nordisk Foundation, Sigrid Jusélius Foundation.

147

Effect of tirzepatide on fasting lipids in patients with type 2 diabetes: meta-analysis of randomised controlled trials

T. Karagiannis1, I. Avgerinos1, A. Tsapas1,2, E. Bekiari1;

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

Background and aims: Tirzepatide is a novel dual glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist that has been shown to reduce HbA1c and body weight in patients with type 2 diabetes. We aimed to assess whether the salutary metabolic effects of tirzepatide extend to improvements in patients’ lipid profile.

Materials and methods: We searched Pubmed, Embase and the Cochrane Library up to January 2022 for randomised controlled trials (RCTs) of at least 12 weeks’ duration that compared tirzepatide 5, 10 or 15 mg once weekly with placebo or other glucose lowering drugs in adults with type 2 diabetes, and reported results on fasting lipids. Outcomes included change from baseline in the concentrations of triglycerides, total cholesterol (T-C), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C) and high-density lipoprotein cholesterol (HDL-C). We conducted random effects meta-analyses pooling mean differences (MDs) with 95% confidence intervals (CIs).

Results: We included data from six RCTs (n = 6527 participants). Comparator was placebo, GLP-1 RA and basal insulin in two, one and two trials respectively, while one trial had both a placebo and a GLP-1 RA control arm. Duration of intervention was 26, 40 and 52 weeks in one, three and two trials respectively. Compared to placebo, tirzepatide induced dose-dependent reductions in fasting triglycerides, T-C, LDL-C and VLDL-C (Table), and had no effect on HDL-C. Compared to GLP-1 receptor agonists (dulaglutide and semaglutide), tirzepatide reduced triglycerides (MDs ranging from -12.51 mg/dL with tirzepatide 5 mg to -24.98 mg/dL with tirzepatide 15 mg) and VLDL-C (MDs ranging from -1.89 mg/dL to -4.08 mg/dL). Compared to basal insulin, tirzepatide improved participants’ lipid profile by reducing triglycerides, T-C, LDL-C and VLDL-C, and by increasing HDL-C.

Conclusion: Based on pooled data from RCTs, treatment with tirzepatide dose-dependently decreased levels of triglycerides, T-C, LDL-C and VLDL-C versus placebo. As such, in addition to its glucose lowering effect, tirzepatide could be used to improve the atherogenic lipoprotein profile in patients with type 2 diabetes by complementing the effect of lipid lowering drugs.

figure ar

Disclosure: T. Karagiannis: None.

148

Lipidomic and metabolomics profiling in low birth weight men reveals a dysmetabolic phenotype associated with increased liver fat

L. Elingaard-Larsen1, C. Brøns1, S. Villumsen1, L. Justesen1, A. Thuesen2, M. Ali1, M. Kim1, C. Legido-Quigley1, G. van Hall3, E. Rubæk Danielsen3, T. Hansen2, A. Vaag1;

1Steno Diabetes Center Copenhagen, Herlev, 2University of Copenhagen, Copenhagen, 3Rigshospitalet, Copenhagen, Denmark.

Background and aims: Being born with a low birth weight (LBW) is associated with an increased risk of developing Type 2 Diabetes (T2D) in adulthood. The subcutaneous adipose tissue (SAT) plays a central role in T2D pathophysiology. We hypothesized, that LBW individuals have an impaired SAT expandability, resulting in ectopic lipid deposition in the liver, thereby contributing to the pathophysiological events linking LBW with T2D development. Using untargeted metabolomics and lipidomics analyses, as well as state-of-the-art magnetic resonance (MR) spectroscopy for liver fat determinations, we aimed to assess the extent to which increased liver fat is associated with dysmetabolic traits in early middle-aged, non-obese LBW men.

Materials and methods: Forty-eight healthy, non-obese males aged 35-39 years, born at term with a LBW (BW<10th percentile, n=26) and age- and BMI-matched NBW controls (BW: 50-90th percentile, n=22) were included in the study. We measured body composition, hepatic fat content using 1H MR spectroscopy and hepatic glucose production determined by isotopic tracers. Untargeted serum metabolomics and lipidomics were performed using mass-spectrometry.

Results: LBW subjects had a significantly increased hepatic fat content compared to NBW controls (P=0.014). Interestingly, 5 of the LBW subjects (20%) and none of the NBW subjects, fulfilled the diagnostic criteria of non-alcoholic fatty liver disease (NAFLD), displaying a median hepatic fat content of 9.45%. This subgroup showed several metabolic derangements compared with NBW and LBW men without NAFLD, including hepatic insulin resistance (P=0.02) and increased fasting levels of triglycerides (TG) (P=0.03). Untargeted serum metabolomics of 65 distinct metabolites, identified 7 metabolites which showed different levels between LBW subjects with NAFLD and NBW controls (P<0.05). This included ornithine and citrulline, suggesting an upregulation of the urea cycle in LBW subjects with NAFLD. Furthermore, a pathway analysis highlighted tRNA charging as the top canonical pathway, driven by differences in amino acid levels between NBW and LBW subjects, in which LBW subjects with NAFLD exhibited elevated levels of nearly all the amino acids. Lipidome profiling included 279 lipids and revealed increased levels of phosphatidylcholines and TGs (P<0.05) in the LBW subjects with NAFLD compared to both LBW without NAFLD and NBW subjects. Both lipid species had differential structural composition in the LBW subjects with NAFLD, including an increased amount of long chain fatty acids and fewer double bonds (0-7 bonds).

Conclusion: Increased liver fat content may play a key role linking LBW with increased risk of developing T2D. The most adverse metabolic phenotype was seen in the LBW subgroup with NAFLD, displaying hepatic insulin resistance and dyslipidemia. Metabolomics and lipidomics analysis emphasize this phenotype by revealing an altered amino acid profile as well as changes in the composition and structural organization of lipids known to be associated with overt T2D.

Clinical Trial Registration Number: NCT02982408

Supported by: NNF, EFSD Organ Crosstalk Program, Trygfonden, Augustinus Fonden and Aase and Ejnar Danielsens Fond, EFSD/Boehringer Ingelheim Research Programme

Disclosure: L. Elingaard-Larsen: None.

149

3-hydroxybutyrate infusion suppresses free fatty acid concentrations in type 1 diabetes patients and healthy men

M. Bangshaab1,2, M.V. Svart3,2, N. Rittig3,2, M.G.B. Pedersen1,2, N. Møller1,3;

1Medical Research Laboratory, Department of Clinical Medicine, Aarhus University, 2Steno Diabetes Center Aarhus, Aarhus University Hospital, 3Department of Internal Medicine and Endocrinology, Aarhus University Hospital, Aarhus, Denmark.

Background and aims: Diabetic ketoacidosis (DKA) remains the most important cause of premature death in younger persons with type 1 diabetes (T1DM). Recent data have revealed the existence of a feedback loop whereby the most prominent ketone body 3-hydroxybutyrate (3-OHB) inhibits adipose tissue release of free fatty acids (FFA), which are the major ketogenic precursors. The current study was designed to test whether this feedback mechanism is disrupted in men with T1DM, as a potential pathophysiological mechanism in DKA.

Materials and methods: We used a 2x2 crossover design to study 10 men with T1DM and 10 age-matched healthy men (CTR) randomly receiving 3-hours intravenous infusion of i) Na-D/L-3-OHB and ii) NaCl, separated by a 1-hour wash-out period. Participants were fasting during examinations. Two-way repeated measures ANOVA analyses were performed to evaluate the effect of time, group and time x group interactions.

Results: During the 3-OHB infusion blood D-3-OHB concentrations rose from 0.2±0.06 to 1.2±0.12 mmol/l in T1DM, a slightly higher concentration compared to CTR, 0.2±0.03 to 1.0±0.1 mmol/l (time, p<0.001 and time x group, p=0.031). 3-OHB infusion decreased circulating FFA concentrations from 0.68±0.12 to 0.15±0.03 mmol/l in T1DM and from 0.48±0.09 to 0.21±0.04 mmol/l in CTR (time, p <0.001 and time x group, p=0.06) while during NaCl infusion, FFA were slowly increasing (time, p=0.002) in both groups. Plasma glucose concentrations were 8.3±0.1 mmol/l in T1DM and 4.7±0.04 mmol/l in CTR (group, p<0.001). Peripheral plasma insulin concentrations were 59.2±2.8 pmol/l in T1DM and 16.3±1.1 pmol/l in CTR (group, p<0.001) and plasma glucagon concentrations were 5.5±0.1 pmol/l in T1DM and 7.8±0.3 pmol/l in CTR (group, p=0.01).

Conclusion: In conclusion our data show similar suppression of blood FFA concentrations by 3-OHB in T1DM compared with CTR, thus strongly suggesting that the inhibition of lipolysis (the negative feedback) by 3-OHB is intact in men with T1DM.

Clinical Trial Registration Number: NCT04656236

Supported by: Novo Nordisk Foundation

Disclosure: M. Bangshaab: None.

150

Inhibition of VEGF-B signalling prevents non-alcoholic fatty liver disease development and progression

A. Falkevall1, A. Mehlem1, E. Folestad1, M. Zeitelhofer1, P. Scotney2, U. Eriksson1;

1Dept of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden, 2CSL Innovations Ltd, Melbourne, Australia.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is a common co-morbidity of type 2 diabetes entailing a range of pathologies from hepatic steatosis to hepatocellular carcinoma. We aimed to explore the role of VEGF-B signaling in this disease complex.

Materials and methods: Mice with adipocyte specific over-expression of VEGF-B, or adipocyte-specific deletion of Vegfb were used to investigate the role of VEGF-B signaling in adipose tissue lipolysis and the development of NAFLD.

Results: We describe a novel function of Vascular Endothelial Growth Factor B (VEGF-B) and show by using mice with adipocyte specific over-expression of VEGF-B, or adipocyte-specific deletion of Vegfb that VEGF-B signaling in adipose tissue regulates lipolysis and directly control hepatic steatosis and NAFLD development in diabetic mice. Inhibiting VEGF-B signaling, using both pharmacological and genetic strategies, in mouse models of NAFLD lowers hepatic steatosis and reduces NAFLD associated-pathologies. Mechanistically we show that VEGF-B signaling in the adipose tissue ameliorates NAFLD by resolving adipose tissue insulin resistance and the activity of the hormon sensitive lipase, a rate-limiting enzyme in lipolysis.

Conclusion: We conclude that VEGF-B antagonism represents a novel approach to combat NAFLD by targeting steatosis through suppressing adipose tissue lipolysis.

Supported by: VR Distinguished Professor Award, EFSD/Boehringer Ingelheim Research Programme

Disclosure: A. Falkevall: Employment/Consultancy; CSL Innovations Ltd. Melbourne.

OP 26 The dark side of diabetes

151

Risk of incident diabetes after hysterectomy: results from the E3N cohort study

F. Bonnet1,2, P. Vaduva1, N. Laouali3,2, G. Fagherazzi4,2, M. Kvaskoff2;

1CHU de Rennes, Rennes, France, 2Centre for Research in Epidemiology and Population Health, Inserm U1018, Villejuif, France, 3Department of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts at Amherst, Amherst, USA, 4Deep Digital Phenotyping Research Unit. Department of Population Health, Luxembourg, Luxembourg.

Background and aims: Hysterectomy has been associated with the risk of hypertension and cardiovascular disease but few studies have examined the relation between hysterectomy and the risk of type 2 diabetes. These studies mostly included post-menopausal women. Furthermore, the potential influence of diet and physical activity has not been addressed in previous reports. The aim of our study was to investigate whether hysterectomy was associated with an increased risk of incident diabetes in a large French cohort of women. In addition, we aimed to examine whether an unhealthy lifestyle influenced the risk of diabetes among women who had a hysterectomy.

Materials and methods: We studied 81 144 women from the E3N cohort who were free of diabetes at baseline; they were followed-up for a mean of 16.4 years. Assessment of diet and physical activity was by questionnaire. Women with gynecological cancers were excluded from the analyses. Cox proportional hazard models with age as the timescale were used to estimate Hazard Ratios (HR) and 95% confidence intervals (CI). Covariates included educational level, physical activity, type of diet, body mass index, smoking status, family history of diabetes, age at menarche, menopausal status, age at menopause, use of oral contraceptives.

Results: A total of 4 367 women developed type 2 diabetes during the follow-up. Women with a history of hysterectomy had an increased risk of incident diabetes, which persisted after adjustment for the main confounding factors (adjusted HR= 1.18, 95% CI 1.10-1.27, p<0.0001). The association was not altered after further adjustment for reproductive factors or hormonal treatments. The type of diet and the level of physical activity did not modify the association with incident diabetes.An increased risk of diabetes was observed irrespective of the cause of hysterectomy; for women who had a hysterectomy for endometriosis or fibroids (adjusted HR= 1.19, 95% CI 1.11-1.28). There was no interaction between the presence of overweight at baseline (BMI≥25 kg/m2) and an increased risk of diabetes. Hysterectomy with (HR: 1.23, 95% CI 1.13-1.35, p<0.001) and without oophorectomy (HR: 1.13, 95% CI 1.03-1.25, p=0.013) were both associated with an increased risk of diabetes. The risk of incident diabetes was greater for women who had a hysterectomy before the age of 50: before 40 years (adjusted HR: 1.27, 95% CI 1.07-1.50, p=0.006); between the age of 40 and 50 years (adjusted HR: 1.27, 95% CI 1.16-1.40, p<0.0001); after age 50 (adjusted HR: 1.06, 95% CI 0.95-1.18, p=0.28) as compared to those without a hysterectomy in these age groups.

Conclusion: Our findings show that women who had a hysterectomy before the age of 50 had an increased risk of developing incident diabetes. This elevated risk appears to be independent of oophorectomy and is not explained by an unhealthy diet or physical inactivity. The underlying mechanisms leading to diabetes among these women remain to be elucidated.

Disclosure: F. Bonnet: None.

152

Association between endometriosis and risk of type 2 diabetes: results of the E3N prospective cohort study

P. Vaduva1, N. Laouali2,3, G. Fagherazzi4, F. Bonnet1,2, M. Kvaskoff2;

1Department of Endocrinology, Diabetes and Nutrition, Rennes University Hospital, Rennes, France, 2Exposome and Heredity Team, Centre for Research in Epidemiology and Population Health, Inserm U1018, Villejuif, France, 3Department of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts at Amherst, Amherst, USA, 4Deep Digital Phenotyping Research Unit. Department of Population Health, Luxembourg Institute of Health, Luxembourg, Luxembourg.

Background and aims: Endometriosis is a chronic inflammatory disease estimated to occur in 10% of women of reproductive age, with an unknown etiology. It is associated with signs and symptoms such as chronic pelvic pain, chronic fatigue and infertility. Several studies suggest an association between this disease and the risk of chronic cardio-metabolic conditions such as hypertension, hypercholesterolaemia, atherosclerosis, and myocardial infarction, but its association with the risk of type 2 diabetes (T2D) is poorly documented. The aim of this study is to analyze the relationship between endometriosis and occurrence of T2D.

Materials and methods: E3N (Etude Epidemiologique après de femmes de l’Education Nationale) is a prospective cohort involving French women born in 1925-1950 followed-up since 1990. Were considered here both prevalent (diagnosed before the initial questionnaire in 1992, retrospectively reported) and incident cases (diagnosed after the initial questionnaire, prospectively reported) of endometriosis. Data on laparoscopically confirmed endometriosis were collected every 2-3 years through self-report. T2D cases were identified using diabetes-specific questionnaires and drug reimbursement insurance databases. Statistical analysis used Cox models, and stratified analyses examined the influence of age, body mass index (BMI) (<25 or ≥25kg/m²), infertility, and menopausal status on the associations between endometriosis and T2D risk.

Results: Among the 83,582 women, free of T2D at baseline, followed up for nearly 17 years, 4606 reported a diagnosis of endometriosis. Age at inclusion was 51 ± 6 years. Compared with prevalent cases, incident cases were younger at inclusion and more likely to be parous and to have ever used hormonal treatments, while they had similar height, BMI, age at menarche, and menstrual cycle length. Women with endometriosis were more likely to have a family history of diabetes (p<0.0001), personal history of elevated cholesterol (p=0.0007) or hypertension (p=0.001), younger age of menarche (p=0.0002), more frequent bilateral oophorectomy (p<0.0001), more frequent oral contraceptives use (p<0.0001) and a higher education level (p=0.017). In a model adjusted for age, BMI, physical activity, smoking, education, age at menarche, and oral contraceptive use, endometriosis was not associated with the risk of T2D (HR=1.09; 95% CI=0.92-1.29). This relationship was similar after further adjustment for family history of diabetes, hypertension, and menopausal status (HR=0.99; 95% CI=0.83-1.19). The relationship between endometriosis and T2D risk did not differ by age at inclusion, BMI, use of infertility treatments, or menopausal status.

Conclusion: Laparoscopically confirmed endometriosis was not associated with risk of type 2 diabetes in this large French cohort. These results are consistent with a recent prospective American study, suggesting that endometriosis is neither a marker nor a risk factor for type 2 diabetes.

Disclosure: P. Vaduva: None.

153

Cardiovascular and metabolic morbidity in women with previous gestational diabetes: a nationwide register-based cohort study

M.H. Christensen1,2, K.H. Rubin3,4, T.G. Petersen4, E.A. Nohr2,3, C.A. Vinter1,2, M.S. Andersen3,5, D.M. Jensen1,2;

1Steno Diabetes Center Odense, Odense University Hospital, 2Department of Gynecology and Obstetrics, Odense University Hospital, 3Department of Clinical Research, University of Southern Denmark, 4OPEN - Odense Patient data Explorative Network, Odense University Hospital, 5Department of Endocrinology, Odense University Hospital, Odense, Denmark.

Background and aims: Gestational diabetes mellitus (GDM) is associated with increased risk of cardiovascular disease (CVD). To our knowledge, the metabolic component of dyslipidemia has not been addressed as an outcome after GDM before, and nor has it been investigated whether the severity of morbidity is associated with previous GDM. Also, insulin treatment during GDM pregnancy and development of manifest diabetes mellitus (as proxies for impaired β-cell function) may further increase the risk of cardiovascular and metabolic morbidity (CVMM). Aims of this study were to investigate incidence and severity of CVMM in women with previous GDM in a Danish population and to study whether proxies of impaired β-cell function influence the incident CVMM risk.

Materials and methods: This study is a nationwide register-based cohort study on the complete cohort of 700,648 women delivering in Denmark during 1997-2018. Exposure was GDM based on ICD-10 diagnosis code. Primary outcome was an overall CVMM outcome consisting of ICD-10 diagnosis code for ischemic heart disease, heart failure, stroke/transient cerebral ischemia (TCI), hypertension, dyslipidemia, venous thrombosis, cardiac arrhythmia, and/or redemption of prescribed medication within antihypertensive, antithrombotic and/or lipid modifying agents. Secondary outcomes were: major CVD (ischemic heart disease, heart failure, stroke/TCI), hypertension (diagnosis code and/or medication), dyslipidemia (diagnosis code and/or medication), and venous thrombosis. Insulin treatment during GDM pregnancy was defined as diagnosis code of insulin treated GDM and/or redemption of insulin during GDM pregnancy. The associations were studied using multiple cox regression models with GDM entering as a time-varying exposure. Severity of morbidity was investigated as number of CVMM related hospital contacts and of redemptions of prescribed CVMM related medication in women with incident CVMM.

Results: The median follow-up period was 10.2-11.9 years depending on specific outcome (total range 0-21.9 years). GDM was associated with significantly increased risk of any CVMM (adjusted hazard ratio (aHR) 2.13 [95% CI 2.07-2.20]), major CVD (aHR 1.69 [95% CI 1.55-1.84]), hypertension (aHR 1.89 [95% CI 1.82-1.96], dyslipidemia (aHR 4.48 [95% CI 4.28-4.69]), and venous thrombosis (aHR 1.32 [95% CI 1.16-1.50]). Insulin treatment during pregnancy and development of manifest diabetes exacerbated risk estimates. Previous GDM was associated with significantly more hospital contacts with CVMM diagnosis codes and more redeemed prescriptions of CVMM related medication in women with incident CVMM within 3 years after initial hospital contact/redemption (p<0.001).

Conclusion: Previous GDM was associated with significantly higher risk of cardiovascular and metabolic morbidity, especially for incident dyslipidemia where a 4.5-fold increased risk was found. The risk was exacerbated by proxies of β-cell impairment. Severity of morbidity was significantly worse, if GDM preceded CVMM.

Supported by: Research grant from the Danish Diabetes Academy which is funded by the Novo Nordisk Foundation

Disclosure: M.H. Christensen: Grants; Danish Diabetes Academy, Region of Southern Denmark, University of Southern Denmark.

154

Association of type 2 diabetes, according to the number of risk factors within target range, with incident major depressive disorder and incident depressive symptoms

A.C.E. van Gennip1, T.T. van Sloten1, A.A. Kroon1, S. Köhler2, A. Koster3, S.J.P. Eussen4, B.E. de Galan1,5, M.T. Schram6, C.D.A. Stehouwer1;

1Internal Medicine, MUMC+, CARIM, Maastricht, 2Psychiatry and Neuropsychology, MUMC+, MHENS, Maastricht, 3CAPHRI, Social Medicine, Maastricht, 4Epidemiology, MUMC+, CARIM, Maastricht, 5Internal Medicine, Radboud UMC, Nijmegen, 6Internal Medicine, Heart and Vascular Centre, MUMC+, CARIM, MHENS, Maastricht, Netherlands.

Background and aims: Type 2 diabetes is associated with increased risks of major depressive disorder (MDD) and depressive symptoms. The extent to which risk factor modification can mitigate these excess risks is unclear. We investigated the associations between incident MDD and clinically relevant depressive symptoms among individuals with type 2 diabetes, according to the number of risk factors on target, compared to controls without diabetes.

Materials and methods: Prospective data were from UK Biobank of 77,786 individuals (n=9,047 type 2 diabetes; n=68,739 controls; baseline 2006-2010; follow-up for MDD until February, 2022). Incident depressive symptoms, defined as a Patient Health Questionnaire (PHQ-9) score of ≥10, were determined on average 7 years after baseline. Analysis was replicated using data from the Netherlands (the Maastricht Study; cohort with oversampling of type 2 diabetes; examination 2010-2017, incident depressive symptoms (PHQ-9 ≥10) assessed annually until 2017). Individuals with type 2 diabetes were categorized according to the number of seven selected risk factors within target range (nonsmoking; guideline-recommended levels of HbA1c, blood pressure, BMI, albuminuria, physical activity and diet).

Results: In UK Biobank, after a mean follow-up of 12 years, 493 (5.5%) individuals with type 2 diabetes and 2,574 (3.7%) controls had incident MDD. Compared to controls, individuals with type 2 diabetes had a higher risk of MDD (HR 1.61 (95% CI, 1.46; 1.77)). Among individuals with type 2 diabetes, excess risk of MDD decreased stepwise for a higher number of risk factors on target (Figure). Similarly, risk of depressive symptoms decreased stepwise for a higher number of risk factors on target (Figure). Individuals with type 2 diabetes who had 5-7 risk factors on target had no excess risk of depressive symptoms (OR for depressive symptoms 1.12 (0.76; 1.66)). These results were replicated in the Maastricht Study. In the Maastricht Study, compared to controls, among individuals with type 2 diabetes, the HR for depressive symptoms was 3.53 (2.55; 4.87) for individuals who had 0-2 risk factors on target, 2.19 (1.61; 2.98) for individuals who had 3 risk factors on target, 1.71 (1.25; 2.35) for individuals who had 4 risk factors on target and 1.03 (0.63; 1.68) for individuals who had 5-7 risk factors on target, respectively.

Conclusion: Among individuals with type 2 diabetes, excess risk of MDD and depressive symptoms compared to controls without diabetes decreased stepwise for a higher number of risk factors on target.

figure as

Supported by: ERDF, PoL, SDW, PSID, CVC, CARIM, CAPHRI, NUTRIM, SA, HFL, JCBV, NNF, SAN, ZonMW, NCDC, MP, DHF

Disclosure: A.C.E. van Gennip: None.

155

Pre(diabetes) and a higher level of glycaemia are continuously associated with corneal neurodegeneration: The Maastricht Study

S.B.A. Mokhtar1,2, F.C.T. van der Heide1,2, K.A.M. Oyaert1, C.J.H. van der Kallen1,2, T.T.M. Berendschot3,4, F. Scarpa5, A. Colonna5, B.E. de Galan1,2, M.M.J. van Greevenbroek1,2, C.G. Schalkwijk1,2, R.M.A. Nuijts3, M.T. Schram1,2, C.A.B. Webers3,4, C.D.A. Stehouwer1,2;

1Department of Internal Medicine, Maastricht University Medical Center+ (MUMC+), Maastricht, Netherlands, 2CARIM School for Cardiovascular Diseases, Maastricht, Netherlands, 3University Eye Clinic Maastricht, Maastricht University Medical Center+ (MUMC+), Maastricht, Netherlands, 4MHeNS School of Mental Health and Neuroscience, Maastricht, Netherlands, 5Information Engineering, University of Padova, Padova, Italy.

Background and aims: Diabetic neuropathy is a hallmark complication of type 2 diabetes. In the cornea, early morphological changes of small nerve fibers are demonstrable using corneal confocal microscopy. We aim to assess the association of glucose metabolism status and different continuous measures of glycemia with corneal nerve fiber measures.

Materials and methods: Population-based cross-sectional data from The Maastricht Study of N= 3,471 participants (mean age 59.4 years, 48.4% were men, 14.7% with prediabetes, and 21% with type 2 diabetes). We studied the associations, after adjustment for demographic, cardiovascular risk, and lifestyle factors, between glucose metabolism status (prediabetes and type 2 diabetes vs normal glucose metabolism status) and measures of glycemia (fasting plasma glucose, 2-hour post-load glucose, HbA1c, skin autofluorescence, and duration of diabetes) with a Z-score of corneal nerve fiber measures or with individual corneal nerve fiber measures (corneal nerve branch density, fiber density, fiber length, and fractal dimension). We used linear regression analyses and, for glucose metabolism status, P for trend analyses.

Results: After full adjustment, a more adverse glucose metabolism status was associated with a lower Z-score of corneal nerve fiber measures (beta [95%CI], prediabetes vs normal glucose metabolism status -0.08 [-0.17; 0.03]; type 2 diabetes vs normal glucose metabolism status -0.14 [-0.25; -0.04], P for trend = 0.001). Analogously, higher levels of fasting plasma glucose (per SD, -0.09 [-0.13; -0.05]), 2-hour post-load glucose (-0.07 [-0.11; -0.03]), HbA1c (-0.08 [-0.11; -0.04]), skin autofluorescence (-0.05 [-0.08; -0.01]), and duration of diabetes (-0.09 [-0.17; -0.00]) were all significantly associated with a lower Z-score of corneal nerve fiber measures. In general, directionally similar associations were observed for individual corneal nerve fiber measures.

Conclusion: This is the first population-based study to show that a more adverse glucose metabolism status and higher levels of glycemia are linearly and independently associated with corneal neurodegeneration; These data suggest that glycemia-associated corneal neurodegeneration is a continuous process that already starts before the onset of type 2 diabetes. Further research is needed to investigate whether early reduction of hyperglycemia can prevent corneal neurodegeneration.

Supported by: ERDF, PoL, DMoEA, SDW, PSID, CVC, CARIM, MHeNS, CAPHRI, NUTRIM, SA, HFL, PERIMED,UGfJ-C, NNF, SAN

Disclosure: S.B.A. Mokhtar: None.

156

Multimorbidity in type 1 diabetes is common and associated with increased mortality

A. Ylinen1,2, S. Hägg-Holmberg1,3, S. Mutter1,3, S. Satuli-Autere1,2, V. Harjutsalo1,3, C. Forsblom1,4, P.-H. Groop1,4, L.M. Thorn1,2, The FinnDiane Study Group;

1Folkhälsan Institute of Genetics, Folkhälsan Research Center, 2Department of General Practice and Primary Health Care, University of Helsinki, 3Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, 4Department of Nephrology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background and aims: Multimorbidity is commonly defined as the coexistence of two or more chronic conditions or diseases, and it is associated with increased mortality and decreased quality of life. Type 1 diabetes is an example of a disease accompanied by other chronic conditions at a relatively young age. The number of chronic conditions correlates with the probability of depression and severe hypoglycemic episodes, but otherwise, data on multimorbidity in type 1 diabetes are scarce. Our aim was, therefore, to study the prevalence of multimorbidity and its impact on mortality in type 1 diabetes.

Materials and methods: This study included 4,069 adults (51.4% men) with type 1 diabetes from the Finnish Diabetic Nephropathy Study, mean age at baseline 38.1±12.0 and duration of diabetes 22.2±12.4 years. The accumulation of diseases was defined based on the number of chronic conditions at baseline from a list of 32 conditions. Conditions were grouped into three subcategories: vascular comorbidities, autoimmune disorders, and other conditions. Data on conditions were collected from clinical records, questionnaires, and registers. Data on mortality were retrieved until the end of 2017. Hazard ratios for all-cause mortality were calculated in sex and age adjusted Cox regression models.

Results: The prevalence of multimorbidity was 60.4% (n=2,458) at baseline. We did not observe a difference between women and men, but an evident association with age: 31.1% in those below 30 years, 59.8% in the 30-40 years age group, 74.8% in the 40-50 group, 84.3% in the 50-60 group, and 93.2% in those above 60 years (p<0.001). We observed vascular comorbidities in 49.2% (n=2,000), autoimmune disorders in 12.7% (n=515), and other conditions in 19.8% (n=807). During a median follow-up of 16.7 (IQR 13.6-18.8) years, 784 (19.3%) participants died. Multimorbidity clearly increased mortality (HR 6.0 [95% CI 4.6-7.9], p<0.001). The figure (showing baseline prevalence of different disease combinations and their mortality HR [95% CI], compared to those with diabetes alone) highlights the increased mortality risk in those with vascular comorbidities from a HR of 5.9 (4.4-7.9) when only vascular comorbidities are present to 11.0 (7.2-16.8) when all three are present.

Conclusion: The prevalence of multimorbidity in individuals with type 1 diabetes is high and increases with age. The increased mortality related to multimorbidity is largely driven by vascular comorbidities, and the risk is further attenuated by the presence of other chronic conditions, but not by autoimmune disorders.

figure at

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

Disclosure: A. Ylinen: Grants; Biomedicum Helsinki Foundation, Stiftelsen Dorothea Olivia, Karl Walter och Jarl Walter Perkléns Minne, Finska läkaresällskapet.

OP 27 Improving your insulin sensitivity: lessons from human studies

157

Circulating microRNA signatures in prepubertal children with obesity and insulin resistance

D. Santos1,2, P. Porter-Gill3, S. Bennuri3, G. Good3, L. Delhaey3, A.E. Sørensen4, R. Shannon3,5, L.T. Dalgaard4, B. Elisabet3,5, E. Carvalho1,6;

1Obesity, Diabetes and Complications, CNC - Center for Neurosciences and Cell Biology, University fo Coimbra, Coimbra, Portugal, 2PhD program in Experimental Biology and Biomedicine, Institute for Interdisciplinary Research, Coimbra, Portugal, 3Arkansas Children’s Research Institute, Little Rock, USA, 4Department of Science and Environment, Roskilde University, Roskilde, Denmark, 5Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, USA, 6Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, USA.

Background and aims: The higher rates of diabetes in childhood reflect unhealthy life-style habits, observed by the high prevalence of obesity and related complications. Circulating microRNAs (miRs) have been suggested as biomarkers and predictors of insulin resistance. We aimed to identify an insulin resistance miR profile, in prepubertal children before development of obesity-induced glucose intolerance.

Materials and methods: Sixty-three prepubertal children (5-9 years of age) were included in a cross-sectional study. Peripheral blood was collected, and plasma miRs were evaluated using TaqMan Advanced miRNA Human Serum/Plasma plates and then were validated by RT-qPCR. Subjects were first divided into normal weight (n=20, NW) and overweight or obese (n=43, OW/OB) according to the BMI z-score. The OW/OB were subdivided into insulin sensitive or metabolically healthy obese (n=26, MHO) and insulin resistant or metabolically unhealthy obese (n=17, MUO), based on HOMA-IR≥1.95.

Results: Fasting plasma glucose levels were normal across the groups [NW: 4.92 ± 0.35 vs OW/OB: 5.07 ± 0.65 vs MHO: 4.98 ± 0.52 vs MUO: 5.23 ± 0.81, p>0.05]. Insulin levels were significantly decreased in the NW when compared with OW/OB [NW: 3.49 (2.38 - 4.71) vs OW/OB: 7.27 (4.91 - 11.10), p ≤ 0.001] and with MHO groups [NW: 3.65 ± 1.55 vs MHO: 5.43 ± 1.77, p ≤ 0.001]. Insulin levels were also different among the OW/OB group when stratified by HOMA-IR≥1.95 [MHO: 5.43 ± 1.77 vs MUO:18.16 ± 10.38, p ≤ 0.001]. Higher levels of miR-146a-5p and miR-18a-5p were observed in the OW/OB group [NW:0.03 (0.01- 0.13) vs OW/OB: 0.04 - 0.30, p = 0.003 and NW: 0.04 (0.04 - 0.07) vs OW/OB: 0.08 (0.04 - 0.18), p = 0.017]. Both miR-18a-5p and miR-146-a were positively correlated with BMI z-score (r = 0.32, p = 0.010 and r = 0.30, p = 0.018). miR-146a-5p was also correlated with the HOMA-IR (r = 0.26, p = 0.038). However, only miR-18a-5p was correlated with BMI z-score independently of the degree of insulin sensitivity [B = 0.42 (0.19), p = 0.032], while miR-146-5p [B = 0.16 (0.14), p = 0.28], miR-423-3p [B = -0.01 (0.11), p = 0.94] and miR-152-3p [B = -0.14 (0.12), p = 0.26] were associated with insulin resistance. Twenty genes, regulators of key metabolic pathways in insulin resistance development, with a positive enrichment by more than 3-fold, p ≤ 0.001, including Metal Regulatory Transcription Factor 1 (MTF1), RAR Related Orphan Receptor A (RORA) and Nuclear Receptor Coactivator 1 (NCOA1), were identified as predicted targets from at least two or more of the identified miRs.

Conclusion: We identified four miRs associated with obesity and insulin resistance, providing evidence of key alterations that occur early in prepubertal obesity. These miRs could potentially be used as biomarkers of insulin resistance.

Clinical Trial Registration Number: NCT03323294

Supported by: FCT, NIH/NIGMS, USDA/ARS, DDA

Disclosure: D. Santos: None.

158

Insulin resistance and visceral or ectopic fat deposition may independently determine metabolic and cardiovascular complications in type 1 diabetes

J.R. Snaith1,2, D.J. Holmes-Walker3,4, J.R. Greenfield1,2;

1Diabetes and Metabolism, Garvan Institute of Medical Research, Darlinghurst, 2Faculty of Medicine and Health, St Vincent's Health Care Campus, Sydney, 3Diabetes and Endocrinology, Westmead Hospital, Westmead, 4Western Clinical School, University of Sydney, Sydney, Australia.

Background and aims: Insulin resistance is an under-recognised cardiovascular risk factor in type 1 diabetes (T1D). The mechanisms linking insulin resistance to increased cardiovascular risk in T1D remain unknown. Our aim was to determine if insulin resistance is associated with adverse body composition in T1D. We hypothesised that adults with type 1 diabetes would display insulin resistance, increased visceral adipose tissue (VAT) and hepatic steatosis.

Materials and methods: Forty adults with T1D (age 37.4±8.8 years, HbA1c 7.5±0.9%, diabetes duration 22.9±8.8 years, BMI 26.4±0.9 kg/m2) and 20 age- gender- and BMI-matched non-diabetic controls (age 37.0±8.4 years, HbA1c 5.1±0.3%, BMI 26.2±4.3 kg/m2) participated in the cross-sectional component of INTIMET (Insulin Resistance in Type 1 Diabetes Managed with Metformin), a randomised double-blind placebo-controlled study. Insulin sensitivity was determined by the hyperinsulinaemic-euglycaemic clamp (60 mUm2 per min, 5.5 mmol/L). Body composition was assessed by dual-energy x-ray absorptiometry (DXA) and abdominal and anterior thigh magnetic resonance imaging (MRI). Hepatic steatosis was estimated by transient elastography and arterial stiffness by radial artery applanation tonometry. Data was expressed as mean ± standard deviation. Skewed data was log-transformed prior to analysis. T-tests and Mann-whitney U tests were used to detect between group differences. Pearson correlation was used to determine linear correlations.

Results: Participants with T1D had 29.5% lower insulin sensitivity than adults without diabetes (glucose infusion rate [GIR] 62±20 vs 88±18 μmol/min·kgFFM respectively; p=0.0004). Despite the difference in insulin sensitivity, there was no difference in DXA VAT (593±530 vs 665±590g T1D vs controls; p=0.6) or degree of hepatic steatosis (controlled attenuation parameter 229±37.0 vs 231±35.7 dB/m, respectively; p=0.9). MRI-determined liver fat (2.8±5.8 vs 2.5±2.3%, respectively; p=0.9) and anterior thigh muscle fat infiltration (4.0±1.0 vs 4.5±1.5%, respectively; p=0.3) were also comparable. T1D had increased arterial stiffness (augmentation index [AIx] 11.2±11.4 vs 4.7±13.1%, respectively; p=0.05). AIx did not correlate significantly with GIR. However, AIx correlated with VAT mass (r=0.281, p=0.03) and anterior thigh fat infiltration (r=0.473, p=0.004), largely driven by controls (r=0.716, p=0.013 vs T1D: r=0.391, p=0.059).

Conclusion: Adults with T1D demonstrated 1) a unique phenotype of insulin resistance, without the expected features of increased visceral adiposity, ectopic liver and muscle fat; 2) increased arterial stiffness; and 3) an association between aberrant fat distribution and arterial stiffness. These findings raise the possibility that insulin resistance and altered fat distribution exert independent effects on cardiometabolic complications in T1D, with visceral and arterior thigh fat being possible important drivers of cardiovascular risk. Future analyses in INTIMET will determine the relative effects of muscle vs liver insulin resistance on these metabolic parameters.

Clinical Trial Registration Number: ACTRN12619001440112

Supported by: Diabetes Australia, St Vincent's Clinic Foundation, NHMRC, UNSW

Disclosure: J.R. Snaith: None.

159

Circulating succinate response is associated with insulin sensitivity and glucose tolerance

B.D. Astiarraga1, L.M. Guasch1, M. Arnoriaga-Rodriguez2, J. Fernandez-Real2, J. Vendrell1, S. Fernandez-Veledo1;

1Diabetes and associated metabolic diseases research group - DIAMET, Hospital Universitari de Tarragona Joan XXIII, Tarragona, 2Nutrition, Eumetabolism and Health Group, Dr. Josep Trueta University Hospital, Girona, Spain.

Background and aims: Succinate emerged as a key player in metabolic processes. Fasting and meal-stimulated succinate levels are altered in obesity and type 2 diabetes (T2D) and recovery after weight loss. Moreover, circulating succinate response depends on the route of glucose administration in an incretin-like manner. We aim to explore metabolite behavior in prediabetes and its dependence on insulin resistance (IR) status.

Materials and methods: Cohort I, twenty-six volunteers received a 3-h OGTT and, on a separate day, an isoglycemic variable i.v. glucose infusion (ISO) mimicking the glycemia recorded during OGTT. Subjects were classified according to ADA criteria in normal glucose tolerance (NGT - 39 ± 11 years, BMI 28.4 ± 3.1 kg/m2, HbA1c 5.2 ± 0.1%) or impaired glucose tolerance (IGT - 48 ± 10 years, BMI 30.4 ± 3.1 kg/m2, HbA1c 5.5 ± 0.4%) groups, matched by sex-age-BMI (n=13 for both). Cohort II, ten lean (CT - 45 ± 12 years, BMI 22.1 ± 2.2 kg/m2, HbA1c 5.3 ± 0.3%) and ten sex-age matched subjects with obesity (OB - 48 ± 10 years, BMI 36.6 ± 3.0 kg/m2, HbA1c 5.5 ± 0.2%) received a 2-h euglycemic hyperinsulinemic clamp (EHC, 240 pmol.min-1.m-2). Anthropometric and clinical data were collected at baseline, and glucose, insulin, and succinate were determined during the tests for both cohorts.

Results: Cohort I, FPG was 5.4 ± 0.3 vs 6.0 ± 0.6 mmol/L, p=0.002, and 2-h glycemia was 7.1 ± 1.3 vs 8.8 ± 2.5, p=0.04, respectively for NGT and IGT groups. Further, the IGT group was hyperinsulinemic and insulin resistant (OGIS, 392.5 ± 33.5 vs 316.2 ± 49.3 ml.min-1.m-2) in comparison to NGT group (p<0.0001 for both). Fasting succinate was 52 ± 2% higher in the IGT group (p=0.01) as well as, the AUC of succinate during OGTT (50.8 ± 40.0%, p=0.003). I.V. glucose elicits a smaller succinate response by 19.6 ± 11.8% in both groups (Panel A). Cohort II, no differences were found for FPG or HbA1c between groups. At fasting, the OB group showed high insulinemia 70.0 ± 29.1 vs 22.5 ± 8.9 pmol/L (p=0.0001), and succinate levels from 66.9 ± 23.1 vs 44.9 ± 9.0 μmol/L (p=0.03), for OB and CT groups, respectively. During EHC, glucose, and insulin levels were similar between groups whereas, the OB group displayed diminished rates of glucose uptake (M/I) from 38 ± 11.6 vs 22.5 ± 13.0 mg.min-1.kgffm-1.pmol.L-1, for CT and OB groups respectively (p=0.02). Succinate levels rose in parallel to insulin in both groups (Panel B), the fold increase was doubled in CT group (p=0.003). In the pooled data, the M/I was associated with BMI (r=-0.639, p=0.002), fasting succinate (r=-0.579, p=0.007), and succinate response during EHC (r=0.504, p=0.02).

