Euglycemic Clamp Research Articles

868-P: Empagliflozin (EMPA) Improves Vascular Insulin Sensitivity and Enhances Skeletal and Cardiac Muscle Microvascular Perfusion in Persons with Type 2 Diabetes

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) decreased cardiac events in large clinical trials; however, an understanding of the physiology driving this benefit is lacking. To investigate this, we measured micro- and macrovascular insulin sensitivity in 10 adults with T2DM before and after 12 weeks of EMPA 10 mg daily. We also measured endothelial function (FMD), peripheral and central aortic pressures, and skeletal (SM) and cardiac (CM) muscle microvascular perfusion (MBV- microvascular blood volume; MFV-microvascular flow velocity; MBF-microvascular blood flow) before and during a 120 min euglycemic insulin clamp at weeks 0 and 12. We hypothesized that EMPA would diminish micro- and macro-vascular insulin resistance and improve vascular function. At week 0, insulin infusion lowered peripheral SBP (132±6 vs 125±5, p<0.05) but diminished SM perfusion MBV (1.75±0.2 vs 1.29±0.1, p<0.01) MFV (0.08±0.01 vs 0.05±0.01, p<0.001) and MBF (0.14±3 vs 0.07±0.01, p<0.01). After 12 weeks of EMPA, insulin infusion improved FMD (7.6 ± 1.2 vs 8.7 ±1.6%, p <0.03), lowered peripheral SBP (127±6 vs 107±5, p<0.001); DBP (74±4 vs 65±3, p<0.04); MAP (92±3 vs 79±3, p<0.01); and PP (53±5 vs 42±3, p<0.05) as well as aortic SBP (117±5 vs 96±4, p<0.001); DBP (75±4 vs 66±3, p<0.03); MAP (89±3 vs 76±3, p<0.01); and PP (43±4 vs 30±2, p<0.01). In addition, EMPA improved SM perfusion (MBV: 1.29±0.1 vs 2.02±0.3, p<0.02; MFV: 0.05±0.01 vs 0.08±0.01, p<0.02; MBF: 0.07±0.01 vs 0.18±0.04, p<0.03) and CM perfusion (MBV: 4.51±0.6 vs 5.11±0.4, p=0.18; MFV: 0.37±0.04 vs 0.52±0.06, p<0.05; MBF: 1.80±0.4 vs 2.74±0.5, p<0.05). In conclusion, 12 weeks of EMPA in adults with T2D improved endothelial function (FMD), reduced central and peripheral blood pressures, and augmented SM and CM microvascular perfusion in response to insulin. These changes may contribute to the improved CV outcomes seen with SGLT2i therapy. Disclosure L.Jahn: None. L.Hartline: None. K.W.Aylor: None. W.B.Horton: None. E.Barrett: None. Funding National Institutes of Health (1R01HL142250-01A1)

143-OR: Prevailing Glucose Levels Alter Left Ventricular Contractions Pattern, Myocardial Performance, and Myocardial Energy Efficiency

Mechanical dispersion (MD) describes heterogeneity in ventricular contraction patterns, and myocardial work (MW) indices quantify ventricular performance. Investigating the myocardial and functional response to acute hypoglycemia and hyperglycemia may aid in identifying putative mechanisms linking glycemia and cardiovascular disease (CVD). From echocardiography performed during euglycemic, hyperglycemic and hypoglycemic clamps, we explored the relationship between glycemia and MD and MW indices in individuals with type 1 diabetes, type 2 diabetes and without diabetes. MD was measured by speckle-tracking echocardiography, and MW measures were derived from pressure-strain loop analyses. We analyzed data (mean±SD) from 84 individuals: 20 young individuals with type 1 diabetes (age: 30±8 years); 24 middle-aged individuals with type 1 diabetes (age: 53±12 years); 21 older individuals with type 2 diabetes (63±7 years), and 21 controls (62±8 years). Results for MD and MW indices are illustrated in Figure 1. In conclusion, acute hyperglycemia leads to an energy-effective myocardium with homogeneous left ventricular contractions. Hypoglycemia increases constructive myocardial work, followed by a less energy-efficient myocardium and more heterogeneous left ventricular contractions, perhaps alluding to a possible link between hypoglycemia and CVD. Disclosure C.R.Andreasen: None. A.Andersen: None. P.G.Hagelqvist: None. K.Maytham: None. M.Sengeløv: None. F.K.Knop: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Consultant; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Research Support; Novo Nordisk, Zealand Pharma A/S, Speaker's Bureau; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Lundbeck. F.J.Olsen: None. T.Vilsbøll: Consultant; AstraZeneca, Boehringer Ingelheim Inc., Gilead Sciences, Inc., Eli Lilly and Company, Mundipharma, Merck & Co., Inc., Novo Nordisk A/S, Sanofi, Sun Pharmaceutical Industries Ltd., Bristol-Myers Squibb Company. Funding Danish Diabetes Academy (17SA0031406); Novo Nordisk Foundation (16230)

853-P: Effect of Ketogenic Diet with Weight Maintenance in Type 2 Diabetes (T2D)

