Metabolic effects of insulin in a human model of ketoacidosis combining exposure to lipopolysaccharide and insulin deficiency: a randomised, controlled, crossover study in individuals with type 1 diabetes
Diabetic ketoacidosis (DKA) is often caused by concomitant systemic inflammation and lack of insulin. Here we used an experimental human model to test whether and how metabolic responses to insulin are impaired in the early phases of DKA with a specific focus on skeletal muscle metabolism.
Nine individuals with type 1 diabetes from a previously published cohort were investigated twice at Aarhus University Hospital using a 120 min infusion of insulin (3.0/1.5 mU kg−1 min−1) after an overnight fast under: (1) euglycaemic conditions (CTR) or (2) hyperglycaemic ketotic conditions (KET) induced by an i.v. bolus of lipopolysaccharide and 85% reduction in insulin dosage. The primary outcome was insulin resistance in skeletal muscle. Participants were randomly assigned to one of the two arms at the time of screening using www.randomizer.org. The study was not blinded.
All nine volunteers completed the 2 days and are included in the analysis. Circulating concentrations of glucose and 3-hydroxybutyrate increased during KET (mean ± SEM 17.7 ± 0.6 mmol/l and 1.6 ± 0.2 mmol/l, respectively), then decreased after insulin treatment (6.6 ± 0.7 mmol/l and 0.1 ± 0.07 mmol/l, respectively). Prior to insulin infusion (KET vs CTR) isotopically determined endogenous glucose production rates were 17 ± 1.7 μmol kg−1 min−1 vs 8 ± 1.3 μmol kg−1 min−1 (p = 0.003), whole body phenylalanine fluxes were 2.9 ± 0.5 μmol kg−1 min−1 vs 3.1 ± 0.4 μmol kg−1 min−1 (p = 0.77) and urea excretion rates were 16.9 ± 2.4 g/day vs 7.3 ± 1.7 g/day (p = 0.01). Insulin failed to stimulate forearm glucose uptake and glucose oxidation in KET compared with CTR (p < 0.05). Glycogen synthase phosphorylation was impaired in skeletal muscle.
In KET, hyperglycaemia is primarily driven by increased endogenous glucose production. Insulin stimulation during early phases of DKA is associated with reduced glucose disposal in skeletal muscle, impaired glycogen synthase function and lower glucose oxidation. This underscores the presence of muscle insulin resistance in the pathogenesis of DKA.
Trial registration www.clinicaltrials.gov (ID number: NCT02157155).
Funding This work was funded by the Danish Council for Strategic Research (grant no. 0603-00479B).
KeywordsClinical diabetes Endogenous glucose production Glucose transport Hyperglycaemia Insulin action Insulin resistance Insulin sensitivity Skeletal muscle
Eukaryotic translation initiation factor 4E binding protein 1
Akt substrate of 160 kDa
Euglycaemic control conditions
Glycogen synthase kinase 3
Hyperglycaemic ketotic conditions
Mammalian target of rapamycin
Ribosomal protein S6 kinase
Respiratory exchange rate
Diabetic ketoacidosis (DKA) is a severe complication of diabetes caused by a deficiency of insulin often in combination with an infection. The insulin deficit has tissue-specific effects that mirror insulin action. In adipose tissue, the release of NEFA is increased owing to impaired inhibition of lipolysis . In the liver, ketogenesis is stimulated and glucose output is increased due to reduced glycogen synthesis and accelerated gluconeogenesis. In skeletal muscle, glucose disposal is diminished due to reduced insulin-stimulated glucose uptake [2, 3].
Conversely, insulin therapy reverses many of the processes underlying DKA, and immediate insulin treatment is important for a positive outcome. In adipose tissue, insulin treatment suppresses lipolysis, thereby limiting the supply of NEFA to the liver . Concurrently, hepatic glucose production and ketogenesis are reduced . The effects of insulin treatment on skeletal muscle during DKA are less well understood.