Conclusion: In prediabetes, succinate levels paralleled hyperinsulinemia and IR status. These findings support the role of succinate as an early marker of IR before beta-cell failure in T2D.

figure au

Supported by: ISCIII, MINECO, Ciberdem, FEDER, ThinkGut, ERDF

Disclosure: B.D. Astiarraga: None.

160

Repeated exposure to cold-induced shivering thermogenesis improves glucose homeostasis in overweight and obese adults

A.J. Sellers1, S.M.M. van Beek1, D. Hashim1, H. Pallubinsky1, E. Moonen-Kornips1, G. Schaart1, A. Gemmink1, T. van de Weijer2, M.K.C. Hesselink1, P. Schrauwen1, J. Hoeks1, W. van Marken Lichtenbelt1;

1Department of Nutrition and Movement Sciences, Maastricht University, 2Department of Radiology and Nuclear Medicine, Maastricht University, Maastricht, Netherlands.

Background and aims: Previously, we demonstrated that 10 days of mild cold acclimation (14-15°C, 6h/day) robustly improved insulin sensitivity in patients with type 2 diabetes, partly via an enhanced glucose transporter 4 (GLUT4) translocation in skeletal muscle, as assessed in the overnight fasted state. Although non-shivering thermogenesis is involved in mild cold acclimation, a follow-up study indicated that some level of (mild) muscle activity/shivering appears crucial in provoking the beneficial metabolic effects of cold acclimation. Therefore, we here investigated the effects of repeated bouts of cold-induced shivering thermogenesis on glucose homeostasis.

Materials and methods: In a single-arm intervention study, 15 overweight/obese men and (postmenopausal) women (n = 11 and 4, respectively, 40-75 years, BMI: 27-35 kg/m²) were exposed to 10 consecutive days of intermittent cold-induced shivering thermogenesis (10°C with 1h of shivering per day) via a water-perfused suit. Shivering thermogenesis was confirmed, by surface electromyography and visual observation, and the 1h of shivering was started when resting energy expenditure increased by 50%. Before and after the intervention, a 2-hour oral glucose tolerance test (OGTT) was performed in the overnight fasted state, under thermoneutral conditions. Prior to the OGTTs, heart rate and blood pressure were measured and muscle biopsies were taken.

Results: Repeated exposure to cold-induced shivering thermogenesis significantly reduced fasting plasma glucose concentrations (5.84 ± 0.38 vs 5.67 ± 0.32 mmol/L, p=0.013) and improved glucose tolerance during the OGTT by 6% (total area under the glucose curve: p=0.041). Plasma insulin concentrations at baseline and during the OGTT were unaffected. Interestingly, fasting plasma triglyceride and free-fatty acid concentrations were robustly decreased by 32% and 11% (p=0.001 and p=0.036, respectively). Additionally, repeated cold exposure markedly reduced systolic and diastolic blood pressure by 7.4% (p<0.001) and 8.1% (p<0.001), respectively, and tended to decrease resting heart rate (p=0.062) when measured at thermoneutrality. Analyses of muscle GLUT4 translocation are currently ongoing and will be presented during the meeting.

Conclusion: Repeated exposure to cold, leading to cold-induced shivering thermogenesis, improved glucose homeostasis and other clinically relevant metabolic health parameters in overweight/obese individuals, and hence presents an alternative strategy for the treatment and prevention of type 2 diabetes.

Clinical Trial Registration Number: NCT04516018

Supported by: ZonMW PTO

Disclosure: A.J. Sellers: None.

161

Thromboxane is elevated in men after exercise and improves skeletal muscle glucose uptake and whole-body glucose homeostasis

A.M. Abdelmoez1, M. Borg2, L. Dollet2,3, J.A.B. Smith2, A. Chibalin1, A. Krook2, J.R. Zierath1,3, N.J. Pillon2;

1Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden, 2Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 3Center for Basic Metabolic Research, Copenhagen, Denmark.

Background and aims: Prostanoids (thromboxane and prostaglandins) are lipid mediators that signal through cell surface receptors expressed in multiple cell types, including skeletal muscle. In men, exercise is associated with increased levels of prostanoids in the circulation and in skeletal muscle. Additionally, subjects with type 2 diabetes show altered levels of prostanoids in the circulation compared to healthy controls. However, the role of prostanoids in the adaptive response of skeletal muscle to exercise or metabolic disease is unknown. We hypothesized that prostanoids play a role in skeletal muscle remodelling and metabolism.

Materials and methods: The exercise response of genes involved in the synthesis of prostanoids was assessed using the MetaMEx database (metamex.eu). Blood samples were obtained from men and women with type 2 diabetes and their matched healthy controls before and after an oral glucose tolerance test. Later, the subjects underwent an acute (30 minutes) aerobic exercise bout, and blood samples were collected before and after exercise. Levels of prostanoids in plasma were measured using ELISA and LC-MS. Primary human myotubes were incubated with the thromboxane receptor agonist I-BOP and levels of glucose uptake, oxidation, and incorporation into glycogen were measured using radiolabelled substrates. Western blot was performed to track signalling events. EDL and soleus muscles from male mice were incubated ex-vivo with I-BOP, and glucose oxidation was measured using [14C]-glucose. Glucose tolerance test in mice was performed after an acute administration of I-BOP.

Results: Skeletal muscle levels of cyclooxygenase-2 (PTGS2), the enzyme required for the first step in prostanoids synthesis from arachidonic acid, was higher in women after exercise but not in men. The thromboxane synthase (TBXAS1) mRNA was elevated after exercise in men but not women. Concomitantly, levels of thromboxane B2 in plasma were higher after exercise only in men (+58%, p=0.033, n=5). Activating the thromboxane receptor with I-BOP in skeletal muscle cells resulted in increased glucose uptake (+53%, p=0.0019, n=5), oxidation (+28%, p=0.017, n=5), and incorporation into glycogen (+398%, p<0.001, n=4). This coalesced with signalling events indicative of active actin cytoskeleton remodelling and GLUT4 translocation to the plasma membrane. In isolated mouse skeletal muscle, I-BOP increased ex-vivo skeletal muscle glucose oxidation (+118%, p<0.001, n=7-8). Finally, acute administration of the thromboxane receptor agonist in mice improved glucose tolerance (AUC -34%, p=0.009, n=7-8).

Conclusion: Endogenous production of prostanoids represents a novel sex-dependent physiological adaptation to exercise. Activating the thromboxane receptor in skeletal muscle improves whole body glucose tolerance, suggesting a potential role of prostanoid in promoting the metabolic health benefits of physical activity. Our findings implicate that drugs targeting the production of prostanoids and sex-specific exercise regimens have potential to improve glucose control and overall metabolic health in individuals with type 2 diabetes.

Supported by: EFSD/Novo Nordisk Foundation Future Leaders Award NNF21SA0072747, Diabetes Wellness Sverige PG21-6524

Disclosure: A.M. Abdelmoez: None.

162

High dietary fat intake increases glucagon levels and the glucagon-to-insulin-ratio in healthy lean subjects

B. Schuppelius1, R. Schüler2, O. Pivovarova-Ramich3,4, S. Hornemann2, A. Busjahn5, J. Machann4,6, M. Kruse2, A.F.H. Pfeiffer1;

1Department of Endocrinology and Metabolism, Charité - University Medicine Berlin, Berlin, 2Department of Clinical Nutrition, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, 3Reseach Group Molecular Nutritional Medicine, Dept. of Molecular Toxicology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, 4German Center for Diabetes Research (DZD), München-Neuherberg, 5HealthTwiSt GmbH, Berlin, 6Institute of Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Centre Munich, Tübingen, Germany.

Background and aims: Emerging data support an essential role of glucagon (GCGN) for lipid metabolism. However, data on the role of dietary fat intake on GCGN secretion is limited, particularly in humans. We analyzed whether altering nutritional fat intake affects glucagon levels in healthy lean subjects.

Materials and methods: 92 twins (age: 31 ± 14 years BMI: 22.8 ± 2.7 kg/m2) consumed two 6-weeks diets: a low fat healthy diet (LFD: 30 %Energy (%E) fat, 15 %E protein, 55 %E carbohydrate) followed by an isocaloric high fat diet (HFD: 45 %E fat, 15 %E protein, 40 %E carbohydrate). 24 twins additionally continued with a high protein diet (HPD: 30 %E protein, 30 %E fat, 40 %E carbohydrate). Clinical investigation days were performed after 6 weeks of LFD, after 1 and 6 weeks of HFD and after 6 weeks of HPD. GCGN and insulin were measured with specific Mercodia sandwich assays. Liver fat content was determined by MR spectroscopy. For estimation of heritability, the “ACE” structural equation model was applied.

Results: The LFD caused a significant decrease of basal GCGN (-27 %, p ≤ 0.001) compared to screening. After 6 weeks of HFD and minimal weight gain (0,6 %, p = 0.124) GCGN increased significantly (117 %, p ≤ 0.001) while triglycerides remained similar and FFA decreased. 6 weeks of HPD further increased GCGN levels ~72 % (p = 0.502). Meal tolerance tests in a subgroup of 14 twins showed increased postprandial GCGN responses after 1 week of HFD that further increased after 6 weeks. Fasting insulin and HOMA-IR increased moderately after one week of HFD, while 6 weeks of HPD significantly decreased both. The fasting GCGN-to-insulin ratio decreased through LFD (p ≤ 0.001) but increased after HFD (p ≤ 0.001) and even further after HPD (p = 0.018). Liver fat content did not increase during the HFD and did not correlate with GCGN levels after the different diets. Heritability of GCGN was 45% after the LFD. The change of GCGN through the diets was not heritable at any time point.

Conclusion: Under isocaloric conditions high fat intake strongly increases basal and postprandial GCGN levels in healthy young subjects. The increased GCGN-to-insulin ratio suggests a reduced insulin-mediated inhibition of GCGN secretion as one potential mechanism. Nevertheless, the postprandial elevated GCGN levels indicate an additional effect of dietary fat intake. As body weight, liver fat and insulin resistance showed no clinically relevant increases, the observed rise in glucagon may represent a protective metabolic response to high dietary fat intake which increases fat oxidation to prevent fatty liver.

Clinical Trial Registration Number: NCT01631123

Supported by: BMBF (NUGAT 0 315 424 OP-R, MK, AB, AFHP), DFG grant (KFO218 PF164/16–1 OP-R, AK, AFHP)

Disclosure: B. Schuppelius: None.

OP 28 Desirable diets

163

Undesired side effects of a formula diet on erythropoetic parameters: data from two randomised controlled trials

S. Kabisch1,2, S.L. Jahn1, U. Dambeck3, M. Kemper3,2, C. Gerbracht3, C. Honsek3, N.M.T. Meyer1,2, M.A. Osterhoff3, A.F.H. Pfeiffer1,2, J. Spranger1,2;

1Charité University Hospital Berlin, Berlin, 2German Center of Diabetes Research e.V., Neuherberg, 3Clinical Nutrition, German Institute of Human Nutrition, Nuthetal, Germany.

Background and aims: Prevention and therapy of type 2 diabetes are based on lifestyle interventions. Apart from classic low-fat or low-carb dietary concepts, formula diets have been proven to be very effective due to rapid and strong weight loss, even supporting diabetes remission. These drastic dietary approaches elicit widespread benefits on various fat depots, lipid profile, glycemia and blood pressure, depending on the achieved weight loss. Thus, long-term meal replacement of up to three months is proposed to allow rapid weight reductions of >15 kg. However, the spectrum of undesired side effects of formula diets is still insufficiently described. We intended to assess detrimental effects on iron metabolism and erythropoesis in two comparable dietary RCTs using a formula diet and/or conventional food products.

Materials and methods: 269 subjects with with high-risk prediabetes and 177 patients with overt type 2 diabetes participated in our two studies on DIabetes Nutrition Algorithms for Prediabetes / Diabetes“ (DiNA-P/DiNA-D). Both trials started with a 3-week 1:1-randomised hypocaloric diet phase (1200-1500 kcal/d), being characterised as either low-carb (< 40 g/d) or low-fat (< 30 kcal%). DiNA-P used conventional food products for both diets, while in DiNA-D the low-fat approach was designed as formula diet (MODIFAST/Vitalkost Nr. 1) with small amounts of vegetables. Clinical tests included a full metabolic assessment with oGTT/MMTT, body imaging and the analysis of blood counts and iron parameters as safety outcomes. In both studies, blood sampling amounts were 180 ml before and after the diet. The statistical analysis was done by two-sided paired (within-group) and unpaired (between-group) comparisons according to the respective data distribution pattern (t-test or Mann-Whitney-U test). P values below 0.05 were considered statistically significant.

Results: In DiNA-P, both diet groups showed significant, but clinically possibly irrelevant reductions of hemoglobin, hematocrit, MCV and iron after 3 weeks, among which the effect on iron was stronger for the low-carb diet. MCH and ferritin decreased under low-fat regime, only. Erythrocyte counts remained unchanged. No patient fulfilled criteria of anaemia. In DiNA-D, both diets led to a significant reduction of erythrocyte count, hematocrit, iron and transferrin saturation. Additionally, the low-fat formula diet markedly reduced hemoglobin levels and RDW-CV, while only the low-carb diet decreased ferritin. The low-fat formula diet had a significantly stronger impact on erythrocyte count (-0.14±0.26 vs. -0.05±0.24 Tpt/L, p=0,032), hematocrit (-1.4±2.5 vs. -0.6±2.1 %, p=0,044), hemoglobin (-0.44±0.77 vs. -0.16±0.69 g/dL, p=0,020) and RDW-CV (-0.3±0.5 vs. -0.0±0.7 %, p<0,001).

Conclusion: To our knowledge, this is the first report of a consistent, qualitative alteration of erythropoetic parameters as a specific, early-onset side effect of a formula diet. In prolonged regimes - as done in trials and clinical practice for up to three months - this could lead to anaemia. Rheologically desired hemodilution and a potential impact on the validity of HbA1c measurements in these patients are additional aspects worth considering. Further clinical trials are warranted.

Clinical Trial Registration Number: NCT02459496

Supported by: German Center of Diabetes Research (DZD), German Diabetes Association (DDG), California Walnut Comm.

Disclosure: S. Kabisch: Employment/Consultancy; German Center of Diabetes Research e.V. (DZD). Grants; German Center of Diabetes Research e.V. (DZD), German Diabetes Association (DDG). Lecture/other fees; Sanofi, Lilly Deutschland, Berlin Chemie. Non-financial support; California Walnut Commission.

164

An isoenergetic multifactorial diet reduces pancreatic fat and increases postprandial insulin response in patients with type 2 diabetes: a randomised controlled trial

G. Della Pepa1, G. Costabile1, V. Brancato2, D. Salamone1, A. Corrado1, M. Vitale1, C. Cavaliere2, M. Mancini3, M. Salvatore2, D. Luongo3, G. Riccardi1, A.A. Rivellese1, G. Annuzzi1, L. Bozzetto1;

1Federico II University, 2IRCCS Synlab SDN, 3Institute of Biostructure and Bioimaging of National Council of Research, Naples, Italy.

Background and aims: Very little is known about the effect of diet composition per se, indipendently of body weight reduction, on pancreatic fat (PF). We evaluated the effect of an isocaloric multifactorial diet with a diet rich in monounsaturated fatty acids (MUFA) and similar macronutrient composition on PF and postprandial insulin response in Type 2 Diabetes (T2D).

Materials and methods: According to a randomized controlled parallel group design, 39 individuals with T2D, 35-75 years-old, in satisfactory blood glucose control, were assigned to an 8-week isocaloric intervention with a multifactorial diet rich in MUFA, polyunsaturated fatty acids, fibre, polyphenols, and vitamins (n=18) or a MUFA rich diet (n=21). Before/after the intervention, PF content was measured by the proton-density fat fraction using a 3D mDixon MRI sequence, plasma insulin and glucose concentrations were measured over a 4h test-meal with a similar composition as the assigned diet.

Results: After 8 weeks, PF significantly decreased after the multifactorial diet (15.7±6.5% vs. 14.1±6.3%, p=0.024) while it did not change after the MUFA diet (17.1±10.1% vs. 18.6±10.6%, p=0.139) with a significant difference between diets (p=0.014). Postprandial glucose response was similar in the two groups. Early postprandial insulin response (iAUC0-120) significantly increased with the multifactorial diet (36340±34954 vs. 44138±31878 pmol/L·min, p=0.037), while it did not change significantly in the MUFA diet (31754±18446 vs. 26976±12265 pmol/L·min, p=0.178), with a significant difference between diets (p=0.023). Changes in PF inversely correlated with changes in early postprandial insulin response (r=-0.383, p=0.023).

Conclusion: In T2D patients, an isocaloric multifactorial diet including several beneficial dietary components markedly reduced PF. This reduction was associated with an improved postprandial insulin response.

Clinical Trial Registration Number: NCT03380416

Disclosure: G. Della Pepa: None.

165

The role of glucagon in type 2 diabetes remission by weight loss

E. Lalama1, K. Ruether1, J. Zhang1, B. Schuppelius1, N. Kraenkel2, M. Csanalosi1, S. Kabisch1, E. Latz3, A. Christ3, A.F.H. Pfeiffer1;

1Department of Endocrinology and Metabolic Diseases, Charité, Berlin, 2Medizinische Klinik für Kardiologie, Charité, Berlin, 3University of Bonn, Bonn, Germany.

Background and aims: Remission of type 2 diabetes (T2D) was achieved by weight loss of 15kg in over 80% of patients within 6 years after diagnosis in the DIRECT study due to improvements of insulin sensitivity and insulin secretion. The role of Glucagon (GCG) has not been evaluated although GCG is thought to play a central role in the early development of diabetes by increasing hepatic glucose production and thereby insulin secretion resulting in a feed forward cycle of hyperinsulinemia and insulin resistance. The opposite view emphasizes that intra-islet alpha- to beta-cell cross talk is essential for intact insulin secretion and GCG determines hepatic fat oxidation, thus supporting intact metabolism. We investigated the role of GCG in diabetes remission within our “FAIR” study - Fasting-Associated Immune-metabolic Remission of Diabetes.

Materials and methods: Participants (n=36) with overt T2D and BMI over 27 kg/m² were studied before (V1) and 3 months after (V3) consuming an 800 (males) or 600 kcal/day (females) formula diet. Mixed Meal Tests (MMT) were done at V1 and V3, GCG, insulin, and C-peptide (all Mercodia Assays) and glucose as well as clinical routine and anthropometric values were collected. We compared tertiles of low, middle and high GCG levels and areas under the curve (AUC) in the MMTs regarding GCG, insulin, glucose, as well as HbA1c and calculated insulin sensitivity and secretion.

Results: The body weight loss of 16 ± 0.8 kg of 34 participants was associated with strong improvements of insulin sensitivity (HOMA, Matsuda and PREDIM indices) and insulin secretion (disposition index, DIO2). GCG baseline levels did not change in the lowest tertile (V1: 3.5 ± 0.4 to V3: 3.0 ± 0.5 pmol/L, ns) but decreased significantly in the middle (V1: 7.1 ± 0.3 to V3: 5.1 ± 0.6, p=0.016) and highest tertile (V1: 13.5 ± 1.1 to V3: 8.0 ± 1.2 pmol/L, p=0.003). Insulin secretion (DIO2) improved significantly in the middle (V1: 63.2 ± 15 to V3: 172.3 ± 40.2, p=0.001) and higher tertile (V1: 44 ± 7.4 to V3: 109.3 ± 18.1, p= 0.006) but not in the lower tertile. The fasting and MMT-AUC glucose, HbA1c, HOMA and Matsuda index significantly improved in all groups and insulin decreased throughout the cohort, although the changes were more pronounced in the higher GCG tertiles. Of note, we identified a subgroup of 9 participants who showed a small to no change in MMT-AUC insulin (-50 to 50 mU/L) secretion but large decreases of fasting and MMT-AUC GCG, HbA1c, HOMA-IR, Matsuda-index and fasting and MMT-AUC glucose.

Conclusion: GCG responses to weight loss-induced diabetes remission vary extensively. Higher GCG before intervention is associated with greater decreases of fasting and postprandial GCG and greater improvements of insulin secretion while improvements of insulin sensitivity were independent of GCG dynamics. Diabetes remission may be primarily GCG-dependent in a subgroup of patients which will be further characterized. Overall, our data support a protective role of GCG in early diabetes.

Clinical Trial Registration Number: NCT05295160

Supported by: EFSD/Boehringer Ingelheim Research Programme

Disclosure: E. Lalama: Grants; EFSD Boehringer Ingelheim European Research Programme.

166

Determinants of blood glucose concentrations after a high carbohydrate meal in type 2 diabetes: a multiple linear regression analysis

C. Xie, R.J. Jalleh, W. Huang, Y. Sun, K.L. Jones, M. Horowitz, C.K. Rayner, T. Wu;

Adelaide medical school, The University of Adelaide, Adelaide, Australia.

Background and aims: Postprandial glycaemia is a key determinant of overall glycaemic control in type 2 diabetes (T2D), particularly when glycated haemoglobin (HbA1c) is < 8.5%. Understanding the determinants of postprandial hyperglycaemia in T2D is therefore of major importance to optimising management. We have employed a multiple linear regression model to determine the association of blood glucose concentrations over 4 hours after a high carbohydrate meal with fasting blood glucose concentrations, the rate of gastric emptying, insulin sensitivity, and postprandial insulin, glucagon and glucagon-like peptide-1 (GLP-1) responses, in T2D patients with relatively good glycaemic control.

Materials and methods: 71 patients with T2D managed by diet and/or metformin monotherapy (39 male, age 64.7 ± 0.8 years, BMI 30.1 ± 0.6 kg/m2, HbA1c 6.6 ± 0.1 %, duration of known diabetes 5.4 ± 0.6 years) consumed a mashed potato meal (1541.8 kJ, labelled with 100 μL 13C-octanoic acid) between t = 0 to 5 min, after an overnight fast. Venous blood was sampled at t = 0, 15, 30, 60, 90, 120, 180 and 240 min for the measurement of blood glucose and plasma insulin, glucagon and total GLP-1 concentrations. Gastric emptying (expressed in kcal/min) was measured by a breath test, and insulin sensitivity by the Matsuda index. Multiple linear regression analysis was performed to examine relationships of blood glucose concentrations at each time point with fasting blood glucose, gastric emptying, insulin sensitivity, and changes from baseline in plasma insulin, glucagon and total GLP-1, with adjustment for age, sex and BMI. Data are mean values ± SEM. P < 0.05 was considered statistically significant.

Results: Postprandial blood glucose concentrations were positively associated with fasting blood glucose between t = 15 to 240 min (P < 0.001 each), and the rate of gastric emptying between t = 30 to 60 min (P < 0.001 each), and inversely associated with the increments in plasma GLP-1 between t = 90 to 180 min (P = 0.04 at t = 90 min, 0.07 at t = 120 min and < 0.001 at t = 180 min, respectively). In contrast, blood glucose was negatively associated with the rate of gastric emptying at t = 180 min (P = 0.03). At t = 240 min, blood glucose was directly associated with the Matsuda index and change in plasma insulin, but inversely with the change in plasma glucagon (P < 0.05 each).

Conclusion: In relatively well-controlled T2D, postprandial glycaemia is predictably related to fasting blood glucose concentrations, but also to the rate of gastric emptying in the early postprandial phase (within the first hour), whereas in the late phase blood glucose is dependent on GLP-1 concentrations. These observations support the concept of slowing gastric emptying and stimulating GLP-1 secretion, to minimise postprandial excursion in T2D.

Clinical Trial Registration Number: ACTRN12614001131640)

Supported by: NHMRC

Disclosure: C. Xie: None.

167

The 'early' postprandial glucagon response to a mixed meal is dependent on the rate of gastric emptying in type 2 diabetes

W. Huang1, C. Xie1, N.J.W. Albrechtsen2, K.L. Jones1, M. Horowitz1, C.K. Rayner1, T. Wu1;

1Adelaide Medical School, The University of Adelaide, Adelaide, Australia, 2Department of Clinical Biochemistry, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark.

Background and aims: Gastric emptying (GE), which exhibits a substantial inter-individual variation, is a major determinant of postprandial glycaemic and insulinaemic responses in both health and type 2 diabetes (T2D). T2D is characterised by attenuated suppression of plasma glucagon after meals, which contributes to postprandial hyperglycaemia. The relationship between the postprandial glucagon response and GE has not been reported. We examined the relationship between plasma glucagon and GE of a standardised mixed meal in well-controlled T2D.

Materials and methods: 94 patients with T2D managed by diet and/or metformin monotherapy (61 male, age 64.6 ± 0.7 years, BMI 29.8 ± 0.5 kg/m2, HbA1c 6.6 ± 0.1% and duration of known diabetes 5.3 ± 0.5 years) were evaluated on a single study day. After an overnight fast, participants consumed a mashed potato meal (1541.8 kJ: 61.4g carbohydrate, 7.4g protein and 8.9g fat, labelled with 100 μL 13C-octanoic acid) between 0-5 min. Venous blood was sampled at t = 0, 15, 30, 60, 90, 120, 180, 240 min for measurements of blood glucose (glucometer) and plasma glucagon (radioimmunoassay). Gastric emptying was assessed by breath test. Data are mean values ± SEM. P < 0.05 was considered statistically significant.

Results: After the meal, blood glucose concentrations increased progressively from 8.2 ± 0.1 mmol/L to the peak of 14.0 ± 0.31 mmol/L at t = 90 min, followed by a decline towards baseline. Plasma glucagon increased from a fasting level of 76.1 ± 2.1 pg/ml to a peak of 92.7 ± 2.6 pg/ml at t = 30 min and then decreased to a nadir of 65.6 ± 1.9 pg/ml at t = 180 min. The gastric half-emptying time (T50) was 68.2 ± 1.4 min (range 39-116 min). The incremental area under the plasma glucagon curve between t = 0-30min (glucagon iAUC0-30min) was inversely related to the T50 (r = -0.3, P = 0.007). The magnitude of increases in blood glucose from baseline at t = 30 (r = -0.3, P = 0.0003), 60 (r = -0.5, P < 0.0001) and 90 min (r = -0.3, P = 0.004) were related inversely to the T50. The increase in blood glucose at t = 30 min was related directly to the glucagon iAUC0-30min (r = 0.3, P = 0.008).

Conclusion: In well-controlled T2D, the early postprandial glucagon response to a mixed meal is related to the rate of gastric emptying, and predictive of the initial glycaemic response. These observations support the concept of slowing of gastric emptying to minimise postprandial glycaemic excursions in T2D.

figure av

Clinical Trial Registration Number: ACTRN12614001131640

Supported by: NHMRC GNT1147333

Disclosure: W. Huang: None.

168

Disparities in blood glucose and incretin responses to intraduodenal glucose infusion in healthy young males and females

T. Wu, C. Xie, W. Huang, Y. Sun, M. Horowitz, K.L. Jones, C.K. Rayner;

Adelaide medical school, The University of Adelaide, Adelaide, Australia.

Background and aims: Premenopausal women are at lower risk of type 2 diabetes compared to men, but the underlying mechanism(s) remain elusive. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) released from the small intestine, modulate insulin and glucagon secretion and play a key role in the regulation of glucose homeostasis. Incretin hormone responses to small intestinal nutrient stimulation are known to vary substantially between individuals, but the potential influence of sex has not been evaluated. We have compared blood glucose and plasma insulin, GIP and GLP-1 responses to intraduodenal glucose infusions in healthy young males and females.

Materials and methods: Data from two independent studies were included in the analysis. In Study 1, 9 females and 20 males, matched for age (female vs. male: 25.0 ± 1.5 vs. 26.3 ± 2.5 years, P = 0.2) and BMI (21.6 ± 0.6 vs. 23.5 ± 0.8 kg/m2, P = 0.3), received an intraduodenal glucose infusion at 2 kcal/min between t = 0 to 60 min. In Study 2, 10 females and 26 males, matched for age (26.4 ± 2.3 vs. 25.2 ± 1.2 years, P = 0.5) and BMI (25.2 ± 2.0 vs. 26.3 ± 1.3, P = 0.5), received an intraduodenal glucose infusion at 3 kcal/min between t = 0 to 60 min. Blood was sampled at t = 0, 15, 30, 45 and 60 min for measurements of blood glucose and plasma insulin, total GLP-1 and GIP levels. Data are means ± SEM. P < 0.05 was considered significant.

Results: Fasting blood glucose, and plasma insulin, GLP-1 and GIP, did not differ between male and female subjects in either study. In Study 1, the incremental areas under the curve between t = 0 to 60 min (iAUCs0-60min) for blood glucose (females vs. males: 135.7 ± 13.1 vs. 140.6 ± 8.5 mmol/L*min, P = 0.8) and plasma GIP (females vs. males: 1937.9 ± 159.5 vs. 1946.2 ± 98.7 pmol/L*min, P = 0.9) during intraduodenal glucose infusion (2kcal/min) did not differ between males and females. However, the iAUC0-60min for plasma GLP-1 (females vs. males: 524.8 ± 89.7 vs. 264.3 ± 57.2 pmol/L*min, P = 0.03) and insulin (females vs. males: 1787.4 ± 229.5 vs. 1030.0 ± 153.4 mU/L*min, P = 0.005) were ~2-fold higher in females than males. In Study 2, the iAUCs0-60min for blood glucose (females vs. males: 99.8 ± 11.7 vs. 110.4 ± 8.0 mmol/L*min, P = 0.5), plasma GIP (females vs. males: 1006.2 ± 124.8 vs. 1030.3 ± 67.3 pmol/L*min, P = 0.9) and plasma insulin (females vs. males: 2829.8 ± 746.1 vs. 2739.9 ± 433.8 mU/L*min, P = 0.9) during intraduodenal glucose infusion (3kcal/min) did not differ between males and females. However, the iAUC0-60min for plasma GLP-1 was 2.5-fold higher in females than males (645.3 ± 135.7 vs. 262.4 ± 54.2 pmol/L*min, P = 0.01).

Conclusion: Healthy young females exhibit comparable GIP but a markedly greater GLP-1 response to small intestinal glucose infusion than males. This disparity warrants further studies to delineate the underlying mechanisms and may also be of relevance to the reduced risk of diabetes in premenopausal women than men.

figure aw

Supported by: NHMRC

Disclosure: T. Wu: None.

OP 29 Saving sweet souls

169

Psychological resilience is predictive of future HbA 1c and mental health status in adults with new onset type 1 diabetes

S.M. Brackley1,2, N. Thomas1,2, A. Hill1,2, B. Shields1, T. McDonald1, C. Fox3, J. Huber4, A. Jones1,2;

1College of Medicine & Health, National Institute for Health Research (NIHR) Exeter Clinical Research Facility, Exeter, 2Research and Development, Royal Devon and Exeter NHS Foundation Trust, Exeter, 3Leicester Diabetes Centre, University of Leicester, Leicester, 4School of Sport and Health Sciences, University of Brighton, Brighton, UK.

Background and aims: Psychological resilience is the successful adaptation in the face of adversity. It is thought to be important for effective self-management of Type 1 diabetes, and is associated with glycaemic control in cross-sectional studies. We aimed to determine the relationship between baseline resilience following a recent diagnosis of diabetes with future glycaemic control and perceived health status, in Type 1 and 2 diabetes.

Materials and methods: We prospectively assessed the relationship between resilience with future HbA1c and health status in 1267 participants with recently diagnosed Type 1 and 2 diabetes (duration <12 months, age >=18 years) in the StartRight Study. Psychological resilience was measured using the Connor-Davidson Resilience Scale (CD-RISC) 10-item questionnaire and health status was measured using the Short Form 12-item (SF12) questionnaire. These questionnaires and HbA1c were assessed annually for up to 3 years.

Results: Mean resilience was 30.3 (SD 7.3, scale 0-40) at the first study visit (median 4 months diabetes duration) and reduced by one point over a median 24 months of follow-up [p<0.001]. At baseline, resilience was strongly correlated with the mental health component of the SF12 [Pearson’s r=0.57] and weakly correlated to the physical health component [Pearson's r=0.20]. In all participants, a difference of +10 points in baseline CD-RISC was associated with a 1.8mmol/mol lower HbA1c at 2 years [95% CI 0.7-2.8, p<0.01]. In type 1 diabetes (n=462), a difference of +10 points associated with a 4.1mmol/mol lower 2-year HbA1c [95% CI 2.0-5.7, p<0.001] and in the subgroup developing insulin deficiency (urinary c-peptide : creatinine ratio <0.2nmol/mmol, n=91) tended towards a numerically stronger association of 6.6mmol/mol lower HbA1c [95% CI -0.5 to 9.8, p=0.07]. In type 2 diabetes (n=573), CD-RISC was associated with a 1.1mmol/mol lower follow-up HbA1c per +10pts CDRISC [95% CI 0.0-2.2, p=0.06]. In all subgroups, lower baseline resilience was associated with greater decline in SF-12 assessed mental health (Standardised beta = +0.38, 95% CI 0.32-0.45, p<10-15) but not physical health (Standardised beta = +0.07, 95% CI 0.00-0.14, p=0.06).

Conclusion: A short, validated psychological resilience questionnaire close to diagnosis of type 1 diabetes is predictive of future glycaemic control and decline in perceived mental health status. This supports a need for studies to determine whether targeted interventions may improve outcomes for people with type 1 diabetes and low resilience.

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Clinical Trial Registration Number: NCT03737799

Supported by: Diabetes UK, NIHR

Disclosure: S.M. Brackley: None.

170

Caregiver-report adherence in diabetes questionnaire is predictive of 10-year HbA 1c trajectories in children and adolescents with type 1 diabetes: a population-based study

K.P. Marks1,2, N.H. Birkebæk3,2, F. Pouwer4,5, E.H. Ibfelt6, M. Thastum7, M.B. Jensen8;

1Department of Clinical Medicine - Paediatrics, Aarhus University, Aarhus N, 2Steno Diabetes Center Aarhus, Aarhus N, 3Department of Paediatrics, Aarhus University Hospital, Aarhus N, 4Department of Psychology, University of Southern Denmark, Odense, 5Steno Diabetes Center Odense, Odense, 6Steno Diabetes Center Copenhagen, Copenhagen, 7Department of Psychology and Behavioural Sciences, Aarhus University, Aarhus N, 8Department of Economics, Aarhus University, Aarhus N, Denmark.

Background and aims: Adherence or self-care skills comprise of all child/adolescent or family’s efforts that are required to fulfill an array of diabetes-specific recommendations, in collaboration with health care providers. We seek to identify distinct 10-year HbA1c trajectories in children/adolescents with type 1 diabetes (T1D) and determine whether the caregiver- and/or child/adolescent-reported answers to Adherence in Diabetes Questionnaire (ADQ) predict membership to these HbA1c trajectories, controlling for sex, age of T1D diagnosis, and insulin pump status.

Materials and methods: We analyzed longitudinal, population-based data from a 2009 Danish national survey cohort (N=672, ages 10-17 years). The survey included the caregiver- and child/adolescent-report on ADQ. HbA1c levels were obtained annually (2010-2020) from the Danish Registry of Childhood and Adolescent Diabetes and Danish Adult Diabetes Registry. First, four distinct HbA1c trajectories from early adolescence to emerging adulthood (12-27 years) were identified with group-based trajectory modeling. Second, associations of baseline ADQ scores with HbA1c trajectory/group membership were investigated by use of a multinomial logit model. We controlled for sex, age at T1D onset, and insulin pump status.

Results: Baseline participant characteristics were: n= 672 (321 [47.8%] males); mean (SD) age 14.4 (2.2) years; mean (SD) age of T1D diagnosis 8.46 (3.50) years; n= 282 (42%) insulin pump users. Four HbA1c trajectories/groups were identified: group 1 (“stable on target [HbA1c 53 mmol/mol (7.0 %)], gradual decrease”, 30% of the sample), group 2 (“above target, small wave”, 40% of the sample), group 3 (“well above target, moderate wave”, 22% of the sample), and group 4 (“well above target, large wave”, 8% of the sample). A higher caregiver-reported ADQ score was predictive of less likely membership to group 2 (coeff.= -0.68, SE= 0.24, p= 0.005), group 3 (coeff.= -1.27, SE= 0.24, p< 0.001) and group 4 (coeff.= -1.74, SE= 0.29, p< 0.001) as compared to group 1. The child/adolescent-reported ADQ score was not a significant predictor once the caregiver-reported ADQ was included in the model. Males were less likely than females to be members of group 2 (coeff.= -0.45, SE= 0.23, p< 0.05) compared to group 1, whereas age of T1D diagnosis, and insulin pump status were not predictive of any group membership.

Conclusion: About 30% of the children and adolescents had a very unfavorable HbA1c trajectory. Caregiver-reported ADQ should be used to identify children at risk of a high or increasing HbA1c trajectory across adolescence and young adulthood.

Supported by: KPM: grant # NNF17SA0031406, Danish Diabetes Academy. NHB: grant from Poul and Erna Sehested's Fond

Disclosure: K.P. Marks: Grants; KPM’s PhD scholarship and work was supported by a research grant from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation, grant number NNF17SA0031406. Other; MT and NHB are coauthors of the Adherence in Diabetes Questionnaire, which is freely available and in the public domain. MT and NHB have no financial conflicts of interest.

171

Associations between generalised anxiety disorder, glycaemic management, and demographic factors among adults with diabetes in Europe

E. Cox, E. Ye, R. Wood, C. Pang;

dQ&A, San Fransisco, USA.

Background and aims: Previous research shows that people with diabetes (PWD) have higher rates of mental health disorders, such as generalized anxiety, compared to the general population. While mental health support, such as therapy or psychiatric medication, has been shown to effectively address mental health concerns among PWD, extant research on the relationship between diabetes management and generalized anxiety is limited. Thus, the present study aims to investigate the relationship between diabetes management metrics and anxiety among PWD in Europe.

Materials and methods: From October to November 2021, 3,077 adults living with diabetes in France, Germany, Italy, Netherlands, Sweden, and the United Kingdom took an online survey in which they reported their most recent HbA1c, if they knew it (n=2,561). Glucose sensor users (n=2,011) also reported the percentage of time in a typical day spent in the target range (70-180 mg/dl), otherwise known as Time in Range (TIR). All respondents completed the Generalized Anxiety Disorder diagnostic tool (GAD-7), a 7-item validated measure assessing the severity of generalized anxiety disorder (GAD). The subsequent responses (66% type 1, 52% female) were scored and analyzed. Statistical testing was conducted using two-proportion Z-tests.

Results: PWD in Italy and the UK report the highest rates of anxiety (63% and 51%, respectively), while PWD in the Netherlands report the lowest rates (39%). Across all European countries studied, women with diabetes are more likely to report experiencing anxiety than men with diabetes (57% vs. 39%, p<0.001). Anxiety is more prevalent among adults with diabetes under 45 years of age than those 45 and above (59% and 34%, p<0.001). PWD with HbA1c levels greater than 7% are significantly more likely than those with HbA1c levels less than or equal to 7% to have moderate (13% vs. 10%, p=0.03) or severe anxiety (6% vs. 4%, p=0.04). PWD using glucose sensors with TIR under 70% have significantly higher rates of moderate or severe anxiety relative to those who spend 70% or more of time in the target range (22% vs. 14%, p<0.001).

Conclusion: This research reveals mental health disparities in nationality, gender, and age among PWD in Europe. Further, these findings highlight a link between the glycemic management of diabetes and anxiety severity. To both minimize anxiety and improve diabetes management, this study emphasizes the need for an integrated approach to mental health support and diabetes management, targeted specifically at at-risk demographic groups.

Disclosure: E. Cox: Employment/Consultancy; EC, EY, RW, and CP are employees of dQ&A, a company that provides research services for a fee to several clients (>10) in the diabetes field.

172

Relationship between elevated diabetes distress and DSM-5 personality traits: evidence from the Czech validation sample

J. Konecna1, D. Lacko2, K.D. Riegel3;

13rd Department of Medicine - Department of Endocrinology and Metabolism, General University Hospital in Prague, Praha, 2Czech Academy of Sciences, Institute of Psychology, Brno, 3Department of Addictology, 1st Faculty of Medicine, General University Hospital in Prague, Praha, Czech Republic.

Background and aims: The level of subjectively experienced Diabetes Distress has an impact on the diabetes management and treatment outcomes. Howerer, there are more psychosocial factors other than DD which could also increase the experienced burden. An example could be the lack of ability to regulate emotions or to regulate negative emotional experiences which is one of the personality traits. Personality traits are also discussed in the association of treatment outcomes or adherence to the diabetes mellitus treatment. The level of Diabetes Distress (DD) captured with a Czech version of the Diabetes Distress Scale (DDS) is observed through the prism of the DSM-5 personality traits according to the Alternative model for personality disorders (AMPD).

Materials and methods: The sample comprised 358 participants with diabetes mellitus (DM) (56.2% female, age M = 42.33, SD = 14.33 years). The subjects have completed both the Czech version of the DDS and the shortened 160-item version of the Personality Inventory for DSM-5 (PID-5). The association between the DDS and PID-5 was analyzed with multiple regressions. The DDS psychometric properties were analyzed in a structural equation modeling framework with a set of confirmatory factor analyses.

Results: Our findings in the matter of the relationship between the levels of DD and personality traits suggest a high association between the PID-5 Negative Affectivity domain and the Emotional burden DDS subscale (β = .852, pHolm < .001), and also between Negative Affectivity and the Regimen Distress DDS subscale (β = .435, pHolm = .006). Furthermore, the Czech version of the DDS showed satisfactory psychometric properties in its factor structure, internal consistency, and measurement invariance between genders and across age. The McDonald’s omega values of subscales varied from .81 in case of Regimen distress up to .92 in case of Emotional distress.