Aim: To examine the effect of ketogenic diet in T2D patients on glucose tolerance, beta cell function, insulin sensitivity and body fat content. Methods: 29 T2D subjects were randomized to receive for 10 days a weight maintaining diet containing: GROUP I - 30% protein, 50% CHO, 20% fat (n=8); GROUP II - isocaloric ketogenic diet with 15% protein, 5% CHO, 80% fat (n=10); GROUP III - isocaloric ketogenic diet plus ketone ester of β-OH-B, 8 grams every 8h (n=11). Subjects ate breakfast daily in the TDI Metabolic Kitchen and picked up food for lunch and dinner. Results: After 10 days, body weight remained constant: Group I (89.0 vs 89.0 kg), II (93.0 vs 92.5) and III (96.0 vs 97.0), as did body fat content. HbA1c and fructosamine did not change in any of the 3 groups. During OGTT, FPG, 2-h PG, mean PG, fasting PI, mean PI, [Delta]I/[Delta]G, and Matsuda index of insulin sensitivity did not change in Groups I, II, III. Subjects received a 2-step euglycemic insulin clamp (20 and 60 mU/m2.min) with 3-3H-glucose and indirect calorimetry. Before and after 10 days basal HGP and suppression of HGP (step I) were similar in all 3 groups. Insulin-stimulated glucose disposal (step 2) did not change in group I (4.23 vs 4.38 mg/kg.min), II (3.62 vs 3.55), or III (3.26 vs 3.35). After 10 days, basal lipid oxidation increased, while CHO oxidation decreased (both P<0.01) in groups II and III and was unchanged in group I. Disclosure A.Merovci: None. B.Finley: None. A.Chavez: None. A.A.Hansis-diarte: None. S.Neppala: None. D.Tripathy: None. R.A.Defronzo: Advisory Panel; AstraZeneca, Bayer Inc., Boehringer-Ingelheim, Novo Nordisk, Research Support; AstraZeneca, Boehringer-Ingelheim, Merck & Co., Inc., Speaker's Bureau; AstraZeneca.

345-OR: Exercise and Liraglutide Synergistically Attenuate Vascular Inflammation and Enhance Metabolic Insulin Action in Diet-Induced Obesity

Endothelial oxidative stress and vascular inflammation induce microvascular insulin resistance which is an early event in diet-induced obesity and contributes to metabolic insulin resistance. To examine whether exercise and glucagon-like peptide 1 (GLP-1) receptor agonism, alone or in combination, modulate vascular and metabolic insulin actions during obesity development, we performed an euglycemic insulin clamp, in conjunction with contrast enhanced ultrasound, in adult male rats after 2 weeks of high-fat diet feeding with either access to running wheel (exercise), liraglutide (200 mg/kg subcutaneously twice daily), or the combination of both. Rats exhibited increased visceral adiposity and blunted microvascular responses (microvascular blood volume, flow velocity, and total flow) and metabolic insulin actions (glucose infusion rates). Exercise and liraglutide alone each improved muscle insulin sensitivity but their combination fully restored insulin-mediated glucose infusion rates. The combined exercise and liraglutide intervention enhanced insulin-mediated muscle microvascular perfusion, reduced perivascular macrophage accumulation and oxidative stress in the muscle, attenuated blood vessel inflammation and improved endothelial function, along with increased nucleus translocation of nuclear factor erythroid 2-related factor 2 (NRF2) and phosphorylation of AMP-activated protein kinase (AMPK) and nitric oxide synthase (eNOS) in endothelial cells. We conclude that exercise and liraglutide synergistically enhance the metabolic actions of insulin and reduce vascular oxidative stress and inflammation in the early stage of obesity development. Our data suggest that early combination use of exercise and GLP-1 receptor agonism might be an effective strategy in preventing microvascular and metabolic insulin resistance and associated complications during the development of obesity. Disclosure J.Liu: None. K.W.Aylor: None. Z.Liu: None. Funding National Institutes of Health (R01DK125330), (R01DK1124344 to Z.L.)

1517-P: Interactive Calculator to Estimate Insulin Sensitivity (IS) in Type 1 Diabetes

Introduction: Euglycemic hyperinsulinemic clamps, although costly, time- and labor-intensive, are “gold standard” in measuring insulin sensitivity (IS). Several formulae to estimate IS in people with Type 1 diabetes (T1D) exist (DOI: 10.1007/978-3-030-81303-1_18-1), using various routine and research assays, and with wide ranging IS results. Aim: To develop a free online tool to estimate IS using different metrics and existent formulae and compare estimated IS with measured glucose disposal rate (GDR) from 104 clamp studies. Methods: We prepared an online tool for calculating IS using 17 formulae. Suitable formula(e) are suggested based on available (clinical and research) data. We also compare calculated IS with measured IS (GDR) from clamp studies in 104 adults with T1D (mean±SD) age 34±7 yrs, T1D duration 10±4 yrs, HbA1c 7.7±1.5%, 33 with microvascular complications). Logistic regression was used to infer probability of calculated IS being below GDR=4.45 mg/kg/min or above GDR=6.5 mg/kg/min, which represent respectively the median and 75th percentile of measured GDR values. Results: A calculator is available at www.bit.ly/estimated-GDR and an example result in the Figure 1. Estimated IS varied widely, but results interpretation is generally consistent. Conclusion: We developed an interactive tool to estimate IS in T1D for clinical and research use. Disclosure A.S.Januszewski: None. P.Niedzwiecki: None. N.Sachithanandan: None. G.M.Ward: None. D.N.O'neal: None. D.Zozulinska-ziolkiewicz: Advisory Panel; Abbott, Lilly, Lilly, Novo Nordisk, Medtronic, Speaker's Bureau; Abbott, Ascensia Diabetes Care, Novo Nordisk, Medtronic, Boehringer-Ingelheim, AstraZeneca. A.Uruska: Other Relationship; Lilly, Boehringer-Ingelheim, AstraZeneca, Ascensia Diabetes Care, Sanofi. A.Jenkins: Advisory Panel; Insulet Corporation, Board Member; Insulin for Life, Research Support; Abbott Diabetes, Medtronic, Hemsley Charitable Trust, Juvenile Diabetes Research Foundation (JDRF), National Institutes of Health.