Under physiological conditions, muscle accounts for as much as 80% of glucose disposal after a glucose load . The uptake of glucose into muscle is initiated by insulin-stimulated translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell membrane. After transport into the muscle cell, glucose is either oxidised in the tricarboxylic acid cycle or incorporated into glycogen . Several factors that are commonly associated with DKA, including increased NEFA levels and prolonged fasting, are potent inducers of insulin resistance in skeletal muscle [7, 8]. The associated hyperglycaemia further impairs insulin action on skeletal muscle causing a vicious circle accelerating insulin resistance . The capacity to increase glucose uptake in skeletal muscle therefore plays an essential role when reversing the hyperglycaemia, and the associated insulin resistance complicates treatment .
Previous investigations into insulin action during ketoacidosis have predominantly used animal models. These studies show that elevated circulating ketone body levels impair glucose uptake in muscle [11, 12]. However, the toxic compound streptozocin is typically used in these animal models to induce hypoinsulinaemia. This reduces insulin production effectively, but as streptozocin also has toxic effects on the liver and the small intestine the model only reflects some aspects of type 1 diabetes mellitus. This, in combination with differences in substrate metabolism between rodents and humans, limits the clinical impact of these studies. Therefore, we have established a human model of DKA by combining insulin deprivation and lipopolysaccharide (LPS) injection in individuals with type 1 diabetes . Using this approach, we aimed to investigate the mechanisms regulating glucose disposal in human skeletal muscle under the hypothesis that early phases of DKA are complicated by impaired insulin action in skeletal muscle.
Study design and participants
The present study was designed as a randomised, controlled, crossover trial consisting of two experimental days, separated by at least 3 weeks, characterised by: (1) euglycaemic control conditions and a bolus of saline (154 mmol/l NaCl) (CTR) and (2) hyperglycaemic ketotic conditions (KET) induced by LPS administration combined with insulin deprivation, as previously described in detail . The study was not blinded. Briefly, nine male volunteers with type 1 diabetes mellitus were selected using the following inclusion criteria: type 1 diabetes mellitus (c-peptide negative), male sex, 20–40 years of age, no medication other than insulin, BMI 19–26 kg/m2 and without any comorbidities or diabetic complications. The study was carried out at Aarhus University Hospital.
Long-acting insulin was replaced by rapidly acting insulin (Insulin Actrapid; Novo Nordisk, Copenhagen, Denmark) to ensure adequate glycaemic control 24 h before each trial and to facilitate a switch to i.v. insulin on the experimental days. During CTR conditions, i.v. insulin was titrated to keep blood glucose within 5–7 mmol/l. During KET, insulin dosing was reduced to 15% of the participants’ regular basal insulin need. LPS (10,000 USP Endotoxin, lot HOK354; United States Pharmacopeial Convention, Rockville, MD, USA) was given as a bolus infusion of 1 ng/kg body weight at 0 min. LPS is used to mimic the presence of Gram-negative bacteria, causing an inflammatory immune response that results in a large increase in proinflammatory cytokines, such as TNF-α, IL-6 and IL-10 .
The project was approved by the local ethics committee (1-10-72-98-14) in accordance with the Declaration of Helsinki and was registered at www.clinicaltrials.gov (ID number: NCT02157155). Participants were randomly assigned to one of the two arms at the time of screening using www.randomizer.org. All participants gave written informed consent prior to participation.
Preconditioning and hyperinsulinaemic infusion periods
Outcomes and analytical methods
We examined glucose disposal in skeletal muscle during the HI period using indirect calorimetry, plethysmography, muscle tissue biopsies and plasma substrate concentrations. Indirect calorimeter with a canopy (Oxycon Pro; Intra medic, Gentofte, Denmark) was applied at 210 min and 400 min to collect respiratory gases and estimate whole body energy expenditure, respiratory exchange rates (RER) and glucose oxidation rates as previously described .
d-[3-3H]glucose (GE Healthcare, Brøndby, Denmark) was administered as a bolus injection of 0.74 MBq followed by continuous infusion of 0.44 MBq/h for 4 h until triplicate samples were drawn and analysed as described previously . These samples were used to estimate endogenous glucose production at the end of the preconditioning period.