Conclusion: Several specific personality traits according to the AMPD deserve attention in the relation of the subjectively experienced levels of DD. The level of Negative affectivity among the patients with DM could affect their emotional burden level and perception of regimen distress. The DDS is a reliable scale for measuring DD in terms of research and clinical practice within Czech samples.

Supported by: Ministry of Health, Czech Republic [GJIH-1599-04-1-180] [64165]

Disclosure: J. Konecna: Grants; This work was supported by the Ministry of Health, Czech Republic [GJIH-1599-04-1-180] – conceptual development of research organization [64165] General University Hospital in Prague, Czech Republic.

173

High burden of depression, anxiety and severe obesity in young women with newly diagnosed type 2 diabetes: reports from a Swedish multicentre study of 1027 patients

E.O. Melin1, P. Wanby2, T. Neumark3, S. Holmberg2, A.-S. Nilsson Neumark4, K. Johansson5, M. Landin-Olsson1, H. Thulesius6, M. Hillman7, M. Thunander1;

1Diabetology and Endocrinology, Lund University, Lund, 2Medicine and Optometry, Linnaeus University, Kalmar, 3RaD, Region Kronoberg, Region Kalmar, Regional Executive Office - Coordination of Health Care, Kalmar, 4Department of Research, Region Kalmar County, Kalmar, 5Department of Health and Caring Sciences, Linnaeus University, Växjö, 6Linnaeus University, Kalmar, 7Lund University, Lund, Sweden.

Background and aims: Depression is a risk factor for type 2 diabetes mellitus and cardiovascular disease. The aims were to explore the prevalence of depression, anxiety, antidepressant use, obesity, Hemoglobin A1c, life-style factors, and pre-existing cardiovascular disease, in patients with newly diagnosed T2D; to explore associations with depression; and to compare with general population data.

Materials and methods: Multicentre, cross-sectional study. Inclusion criteria: adults with serologically verified newly diagnosed type 2 diabetes mellitus. Data collection and analyses included age, sex, current depression and anxiety (Hospital Anxiety and Depression Scale), previous depression, antidepressant use, body mass index (BMI), Hemoglobin A1c, and pre-existing myocardial infarction and stroke.

Results: In all 1027 participants, aged 18-94 years, the depression prevalence was 12%, and depression was associated with age (per year) (inversely) (odds ratio (OR) 0.97), anxiety (OR 12.2), previous depression (OR 7.1), antidepressant use (OR 4.2), obesity (BMI ≥30 and ≥40 kg/m2) (OR 1.7 and OR 2.3 respectively), smoking (OR 1.9), physical inactivity (OR 1.8), and women (OR 1.6) (all p ≤0.013). Younger, ≤59 years, women (n=113) compared to younger men (n=217) had higher prevalence of current depression (31% vs 12%), previous depression (43 vs 19%), anxiety (42% vs 25%), antidepressant use (37% vs 12%), obesity (BMI ≥ 30 and ≥ 40 kg/m2) (73% vs 60% and 18% vs 9% respectively), and smoking (26% vs 16%) (all p ≤0.029). Older, ≥60 years, women (n=297) compared to older men (n=400) had higher prevalence of previous depression (45% vs 12%), anxiety (18% vs 10%), antidepressant use (20% vs 8 %), and obesity (BMI ≥30 and ≥40 kg/m2) (55% vs 47% and 7% vs 3% respectively) (all p ≤0.048). Compared to the Swedish general population (prevalence of depression (women 11.2%/men 12.3%) and antidepressant use (women 9.8%/men 5.3%)), younger women with T2D had higher prevalence of current depression, and all patients had higher prevalence of antidepressant use.

Conclusion: Three risk factors for cardiovascular disease, obesity, smoking, and physical inactivity, were associated with depression in patients with newly diagnosed type 2 diabetes mellitus. The younger women had the highest prevalence of depression, anxiety, and severe obesity. All patients had higher prevalence of antidepressant use than people in the Swedish population.

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Supported by: FORSS, Region Kronoberg

Disclosure: E.O. Melin: None.

174

Let the patient choose! Patient preferences for type 2 diabetes therapy in the trimaster double-blind three-way randomised crossover trial

B. Shields1, C. Angwin1, A.G. Jones1, R.R. Holman2, N. Sattar3, N. Britten1, E. Pearson4, M. Shepherd1, A.T. Hattersley1;

1University of Exeter Medical School, Exeter, 2University of Oxford, Oxford, 3University of Glasgow, Glasgow, 4University of Dundee, Dundee, UK.

Background and aims: Determining optimal glucose lowering drugs for a person with type 2 diabetes requires balancing the benefits and side effects of alternative therapies. No randomised trials have allowed patients to compare their own experience on different drugs to decide their own preference. We aimed to examine patient preference to second/third line glucose-lowering drugs in a three-way double blind randomised crossover trial.

Materials and methods: People with poorly controlled type 2 diabetes (HbA1c >58mmol/mol) on stable metformin +/- sulfonylurea therapy received, in randomly assigned order, 16 weeks each of pioglitazone 30mg, sitagliptin 100mg, and canagliflozin 100mg. At the end of each treatment period, HbA1c, and weight were measured, and patient-reported benefits, side effects, and willingness to take the drug long-term were recorded. At the end of the study, patients were asked to rank the three drugs in order of preference and state why. Reasons were coded, by 2 independent observers, as “feeling better” or “lack of side effects” compared with the other drugs.

Results: 457 participants tried all three study drugs. For each drug, achieved HbA1c was similar (pioglitazone 59.5, sitagliptin 59.9 canagliflozin 60.5 mmol/mol, p=0.19) and similar proportions of participants stated they would take it long term (54%, 58%, 59% respectively, p=0.48). 448/457 stated a drug preference. 115 (25%) preferred pioglitazone, 158 (35%) sitagliptin, 175 (38%) canagliflozin. The drug chosen as preferred, compared with the other two, was associated with a lower HbA1c (4.8[95%CI 4.1, 5.5]mmol/mol lower, p<0.001) and less side effects (0.5[0.36, 0.65] fewer, p<0.001). Regardless of preference, pioglitazone was associated with the highest weight on therapy (2.6 [2.3, 2.9]kg higher, p<0.001). Overall, 51% chose the preferred drug because of “feeling better”, whereas 39% chose it due to “lack of side effects”, with 10% unclassifiable. Canagliflozin was less often chosen by patients because of lack of side effects (27%) compared to pioglitazone (50%) and sitagliptin (42%). However, it was most often chosen due to “feeling better” (68%) compared with sitagliptin (48%) or pioglitazone (39%).

Conclusion: After trying all three drugs, that were equally effective overall, patients preferred the drug that gave them lowest HbA1c and least side effects. We propose that in the absence of a specific indication for a particular drug, patients should be offered a brief trial of potential alternative therapies to allow them to decide which they prefer.

Clinical Trial Registration Number: NCT02653209

Supported by: MRC

Disclosure: B. Shields: None.

OP 30 Novel ways of beta cell replacement

175

A highly oxygenated hydrogel enhanced the survival of human islets encapsulated within macroencapsulation devices

D.A. Domingo-Lopez1, D. Brandhorst2, E. O’Cearbhaill3, F. Coulter3, L. McDonough4, H. Brandhorst2, S. Deotti3, P. Johnson2, H. Kelly4, G.P. Duffy1;

1College of Medicine, Nursing and Health Sciences, Anatomy and Regenerative Medicine Institute, National University of Ireland Galway, Galway, Ireland, 2Nuffield Department of Surgical Sciences, John Radcliffe Hospital, Research Group for Islet Transplantation, University of Oxford, Oxford, UK, 3School of Mechanical Engineering, Centre for Biomedical Engineering, University College of Dublin, Dublin, Ireland, 4School of Pharmacy & Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.

Background and aims: Islet transplantation aims to reverse type 1 diabetes by restoring insulin production. Post-transplantation islet survival is limited by the lack of suitable support matrix and insufficient oxygen supply (hypoxia), which is aggravated when using macroencapsulation devices. Graft failure can be overcome by cell encapsulation in highly oxygenated biomaterials, like Oxygel (OxG), able to provide O2 and extracellular matrix (ECM) support when incorporated in macroencapsulation devices.

Materials and methods: OxG was formulated by the shear mixing of Hyaluronic acid hydrogel (HA) with a Perfluorocarbon nanoemulsion and oxygenated by simultaneous O2 and gel infusion using a customized set-up (n=3). Mechanical properties; viscosity (n), storage (G’) and loss (G”) modulus, were characterized using oscillatory rheology and creep recovery test. O2 levels of oxygenated OxG (O2-OxG) and controls were monitored by oxygen microsensors. Oxygen diffusion coefficient (Dv) was estimated by experimentally fitting the O2 release to a Fickian diffusion model. Human islets were isolated (n = 7) and mixed with different matrices: (A) supplemented CMRL; (B) HA; (C) OxG or (D) O2-OxG. Afterwards, silicone macrodevices were loaded with 600 IEQ and cultured for 5 days. Islets were recovered and islet death was characterized by FDA-PI staining. Data was normalized to IEQ and related to preculture (PC) data.

Results: OxG mechanical properties showed a shear thinning behavior with a transition from viscoelastic solid to viscoelastic liquid behavior at 3-4 Pa. OxG recovered its initial viscosity after 4 min of stress removal. High O2 tension was achieved in O2-OxG and O2-PFD emulsion (475-641 torr) that was released for 90 h compared to a fast oxygen release found in O2-PBS (14 hours). Significantly smaller Dv was found in OxG (2.71 ± 0.04 x 10-10 m2 s-1) and PFD emulsion (2.75 ± 0.05 x 10-10 m2 s-1, p > 0.05 vs OxG) compared to PBS (3.93 ± 0.17 x 10-9 m2 s-1, p < 0.001 vs OxG and PFD emulsion). Human islets loss was reduced when encapsulated in O2-OxG (20.10 ± 6.36%, p > 0.05 vs PC) compared to OxG (36.80 ± 4.87 %, p < 0.001 vs PC), HA (65.40 ± 7.80 %. p < 0.001 vs PC) and CMRL (89.07 ± 2.88%, p < 0.001 vs PC). A decrease in cell death was also observed in O2-OxG (37.57 ± 1.96%, p > 0.05 vs PC [31.71 ± 2.14%]) compared to OxG (42.79 ± 1.37 %, p < 0.001 vs PC), HA (43.23 ± 2.07 %, p < 0.001 vs PC) and CMRL (49.19 ± 1.57%, p < 0.001 vs PC).

Conclusion: Oxygel was developed as an ECM-based hydrogel with optimal mechanical properties (shear-thinning and self-healing), high O2 loading capacity and slow O2 diffusion ability. The preliminary in vitro evaluation of Oxygel’s influence on human islets indicates that the incorporation of a suitable ECM within macrodevices helps maintaining the integrity of encapsulated islets/cells over time. Additionally, O2 delivery within macroencapsulation devices appears to be highly beneficial in overcoming hypoxia-mediated islet death.

Supported by: DELIVER EU Horizon 2020 MSCA programme (812865)

Disclosure: D.A. Domingo-Lopez: None.

176

Microsphere-based bioartificial islet with beta cells and mesenchymal stem cells co-encapsulation for diabetes treatment

J. Sun, L. Li;

Department of Endocrinology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.

Background and aims: Recent therapeutic approaches of type 1 diabetes mellitus (T1DM) target restoration of endogenous insulin production rather than the traditional insulin injection therapy. Thus, β-cell replacement therapy is the practical cell treatment option for the management of T1DM. However, the broad application of β-cell transplantation is greatly limited by donor shortage, side effects of immunosuppressants and persistent post-transplantation loss of β-cells due to hypoxia. Co-transplantation with mesenchymal stem cells (MSCs) could contribute to enhancing function and survival of β-cells. Biocompatible hydrogels have potential to facilitate implantation, which could eliminate direct contact of β-cells with immunocompetent cells. This study aimed to develop porous hydrogel microspheres to co-encapsulate β cells and MSCs to construct novel biological artificial islets for hyperglycemia treatment.

Materials and methods: We used microfluidic technology to co-encapsulate β-cells and MSCs in porous hydrogel microspheres based on the hybrid solution of gelatin methacrylate (GelMA) and poly (ethylene oxide) (PEO) within fast response time. The β-cell viability and insulin secretory function was examined in vitro. Naked β cells and MSCs, microspheres encapsulated with β cells alone and microspheres co-encapsulated with β cells and MSCs were implanted into the omentum of diabetic mice, and blood glucose and body weight changes were monitored. The glucose tolerance test was performed at 4 weeks of implantation and cell-laden microspheres were removed at six weeks post-transplantation to assess β-cell function.

Results: Three dimensional (3D) porous structure in microspheres facilitated β-cell proliferation in clusters. β-cells co-encapsulated with MSCs increase glucose-stimulated insulin secretion and content in vitro. The transplantation results suggested that bioartificial islets significantly reversed diabetes in diabetic mice within a week post-transplantation and continuously regulate the dynamic balance of blood glucose levels in vivo. Fluorescence microscopy showed distinct insulin-expressing cells in bioartificial islets removed after 6 weeks of implantation.

Conclusion: The bioartificial islet could be endowed with the high glucose responsiveness and ability of insulin secretion, achieving highly simulation of islet survival environment. The results of transplantation further confirmed the therapeutic potential of bioartificial islets to significantly improve hyperglycemia and achieve stable blood glucose control in diabetic mice. Overall, the novel bioartificial islet has a distinctive function in the treatment of diabetes, and could suggest a suitable platform for β cell transplantation in clinical application.

Supported by: National Natural Science Foundation of China (No 6590009658)

Disclosure: J. Sun: None.

177

Parallel single cell RNA sequencing and spatial transcriptomics analysis of the developing human pancreas

O. Olaniru1, U. Kadolsky2, S. Kannambath2, H. Vaikkinen2, K. Fung2, P. Dhami2, S.J. Persaud1;

1Department of Diabetes, King's College London, 2BRC, King's College London, London, UK.

Background and aims: There is a global research effort to generate unlimited amounts of human beta-cells in vitro for type 1 diabetes transplantation therapy but current stem cell differentiation protocols are mostly derived from mouse pancreas development, and they do not generate fully functional beta cells. We have therefore generated and integrated single cell RNA sequencing (scRNA-seq) and spatial transcriptomics of the developing human pancreas at multiple timepoints to provide detailed transcriptomic analysis of the various pancreatic cell types.

Materials and methods: Using scRNA-seq, we determined the transcriptome of over 53,000 human fetal pancreatic cells at 8, 10, 12, 13, 14, 15, 18, 19 and 20 post conception weeks. We performed 10x Visium spatial transcriptomics on 8 pancreas sections at four developmental time points covering over 10,000 barcoded spots to spatially localise the pancreatic cells. scRNA-seq and spatial transcriptomics data were integrated by canonical correlation analysis. Using time-series and pseudotime trajectory inferences including a deep-learning based spatial trajectory analysis, we uncovered differentiation transitions occurring in temporal and spatial contexts. We validated novel genes driving endocrine differentiation by smFISH and analysed the influence of pancreas microenvironment on endocrine progenitor differentiation by a connectome-based network analysis.

Results: scRNA-seq revealed distinct clusters of acinar, ductal, endocrine progenitors, alpha, beta, delta, immune, endothelial, Schwann and mesenchymal cells. Spatial transcriptomics samples were sequenced to a median depth of 177.5 × 106 reads (interquartile range 116.9-294.4 × 106), which yielded a mean of 1692 genes and 3395 unique molecular identifiers per spot. We identified spatially correlated genes and spatially proximal cells and found that spatial neighbourhoods were shared by acinar and endocrine cells, by endocrine, ductal, acinar and pancreatic progenitors, and also by endothelial and mesenchymal cells, suggesting that these neighbouring cell populations are more likely to interact together. Cell trajectory inference identified three endocrine progenitor populations and novel branch-specific genes as the endocrine progenitors differentiate towards alpha and beta cells, which were confirmed by smFISH. By integrating scRNAseq with spatial transcriptomics, we showed that mesenchymal cells undergo transition in the presence of immune cells to increase acinar cell number, with upregulation of CTRB2, SYCN, CEL and CPA1 (p<2.8x10e-4) and downregulation of COL3A1, EEF1A1, SNX3, COL1A2, RPL9 (p<3.9 x10e-5). Spatial differentiation trajectories in situ indicated that Schwann precursor cells are spatially co-located with endocrine progenitors and contribute to beta cell maturation via the L1CAM-EPHB2 pathway.

Conclusion: We have characterised and spatially resolved multiple human pancreatic cell populations at multiple developmental stages. We have identified sub-populations of human endocrine progenitors, novel genes that may direct their differentiation to beta or alpha cell lineage, and the influence of pancreas microenvironment on endocrine progenitor differentiation. Our data identified the roles of Schwann precursor cells and mesenchymal cells in the differentiation of endocrine progenitors and acinar cells, respectively.

Supported by: Novo Nordisk UK Research Foundation and NC3Rs

Disclosure: O. Olaniru: None.

178

Elucidating the role of TFB1Mgenetic variants in diabetogenic mechanisms using genome-edited stem cell models

F. Roberts, S. Hladkou, T. Singh, R. Prasad, M. Fex, H. Mulder;

Clinical Research Center, Malmö, Sweden.

Background and aims: Mitochondria play a pivotal role in linking the metabolism of nutrients and insulin release from pancreatic beta (β)-cells via metabolic coupling factors. It is established that Type 2 Diabetes (T2D) arises when insulin secretion fails due to β-cell dysfunction. Transcription Factor B1 Mitochondrial (TFB1M) is a nuclear-encoded methyltransferase, essential for ribosomal stability and translation of mitochondrially encoded genes. In this context, TFB1M function is important for physiological insulin release via proper mitochondrial function. We previously described the human TFB1M gene intronic SNP rs950994 (A/G) as an expression quantitative trait locus (eQTL). The risk (AA) allele is associated with reduced islet TFB1M expression, reduced β-cell mass, metabolic coupling and impaired insulin secretion. The rs950994 SNP is also prognostically associated with T2D traits. Therefore, we aimed to understand the pathogenetic effects of the rs950994 SNP specifically on β-cell dysfunction in T2D.

Materials and methods: We created a humanized β-cell model by utilizing human induced pluripotent stem cells (hIPSCs) differentiated into β-like cells. Fibroblasts in skin biopsies from one female and one male TFB1M rs950994 risk (AA) carrier were reprogrammed to hIPSCs and subsequently differentiated into β-like cells. The efficiency of differentiation was determined by stage-specific RNA/protein marker expression via FACS, qPCR, Western and ICC. Genome editing by CRISPR/Cas12a was performed in donor hIPSCs via homology-directed repair and the risk (AA) alleles were successfully edited to non-risk (GG) alleles. Insulin-expressing β-like cells from risk and non-risk donors were FACS-sorted using a lentivirus GFP reporter and analyzed for TFB1M mRNA expression. Insulin secretion was quantified by insulin ELISA.

Results: The hIPSCs from both donors were differentiated to insulin-expressing β-like cells with 3-5% efficiency. Rs950994 risk (AA) to non-risk (GG) allele correction was 41%, and 59% efficient in male and female hIPSCs, respectively. Compared to non-risk (GG) allele β-like cells, the insulin-producing β-like cells from a heteroclonal allele-corrected population of risk (AA) background exhibited decreased IBMX-induced insulin secretion (3.76/fold change over basal versus a 4.66-fold change) and total insulin content (486.3 versus 1297.5 mU/g total protein) and a 22% decreased TFB1M mRNA expression. Insulin data was derived from β-like cells differentiated from one female donor.

Conclusion: These data demonstrate a reduced expression of TFB1M mRNA in risk (AA) allele β-like cells coupled with reduced insulin secretion capacity and insulin content compared to non-risk (GG) variants. Thus, we have provisionally shown that theTFB1M risk variant (AA) is indeed an eQTL, which may underlie the increased risk of T2D conferred by rs950994. Further mechanistic explanations are warranted to explain this effect at the β-like cell level, including analysis of additional donors

Disclosure: F. Roberts: None.

179

Stem cell-derived islets display functional and metabolic maturation post-engraftment

T. Barsby, E. Vähäkangas, H. Montaser, J. Ustinov, J. Saarimäki-Vire, T. Otonkoski;

University of Helsinki, Helsinki, Finland.

Background and aims: Pluripotent stem cell-derived islets (SC-islets) are a promising tool for the development of translational therapies for diabetes. Following murine engraftment, SC-islets respond dynamically to glucose challenge, humanize blood glucose levels, and display gene network expression patterns closely related to those of primary human islets. However, it is unknown to what extent SC-islets maintain immature features of beta cell function post-engraftment and little is known about how nutrient-sensitive metabolic pathways develop during in vivo maturation. Here we generate SC-islets and follow their functional acquisition in parallel with metabolite tracing analyses and mitochondrial morphology throughout in vivo engraftment in mice.

Materials and methods: SC-islets were differentiated in vitro using an optimised 7-stage protocol and engrafted into the kidney capsules of NOD-SCID-Gamma mice for up to 4 months. Dynamic graft function assays as well as blood glucose and C-peptide measurements were conducted throughout the engraftment time course. Grafts were retrieved at 1- and 4-months post-implantation for immunohistochemical and electron microscopic assays of insulin granule and mitochondrial morphology. Retrieved grafts were also acutely exposed to basal and stimulatory concentrations of [U-13C6] glucose for LC-MS based metabolite flux analyses.

Results: SC-islets demonstrated increasing levels of C-peptide release and improved dynamic function during 4 months of murine engraftment. Metabolite tracing assays of SC-islets (pre- and post- humanization of murine blood glucose levels) showed that engrafted SC-islets progressively acquired glucose-responsive TCA metabolite flux patterns more closely resembling those of primary human islets. However, the labelling patterns of other TCA-derived metabolites (such as glutamine and proline) still differed from primary islets even after extended engraftment times. Furthermore, the directionality and flux of the TCA cycle in basal glucose conditions was also aberrant in SC-islet derived grafts. From this data we found that de novo aspartate production (derived from glucose metabolism) correlated with the acquisition of improved graft function. We further correlated these metabolic findings with assays of mitochondrial morphology and candidate marker proteins as proxies of functional maturity.

Conclusion: Engrafted SC-islets display progressive functional maturation within the first 4 months following implantation. Functional improvements correlated with enhanced glucose-responsive TCA-metabolite flux, although differences in the production of TCA-derived metabolites and directionality within the TCA cycle still differed between late stage grafted SC-islets and primary adult islets. The profiling of SC-islets post-engraftment is a key step in gauging the efficacy and safety of utilizing SC-islets in cell replacement therapies.

Supported by: STEMM Research Programs Unit and the Academy of Finland

Disclosure: T. Barsby: None.

180

MicroRNAs predictive of islet graft function in islet transplant recipients

W.K.M. Wong1, M.V. Joglekar1, V. Saini1,2, C.X. Dong1, P.S. Kunte3, B.L. Anderson4, K.Z. Dajani4, A.M. Simpson2, P.E. MacDonald4, P.A. Senior4, K.K. Danielson5, A.M.J. Shapiro4, A.A. Hardikar1;

1School of Medicine, Western Sydney University, Campbelltown, Australia, 2School of Life Sciences and the Centre for Health Technologies, University of Technology Sydney, Sydney, Australia, 3KEM Hospital and Research Center, Pune, India, 4University of Alberta, Edmonton, AB, Canada, 5University of Illinois, Chicago, USA.

Background and aims: Human islet transplantation is the only approved cell-based therapy for type 1 diabetes in some countries. However, several pancreatic islets die immediately after transplantation. The success of islet transplantation depends on the number of surviving and functional islet cells, which have been difficult to estimate using current methodologies. We aimed to assess microRNAs as surrogate markers of beta-cell function/death, and identify their potential to predict subsequent graft function in islet transplant recipients.

Materials and methods: Plasma samples were collected from islet transplant recipients (n=15) before and at 1-hour, 24-hours (in Edmonton) or from n=18-23 recipients at 1-week and 20-weeks (in Chicago) after transplantation of allogeneic human islets. Real-time qPCR was used to measure a panel of microRNAs that we had identified to be associated with insulin gene transcription. Significantly altered circulating microRNAs were identified using appropriate statistical methods based on data distribution and variance. We also employed machine-learning (ML) workflows to identify important microRNAs associated with fasting C-peptide levels and exogenous insulin requirement at one month post-transplant.

Results: Nineteen of the insulin transcript-associated microRNAs demonstrated an overall increase in abundance at 1-hour post-transplantation, compared to their pre-transplant levels. Two of the measured microRNAs (miR-375-3p, and miR-216b-5p) had significant increase (p<0.05) at the 1-hour post-transplant stage and then reduced at 24-hours post-transplant. Interestingly, these two microRNAs continued to remain low at 1-week and 20-weeks post-transplant. ML workflows (penalised regression with bootstrapping) identified microRNAs measured at 1-hour post islet transplant that could predict post-transplantation C-peptide levels at 1-month (spearman R=0.79, p=0.0008). Insulin requirement at 1-month post-transplant was determined with microRNAs measured at 1-hour post islet transplant using ML methodologies. The important microRNAs for determining insulin requirement at 1-month yielded an area under the curve of 0.85 (n=15, Sensitivity=0.60, specificity=0.83) in a Receiver Operating Characteristics curve analysis using leave one out cross-validation ML technique.

Conclusion: Upregulation of insulin transcript-associated microRNAs in just 1-hour post-transplant, indicates that they may be released as a result of islet cell death and could be used as biomarkers to assess beta-cell function in islet transplant recipients. Validation of these microRNAs in other sample sets will confirm the utility of these microRNAs in predicting islet graft function within an hour post-transplantation.

Supported by: JDRF International, JDRF Australia and Helmsley Trust.

Disclosure: W.K.M. Wong: None.

OP 31 Diabetes: size matters

181

IDF diabetes atlas: global prevalence estimates of prediabetes for 2021 and projections for 2045

D.J. Magliano1, M. Fang2, M. Rooney2, K. Ogurtsova3, E. Boyko4, E. Selvin2;

1Baker Heart and Diabetes Institute, Melbourne, Australia, 2Epidemiology, John Hopkins University, Baltimore, USA, 3German Diabetes Center of the Leibniz Association, Dusseldorf, Germany, 4Epidemiology, University of Washington, Seattle, USA.

Background and aims: Persons with prediabetes—impaired glucose tolerance (IGT) or impaired fasting glucose (IFG)—are at high risk for developing diabetes and microvascular and macrovascular complications. We aimed to estimate the global prevalence of IGT and IFG.

Materials and methods: We systematically reviewed 7,014 articles and reports published after 2000 to identify reliable estimates of IGT (2-hour glucose concentration of 7.8-11.0 mol/L ) and IFG (fasting glucose of 6.1-6.9 mmol/L) for each country. Studies were assessed for quality using pre-established criteria. Extracted data were modelled using logistic regression to produce smoothed age-specific prevalence estimates of IGT and of IFG for 2021, and for 2045. For countries without in-country data, estimates were extrapolated from countries with similar economies, ethnicity, geography, and language.

Results: There were 51 high-quality studies for IGT (from 43 countries) and 42 high-quality studies for IFG (from 39 countries). Approximately 80% of countries did not have high quality IFG or IGT data. The global prevalence of IGT in 2021 was 9.1% (464 million adults) and is projected to increase to 10.0% (640 million) in 2045 (Figure A). The global prevalence of IFG in 2021 was 5.6% (286 million) and is projected to increase to 6.2% (397 million) in 2045 (Figure B). The prevalence of IGT and IFG was highest in high income countries.

Conclusion: The global burden of prediabetes is substantial and growing. There is pressing need to implement interventions which will halt this increase, and to avoid the future diabetes epidemic that currently threatens to overwhelm global healthcare systems.

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Disclosure: D.J. Magliano: None.

182

Body size change from childhood to adulthood and type 2 diabetes risk

G.D. Carrasquilla, T.O. Kilpeläinen, R.J.F. Loos;

NNF Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Obesity prevalence is rising worldwide among children and adults. The high prevalence is particularly alarming among children, given the long-term health consequences and risk for chronic diseases, including type 2 diabetes (T2D). Little is known as to whether excess adiposity in childhood affects people’s risk of T2D in adulthood, independent of adult BMI or obesity status. Here, we examine whether relative body size in childhood affects T2D, independent of body size in adulthood. We also examine whether a change in relative body size from childhood to adulthood independently affects T2D risk.

Materials and methods: We used data from 378,873 individuals of European ancestry from the UK Biobank who did not have T2D in adulthood at baseline. Based on self-reported relative body size at age 10 (comparative body size higher, average, or lower than peers), we classified individuals in three sex-stratified groups: low (LowC), average (AverageC), or high (HighC). We created similar-sized sex-stratified groups for adult body size based on BMI: LowA, AverageA, and HighA. By combining childhood and adulthood body size categories, the population was divided in nine categories (Figure 1A), with most individuals being average in both childhood and adulthood. However, a substantial number of individuals changed categories between childhood and adulthood. Over an average follow up of 8.38 years, 7,042 individuals developed T2D. We applied multivariate Cox proportional regression models to obtain hazard ratios and 95% confidence intervals of incident T2D risk across the nine body size groups. Individuals with an average body size during childhood and adulthood were defined as the reference population. Analyses were adjusted for sex, age, principal components, socioeconomic factors, and various lifestyle factors including diet, physical activity, smoking and alcohol.

Results: Above average body size in adulthood (HighA) was a major risk factor for T2D (Figure 1B). Interestingly, among the “above average” (HighA) adults, those whose body size was below average during childhood (LowC-HighA) had a higher risk (HR: 4.9, 95%CI 4.4, 5.4) than those whose body size during childhood was average (HR: 3.6 95%CI 3.3, 4.0) or even above average (HR: 3.7, 95%CI 3.4, 4.2) (Figure 1B). A similar trend, although less pronounced, was observed in the other adult body size categories as well. Specifically, a below average body size during childhood increased risk of T2D in adulthood at each level of the adult body size (Figure 1B).

Conclusion: We show that below average body size in childhood increases risk of T2D in adulthood at each level of adult body size. The increase in risk is most evident among those who gain above average body size in adulthood. We speculate that lean children may have an increased susceptibility for T2D because of a limited capacity for triglyceride storage in the adipose tissue, which may lead to metabolic complications upon weight gain due to lipotoxicity in adipose tissue and systematically.

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Supported by: Novo Nordisk Foundation (NNF18CC0034900, NNF17OC0026848 and NNF17SA0031406) and MSCA (846502)

Disclosure: G.D. Carrasquilla: None.

183

Type 2 diabetes, and its modulating effect on the aging body, studied using voxel-wise analysis of whole-body MR images

J. Kullberg1,2, A. Martinez Mora1, N. Ahmad1, F. Malmberg1,3, R. Strand1,3, L. Johansson2, T. Fall4, S.C. Larsson1,5, L. Lind6, H. Ahlström1,2;

1Department of Surgical Sciences, Uppsala University, Uppsala, 2Antaros Medical, Mölndal, 3Department of Information Technology, Uppsala University, Uppsala, 4Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory, Uppsala University, Uppsala, 5Unit of Cardiovascular and Nutritional Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, 6Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Background and aims: Morphology of multiple organs and tissues have been associated with type 2 diabetes (T2D) and aging. Magnetic Resonance Imaging (MRI) can be used for detailed studies of body composition. Studies are typically performed after explicit quantification of targets of interest. Here we studied perturbations in body composition in T2D and aging at the voxel-level using a statistical image analysis approach (Imiomics) that utilizes all image data collected.

Materials and methods: Neck-to-knee water-fat MRI from participants in the UK Biobank imaging study (18 778 males, age 65.0±7.7, T2D 6.5% and 20 120 females, age 63.7±7.4, T2D 2.8%) were investigated. T2D was classified using a previously validated algorithm. Image registrations of the water-fat MRI scans were used for sex-stratified voxel-wise analysis of tissue volume and fat content throughout the body. Multiple regression was used to model voxel-wise association to T2D and age using what can be described as association imaging. Interactions between T2D and age were also studied. Key findings were confirmed using explicit measurements. This research used the UK Biobank resource (applic. no. 14237) and SNIC (sens2019016).

Results: Volume and fat content of multiple tissues were associated with both T2D (Figure 1) and age. Findings of particular interest included findings in liver, skeletal muscle, and bone marrow as well as regional differences within the subcutaneous adipose tissue (SAT), including differences in the upper vs lower body and in the anterior vs posterior lower abdomen. A lower fat content in long bones was found in T2D, especially in females and was confirmed by explicit measurements. Most tissue volumes correlated negatively with age. However, visceral adipose tissue (VAT) and lower anterior SAT show positive correlations. Liver and muscle showed higher fat contents in T2D and muscle fat fraction increased with age. T2D patterns were relatively similar in both sexes except for lower thigh muscle volumes in males. T2D and age interactions were found in upper body SAT and VAT, especially in females, as well as in liver volume, liver fat and bone marrow fat content in long bones in both sexes. Most deviations seen in T2D in middle aged subjects tended to normalize during aging.

Conclusion: This voxel-wise study of T2D and age confirmed many previous reported findings but also identified and visualized multiple findings that would be very difficult to study using other techniques.

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Supported by: Swedish Research Council, Heart-Lung Foundation, EXODIAB

Disclosure: J. Kullberg: Employment/Consultancy; Antaros Medical. Stock/Shareholding; Antaros Medical.

184

Low birth weight is associated with lower age and BMI at the time of type 2 diabetes diagnosis in the Danish DD2 cohort

A.L. Hansen1, R.W. Thomsen2, C. Brøns1, H.M.L. Svane2, J.S. Nielsen3, P. Vestergaard4, K. Højlund3, N. Jessen5, M.H. Olsen6, T. Hansen7, H.T. Sørensen2, A.A. Vaag1;

1Steno Diabetes Center Copenhagen, Herlev, 2Dept. of Clinical Epidemiology, Aarhus University, Aarhus, 3Steno Diabetes Center Odense, Odense, 4Steno Diabetes Center North, Aalborg, 5Steno Diabetes Center Aarhus, Aarhus, 6Steno Diabetes Center Zealand, Holbæk, 7Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Low birthweight (LBW) is a risk factor for type 2 diabetes (T2D), but the extent to which LBW influences the clinical presentation of T2D is unknown. We examined whether LBW is associated with a range of key clinical characteristics, including age at diagnosis, family history of diabetes, obesity, hypertension, subclinical inflammation, HBA1c, insulin secretion and action, as well as comorbidities in patients with newly diagnosed T2D in the nationwide Danish Centre for Strategic Research in Type 2 diabetes (DD2) cohort.

Materials and methods: Our analysis included 7,297 newly diagnosed T2D patients enrolled in the DD2 cohort by general practitioners and outpatient hospital clinics in Denmark during 2010-2018. We retrieved birthweight data from midwife records in the Danish National Archives. Birthweight was divided into three groups: the lowest 25% (<3000g = LBW), the middle 50% (3,000-3,700 g = normal birthweight (NBW)), and the highest 25% (>3,700g = high birthweight (HBW). Log-binomial and Robust Poisson regression analyses were used to calculate sex- and age-adjusted prevalence ratios (PRs) with 95% confidence intervals (95% CIs) of T2D characteristics associated with LBW and HBW exposure, using NBW as the reference category.

Results: In total, 1,556 (21.3%) patients had LBW, 3,279 (44.9%) NBW, and 1,544 (21,1%) HBW. Compared with NBW (median age at T2D onset = 62.3 years), LBW was associated with a lower age (59.6 years) and HBW conversely with a higher age at T2D onset (64.8 years), as reflected by opposite directed PR´s in LBW versus HBW in patients with onset before age 45 versus after 75 years (LBW: PR 1.36 (95% CI, 1.13-1.64) at age <45 versus 0.74 (95% CI, 0.59-0.92) at age >75 years. HBW: PR 0.65 (95% CI, 0.52-0.83) at age < 45 versus 1.48 (95% CI, 1.24-1.76) at age > 75 years). At T2D onset, patients with LBW were leaner (BMI <25: PR 1.11 (95% CI, 1.03-1.21)), less often severely obese (BMI >40: PR 0.55 (95% CI, 0.42-0.72), and had lower waist circumference (<94/80 cm for men/women: PR 1.34 (95% CI, 1.13-1.59)). After adjustments for sex, age and BMI, LBW patients had a higher Charlson Comorbidity Index score (≥3: PR 1.36 (95% CI, 1.06-1.75)), used more antihypertensive medications (≥3 antihypertensive drugs: PR 1.33 (95% CI, 1.06-1.67)), had increased subclinical inflammation (hsCRP >3 mg/L: PR 1.09 (95% CI, 1.00, 1.19)), and were less likely to have a family history of diabetes (no affected first-degree relatives: PR 1.08 (95% CI, 1.02-1.15)) versus 3 or more affected relatives: PR 0.68 (95% CI, 0.52-0.90)).

Conclusion: LBW was associated with younger age, less obesity and less genetic predisposition at time of T2D diagnosis. Furthermore, LBW was associated with increased subclinical inflammation, hypertension and overall comorbidity burden. There is a need for prospective studies of disease trajectories, comorbidities, complications, and mortality in T2D patients with LBW.

Supported by: Novo Nordisk Foundation

Disclosure: A.L. Hansen: Grants; Novo Nordisk Foundation.

185

Four weeks of carbohydrate overfeeding induce widespread dysmetabolic traits in men born with a low birth weight compared to matched normal birth weight controls

C. Brøns1, A.B. Thuesen2, S.O. Villumsen1, L. Justesen1, L.O. Elingaard-Larsen1, J. Størling1, M. Kim1, C. Legido-Quigley1, G. van Hall3, E.R. Danielsen3, T. Hansen2, A.A. Vaag1;

1Steno Diabetes Center Copenhagen, Herlev, 2University of Copenhagen, Copenhagen, 3Rigshospitalet, Copenhagen, Denmark.

Background and aims: Low birth weight (LBW) is a well-known risk factor for development of type 2 diabetes (T2D) later in life. This is particularly apparent when exposed to an affluent dietary lifestyle. Data from our group has indicated that impaired subcutaneous adipose tissue expandability, associated with increased ectopic (hepatic) fat deposition, may be on the critical path of T2D development in LBW subjects. We aimed to study whether a 4-week overfeeding (+25% energy) challenge, consisting of simple carbohydrates (COF), would result in differential dysmetabolic effects, including increased hepatic fat content, in LBW men compared with normal birth weight (NBW) controls.

Materials and methods: We included 22 healthy, early middle-aged, and non-obese LBW men (mean BW 2797±175 g) as well as 21 NBW controls (mean BW 3807±176 g) matched for age and BMI. We measured body composition by DXA, hepatic fat content by magnetic resonance spectroscopy, glucose and insulin metabolism, hepatic glucose production (HGP) by deuterium glucose, energy metabolism by indirect calorimetry, selected plasma biomarkers by multiplex Mesoscale Discovery technology as well as untargeted serum metabolomics and lipidomics by mass spectrometry before and after 4 weeks COF.

Results: In response to COF LBW, but not NBW subjects, displayed increased fasting plasma levels of glucose (P=0.03), C-peptide (P=0.01), leptin (P=0.0002) and FGF21 (P=0.005), as well as increased energy expenditure (P=0.0002) and fat oxidation rates (P=0.02) compared to baseline. COF resulted in similar significant increases in body weight in both groups. Hepatic fat content increased slightly in both LBW (P=0.023) and NBW (P=0.018) subjects after COF, however, with the LBW group having significantly increased hepatic fat content before and after COF compared with NBW controls. Fasting plasma adiponectin was significantly reduced in LBW compared with NBW subjects both before and after COF. Among 65 metabolites, 8 were differentially abundant between the groups (P<0.05) after COF. Ingenuity pathway analyses (IPA) revealed accumulation and increased lipid peroxidation in LBW subjects after COF. Further, IPA showed activation of the PPARGC1A pathway in LBW subjects contrasting a minor downregulation of the pathway in NBW subjects after COF. Finally, among 279 lipids identified by lipidomics, the LBW subjects exhibited a significantly (PFisher=0.0004) higher number of lipids to be increased after COF (n=26) compared to NBW subjects (n=6). This finding was further supported by a network analysis showing a general upregulation of the lipid profile in LBW subjects opposing a slight downregulation in NBW controls after COF.

Conclusion: LBW subjects at increased risk of T2D respond to COF with differential increases in plasma glucose, C-peptide, leptin and FGF21 levels, as well as with increased fat oxidation rates and severe differential perturbations of lipid metabolism, compared to NBW controls. The findings stress the importance of LBW individuals refraining from overeating simple carbohydrates. Additional studies are needed to further understand the role of increased hepatic fat accumulation in LBW subjects in T2D development.

Clinical Trial Registration Number: NCT02982408

Supported by: EFSD/Lilly Research Programme, Organ Cross-talk, NNF, Augustinus Foundation, Aase and Ejnar Danielsen Foundation, Trygfonden

Disclosure: C. Brøns: Grants; European Foundation for the Study of Diabetes, Novo Nordisk Foundation, Aase and Ejnar Danielsens Fond, Augustinus Foundation, TrygFonden.

186

Efficacy of metformin in preventing progression to diabetes in Chinese subjects with impaired glucose regulation: results from a multicentre, open-label RCT

L. Zhang1, Y. Zhang2, S. Shen3, X. Wang4, Q. Li5, L. Ji6, N. Sun6, G. Li7, CDPP study group;

1The Second Hospital of Hebei Medical University, Shijiazhuang, 2Baoding First Central Hospital, Baoding, 3Yanji Hospital, Yanji, 4Jinzhou Central Hospital, Jinzhou, 5Kailuan General Hospital, Tangshan, 6Peking University People’s Hospital, Beijing, 7Chinese Association of Geriatric Research, Beijing, China.