147-OR: Revisiting the Impact of Opioid Receptor Blockade on Hypoglycemia-Associated Autonomic Failure (HAAF)

HAAF, which has been defined as >20% reduction in average epinephrine (Epi) levels following recurrent hypoglycemia (PMID: 28911152), complicates insulin therapy in T1D. We previously showed that activation of opioid receptors induces HAAF, while intravenous (IV) administration of the opioid receptor antagonist naloxone prevents HAAF. Because of its wide clinical use and possible direct CNS effects, we examined the effects of intranasal naloxone (IN) on hypoglycemia and HAAF. Seventeen (15M, age 43.8±8.7) healthy adults were enrolled in a randomized, placebo-controlled, cross-over, double-blinded study in which they underwent three hyperinsulinemic hypoglycemic clamp studies (duration 2h, glucose nadir 54 mg/dL), with administration of IN (4mg) or placebo during the first two clamps. Two subjects were excluded for lack of Epi response to hypoglycemia. Nine of the remaining 15 subjects developed HAAF, confirming inter-individual variability in susceptibility to HAAF (PMID: 32915987, 35475026). Among the 15 subjects included in the analysis, Epi levels trended downward between the 1st and 3rd clamps in placebo studies (mean ± SEM: 677 ± 114 vs. 512 ± 78 pg/mL, p=0.054), whereas Epi levels remained stable in IN studies (1st vs. 3rd: 519 ± 70 vs. 487 ± 77 pg/mL, p= 0.69). Among the 9 subjects who developed HAAF, Epi levels during the 1st clamp were significantly lower with IN vs. placebo (493 ± 102 vs. 858 ± 161 pg/mL, p=0.03), but there was no further reduction in Epi with IN (1st vs. 3rd: 493±102 vs. 539±101 pg/ml, p=0.67). In summary, these studies suggest differential effects of intranasal naloxone on initial vs. repeated hypoglycemic episodes. Specifically, IN may be effective in preventing Epi blunting during repeated exposure to hypoglycemia. In subjects susceptible to HAAF, IN may paradoxically blunt the Epi response to initial hypoglycemic exposure. Future studies should investigate the clinical significance of initial vs. ongoing epinephrine responses to hypoglycemia. Disclosure C.E.Boyle: None. M.Hawkins: None. E.Lontchi yimagou: Employee; Sanofi. S.Aleksic: None. P.M.Mathias: None. M.Nogueira cordeiro: None. A.Manavalan: None. M.Carey: None. N.Jiang: None. O.Sandu: None. I.Gabriely: None. K.Zhang: None. Funding National Institutes of Health (R01DK079974)

265-LB: Human Skeletal Muscle Mitochondrial Dynamics across the Insulin-Sensitivity Spectrum

Introduction: There is immense dispute as to whether perturbations in skeletal muscle mitochondrial function contribute to the onset and progression of type 2 diabetes (T2D). The purpose of this study was to examine differences in mitochondrial dynamics, structure, and energetic function in humans across the spectrum of insulin sensitivity. Methods: 58 sedentary adults (37±12 years) were enrolled into one of three groups based upon the following criteria: (1) healthy weight without T2D (HW; BMI: 22.6±1.8 kg/m2 and HbA1c: 5.0±1.1 %); (2) overweight/obesity without T2D (Ov/Ob; BMI: 32.6±3.3 kg/m2 and HbA1c: 5.6±0.4 %); or (3) overweight/obesity with T2D (T2D; BMI: 36.1±6.5 kg/m2 and HbA1c: 6.9±0.9 %). Participants underwent a 3-day inpatient stay consisting of body composition (DXA), aerobic capacity (VO2MAX), and insulin sensitivity (hyperinsulinemic euglycemic clamp). Prior to insulin sensitivity testing, a skeletal muscle biopsy was obtained for determination of mitochondrial dynamics (Western blot), DNA content (qPCR), ultrastructure (electron microscopy), and respiratory capacity (respirometry). Comparisons were made using a one-way ANOVA with contrasts. Results: Insulin sensitivity and aerobic capacity were lower in Ov/Ob and T2D compared to HW. Markers of mitochondrial fission were higher in T2D (Drp1Ser616, MiD49), and fusion lower in T2D and Ov/Ob (Mfn2). The mitophagy marker Parkin was higher in T2D only while Pink1 was lower in T2D relative to Ov/Ob but not HW. The autophagy marker LC3II was higher in Ov/Ob. Mitochondrial content was lower in Ov/Ob and T2D relative to HW. Mitochondrial respiratory capacity was similar between groups. Conclusions: T2D is associated with heightened expression of proteins required for mitochondrial quality control and reduced mitochondrial volume despite intact respiratory function. These data support the notion that mitochondria adapt to progressive insulin resistance by increasing fragmentation to maintain bioenergetic capacity. Disclosure C. L. Axelrod: None. C. Fealy: Employee; Mission Therapeutics. C. L. Hoppel: None. J. P. Kirwan: None. W. S. Dantas: None. E. R. M. Zunica: None. K. Belmont: None. E. C. Heintz: None. G. Davuluri: None. J. T. Mey: None. M. Erickson: None. H. Fujioka: None. Funding National Institutes of Health (DK108089, GM104940)

1648-P: Triagonism Targeting GCGR/GLP-1/GIP-1 Receptors Improves Insulin Sensitivity in Obese Mice