At 0 min, [15N]phenylalanine (prime 0.75 mg/kg, infusion rate 0.75 mg kg−1 h−1), [13C]urea (prime 390.6 mg, infusion rate 42 mg/h), l-[2H4]tyrosine (prime 0.6 mg/kg, infusion rate 0.6 mg/kg/h) and a bolus of [15N]tyrosine (prime 0.3 mg/kg) were started. Triplicate samples were drawn on arterial and venous blood during the preconditioning period at 240–260 min. Isotopic enrichments of these blood samples were measured using GC-MS. Immediately following the collection of each blood sample, blood flow was measured using venous occlusion plethysmography across the forearm. In addition, urine was sampled throughout each study day to estimate urea excretion rates. Protein kinetics in muscle were calculated using the forearm model .
Muscle tissue biopsies
Muscle biopsies were obtained from each participant from the vastus lateralis of the quadriceps femoris at 290 min (preconditioning period) and 360 min (HI period).
Muscle tissue was immediately snap-frozen in liquid nitrogen before storage at −80°C. All samples were subsequently homogenised in a buffer (pH 7.4) containing 50 mmol/l HEPES, 137 mmol/l NaCl, 20 mmol/l NaF, 10 mmol/l Na4P2O7, 5 mmol/l EDTA, 1 mmol/l MgCl, 1 mmol/l CaCl2, 2 mmol/l sodium orthovanadate, Halt (Halt protease inhibitor cocktail no. 78438, Thermo Scientific, Waltham, MA, USA), 5 mmol/l nicotinamide, 10 μmol/l trichostatin A, 1% (vol./vol.) NP-40, and 10% (vol./vol.) glycerol. Samples were centrifuged after homogenisation at 14,000 g for 20 min and insoluble material was removed.
Protein samples were resolved by SDS-PAGE (4–12% Bis-Tris gels, Criterion XT System; BioRad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes according to the manufacturer’s instructions. Western blot analysis was performed using the following primary antibodies: v-Akt murine thymoma viral oncogene homologue (Akt) (total no. 4691, Ser473 no. 9271), Akt substrate of 160 kDa (AS160) (Thr642 no. 4288), glycogen synthase kinase 3 (GSK3) (total no. 5676, Ser21/9 no. 9331), glycogen synthase (GS) (total no. 3886, Ser641 no. 3891), mammalian target of rapamycin (mTOR) (total no. 2972, Ser2448 no. 2971), eukaryotic translation initiation factor 4E binding protein 1 (4eBP1) (total no. 9644, Thr46 no. 4923), ribosomal protein S6 kinase (p70s6K) (total no. 9202, Thr389 no. 9205) and prohibitin 1 (PHB1) (no. 2426) from Cell Signaling (Danvers, MA, USA); and AS160 (total no. 07-741) from Merck Milipore (Darmstadt, Germany). Horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham, GE Healthcare, Pittsburgh, PA, USA) was used as a secondary antibody. Proteins were visualised by chemiluminescence (Clarity Western ECL, BioRad) and ChemiDoc MP imaging system (BioRad), and quantified with Image Lab 5.0 (BioRad). Precision Plus All Blue protein standards were used as a marker of molecular mass (BioRad). Equal loading was ensured using stain-free technology . Data are presented as individual dot plots showing a ratio of phosphorylated protein compared with total level of the same protein. Western blot membranes were stripped after analysing phosphorylated proteins to allow for total protein analysis on the same membranes. Further information is given in the electronic supplementary material (ESM) Table 1.