Background and aims: Impaired glucose regulation (IGR) is an important risk factor for the development of diabetes, and includes impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). Pharmacological agents can decrease the rate of progression from IGT to diabetes, with the ADA stating that metformin currently has the strongest body of evidence. The objectives of this study were to determine whether lifestyle intervention (LSI) plus metformin was superior to LSI alone in preventing diabetes, and to assess the effect of metformin on weight loss and blood pressure in Chinese subjects with IGR.

Materials and methods: In this study, 1706 subjects with IGR, including IFG (fasting plasma glucose 6.1-6.9 mmol/L and 2-h postprandial glucose <7.8 mmol/L) and IGT (fasting plasma glucose <7.0 mmol/L and 2-h postprandial glucose 7.8-11.1 mmol/L) by OGTT, were randomly assigned (1:1) to LSI plus metformin (n=848) or to LSI only (n=858) . The primary endpoint was the incidence of diabetes based on OGTT. Secondary endpoints were weight and blood pressure reduction. Safety was assessed by the incidence of adverse events (AEs).

Results: The mean follow-up time was 1.99±0.62 and 1.94±0.60 years in the LSI plus metformin and LSI groups, respectively ; incidence of diabetes was 18.23 and 20.72 per 100 person-years. The risk of diabetes was 17% lower in the LSI plus metformin group vs the LSI group (HR 0.83; 95% CI 0.70-0.99); however, in a prespecified analysis of subjects with IFG alone, the risk of diabetes was not significantly different between two groups (HR 0.84; 95% CI 0.55-1.30). Progression to diabetes was significantly delayed in the LSI plus metformin group (Figure ). The LSI plus metformin group showed a greater weight reduction after 2 years (1.03 kg) than the LSI group (−2.43±0.28 vs −1.40±0.27 kg respectively, p=0.0002). In subjects who were hypertensive at baseline but not receiving an anti-hypertensive agent, the mean change in systolic/diastolic BP was not significantly different between groups: −9.4/−3.9 mmHg (LSI plus metformin group) and −8.97/−3.4 mmHg (LSI group). The incidences of AEs and serious AEs were 34.55% and 1.71% in the LSI plus metformin group, and 22.68% and 1.29% in the LSI group, respectively.

Conclusion: This study demonstrated that LSI plus metformin is more effective than LSI alone in reducing the risk of diabetes, delaying progression to diabetes, and reducing body weight in Chinese adults with IGR. The effects for subjects with IFG alone remain uncertain.

figure bc

Clinical Trial Registration Number: ClinicalTrials.gov ID: NCT03441750

Supported by: Chinese Association of Geriatric Research (CAGR) and Merck Serono China Co. Ltd.

Disclosure: L. Zhang: Other; This study was sponsored by the Chinese Association of Geriatric Research (CAGR) and was funded by Merck Serono China Co. Ltd., an affiliate of Merck KGaA, Darmstadt, Germany.

OP 32 Pain or no pain?

187

Painful diabetic peripheral neuropathy in type 1 diabetes in the Epidemiology of Diabetes Interventions and Complications (EDIC) study

B.H. Braffett1, L. El ghormli1, J.W. Albers2, E.L. Feldman2, R.A. Gubitosi-Klug3, W. Herman2, C.L. Martin2, T.J. Orchard4, B.A. Perkins5, N.H. White6, J.M. Lachin1, R. Pop-Busui2, DCCT/EDIC Research Group;

1George Washington University, Rockville, USA, 2University of Michigan, Ann Arbor, USA, 3Case Western Reserve University, Cleveland, USA, 4University of Pittsburgh, Pittsburgh, USA, 5University of Toronto, Toronto, Canada, 6Washington University St. Louis, St Louis, USA.

Background and aims: We evaluated the prevalence and incidence as well as risk factors associated with painful diabetic peripheral neuropathy (DPN) in participants with type 1 diabetes (T1D) enrolled in the Epidemiology of Diabetes Interventions and Complications (EDIC) study, an observational follow-up of the Diabetes Control and Complications Trial (DCCT).

Materials and methods: Data were obtained from 1,401 participants followed for 26 years during EDIC. The Michigan Neuropathy Screening Instrument (MNSI) questionnaire, which includes specific neuropathic pain questions (Q2: “Do you ever have any burning pain in your legs and/or feet?” and Q6: “Does it hurt when the bed covers touch your skin?”), was administered annually. Painful DPN was defined as a positive response to Q2 and/or Q6 plus a MNSI examination score >2. Sustained painful DPN was defined as occurring on 2 or more consecutive annual visits. In addition, Kaplan-Meier estimates were used to describe the 26-year cumulative incidence of the first occurrence of any sustained painful DPN.

Results: Over the 26-year duration in EDIC, the prevalence of neuropathic pain (Q2 and/or Q6) increased from 8.5% to 19.8% while the prevalence of a MNSI examination score >2 increased from 22.9% to 43.5%. During EDIC, 889 (63.5%) participants did not experience any painful DPN while 512 (36.5%) experienced painful DPN at least once, of which 263 (18.8%) met the criteria for sustained painful DPN. After adjusting for age at DCCT closeout and DCCT treatment group, participants with sustained painful DPN had significantly higher BMI, waist circumference, systolic blood pressure, pulse pressure, heart rate, total and LDL cholesterol, triglycerides and HbA1c levels over time versus participants without any painful DPN during the study (p<0.0005 for all). Among the 1,348 participants without painful DPN at EDIC year 1, the 26-year cumulative incidence of sustained painful DPN was 18% (Figure).

Conclusion: In this large cohort of well-characterized T1D participants, the prevalence of neuropathic pain and DPN increased steadily over the 26-year follow-up. These data also suggest that differences in several cardiometabolic risk factors, in addition to glucose control, may contribute to differences in painful DPN risk.

figure bd

Clinical Trial Registration Number: NCT00360893

Supported by: NIH/NIDDK

Disclosure: B.H. Braffett: None.

188

Increased functional connectivity of the Thalamus and primary Somatosensory cortex and insular cortex following treatment withdrawal: a potential biomarker of painful-DPN

G. Sloan1, D. Selvarajah2, K. Teh3, I. Wilkinson3, S. Tesfaye1;

1Sheffield Teaching Hospitals, 2Department of Oncology and Human Metabolism, 3Academic Unit of Radiology, Sheffield, UK.

Background and aims: Altered functional connectivity has been identified in key brain regions involved in somatosensory perception in patients with painful diabetic peripheral neuropathy (painful-DPN), using resting state functional-magnetic resonance imaging (fMRI). However, these studies have not looked at the impact of neuropathic pain treatments. A greater understanding of treatment upon these pain processing areas might lead to greater understanding of the mechanisms of these pharmacotherapeutic agents and also development of new treatments that target these brain regions.

Materials and methods: A total 15 participants (Age, 62.1 ± 9.0; HbA1c 65.4 ± 16.2 mmol/mol; 13 type 2 diabetes, 1 type 1 diabetes and 1 MODY; 13% female) enrolled in the OPTION-DM clinical trial (ISRCTN17545443) underwent neuroimaging. All participants had clinical and neurological assessments, including the modified Toronto Clinical Neuropathy Score, Doleur Neuropathique 4 and Neuropathic Pain Symptom Inventory. Participants underwent fMRI imaging using 3T (Achieva, Phillips Healthcare) when the participants were on maximum tolerated medication (Treatment Scan) and one week after washout of these medications (Washout Scan). The data was analysed using Conn Functional Connectivity Toolbox in SPM.

Results: There was a significant increase in Pain Numeric Rating Scale (NRS) from Treatment Scan (4.0 ± 2.1) to the Washout Scan (6.1 ± 2.4, p=0.044). There was a significantly greater functional connectivity between the Primary Somatosensory Cortex (S1) and the Thalamus, and the Insular Cortex and Thalamus (p false discovery rate [FDR] = 0.041) during the Treatment Scan compared with the Washout Scan. Moreover, there was a significant difference in the change between scans in S1 - Thalamic functional connectivity in participants with Severe-Pain (NRS ≥8 at baseline: Age, 64.5 ± 10.1; 10% Female; -0.372 ± 0.275) compared to participants with Moderate-Pain (NRS ≤7: Age, 57.4 ± 3.0; 20% Female; -0.051 ± 0.180, p=0.035). The change in S1-Thalamic connectivity also correlated with a number of variables including baseline pain (r -0.585, p=0.022), NPSI (r -0.597, p=0.019) and the difference in NRS at the Treatment Scan and Baseline pain (r -0.513, p=0.050).

Conclusion: This is the first study to look at the impact of neuropathic pain medication withdrawal on functional connectivity of pain matrix brain regions. On neuropathic pain medication withdrawal there is an increase S1 - Thalamus and Insular Cortex - Thalamus functional connectivity. Moreover, the change in S1 - Thalamus functional connectivity from the Treatment Scan to Withdrawal Scan differentiated participants with high and low baseline neuropathic pain and correlated with baseline pain. This study further demonstrates that the thalamus is a key area for the central mechanisms of painful-DPN and its functional connectivity to the S1 and Insular Cortex has the potential to act as a biomarker of painful-DPN.

Disclosure: G. Sloan: None.

189

Better target engagement of opioid receptor systems in responders to neuropathic pain treatment: a neurotransmitter-enriched functional connectivity mapping study

D. Selvarajah1, K. Teh2, J. Mcallister1, A. Anandhanarayanan1, J. Fan1, G. Sloan3, S. Tesfaye3;

1Department of Oncology and Metabolism, University of Sheffield, 2Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, 3Diabetes Research Department, Sheffield Teaching Hospitals, Sheffield, UK.

Background and aims: The neurobiological mechanisms underlying treatment response in painful diabetic neuropathy are poorly understood. We have demonstrated that responders to neuropathic pain treatment have greater functional connectivity between the insula cortex and the corticolimbic system compared to non-responders. Activity within these networks is mediated by endogenous opioid receptor systems and may hold clues to possible future treatment targets.

Materials and methods: 43 painful-DN subjects [responders (VAS<4; n=29) and non-responders (VAS>4; n=14)] underwent detailed clinical and neurophysiological assessment, and RS-fMRI. Data analysis was performed using the NITRC Functional ConnectivityToolbox and SPM8 in MATLAB. RS-fMRI data was masked and binarised using an opioid receptor atlas to restrict the analysis to the voxels with high receptor density. Subject-specific spatial maps of responders and non-responders were compared.

Results: Compared to painful-DN non-responders, responders had greater functional connectivity between the corticolimbic system with the opioid receptor networks [F(2)(41)=43.53;intensity=128.7; R-amygdala beta=0.48; p-FDR<0.0001; R-putamen beta=0.3; p-FDR<0.0001; dorsal lateral prefrontal cortex beta=0.25; p-FDR=0.0002 and the posterior parietal cortex networks beta0.25; p-FDR=0.0002].

Conclusion: Painful DN treatment responders have better target engagement of opioid receptors systems compared to non-responders. Whilst further clinical validation in larger cohorts is warranted, our findings suggests that a functioning/intact descending pain inhibition network is crucial for a better pain response. Interventions targeted at this network could provide better pain relief in non-responders to neuropathic pain treatment.

Supported by: EFSD/Lilly Research Programme

Disclosure: D. Selvarajah: None.

190

Declining incident rates of distal polyneuropathy in a cohort study, but with distinct age-related patterns between diabetes types in the period 1996-2018

H. Mizrak, H. Amadid, P. Rossing, D. Vistisen, C. Hansen;

Steno Diabetes Center Copenhagen, Herlev, Denmark.

Background and aims: Distal symmetric sensorimotor polyneuropathy (DSPN) is irreversible and debilitating for people with diabetes and imposes a considerable burden on health care systems. The incidence of major diabetic complications, such as cardiovascular disease, renal disease, blindness and amputation, has been decreasing in many countries over the last few decades. However, it is unknown if this applies to diabetic neuropathy. The aim of this study was to estimate temporal changes in the incidence rates of DSPN in individuals with type 1 diabetes (DM1) and type 2 diabetes (DM2) in a longitudinal cohort at a tertiary diabetes treatment facility. In addition, differences in age-related incidence rates were investigated.

Materials and methods: In the period between 1996-2018, we identified 13,650 individuals with DM1 and DM2, with repeated measures of vibration perception threshold (VPT) and a height measure. Individuals with prevalent neuropathy at the first performed foot exam were excluded. In total 2783 individuals, 1071 individuals with DM1 and 1712 individuals with DM2 were included in the further incidence analysis. VPT was measured with Bio-Thesiometer and age- sex- and height-specific cut-off values were used to assess the presence of DSPN. DSPN was defined as decreased VPT in both feet. Incidence rates of DSPN, sex, age and calendar time were modelled by Poisson regression for time-split data and separate for DM1 and DM2.

Results: From 1996 to 2018, 737 cases of incident DSPN occurred during 18,677.5 person-years (PY) among individuals with DM1 corresponding to an overall incidence rate of 19.5/100 PY, and 709 cases of incident DPSN occurred during 18,677.5 PY among individuals with DM2 corresponding to an overall incidence rate of 15.6/100PY. For both DM1 and DM2 we observe a decreasing incidence rate in the period 2007-2017, similar in all ages (data not shown). For DM1 decreasing incidence rate occurs with increasing age, and there are no sex differences (figure 1A). However, we observe an increasing incidence rate with increasing age for DM2, and we see no sex differences (figure 1B).

Conclusion: This cohort study shows that the incidence rate for DSPN in both DM1 and DM2 has been declining from 2007 to 2017. With increasing age, individuals with DM1 and DM2 have, respectively, declining and increasing incidence rates of DSPN.

figure be

Disclosure: H. Mizrak: None.

191

Type 2 diabetes phenotypes and polyneuropathy: a prevalence study in the DD2 Cohort

F.P. Kristensen1, D.H. Christensen1, B.C. Callaghan2, J. Stidsen3, J.S. Nielsen3, K. Højlund3, T.S. Jensen4, P. Vestergaard5, N. Jessen6, T. Hansen7, R.W. Thomsen1;

1Dept. of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark, 2Dept. of Neurology, University of Michigan, Ann Arbor, USA, 3Steno Diabetes Center Odense, Odense University Hospital, Odense, Denmark, 4Dept. of Neurology, Aarhus University Hospital, Aarhus, Denmark, 5Steno Diabetes Center North Denmark, Aalborg University Hospital, Aalborg, Denmark, 6Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark, 7Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Hyperinsulinemia may cause diabetic polyneuropathy (DPN), but large-scale studies are scarce. Three phenotypes of type 2 diabetes (T2D) have recently been proposed: hyperinsulinemic (low insulin sensitivity, high beta cell function), classical (low insulin sensitivity, low beta cell function), and insulinopenic (high insulin sensitivity, low beta-cell function). We aimed to investigate the association of hyperinsulinemia with DPN.

Materials and methods: We included 3,397 recently diagnosed T2D patients prospectively enrolled from general practitioners and outpatient hospital clinics in Denmark, 2010-2015. Insulin sensitivity and beta-cell function were quantified with the HOMA2 model based on fasting serum C-peptide and plasma glucose levels measured at enrollment. DPN was defined as a score of ≥4 on the Michigan Neuropathy Screening Instrument questionnaire sent out median 3 years after study enrollment. We imputed missing values of potential confounders and applied Poisson regression to calculate adjusted prevalence ratios (PR) of DPN.

Results: We identified 900 (27%) hyperinsulinemic, 2,150 (63%) classical, and 347 (10%) insulinopenic T2D patients. Hyperinsulinemic patients had the highest prevalence of central obesity (waist circumference ≥88/102 cm [F/M]; 89% of hyperinsulinemic, 75% of classical, and 36% of insulinopenic) and had more dyslipidemia and hypertension, but less dysregulated HbA1c (≥53 mmol/L; 9%, 16%, 22%). The age-, sex-, and diabetes duration adjusted PR of DPN was 1.44 (95% CI 1.23-1.68) for hyperinsulinemic T2D patients, compared with the classical phenotype. The prevalence remained elevated (1.32 [1.13-1.55]) after further adjustment for waist circumference, dyslipidemia, hypertension, and HbA1c. For the insulinopenic patients, the adjusted PRs of DPN were 0.87 (0.65-1.15) and 1.16 (0.86-1.55), respectively. In spline analyses, both hyperinsulinemia (ie., higher beta-cell function) and lower insulin sensitivity associated with increased prevalence of DPN. The association between increasing hyperinsulinemia and increasing DPN prevalence persisted among patients with low insulin sensitivity (classical or hyperinsulinemic patients). In contrast, among hyperinsulinemic (high beta-cell function) patients, decreasing insulin sensitivity was not associated with DPN.

Conclusion: The prevalence of DPN is increased in T2D patients with the hyperinsulinemic phenotype. Hyperinsulinemia per se is associated with DPN, irrespectively of insulin resistance and other metabolic factors.

Supported by: Novo Nordisk Foundation.

Disclosure: F.P. Kristensen: None.

192

Environmental risk factors of incident distal sensorimotor polyneuropathy: results from the population-based KORA F4/FF4 study

C. Herder1,2, S. Zhang3, K. Wolf3,2, H. Maalmi1,2, G.J. Bönhof1,4, W. Rathmann1,2, L. Schwettmann3,5, B. Thorand3,2, M. Roden1,4, A. Schneider3, D. Ziegler1,4, A. Peters3,2;

1German Diabetes Center, Düsseldorf, 2German Center for Diabetes Research, München-Neuherberg, 3Helmholtz Zentrum München, Neuherberg, 4Heinrich Heine University Düsseldorf, Düsseldorf, 5Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

Background and aims: Distal sensorimotor polyneuropathy (DSPN) is a common condition in older populations with high prevalences of obesity and type 2 diabetes. We hypothesised that the risk of DSPN is affected by ubiquitous environmental risk factors, particularly in people with obesity.

Materials and methods: The study was based on 423 participants aged 62-81 years without DSPN who participated in the population-based Cooperative Health Research in the Region of Augsburg (KORA) F4 survey (2006-2008) in Southern Germany. During a mean follow-up of 6.5 years, 188 participants developed DSPN, which was defined using the Michigan Neuropathy Screening Instrument. Environmental exposures, including annual and seasonal mean air temperature, greenness (assessed with the normalised difference vegetation index [NDVI]), road traffic noise and annual mean air pollution, were assessed at participants’ residences. The cumulative risk index (CRI) was calculated to evaluate the joint effects of co-occurring exposures on the risk of DSPN based on effect estimates from multi-exposure Poisson regression models. The models were adjusted for age, sex, height, waist circumference, smoking, alcohol consumption, physical activity, education and neighbourhood socioeconomic status.

Results: In the total study sample, the co-occurrence of an interquartile range (IQR) decrease in temperature of the warm season and NDVI in a 100-m buffer and an IQR increase in night-time average traffic noise and particle number concentration (PNC) was associated with an increased risk of DSPN (CRI [95% CI] 1.39 [1.02, 1.91]). Additional adjustment for HbA1c and other covariables did not change associations between the exposures and DSPN. Effect estimates for exposure combinations were generally higher in individuals with obesity (CRI between 1.34 and 2.01) than in those without obesity (CRI between 0.90 and 1.33). The four-exposure model showed a twofold increased risk of DSPN among obese (CRI [95% CI] 2.01 [1.10, 3.67], but not among non-obese individuals (CRI [95% CI] 1.18 [0.83, 1.67], P for interaction=0.13).

Conclusion: Ubiquitous environmental exposures modulate the risk of DSPN in the older population. The joint effects of lower air temperature in the warm season, less greenness close to participants‘ residences, and higher noise and air pollution levels identified people with obesity as a particularly vulnerable subgroup.

Supported by: DZD

Disclosure: C. Herder: None.

OP 33 Therapy outside the box

193

Discovery of malonic acid as a novel adipocyte beiging molecule

C. Luk, N.J. Haywood, K.I. Bridge, K.J. Simmons, N.Y. Yuldasheva, P. Sukumar, L.D. Roberts, S.B. Wheatcroft, R.M. Cubbon, M.T. Kearney;

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

Background and aims: Pilot data from our group show that endothelial insulin-like growth factor 1 receptor knockdown (ECIGF-1RKD) mice are protected from diet-induced insulin resistance. In vitro, we found their endothelial cells secrete a small molecule capable of inducing adipocyte beiging and used metabolomics to identify candidate small molecule metabolites. With the hypothesis that adipose remodelling is the primary contributor to the improved metabolic phenotype and is mediated by an endothelial-derived small molecule, we aim to screen the adipocyte beiging effect of these hits.

Materials and methods: Mature murine 3T3-L1 adipocytes were stimulated with the different metabolites before measuring gene expression of various white and beige adipocyte markers by qPCR. To probe the beiging mechanism, adipocytes were stimulated with malonic acid at different doses or for different duration, with or without pre-treatment with relevant inhibitors. To begin to examine functional readouts, the concentration of adiponectin secreted by stimulated adipocytes was measured by ELISA. Finally, to determine the translational potential, mature human white adipocytes were stimulated with malonic acid and its derivative, di-tert-butyl malonate (DBM). To determine the dose-dependent effect of DBM, different doses of DBM were applied to human adipocytes. Mitochondrial respiration of stimulated adipocytes was measured by Seahorse respirometry.

Results: Among 28 screened metabolites, only malonic acid induced gene expression of beige adipocyte markers in mature 3T3-L1 adipocytes. 24-hour treatment with 10mM malonic acid induced gene expression of various beige adipocyte markers including Ucp1 (15-fold, n=6-7, p<0.05), Cited1 (12-fold, n=5, p<0.01) and Fgf21 (by 6-fold, n=5, p<0.05). Time-course study showed malonic acid-induced Fgf21 gene upregulation (first determined at 6-hour time-point, p<0.05) preceded Ucp1 gene upregulation (first determined at 8-hour time-point, p<0.05). Lower doses of malonic acid did not induce beige marker gene expression. Malonic acid-induced Ucp1 gene upregulation is partly attenuated in adipocytes pre-treated with either 100nM MitoQ (-59.3%, n=5, p<0.001), 20nM PD173074 (-50.0%, n=5, p<0.05) or 3μM A485 (-47.7%, n=5, p<0.01), suggesting malonic acid-induced beiging is partly mediated by mitochondrial oxidative stress, FGF receptors and Cbp/p300 signalling, respectively. In addition, malonic acid enhanced adipocyte secretion of adiponectin (+21.5%, n=3, p<0.05). 24-hour treatment with 10mM DBM recapitulated malonic acid-induced UCP1 gene upregulation in human adipocytes (13-fold, n=5, p<0.05). Dose-response study showed 100μM DBM was sufficient to induce UCP1 gene upregulation in human white adipocytes (+143%, n=5, p<0.05). Preliminary data from respirometry study suggest DBM may increase basal respiration in adipocytes (+78.75%, n=3, p=ns).

Conclusion: Malonic acid can induce adipocyte beiging and adiponectin secretion in vitro. Malonic acid-induced beiging is partly mediated by mitochondrial oxidative stress, FGF receptors and Cbp/p300 signalling. DBM may have therapeutic potential in diet-induced diabetes. Future work will focus on examining the pathway by which IGF-1R deficiency leads to increased endothelial malonic acid secretion and the translational relevance of DBM in treating diabetes.

Supported by: BHF 4-year PhD studentship

Disclosure: C. Luk: Grants; British Heart Foundation 4-year PhD studentship.

194

Hepatic S100A9-TLR4-MTORC1 axis normalises diabetic ketogenesis

G. Ursino1, G. Ramadori1, A. Höfler2, S. Odouard1, P. Teixeira1, F. Visentin1, C. Veyrat-Durebex1, G. Lucibello1, R. Firnkes1, S. Ricci1, J. Elmquist3, T. Vogl4, A. Boland5, R. Coppari1;

1Physiology & Cell Metabolism, University of Geneva, Geneva, Switzerland, 2Molecular Biology, University of Geneva, Geneva, Switzerland, 3Centre of Hypothalamic Research, University of Texas Southwestern Medical Centre, Dallas, USA, 4Institute of Immunology, University of Munster, Munster, Germany, 5Molecular Biology, Universtiy of Geneva, Geneva, Switzerland.

Background and aims: Type 1 Diabetes (T1D) afflicts millions of people and is usually diagnosed in children and young adults with an incidence that has been increasing at an alarming annual rate of ~3%. T1D is mainly characterized by hyperglycaemia, however β-cell loss also leads to other serious metabolic derangements such as hypertriglyceridemia, hyperglucagonemia and diabetic ketoacidosis (DKA). DKA is often life threatening (accounting for up to 263 yearly hospitalization events for each 1000 patients) and occurs due to unrestrained ketogenesis, whose incidence is high in diabetic pateints. While insulin therapy reduces ketogenesis this approach is sub-optimal and currently available insulin adjuvants actually increase the risk of developing DKA. To this end, we have identified a ketone normalizing action of S100A9 in T1D. The aims of our study were to uncover where (cell types and tissues) and how (molecular mechanism) S100A9 was exerting this beneficial effect on ketogenesis and to assess its therapeutic potential and clinical relevance.

Materials and methods: We generated insulin deficient mice lacking or re-expressing Toll-Like Receptor 4 (TLR4) only in liver or hepatocytes and mice with hepatic-restricted and hepatocyte restricted loss of Tuberous Sclerosis Complex 1 (TSC1, an mTORC1 inhibitor) in the context of liver overexpression of S100A9. We assessed cellular signaling pathways and circulating metabolic parameters in-vivo and ex-vivo fatty acid oxidation in these mice to determine the mechanism of action of S100A9. We tested the translational feasibility and safety of an S100A9 based therapeutic in insulin deficient mice through administration of recombinant mouse and human S100A9 (acute and chronic treatment)We analyzed plasma samples of decompensated diabetic patients to determine clinical feasibility of S100A9 treatment.

Results: S100A9 suppresses diabetic ketogenesis via activating mTORC1 signaling downstream of the TLR4-Akt pathway in hepatic non-parenchymal cells. This activation suppresses fatty acid oxidation in the liver. Extracellular S100A9 activates mTORC1 signaling in a cell-autonomous fashion. Administration of recombinant mouse and human S100A9 suppresses DKA in insulin deficient rodents.Chronic treatment of recombinant S100A9 suppresses DKA and displays a promising safety profile in insulin deficient rodents. Plasmatic S100A9 content was only modestly increased in decompensated diabetic patients (possibly indicating a compensatory mechanism to reduce ketogenesis) therefore providing scope for further increasing plasmatic S100A9 as a potential therapeutic clinical avenue in diabetes.

Conclusion: Our results indicate that it acts through the hepatic TLR4-mTORC1 axis in non-parenchymal cells; hence suggesting the existence of an insulin-independent intra-hepatic network able to regulate lipid and ketone metabolism in TID. S100A9 shows promise as a therapeutic,holding potential to reduce insulin needs and consequently diminish unwanted side effects of insulin treatment while improving metabolic control.

Supported by: B&KH, GVM, ERC, JDRF, INNOGAP, SNF

Disclosure: G. Ursino: None.

195

Validation of duodenal targeting by oral pharmacologic duodenal exclusion therapy for treatment of type 2 diabetes

T. Carlson1, A. Nimgaonkar2, T. Guerina1, K. Colbert1, S. Polomoscanik1, J. Petersen1, T. Jozefiak1, M. Fineman1;

1Glyscend Inc, Lowell, 2Glyscend Inc, Baltimore, USA.

Background and aims: Metabolic surgery is the most successful long-term therapy for T2DM and obesity, reducing macro, microvascular complications, and mortality. The acute and chronic metabolic improvements observed with these surgeries are believed to be, at least in part, a direct consequence of preventing nutrient exposure to the proximal small intestine (duodenum and proximal jejunum exclusion). The use of metabolic surgery and duodenal exclusion devices is limited, however, due to their invasive nature. We developed oral, non-absorbed polymers (GLY100, GLY200) designed to mimic the effects of metabolic surgery non-invasively by augmenting the natural mucus lining of the proximal small intestine to create a temporary barrier. In chronic rodent models of T2DM (GK-Rat and ZDF-Rat), these polymers produced progressive reductions in post-prandial glucose of up to 70% following standardized caloric loads. Improvements in fasting plasma glucose, HOMA-IR, and bodyweight were also observed. The aim of this study was to visualize the retained duodenal barrier in rats using appropriate imaging modalities.

Materials and methods: Imaging of GLY200 after gavage administration to fasted Sprague-Dawley rats was performed using 2 methods: Computed Tomography (CT), and an In Vivo Imaging System (IVIS). For CT imaging, GLY200 was covalently conjugated with diatrizoate via an efficient amide forming reaction and unconjugated diatrizoate was administered as a control. CT images were obtained after transient isoflurane administration 1, 2, and 4 hours after gavage. For IVIS imaging, GLY200 was reacted with FITC to produce a fluorescein-GLY200 conjugate, and FITC-dextran70 was administered as a control. Rats were euthanized 0.5, 1.0, 2, 4, and 8 hours after dosing and the GI-tracts were immediately removed for imaging.

Results: CT imaging (see figure) clearly demonstrated retention of GLY200-diatrizoate within the proximal small intestine 1- and 2-hours after administration, with an intense signal only in the duodenum. In comparison, the diatrizoate control was broadly distributed over the small intestine distal to the duodenum by 1-hour. In IVIS imaging, GLY200-FITC showed intense fluorescence in the proximal small intestine that persisted for 4-hours after administration and was undetectable by 8-hours. In comparison, FITC-dextran70 was distributed broadly throughout the distal intestine and cecum by 30 minutes with no retention in the duodenum.

Conclusion: Our imaging studies confirm that GLY200 conjugate is effectively retained in the duodenum after oral administration. These observations, along with previously reported glycemic improvements in chronic rodent models of T2DM, support the hypothesis that orally administered polymers designed to target the proximal small intestinal gut wall could potentially be used to non-invasively recapitulate the glycemic and weight effects observed with metabolic surgery and duodenal exclusion devices.

figure bf

Disclosure: T. Carlson: None.

196

Human engineered skeletal muscle to treat insulin resistance in type 2 diabetes

H. Shoyhet1, Y. Herman1, M. Beckerman1, E. Karnieli2, S. Levenberg1;

1Biomedical engineering, Technion- Israel Technion Israel of Technology, 2Medicine, Technion- Israel Technion Israel of Technology, Haifa, Israel.

Background and aims: Diabetes Mellitus type II (DM2) is the most common type of diabetes, widely spread in developed countries and responsible for more than 1 million deaths every year. It is characterized by skeletal and adipose tissues insulin resistance and is usually non-insulin dependent. However, progression of the disease can cause various complications, including insulin impairment. Glucose transporter type 4 (GLUT4), mainly expressed in skeletal muscle tissue, is the key factor in glucose homeostasis overall and in DM2 specifically; Many studies have shown reduction in GLUT4 expression and translocation levels in DM2 patients. Therefore, we hypothesize GLUT4 can be part of the solution. Previous study in our group showed that implantation of GLUT4 over-expressing (GLUT4 OE) mouse skeletal muscle tissue in diabetic mice can improve blood glucose levels. To bring the technology closer to the clinic, we aim to prove that the concept works in human cells as well. Moreover, to improve patient’s compliance, less invasive delivery methods must be developed. In this study we have two aims: 1. Engineer GLUT4 OE human skeletal muscle tissue and implant it in diabetic mice. 2. Develop a new delivery method of the engineered skeletal muscle tissue using minimally invasive procedure

Materials and methods: Human myoblasts are genetically modified to overexpress GLUT4 via viral transduction. The cells are seeded on 3D PLLA/PLGA scaffolds for differentiation and maturation. The 3D tissue is characterized using immunostaining and glucose uptake assays to assess their anti-diabetic properties. The scaffolds are implanted in diabetic mice, and their blood glucose levels are monitored prior and following implantation. We have also developed a novel scaffold to deliver the functional muscle tissue using non-invasive procedure. The scaffold is characterized using the same methods listed above. We are also assessing the scaffolds mechanical properties and injectability.

Results: We have genetically modified human myoblasts to overexpress GLUT4. The modified cells can be grown on 3D PLLA/PLGA scaffolds and differentiated to functional skeletal muscle tissue. Immunostaining and molecular assays are used to characterize and quantify the GLUT4 expression. Glucose uptake assays show the engineered tissue has ~ 50% (p>0.05) enhanced uptake capacity compared to WT cells. We also developed new scaffold to deliver functional skeletal muscle tissue via injection. The novel injectable scaffold was fine-tuned to enable skeletal muscle growth and differentiation which was confirmed via immunostaining for myogenic markers. Tissue survival and functionality following injection were assessed using Alamar-Blue and glucose uptake assays showing no singnificant differences prior and following injecion. Glucose uptake assays also confirm that GLUT4 OE skeletal muscle tissue can develop on the scaffold.

Conclusion: New routes to treatment for type II diabetes are needed to fight this global issue. We present a concept of therapy based on GLUT4 and skeletal muscle tissue engineering. Preliminary results indicate this approach has great potential to tackle one of the key issues in type II diabetes and insulin resistance.

Supported by: RAS fund, TEVA fellowship

Disclosure: H. Shoyhet: None.

197

Oral long-acting menin inhibitor normalises type 2 diabetes in two rat models

T. Butler, S. Mourya, W. Li, B. Law, T. Archer, T. Kinoshita, P. Somanath;

Biomea Fusion, Inc., Redwood City, USA.

Background and aims: Menin is a scaffold protein that has been recognized for its role in T2DM as a key regulator of β-cell proliferation. Menin inhibition has previously been shown to improve glycemic control in diabetic mice. Herein, we report the first evidence that BMF-219, an orally bioavailable, selective, covalent menin inhibitor, restores glycemic control in Zucker Diabetic Fatty (ZDF) Rat and Streptozotocin-induced (STZ) Rat models of T2DM.

Materials and methods: Rats were treated daily with BMF-219, vehicle, or pioglitazone for 16 days and analyzed for fasting and non-fasting blood glucose levels, insulin, c-peptide, and blood lipemic levels. Oral Glucose Tolerance Test (OGTT) was conducted up to Day 15 in both models and two-weeks post-treatment in the ZDF model. Body weight of all rats was also monitored.

Results: BMF-219 was well tolerated throughout the conduct of the study. BMF-219 treatment resulted in a significant 50% reduction in fasting and non-fasting blood glucose levels, reduced serum insulin and c-peptide levels (p<0.05), and reduced HOMA-IR (p<0.001) after two weeks of treatment in ZDF rats. BMF-219 decreased glucose levels at all timepoints during an OGTT at Day 15 (AUC reduction of 54%, p<0.001) and at Day 29 (AUC reduction of 40%, p<0.05, ~2 weeks after the last dose in the ZDF model, indicating prolonged glycemic control. Strikingly, BMF-219, but not pioglitazone, reduced blood glucose levels during an OGTT in STZ animals (AUC reduction of 41%, p<0.05, see figure). Significant reductions in blood lipemic levels (p<0.01) and body weight were observed in both models.

Conclusion: Collectively, our data indicate the novel and marked potential of BMF-219 as an oral, long-acting treatment for T2DM.

figure bg

Disclosure: T. Butler: Employment/Consultancy; Biomea Fusion, Inc. Stock/Shareholding; Biomea Fusion, Inc.

198

Glp-1/gdf15 dual agonist to treat obesity and hyperglycaemia in mice and non-human primates

Y.Y. Zhang, A. Kharitonenkov, X.Y. Zhao, X.N. Dong, Y.Y. Zhang, H.X. Zou, Y.G. Jin, W. Guo, P. Zhai, X. Chen;

Beijing QL Biopharmaceutical Co., Ltd., Beijing, China.

Background and aims: Obesity and its associated co-morbidities such as Type 2 Diabetes, cardiovascular disease and NASH are considerable health concerns that are exponentially rising worldwide. Only limited medicinal options to treat these diseases are currently available. Recent data is suggestive that polypharmacy-based molecules are capable to provide augmented efficacy. Utilizing protein engineering strategies we designed here long-acting GLP-1/GDF15 fusion protein, QL1005, and explored its therapeutic potential in animals.

Materials and methods: The in vitro assays in this report were conducted in the absence and presence of human serum albumin (HSA) to demonstrate the impact of fatty acid-protraction on molecules’ potencies. In vivo tests were carried in DIO and db/db mice, and obese cynomolgus monkeys to demonstrate the pharmacological efficacy of QL1005 on body weight, food intake, plasma glucose, circulating lipids and other metabolic parameters as compared to the benchmark molecules.

Results: QL1005 is an engineered dual agonist protein, designed via fusing GLP-1 and GDF15 analogs by a peptide linker conjugated with a fatty acid for time-action extension. In cell-based assays QL1005 is superior in potency to GLP-1 analog semaglutide with its pharmacokinetic parameters are comparable to this GLP-1 agonist. When tested in obese mice, QL1005 demonstrated dose-dependent lowering of body weight, food intake, fasting glucose and triglyceride with all these effects being greater in magnitude to the efficacy of semaglutide or long-acting GDF15. Of importance, QL1005-induced metabolic improvements come as the result of activity emanating from both therapeutic components within this dual agonist molecule, GLP-1 and GDF15. Indeed, the individual pathway-inactivated QL1005 analogs are approximately 50% efficacious than parental QL1005 suggestive that this co-agonist in animals is balanced in activation of both GLP-1 and GDF15 receptors. When dosed to cynomolgus obese monkeys, QL1005 is more efficacious in body weight and glucose lowering but similar in the incidence of GI side-effects as compared to semaglutide treatment.

Conclusion: Altogether, this novel GLP-1/GDF15 fusion protein in animals reveals itself therapeutically efficacious to treat obesity, diabetes and related co-morbidities thus demonstrating the promise of the poly-pharmaceutical approach in metabolic drug discovery and developments.

Disclosure: Y.Y. Zhang: None.

OP 34 Insulin signalling, novelties from the petri dish!

199

Finnish-specific AKT2 gene variant leads to impaired insulin signalling in human myotubes

S. Mäkinen1,2, N. Datta1,2, Y.H. Nguyen1,2, V.M. Olkkonen1,3, A. Latva-Rasku4, P. Nuutila4, M. Laakso5, H.A. Koistinen1,2;

1Minerva Foundation Institute for Medical Research, Helsinki, 2Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, 3Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, 4Turku PET Centre, University of Turku and Turku University Hospital, Turku, 5Institute of Clinical Medicine, Internal Medicine, University of Eastern Finland, Kuopio, Finland.

Background and aims: Finnish-specific gene variant p.P50T/AKT2 (MAF=1.1%), is associated with higher fasting insulin concentrations, in vivo insulin resistance, and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signaling in human primary myotubes, that were established from 14 male p.P50T/AKT2 variant carriers and 14 controls.

Materials and methods: Insulin-stimulated glucose uptake and its incorporation into glycogen were detected with [3H]-2-Deoxy-D-glucose and [14C]-D-Glucose, respectively, and the rate of glycolysis was measured with Seahorse XFe96 analyzer. Insulin signaling was investigated with western blotting. Binding of the p.P50T/AKT2 and control-form PH domains to phosphatidylinositol (3,4,5)-trisphosphate ((PI(3,4,5)P3)) was assayed using PIP Strips™ Membranes.

Results: Insulin increased glucose uptake and glycogen synthesis in human primary myotubes, with no difference between p.P50T/AKT2 variant carriers (n=10) and controls (n=8) (2-way ANOVA with repeated measures). Insulin-stimulation led to a significant increase in glycolytic rate (p<0.001) and in compensatory glycolysis (p<0.01) in control myotubes (n=14), but not in p.P50T/AKT2 variant myotubes (n=14, p=NS). Comparison between the genotypes revealed a significant reduction in insulin-stimulated glycolysis and compensatory glycolysis in myotubes from p.P50T/AKT2 variant carriers (p<0.05, 2-way ANOVA). Insulin-stimulated phosphorylation of AKT-Thr308 (p<0.05), AS160-Thr642 (p<0.05) and GSK3β-Ser9 (p<0.05) was reduced in p.P50T/AKT2 variant carriers (n=10) compared to controls (n=8) (2-way ANOVA with repeated measures). Binding of the variant form of p.P50T/AKT2-PH domain to PI(3,4,5)P3 was reduced when compared to the control protein (p<0.001, n= 4 repeated assays, unpaired t-test).

Conclusion: p.P50T/AKT2 leads to impaired insulin signaling in human primary myotubes, which may be a result of a defective PI(3,4,5)P3-binding capacity of the p.P50T/AKT2-PH domain.

Supported by: Diabetes Wellness Sverige, Finnish Diabetes Research Foundation, Finska Läkaresällskapet

Disclosure: S. Mäkinen: None.

200

Role of Diacylglycerol kinase δ in skeletal muscle glucose metabolism in type 2 diabetes

F. Tramontana, M. Kuefner, A. Krook, J.R. Zierath;

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Reduced Diacylglycerol Kinase δ (DGKδ) protein abundance and activity have been observed in skeletal muscle from people with type 2 diabetes (T2D), resulting in increased diacylglycerol (DAG) levels - a widely recognized lipotoxic mediator of insulin resistance. Thus, downregulation of DGKδ may play a role in the regulation of DAG and the development of insulin resistance in type 2 diabetes. However, the underlying mechanisms by which reduction of DGKδ levels lead to the development of insulin resistance are still unknown. We hypothesized that lower of DGKδ in skeletal muscle impairs glucose homeostasis by disrupting the canonical insulin signaling pathway, thus contributing to skeletal muscle insulin resistance.

Materials and methods: Primary human skeletal muscle cells were isolated from vastus lateralis biopsies obtained from people with type 2 diabetes (n=6) or normal glucose tolerance (NGT) (n=6). Cells were differentiated to mature myotubes and transfected with DGKδ siRNA (siDGKδ). Cells were harvested 48 hours after transfection, under basal and insulin-stimulated (120nM) conditions. Western blot, qPCR analysis, glycogen synthesis assay, and lactate measurements were performed to assess glucose homeostasis and insulin signaling.