Glucagon is best known as the primary counter-regulatory hormone against insulin action. Glucagon secretion is likewise elevated in Diabetes and thus it is often implicated as a diabetogenic hormone. However, emerging data from our group and others have uncovered that Glucagon receptor (GCGR) agonism improves insulin sensitivity in both lean and obese mice. Mechanistically, this improvement is associated with enhanced AKT phosphorylation and mTOR signaling in the liver. Novel, single-molecule, triagonists targeting GCG/GLP-1/GIP receptors (GGG) have recently been described to exert stronger anti-obesity effects than dual or single agonism in obese mice. Thus, we evaluated the effect of these agonists on insulin sensitivity using hyperinsulinemic euglycemic clamp. Diet-induced obese (DIO) mice were randomly assigned to 4d treatment (10 nmole/kg subcutaneous injection) of vehicle, GGG, GCGR agonism (IUB288), GLP-1R agonism (Semaglutide), or a long-acting GIP-1R agonist (GIP-FA 085). On the fourth day, mice were fasted for 5 hours, continuously infused with 1.2mU/kg/min insulin, and blood glucose levels clamped around 80-100 mg/dL. Under these conditions Semaglutide and GGG treatments stimulated the greatest glucose infusion rate (p<0.0001) and significantly improved insulin-mediated suppression of Endo Ra (p<0.01). Smaller, but significant, enhancement was also observed in the IUB288-treated mice. Moreover, [14C]-2GDp uptake into brown adipose tissue was improved in GGG, Semaglutide, and IUB288 treated mice compared to Vehicle treated mice. Importantly, these improvements in insulin sensitivity and glucose dynamics were body-weight independent. Therefore, GCGR and GLP-1R signaling combine in GGG triagonism to improve insulin sensitivity mainly via suppression of hepatic glucose production. Disclosure T.Kim: None. S.Nason: None. J.Antipenko: None. K.M.Habegger: Consultant; Glyscend Inc., Research Support; Eli Lilly and Company, Novo Nordisk, Glyscend Inc., Stock/Shareholder; Glyscend Inc. Funding Eli Lilly and Company

1311-P: Insulin Resistance in Non-obese Young Adults with Type 1 Diabetes

Insulin resistance has been noted in youth and adults with type 1 diabetes (T1D) and is associated with higher levels of cardiovascular and kidney complications. Obesity is increasing in populations with T1D, and the degree of insulin resistance in younger adults with T1D who are free from complications and non-obese isn’t clear. We performed hyperinsulinemic euglycemic clamps in non-obese adults aged 20-45 with and without T1D. Inclusion criteria included diabetes duration > 5 years and HbA1c <9% among people with T1D, or no diabetes based on fasting glucose and HbA1c among controls; Body mass index (BMI) < 30 kg/m2, no use of statins or other medication affecting insulin sensitivity, non-smokers and normal thyroid function. Participants were given a fixed macronutrients study diet for 2 days and were asked to refrain from vigorous exercise for 72 hours. Clamps were performed by a single investigator after an overnight stay. A sliding scale insulin infusion was used overnight for participants with T1D. Arterial stiffness (brachial artery distensibility, pulse wave velocity and reactive hyperemia index) was assessed. There were 47 participants, 24 with T1D and 23 without diabetes. 53% were women (25/47), and the average age did not differ between controls and people with T1D (31 ± 6 years vs. 28 ± 6 years, p=0.12). Whole body insulin sensitivity estimated by glucose infusion rate (GIR) per kg fat free mass was reduced by more than half in adults with T1D (LS mean ± SE =5.4 ± 1.0 mg/kg FFM/min) compared to adults without diabetes (12.7 ± 1.0 mg/kg FFM/min, p<0.0001). In contrast, there were no differences in BMI, body fat, urinary albumin, or measures of arterial stiffness by diabetes status. Young, non-obese adults with T1D are profoundly insulin resistant compared to nondiabetic adults, even when no differences in vascular stiffness or kidney complications are apparent. These results suggest that insulin resistance may occur earlier than vascular or kidney complications in people with T1D and is independent of obesity. Disclosure C.J. Chartier-Logan: None. I.E. Schauer: None. M. Cree-Green: Consultant; Pollie Inc. Research Support; Amino Corp LLC. L. Pyle: None. B.C. Bergman: Research Support; Eli Lilly and Company. J.K. Snell-Bergeon: None. Funding JDRF (2-SRA-2019-849-S-B)

1660-P: Lactate Inhibits Lipolysis in Healthy Males—A Randomized Crossover Trial

Background: The intermediary metabolite lactate has signaling capacities through the lactate specific receptor GPR81/HCA1. Ex-vivo, lactate inhibits lipolysis through GPR81, but in-vivo human data are scarce. Lactate-mediated inhibition of lipolysis could improve insulin sensitivity. The aim of the study was to investigate if hyperlactatemia at physiological concentrations inhibits palmitate lipolysis and affects glucose metabolism in healthy males. Methods: In a randomized cross-over design, eight healthy men were studied after an overnight fast on two occasions 1) during a sodium-lactate infusion (LAC) and 2) during a NaCl infusion (CTL). Both study days comprised a 3-hour basal period followed by a 3-hour hyperinsulinemic euglycemic clamp (HEC) period. Lipolysis rate and endogenous glucose production (EGP) were evaluated using [9,10-3H]-palmitate and [3H]-glucose tracers. Average glucose infusion rate (mg/kg/min) during the last 30 min of the HEC (M-value) was used as a measure of insulin sensitivity. Data are mean±SD Results: Plasma lactate increased to 2.6 ±0.6 mmol/L during LAC vs. 0.8 ±0.3 mmol/L during CTL (p<0.001). At the end of the basal period, palmitate flux was 30% lower during LAC compared with CTL (84 ±32 μmol/min vs. 120 ±35 μmol/min, p=0.003). At the end of the HEC, palmitate flux was similarly suppressed during both interventions (p=0.7). Plasma FFA were lower during LAC compared with CTL (0.2 ±0.1 mmol/L vs. 0.3 ±0.1 mmol/L, p=0.03) at the end of the basal period and equally suppressed during both interventions at the end of the HEC (p=0.7). EGP was unaffected by LAC compared with CTL at the end of the basal period (p=1.0) and suppressed at the end of the HEC during both interventions (p=1.0). M-value was unaffected by LAC compared with CTL (p=0.8). Conclusion: Lactate inhibits palmitate lipolysis by 30 %, an effect which per se may improve insulin sensitivity. Hyperlactatemia did not affect EGP or insulin sensitivity, possibly because lactate intrinsically may induce insulin resistance. Disclosure M.G.B.Pedersen: None. N.Rittig: Research Support; ARLA. E.Søndergaard: None. L.C.Gormsen: None. N.Møller: Other Relationship; Aarhus University/Ketlace.