Skeletal muscle glycogen levels were assessed by a hexokinase enzymatic reaction as previously described . Briefly, pulverised muscles were solubilised in HCl at 95°C, subsequently neutralised with NaOH and centrifuged. Muscle glucose levels were assessed using hexokinase reagent (CIMA Scientific, De Soto, TX, USA) in the supernatant fraction.
Statistics and power calculations
The primary endpoint was glucose uptake in skeletal muscle estimated from arteriovenous glucose difference in mmol/l and blood flow. The power calculation for the primary endpoint as a comparison of two paired means with significance level = 0.05, power = 0.90, difference of means =1.0 mmol/l and SD of the mean = 0.3 mmol/l. This gave a sample size of n = 4. We included nine participants to ensure secondary outcomes as well.
Results are presented as means ± SEM, unless otherwise specified. Normal distribution of data was ensured by inspection of QQ-plots. Statistical associations between the two trial days was tested using a paired t test and two-way repeated measure ANOVA analysis when relevant. A p value <0.05 was considered significant. Graphing and statistical analyses were completed using Sigmaplot 11 (Systat Software, San Jose, CA, USA) and Stata 13 (Stata, College Station, TX, USA), respectively.
Metabolic status at t = 0 and right before hyperinsulinaemic infusion was started
Metabolic status at t = 0
Metabolic status at t = 270
7.41 ± 0.01
7.4 ± 0.01
7.39 ± 0.01
Base excess (mmol/l)
1.1 ± 0.6
−0.03 ± 0.4
−2.9 ± 0.6*
6.9 ± 0.7
6.8 ± 0.4
17.7 ± 0.6*
0.5 ± 0.1
0.6 ± 0.1
1.4 ± 0.1*
40.4 ± 4.5
38.9 ± 5.8
95.1 ± 18.8*
0.1 ± 0.1
0.3 ± 0.1
1.6 ± 0.2*
139.7 ± 53.1
92.9 ± 30.7
291.4 ± 34.4*
Insulin-stimulated glucose disposal is impaired in KET
During the HI, the glucose oxidation rate was 40% higher in CTR (1268 kJ/day; p < 0.05) (Fig. 2c). Energy expenditure was similar during the HI period on both experimental days. The RER was higher in CTR (0.87 ± 0.03) compared with KET (0.81 ± 0.01), but the difference was only borderline significant (p = 0.07; Fig. 2d). We found no alteration in mitochondrial content estimated from PHB1 levels (Fig. 2e).
At the end of the preconditioning period, cortisol levels were threefold higher in KET (291 ± 34 ng/ml) compared with CTR (93 ± 31 ng/ml; p < 0.001), adrenalin levels did not differ significantly between KET (133 ± 88 pg/l) and CTR (42 ± 11 pg/l; p > 0.05) and glucagon levels were nearly threefold higher during KET (95 ± 19 pg/ml) compared with CTR (38 ± 6 pg/ml; p < 0.05). During the HI, cortisol decreased in KET, while no effect of hyperinsulinaemia was observed in CTR. Glucagon remained elevated by 50% in KET (48.9 ± 5.0 pg/ml) compared with CTR (31.3 ± 4.7 pg/ml; p < 0.01) during the HI, while adrenalin was unaffected.
Insulin signalling through Akt/AS160 is increased in KET
Insulin-stimulated regulation of GS phosphorylation is impaired in KET
GS, the rate-limiting enzyme for glycogen synthesis, is inactivated by phosphorylation at Ser641 . During the HI period, insulin reduced GS Ser641 phosphorylation in both CTR and KET, but as shown in Fig. 4c insulin-induced reduction in phosphorylation was ∼50% lower for KET. GSK3 is the upstream kinase for GS Ser641 but, similar to Akt and AS160, insulin-induced GSK3 phosphorylation was enhanced in KET (Fig. 4d). We did not find any statistically significant difference in muscle tissue glycogen content.