Results: Cells transfected with DGKδ were characterized. DGKδ gene expression was reduced 80% after silencing. Further characterization revealed that siDGKδ silencing in myotube cultures derived from people with type 2 diabetes showed impaired abundance of proteins involved in the insulin signaling cascade as compared to myotube cultures derived from people with NGT. Insulin signal transduction was also impaired, including reduced pAKTT308 (-53.8%, p=0.03) and its downstream substrate, pAS160T642 (-47.5%, p=0.02), along with a trend towards increased levels of the inactive form of glycogen synthase (pGSS641) (+121%, p=0.07). Moreover, insulin-stimulated levels of PKC substrates were reduced in myotube cultures from people with T2D transfected with siDGKδ as compared to myotube cultures from people with NGT (+17.6%, p=0.07), suggesting increased PKC activity. DGKδ silencing decreased insulin-stimulated glucose incorporation into glycogen as compared to control cells (-8.9%, p=0.05), although no differences were seen between diagnosis groups. siDGKδ treatment of myotube cultures from people with type 2 diabetes displayed a 75% increase in lactate production under basal conditions as compared to myotube cultures from people with NGT (p<0.001).

Conclusion: Reduced levels of DGKδ may contributes to dysregulation of glucose metabolism in skeletal muscle through impairments in insulin signaling. Therefore, reductions in DGKδ abundance may further aggravate glycemic control and insulin sensitivity in type 2 diabetes. Level and activity of DGKδ may influence cellular processes as diverse as insulin signaling, glucose metabolism and energy homeostasis.

Supported by: EFSD/Novo Nordisk Programme for Diabetes Research in Europe

Disclosure: F. Tramontana: None.

201

Pharmacological inhibition of Integrin a5b1 improves insulin sensitivity in H9C2 cells and cardiac performance in obese mice

A.K. Banah, V. Musale, C.K. Hennayake, C.E. Murdoch, L. Kang;

University of Dundee, Dundee, UK.

Background and aims: Extracellular matrix (ECM) remodelling has recently emerged as a key contributor to the development of insulin resistance (IR). Inhibition of ECM collagen and hyaluronan deposition by clinical and preclinical antifibrotic drugs attenuated cardiac IR and cardiac dysfunction in diet-induced obese mice. Fibronectin is a multifunctional glycoprotein in the ECM known to communicate with cells mainly via the integrin α5β1 receptor. The present study investigated the effects of CLT-28643, an α5β1 integrin antagonist, on insulin sensitivity in H9C2 cardiomyocytes, and cardiac function in high-fat-high-sucrose (HFHS) fed obese mice.

Materials and methods: In vitro effect of ECM on insulin signalling was investigated by culturing H9C2 cells on fibronectin coated plate for 5 days followed by 15 min treatment with insulin (10nM). Furthermore, insulin resistant H9C2 cells, induced by high glucose (33.3 mM) and palmitic acid incubation (100 μM) for 24-h, were subjected to CLT-28643 treatment for 3 hrs followed by 15 min incubation with insulin (100nM). Insulin sensitivity of treated cells was examined by phosphorylation of Akt using western blot. In vivo, male C57BL/6 mice fed 45% high-fat diet and 30% sucrose water for 10 weeks received twice-daily oral gavage of either vehicle or CLT-28643 (75 mg/kg body weight) for 4 weeks. A separate group of mice fed with chow diet for 14 weeks were used as lean controls (n=8). Cardiac function was measured by Pressure-Volume (PV) loop analysis (Transonic) using PV conductance catheter in closed-chest preparation. Immunohistochemical analysis was performed to assess changes in collagen and alpha-smooth muscle actin (α-SMA) expression in the left ventricle (LV) of the heart.

Results: In H9C2 cells, FB coating decreased insulin-stimulated phosphorylation of Akt (Ser473); CLT-28643 prevented high glucose & palmitate-induced IR as indicated by an increased ratio of the phosphorylated Akt (Ser473) to total AKT compared to vehicle-treated cells (2.67±1.55 fold increase, P<0.05). In mice, HFHS diet resulted in significant increases in body weight (29.05 ± 0.82 (chow) vs 40.86 ± 1.34 (HFHS) gram, P<0.0001), %fat mass (15.52 ± 0.80 vs. 51.30 ± 5.89%, P<0.0001) and decreases in %lean mass (78.29 ± 0.98 vs. 58.61 ± 1.01%, P<0.0001) compared to chow-fed mice. CLT-28643 displayed a trend of decrease in %fat mass, despite no changes in body weight or %lean mass. HFHS diet impaired cardiac function in mice as evidenced by a decrease in end-systolic elastance (31.06 ± 6.23 (chow) vs 10.88 ± 1.47 (HFHS) mmHg/μL, P<0.01), ejection fraction (86.14 ± 4.24 vs 72.82 ± 3.60%, P<0.05) and an increase in heart rate (554.3 ± 21.41 vs 603.7 ± 9.72bpm, P<0.05) and ventricular arterial coupling (VAC) index (0.19 ± 0.06 vs 0.46 ± 0.07, P<0.05) compared to lean controls. Interestingly, elevated heart rate was reversed by CLT-28643 treatment, suggesting an improved cardiac performance; while the slope of the end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relation (EDPVR) were not different among groups. Immunohistochemical analysis revealed no significant changes in LV collagen and α-SMA expression in experimental mice.

Conclusion: Pharmacological inhibition of integrin α5β1 improved insulin signalling in H9C2 cells and cardiac performance in obese mice. These findings suggest that therapies targeting FB-α5β1 interaction may be beneficial to obesity-associated cardiometabolic complications.

Supported by: BHF

Disclosure: A.K. Banah: None.

202

Impact of protein kinase D2 in the regulation of hepatic insulin sensitivity

P. Rada1,2, A.B. Hitos1,2, E. Rey3, E. Carceller-López1, J. Pose-Utrilla1,4, C. García-Monzón3, G. Sabio5, T. Iglesias1,4, Á. González-Rodríguez2,3, Á.M. Valverde1,2;

1Instituto de Investigaciones Biomédicas Alberto Sols (CSIC/UAM), 2Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 3Instituto de Investigación Sanitaria Princesa, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), 4Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 5Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.

Background and aims: Protein Kinase D2 (PKD2) is a Ser/Thr kinase of the Ca2+-Calmodulin kinase superfamily. Recently, emerging evidences indicate that PKD2 regulates glucose homeostasis in several tissues. Two previous studies, one in global PKD2-null mice, and other in mice lacking PKD2 in intestine, reported opposite results with metabolic dysfunction or protection against High Fat Diet (HFD)-induced obesity, respectively. Given that insulin resistance is a key initial trigger for NAFLD progression, unravelling PKD2 contribution to the initial stages of this disease will add fundamental knowledge to metabolic liver dysfunction with potential therapeutic value.

Materials and methods: PKD pharmacological and genetic inhibition was analyzed in primary hepatocytes and in Huh7 cells. Huh7 cells were transfected with EGFP-PKD2-CA, a constitutively active PKD2 fused to EGFP. Insulin signalling was examined in hepatocytes stimulated with insulin (10 nM, 5-15 min). As an in vivo model of hepatic insulin resistance, mice with a liver-specific PKD2 depletion (PKD2∆Hep) were fed HFD. Parameters assessing glucose homeostasis and hepatic insulin sensitivity were analyzed. PKD2 was overexpressed in liver by an injection of AAV-EGFP-PKD2-CA and insulin sensitivity was evaluated. PKD signature was analyzed in liver biopsies from NAFLD patients.

Results: PKD pharmacological inhibition resulted in higher AKT phosphorylation upon insulin stimulation in both primary mouse hepatocytes (p<0.001, n=6) and Huh7 cells (p<0.01, n=3). Similar results were obtained in PKD2∆Hep hepatocytes (p<0.05, n=5) in which IRS1 was upregulated (p<0.001, n=6). Moreover, PKD2 knocking down by shRNA enhanced the insulin response (p<0.01, n = 8-10). Alternatively, EGFP-PKD2-CA overexpression reduced insulin-stimulated AKT phosphorylation compared to EGFP-transfected cells (p<0.01 t=5 min; p<0.001 t=15 min; n=4-10). In this line, in vivo injection of AAV-EGFP-PKD2-CA resulted in moderate impairment of glucose homeostasis and reduced IR and AKT phosphorylation in liver (p<0.05 pIR, pAKT(Ser473); p<0.001 pAKT(Thr308), n = 5-6 mice/group). Importantly, HFD-fed PKD2∆Hep mice displayed a tendency to improve glucose tolerance and insulin sensitivity compared to control mice, being statistically significant at different time points (GTT: p<0.05 at 15 min; ITT: p<0.05 at 30, 60 and 90 min; PTT: p<0.05 at 60 min) (n=7-10 mice/group). Our preliminary data showed an increase in insulin-induced AKT phosphorylation in livers from HFD-fed PKD2∆Hep mice compared to controls. Moreover, PKD immunostaining showed that PKD2 was increased in NAFLD patients.

Conclusion: Results herein strongly suggest a role of PKD2 in the control of hepatic insulin signalling and point PKD2 as a new therapeutic target in the progression of hepatic insulin resistance-related pathologies.

Supported by: MICINN RTI2018-094052-B-100; CAM S2017/BMD-3684; CIBERDEM (ISCIII)

Disclosure: P. Rada: None.

203

Alpha-melanocyte stimulatory hormone (αMSH): a novel and potent regulator of glucose tolerance in humans

N.G. Docherty1, P. Swan1, B. Johnson2, S. Samarasinghe3, M. Cowley4, C.W. le Roux1,5, A.D. Miras2;

1School of Medicine & Medical Science, University College Dublin, Dublin, Ireland, 2Department of Metabolism, Digestion and Reproduction Imperial College London, London, UK, 3School of Medicine & Medical Science, Department of Metabolism, Digestion and Reproduction Imperial College London, London, UK, 4Monash Biomedicine Discovery Institute, Melbourne, Australia, 5Centre for Diabetes, Coleraine, UK.

Background and aims: Intravenous administration of exogenous αMSH lowers glucose excursions during oral glucose tolerance testing (OGTT) in pre-clinical studies in mice. We set out to interrogate whether this action is preserved in human physiology, both in vivo and in vitro.

Materials and methods: Two cohorts, each of fifteen healthy volunteers received infusions of physiological saline, 15, 150, and 1500 ng/kg/hr αMSH initiated 30 minutes prior to the administration of a standard OGTT. Plasma glucose and insulin were measured during the OGTT. To assess the effect of αMSH on skeletal muscle glucose disposal, subjects in cohort 1 underwent sequential hyperinsulinaemic-euglycaemic clamp studies with saline and 150ng/kg/hr αMSH infusion. In a separate cohort of healthy volunteers (n=6), primary human myotube cultures were generated from vastus lateralis muscle biopsies and used to directly assess glucose uptake in response to αMSH.

Results: Infusion of αMSH (1500ng/kg/hr) led to a 45% reduction in the 2-hour incremental area under the curve (iAUC) for plasma glucose (p<0.001). Accordingly in cohort 1, the iAUC for plasma insulin was reduced by 20% (p=0.006). In clamp studies, αMSH increased glucose requirements for the maintenance of euglycaemia. Primary human myotube cultures expressed melanocortin receptor subtypes (MC1R>MC3R≈MC4R). A sixty-minute incubation of myotube cultures with 10nM αMSH increased glucose uptake by two-fold versus vehicle (p=0.001), this being equipotent to the effect obtained with insulin. The glucose uptake promoting effects of insulin and α-MSH in myotubes were additive.

Conclusion: These findings substantiate a role for peripheral αMSH as a hitherto undescribed component of the endocrine control of glycaemia in human physiology. The αMSH-skeletal muscle axis offers a novel target for the development of diabetes pharmacotherapy.

Clinical Trial Registration Number: ISRCTN26265036

Supported by: Health Research Board Ireland and EFSD Albert Renold Travel Fellowship

Disclosure: N.G. Docherty: None.

204

Abrogation of circular dorsal ruffles by hyper insulinemia impairs insulin receptor internalisation and recycling

A. Teshima1, M.A. Correia1, L.M. Oliveira1, D.C. Barral1, R.M. de Oliveira1, M.P. Macedo1,2;

1Chronic Diseases Research Centre (CEDOC), NOVA Medical School, Faculdade de Ciencias Medicas, Universidade NOVA de Lisboa, Lisbon, 2Departament of Medical Science, Departamento de Ciencias Medicas, Universidade de Aveiro, Aveiro, Portugal.

Background and aims: Insulin resistance is a hallmark for type 2 diabetes mellitus (T2DM) and occurs when the body cannot respond appropriately to circulating insulin levels. Hyperinsulinemia is both a trigger and a consequence of insulin resistance. Insulin mediates its physiological effects through binding to the extracellular subunit of the insulin receptor (lnsR) at the plasma membrane. Impairing the activity of any component of this complex, from insulin binding to InsR internalization, trafficking and signal propagation, can lead to insulin resistance. However, how altered InsR internalization contributes to the development of insulin resistance and T2DM is understudied. Ligand-activated InsR is internalized and trafficked to early endosome (EE), where InsR is dephosphorylated and sorted. InsR can be subsequently conducted to lysosome for degradation or recycled back to the plasma membrane. Previous results from our group showed that lnsR is internalized through actin-rich ring-shaped structures circular dorsal ruffles (CDRs), which form exclusively at the dorsal surface of hepatocytes, upon insulin stimulation. Our group also showed that chronic exposure to elevated insulin concentration inhibits CDRs formation. Having these findings in mind, we hypothesized that inhibition of CDRs formation by exposing hepatocytes to hyper insulin concentration compromises InsR internalization and subsequent trafficking to EE and recycling to membrane.

Materials and methods: Hepa 1-6 mouse hepatoma cells were used to study lnsR internalization and subsequent trafficking. Cells were maintained in normal medium or, to mimic hyperinsulinemia condition, they were maintained in medium supplemented with 200 nM of insulin for 48h. Cells were serum-starved for 3 hours, then stimulated with 100nM insulin for 1, 5, 15 and 30 mins. To observe EE and recycling endosomes (RE), cells were fixed and processed for immunofluorescence, being labeled with EEA1, a marker for EE, and Rab11a a marker for RE and colocalization was measured respectively.

Results: We observed that in normal condition, InsR colocalizes with EEA1 peaking at 5 minutes, then we observed a decrease of colocalization along time, suggesting that the vesicles containing InsR are maturating along the endocytic route. On the other hand, hyper insulin incubated cells, mimicking prediabetes, showed significant decrease of InsR colocalization with EEA1 in every timepoint, indicating an abrogation of the internalization in early endocytosis. Subsequently, we looked at recycling endosomes by staining for Rab11a. While in control condition Rab11a co-localizes with InsR at 15 minutes upon insulin stimulation, when CDR formation is blocked in cells exposed to chronic hyperinsulin, recycling to the plasma membrane is impaired.

Conclusion: Here we show for the first time that hyperinsulinemia inhibits lnsR internalization through CDRs and subsequent trafficking to EE and RE. In toto, our results suggest that abrogation of CDRs formation in a condition mimicking prediabetes impairs InsR internalization and thus contributing to the onset and/or exacerbation of insulin resistance.

Supported by: PTDC/MEC-MET/29314/2017, UIDB/Multi/04462/2020, Programa Gilead GÉNESE─Edição de 2019

Disclosure: A. Teshima: None.

OP 35 Different pathways involved in killing the beta cell

205

IER3IP1 mutations lead to ER stress-induced neonatal diabetes

H. Montaser, S. Leppänen, H. Ibrahim, S. Eurola, J. Saarimäki-Vire, T. Otonkoski;

University of Helsinki, Helsinki, Finland.

Background and aims: IER3IP1, a highly conserved protein in humans, is highly expressed in the developing brain cortex and β-cells. The endoplasmic reticulum-transmembrane protein is thought to play a major role in regulating ER secretion and cell apoptosis. Homozygous mutations in IER3IP1 have been associated with neonatal diabetes and microcephaly. The aim of this study is to characterize the role of IER3IP1 in the development, function, and survival of human β-cells using human embryonic stem cell (hESC)-derived pancreatic islets (SC-islets).

Materials and methods: Utilizing CRISPR/Cas9, we deleted the first exon of IER3IP1 in hESCs to generate a knockout (KO) model. Alongside their wild-type (WT) counterparts, the KO clones were differentiated into SC-islets using an optimized 7-stage protocol. The effect of IER3IP1-KO on β-cell development was characterized using flow cytometry, qPCR, and immunohistochemistry for β-cell and ER stress markers. Additionally, the survival of IER3IP1-KO β-cells was tested by treating the cells with chemical ER-stress inducers and measuring their apoptosis using TUNNEL assay. Furthermore, the functionality of SC-islets was tested in-vitro through glucose-stimulated insulin secretion assay, and in-vivo after implanting the SC-islets under the kidney capsule of immunodeficient mice.

Results: We confirmed the successful generation of IER3IP1-KO hESC clones using Sanger sequencing and qPCR. Intriguingly, IER3IP1-KO SC-islets showed significantly lower percentage of β-cells (41% in WT vs 21% in KO, p= 0.001, n= 3), reduced INS transcript levels, and drastically diminished insulin content (766 ng hINS/μg DNA in WT vs 111 ng hINS/μg DNA, p= 0.02, n= 3). Additionally, immunostaining of IER3IP1-KO β-cells showed notably higher accumulation of proinsulin, and significantly higher levels of the ER stress marker BiP when compared to their wild-type controls. Furthermore, IER3IP1-KO β-cells were more vulnerable to apoptosis upon inducing ER stress using chemical stressors. Testing their functionality, IER3IP1-KO SC-islets were able to respond to high glucose treatment by increasing their insulin secretion; however, their total insulin secretion was substantially lower than WT SC-islets. Moreover, the levels of circulating human C-peptide detected in mice implanted with WT SC-islets were significantly higher than those detected in the mice with KO implants, since the first month of implantation (606 pmol/L human C-peptide in WT-grafted mice vs 82 pmol/L human C-peptide in KO-grafted mice, p= 0.009, n= 3). After three months of implantation, the WT grafts were able to regulate the blood glucose during an intraperitoneal glucose tolerance test, while the KO grafts failed to upregulate their C-peptide secretion and the mice showed impaired glucose tolerance.

Conclusion: Our results indicate the essential role of IER3IP1 in maintaining normal ER homeostasis and regulating the function and survival of human beta cells.

Supported by: ILS doctroal program, UH and Orion research foundation.

Disclosure: H. Montaser: None.

206

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates tumor necrosis factor alpha-induced human beta cell dysfunction and death

A. Roca Rivada, S. Marín Cañas, M.L. Colli, D.L. Eizirik;

ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium.

Background and aims: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine produced by macrophages/monocytes during acute inflammation. The cytokine plays a role in insulitis and pancreatic beta-cell loss in type 1 diabetes mellitus (T1D) and a phase 2 clinical study showed that TNF-α inhibition in newly diagnosed T1D patients preserves C-peptide production (PMID: 33207093). In this study, we evaluated the effects of TNF-α (as compared to type I IFNs, previously shown by us to be affected by PTPN2) in human pancreatic beta-cells silenced for the protein tyrosine phosphatase PTPN2, a candidate risk gene for T1D.

Materials and methods: EndoC-βH1 cells, dispersed human islets, and iPSC-derived beta-like cells were transfected with siCTRL, siPTPN2, siJNK1, or siBIM. After 24h of recovery cells were left untreated (vehicle) or treated for 48h with IFN-α (2000 units/mL), TNF-α (1000 units/mL), or the combination IFN-α + TNF-α (2000 and 1000 units/mL respectively). Apoptosis was evaluated using Hoechst and propidium iodide staining; mRNA levels were assessed by RT-PCR. PTPN2, pJNK1, pP38, pBIM, and GAPDH protein expression were examined by immunoblot. Results are the mean of 6-7 independent experiments; statistical analysis was done by one-way ANOVA with Bonferroni correction.

Results: 48 hours treatment with IFN-α or TNF-α in EndoC-βH1, dispersed human islets or iPSC-derived β-like cells silenced for PTPN2 induced a significant increase in cellular apoptosis (17.7-50.5% for IFN-α, and 26.5-50.5% for TNF-α; P < 0.01 compared to cells exposed to the cytokines without PTPN2 silencing); a similar result was observed with the combination of IFN-α + TNF-α (24.7-43.8%; P < 0.01 in iPSC-derived β-like cells, and P < 0.001 in EndoC-βH1 cells and dispersed human islets). There was no potentiation between IFN-α + TNF-α suggesting that these cytokines may act through a similar intracellular pathway. To test this hypothesis, we co-silenced PTPN2 and JNK1 or BIM, known to be involved in IFN-induced apoptosis, in EndoC-βH1 and iPSC-derived β-like cells (>50-60% silencing each). This abolished the proapoptotic effects of IFN-α, TNF-α, or the combination of both cytokines with (P < 0.05) or without PTPN2 inhibition (P < 0.01). Interestingly, silencing PTPN2 increased by 2-fold TNFα-induced phosphorylation of JNK1 (P < 0.05), P38 (P < 0.01) and BIM (P < 0.01), suggesting a critical and protective role for PTPN2 against TNF-α toxic effect in beta-cells. These findings also point to an unexpected common pathway for signaling between IFNα and TNFα.

Conclusion: TNF-α has been shown to play a role in the pathogenesis of human T1D. We presently show that PTPN2 is a key regulator of the deleterious effects of TNF-α in human beta-cells. PTPN2 is a candidate risk gene for T1D, and it is conceivable that patients carrying risk-inducing PTPN2 polymorphisms may particularly benefit from therapies targeting TNF-α.

Disclosure: A. Roca Rivada: None.

207

GDF11 improve age-dependent beta cell deterioration in mouse and man

R. Wu;

Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.

Background and aims: GDF11 is a member of the transforming growth factor (TGFβ) superfamily. An age-dependent decline in serum GDF11 levels can be observed in multiple mammalian species. In mice, GDF11 treatment protects glucotoxicity-induced beta-cell dysfunction and apoptosis. GDF11 deficient mice exhibit decreased beta-cell mass and reduced expression of MafA in beta cells during embryonic development. However, the roles of GDF11 in the regulation of ageing-related cell functions are rather controversial. Here we aim to explore the underlying function of GDF11 in controlling beta-cell deterioration in ageing rodents and islets from human donors.

Materials and methods: All human islets were collected from the Nordic Network for Islet Transplantation (Uppsala University, Sweden) via EXODIAB Human Tissue Lab in Lund University Diabetes Centre. In vitro insulin secretion experiments and immunostaining were performed in human islets from 7 non-diabetic and 3 diabetic donors with > 50 years of age. Aged diabetic db/db mice (24 weeks) and GK rats were used in the study. The db/db mice were intraperitoneally injected with GDF11 (100 ug/kg body weight) or vehicle (12 mice in each group) every other day for 30 days. Blood samples were collected from vena in the mice, and both blood glucagon and insulin were analyzed by ELISA. Blood glucose was also measured during the period of GDF11 injection and continued for 5 months. A 100 ng/ml GDF11 was used for in vitro experiments in the insulin-secreting INS-1 832/13 cells, primary rodent and human islets for three days.

Results: In our study, we found GDF11 expression was reduced by more than 80% in the aged human donors (~66 years old) with diabetes in comparison with non-diabetic donors (n=3, p<0.01). Similarly, GDF11 expression was 50% lower in diabetic GK rats than Wistar rats (n=3, p<0.01). After GDF11 treatment, Pdx1, the transcription factor indicating pancreatic beta cell identity, significantly increased (n=3, p<0.01). Notably, injection of GDF11 in aged db/db mice decreased both blood glucose and blood insulin significantly, while the blood glucose continued to be improved even 150 days after withdrawal of treatment (n=3, p<0.05). GDF11 treatment elevated insulin content in both diabetic human islets (n=3, p<0.01) and INS1 832/13 cells (n=3, p<0.005). Notably, glucose-stimulated insulin secretion appeared to be affected differentially in the GFD11-treated isolated islets from db/db mice: increased at low secretion rates (n=4, p<0.05) but decreased at high secretion rates (n=4, p<0.01)

Conclusion: Our findings suggest a novel role of GDF11 in reversing the age-dependent decline in beta-cell function and therefore hold promise for therapy of age-related diabetes.

Supported by: The Swedish Research Council (2019-01567, 2018-03258); Clinical research (ALF); Crafoord foundation

Disclosure: R. Wu: None.

208

Insulin, proinsulin and PC1/3 expression heterogeneity and beta cell function in diabetes

P.S. Apaolaza Gallegos1, Y.C. Chen2, K. Grewal2, Y. Lurz1, B. Verchere3, T. Rodriguez-Calvo1;

1Helmholtz Zentrum München, Munich, Germany, 2Department of Surgery, University of British Columbia, Vancouver, Canada, 3Department of Surgery and Pathology & Laboratory Medicine, University of British Columbia, Vancouver, Canada.

Background and aims: The detection of proinsulin (PI), insulin (INS), or C-peptide in the blood may help predict the decay in beta-cell mass and function during diabetes progression. However, their expression in the pancreas is still not well-understood. Thus, we aimed to provide an in-depth characterization of INS, PI, and the prohormone convertase PC1/3 (PC1) expression in the islets of non-diabetic (ND), autoantibody-positive (AAb+), type 1 diabetic (T1D) and type 2 diabetic (T2D) donors using confocal imaging and state-of-the-art image analysis. Moreover, we investigated islet beta-cell phenotype and islet morphology during disease progression.

Materials and methods: FFPE-Pancreatic sections from 20 age-matched ND, 7 AAb+ (4 single (s) and 3 double (d)), 8 T1D with short disease duration (<5y), 9 with long-duration T1D (>15y), and 6 T2D donors were included in the study. Confocal microscopy images for INS, PI, and PC1 from up to 30 islets/donor were analyzed using the software QuPath. Different cell populations were defined as follows: 1) INS+PI+PC1+, triple+, 2) INS+PI+PC1-, INS+PI-PC1+, INS-PI+PC1+ double+ and 3) INS+PI-PC1-, INS-PI+PC1-, INS-PI-PC1+ single+ cells. Proportion and density (number of cells/islet area) of each population as well as islet cellularity/ morphology were analyzed. The sum of triple+, double+, and single+ cells was used to determine total INS+, PI+, and PC1+ cells.

Results: In dAAb+, in comparison with the ND group, the density, and proportion of INS+PI+PC1- and INS-PI+PC1- cells was increased, while there was a decrease in INS+PI-PC1+ cells, suggesting active PI and insulin cellular accumulation without high PC1 expression. After onset, the proportion and density of total+ and triple+ (INS+PI+PC1+) cells were low in most T1D donors due to beta-cell loss. Conversely, INS+PI-PC1+ doubled, and INS-PI-PC1+ increased between 16% and 40% in T1D, and 5% in T2D compared to ND donors. No differences were observed in INS-PI+PC1+ cells among groups, with the exception of T1D (>15y) donors (8 times lower values). In general, single PI+ or INS+ cells were rare. The study of islet morphology revealed that the number of cells per islet was comparable in all groups while endocrine cell density was reduced in dAAb+ donors, yet increased in all T1D and T2D donors compared to ND donors. Last, no differences were found between ND and sAAb+ donors in all categories.

Conclusion: Beta-cells undergo functional and morphological changes as diabetes progresses. We observed a decrease in cells expressing INS, PI, and PC1 from dAAb+ to T1D, reflecting the ongoing beta cell loss. Changes in distinct double+ cell subpopulations may represent different stages of beta-cell dysfunction, as suggested by the increase in cells lacking PC1 in the islets of dAAb+ donors. Conversely, the increase in single PC1+ cells in T1D and T2D could be due to an increase in PC1+ alpha cells, possibly driven by dedifferentiation or to the presence of beta cells that cannot produce PI and INS. Lastly, islet morphological analysis suggests that beta-cell hypertrophy first and then atrophy might occur during disease progression. Our findings reveal intriguing dynamics of proinsulin/insulin production and conversion and could contribute to our understanding of diabetes pathogenesis at a beta-cell level.

Disclosure: P.S. Apaolaza Gallegos: None.

209

FFA1/Gq - Gi interaction determines glucose-induced insulin secretion of neonatal beta cells

F. Gerst1,2, E. Lorza-Gil1,2, G. Kaiser1,2, T. Ulven3, E. Kostenis4, H.-U. Häring2, A.L. Birkenfeld1,2, S. Ullrich1,2;

1IDM, Helmholtz Center Munich, Tübingen, Germany, 2German Center for Diabetes Research (DZD e.V.), Tübingen, Germany, 3Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark, 4Institute of Pharmaceutical Biology, Bonn University, Bonn, Germany.

Background and aims: Adequate functional maturation of neonatal beta cells determines the efficacy of glucose metabolism during adult life. Insulin secretion of newborn islets relies on aminoacids and fatty acids metabolism, while glucose-responsive insulin secretion progressively arises days to weeks later after birth. Long chain fatty acids (LCFA) are major components of breastmilk and insulin secretagogues. LCFA are endogenous ligands of FFA1, a Gq/PLC coupled receptor acting on Ca2+ mobilization and actin network in beta cells. Whether FFA1 recruits Gα12/13 and impact on GSIS via downstream RhoA/ROCK-mediated actin remodelling is unknown. We assessed the role of FFA1 in postnatal maturation of neonatal beta cells in regard to the regulation of insulin secretion and beta cell mass.

Materials and methods: WT and Ffar1 KO BL6NCrl mice harboring a RIP-Cre driven EGFP expression were bred and 1, 6, 11 and 26d old offspring (P1, P6, P11, P26) were used for analysis. Pancreatic insulin content was measured in P1-P26 pancreata. Beta cell proliferation and mass were determined in P1-P26 pancreatic sections by estimating the number of Ki67-stained/EGFP-positive islet cells and the EGFP-positive islet area, respectively. Insulin secretion was performed in static incubations with WT and Ffar1 KO P6 islets using palmitate (600 and 60 μM), TUG469 (FFA1 agonist, 10 μM), exendin-4 (GLP-1R agonist, 100 nM), FR900359 (Gq inhibitor, 1 μM), pertussis toxin (PTX, Go/Gi inhibitor, 100 ng/ml), CT3 toxin (RhoA inhibitor, 2 μg/ml) and H1152 (ROCK inhibitor, 1μM).

Results: WT islets displayed glucose (12 mM) stimulated insulin secretion (GSIS, 6-fold change (FC) over 2.8G) mirroring the ongoing functional maturation. WT islets showed also a robust secretion in response to palmitate (600 μM) and exendin-4 (5-FC over 12G). In KO islets both glucose- and palmitate- stimulated insulin secretion were impaired, indicating that FFA1 signalling is critical for both secretagogues. As expected, palmitate (60 μM)- and TUG469-stimulated (2.25- and 3.4-FC over 12G, respectively) insulin secretion was inhibited by FR900359 (0.34- and 0.13-FC over 12G, respectively) in WT islets. Surprisingly, Gq inhibitor canceled also GSIS (from 10.6- to 1.7-FC over 2.8G) of WT islets. When the inhibitory Go/Gi signalling was blocked with PTX, GSIS was massively increased (100-FC over 2.8G), an effect that was up to 90% inhibited by FR900359 in WT islets. Noteworthy, PTX rescued GSIS in KO islets (from 1.6- to 11-FC over 2.8G). In addition, inhibition of RhoA and ROCK improved GSIS in KO islets. The secretory defects of KO islets do not originate in insufficient insulin production, since pancreatic insulin content increased with offspring’s age irrespective of Ffar1 genotype. Deletion of Ffar1 augmented beta cell proliferation in P6 offspring. Nevertheless, we found no genotype-driven differences in beta cell mass of P1-P26 offspring.

Conclusion: These findings indicate that (i) glucose responsiveness of the neonatal islets fully depends on active Gq and (ii) FFA1/Gq inactivation unlocks Go/Gi- and G12/13/RhoA/ROCK-mediated signals with negative impact on GSIS. Thus, FFA1/Gq signaling is essential for the gain of function of neonatal beta cells.

Supported by: 01GI0925

Disclosure: F. Gerst: None.

210

Oral administration of the small molecule cathepsin S inhibitor LY3000328 accelerates disease onset and impairs glucose tolerance in non-obese diabetic mice

T. Fløyel1, C. Frørup1, M. Haupt-Jørgensen2, L.J. Holm2, K. Pedersen2, M.Ø. Mønsted2, K. Buschard2, J. Størling1,3, F. Pociot1,4;

1Translational Type 1 Diabetes Research, Clinical Research, Steno Diabetes Center Copenhagen, Herlev, 2Bartholin Institute, Department of Pathology, Rigshospitalet, Copenhagen, 3Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, 4Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Accumulating data suggest a role for lysosomal cathepsin proteases in β-cell destruction and development of type 1 diabetes (T1D). We recently showed that cathepsin S (CTSS) is upregulated by proinflammatory cytokines in human pancreatic islets and β-cell lines. CTSS is increased in plasma from individuals with T1D compared to healthy individuals. Previous studies have investigated the involvement of CTSS in autoimmune diabetes and regulation of blood glucose, however, whether CTSS has protective or deleterious effects is not clear. The aim of this study was to confirm that circulating CTSS is increased in individuals with T1D and to examine if oral administration of the CTSS inhibitor LY3000328 affects autoimmunity, glucose tolerance, and diabetes incidence in non-obese diabetic (NOD) mice.

Materials and methods: CTSS was measured in serum from 148 children with T1D and 147 healthy siblings by ELISA (CV%<10). NOD mice were fed chow diet with LY3000328 ~10 mg/kg/day (CTSSi) or a standard diet (CTRL) from 3 weeks of age throughout life. Mice were euthanized at diabetes diagnosis (defined as two blood glucose measurements >12 mmol/l with an interval of two days) or at 310 days of age. 13-weeks-old mice were used for investigation of glucose tolerance and insulitis. Three days post intravenous glucose tolerance test, mice were anaesthetized for collection of heart blood (serum), pancreas, and spleen. Insulitis was evaluated from H&E stainings of pancreatic sections. Serum concentrations of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-10, TNFα, IP10, KC/GRO, MCP-1, MIP-1α, and MIP-2 were measured by MSD multiplex immunoassays. The effect of LY3000328 on CTSS activity was examined in serum using CTSS activity assay and in spleen by immunoblotting of the CTSS substrate invariant chain p10 (Iip10).

Results: Serum CTSS was increased by 8% in children with T1D compared to healthy siblings (p<0.01). Oral administration of LY3000328 led to increased diabetes incidence (CTRL, n=20; CTSSi, n=20; p<0.01) and lower glucose tolerance (CTRL, n=7; CTSSi, n=6; p<0.05) in NOD mice. There were no differences in weight, food intake, water intake, or insulitis score (p>0.05). Oral administration of LY3000328 correlated with lower serum levels of IL-6 (CTRL, n=8; CTSSi, n=4; p<0.05). CTSS inhibition was confirmed by detecting lower CTSS activity in serum (CTRL, n=8; CTSSi, n=5; p<0.001) and accumulation of Iip10 in spleen from mice treated with LY3000328 (CTRL, n=8; CTSSi, n=5; p<0.0001).

Conclusion: Our results demonstrate that LY3000328-mediated inhibition of CTSS accelerates onset of autoimmune diabetes and impairs glucose tolerance, suggesting that CTSS may have a protective role during development of T1D. CTSS may regulate β-cell sensitivity via IL-6 which previously has been shown to have a protective effect on β cells.

Supported by: JDRF, DFF/FSS

Disclosure: T. Fløyel: None.

OP 36 Central aspects of diabetes

211

Involvement of hypothalamic de novo ceramide synthesis in central resistin-induced glucose intolerance: impact on hypothalamic and hepatic inflammation

J. Guitton1, C. Alexandre1, M. Taouis1, M. López2, Y. Benomar1, H. Le Stunff1;

1Institut des Neurosciences Paris-Saclay (NeuroPSI), Saclay, France, 2Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Santiago de Compostela, Spain.

Background and aims: In obesity, ectopic lipid accumulation in non-adipose tissues causes functional impairments in several metabolic pathways leading to 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. Besides, in an over-nutrition environment, the hypothalamus is also subjected to changes in circulating factors such as resistin, a key mediator linking obesity with IR and type 2 diabetes. Recently, we have reported that resistin induces whole body IR and promotes hypothalamic inflammation. Moreover, several studies reported resistin as a regulator of peripheral lipid metabolism impairing insulin sensitivity through a central mechanism. In this context, we investigated the potential involvement of hypothalamic de novo ceramide synthesis in central resistin-induced hypothalamic inflammation and dysregulation of glucose homeostasis.

Materials and methods: C57Bl6J mice (n=5/group) were intracerebroventricularly (ICV) infused with resistin for 15 days. To investigate the role of hypothalamic de novo ceramide synthesis in ICV resistin-induced hypothalamic inflammation and glucose intolerance we selectively knocked down the serine palmitoyltransferase (SPT) 1, a de novo ceramide synthesis limiting enzyme, in the hypothalamus through stereotaxic adenovirus injection (shRNA-SPT1) into the third ventricle. In the hypothalamus, we evaluated microgliosis and astrogliosis by immunohistochemistry. Hypothalamic and hepatic inflammation as well as enzymes driving gluconeogenesis were evaluated by RTqPCR. Glucose homeostasis was assessed by an intraperitoneal glucose tolerance test (IP-GTT).

Results: Injection of shRNA-SPT1 adenovirus in the third ventricle decreases hypothalamic SPT1 mRNA expression by 62% (p<0,001). Resistin ICV infusion induces hypothalamic inflammation as evidenced by increased IL-1β expression (p<0,01) in association with significant changes in microglia and astrocyte morphology in the hypothalamus as a sign of reactive gliosis. Concomitantly, ICV resistin-treated mice exhibit marked glucose intolerance and increased hepatic expression of key proinflammatory markers IL-6 (p<0,05), TNFα (p<0,05), IL-1β (p<0,01) and NF-Κβ (p<0,01) in addition to the upregulation of hepatic glucose 6-phosphatase (p<0,05) as a sign of increased hepatic gluconeogenesis. Importantly, the selective deletion of SPT1 in the hypothalamus prevents ICV resistin-induced hypothalamic inflammation. At the periphery, hypothalamic down-regulation of SPT1 improves glucose tolerance by counteracting hepatic inflammation and gluconeogenesis induced by resistin.

Conclusion: These findings reveal hypothalamic de novo ceramide synthesis as a new regulatory pathway of resistin-induced hypothalamic inflammation and dysregulation of glucose homeostasis. Targeting this signaling pathway may constitute a significant breakthrough to overcome obesity-induced hypothalamic inflammation and related metabolic dysfunction.

Supported by: SFD Société Francophone du Diabète

Disclosure: J. Guitton: None.

212

Effect of high fat diet and exendin 4 on inhibitory neurotransmission markers and insulin signalling in the brain in high fat fed mice

V. Sancho1, A. Faraone2, A. Dardano1, S. Del Prato1, M. Mainardi3,2, G. Daniele1;

1Clinic and Experimental Medicine, Pisa University, 2Laboratory of Biology, Scuola Normale Superiore, 3National Research Council, Pisa, Italy.

Background and aims: Brain plasticity (induced by visual deprivation) is modulated by the tone of GABAergic neurotransmission. In a previous study, we have shown that visual cortical plasticity was altered in morbidly obese patients, and it is completely rescued 6 months after bariatric surgery. Brain plasticity improvement was related to increased GLP-1 levels post-RYGB. To shed light on the molecular mediators of this effect, we explored the impact of high-fat diet (HFD) on GABA and insulin signaling in the visual cortex and hippocampus of HFD mice. We also have assessed Exendin-4 (Ex4; a GLP-1 agonist) effects on the same molecular pathways.

Materials and methods: 60-days old male C57BL/6J mice were fed HFD (23% proteins; 42% CHO - 28% starch, 9% sucrose, 5% maltodextrin; 34% fats; 60% fat caloric content) or standard chow diet (SD: 18.5% proteins; 46% CHO - 42% starch, 4% sucrose; 3% fats; 6.55% fat caloric content) for 8 weeks followed by Ex4 (25ng/kg; N=XX) or vehicle (VC; N=XX) intraperitoneal injection administration and blood samples collection 1h post-injection, before animals were sacrifice for hippocampus and visual cortex dissection. Glycemia was determined by YLS 2300 STAT Plus glucometer and insulinemia by ELISA. Western blotting was used to assess expression levels of total and phosphorylated Akt and ERK kinases, and GABAergic markers GAD2 and vGAT, aall djusted for β-Actin expression.

Results: HFD was associated with body weight gain compared to SD (35.2±0.7 vs 32.4±0.4 g; p≤0.003), hyperglycemia (319±18 vs 238.4±16 mg/dl, p≤0.02) and hyperinsulinemia (72.2±19.5 vs 28.4±7.0 pmol/l, p≤0.05). In HDF mice, GAD2 levels were increased by 156±15% as compared to SD (p=0.03) in the visual cortex and reduced in the hippocampus (79±4% of SD, p=0.027) with no difference in vGAT expression. As far as insulin signaling is concerned, in HFD mice, phospho-Akt expression was reduced in the visual cortex (HFD 71±9% of SD, p=0.048) and increased in the hippocampus (HFD-vehicle 139±7% of SD-vehicle, p=0.004). One-hr after Ex-4, both glucose (99.8±12.0 vs 70.8±23.9 mg/dl) and insulin levels 7.9±6.5 vs 33.5±9.3 pmol/l) were decreased in HFD mice as compared to VC (both p≤0.001). Ex4 did not affect Akt phosphorylation in the visual cortex of both SD and HFD mice, while a significant reduction was observed in the hippocampus of HFD mice (HFD-Ex4 99±7 vs HFD-vehicle 139±7% of SD-vehicle, p=0.002). ERK phosphorylation in the visual cortex was not affected by Ex4, but it was increased in SD mice (175±17% of SD-vehicle, p=0.015)

Conclusion: HFD differentially affects the expression of GABAergic markers and activation of insulin-related signaling in the visual cortex and hippocampus. HFD is followed by increased Akt phosphorylation in the hippocampus and Exendin-4 acutely normalizes this alteration, suggesting a potential beneficial effect in rescuing altered insulin-related signaling in the brain caused by high-fat diets.