1630-P: Reduced Fatty Acid Oxidation Does Not Prevent Intralipid-Induced Peripheral Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate is thought to be an important determinant of insulin resistance. Evidence from animal models support insulin signaling defects resulting from either intra-cellular lipid accumulation or by mitochondrial dysfunction and altered fatty acid oxidation (FAO) flux. To explore the relationship between FAO and insulin sensitivity, we measured the change in peripheral glucose disposal (Rd) during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of a control glycerol solution versus a 20% intralipid solution in 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Very long-chain, medium-chain or long-chain 3-hydroxy AcylcoA Dehydrogenase Deficiencies) and 18 age, sex, and weight-matched normal healthy controls. Rd was similar between groups during the glycerol control clamp. Intramyocellular lipid (IMCL) content as measured by MRS increased after the 5 hour intralipid infusion in controls but not in participants with FAOD. Plasma free fatty acids during intralipid infusion were higher in FAOD compared to controls suggesting infused lipid remained in circulation. Muscle fiber type and expression of lipid transporters CD36 and FATP1 were similar in biopsy samples between groups and cannot explain decreased lipid uptake into muscle among FAOD subjects. However, intralipid suppressed Rd equally in subjects with FAOD and controls inducing peripheral insulin resistance in both groups. Impaired FAO decreased lipid accumulation in the muscle during intralipid infusion, but despite lower IMCL, subjects with FAOD developed peripheral insulin resistance similar to controls. Increased IMCL is not essential for, nor is reduced FAO flux protective against, the development of peripheral insulin resistance during intralipid infusion. Disclosure M.B.Gillingham: None. J.Q.Purnell: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk. A.N.Gregor: None. O.Varlamov: None. J.J.Robino: None. C.E.Mccurdy: None. K.T.Greyslak: None. D.Choi: None. E.Baetscher: Consultant; Biogen. W.Rooney: Consultant; Ultragenix, Research Support; Revalesio. Funding National Institutes of Health (DK102813)

191-OR: Deletion of the Type 2 Diabetes Candidate Gene SLC16A11 Reduces Peripheral Insulin Sensitivity in Mice

Background and Aims: Recent GWAS in large human populations identified the monocarboxylate transporter SLC16A11 as a candidate gene for T2D. While previous studies postulated that SLC16A11 plays a role in hepatic lipid and glucose metabolism, the existing data are controversial and exact mechanisms of how SLC16A11 affects T2D are missing. Here, we aimed at elucidating the role of SLC16A11 in the development of metabolic disease. Methods: HEK293 cell lines expressing murine and human SLC16A11 were established. Moreover, using CRISPR/Cas9, a SLC16A11 knockout mouse model was generated. Finally, SLC16A11 mRNA expression was measured in various insulin responsive tissues of ob/ob and db/db mice. Results: In HEK293 cells, substrates of the transporter were identified. mRNA expression of SLC16A11 was reduced in skeletal muscle of ob/ob and db/db mice (-69.8% and -22.8%; p<0.05) compared to littermate controls. SLC16A11 KO and WT littermate mice fed NCD or HFD did not differ in body weight and composition, energy expenditure or food intake. While basal glucose and insulin in SLC16A11 KO and WT littermates on a HFD were not different, glucose infusion rate during hyperinsulinemic euglycemic clamp was lower in SLC16A11 KO mice (SLC16A11 WT= 36.4±3 mg/kg/min; SLC16A11 KO= 24.7±2 mg/kg/min; P<0.01), indicating reduced whole-body insulin sensitivity. No changes were observed in hepatic glucose production, liver fat content or hepatic mitochondrial function using PINTA method. Instead, clamped 2-deoxy-glucose uptake was significantly reduced in skeletal muscle (-41%; p<0.05) of SLC16A11 KO, together with increased DAGs levels and PKCθ activation, indicating skeletal muscle insulin resistance. Conclusions: Our data show for the first time that in contrast to previous assumptions, SLC16A11 impairs skeletal muscle insulin sensitivity without affecting hepatic metabolism. Taking also human GWAS into account, we propose that SLC16A11 may be an interesting target for the treatment of T2D. Disclosure N.N.El-agroudy: None. T.Schumann: None. A.Kurzbach: None. G.Sancar: None. L.Sandforth: None. C.Herrmann: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. A.L.Birkenfeld: None.