Insulin-mediated suppression of lipolysis is impaired in KET
Hyperketonaemia was suppressed to equal levels in both CTR and KET (Fig. 3c). However, during the HI, insulin-stimulated suppression of NEFA levels was impaired in KET compared with CTR (0.26 vs 0.12 mmol/l; p < 0.05) (Fig. 3d). This was associated with increased lipid oxidation in KET (3640 kJ/day) compared with CTR (2356 kJ/day), although this difference was not statistically significant (p = 0.17).
Increased protein turnover and urea excretion but no changes in muscle balances in KET
Phenylalanine, thyrosine and urea tracers together with pletysmography and indirect calorimetry were used to investigate skeletal muscle and whole body protein balance prior to the HI period. Urine nitrogen excretion doubled from 7.3 ± 1.7 g/day (CTR) to 16.9 ± 2.4 g/day (KET; p < 0.05), but this was not associated with detectable differences in skeletal muscle protein synthesis, breakdown or balance; phenylalanine fluxes were 2.9 ± 0.5 μmol kg−1 min−1 vs 3.1 ± 0.4 μmol kg−1 min−1 (p = 0.77). In accordance with the increased nitrogen excretion, whole body protein breakdown was 30% higher in KET (42.7 μmol kg−1 h−1) compared with CTR (31.4 μmol kg−1 h−1; p < 0.001) and synthesis increased by 40% in KET (39.9 μmol kg−1 h−1) compared with CTR (28.4 μmol kg−1 h−1; p < 0.001). Ultimately, these changes did not affect whole body protein balance. Urea turnover tended to be increased in KET (347 ± 44 μmol kg−1 h−1) compared with CTR (279 ± 29 μmol kg−1 h−1; p = 0.13).
In general, hyperglycaemia is an important component of DKA . The major findings of this study are that the early phases of DKA in individuals with type 1 diabetes mellitus are associated with elevated endogenous glucose production, pronounced insulin resistance and impaired glucose disposal in skeletal muscle. In this study, insulin treatment during early phases of DKA elevated circulating insulin levels to ∼600 pmol/l, which is high in the normal range of postprandial levels. The slightly higher insulin level observed during steady state (KET) reflects differences in insulin breakdown and could be a consequence of LPS-induced inflammation of the liver . However, despite significant insulin stimulation we failed to detect an increased glucose disposal over forearm muscle. Therefore, it is probably necessary to impose supraphysiological insulin levels to ensure increased glucose disposal in skeletal muscle during DKA.
Stress hormones, such as cortisol, adrenalin and glucagon, are known to accompany inflammation and contribute to insulin resistance [23, 24, 25]; however, in our model of DKA only increases in glucagon levels persisted throughout the HI period. Glucagon receptors have not been identified on skeletal muscle and the elevated glucagon levels are unlikely to directly induce insulin resistance in skeletal muscle. However, glucagon may increase endogenous glucose production in the liver and thereby contribute to persisting hyperglycaemia . The elevated glucose levels will induce insulin resistance in skeletal muscle and glucagon could therefore contribute to the impaired insulin action in skeletal muscle indirectly. In septic individuals, in general, the aim is to reduce hyperglycaemia in order to prevent oxidative stress and further inflammation . Studies have shown that hyperglycaemia compromises the innate immune system and is additive to the sepsis, increasing both mortality and morbidity rates (both higher in this group) . Our data shows that insulin resistance during early phases of DKA is not solely explained by increases in stress hormone level, but may include other effects on liver and skeletal muscle.
Despite profound inhibition of insulin-stimulated glucose uptake in skeletal muscle, insulin stimulation increased signalling through Akt and AS160. In skeletal muscle, glucose is taken up via GLUT4 and oxidised or stored as glycogen . It is not known if the enhanced insulin-stimulated AS160 Thr642 phosphorylation observed during the HI period under KET would translate into greater GLUT4 translocation. Insulin resistance in type 2 diabetes mellitus is associated with impaired insulin-stimulated GLUT4 translocation in skeletal muscle . Alternatively, glucose uptake could be impaired due to diminished GLUT4 transporter activity or reduced glucose metabolism leading to a lower gradient for passive transport through GLUT4 [30, 31]. However, circulating glucose levels were elevated throughout KET, and this would increase the gradient for glucose transport across the sarcolemma. It is therefore likely that DKA is associated with impaired GLUT4 translocation.