Disclosure: V. Sancho: None.

213

Central action of FGF19 improves energy homeostasis in diet-induced obese mice

L. Zangerolamo, M. Carvalho, C. Solon, G.M. Soares, L.A. Velloso, H.C.L. Barbosa;

University of Campinas, Campinas, Brazil.

Background and aims: Fibroblast growth factor-19 (FGF19) is a gut-derived hormone released postprandially, which is emerging as a potential therapeutic agent for metabolic disorders, including diabetes and obesity. It is known that the central (hypothalamic) action of FGF19 reduces body weight (BW) in diet-induced obese (DIO) mice, however, the mechanisms involved in this phenomenon remain poorly understood. Considering the crucial role of the hypothalamus in activating thermogenic mechanisms in brown adipose tissue (BAT), we aimed to investigate the central actions of FGF19 upon the energy homeostasis of DIO mice, focusing on the thermogenic capacity of BAT.

Materials and methods: In this study, C57BL/6 lean and DIO (8 weeks of high-fat diet) mice were used. For chronic (10 days) central FGF19 administration (2 μg/day), mice received a cannula into the lateral ventricle of the brain. A micro-osmotic pump (Alzet) filled with recombinant FGF19 or saline was subcutaneously implanted in mice and were connected to the newly implanted brain cannula via a catheter. Food intake and BW were monitored throughout the treatment. Energy expenditure (EE) and respiratory quotient (RQ) were measured by indirect calorimetry. Fat depots weights were obtained at the time of euthanasia and the adipocytes area was evaluated by histological analysis. Insulin sensitivity was measured by insulin tolerance test and through the homeostasis model assessment-estimated insulin resistance (HOMA-IR). BAT temperature was acquired by infrared thermography (FLIR Systems) and gene expression was evaluated by qPCR. Data were analyzed by one-way ANOVA and are displayed as mean ± SEM. The difference between the groups was considered statistically significant if P ≤ 0.05.

Results: DIO mice treated with FGF19 displayed reduced BW gain (g) (0.30 ± 0.10 Lean x 2.03 ± 0.08 DIO x 1.18 ± 0.22 DIO+FGF19) during the treatment, which was associated with lower food intake (kcal) (158.50 ± 5.67 Lean x 210.00 ± 16.84 DIO x 152.40 ± 7.11 DIO+FGF19), diminished fat depots (% BW) (epididimal: 0.38 ± 0.04 Lean x 2.81 ± 0.27 DIO x 1.95 ± 0.30 DIO+FGF19; inguinal: 0.40 ± 0.04 Lean x 1.89 ± 0.34 DIO x 0.99 ± 0.16 DIO+FGF19), and smaller adipocyte size (μm2) (1606 ± 213 Lean x 11685 ± 1016 DIO x 6572 ± 179 DIO+FGF19). The central action of FGF19 also improved peripheral insulin sensitivity, kITT (%/min) (4.95 ± 0.22 Lean x 3.10 ± 0.20 DIO x 4.63 ± 0.24 DIO+FGF19), as well as HOMA-IR (0.77 ± 0.07 Lean x 5.61 ± 1.10 DIO x 2.04 ± 0.89 DIO+FGF19) in treated mice. Furthermore, central administration of FGF19 ameliorated EE (Kcal/day/kg^0.75) (191.24 ± 2.56 Lean x 164.19 ± 4.12 DIO x 182.46 ± 2.61 DIO+FGF19) and RQ (0.92 ± 0.01 Lean x 0.79 ± 0.01 DIO x 0.87 ± 0.02 DIO+FGF19) in mice during the dark cycle. This improvement observed in FGF19 treated DIO mice was also associated with higher BAT temperature (°C) (35.58 ± 0.33 Lean x 34.60 ± 0.14 DIO x 36.76 ± 0.35 DIO+FGF19), as well as increased expression of thermogenic marker genes in the BAT (UCP1: 0.27 ± 0.06 DIO x 0.64 ± 0.09 DIO+FGF19; DIO2: 0.25 ± 0.03 DIO x 0.68 ± 0.18 DIO+FGF19; PPARGC1α: 0.52 ± 0.09 DIO x 1.39 ± 0.30 DIO+FGF19; PRDM16: 0.33 ± 0.05 DIO x 0.73 ± 0.15 DIO+FGF19, and CIDEA: 0.35 ± 0.06 DIO x 0.79 ± 0.11 DIO+FGF19, fold change of Lean).

Conclusion: Our results show that central delivery of FGF19 improves energy metabolism in DIO mice, and the increased thermogenic capacity of BAT must be involved, at least in part, in the observed beneficial effects.

Supported by: FAPESP

Disclosure: L. Zangerolamo: Grants; FAPESP - 2020/14020-7.

214

Modulation of hypothalamic AMPK phosphorylation by Olanzapine controls energy balance and body weight

V. Ferreira1, C. Folgueira2, M. Guillén1, P. Zubiaur3, D. Grajales1, A. Sarsenbayeva4, P. López Larrubia1, J.W. Eriksson4, M.J. Pereira4, F. Abad-Santos3, G. Sabio2, P. Rada1, Á.M. Valverde1;

1IIBm Alberto Sols (CSIC-UAM), Madrid, Spain, 2Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, 3Instituto de Investigación Sanitaria La Princesa, Madrid, Spain, 4Clinical Diabetes and Metabolism, Uppsala University, Uppsala, Sweden.

Background and aims: Second-generation antipsychotics (SGAs) are mainstay therapy for psychiatric disorders. SGA-treated patients present risk for weight gain and insulin resistance. Since overweight/obesity favors insulin resistance and Type 2 Diabetes, we evaluated the effect of olanzapine (OLA), a widely prescribed SGA, in mice, focusing on body weight, energy balance and insulin sensitivity. We also explored OLA effects in protein tyrosine phosphatase-1B deficient (PTP1B-KO) mice, a preclinical model of insulin and leptin hypersensitivity.

Materials and methods: Wild-type (WT) and PTP1B-KO mice were fed an OLA-supplemented diet (5 mg/kg/day, 7 months) or treated via intraperitoneal (i.p.) injection (10 mg/kg/day, 8 weeks). Readouts of hypothalamus-periphery crosstalk were assessed by OLA intrahypothalamic administration with or without adenoviruses expressing a constitutive active AMPKα1 mutant.

Results: Although both genotypes of mice treated orally with OLA presented hyperphagia (p<0.001, n=19-33), weight gain was enhanced only in the WT (p<0.001, n=9-10). Unexpectedly, all mice receiving OLA via i.p. lost weight (p<0.001, n=6-11) without changes in food intake, but with an increase in energy expenditure (p<0.05, n=4-6). Elevation in UCP-1 levels (p<0.05, n=5-6) and brown adipose tissue (BAT) temperature (p<0.05, n=7-17) appeared concomitantly with lower hypothalamic phospho-AMPK (p<0.01, n=8-13). Likewise, OLA central injection reduced phospho-AMPK (p<0.05, n=3) and increased BAT UCP-1 (p<0.05, n=5-6), effects abolished by hypothalamic AMPK activation (n=6-7). OLA i.p. treatment was associated with enhanced Tyrosine Hydroxylase (TH)-positive innervation (p<0.05, n=10-14) and less sympathetic neuron-associated macrophages in subcutaneous white adipose tissue (iWAT). Both central and i.p. OLA injections increased UCP-1 (p<0.05, n=8-11) and TH (p<0.05, n=5-11) in iWAT, effects prevented by hypothalamic AMPK activation. Contrarily, BAT thermogenesis was unaltered in dietary-treated WT mice while increased in PTP1B-KO mice (p<0.05 n=6-7). Notably, OLA treatment induced markers of insulin resistance in WT mice by both administration routes. Specifically, after i.p. OLA treatment, WT mice presented insulin (p<0.05, n=5-9) and pyruvate (p<0.05, n=17-24) intolerance, effects absent in PTP1B-KO mice. The insulin signaling analysis showed a reduction insulin receptor (p<0.05, n=4-5) and Akt (p<0.05, n=4-5) phosphorylation in liver, muscle and primary hepatocytes from OLA-treated WT mice concomitantly with inflammatory hallmarks (p<0.05, n=5). Importantly, PTP1B-KO mice were protected against OLA-induced insulin resistance.

Conclusion: Mechanistically, we found that OLA reduces hypothalamic phospho-AMPK inducing weight loss through thermogenesis activation. In conclusion, we unraveled an unexpected metabolic rewiring controlled by hypothalamic AMPK that avoids weight gain in male mice treated i.p. with OLA by activating BAT thermogenesis and iWAT browning, as well as a therapeutic benefit of PTP1B inhibition against OLA-induced insulin resistance and weight gain.

Supported by: ITN-TREATMENT (721236); FCT/FEDER (2020.08388.BD); MICINN/AEI/FEDER, EU (RTI2018-094052-B-100)

Disclosure: V. Ferreira: None.

215

Effect of interesterified fat and its metabolites on inflammation and insulin resistance in hypothalamus

J.E. Miyamoto1,2, R.M.A. Santos1, B.P. Siqueira1, A. Reginato3, J. Guitton4, P.L.R. Menta1, T.G. Ramalheira1, A.S. Torsoni1, L.M. Ignácio-Souza1, M.A. Torsoni1, H. Le Stunff4, C. Magnan2, M. Milanski1;

1Laboratory of Metabolic Disorders, University of Campinas, Limeira, Brazil, 2Biologie Fonctionnelle et Adaptative (BFA) - CNRS UMR 8251, Université Paris Cité, Paris, France, 3Medicine/Endocrinology, Albert Einstein College of Medicine, Bronx, USA, 4Neuroscience Paris-Saclay Institute (NeuroPSI) - UMR CNRS 9197, Université Paris Saclay, Paris, France.

Background and aims: The overconsumption of dietary fats leads to inflammation in peripheral tissues, which is implicated in the development of obesity and insulin resistance (IR). More recently, hypothalamus has become a key target of dietary fat in the development of central and peripheral IR. Nevertheless, the role of different types of dietary lipids, particularly the nature of triacylglycerols (TAGs) in the development of IR are still poorly understood. TAGs occur naturally or they can be manufactured from vegetable oils and animal fats by interesterification. This latter process leads to an enrichment of saturated fatty acids (SAFA) on the sn-2 position of TAGs. Recently, we provided evidence that a normocaloric interesterified lipid diet impaired glucose tolerance in mice. In the present study, we evaluated the role of interesterified palm oil and their metabolites on insulin signaling and inflammation in mice and in hypothalamic neuronal cells.

Materials and methods: Adult male Swiss mice were divided into four experimental groups: nomorcaloric palm oil diet (PO), nomorcaloric interesterified palm oil diet (IPO), PO high fat diet (POHFD) or IPO high fat diet (IPOHFD) during 8 weeks. Glucose homeostasis was assessed by GTT. Hypothalamus were processed and RNA-seq was performed. For in vitro analysis, mHypoA 2/28 hypothalamic cell was treated for 16h with TAG derivatives namely monoacylglycerol either linked to palmitate on sn-2 position (2-palmitoylglycerol (2-PG)) or to oleic acid (Oleic) on sn-2 position (2-oleylglycerol (2-OG)). Free palmitic acid (Palm) or oleic acid were used as controls. Pro-inflammatory mediators expression were assessed by RTqPCR. Insulin signaling pathway was evaluated by quantifying Akt phosphorylation. Involvement of de novo ceramide synthesis was determined by the use of the serine palmitoyl-transferase inhibitor, myriocin.

Results: The interesterification generates new TAGs, which is characterized by increased SAFA on sn-2, induced glucose intolerance. In vivo, normocaloric IPO diet induced the regulation of various genes in hypothalamus including cellular stress markers. In vitro studies with hypothalamic cell lines (mHypoA-2/28) evidenced that the treatment with 2-PG as free palmitate impaired insulin signaling by decreasing AKT phosphorylation. 2-PG also promoted inflammation by increasing IL-6 levels. Interestingly, treatment 2-OG or free oleic acid did not induced inflammation. Importantly, lipid profile analysis revealed that both Palm and 2-PG treatment increased intracellular levels of palmitate in mHypo 2/28 cells. Finally, myriocin treatment counteracted the impairment of insulin signaling induced by 2-PG.

Conclusion: Altogether, our data showed that interesterified fats targets hypothalamus to mediate their metabolic effect. This preliminary data also allowed us to assert that SAFA on sn-2 position of TAG could as free fatty acids to alter insulin signaling and inflammation in neuronal cells and contributed therefore to the development of obesity and IR.

Supported by: FAPESP 2016/24768-3 and 2020/16040-5 (Fellowship), 2017/15925-0 and 2019/26538-3 (Grant)

Disclosure: J.E. Miyamoto: None.

216

A role for GIP in the regulation of food intake and body weight in mice

J.E. Lewis, T. Darwish, F. Reimann, F.M. Gribble;

Institute of Metabolic Science, University of Cambridge, Cambridge, UK.

Background and aims: Hormones from the gut that signal nutrient uptake and availability to the brain are key elements in the control of appetite. Glucagon-like peptide-1 (GLP-1) based pharmacotherapies are licensed for the treatment of type 2 diabetes and obesity and dual agonist peptides stimulating both GLP-1R and glucose-dependent insulinotropic peptide receptors (GIPR) have been shown to promote greater weight reduction. GIP is well established to regulate blood glucose via its insulinotropic and glucagonotropic action on the pancreas, however, the role of GIP in the regulation of food intake and body weight remains controversial. This study addressed the metabolic roles of endogenous GIP by directly activating the GIP-secreting cell population.

Materials and methods: Utilising a novel rodent model (GIP-Cre x Dq), targeting the Designer Receptor Activated by Designer Drugs (Dq-DREADD) to GIP expressing cells, we measured intraperitoneal glucose tolerance, acute food intake and whole-body physiology/feeding behaviour in metabolic cages in the lean and diet induced obese state with and without activation of cells through Dq with the DREADD-ligand CNO in a cross-over design (n=14).

Results: In lean GIP-Cre x Dq mice, CNO resulted in an increase in plasma GIP, akin to that in the postprandial state in mice. In lean and diet induced obese mice, the increase in GIP was associated with improved glucose tolerance (p<0.0001) and a reduction in food intake both in the ad lib fed state at the onset of the dark phase (p<0.01) and in a fast-refeed paradigm (p<0.001). The reduction in food intake was a consequence of reduced feeding time (p<0.05) and an increase in the time between meals (p<0.001). No change in energy expenditure or activity was detected following GIP cell activation, however, a transient reduction in body weight was apparent (p<0.05). In lean mice, the effects on glucose tolerance and food intake were blocked by pre-treatment with a GIPR antagonistic antibody, suggesting the effects were specific to GIP and mediated through an antibody accessible receptor site.

Conclusion: These studies provide new insights into the physiological roles of GIP, suggesting that it contributes to the control of appetite as well as blood glucose levels.

Supported by: EFSD/Lilly European Diabetes Research Programme

Disclosure: J.E. Lewis: None.

OP 37 Insulin deficiencies and cardiovascular disease

217

Incidence of cardiovascular disease in latent autoimmune diabetes in adults

Y. Wei, E. Ahlqvist, T. Andersson, T. Tuomi, S. Carlsson;

Karolinska Institutet, Stockholm, Sweden.

Background and aims: Latent autoimmune diabetes in adults (LADA) is a slowly progressing form of autoimmune diabetes with an adult onset. Our aim was to contribute to the limited knowledge on the risk of CVD in LADA with varying degrees of autoimmunity.

Materials and methods: This population-based Swedish study followed individuals with newly diagnosed LADA (n= 587) and type 2 diabetes (T2D, n=2013) and matched diabetes-free controls (n=2385) between 2007 and 2019 through National Diabetes, Patient and Cause-of-Death Registers. HR (95% CI) for a first occurrence of CVD (ischemic heart disease, stroke, and heart failure) in LADA and T2D compared to controls were estimated by Cox regression and adjusted for age, sex, calendar year and lifestyle factors. LADA patients were stratified based on the median level (250 IU/ml) of glutamic acid decarboxylase antibodies (GADA) into LADAhigh and LADAlow.

Results: During a median follow-up of 6.05 years, we recorded 328 CVD events (37 in LADA and 173 in T2D). The risk of a first occurrence of CVD was increased in T2D (HR: 1.58, 95% CI: 1.22, 2.06) but not in LADA (HR: 1.25, 95% CI: 0.86, 1.83) when compared to people without diabetes. However, stratification by GADA levels revealed that patients with LADAhigh had excess risk of CVD (HR: 1.72, 95% CI: 1.09, 2.71) that was on par with that observed in T2D (Figure 1). LADAhigh patients had worse beta-cell function (median HOMA-B 30.9 vs 46.8, p<0.001) and glycemic control (median HbA1c during follow-up: 49.7 vs 55.3 mmol/mmol, p<0.001), but lower prevalence of CVD (6.7% vs 14.8%, p=0.002) at baseline compared to LADAlow patients.

Conclusion: Among LADA patients free of CVD at the time of diabetes diagnosis, those with high GADA levels have higher future risk of CVD, which might be driven by worse β cell function and glucose control. However, attention should also be paid to LADA patients with low GADA given their much higher CVD prevalence at diabetes diagnosis.

figure bh

Supported by: CSC, Novo Nordisk foundation, Swedish Research Council, FORTE.

Disclosure: Y. Wei: None.

218

Endotrophin is a risk marker of complications in a type 1 diabetes cohort

A.L. Møller1,2, N.H. Tougaard3, P.F. Rønn3, T.W. Hansen3, F. Genovese1, M.A. Karsdal1, D.G.K. Rasmussen1, P. Rossing3,4;

1Nordic Bioscience, Herlev, 2Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 3Steno Diabetes Center Copenhagen, Herlev, 4Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Persons with diabetes have a high risk of complications related to the micro- and macrovascular circulation. Hyperglycemia can trigger pathological pathways leading to fibrosis, an overreaction to tissue injury caused by accumulation of extracellular matrix components. In this study, we investigated, the potential of endotrophin, a pro-fibrotic molecule generated during collagen type VI (COL6) formation, as a risk marker for development of complications and mortality in an unselected type 1 diabetes population.

Materials and methods: We measured endotrophin in serum and urine in 1468 individuals with type 1 diabetes (49% females, mean±SD age of 51±16 years) recruited from a Diabetes Center between 2012-2016. Urine endotrophin values were normalized to urinary creatinine levels. Participants were followed for a median of up to 6.4 years, and information on endpoints was extracted from national registers. Endpoints included: a composite renal endpoint, major adverse cardiovascular event (MACE), all-cause mortality, progression of albuminuria, incident heart failure (HF), and sight-threatening eye disease. Cox proportional hazards models adjusted for conventional risk factors and prevalent disease (endpoint of interest) at baseline were applied.

Results: A doubling of serum endotrophin was independently associated with the composite renal endpoint (n=36, HR: 3.69, 95% CI: 2.24-6.07), MACE (n=140, HR: 1.46, 95% CI: 1.12-1.89), all-cause mortality (n=93, HR: 1.44, 95% CI: 1.03-2.0), and progression of albuminuria (n=80, HR: 1.69, 95% CI: 1.22-2.35), but not with incident HF or sight-threatening eye disease after adjustment for conventional risk factors. The associations remained significant after further adjustment for prevalent disease at baseline: composite renal endpoint (HR: 3.27, 95% CI: 1.89-5.65) and MACE (HR: 1.43, 95% CI: 1.06-1.93). A doubling of urine endotrophin was not associated with any of the endpoints after adjustment.

Conclusion: Serum endotrophin released during COL6 formation is an independent risk marker of mortality, cardiovascular and renal complications in persons with type 1 diabetes.

Supported by: This work was supported by the Innovation Fund Denmark (0172-00270B)

Disclosure: A.L. Møller: None.

219

Predictors of arterial stiffness in patients with type 1 diabetes: importance of long-term glycaemic control and physical activity

S. Helleputte1,2, P. Calders1, J. Marlier3, T. De Backer4,1, B. Lapauw3,1;

1Faculty of Medicine and Health Sciences, Ghent University, Gent, 2Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen, Brussels, 3Endocrinology, Ghent University Hospital, Gent, 4Cardiology, Ghent University Hospital, Gent, Belgium.

Background and aims: In patients with type 1 diabetes, arterial stiffness can add value in estimating cardiovascular disease (CVD) risk. Traditional CV risk factors and glycaemic control as reflected by HbA1c, are main determinants of arterial stiffness. However, the relationship with other markers of glycaemic control, as advanced glycation end products (AGEs) and continuous glucose monitoring (CGM)-derived parameters, and with physical activity levels, is less well explored. This study aimed to examine the relationship of arterial stiffness with short- and long-term parameters of glycaemic control, physical performance and activity level in patients with type 1 diabetes free from overt CVD.

Materials and methods: Cross-sectional study in patients with a type 1 diabetes duration of at least ten years and still free from known CVD. Current level and 10-years history of HbA1c was evaluated, as well as skin AGEs. Arterial stiffness was assessed with carotid-femoral pulse wave velocity (cf-PWV). CGM for 7 days was used to determine time in range (TIR), time in hyper- and hypoglycaemia, and glycaemic variability. Levels of physical activity and exercise capacity were evaluated. Pearson (r) and Spearman (rs) correlations, and multiple linear regression was used to investigate associations with cf-PWV.

Results: 54 patients (M/F: 32/22; age: 46 ± 9.5 yrs; type 1 diabetes duration: 27 ± 8.8 yrs; HbA1c: 7.8 ± 0.83%) were included. cf-PWV showed significant associations with traditional risk factors age (rs= +0.69), type 1 diabetes duration (rs= +0.41) and 24-hours mean arterial pressure (rs= +0.45); cf-PWV was significantly associated with current HbA1c (rs= +0.28), mean 10-years HbA1c (rs= +0.36) and AGEs (rs= +0.40), but not with any of the CGM-derived parameters; and negatively associated with VO2max (rs= -0.41) and physical activity level (rs= -0.60). Multiple linear regression for cf-PWV showed that the model with the best fit included age, type 1 diabetes duration, 24-hour mean arterial pressure and mean 10-years HbA1c (adjusted R2= 0.645); and that VO2max had independent predictive value.

Conclusion: This study demonstrated that long-term glycaemic exposure as reflected by mean 10-years HbA1c and AGEs is a main predictor of arterial stiffness in patients with type 1 diabetes, while no relationship was found with any of the CGM-parameters. Importantly, physical activity level and exercise capacity were inversely associated with arterial stiffness. Our findings stress the importance of early and sustained good glycaemic control and of engagement in physical activity to prevent premature CVD in patients with type 1 diabetes.

figure bi

Supported by: The first author (S.H.) is supported by a PhD grant Fundamental Research from FWO Vlaanderen.

Disclosure: S. Helleputte: Grants; Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen PhD fellowship grant.

220

Hyperglycaemia and hypoglycaemia exposure are differentially associated with micro- and macrovascular complications in adults with type 1 diabetes

A. Mesa1, M. Giménez1,2, I. Pueyo1, V. Perea3, C. Viñals1, J. Blanco1,2, I. Vinagre1,2, T. Serés-Noriega1, L. Boswell1,4, E. Esmatjes1,2, I. Conget1,2, A.J. Amor1;

1Endocrinology and Nutrition Department, Hospital Clínic de Barcelona, Barcelona, 2IDIBAPS (Institut d’investigacions biomèdiques August Pi i Sunyer), Barcelona, 3Endocrinology and Nutrition Department, Hospital Mútua de Terrassa, Terrassa, 4Endocrinology and Nutrition Department, Althaia – Xarxa Assistencial Universitària de Manresa, Manresa, Spain.

Background and aims: Information on the association between continuous glucose monitoring (CGM) data and chronic complications in type 1 diabetes (T1D) is scarce. We explored the relationship between high and low glucose exposure glucometrics obtained by CGM and micro- and macrovascular complications in this population.

Materials and methods: Cross-sectional study in T1D patients without cardiovascular disease (CVD) and with at least one of the following: ≥40 years, diabetic nephropathy or ≥10 years of T1D duration with CVD risk factors. CGM data were obtained from a CGM sensor regularly used by each patient (Dexcom G5, Guardian Sensor 3 or FreeStyle Libre). CGM-derived glucometrics from 14 consecutive days were collected: glucose management indicator (GMI), coefficient of variation and proportion of time (%) <54 (time below range; TBR<54), <70, 70-180 (time in range; TIR), >180 (time above range; TAR). Carotid plaque (intima-media thickness ≥1.5 mm) was evaluated by standardized ultrasonography protocol. Logistic regression models adjusted for CGM sensor, age, sex, diabetes duration and other CVD risk factors were constructed to test the independent association between CGM-derived glucometrics and chronic complications.

Results: We included 152 patients (54.6% men, mean age 48.7±10.0 years, T1D duration 28.6±11.3 years, 5-year mean HbA1c 7.31% (6.86-7.89), insulin pump therapy 36.2%, TIR 61.3±15.3%, TBR<54 1.0±1.5%, TAR 33.3±15.7%, GMI 7.11±0.71%). Sixty-seven patients showed carotid plaque and n=71 microvascular complications (retinopathy and/or nephropathy). TAR (OR 1.28 [1.09-1.51], p=0.003; 5% increase) and GMI (OR 3.05 [1.46-6.36], p=0.003; 1% increase) were directly associated with the presence of microvascular complications, while TIR had an inverse relationship (OR 0.79 [0.66-0.93], p=0.005; 5% increase). TBR<54 was directly associated with the presence of plaques, even after adjusting for 5-year mean HbA1c (p<0.05; Figure).

Conclusion: Glucometrics related to high glucose exposure were independently associated with microvascular complications. Only low glucose exposure glucometrics was significantly associated with preclinical atherosclerosis. Our data supports the role of hypoglycemia in the development of CVD in this population.

figure bj

Supported by: Contracte Clínic Recerca "Josep Font-Emili Letang" and Ajut programa doctorat ACD

Disclosure: A. Mesa: Grants; Contracte Clínic de Recerca "Josep Font-Emili Letang", Ajut per a la realització d'un programa de doctorat de l'Associació Catalana de Diabetis.

OP 38 It is always a D-D-Day: diabetes, digital, device

221

Long term weight loss in a primary care-anchored eHealth lifestyle coaching programme in Denmark: a randomised controlled trial

L. Hesseldal1, J.R. Christensen1, T.B. Olsesen2, M.H. Olsen3, P.R. Jakobsen4, J.T. Lauridsen5, D.H. Laursen6, J.B. Nielsen1, J. Søndergaard1, C.J. Brandt1;

1Research Unit for General Practice,, Research Unit for General Practice, Institute of Public Health; University of Southern Denmark, Odense, 2Steno Diabetes Center Odense, Steno Diabetes Center Odense, Odense University Hospital (OUH), Odense, 3Department of Internal Medicine, Holbaek Hospital and Steno Diabetes Center Zealand, Holbaek, 4Research Unit for General Practice, Research Unit for General Practice, Institute of Public Health; University of Southern Denmark, Odense, 5Department of Business and Economics, University of Southern Denmark, Odense, 6Department of Public Health, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Long-term weight loss among subjects with obesity can reduce the risk and progression of noncommunicable diseases (NCDs) such as cardiovascular disease, respiratory disease, and type 2 diabetes (T2D). Unfortunately, long-term weight loss has been historically difficult for patients with obesity and T2D to achieve and maintain. Observational studies suggest that digital coaching can lead to long-term weight loss and potentially reduce the risk of developing NCDs. To assess whether an eHealth lifestyle coaching program (LIVA) for motivated subjects with obesity with or without T2D leads to significant long-term (more than 6 months) weight loss compared to usual care.

Materials and methods: In an open, randomized controlled trial, 340 subjects with obesity with or without T2D were enrolled from March 2018 to March 2019 and randomized to the intervention (200) and control (140) groups. The digital lifestyle intervention comprised an initial one hour face-to-face motivational interview followed by digital coaching using behavioral change techniques enabled by individual live monitoring.

Results: At 6 and 12 months, data were assessed for 235 participants, 149 from the intervention group and 86 from the control group. After 12 months mean body weight and body mass index were reduced significantly in both groups but significantly more in the intervention group (-4.6(95%CI, -5.7; -3,4) kg vs. -1.4(95%CI,-2.6;-0.1) kg, P<0.005 and -1.5(95%CI,-1.9;-1.2) kg/m2 vs. -0.5(95%CI,-0.9;-0.1) kg/m2, P < 0.005). HbA1c was significantly reduced in both the intervention (-6.0(95%CI,-7.7;-4.3)mmol/mol) and control group (-4.9(95%CI,-7.4;-2.4)mmol/mol) without significant group difference. Blood pressures and lipid profiles did not change significantly.

Conclusion: Compared to usual care, digital lifestyle coaching can induce significant weight loss in obese subjects with or without T2D.

Clinical Trial Registration Number: NCT03788915

Disclosure: L. Hesseldal: None.

222

Telehealth versus in-person standard care in patients with type 1 diabetes treated with multiple doses of insulin: an open-label randomised controlled trial

S. Ballesta1,2, J.J. Chillarón1,2, Y. Inglada1, A. Rodriguez3, E. Climent2, G. Llauradó2, H. Camell4, J.A. Flores1,2, D. Benaiges1,2;

1Endocrinology and Nutrition, Hospital Comarcal de l´Alt Penedès, Vilafranca del Penedès, 2Endocrinology and Nutrition, Hospital del Mar, Barcelona, 3Research department, Hospital Comarcal de l´Alt Penedès, Vilafranca del Penedès, 4Internal Medicine, Hospital Comarcal de l´Alt Penedès, Vilafranca del Penedès, Spain.

Background and aims: Increasing evidence indicates that telehealth (TH) is non-inferior to in-person approach regarding metabolic control in type 1 diabetes (T1D) patients, and offers advantages such as a decrease in travelling-time to outpatient clinic and increased accesibility for shorter and more frequent visits. This point has been even more important during the COVID-19 pandemic in rural zones, where distances are longer and time spent travelling are greater. The primary aim of our study was to compare, in T1D patients assisted in a rural area, the change in HbA1c at 6-month between TH and in-person visits. As secondary objectives we compared glucometric parameters, hypoglycemic events, EsDQoL questionnaire, direct and indirect costs, and patient’s satisfaction.

Materials and methods: Randomized controlled study, open-label, parallel arms, among T1D subjects. In the conventional group, patients were submitted to standard in-person visits (30 minutes) in outpatient clinic (baseline, 3 and 6 months). In the TH group, patients were submitted to teleconsultation (10 minutes) in month 1, 2, 3 and 4; baseline and 6-month visits were in-person. Mean-differences in change of included variables during follow-up were analyzed.

Results: 55 subjects were included (29 conventional, 26 TH). Mean age was 50.6 years ± 12.0, 48% were females, T1D evolution was 21.5 years + 11.6 and mean HbA1c was 7.56 % ± 0.7. No differences in baseline characteristics between groups were observed. At 6-month follow-up, the mean change in HbA1c was 0.2% (range: -1.3 - 1.2) in the control group and -0.05% (range: -0.8 - 1.7) in the TH group (mean difference 0.013, P = 0.932). Changes in EsDQoL questionnaire, glucometry parameters, mild hypoglycemic events and costs are shown in Table 1. No severe hypoglycemia was detected. Total time spent by endocrinologist was 93.2 minutes + 12.0 in conventional group vs 102.9 minutes + 7.6 in TH group (P= 0.001). 46% of patients in TH group preferred alternation between both types of visits. The main concern about telehealth was non-compliance with visiting hours, and main advantages were no need to travel to hospital and time saving.

Conclusion: TH is comparable to in-person visits regarding HbA1c levels at 6-month follow-up among T1D subjects, with significant improvement in time in range (TIR) and EsDQoL. Despite increased costs for national care system, teleconsultations imply a decrease in direct and indirect costs for patients. Further studies about telemedicine in T1D are necessary to evaluate a more efficient timing of the TH visits, as well as long-term cost analysis studies.

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Clinical Trial Registration Number: ClinicalTrials.gov identifier NCT04758884

Disclosure: S. Ballesta: None.

223

Association between HbA 1c and time in range in adults with type 1 diabetes using sensor-based glucose monitoring: a Swedish National Diabetes Register population-based study

E. Kjölhede1,2, J. Nåtman3, J. Ekelund3, S. Salö4, N.F. Nielsen5, B. Eliasson1, K. Eeg-Olofsson1,3;

1Department of Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden, 2Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, Gothenburg, Sweden, 3Centre of Registers, Gothenburg, Sweden, 4Novo Nordisk Skandinavia AB, Malmö, Sweden, 5Novo Nordisk A/S, Søborg, Denmark.

Background and aims: New sensor-based continuous glucose monitoring (CGM) systems used in combination with modern insulin treatment (either multiple daily injections or insulin pump) offer potential to improve glycaemic control and provide new glycaemic measures. HbA1c is an important measure of overall glycaemic control but not of glucose variability. In Sweden today, >85% of type 1 diabetes (T1D) patients use sensor-based glucose monitoring, making it possible to assess new measures of glucose variability and time in range (TIR) in routine diabetes care. This study aimed to describe the relationship between HbA1c and TIR (percentage of time in the range of 4-10 mmol/L).

Materials and methods: This cross-sectional nationwide study included people aged ≥18 years with T1D using CGM (intermittent-scanning or real-time) with at least one registration of TIR in the Swedish National Diabetes Register (NDR) from 1 January 2020 to 23 December 2021. Each TIR value was matched to the HbA1c value closest in time, maximum ±90 days. If a patient had more than one TIR and HbA1c pairing, one observation was selected at random. Other sensor data variables were collected from the same registration as the selected TIR value. For all other characteristics in the NDR, the latest registered value within 365 days before the TIR registration was selected. The linear association was assessed using Pearson correlation coefficient. Linear regression models were estimated between HbA1c and TIR, with and without adjustment for time below range (TBR; <4 mmol/L), diabetes duration, sex and insulin delivery method.

Results: The analysis included 27,980 people with T1D, 46% women, 30% on insulin pump, 7% with previous coronary heart disease and 64% with retinopathy. Mean (±SD) values were: age 48±18 years, diabetes duration 25±16 years, HbA1c 59±13 mmol/mol, TIR 59±19%, TBR 5±5%, sensor glucose 9.2±2.0 mmol/L and sensor glucose SD 3.3±1.0 mmol/L (mean coefficient of variation 36±7%). The distribution of TIR was marginally right-shifted in women compared with men. Figure 1 shows the association between HbA1c and TIR with a correlation coefficient −0.71 (−0.71 in men, −0.71 in women, −0.72 in pen users, −0.69 in pump users). In the crude linear regression, R2 was 0.51 and, in the adjusted model, R2 was 0.57.

Conclusion: This study with real-world data showed a clear association between HbA1c and TIR in adults with T1D, suggesting that TIR may be a relevant complement to HbA1c in everyday practice. Future studies are needed to evaluate if TIR is also associated with risk of complications and costs.

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Supported by: Novo Nordisk A/S

Disclosure: E. Kjölhede: Honorarium; Novo Nordisk: consulting fee for reviewing and revising survey content.

224

Accuracy of continuous glucose monitoring systems during exercise-related hypoglycaemia in patients with type 1 diabetes

K. Maytham1, P.G. Hagelqvist1, U. Pedersen-Bjergaard2, F.K. Knop3, A. Andersen1, T. Vilsbøll1;

1Clinical Research, Steno Diabetes Center Copenhagen, University of Copenhagen, Herlev, 2Department of Endocrinology and Nephrology, Nordsjællands Hospital Hillerød, University of Copenhagen, Hillerød, 3Center for Clinical Metabolic Research, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark.

Background and aims: Hypoglycaemia is common in patients with type 1 diabetes, especially during exercise. Continuous glucose monitoring (CGM) accuracy is clinically important since low sensor precision may lead to undetected events of hypoglycaemia or unnecessary meal intake resulting in hyperglycaemia. We investigated sensor accuracy in available CGM devices during exercise-related hypoglycaemia.

Materials and methods: Fifteen patients with type 1 diabetes participated in two separate euglycaemic-hypoglycaemic clamp days (Clamp-exercise and Clamp-rest) with five different phases: 1) a baseline-euglycemic phase, 2) a plasma glucose decline phase ± exercise, 3) a 15-minute hypoglycaemic phase ± exercise, 4) a 45-minute hypoglycaemic phase (at rest), and 5) a plasma glucose incline phase. Plasma glucose was determined every five minutes, using Dexcom G6 and FreeStyle Libre 1. Yellow Spring Instruments 2900 (YSI) was used as a plasma glucose reference method, enabling mean absolute relative difference (MARD) assessment during each phase and Clarke error grid analysis during each day. We defined ΔMARD = MARDe (Clamp-exercise) - MARDr (Clamp-rest).

Results: We observed an overall higher MARD for Dexcom G6 compared to FreeStyle Libre 1 throughout both clamp days. CGM accuracy during phase 2 was more accurate during exercise compared to rest for Dexcom G6 (ΔMARD = -6.2 percentage points) while FreeStyle Libre 1 showed opposite results (ΔMARD = +5.3 percentage points) (Figure 1). During phase 3, exercise had no effect on Dexcom G6 accuracy, whereas FreeStyle Libre 1 was more accurate during rest (ΔMARD = +13.5 percentage points). During phase 4 (post-exercise versus post-rest) Dexcom G6 was more accurate after exercise than after rest (ΔMARD = -8.4 percentage points), whereas FreeStyle Libre 1 showed no differences between days. Clarke error grid analysis showed a decrease in clinically acceptable treatment decisions during Clamp-exercise for FreeStyle Libre 1 while a corresponding increase was observed for Dexcom G6.

Conclusion: Dexcom G6 had an overall lower accuracy compared to FreeStyle Libre 1. Exercise negatively impacted FreeStyle Libre 1 sensor performance during both declining plasma glucose and hypoglycaemia, whereas it improved Dexcom G6 sensor performance. Thus, physical exercise can affect the accuracy of different sensor systems in different manners, which may be of clinical importance, especially in detecting episodes of hypoglycaemia.

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Clinical Trial Registration Number: NCT04650646

Supported by: DFF

Disclosure: K. Maytham: None.

OP 39 Too little sugar is also bad: understanding hypoglycaemia

225

Tmem117 in AVP neurons regulates the counterregulatory response to hypoglycaemia

S. Gaspari, G. Labouèbe, A. Picard, A. Rodriguez Sanchez-Archidona, B. Thorens;

University of Lausanne, Lausanne, Switzerland.

Background and aims: The counterregulatory response to hypoglycemia (CRR), which ensures a sufficient glucose supply to the brain, is an essential survival function. It is orchestrated by incompletely characterized glucose-sensing neurons, which trigger a coordinated autonomous and hormonal response that restores normoglycemia. A genetic-genomic screen for insulin-induced glucagon (GCG) secretion was conducted using a panel of 36 BXD recombinant inbred mouse strains. Tmem117, located on chromosome 15, was identified as a potential regulator of GCG secretion. Here, we aimed at characterizing the role of Tmem117 in hypoglycemia-induced GCG secretion.

Materials and methods: Immunofluorescence microscopy (IF); physiological characterization of mice with conditional, cell-specific gene inactivation; electrophysiological and in vivo fiber photometry measurements were used.

Results: We found that Tmem117 expression in the hypothalamus of the BXD mice was negatively correlated with the GCG response (Pearson’s R: -0.55; p=0.0003). IF found Tmem117 to be expressed in vasopressin (AVP) magnocellular neurons of the paraventricular and the supraoptic nuclei of the hypothalamus. Inactivation of Tmem117 specifically in AVP neurons (AVPTM117KO) led to higher hypoglycemia-induced copeptin (CPP; surrogate for AVP) secretion and higher plasma GCG levels in male mice [CPP: 89±5 vs 121±13 pg/ml, GCG: 124±19 vs 186±21 pg/ml, in Ctrl and AVPTM117KO mice, respectively; p<0.01; 2way-ANOVA RM]. In female mice this secretory phenotype was present only during the proestrus phase [CPP: 66±9 vs 107±16 pg/ml, GCG: 224±30 vs 377±22 pg/ml, in Ctrl and AVPTM117KO mice, respectively; p<0.05; t-test]. c-Fos IF and electrophysiological recordings performed on acute brain slices revealed that a large proportion (50%) of AVP magnocellular neurons were activated by hypoglycemia (glucose-inhibited, GI neurons). Inactivation of Tmem117 did not affect the glucose responsiveness of the AVP neurons. However, it led to their progressive elimination (number of AVP neurons in the SON: 91±9 vs 54±3 in Ctl and AVPTM117KO mice, respectively; 35 days after Tmem117 inactivation; p<0.05; t-test) and a loss of the increased secretory phenotype (CPP: 55±14 vs 83±6 pg/ml, GCG: 81±11 vs 119±19 pg/ml in Ctl and AVPTM117KO mice, respectively; 34 days after Tmem117 inactivation; p>0.05; 2way-ANOVA RM). Overexpression of Tmem117 in the insulin secreting MIN6B1 cells reduced K+-induced [Ca++]i and insulin secretion (Fluo-4: 35452±1030 vs 30888±2513 RFU, insulin: 145±11 vs 88±11 μg/L in Ctl and Tmem117 overexpressing cells, respectively; p<0.01; t-test). Inversely, fiber photometry experiments showed that Tmem117 inactivation increased [Ca++]i both at baseline (GCaMP7: 15±2 vs 42±10 z-score in Ctl and AVPTM117KO mice, respectively; p<0.01; 2way-ANOVA RM) and during insulin-induced hypoglycemia (GCaMP7: 1961±54 vs 2491±64 AUC in Ctl and AVPTM117KO mice, respectively; p<0.01; 1way-ANOVA).