1579-P: Impact of Adipose Tissue Insulin Sensitivity on Hepatic Insulin Sensitivity Preservation in Longitudinal Observation of Patients with NAFLD

Hepatic insulin resistance associated with NAFLD is a major pathogenesis of type 2 diabetes and its complications. In addition to reducing lipids in the liver, identifying the factors that may preserve hepatic insulin sensitivity (H-IS) is crucial. In this study, we investigated the factors that may preserve H-IS in patients with NAFLD in longitudinal observations. Comprehensively, putative biomarkers in blood and urine, body composition, tissue-specific lipid accumulation and insulin sensitivity by 1H-MRS and hyperinsulinemic euglycemic clamp, respectively, and pathological diagnosis using liver biopsy were assessed in patients with NAFLD (n=62) at baseline. After more than one year of general treatment, consented 36 patients (average age 51.3±11.2, men n=21, diabetes n=23) were subjected to the same assessment as baseline. To investigate the indices associated with H-IS, all indices were compared between upper and lower one third patients of Δ% suppression of endogenous glucose production (EGP) (+11.5±4.0 vs. -10.9±5.1%, respectively). As results, body weight, fasting plasma glucose, HbA1c, muscle and adipose tissue insulin sensitivity, visceral fat, and adiponectin levels were significantly improved in improved H-IS group. Interestingly, Δhepatic lipid content or fibrosis index was not different between the improved and worsened H-IS groups. Furthermore, simple regression analysis with Δ% suppression of EGP and all indices revealed that Δ% suppression of free fatty acid, visceral fat area, and adiponectin levels were significantly associated with Δ% suppression of EGP. Multiple regression analysis with Δ% suppression of EGP and these three indices showed that only Δ% suppression of free fatty acid significantly associated with Δ% suppression of EGP. Taken together, not only lipid volume but also the quality of accumulated lipid is important to preserve hepatic insulin sensitivity in patients with NAFLD. Disclosure F.Shigiyama: None. S.Hiruma: None. N.Kumashiro: Research Support; Terumo Corporation, LifeScan Diabetes Institute, Sumitomo Dainippon Pharma Co., Ltd., Speaker's Bureau; Daiichi Sankyo, Novo Nordisk.

240-LB: Unrestrained Sympathetic Signaling Is a Key Driver of Obesity-Induced Insulin Resistance

Obesity is the major driver of type 2 diabetes. The mechanisms that account for overnutrition induced insulin resistance are yet to be delineated as impaired cellular insulin signaling may or may not account for the impaired insulin action. We find that overnutrition rapidly impairs insulin action and increase plasma norepinephrine, suggesting heightened activation of the sympathetic nervous system (SNS), possibly due to brain insulin resistance as earlier studies from our group suggested. Here, we hypothesize that unrestrained SNS activity (SNA) is a key driver of obesity induced insulin resistance. To test this hypothesis, we examined a mouse model of inducible and peripherally restricted tyrosine hydroxylase (th) gene deletion (THΔper) that have profoundly lower catecholamine levels in peripheral tissues is protected from high fat diet (HFD) induced insulin resistance. Despite similar food intake, obesity and cellular insulin signaling in liver and adipose tissue, THΔper mice were protected from both short-term and long-term HFD feeding-induced glucose intolerance, increases in plasma catecholamines and hyperglucagonemia. Hyperinsulinemic euglycemic clamp studies revealed that overnutrition induced hepatic insulin resistance was markedly ameliorated in the THΔper mice, even though cellular insulin signaling in liver and adipose tissue was comparable. Moreover, THΔper mice were protected from HFD-induced adipose tissue dysfunction characterized by reduced lipogenic capacity, unrestrained lipolysis and increased low-grade inflammation as well as hepatic steatosis, possibly due to decreased lipolytic flux and hepatic de novo lipogenesis. In addition, fibrosis genes were markedly reduced in the liver of the THΔper mice, suggesting that reducing SNA can prevent metabolic dysfunction-associated fatty liver disease. These results demonstrate that unrestrained SNA is a pivotal driver of insulin resistance, hyperglucagonemia and metabolic dysfunction in obesity. Disclosure K. Sakamoto: None. G. Maurizi: None. B. Chen: None. A. Shawkat: None. L. Patlolla: None. A. Tahiri: None. A. Butera: None. C. Buettner: None.

1098-P: Association of Sleep with ß-Cell Response and Insulin Sensitivity in Youth

Poor sleep may be associated with reduced β-cell response or decreased insulin sensitivity in youth. We explored whether sleep duration, sleep quality or obstructive sleep apnea (OSA) risk factors were associated with measures of β-cell response or insulin sensitivity in youth. At baseline, 88 youth (10-19 yrs of age) with recently diagnosed T2D or prediabetes completed validated questionnaires (Sleep Disturbances Scale and Cleveland Adolescent Sleepiness) in the Restoring Insulin Secretion (RISE) Study. Hyperglycemic clamps measured insulin sensitivity (steady state glucose infusion rate/insulin [M/I]) and β-cell responses: acute (0-10 min) C-peptide response to glucose (ACPRg), steady-state C-peptide at a glucose of 11.1 mmol/L (SSCP), and arginine-stimulated maximum C-peptide responses at glucose >25 mmol/L (ACPRmax). Linear regression models explored the independent association between sleep variables and clamp measures, adjusted for age, race/ethnicity, sex, Tanner stage, metformin use and BMI. Models including β-cell responses were adjusted for M/I to account for the role of insulin sensitivity in β-cell function. The cohort was 70% female, 28% white, 25% black, 36% Hispanic; age 14.3±2.0 yrs and BMI 36.9±6.4 kg/m2 (mean±SD). Using ADA criteria, 60% had prediabetes and 40% had T2D; 27% reported metformin use. Sleep duration <8 h was reported in 59%; 57% reported daytime sleepiness; 28% reported poor sleep quality; 29% had high risk for OSA. Low sleep duration (<8 h) was associated with a trend for lower ACPRg (p=0.069). No sleep variables (sleep duration or quality, OSA risk) were associated with clamp-derived outcomes in unadjusted or adjusted linear regression models. In youth with prediabetes or T2D, subjective measures of sleep quantity, sleep quality and OSA risk were not independently associated with β-cell response or insulin sensitivity. Further research using objective measures of sleep may better delineate the relationship between sleep and β-cell function in youth. Disclosure K.A.Temple: None. A.H.Tjaden: None. S.Manchanda: Advisory Panel; Inspire. D.Ehrmann: None. K.J.Nadeau: None. S.Edelstein: None. T.S.Hannon: Advisory Panel; Eli Lilly and Company. B.Mokhlesi: None. Rise consortium: n/a. Funding American Diabetes Association (1-20-RISE-01 to S.E.); National Institute of Diabetes and Digestive and Kidney Diseases; National Heart, Lung, and Blood Institute