KET was associated with compromised glycogen synthesis evident by increased phosphorylation of the inhibiting site 3a . In addition to regulation by phosphorylation, GS activity is dependent on substrate availability, and the ablated glucose uptake in skeletal muscle is likely to contribute further to impairment of glycogen synthesis [33, 34]. In spite of ablated glucose uptake in skeletal muscle, insulin stimulation was associated with increased glucose oxidation. This was determined on a whole body level and glucose oxidation in tissues other than skeletal muscle will have contributed. Prolonged hyperglycaemia and associated increases in oxidative stress can cause mitochondrial dysfunction . We did not find evidence to support during the early phases of DKA a reduction of mitochondrial content in skeletal muscle, although reductions in mitochondrial function cannot be excluded.
Lipid metabolism is profoundly affected by KET and elevated NEFA levels are a potent inducer of insulin resistance in skeletal muscle . This is independent of alterations in insulin signalling for glucose transport and therefore in agreement with the observed insulin signalling during KET [7, 37]. The precipitating steps of DKA include increased ketogenesis ; in rats, elevated ketone bodies impair glucose uptake in skeletal muscle. Therefore, this could have contributed to the insulin resistance during these early phases of DKA . Despite slightly higher insulin levels during KET, suppression of lipolysis was impaired. This demonstrates insulin resistance in adipose tissue during KET. In contrast, the suppression of ketogenesis by insulin was intact indicating that hepatic insulin action was normal.
We did not detect any effect of KET on total protein balance. This was evident both on a whole body level and on a muscular level, and this is in accordance with previous studies [38, 39]. An increased protein turnover on a whole body level was expected as inflammation was induced . This finding was supported by no differences in protein oxidation between the two conditions and equal insulin-stimulated changes in intracellular signalling pathways for protein synthesis as reflected by mTOR, p70s6K and 4eBP1. Skeletal muscle break down is shown to proceed for days following acute inflammatory conditions after surgery if no supplement is given and acidosis promotes negative protein balance primarily by increased break down [41, 42]. We allowed KET and CTR conditions to continue for five consecutive hours after LPS/saline administration. During KET, acidosis was buffered by bicarbonate, and pH did not change to acidotic levels. It is likely that prolonged DKA lasting for days will augment protein balance but this cannot be determined using our model of the early phases of DKA in humans. In addition, LPS mimics a Gram-negative infection and, therefore, our conclusion is limited to this scenario.
In conclusion, early stages of DKA in humans are characterised by increased levels of glucagon and elevated endogenous glucose production. Insulin stimulation during the early phases of DKA is associated with impaired stimulation of glucose uptake in skeletal muscle. The skeletal muscle insulin resistance is associated with increased insulin-stimulated signalling through Akt and AS160 but impaired insulin signalling for glycogen synthesis. Together these findings demonstrate that profound insulin resistance in skeletal muscle complicates insulin treatment of DKA.
The authors would like to thank A. Mengel, K. Mathiassen, H. Pedersen and H. Zibrandtsen from the Department of Clinical Medicine, Aarhus University Hospital, Denmark, for their excellent technical assistance and consistent work throughout the study. Likewise, the authors would like to thank the nine participants for participation and completion of the study days.
Data and original blots are available upon request, but permission must, in selected cases, first be obtained from the Danish Data Protection Agency.
This work was funded by the Danish Council for Strategic Research (grant no. 0603-00479B).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
MVS contributed to the research design and conduct, data analysis, and writing and final approval of the manuscript. TSV, NR and UK contributed to the research design and conduct, revised the manuscript critically and gave final approval of the manuscript. NM and NJ contributed to the research design, data analysis, and writing and final approval of the manuscript. NJ is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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