Conclusion: Our study shows that AVP magnocellular neurons are activated by hypoglycemia to secrete AVP, leading to enhanced GCG secretion. It identifies Tmem117 as a regulator of hypoglycemia-induced CPP and GCG secretion, whose effect depends on the modulation of intracellular calcium dynamics. Finally, as these data were based on an unbiased genetic screen, they highlight the physiologically important contribution of AVP neuroendocrine cells to the counterregulatory response.

Supported by: INTEGRATE 694798, SNSF 310030-182496,

Disclosure: S. Gaspari: None.

226

Molecular investigations of defective hypoglycaemia counter-regulation in a murine model of type-2 diabetes: a multi-omics study

J. Castillo-Armengol1,2, A. Rodriguez Sanchez-Archidona2, C. Fledelius1, F. Marzetta3, B. Thorens2;

1Novo Nordisk A/S, Måløv, Denmark, 2Center for Integrative Genomics (CIG), University of Lausanne, Lausanne, Switzerland, 3Vital-IT Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland.

Background and aims: Repeated exposures to insulin-induced hypoglycaemia in diabetic patients progressively impair the counter-regulatory response (CRR), characterised by reduced secretion of glucagon and other counter-regulatory hormones. Increasing evidence indicates that glucose responsive neuronal networks, in particular located in the hypothalamus, orchestrate the CRR. However, how these neuronal networks become dysfunctional remains to be elucidated. Here we characterised the hypothalamus transcript profiles and chromatin structures in a mouse model of impaired CRR.

Materials and methods: Type-2 diabetes mellitus (T2DM) was induced in C57BL6N mice by high fat diet feeding and low dose streptozotocin injections. Mice were exposed to one (acute hypoglycaemia, AH) or multiple (recurrent hypoglycaemia, RH) insulin-induced hypoglycaemic episodes and plasma glucagon levels were measured. Single nuclei RNA sequencing (snRNAseq) and ATAC sequencing (snATACseq) data were obtained from the hypothalamus and cortex of AH and RH mice.

Results: A defective counter-regulatory response was observed in the T2DM mice exposed to RH as shown by the significant impairment of glucagon secretion in response to insulin injection (AH, n=33, 94.52±9.20 pg/mL vs RH, n=37, 59±pg/mL, p<0.001). Analysis of snRNAseq data revealed identical hypothalamic cell subpopulations in the hypothalamus of AH and RH mice. However, in the neuronal population, a total of 2373 genes were differentially expressed (FDR<0.05) between both mouse groups. Functional enrichment analysis revealed that these genes were strongly associated to the organisation of the synapses and cell projections. Interestingly, numerous genes related to the mitochondrial electron transport chain were also affected. The analysis of open chromatin regions confirmed the observations of the transcriptomic profiling. A subsequent analysis of the cortex of these animals showed that the effects observed in the hypothalamus were also identified in the cortex, suggesting that other brain regions might be similarly affected by the RH. A proteomic profiling of the synaptosomes from the hypothalami of these animals is currently being investigated and will provide additional characterisation of the synaptic proteome.

Conclusion: The present study provides a model of T2DM and defective counter-regulation in mice. Moreover, we show that the impairment of the gluco-regulatory response is related to genes controlling synaptic function and organisation. These changes in synaptic gene expression were observed not only in the hypothalamus, but also in the cortex suggesting that repeated exposure to hypoglycaemia impacts not only glucose sensing cells but may also induce neuronal dysfunctions in multiple brain regions.

Supported by: Novo Nordisk A/S, Innovative Medicines Initiative 2 Joint Undertaking (JU)

Disclosure: J. Castillo-Armengol: Employment/Consultancy; Novo Nordisk A/S. Grants; It has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 777460.

227

Heart rate variability, an index of autonomic function, is associated with C-peptidogenic index, an index of beta cell function: The Maastricht study

E. Rinaldi1, F. van der Heide2, R. Henry3, A. Kroon2, C. van der Kallen2, S. Eussen2, E. Bonora1, R. Bonnadonna4, M. Trombetta1, C. Zusi1, C. Schalkwijk2, M. van Greevenbroek2, C. Stehouwer2;

1Department of Medicine, University Hospital of Verona, Verona, Italy, 2Maastricht University, Maastricht, Netherlands, 3University of Maastricht, Maastricht, Netherlands, 4Department of Medicine, University Hospital of Parma, Parma, Italy.

Background and aims: Beta cell dysfunction is an important contributor to the early pathobiology of type 2 diabetes and its causes are matter of intensive investigation. Autonomic dysfunction may contribute to the pathobiology of beta cell dysfunction. We investigated, using population-based data, whether autonomic function is associated with beta cell function.

Materials and methods: We used cross-sectional data from The Maastricht Study, a population-based cohort study (N=2,007; mean ± SD age 60 ± 8 years; 52% men; and 24% with type 2 diabetes [the latter oversampled by design]). We assessed autonomic function from 24-hour ECGs as time- and frequency-domain heart rate variability (HRV); and we, using data from a 7-point oral glucose tolerance test, estimated beta cell function as C-peptidogenic index, beta cell glucose sensitivity, beta cell potentiation factor, overall insulin secretion, and beta cell rate sensitivity. Then, we used linear regression analyses to study the associations of standardized indices of HRV with standardized indices of beta cell function; and adjusted for age, sex, educational level, insulin sensitivity and major cardiovascular risk factors. In addition, we tested for interaction by sex and glucose metabolism status (type 2 diabetes; prediabetes; normal glucose metabolism) to investigate whether associations differed in strength by these factors.

Results: After full adjustment, greater time and frequency domain HRV were significantly associated with a higher C-peptidogenic index (standardized beta [95%CI], 0.05 [0.00;0.09] and 0.05 [0.00;0.09], respectively) and numerically similarly, though not statistically significantly, associated with higher beta cell glucose sensitivity (0.04 [0.00;0.08] and 0.04 [-0.00;0.08]), higher beta cell potentiation factor (0.04 [-0.00;0.08] and 0.03 [-0.01;0.08]), and higher overall insulin secretion (0.04 [-0.00;0.08] and 0.03 [-0.01; 0.07]). However, neither time nor frequency domain HRV were associated with beta cell rate sensitivity. Last, sex did not modify associations and glucose metabolism did not consistently modify associations.

Conclusion: The present population-based study found that better autonomic function, estimated from greater HRV, was associated with better beta cell function, estimated from C-peptidogenic index. Hence, autonomic dysregulation may contribute to the beta cell dysfunction and, ultimately, to deterioration of glucose metabolism.

Supported by: ERDF, PL, DMEA,SD,PSID,CVM,CARIM, CAPHRI,NUTRIM, SA, HFL, DF, OF, JC, NNF, SA,

Disclosure: E. Rinaldi: None.

228

Agpat5 in AgRP neurons is required for hypoglycaemia-induced glucagon secretion

A. Strembitska1, G. Labouebe1, A. Picard1, X.P. Berney1, D. Tarussio1, M. Jan2, B. Thorens1;

1University of Lausanne, 2Vital-IT, Lausanne, Switzerland.

Background and aims: The counter-regulatory response to hypoglycemia restores blood glucose levels by stimulating glucagon secretion and hepatic glucose release. This response is coordinated by still poorly characterized brain hypoglycemia sensing mechanisms, which control autonomic nervous activity and the hypothalamus-pituitary-adrenal axis. In a previous genetic and genomic screen for hypothalamic regulators of hypoglycemia-induced glucagon secretion we identified Agpat5, located on mouse chromosome 8, as a candidate gene. Here we aimed at characterizing the role of Agpat5, a mitochondrial membrane-associated enzyme that uses fatty acyl-CoAs and lysophosphatidic acid to produce phosphatidic acid.

Materials and methods: We generated mice lacking Agpat5 expression in AgRP neurons of the arcuate nucleus of the hypothalamus (AgRPAgpat5KO). Glucagon secretion was assessed following insulin-induced hypoglycemia (IIH). AgRP neurons activation was assessed by c-Fos immunostaining, patch clamp analysis and fiber photometry. Mitochondrial respiration experiments were conducted using a Seahorse instrument.

Results: We found that AgRPAgpat5KO (KO) mice had reduced glucagon secretion upon IIH as compared to control mice (29.0±4.1pM (Ctrl) vs 16.1±1.7pM (KO), n=18-22, p<0.001, two-way ANOVA, Tukey’s post hoc). c-Fos immunostaining showed reduced number of AgRP neurons activated by hypoglycemia in KO vs. Ctrl mice. Patch clamp analysis performed on acute brain slices revealed that inactivation of Agpat5 reduced by half the percentage of glucose inhibited (GI) AgRP neurons (72.2% (Ctrl) vs 33.3% (KO), n=18 neurons/group, p<0.05, Fisher’s exact test). In vivo fiber photometry revealed that AgRP neurons of KO mice were less activated by hypoglycemia than those of Ctrl mice. These hypoglycemia sensing defects were associated with defective activation of the vagal nerve. To find how Agpat5 could interfere with hypoglycemia sensing, we firs used a hypothalamic cell line. We found that silencing Agpat5 expression increased Cpt1a-dependent mitochondrial fatty acid β-oxidation (FAO), oxygen consumption rate (OCR), and ATP production (49.8% increase in mitochondrial OCR with Agpat5 silencing (p<0.05) vs 3.4% decrease in OCR with Agpat5/CPT1a silencing (p>0.05) when compared to Ctrl, n=5, two-way ANOVA, Tukey’s post hoc). In mice, inactivating Cpt1a in AgRPAgpat5KO mice (AgRPAgpat5KO/CPT1aKO) restored the number of hypoglycemia-activated AgRP neurons as determined by c-Fos immunostaining and patch clamp analysis. This suggests that limiting FAO in AgRP neurons is necessary for effective hypoglycemia detection.

Conclusion: Collectively, these data indicate that Agpat5 is required to partition acyl-CoA away from mitochondrial FAO and ATP generation. This is required to ensure that the hypoglycemia-dependent fall in ATP production, which triggers AgRP firing through inhibition of the Na+/K+ATPase, is not prevented by increased FAO. This mechanism is especially important during the fasted state when circulating free fatty acid concentrations increase. Our study thus describes a so far unknown protective mechanism that ensures proper hypoglycemia sensing by AgRP neurons and the control of glucagon secretion.

Supported by: FNS, ERC-INTEGRATE, Hypo-Resolve

Disclosure: A. Strembitska: None.

OP 40 When the clock ticks

229

Time-of-day effects of exercise training on multi-tissue metabolome and skeletal muscle proteome profiles in men with type 2 diabetes

M. Savikj1, S. Ben2, S. Shogo3, K. Caidahl1,4, A. Krook5, A.S. Deshmukh2,6, J.R. Zierath1,2, H. Wallberg-Henriksson5;

1Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden, 2Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 3Center for Epigenetics and Metabolism, INSERM U1233, Department of Biological Chemistry, School of Medicine, University of California, Irvine, USA, 4Department of Clinical Physiology, Karolinska University Hospital, Stockholm, Sweden, 5Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 6The Novo Nordisk Foundation Center for Protein Research, Clinical Proteomics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Metabolic effects of exercise may partly depend on the time-of-day when exercise is performed. We tested the hypothesis that exercise timing affects the adaptations in multi-tissue metabolome and skeletal muscle proteome profiles in men with type 2 diabetes.

Materials and methods: Men fitting the inclusion (type 2 diabetes, age 45-68 years and body mass index 23-33 kg/m2) and exclusion criteria (insulin treatment, smoking, and concurrent systemic disease) were included in a randomized crossover trial (n=15). Participants that fully completed all exercise sessions (n=8) were analysed. The trial consisted of two weeks of high-intensity interval training (HIT) (three sessions/week) either in the morning (08:00, n=5) or afternoon (16:45, n=3), a two-week wash-out period, and an additional two weeks of HIT at the opposing time. Blood, skeletal muscle and subcutaneous adipose tissue were obtained before the first, and after each training period. Broad-spectrum, untargeted proteomic analysis was performed on skeletal muscle, and metabolomic analysis was performed on all biosamples. Differential content was assessed by linear regression and pathway set enrichment analyses were performed. Coordinated metabolic changes across tissues were identified by Spearman correlation analysis.

Results: Metabolic and proteomic profiles remained stable after two weeks of HIT, and individual metabolites and proteins were not altered. However, coordinated changes in relevant metabolic pathways and protein categories were identified. Morning and afternoon HIT similarly increased plasma diacylglycerols, skeletal muscle acyl-carnitines, and subcutaneous adipose tissue sphingomyelins and lysophospholipids. Acyl-carnitines were central to training-induced metabolic cross-talk across tissues. Plasma carbohydrates, via the penthose phosphate pathway, were increased and skeletal muscle lipids were decreased after morning compared to afternoon HIT. Skeletal muscle lipoproteins were higher, and mitochondrial complex III abundance was lower after morning compared to afternoon HIT.

Conclusion: We provide a comprehensive analysis of a multi-tissue metabolomic and skeletal muscle proteomic responses to training at different times of the day in men with type 2 diabetes. Increased circulating lipids and changes in adipose tissue lipid composition were common between morning and afternoon HIT. However, afternoon HIT increased skeletal muscle lipids and mitochondrial content to a greater degree than morning training. This diurnal component in the metabolomic and proteomic training response may be clinically relevant and warrants further investigation.

Clinical Trial Registration Number: Pilot study for: NCT03553524

Supported by: KAW, Swedish Diabetes and HLF Foundations, Vetenskapsrådet, SLL, EFSD/Lilly and SRP Programmes

Disclosure: M. Savikj: None.

230

Beta cell functional heterogeneity underpinning coordinated oscillatory activity is not fixed

K. Suba1, B. Hansen1, Y. Patel2, B.M. Owen3, W. Distaso4, V. Salem1;

1Department of Bioengineering, Imperial College London, London, 2PerkinElmer, Newport, Wales, 3Department of Metabolism, Digestion and Reproduction, Imperial College London, London, 4Imperial College London, London, UK.

Background and aims: There is increasing recognition that pancreatic islets work as functional units. Electrically coupled beta-cells subserve pulsatile insulin release, co-ordinated by sub-populations of leader cells in vivo. It remains unknown whether this beta-cell functional heterogeneity is associated with a fated or fixed identity of these highly connected hubs. We identified the same cross-section of pancreatic islets in vivo, at single-cell resolution, in repeated imaging sessions over months to longitudinally track the activity of leader cells.

Materials and methods: We implanted Ins1CreGCaMP6ffl/fl-expressing islets into the anterior eye chamber of wild-type C57BL/6 syngeneic recipients. Calcium dynamics of reporter islets were imaged over a period of 4months; at baseline (normal chow), after high-fat diet and following daily injections of gut-hormone analogues or weight-matching intervention. The same islet cross-section was re-identified during each imaging session and calcium oscillations of individual beta-cells were recorded. First responder cells during a wave of calcium activity were identified and all beta-cell calcium traces were subjected to independent Granger causality analysis

Results: We found that first responder cells were among the top 25% of cells with the highest number Granger links. Granger leaders emerged from the same area in the islet cross-section during our recordings (<4minutes) however their identity changed dynamically over a longer period of time (months) in vivo.

Conclusion: The identity of Granger leader cells remains stable in the acute setting (minutes to hours) but changes dynamically over longer periods of time (months). Investigating the mechanisms underlying these changes will improve our understanding of pulsatile insulin release and how to sustain it.

Disclosure: K. Suba: None.

231

Time-of-day influences post-exercise metabolism in mouse adipose tissue

L.A. Pendergrast1, L. Dollet2,3, L.S. Lundell3, A.M. Ehrlich3, S.P. Ashcroft3, J.T. Treebak3, A. Krook2, J.R. Zierath1,3;

1Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden, 2Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 3Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Background and aims: Exercise is an effective approach to reduce the risk of type 2 diabetes, as well as to improve glucose uptake and mitochondrial activity in adipose tissue. Physiological events such as hormonal secretion, feeding behavior, and tissue metabolism follow a diurnal pattern regulated by central and peripheral circadian clocks. We hypothesized that the circadian clock may interact with exercise in regulating adipose tissue function and thus investigated the time-of-day response to exercise on the adipose tissue transcriptome and metabolism.

Materials and methods: Mice performed a 60-minute exercise bout (or sham) during the early rest (day, ZT3) or early active (night, ZT15) phase. Tissue and serum samples were collected at 0, 4, 8, 12, 16, and 20h post-exercise (n=6 per group), and RNA sequencing was performed on inguinal adipose tissue at t=0h after exercise. An additional group of mice was exercised (or sham) during the early rest phase in either an ad libitum or 10h-fasted state, with samples collected immediately following intervention (n=7 per group). Adipose tissue response and whole-body metabolic adaptation were evaluated by measuring gene expression, hepatic and skeletal muscle glycogen, serum lipid, and hormone levels.

Results: The exercise intervention did not modulate rhythmic expression of core clock genes Arntl and Nr1d1 in adipose tissue. Exercise-mediated adrenaline and corticosterone levels were similar between the early rest and early active phase, yet plasma non-esterified fatty acids (NEFA) were increased with early active phase exercise only (two-way ANOVA; p<0.05). Transcriptomic data revealed a time-of-day specific exercise response in adipose tissue, with changes specific to active phase only (79 transcripts altered in active phase versus 0 in rest phase, FDR<0,05). Among genes altered by early active phase exercise were those related to stress and thermogenesis, such as Nr4a3 and Ucp1. To decipher if feeding status and differential substrate availability are responsible for these time-of-day specific effects, we replicated the “unfed” state of the early active phase mice by fasting mice 10-hours prior to the early rest phase. Fasted early rest mice showed an increase in plasma NEFA (0.94± 0.06 mmol/L versus 0.66± 0.05 mmol/L; p=0.0001) and a depletion of liver glycogen (26.12±9.5 μmol/g versus 98.31±14.3 μmol/g; p=0.0001) similar to early active phase mice. However, fasted early rest phase exercise did not recapture expression patterns of early active phase exercise-responsive genes.

Conclusion: Exercise in the active phase influences adipose tissue gene expression and increases exercise-induced lipolysis. However, fasted early rest phase exercise does not replicate the time-of-day profile in gene expression observed with early active phase exercise, and implicates a role of the circadian clock in the adipose tissue post-exercise transcriptome. Our results provide evidence to suggest that exercise timing can fine-tune the metabolic benefits of exercise—particularly within adipose tissue. Time of day is a central variable to consider in prescribing exercise interventions for humans with metabolic syndrome.

Supported by: KID funding from Karolinska Institutet. Novo Nordisk Foundation Challenge Program

Disclosure: L.A. Pendergrast: None.

232

The effects of hyperglycaemia on the circadian clock in TALLYHO/JngJ mice

S.P. Ashcroft1, A.M. Ehrlich1, T.S. Nielsen1, S. Larsen2, J.T. Treebak1, J.R. Zierath3,1;

1Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark, 2Center for Healthy Aging Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark, 3Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Perturbed circadian rhythms have been associated with metabolic dysfunction and type 2 diabetes (T2D) however, little is known regarding how hyperglycemia influences the circadian clock in peripheral tissues. To investigate this further, we utilised the TALLYHO/JngJ mouse which is a polygenic rodent model of T2D characterised by hyperglycemia, hyperinsulinemia, peripheral insulin resistance and hepatic steatosis. Interestingly, the hyperglycemic phenotype is not 100% penetrant allowing us to segregate these mice into normoglycemic (TALLY-Low) and hyperglycemic (TALLY-High) groups. Therefore, the aim of the study is to determine how the degree of hyperglycemia influences the circadian clock in TALLY-Low and TALLY-High mice. To do this, we performed a multi-tissue transcriptomic analyses of skeletal muscle, adipose tissue and liver samples collected across a circadian cycle.

Materials and methods: Male TALLY-Low and TALLY-High mice were studied between the ages of 6 to 18 weeks. Age and sex matched C57BL/6NTac mice were utilised as a healthy control mouse in all experiments. To characterise the development of hyperglycaemic phenotype, body weight, blood glucose and insulin were monitored on a weekly basis from 6 to 18 weeks of age (n=10/group). To assess tissue specific alterations to the circadian clock, liver, quadricep and white adipose tissue samples were collected across a 24-hour period at zeitgeber times (ZT) 0, 4, 8, 12, 16 and 20 for RNA-sequencing (n=4/group) in 10-week-old mice. Finally, metabolic caging was utilised to assess daily rhythms in whole-body metabolism (n=12/group).

Results: TALLY-High mice displayed overt hyperglycaemia by 9 weeks of age (20.9 ± 7.2 mmol) in comparison to TALLY-Low (10.9 ± 1.1 mmol) and C57BL/6NTac (8.9 ± 0.5 mmol) mice (P < 0.05). At 8 weeks of age, both TALLY-Low (1.4 ± 0.4 ng/ml) and TALLY-High (1.0 ± 0.4 ng/ml) display hyperinsulinemia in comparison to C57BL/6NTac mice (0.6 ± 0.1 ng/ml) (P < 0.05). Insulin production is, however, almost completely reduced in TALLY-High mice by 18 weeks of age (0.5 ± 0.4 ng/ml) whereas hyperinsulinemia is maintained in TALLY-Low mice of the same age (1.6 ± 0.3 ng/ml). Both TALLY-Low and TALLY-High mice display reduced activity, energy expenditure and RER in comparison to C57BL/6NTac mice (P < 0.05). Finally, preliminary data revealed time-of-day dependent alterations in the expression of circadian clock genes Bmal1, Cry1 and Cry2 in TALLY-High liver samples collected at ZT4 and ZT16. Further analyses will determine the effects of hyperglycemia on the circadian clock in skeletal muscle and adipose tissue.

Conclusion: In conclusion, the TALLYHO/JngJ mouse is characterised by the divergent development of the hyperglycemic phenotype. This divergence is exacerbated by a reduction in insulin production in TALLY-High mice and preliminary analyses have revealed disruptions to the circadian transcriptome in the liver of TALLY-High mice.

Disclosure: S.P. Ashcroft: None.

OP 41 Viruses and diabetes: more than COVID-19

233

SARS-CoV-2 infection and subsequent risk of type 1 diabetes in 1.2 million children

H.L. Gulseth1, P.L.D. Ruiz1,2, K. Størdal3, Ø. Karlstad1, N. Gunnes1,2, N.A. Lund-Blix1, H. Bøås1, L.C. Stene1, G. Tapia1;

1Norwegian Institute of Public Health, 2Oslo University Hospital, 3University of Oslo, Oslo, Norway.

Background and aims: Viral infections, including respiratory viruses, are hypothesized to increase the risk of type 1 diabetes. Several case reports suggest an association between new onset type 1 diabetes and SARS-CoV-2 infection. In this nationwide register-based study we aimed to test whether SARS-CoV-2 infection was associated with an increased risk of developing type 1 diabetes in children and adolescents.

Materials and methods: We linked individual-level data from national health registries for all children and adolescences in Norway (1.2 million individuals). Data were obtained from the Norwegian preparedness register that is updated daily with individual-level data on PCR-confirmed SARS-CoV-2 infections, COVID-19 vaccinations and disease diagnoses from primary and secondary health care. Children were followed from March 1st 2020 (the start of the pandemic) until diagnosis of type 1 diabetes, age 18 years, death, or March22th 2022, whichever occurred first. In a full population cohort and a test-negative design we used Cox regression with SARS-CoV-2 PCR positivity as a time-dependent exposure to estimate hazard ratios (HR) with 95% confidence intervals (CI), both unadjusted and adjusted for age, sex, non-Nordic country of origin, geographical area and socio-economic factors. Analyses were done separately for first type 1 diabetes diagnosis within or after 30 days post-SARS-CoV2 infection.

Results: We identified a total of 424,354 children with SARS-CoV-2 infection (of 1,202,174 children included at study start) and 990 incident cases of type 1 diabetes. The adjusted HR for type 1 diabetes at least 31 days after SARS-CoV-2 infection was 1.63 (95% CI 1.08, 2.47) in a test-negative design and 1.57 (95% CI 1.06, 2.33) in the full-cohort (figure).

Conclusion: Our findings suggest that SARS-CoV-2 infection is associated with increased risk of subsequent type 1 diabetes. Future studies should include long-term follow-up and SARS-CoV2 virus variants.

figure bn

Disclosure: H.L. Gulseth: None.

234

Relation of incident type 1 diabetes to recent COVID-19 infection: cohort study using e-health record linkage in Scotland

H.M. Colhoun1, S. McGurnaghan1, L. Blackbourn1, L.E. Bath2, D.A. McAllister3, T.M. Caparrotta1, S.H. Wild4, S.N. Wood4, D. Stockton5, P.M. McKeigue4;

1Institute of Genetics & Cancer, University of Edinburgh, Edinburgh, 2Royal Infirmary of Edinburgh, Edinburgh, 3University of Glasgow, Glasgow, 4University of Edinburgh, Edinburgh, 5Public Health Scotland, Edinburgh, UK.

Background and aims: Studies using claims databases reported that SARS-CoV-2 infection >30 days earlier increased the incidence of type 1 diabetes. Using exact dates of diabetes diagnosis from the national register in Scotland linked to virology laboratory data we sought to replicate this finding.

Materials and methods: A cohort of 1849411 individuals aged <35 years without diabetes, including all those in Scotland who subsequently tested positive for SARS-CoV-2, was followed from 1 March 2020-22 November 2021. Incident type 1 diabetes was ascertained from the national registry. Using Cox regression we tested the association of time-updated infection with incident diabetes. Trends in incidence of type 1 diabetes in the population from 2015-2021 were also estimated in a generalised additive model.

Results: There were 365080 in the cohort with at least one detected SARS-CoV-2 infection during follow-up and 1074 who developed type 1 diabetes. The rate ratio for incident type 1 diabetes associated with first positive test for SARS-CoV-2 (reference category: no previous infection) was 0.88 (95% CI 0.63 to 1.23) for infection more than 30 days earlier and 2.62 (95% CI 1.81 to 3.79) for infection in the previous 30 days. However negative and positive SARS-CoV-2 tests were more frequent in the days surrounding diabetes presentation. In those aged 0-14 years incidence of type 1 diabetes during 2020-2021 was 20% higher than the 7-year average.

Conclusion: Type 1 diabetes incidence in children increased during the pandemic. However the cohort analysis suggests that SARS-CoV-2 infection itself was not the cause of this increase.

Supported by: DiabetesUK 17/0005627

Disclosure: H.M. Colhoun: None.

235

Modifiable risk factors including HbA 1c and BMI are consistently associated with severe influenza, pneumonia, and Covid-19 infection outcomes in people with type 2 diabetes

R. Hopkins, K.G. Young, J. Godwin, D. Raja, N.J. Thomas, B.M. Shields, J.M. Dennis, A.P. McGovern;

Institute of Biomedical & Clinical Science, University of Exeter Medical School, Exeter, UK.

Background and aims: Previous UK population-based research has identified risk factors for severe Covid-19 outcomes in people with type 2 diabetes, but it is unclear whether these are specific to Covid-19 or general to respiratory infections. We aimed to compare risk factors for hospitalisation from Covid-19 (pre-vaccination roll-out) to those for pneumonia and influenza.

Materials and methods: UK routine primary care data were accessed from the Clinical Practice Research Datalink (CPRD) and linked to Hospital Episode Statistics (HES). We followed adults with type 2 diabetes from 01/09/2018-31/05/2019 (influenza and pneumonia hospitalisation cohort, n = 655,677) and from 01/02/2020-31/10/2020 (Covid-19 hospitalisation cohort, n = 583,185). We used multivariable Cox proportional hazard models to identify sociodemographic risk factors (sex, age, ethnicity, index of multiple deprivation quintile) and modifiable risk factors (HbA1c and BMI) for hospitalisation in each cohort. Models were adjusted for macrovascular and microvascular complications, and other key comorbidities.

Results: We observed 6,061 (1.04%) hospitalisations for Covid-19, 1,358 (0.21%) for influenza, and 13,987 (2.13%) for pneumonia. When assessing sociodemographic risk factors, for Covid-19 we replicated previously reported associations between male sex, older age, greater deprivation, non-white ethnicities, and severe outcomes. However, these differed from the associations found for the other respiratory infections. We observed a differential effect of ethnicity, where compared to people of white ethnicity, black and south Asian groups had increased Covid-19 hospitalisation (adjusted hazard ratio [aHR] 1.84 [95%CI 1.67-2.03], p<0.001, and 1.42 [1.30-1.54], p<0.001, respectively), but lower hospitalisation for pneumonia (aHR 0.74 [95%CI 0.68-0.82], p<0.001, and 0.87 [0.81-0.93], p<0.001, respectively). There was a stronger association between age and hospitalisation for pneumonia (aHR per 10 year increase in age: 1.51 [95%CI 1.48-1.54], p <0.001) than for Covid-19 (aHR 1.23 [1.19-1.26], p<0.001) and influenza (aHR 1.15 [1.08-1.22], p<0.001). Assessing modifiable risk factors, high HbA1c (>86 mmol/mol) was a consistent risk factor for all three respiratory infections: aHR for HbA1c >86 vs 48-53 mmol: 1.48 [95%CI 1.33-1.65], p<0.001 for Covid-19, 1.62 [1.29-2.05], p<0.001 for influenza, 1.33 [1.23-1.44], p<0.001 for pneumonia. Similarly, marked obesity (BMI>40) was a consistent risk factor: BMI >40 vs 25-29.9 kg/m² aHR 1.50 [1.36-1.67], p<0.001 for Covid-19, 1.48 [1.21-1.81], p<0.001 for influenza, 1.34 [1.24-1.44], p<0.001 for pneumonia.

Conclusion: In people with type 2 diabetes, sociodemographic risk factors, such as ethnicity and age, are differentially associated with hospitalisation for Covid-19, influenza, and pneumonia. Therefore existing Covid-19 risk models cannot be assumed to be entirely applicable to new respiratory infections. However, higher HbA1c and BMI are consistently associated with hospitalisation for all three infections studied. This supports that good glycaemic and weight control may lower the risk of severe respiratory infection outcomes.

Supported by: Diabetes UK (20/0006220)

Disclosure: R. Hopkins: None.

236

Enterovirus infection and risk of islet autoimmunity and type 1 diabetes: systematic review and meta-analysis of molecular studies

S.R. Isaacs1,2, A. Roy3, B. Dance1, D.B. Foskett1,2, A.J. Maxwell1,2, W.D. Rawlinson1,2, K.W. Kim1,2, M.E. Craig1,4;

1School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, 2Serology and Virology Division, NSW Health Pathology, Virology Research Laboratory, Prince of Wales Hospital, 3School of Medical Sciences, Faculty of Medicine, University of New South Wales, 4Institute of Endocrinology and Diabetes, Children's Hospital at Westmead, Sydney, Australia.

Background and aims: Molecular methods of enterovirus (EV) detection have been widely adopted in recent years, especially within large prospective cohort studies of islet autoimmunity (IA) and type 1 diabetes (T1D). Here, we present a major update to our previous systematic review of molecular detection of EV infection and (i) IA or (ii) T1D.

Materials and methods: A systematic search of controlled observational human studies was performed using PubMed and Embase databases until August 2021 (PROSPERO #CRD42021236044), with no language restrictions. Eligible studies included cohort or case-control studies measuring EV RNA or protein in blood, stool, or tissue of individuals with IA or T1D, and in controls. Studies were assessed for bias using the Newcastle-Ottawa Scale and NHMRC levels of evidence, with meta-analysis performed in RevMan v5.4 using the Mantel-Haenszel method and random effects models, producing ORs with 95% CIs and p-values for each outcome.

Results: The initial search returned 3266 publications; removal of 570 duplicates and 1799 irrelevant studies left 897 relevant articles for full-text screening. After consolidation of participant overlap, the 113 records meeting eligibility criteria were assigned to 61 study units (50 case-control/NHMRC-III, 11 nested case-control/NHMRC-II), where 41 examined T1D, nine IA, and 11 both outcomes. NOS scores ranged from 2 to 8, with 34 studies scoring ≥6, indicating good methodological quality. For 25 studies, cases could be further separated into multiple groups, resulting in 91 case sub-groups overall (24 for IA, 67 for T1D). There were 11975 participants altogether, including 6003 cases (900 with IA, 5103 with T1D) and 5972 controls. Study design, quality, and method of EV detection demonstrated high levels of statistical heterogeneity. Meta-analysis of 56 studies demonstrated a significant association between EV infection and IA (17 studies, OR 2.16; 1.28-3.64; p=0.004; χ2/df=2.77) and T1D (48 studies, OR 8.41; 5.16-13.69; p<0.00001; χ2/df=6.59); within one month of T1D onset (27 studies, OR 18.01; 9.37-34.60; TBC p<0.00001; χ2/df=3.50).

Conclusion: There is a clinically significant association between EV infection, and both IA and T1D. This further supports the rationale for development of EV-targeted vaccines and antiviral therapy to prevent and reduce the impact of T1D. Large prospective studies commencing from pregnancy (e.g., ENDIA and DIPP-novum) will provide insight into the temporal relation between EV infection and the development of IA and T1D in early life.

figure bo

Disclosure: S.R. Isaacs: None.

OP 42 Moving towards the beta cell plasma membrane

237

OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion

S. Panagiotou1, P.M. Nguyen1, A. Wendt2, L. Eliasson2, A. Tengholm1, O. Idevall-Hagren1;

1Medical Cell Biology, Uppsala University, Uppsala, 2Clinical science, Lund University Diabetes Centre, Malmö, Sweden.

Background and aims: Insulin granule biogenesis is a multi-stage process that involves cargo loading and processing and intracellular transport. Mature granules dock with the plasma membrane and undergo exocytosis to release insulin. We recently identified phosphatidylinositol-4 phosphate (PI[4]P) dephosphorylation by Sac2 on the surface of insulin granules as a key step in granule docking and found that Sac2 levels were reduced in islets from type-2 diabetics. The aim of this study was to determine how Sac2 and its lipid substrate PI(4)P contribute to the regulation of insulin secretion from β-cells.

Materials and methods: Clonal MIN6 β-cells expressing fluorescently tagged proteins were imaged by spinning disk confocal and TIRF microscopy. Cellular cholesterol distribution was determined using fluorescently labeled cholesterol (TopFluor-chol; live cells) or Filipin (fixed cells). Insulin secretion from MIN6 cells and mouse islets was determined by ELISA. Ultrastructural analysis of mouse and human islet cells was performed by transmission electron microscopy. For visualization of ER-granule contact sites in living cells, we developed a method based on dimerization-dependent red fluorescent protein monomers.

Results: Sac2 knockdown resulted in pronounced cholesterol accumulation on insulin granules, seen as increased TopFluor-chol fluorescence (0.117 for control, 0.161 for Sac2-KD, n = 25-26 cells, P < 0.05) and enhanced Filipin staining (control = 98 ± 3, Sac2-KD = 136 ± 5, n = 54, P < 0.001). Oxysterol-binding protein (OSBP) can exchange PI(4)P for cholesterol at membrane contact sites, and we found that OSBP knockdown normalized insulin granule cholesterol levels in Sac2 knockdown cells. OSBP localized to the trans-Golgi compartment under resting conditions but redistributed to insulin granules following inhibition of lipid exchange with 20 nM OSW-1 (n=6, P<0.001 for comparison to control). Similar redistribution was seen in mouse islets immunostained for OSBP and insulin. Depolarization of MIN6 cells also resulted in enrichment of OSBP at insulin granules (0.02±0.02 vs 0.13±0.03, n=30, P=0.0008) through a mechanism involving acidification of the cytosol, since it was prevented by the addition of 20 mM NH4Cl (-0.01±0.03, n=30, P=0.003). Ultrastructural examination showed many insulin granules in close proximity to the ER, and expression of an ER-insulin granule proximity reporter in mouse islets confirmed the existence of physical contacts between these two organelles and revealed that OSBP resides at these locations. siRNA-mediated knockdown of OSBP in MIN6 cells caused a 16±2% (n=4, P<0.05) reduction in GSIS and acute inhibition of OSBP with OSW-1 impaired second phase insulin secretion from mouse islets (n=6, P<0.05) without affecting cytosolic Ca2+.

Conclusion: Sac2 controls granule PI(4)P levels, which in turn fuel cholesterol transport to granules through the action of OSBP at ER-insulin granule contact sites. Defects in this lipid exchange result in impaired insulin secretion. This study identifies ER-granule contacts as important reaction centers in β-cells where lipid exchange occurs, but these sites are likely involved in additional processes, and identifying these is an important future goal.

Supported by: Swedish Research Council, NNF

Disclosure: S. Panagiotou: None.

238

Presynaptic scaffold protein, liprin, regulates glucose stimulated insulin secretion and the spatial organisation of exocytosis in pancreatic beta cells

K. Deng, N. Hallahan, P. Thorn;

Charles Perkins Centre, The University of Sydney, Sydney, Australia.

Background and aims: A key feature of insulin secretion is that it is targeted towards specialised regions (or hot spots) of the β-cell membrane. Recent evidence has shown that these exocytotic hotpots lie at the interface between β-cells and extracellular matrix (ECM) proteins of the islet capillaries. However, how insulin secretion is targeted towards these regions of cell-capillary contact remains elusive. Analogous to targeted insulin exocytosis in the β-cell, neurotransmitter release in neurons is confined to a specialized region of the presynaptic membrane known as the active zone; here, exocytosis is spatially regulated by a large multiprotein complex consisting of presynaptic scaffolds which dock and tether synaptic vesicles to specific sites for fusion. Interestingly, several of these presynaptic scaffold proteins (liprin, ELKS, RIM and piccolo) that are typically associated with the neuronal active zone have also been identified in β-cells and are enriched at the β-cell-capillary interface where secretion is targeted; however, whether these scaffold proteins facilitate targeted exocytosis in β-cells, as they do in neurons, is unknown. This research investigates presynaptic-like mechanisms for the spatial regulation of exocytosis in the β-cell, particularly, the role of liprin in positioning sites of β-cell insulin exocytosis.

Materials and methods: β-cells were isolated from humanely sacrificed C57BL/6 mice (approved by local and national ethics). We performed 3D live-cell two-photon imaging on β-cells expressing GFP-tagged liprin, to visualise the precise location of liprin with respect to sites of insulin exocytosis. We then optimised a protocol for the knockdown of liprin using an adenoviral-mediated shRNA system. We measured glucose-stimulated insulin secretion (GSIS) in control (scramble shRNA) and knockdown (liprin shRNA) β-cells using homogenous time resolved fluorescence and performed immunofluorescence staining. Finally, we expressed various liprin mutant constructs (designed to specifically interfere with binding to other presynaptic scaffold proteins) in β-cells to test whether liprin interacts or assembles with other scaffold proteins in a β-cell ‘presynaptic-like’ complex.

Results: We show that liprin assembles in small ‘islands’ or microdomains at the β-cell-capillary interface, where insulin granules preferentially fuse near (<0.3 μm) liprin structures (p<0.01) but are excluded from regions directly overlapping with liprin (p<0.01), suggesting that liprin may be involved in tethering insulin granules to specific sites for fusion. Western blot confirmed shRNA-knockdown of liprin (42.1%±4.0) in β-cells. Knockdown of liprin abolishes GSIS (p<0.05) and disrupts localisation of ELKS at the β-cell-capillary interface. Rescue of liprin knockdown using adenoviral-mediated overexpression rescues secretory phenotype. Finally, we show that expression of the N-terminus of liprin (ELKS- and RIM- binding domains) alone is sufficient for normal GSIS. Deletion of N-terminus significantly reduces GSIS (p<0.05), suggesting that liprin may function through interactions with ELKS and RIM in an ‘presynaptic-like’ complex at the β-cell-capillary interface.

Conclusion: We conclude that liprin is involved in spatially regulating insulin exocytosis through mechanisms analogous to a neuronal synapse.

Supported by: NHMRC, The Bioscientifica Trust

Disclosure: K. Deng: Grants; National Health and Medical Research Council, The Bioscientifica Trust.

239

Vamp8 is an endosomal v-SNARE involved in GLP1R trafficking and inhibits insulin exocytosis

L. Liu, M. Marshall, J. Saras, S. Barg;

Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Background and aims: Insulin is released by Ca2+-dependent-dependent exocytosis of secretory granules, while many receptors, ion channels and transporters reach the membrane by constitutive exocytosis of vesicles that belong to the endosomal system. Both processes rely on distinct sets of SNARE proteins for membrane fusion. Beta cells express both VAMP2 on insulin granules and VAMP8 (endobrevin), which is an endosomal vesicular SNARE that has been proposed to have an additional role in “newcomer” exocytosis of insulin granules. The differential role of VAMP isoforms in insulin secretion is not well understood.

Materials and methods: Live cell TIRF-microscopy was performed in INS1 cells and in dispersed beta cells of cadaveric human donors. Vesicle behavior, exocytosis, and protein location was studied by expressing EGFP- or mCherry-tagged versions. Exocytosis was evoked by elevated K+ in presence of 10mM glucose/200μM diazoxide. Association of proteins with vesicular compartments was analyzed by quantitative image analysis. Gene expression was studied in publicly available expression data.

Results: VAMP8-EGFP was found on rab5-, rab7, and rab11-positive endosomal vesicles, but not on rab3-or NPY-labeled insulin granules. Depolarization with elevated K+ promoted exocytosis of the VAMP8/rab11-positive vesicles; these vesicles were distinct from insulin granules since exocytosis proceeded with a slower monophasic time course, was insensitive to tetanus toxin cleavage of VAMP2, and was accelerated by somatostatin but not exendin-4. VAMP8-positive vesicles underwent exocytosis within seconds of their arrival at the plasma membrane, in contrast to minutes for secretory granules. These vesicles also contained GLP1-receptor, GLUT2, and the autophagosome marker LC3. Expression of full-length VAMP8, VAMP8 lacking its transmembrane domain (but not VAMP2) inhibited exocytosis of insulin granules. The latter is consistent with negative correlation between VAMP8 gene expression and secretory index (SI) in human donor islets (puncorr.=0.00016, n=125).