22-OR: High Glucose Metabolism Fuels Cathepsin L Maturation to Promote SARS-CoV-2 Infection in Patients with Diabetes

Patients with diabetes are more likely to develop severe COVID-19 and deaths. However, the underlying mechanism remains poorly understood. Previously, we identified a widely expressed lysosome protease Cathepsin L (CTSL) promotes SARS-CoV-2 infection and may be a promising drug target (PMID: 33774649, 35668062). Here, we show that patients with diabetes have elevated CTSL concentration and activity in patients with or without COVID-19. The CTSL levels are positively correlated with HbA1c in humans. Hyperglycemic clamp study suggests that high glucose increases CTSL activity in healthy individuals. In vitro, we find that high glucose, but not high insulin, level promotes SARS-CoV-2 infection in wildtype (WT) cells. However, these promoting effects are largely reduced in CTSL-knockout (KO) cells. By using lung tissues from healthy, diabetic patients, WT and db/db mice, we find diabetes promotes CTSL maturation. Further, we identify that high glucose level, but not high insulin level, promotes CTSL activity. The matured CTSL levels are significantly increased in diabetic tissues. High glucose promotes CTSL translocation from the endoplasmic reticulum (ER) to lysosome through the ER-Golgi-lysosome axis (Fig.1). Our study reports the biological and potential clinical significance of glucose metabolism in CTSL maturation to promotes SARS-CoV-2 infection in patients with diabetes. Disclosure M.Zhao: None. Q.He: None. J.Yang: None. Funding National Natural Science Foundation of China (81930019)

36-OR: The Effect of Glucagon-Like Peptide-1 Receptor Blockade on the ß-Cell Response to Glucagon Bolus in People With and Without Type 2 Diabetes

Glucagon acts as an insulin secretagogue in part via the glucagon-like peptide-1 receptor (GLP1R) expressed on β-cells. Type 2 diabetes (DM2) is characterized by increased (and inappropriate) glucagon secretion for the prevailing hyperglycemia. Increased islet glucagon staining has also been observed in DM2. This suggests that increased glucagon signaling via GLP1R is a mechanism to maintain β-cell secretion in DM2. To ascertain if this is the case, we quantified the relative contribution of GLP1R signaling to the insulin response to glucagon in the presence and absence of DM2. We studied 12 individuals without DM2 (54 ± 2 yrs, 33 ± 1 kg/m2) and 11 with DM2 (58 ± 2 yrs, 34 ± 2 kg/m2) on 2 occasions in random order. On one occasion Exendin-9,39 (300pmol/kg/min) was infused to block GLP1R, while on the other study day saline was infused. Following an overnight fast and a 3-hour hyperglycemic clamp (~9 mmol/L), 1 mg glucagon bolus was administered intravenously with subsequent measurement of insulin, and C-peptide over 30 minutes during hyperglycemia. Exendin-9,39 infusion decreased peak and integrated insulin and C-peptide concentrations in response to the glucagon bolus in both those with and without DM2. The symmetric % change in insulin (-49 ± 8 vs -32 ± 8, DM2 vs. subjects without diabetes respectively, p = 0.2) and C-peptide (-24 ± 5 vs -15 ± 4, p = 0.18) did not differ between groups. In addition, six of the subjects without DM2 completed two additional study days (± exendin-9,39) with infusion of Intralipid® and heparin to raise free fatty acids (FFA) and cause acute insulin resistance. Acute FFA elevation also did not alter the net contribution of GLP1R stimulation to the response to glucagon in individuals without DM2 (% change insulin: -34 ± 10, p = 0.42; C-peptide: -23 ± 7, p = 0.38). These data suggest that the % contribution of glucagon to the stimulation of β-cell secretion via GLP1R does not change significantly in the presence or absence of DM2 or of FFA elevation. Disclosure A.M.Egan: None. M.Zeini: None. A.A.Welch: None. R.A.Farahani: None. M.C.Laurenti: None. C.Cobelli: None. C.Dalla man: Research Support; Sanofi-Aventis Deutschland GmbH, Becton, Dickinson and Company. A.Vella: Advisory Panel; Rezolute, Inc., Consultant; Crinetics Pharmaceuticals, Inc., Hanmi Pharm. Co., Ltd., Zealand Pharma A/S, Other Relationship; Novo Nordisk. Funding National Institutes of Health (DK126206)

1554-P: Carnosic Acid Protects against Short-Term Lipid-Infusion–Induced Glucose Intolerance in Mice