Conclusion: We conclude that VAMP8 associates with early, late and recycling endosome compartment, but not insulin containing secretory granules. VAMP8 traffics to the plasma membrane with, and likely promotes, exocytosis of a rab11-positive endosomal recycling compartment. While Ca2+-dependent exocytosis of these vesicles controls surface expression of important membrane proteins (GLP1R and GLUT2), the associated release of VAMP8 may also regulate insulin secretion. Finally, the data suggest that the previously described “newcomer” exocytosis reflects release from an endosomal compartment, rather than exocytosis of insulin containing secretory granules.

Supported by: VR, NNF, DiabF, Ernfors, Rudberg

Disclosure: L. Liu: None.

240

Villin interacts with Snap25 and its expression increases in tamoxifen-treated EndoC-βH3 cells

H. Mziaut1, I. Kalaidzidis1, J. Dehghany2, A. Gheisari3, M. Herbig4, J.-C. Escolano4, A. Sönmez1, M. von Bülow5, M. Lohmann5, Y. Kalaidzidis6, M. Meyer-Hermann2, J. Guck4,7, A. Schulte5, R. Scharfmann8, M. Solimena1,6;

1Paul Langerhans Institute Dresden, Dresden, Germany, 2Helmholtz Centre for Infection Research, Braunschweig, Germany, 3Biopolis Dresden Imaging Platform (BioDIP), TU Dresden, Dresden, Germany, 4Biotechnology Center, Center for Molecular and Cellular Bioengineering, Dresden, Germany, 5Diabetes, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany, 6Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 7Max Planck Institute for the Science of Light & Max-Planck-Zentrum für Physik und Medizin, Erlangen, Germany, 8INSERM, U1016, Institut Cochin, Paris, France.

Background and aims: Ica512/Ptprn is a transmembrane cargo of the insulin secretory granules (ISGs). In Ica512-/- mouse islets, ISG stores as well as mRNA and protein levels of the F-actin modifier villin are strongly reduced. In rat insulinoma INS-1 cells, villin dynamically modulates the size of actin cages surrounding cortical ISGs, hence regulating their mobility and exocytosis. Evidence that villin acts downstream of Ica512 suggests that ISGs directly influence the remodelling of the cortical cytoskeleton for tight control of glucose-stimulated insulin secretion (GSIS). In the present studies we tested this hypothesis further.

Materials and methods: ISG motility in siRNA villin-depleted INS-1 cells was analyzed by total internal reflection fluorescence microscopy and in silico modelling. Protein-protein interactions in INS-1 cells were investigated by Förster-resonance energy transfer (FRET) and microscale thermophoresis (MST). Gene expression and protein localization in tamoxifen (TAM)-treated and untreated EndoC-βH3 cells, a surrogate model of human pancreatic β-cells, were assessed by next generation sequencing and confocal microscopy, respectively. Stiffness of TAM-treated and untreated EndoC-βH3 cells was quantified by atomic force microscopy (AFM) and real-time deformability cytometry (RT-DC).

Results: In silico modelling of siRNA villin-depleted insulinoma INS-1 cells suggested that increased ISG motility could not account alone for enhanced basal insulin release unless more docking sites for ISG exocytosis were accessible. Overexpression in INS-1 cells of villin and Snap25-mCherry for MST, and villin-TQ2, Snap25-Venus and positive and negative control reporters for FRET indicated that villin interacts with the SNARE Snap25. This finding is reminiscent of previous studies indicating that the villin paralogue gelsolin interacts with the SNARE Syntaxin4. Accordingly, in siRNA villin-depleted INS-1 cells, Snap25 was less restricted at the plasma membrane. Reduced proliferation as well as enhanced ISG stores and GSIS of TAM-treated EndoC-βH3 cells correlated with increased mRNA and protein expression of ICA512 and villin. Furthermore, TAM-treated EndoC-βH3 cells, as measured by RT-DC and AFM were 32% larger and had significantly higher apparent elastic modulus, while the processive mobility of ISGs was increased.

Conclusion: Based on these data we propose that: a) villin-induced remodelling of the F-actin cytoskeleton modulates ISG exocytosis by regulating their mobility and access to Snap25; b) upon β-cell differentiation villin expression is upregulated through Ica512 to adapt the plasticity of the cortical cytoskeleton to the enlarged ISG stores, thus enabling a tight control of GSIS.

Supported by: RHAPSODY, EFPIA, SERI, BMBF, SMWK and DZD

Disclosure: H. Mziaut: None.

OP 43 Microvascular cocktail

241

Prognostic value of diabetes microvascular complication for 21-years all-cause mortality

L. Sacchetta1, M. Chiriacò1, G. Forotti1, S. Leonetti2, L. Nesti1, A. Natali1, A. Solini1, D. Tricò1;

1University-Hospital of Pisa, 2Sant'anna School of advanced studies, Pisa, Italy.

Background and aims: Diabetes mellitus is associated with microvascular complications that impair patients’ prognosis and quality of life. Here we aimed to evaluate the impact of directly-measured diabetes microvascular complications, alone or in combination, on long term all-cause mortality.

Materials and methods: 497 subjects were screened for microvascular complications from 1999 to 2001. Diabetic kidney disease (DKD) was defined as glomerular filtration rate (GFR) <60 ml/min measured by dynamic renal scintigraphy and/or overnight albuminuria >20 μg/min; diabetic retinopathy (DR) by presence of pre-proliferative or proliferative retinopathy at dilated fundus oculi examination; and cardiac autonomic neuropathy (CAN) by presence of at least 2 cardiovascular test abnormalities and/or postural hypotension. Mortality data were retrieved from administrative databases for 303 (61.0%) patients in April 2021. Multivariate models were adjusted for age, sex, BMI, HbA1c, type and duration of diabetes.

Results: After 5,244 person-years of follow-up among 303 participants (median follow-up 21.0 [14.0-21.0] years, age 55.5±13.8 years, 155 [51%] women, BMI 28.6±5.9 kg/m2, HbA1c 9.0±2.1%, 218 [71.9%] with type 2 diabetes and 85 [28.1%] with type 1 diabetes), a total of 133 (43.9%) deaths occurred. The prevalence of DKD, DR, and CAN at baseline was 23.1%, 33.3%, and 24.1%, respectively. The presence of DKD reduced the mean overall survival (OS) by 2.6 years (-14.4%; log-rank test p<0.0001) and increased the adjusted all-cause mortality risk by 117% (aHR 2.17 [1.45-3.26]) (panel A). Patients with CAN showed 8.1% reduction in mean OS (-1.4 years; log-rank test p=0.046) and 54% increase in the adjusted risk for all-cause mortality (aHR 1.54 [1.01-2.36]). DR showed a mean OS reduction of 1.3 years (-7.4%; log-rank test p=0.02) but only numerically higher mortality risk (HR 1.23 [0.82-1.84]). A significant interaction emerged between CAN and type of diabetes (p=0.04), with CAN increasing all-cause mortality risk only in type 2 diabetes patients (HR 1.78 [1.32-2.81]). Patients with one, two, or three complications showed, respectively, a reduction in mean OS of 0.48 years (-2.7%), 2.52 years (-14%), and 2.96 years (-16.4%) (log-rank test p=0.0016). The presence of two or three concomitant microvascular complications increased the adjusted risk for all-cause mortality by 203% (HR 3.03 [1.62-5.68]) and 692% (HR 7.92 [2.93-21.37]), respectively (panel B).

Conclusion: Chronic microvascular complications significantly increase the 21-year all-cause mortality risk in patients with diabetes, regardless of age, sex, BMI, glycaemic control, diabetes type and duration. Multiple microvascular complications reduce life expectancy and have additive effects on the incidence of all-cause death in a real-life population with diabetes.

figure bp

Supported by: EFSD Rising Star Fellowship

Disclosure: L. Sacchetta: None.

242

Three-years follow up of retinal neurodegeneration and neuropathic characteristics in pediatric type 1 diabetic patients

M. Menduni1, F. Picconi1, M.C. Parravano2, B. Russo1, A. Maiorino1, L. Chioma3, S. Cianfarani3, D. Ylli4, P.I. Patera3, S. Frontoni1;

1Unit of Endocrinology, Diabetes and Metabolism, S. Giovanni Calibita Fatebenefratelli Hospital, Rome, Italy, 2IRCCS-G.B. Bietti Foundation, Rome, Italy, 3Diabetes Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 4Division of Endocrinology, MedStar Washington Hospital Center, MedStar Health Research Institute, Washington DC, USA.

Background and aims: Retinal neurodegeneration (RN) is considered an early marker of diabetic retinopathy. Few data are available on the possible association between RN and diabetic neuropathy (DN) and the predictive role of glycemic variability (GV) and lipid profile on early RN signs in the pediatric population with type 1 diabetes mellitus (T1DM). We previously demonstrated, in adult T1DM patients, a significant association between GV and triglyceride (TG) with early RN and between TG and the risk for early peripheral DN, independent of glucose control. The aim of our study is to evaluate the 3-year progression of structural alteration of neuroretina, the possible association between these early alterations and DN and the predictive role of GV and lipids on these precocious changes in pediatric T1DM subjects.

Materials and methods: 25 patients with T1DM (ages 10-20 years), using continuous glucose monitoring and continuous subcutaneous insulin infusion, without any complication, and 18 healthy controls (C), comparable in age and gender, were enrolled and followed for 3 years. All subjects underwent an Optical Coherence Tomography Heidelberg Spectralis, with analysis of macular neuroretinal layers. In T1DM, metabolic parameters, GV indexes, peripheral (Michigan Neuropathy Screening Instrument-MNSI, thermal threshold and vibration perception threshold-VPT) and autonomic assessment (using cardiovascular autonomic reflex tests) were investigated. Data were collected at baseline (V0) and after 12 (V1), 24 (V2) and 36 months (V3).

Results: During tthe 3-year follow up, among neuroretinal layers, Retinal Nerve Fiber Layer (RNFL), Outer Plexiform Layer (OPL) and Inner Retinal Thickness (IRT) were significantly thinner in T1DM versus C and a progressive reduction in OPL was observed in T1DM (p<0.05). In T1DM patients, negative correlations were observed between GV and neuroretinal layers, specially between continuous overall net glycemic action-1h and RNFL at V0 (r=-0.4, p=0.05), between mean absolute glucose (MAG) and OPL (r=-0.5, p=0.04) at V2 and between IRT delta thickness (V2-V1) and Lability Index (r=-0.6, p=0.01). No significant correlation between HbA1c and macular layer thickness was observed (p>0.05). Among metabolic parameters, a negative correlation between TG and IRT delta thickness (V2-V1) r=-0.5, p<0.01) was found. Among neuropathic characteristics, at V3, a negative correlation between the systolic blood pressure fall and OPL (r= -0.9 p=0.04) and a positive correlation between the VPT and RNFL (r=0.2 p=0.05) were observed. Moreover, there was a positive correlation between low blood glucose index and VPT (r=0.7 p=0.01) and between MAG and MNSI (r=0.7 p=0.02).

Conclusion: Very early morphological alterations of neuroretina are already present in pediatric T1DM patients without both vascular retinopathy and DN, and there is a possible association between these variations and early signs of peripheral and autonomic DN. These data corroborate the hypothesis that RN is, in fact, an early sign of DN and GV and TG should be efficaciously addressed in the early stage of T1DM.

Disclosure: M. Menduni: None.

243

Progression of diabetic retinopathy in a prospective randomised trial comparing Everolimus versus Mycophenolate therapy in pancreas and kidney transplant recipients

B. Hagerf (Voglova)1, Z. Hladikova1, L. Nemetova1, M. Zahradnicka1, K. Kesslerova2, T. Sosna2, F. Saudek1;

1Institute for Clinical and Experimental Medicine, 2Ophtalmology Clinic of Thomayer Hospital, Prague, Czech Republic.

Background and aims: Successful pancreas and kidney transplantation (SPK) in type 1 diabetic patients does not initially halt progression of diabetic retinopathy (DR), but tends to stabilize it in the long run. mTOR inhibitors are known for their antiangiogenic effect, but the impact of long-term systemic immunosuppression on DR had not yet been prospectively studied. Therefore we initiated a prospective randomized trial comparing the effect of either everolimus (E) or mycophenolic acid (MPA) on the course of DR in SPK recipients.

Materials and methods: Waitlisted Type-1 diabetic subjects were randomized to treatment with MPA or E together with tacrolimus, 6 weeks steroids and ATG induction. Eye examination including optical coherence tomography was done at the baseline, 6, 12 and 24 months. The composite primary endpoint comprised new need for laser therapy, newly diagnosed proliferation, clinically significant macular edema (CSME), best corrected visual acuity (BCVA) worsening. For statistical evaluation we used t-test, Mann-Whitney test, Fisher’s exact test, Kaplan-Meier test and log-rank test. Endpoints were evaluated per patient and per eye.

Results: Out of 64 enrolled patients, 55 (MPA 29, E 26) completed the follow-up. Most of these had proliferative DR (82.4% eyes in MPA and 78.2% in E group) with previous laser treatment. 2-year patient and death-censored pancreas and kidney graft survival rates did not differ between the groups. 59% of the patients in the MPA group and 50% in E group met the primary endpoint (p=0.6), mostly due to new proliferation, need for laser treatment and BCVA worsening. When analyzed per eye, the need for laser therapy was more frequent in the MPA group, reaching statistical significance in the second year post-transplant (22.8% vs 5.8% eyes, p=0.015), which corresponded with BCVA worsening (33.3% and 15.7%, p=0.045). Retinopathy-related BCVA worsening rate was significantly higher in the MPA than E group (22.8% vs 5.8%, p=0.015). Other causes of vision worsening were cataract and glaucoma. Total occurrence of bleeding was 19.3% in MPA group vs 3.9% in the E group (p=0.0154). There was no case of new blindness. Central retinal thickness increased in the 6th month post-transplant in both groups with subsequent restoration at 12 and 24 months. CSME rate was almost identical in both groups (MPA 7%, E 7.8%, p=0.9). Patients treated with laser less than 12 months pre-transplant were more likely to suffer from further DR progression meeting the primary endpoint in 72.7% (MPA) and 66.7% (E).

Conclusion: We observed retinopathy progression in both groups, with slightly more favorable outcomes in the everoliumus-treated group. We did not observe typical early worsening, rather ongoing natural course of advanced diabetic retinopathy in both groups.

Clinical Trial Registration Number: EUDRACT No 2013-004934-14

Supported by: Ministry of Health of Czech Republic, grant no.15-26746 A

Disclosure: B. Hagerf (Voglova): None.

244

Circulating proteins associated with risk of progression to ESKD in type 1 diabetes: results of multi-cohort study

J.K. Haukka1,2, Z.M. Dom3,4, V. Curovic5, K. Ihara3, E. Satake3,4, S. Mutter2, C. Forsblom1,2, A. Doria3,4, N. Sandholm1,2, P. Rossing5, P.-H. Groop1, A. Galecki3,6, A. Krolewski3,4;

1Folkhälsan Research Center, Helsinki, Finland, 2Abdominal Center, Nephrology, Helsinki, Finland, 3Research Division, Joslin Diabetes Center, Boston, USA, 4Harvard Medical School, Boston, USA, 5Steno Diabetes Canter Center, Copenhagen, Herlev, Denmark, 66Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, USA.

Background and aims: Recently, many circulating proteins were found to be associated with the development of diabetic kidney disease (DKD) and its progression to ESKD. To facilitate research on the development of prognostic tests and on the role of circulating proteins in the etiology of DKD, a small set of 21 important proteins, referred to as the Joslin Kidney (JK) Panel, was developed. The assays to quantify these proteins were developed by OLINK Inc (Uppsala Sweden). The aim of this study was to examine whether the proteins included in the JK Panel were associated with progression to ESKD.

Materials and methods: Three international cohorts of patients with T1D, macro-albuminuria and eGFR >45 ml/min had been assembled and followed for 7 - 20 years to ascertain onset of ESKD: at Joslin (Boston) 387 patients (127 ESKD), at Steno (Denmark) 435 patients (104 ESKD), and at FinnDiane (Finland) 387 patients (127 ESKD). Methods of the assembly and follow-up of these cohorts were described previously (Skupien et al, 2019). Serum concentrations of the 21 proteins were measured with the OLINK platform. Association with DKD progression to ESRD was analyzed with Cox regression adjusted for baseline urinary albumin excretion rate, eGFR, and HbA1c.

Results: On average, patients who progressed to ESKD in comparison with those who did not progress had shorter duration of diabetes (26 vs. 28 years), higher baseline HbA1c (9.5 % vs. 8.9 %) and lower baseline eGFR (69 vs. 89 ml/min/1.73m2). Across the 3 cohorts all 21 proteins with nominally significant P-values show positive association with DKD progression (Figure 1). Strikingly, in the Joslin and the FinnDiane Cohorts almost all circulating proteins included in the JK Panel had strong association with progression to ESKD except for TNFRSF6B and PI3. In the Steno cohort, examined proteins showed weaker association with progression to ESKD compared to the two other cohorts. We are presently using the above data to develop a prognostic algorithm to estimate time to onset of ESKD in patients with DKD.

Conclusion: The JK Panel of circulating proteins measured using a custom-made OLINK platform showed extremely similar findings in two international cohorts. The weaker association with progression to ESKD of the majority proteins examined in Steno Cohort is difficult to explain at present. The JK Panel should be examined further not only for prediction of time to ESKD but also for identification of patients who will respond to specific reno-protective therapies and for monitoring the effectiveness of such therapies.

figure bq

Disclosure: J.K. Haukka: None.

OP 44 Grading insulin therapy: simple, simpler, the safest

245

Delayed prandial insulin boluses are frequent and associated with glucose control in type 1 diabetes patients on advanced technologies

R. Triggiani, R. De Angelis, L. Bozzetto, G. Annuzzi;

Federico II University, Naples, Italy.

Background and aims: Advanced technologies (Hybrid Closed Loop Systems (HCLS) and Sensor Augmented Pump (SAP)) significantly improve blood glucose control in patients with type 1 diabetes. However, the optimization of their performances requires patient’s compliance to proper pre-meal insulin bolus administration. We explored how timely patients on HCLS or SAP inject the pre-meal bolus and whether delayed boluses may affect blood glucose control.

Materials and methods: One-hundred fifty-three type 1 diabetes patients, 79 women and 54 men, aged 41.9±14.2 on HCLS (n=121) or SAP (n=32) were consecutively recruited for this study. Two diabetologists (RT, RDA) independently reviewed two-week CGM and pump reports. Delayed boluses were assigned when a prandial bolus was preceded on CGM by a steep increase in blood glucose clearly indicating a postprandial response. An online questionnaire on Fear of Hypoglycemia was completed by participants Two-week CGM metrics were evaluated in relation to the number of delayed boluses by Pearson correlation

Results: At least one delayed bolus was identified in all patients, with a mean total number over two weeks of 9.2±4.4 delayed boluses. The number of delayed boluses was directly associated with GMI (r= 0.356, p<0.001), Coefficient of variation (r= 0.290, p<0.001), TAR>250 (r= 0.412, p<0.001), TAR>180 (r= 0.303, p<0.001), TBR<70 (r= 0.063, p=0.436), TBR<54 (r= 0.082, p=0.311), and inversely associated with TIR (r= -0.441, p<0.001). The number of delayed boluses was directly associated with fear of hypoglycemia at work, during night and being alone.

Conclusion: Delayed insulin boluses in patients on advanced technologies are very common (on average 1 out of 5 meals) and are associated with a significant worsening of blood glucose control. Adequate attention should be given to the timing of bolus injection also in patients on advanced technologies, mainly acting on fear of hypoglycemia.

Disclosure: R. Triggiani: None.

246

With which dose of NPH insulin to start at bedtime? Study in a cohort of 1006 patients with type 2 diabetes and failure of oral antidiabetic drugs

B. Mertes1, S. Gödde1, G. Egidi2, T. Uebel3, G. Kramer4, N. Kuniß4,5;

1Abteilung Diabetes, Neuropathie, Fußsyndrom, Cardioangiologisches Centrum Bethanien, Frankfurt am Main, 2Hausarztpraxis, Bremen, 3Hausarztpraxis, Neckargemünd, 4FB Endokrinologie und Stoffwechsel, Universitätsklinikum Jena, Klinik für Innere Medizin III, Jena, 5Dr. med. Kielstein Ambulante Medizinische Versorgung GmbH, Erfurt, Germany.

Background and aims: In the updated German National Health Care Guideline “Therapy of diabetes type 2”, for the first time, NPH insulin (Neutral Protamine Hagedorn, isophane insulin) at bedtime is recommended, if the HbA1c therapy target is missed after failure of oral medication. As a starting dose, various research groups worldwide recommend 0.1-0.2 IU/kg body weight. However, systematic studies investigating the starting dose of an insulin therapy with NPH insulin do not exist.

Materials and methods: A cohort of 1006 patients with diabetes type 2 and failure of oral antidiabetic drugs was treated additionally with NPH insulin at bedtime (63% male, age 59.5±11.5 years, weight 91.8±19.1 kg, body mass index 31.4±5.9 kg/m², diabetes duration 7.5±5.7 years, HbA1c 8.6±1.1%, number of different oral antidiabetic agents 1.2±0.5/patient). Blood glucose levels were checked twice during the night (22:00, 2:00, 5:00, and 7:00 h) to detect hypoglycaemia from a dose of 12 IU. All patients participated in structured education programme for individuals with insulin therapy at baseline. The combination of oral antidiabetic agents remained unchanged. Multiple linear regression was used to determine which factors influence final insulin dose.

Results: The NPH insulin dose of initially 9.4±2.0 IU was adapted within 20.2±24.9 days to 9.8±2.7 IU. This corresponds to a final insulin dose of 0.11±0.03 IU/kg body weight. In 77 patients (7.7%) the initial insulin dose had to be reduced by 2.44±1.09 IU due to low blood glucose levels during the night. Severe hypoglycaemia did not occur. Body weight and HbA1c were significantly associated with the final insulin dose. The lower the BMI (p<0.001) and the higher the HbA1c (p<0.001), the higher the insulin dose.

Conclusion: The NPH insulin dose of up to 0.2 IU/kg recommended by various research groups worldwide is too high. For example, a patient with body weight of 90 kg would need to inject 18 IU NPH insulin at baseline, but only 10 IU according to our study. A too high dose can be associated with nocturnal hypoglycaemia. A limitation of this study is the retrospective setting. If oral medication fails in type 2 diabetes, NPH insulin should be started at night with 0.1 IU/kg body weight. At a NPH insulin dose of 12 IU and above, monitoring blood glucose levels during the night is recommended.

Disclosure: B. Mertes: None.

247

Comparison of the basal insulin analogues Gla-300 and IDeg 100 using continuous glucose monitoring in people with type 1 diabetes: the InRange randomised controlled trial

T. Battelino1, T. Danne2, S. Edelman3, P. Choudhary4, E. Renard5, J. Westerbacka6, B. Mukherjee6, P. Picard7, V. Pilorget6, R. Bergenstal8;

1UMC–University Children’s Hospital, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 2Diabetes Centre for Children and Adolescents, Children’s and Youth Hospital ”Auf Der Bult”, Hannover, Germany, 3University of California, San Diego, USA, 4Diabetes Research Centre, University of Leicester, Leicester, UK, 5Department of Endocrinology, Diabetes and Nutrition, Montpellier University Hospital, University of Montpellier, Montpellier, France, 6Sanofi, Paris, France, 7IviData LIFE SCIENCES, Levallois-Perret, France, 8International Diabetes Center at Park Nicollet, Minneapolis, USA.

Background and aims: InRange is the first large randomised controlled trial to use continuous blood glucose monitoring (CGM) time-in-range (TIR) as a primary efficacy endpoint to compare second-generation basal insulin (BI) analogues, insulin glargine 300 U/mL (Gla-300) and insulin degludec 100 U/mL (IDeg-100) in adults with type 1 diabetes (T1D).

Materials and methods: Multicentre, randomised, active-controlled, parallel-group, 12-week open-label study comparing efficacy of Gla-300 and IDeg-100 using 20-day CGM profiles (≥10 days evaluable) at Week 12. Inclusion: adults with T1D treated with multiple daily injections, using BI analogues once daily and rapid-acting insulin analogues for ≥1 year; HbA1c ≥7 (≥53 mmol/mol) and ≤10% (≤86 mmol/mol) at screening; not requiring CGM for routine care during study.

Results: In total, 343 participants were randomised (172 Gla-300, 171 IDeg-100): mean (SD) age was 42.8 (13.3) years, BMI 27.3 (4.8) kg/m2, T1D duration 20.5 (12.8) years, 33.8% had ≥1 diabetic complication. Non-inferiority of Gla-300 versus IDeg-100 was demonstrated on percent TIR 3.9-10 mmol/L (primary endpoint, Table). Non-inferiority was demonstrated on glucose total coefficient of variation (CV, main secondary endpoint) with lower CV for Gla-300. Following demonstration of non-inferiority on TIR and glucose CV, superiority of Gla-300 over IDeg-100 on TIR 3.9-10 mmol/L was tested and not demonstrated. Rates of hypoglycaemia at <3.9 to ≥3.0 mmol/L and <3.0 mmol/L thresholds did not differ between groups. Safety profiles were consistent with known profiles of Gla-300/IDeg-100 and no unexpected safety findings were identified. No treatment-emergent adverse event (TEAE) leading to permanent treatment discontinuation and no death due to TEAE was reported.

Conclusion: Using clinically relevant CGM metrics, the InRange study shows that Gla-300 is non-inferior to IDeg-100 in people with T1D, with similar hypoglycaemia and safety profiles.

figure br

Clinical Trial Registration Number: NCT04075513

Supported by: Sanofi

Disclosure: T. Battelino: Grants; Abbott Diabetes Care, Medtronic, Novo Nordisk, GluSense, Sanofi, Sandoz, Novartis and Zealand. Honorarium; Participation on advisory boards for Novo Nordisk, Sanofi, Eli Lilly, Boehringer Ingelheim, Indigo and Medtronic. Stock/Shareholding; DreaMed Diabetes. Other; Speaker for AstraZeneca, Eli Lilly, Novo Nordisk, Medtronic, Pfizer, Sanofi, Dexcom, Abbott and Roche.

248

Switching from multiple insulin injections to a fixed combination of degludec and liraglutide in patients with type 2 diabetes: Simplify study: results after 3 months

E. Martinka1, I. Dravecka2, I. Tkac3;

1Diabetology, National institute of endocrinology and diabetology, Lubochna, 2Department of Internal Medicine 1, Faculty of Medicine, P.J. Safarik University, Faculty of Medicine, L. Pasteur University Hospital, Kosice, 3Department of Internal Medicine 4, Faculty of Medicine, P.J. Safarik University, Faculty of Medicine, L. Pasteur University Hospital, Kosice, Slovakia.

Background and aims: A non-randomized, open-label, multicenter, single-arm prospective study was performed with the aim to evaluate whether switching from basal insulin + prandial insulin boluses (IIT) to fixed-combination of the basal insulin analog degludec and the GLP-1 receptor agonist liraglutide (IDegLira) in patients with type 2 diabetes mellitus (DM2T) is at least as effective as previous IIT treatment in terms of glycemic control, body weight, blood pressure, and blood lipid levels. The analysis was performed after the first 3 months.

Materials and methods: The study enrolled 147 patients with DM2T with duration > 5 years, on IIT treatment > 12 months, with HbA1c > 7%, with preserved insulin secretion and total daily insulin dose (TDDI) <0.7 U/kg body weight or <70 U/day. Parameters of glycemic control (HbA1c, glycemia in glycemic profiles) were compared during the treatment of IIT and subsequently after 3 months of treatment after switching to IDegLira. In a subgroup (n=31) of patients, continuous glucose monitoring (CGM) was performed and parameters time in range (TIR), time above range (TAR), and time below range (TBR) were evaluated. Other endpoints were body weight (BW), body mass index (BMI), blood pressure, lipids, the prevalence of hypoglycemia, and insulin doses. Switching from IIT to IDegLira was performed in a single ambulatory session with an initial dose of IDegLira up to 16 U followed by titration of ± 2-4 U/3 days.

Results: Analysis of the results after the first 3 months of treatment showed that switching from IIT to IDegLira was associated with a significant improvement both with HbA1c (8.6±0.9 vs 7.7±1.2, p<0,0001) and plasma glucose levels in glycemic profiles. TIR and TAR values were also improved, but the difference was not statistically significant. Change of treatment has also been associated with significant reductions in BW (97.7±18.3 vs 94.2±17.9 kg, p<0,0001), BMI, systolic blood pressure, triglycerides, total cholesterol, and LDL-cholesterol. There was also a significant reduction in frequency of hypoglycaemia both in self-monitoring (0.52 vs 0.03 episodes per patient, 19,7% vs 3,4% of patients) and TBR (2.8% vs 0.8%, p<0,05 in CGM), and a significant reduction in insulin doses (55.6±14.3 vs 30.9±9.4 U, p<0,0001).

Conclusion: Switching from IIT to IDegLira in DM2T with HbA1c> 7%, preserved insulin secretion and TDDI <70U was a safe, effective, and less demanding form of treatment, the benefit of which manifests already after three months of treatment.

Disclosure: E. Martinka: Lecture/other fees; Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Sanofi.

OP 45 Newer agents, better outcomes

249

Effect of empagliflozin on left ventricular contractility and peak oxygen uptake in subjects with type 2 diabetes without heart disease: results of the EMPA-HEART trial

L. Nesti1, N.R. Pugliese1, P. Sciuto1, D. Tricò1, A. Dardano1, I. Fabiani2, A. Natali1;

1Università di Pisa, 2Fondazione Toscana G. Monasterio, Pisa, Italy.

Background and aims: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are effective in primary prevention of hospitalization for heart failure through mechanisms that still are uncertain. The EMPA-HEART trial is a phase III, active-controlled, parallel groups, exploratory study aiming at verifying whether the SGLT2i empagliflozin is associated with improved myocardial contractility and/or cardiopulmonary fitness in patients with type 2 diabetes (T2D) without heart disease, in comparison with sitagliptin as an active control, an equally effective glucose lowering agent that has been shown to be neutral on the prevention of heart failure-related events.

Materials and methods: Patients with T2D of either sex, age 40-80 years, on stable therapy with metformin and/or basal insulin, suboptimal glycaemic control (HbA1c 7.0-8.5%), and normal biventricular systo-diastolic function, no ischemic or valvular heart disease, and devoid of micro- or macrovascular complications were randomized to either empagliflozin 10 mg/die or sitagliptin 100 mg/die for 6 months and were subjected to resting and exercise echocardiography with speckle-tracking technology (left ventricle global longitudinal strain, LV-GLS, a more sensitive measure of systolic function than 2D-left ventricular ejection fraction), cardiopulmonary exercise testing (measurement of peak oxygen uptake, VO2peak) , and an extensive vascular, endothelial, autonomic, and biohumoral characterization.

Results: Forty-four patients completed the study, 22 per arm. While glycaemic control similarly improved in both groups, a relative reduction in body weight (-1.6; [-2.7/-0.5] kg, p=0.03) and plasma uric acid (-1.5; [-2.3/-0.6], p=0.002), as well as an increase in haemoglobin (+0.7; [+0.2/+1.1] g/dL, p=0.0003) were evident with empagliflozin. No difference between the treatments was detectable in the absolute changes of either LV-GLS at 1 month (empagliflozin vs sitagliptin: +0.44; [-0.10/+0.98] %, p=0.11) and 6 months of therapy (+0.53; [-0.56/+1.62] %), or in VO2peak (+0.43; [-1.4/+2.3] ml/min/kg, p=0.65), as well as in vascular function and plasma biomarkers of ventricular strain and damage, extracellular matrix remodeling, and inflammation. Nevertheless, with empagliflozin, the subgroup with baseline LV-GLS below the median experienced a significantly greater increase (time*drug p<0.05) in LV-GLS at both 1 month (+1.22; [+0.31/+2.13] %) and at 6 months (+2.05; [+1.14/+2.96] %), while sitagliptin only induced a modest improvement in LV-GLS at 6 months (+0.92; [+0.21/+0.62] %).

Conclusion: Empagliflozin has neutral impact on both LV-GLS and exercise tolerance in subjects with T2D and normal left ventricular function. Nonetheless, in patients with subclinical systolic dysfunction (baseline LV-GLS <16.5%) it produces a rapid and persistent amelioration of LV contractility.

figure bs

Clinical Trial Registration Number: EUDRACT Code 2016-0022250-10

Supported by: Investigator-initiated study supported at 49% by an unrestricted grant from Boehringer Ingelheim

Disclosure: L. Nesti: None.

250

Novel subgroups of patients with type 2 diabetes show differential cardiovascular and kidney benefits with canagliflozin: a data-driven proteomic cluster analysis

M. Schechter1, A. Koshino1, N. Jongs1, B. Neal2, C. Arnott2, V. Perkovic2, M.K. Hansen3, H.J. L. Heerspink1,2;

1Dept of Clinical Pharmacy and Pharmacology, University of Groningen, Groningen, Netherlands, 2The George Institute for Global Health, UNSW Sydney, Sydney, Australia, 3Janssen Research and Development, LLC, Spring House, USA.

Background and aims: Canagliflozin (CANA) improves cardiovascular (CV) and kidney outcomes in patients with type 2 diabetes (T2D) at high CV or kidney risk. By applying a data-driven approach, we tested whether a combination of novel protein biomarkers could identify patients that are more likely to experience CV or kidney benefits with CANA treatment.

Materials and methods: The CANVAS trial randomized 4330 participants with T2D at high CV risk to receive CANA or placebo. The primary CV outcome was a composite of non-fatal myocardial infarction or stroke or CV death. The prespecified kidney outcome was a composite of sustained ≥40% estimated glomerular filtration rate (eGFR) decline, end-stage kidney disease, or death due to kidney failure. K-mean clustering, relying on eight novel blood (IL-6, TNFR1, TNFR2, KIM1, GDF15, MMP7) and urine (EGF, MCP1) protein biomarkers, was used to classify patients into subgroups. The elbow, silhouette, and gap statistic methods were applied to determine the optimal clusters’ number. Treatment effects of CANA on CV and kidney outcomes across biomarker clusters were assessed using Cox proportional hazard models.

Results: Complete biomarkers values at baseline were available in 3381 (78.1%) participants, followed for a median [IQR] of 6.1 [5.9, 6.4] years. K-mean clustering, based on the eight novel biomarkers, discovered two different patients’ clusters (Cluster 1, N=1957; Cluster 2, N=1424). Compared with Cluster 1, participants in Cluster 2 had lower baseline eGFR (mean [SD]= 69.6 [16.6] vs 86.2 [13.8] mL/min/1.73 m2), higher urine albumin to creatinine ratio (UACR; median [IQR]= 19.6 [8.3,82.7] vs 8.9 [5.8,18.5] mg/g), and were more likely to have established CV disease (61.4% vs. 57.5% of patients). All biomarkers’ levels were higher in Cluster 2, except for urine EGF which was higher in Cluster 1. Within each cluster, baseline characteristics were balanced between the CANA and placebo groups. Compared with placebo, CANA improved the cardiovascular outcome in Cluster 2 but not in Cluster 1 (P-interaction =0.011; table). In contrast, its effect on the kidney outcome was statistically significant in Cluster 1 but not in Cluster 2 (P-interaction =0.004). The interaction remained statistically significant after model adjustment for other baseline clinical characteristics.

Conclusion: This data-driven analysis, relying on a combination of novel biomarkers, suggested two separate clusters among participants in the CANVAS trial - in one cluster CANA improves the CV outcome, and in the other CANA improves the kidney outcome. These hypothesis-generating findings warrant validation in a separate clinical trial.

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Clinical Trial Registration Number: NCT01032629

Supported by: Janssen Research & Development

Disclosure: M. Schechter: None.

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SGLT-2 inhibitors and GLP-1 receptor agonists are associated with lower mortality in younger people with type 2 diabetes and a first myocardial infarction between 2016 and 2020

M. Löndahl1, S. Puvaneswaralingam2, E. Uddman1, K. Filipsson1;

1Endocrinology, Clinical Sciences, Lund University, 2Endocrinology, Skane University hospital, Lund, Sweden.

Background and aims: Type 2 diabetes is a significant risk factor for cardiovascular disease and death. In people with type 2 diabetes and higher risks of cardiovascular disease, several large randomised controlled trials have identified a reduction in cardiovascular mortality and non-fatal cardiovascular complications after initiating treatment with sodium-glucose cotransporter-2 (SGLT-2) inhibitors and glucagon-like peptide-1 receptor agonists (GLP1-RA). Although the first data were published in 2015, the prescription of these drugs in people with high-cardiovascular risk has been modest. This study aimed to evaluate the effect of prescription of SGLT-2 inhibitors and /orGLP1-RA on all-cause mortality in people younger than 70 years with type 2 diabetes having their first myocardial infarction.

Materials and methods: This study is an observational, population-based cohort study of individuals with type 2 diabetes, aged between 40 and 69 years, and the first diagnosis of myocardial infarction between January 2016 and December 2020 in Sweden using data from national registries. People with type 2 diabetes were identified in the National Drug Prescription Registry and defined as people who at least twice had been prescribed an anti-diabetic drug. Prescription of anti-diabetic and antihypertensive drugs and date of the collection were registered. Data on comorbidities and cardiovascular events were collected from the National Patient Registry, which contains nationwide hospital discharge information since 1987. Information on the time of death was retrieved from the Swedish Cause of Death Register. Patients were grouped according to the use of SGLT-2 inhibitors, GLP1-RA, or no prescription of any of these drugs. The primary endpoint, all-cause mortality, is presented as Kaplan-Meier curves, and statistical differences were evaluated using the Log-Rank test.

Results: Of the 9117 identified patients below 70 years of age with a history of type 2 diabetes and a first myocardial between 2016 and 2020, 4584 (50.3%) had been prescribed neither an SGLT-2 inhibitor nor a GLP1-RA, while 26.8% was prescribed an SGLT-2 inhibitor, 9.4% a GLP-1 RA and 13.6% drugs from both these classes. During the follow-up period (median three years), 1001 events (11.0%) occurred. The five-year mortality is given in Figure 1.

Conclusion: In this population of younger people with type diabetes and a first myocardial infarction between 2016 and 2020, prescription of an SGLT-2 inhibitor or a GLP1-RA was associated with lower five-year mortality. The number of high-risk patients with access to the drugs still seems too low.

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Supported by: Unrestricted grant from Boehringer-Ingelheim.

Disclosure: M. Löndahl: Grants; Unrestricted grant from Boehringer-Ingelheim. Lecture/other fees; AstraZeneca, Boehringer-Ingelheim, EliLilly, NnovNordisk.

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Efficacy of ertugliflozin (ERTU) on hospitalisation for heart failure (HHF) across the spectrum of pre-trial ejection fraction (EF): post hoc analyses of VERTIS CV

A. Pandey1, A.A. Kolkailah1, F. Cosentino2, C.P. Cannon3, R. Frederich4, D.Z.I. Cherney5, S. Dagogo-Jack6, R.E. Pratley7, N.B. Cater8, I. Gantz9, J.P. Mancuso10, D.K. McGuire1;

1Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, USA, 2Unit of Cardiology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden, 3Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA, 4Pfizer Inc., Collegeville, USA, 5University of Toronto, Toronto, Canada, 6University of Tennessee Health Science Center, Memphis, USA, 7AdventHealth Translational Research Institute, Orlando, USA, 8Pfizer Inc., New York, USA, 9Merck & Co., Inc., Kenilworth, USA, 10Pfizer Inc., Groton, USA.

Background and aims: There is controversy over whether SGLT2 inhibitors have efficacy in high-risk patients (pts) with HF and EF >60% with or without T2D. VERTIS CV studied pts with T2D and atherosclerotic CVD (ASCVD); 23% had a history of HF. In VERTIS CV, ERTU significantly reduced risk of first and total HHF vs placebo (PBO). Whether efficacy in VERTIS CV is consistent across the spectrum of pre-trial EF is unknown, particularly for pts with EF >60%. These post hoc analyses explored the effects of ERTU vs PBO on time to first and total HHF in VERTIS CV across pre-trial EF.

Materials and methods: ERTU 5 and 15 mg groups were combined for all analyses. Treatment effects of ERTU vs PBO on risk of first and total HHF were analysed using adjusted Cox models for first and Andersen-Gill models for total (i.e., first + recurrent) events. Data on pre-trial EF were from medical records at trial entry. Multiplicative interaction terms (EF * treatment arm) were used to determine if ERTU efficacy was modified by pre-trial EF.

Results: 8246 pts were randomised to ERTU 5 or 15 mg or PBO (mean follow up 3.5 years). Overall, 5006 pts had pre-trial EF data available; 959 had EF ≤45%, 2860 had EF >45-60%, and 1187 had EF >60%. Overall, the event rate for first HHF was lower with ERTU vs PBO (HR 0.70; 95% CI 0.54-0.90). The findings were generally consistent across pre-trial EF (P-interaction=0.26; Figure), including pts with pre-trial EF >60% (HR 0.72; 95% CI 0.34-1.55). Overall, the event rate for total HHF was lower with ERTU vs PBO (HR 0.70; 95% CI 0.56-0.87). A significant interaction was observed between pre-trial EF and treatment arm for the risk of total HHF events (P-interaction=0.02), with a greater magnitude of risk reduction in pts with a low pre-trial EF (≤45%; HR 0.39; 95% CI 0.26-0.57). However, the 95% CIs for the HR for total HHF for those with EF >45-60% and >60% nearly entirely or entirely contained the 95% CI of the overall population, respectively (Figure).

Conclusion: In the VERTIS CV trial of pts with T2D and ASCVD, the efficacy of ERTU in preventing first HHF was generally comparable across the spectrum of pre-trial EF. The trend for greater benefit at lower EF was statistically significant for total HHF events. Findings for pts with EF >45-60% and >60% appeared quantitively consistent with the overall findings for both first and total HHF.

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