Background: Obesity and type 2 diabetes (T2D) are associated with elevated plasma free fatty acids (FFA). In vivo exposure to FFAs, such as palmitate, by lipid infusion results in insulin resistance as observed in obesity and T2D. In recent years, compounds found in herbs and plants have attracted attention for prevention and treatment of insulin resistance and T2D. In recent studies by our group, carnosic acid (CA), found in rosemary, increased glucose uptake and attenuated the FFA (palmitate)-induced insulin resistance in muscle cells and adipocytes. In addition, CA caused a robust activation of AMP-activated protein kinase (AMPK). Our present study aimed to examine the effects of CA in short-term FFA-induced glucose intolerance in mice. Material and Methods: C57Bl/6J mice were cannulated in their jugular veins and infused for 48h with ethylpalmitate (EtP, nontoxic method of elevating circulating palmitate because of hydrolysis to palmitate and ethanol by plasma esterases), mimicking elevated FFA levels seen in obesity. In addition, a group of mice were infused with EtP plus 50 mg/kg CA for 48h. Ethanol vehicle with or without CA were two additional control groups. At the end of the infusions, the mice were subjected to a 2 hr hyperglycemic clamp (20 mM glucose) and the glucose infusion rate was measured to assess glucose tolerance. Results: Preliminary data revealed that mice infused with EtP and CA had a higher glucose infusion rate (GIR; mg/kg*min) (66.5±10, mean±SEM) than mice infused with EtP alone (22.7±10). The EtP and CA was comparable to the GIR of ethanol vehicle infused mice (73±9). Future Directions: Further analysis of collected plasma and tissue will be used to assess insulin resistance and beta cell function in vivo and to elucidate the mechanisms of CA alleviation of FFA-induced insulin resistance. Conclusions: Our data indicate that CA has the potential to counteract FFA-induced glucose intolerance in mice. Disclosure N.Tsakiridis: None. F.Vlavcheski: None. E.Tsiani: None. A.Giacca: None.

Crosstalk between genetic variability of adiponectin and leptin, glucose-insulin system and subclinical atherosclerosis in patients with newly diagnosed type 2 diabetes. The Verona Newly Diagnosed Type 2 Diabetes Study 14.

To evaluate the relationship of genetic variability of adiponectin (ADIPOQ), leptin (LEP) and leptin receptor (LEPR) genes with glucose-insulin system and markers of subclinical atherosclerosis (ATS) in patients with newly diagnosed type 2 diabetes. In 794 subjects we performed: 1) euglycemic hyperinsulinemic clamp to assess insulin sensitivity; 2) mathematical modelling of a 5h-OGTT to estimate β-cell function; 3) resting ECG; 4) carotid artery and lower limb artery eco-doppler sonography to identify ATS; 5) genotyping of tag-SNPs within ADIPOQ, LEP and LEPR gene. Regression analyses showed: 1) adiponectin levels were negatively associated with BMI, waist-to-hip ratio and triglycerides and positively with HDL and insulin sensitivity (p-all<0.03); 2) leptin levels were positively associated with BMI, HDL-cholesterol and plasma triglycerides and negatively with insulin sensitivity (p-all<0.001). Two SNPs (rs1501299 and rs2241767) within ADIPOQ gene were associated with circulating levels of adiponectin. The ADIPOQ-GAACA haplotype was associated with plasma adiponectin (p=0.034; β=-0.24), ECG abnormalities (p=0.012; OR=2.76), carotid ATS (p=0.025; OR=2.00) and peripheral limb artery ATS (p=0.032; OR=1.90). The LEP-CTA haplotype showed an association with ischemic ECG abnormalities (p=0.017; OR=2.24). Finally, LEPR-GAACGG was associated with circulating leptin (p=0.005; β=-0.31) and worst β-cell function (p=0.023; β=-15.10). Omnibus haplotype analysis showed that ADIPOQ haplotypes were associated with levels of adiponectin and common carotid artery ATS, LEP with peripheral limb artery ATS, whereas LEPR haplotypes influenced circulating levels of leptin. Results of this study reinforce knowledge on adipokines' role in regulating glucose metabolism; in particular highlighted the potential atherogenic role of leptin and the anti atherogenic role of adiponectin.

A Short-Term High-Fat Diet Worsens Insulin Sensitivity with Changes in Metabolic Parameters in Non-Obese Japanese Men.

A short-term high-calorie high-fat diet (HCHFD) impairs insulin sensitivity in non-obese South Asian but not Caucasian men; however, the effect of short-term HCHFD on insulin sensitivity in East Asians is unknown. We recruited 21 healthy non-obese Japanese men to evaluate metabolic parameters and gut microbiota before and after 6-day HCHFD consisting of a regular diet plus a 45% energy excess with dairy fat supplementation. We evaluated tissue-specific insulin sensitivity and metabolic clearance rate of insulin (MCRI) using a two-step hyperinsulinemic euglycemic clamp, glucose tolerance using the glucose tolerance test, and measured ectopic fat in muscle and the liver using ¹H-magnetic resonance spectroscopy. The primary outcome of this study was insulin sensitivity measured by the clamp study. The secondary/exploratory outcomes were other metabolic changes. After HCHFD, levels of circulating lipopolysaccharide binding protein (LBP), a marker of endotoxemia, increased by 14%. In addition, intramyocellular lipid levels in the tibialis anterior and soleus and intrahepatic lipid levels increased by 47%, 31%, and 200%, respectively. Insulin sensitivity decreased by 4% in muscle and 8% in liver. However, even with reduced insulin sensitivity, glucose metabolism was maintained by increased serum insulin concentrations due to lower MCRI and higher endogenous insulin secretion during the clamp. Glucose levels during the meal tolerance test were comparable before and after HCHFD. In conclusion, short-term HCHFD impaired insulin sensitivity in the muscle and livers of non-obese Japanese men with increased LBP and ectopic fat accumulation. Elevated insulin levels from modulated insulin secretion and clearance might contribute to the maintenance of normal glucose metabolism during the clamp and meal tolerance test.