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Glucokinase (GCK) mediates hepatic glucose uptake during hyperglycemia and is crucial for the regulation of glucose-responsive glycolysis genes. Mutations in GCK are responsible for maturity-onset diabetes of the young, and glucokinase regulatory protein (GKRP) , the regulator of hepatic GCK, is also a diabetes susceptibility locus. Recently in in vivo experiments, we used hepatic lactate export as a surrogate for GCK activity, which was a measure of “glucose effectiveness.” We hypothesized that in vitro GCK and GKRP suppression in hepatocytes promotes a reduction in lactate and pyruvate export from the liver. We used cryopreserved canine hepatocytes and knocked down GCK and GKRP via lipid delivery of siRNA. A control group did not receive siRNA. We measured lactate dehydrogenase D (LDHD) and pyruvate kinase (PK) gene expression and lactate and pyruvate release into the medium 24 and 48 h after siRNA delivery. Selective blocking of GCK and GKRP did not change cell proliferation but reduced the expression of these genes by 90% and 85%, respectively (P Disclosure M.Kabir: None. M.Ader: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research & Development, LLC. Funding National Institutes of Health R01- DK027619-28

Estimating insulin resistance from the oral glucose tolerance test (OGTT) has long been a challenge in diabetes research and practice. Because the time courses of glucose and insulin during the OGTT are similar in shape, the estimates of insulin sensitivity per se may be corrupted by other metabolic responses such as insulin release or glucose absorption. We have previously shown insulin injection during the intravenous test yields accurate values of insulin sensitivity (SI) and glucose effectiveness (SG) . Here we examine an analogous approach during the OGTT - in which exogenous insulin is administered during the test. Methods: For 100 simulated subjects (NGT: n=25, IGT: n=25, T2D: n=25, T1D: n=25) , glucose and insulin data were calculated using Dalla Man Mixed Meal Simulator. For each simulated subject, several protocols were examined by varying the time of insulin bolus (30, 60, or 90 min) and the dose of the bolus (0, 0.01, 0.02, or 0.03 IU/kg) . The resulting 3600 simulated datasets were analyzed with Dalla Man’s OGTT model to estimate SI. Results: When adjusted for the dose of insulin, the time of the dose did not significantly impact the estimation of SI. The 0.IU/kg insulin dose resulted in significantly (P Disclosure D.Stefanovski: None. F.Piccinini: Employee; Medtronic. M.Ader: None. M.Kabir: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research & Development, LLC.

The prodromal prediabetic state is characterized by glucose intolerance. Both insulin-dependent (sensitivity, secretion, clearance) and -independent factors (“glucose effectiveness”) contribute to glucose homeostasis. The relative importance of glucose effectiveness is not known. Healthy dogs (n=18) were tested at baseline (BL), after 6 wk on a high-fat diet (HFD), and 2 wk, after a sub-diabetogenic dose of streptozotocin (STZ, 18.5 mg/kg); HFD was continued. We performed intravenous glucose tolerance tests (IVGTTs)at 0, 6, and 8 wks. Minimal model analysis quantified insulin sensitivity (SI), acute insulin response to glucose (AIRg), disposition index (DI, = SI x AIRg), and glucose tolerance (KG). Glucose effectiveness (SG) was measured both by minimal model analysis and directly by multi-step hyperglycemic clamps at basal insulin in a subset of the dogs (n=5). Consumption of HFD induced 7.7±1.0% weight gain (P Disclosure C. M. Kolka: Research Support; Self; AstraZeneca. J. Porter: None. M. Ader: None. R. N. Bergman: None. Funding National Institutes of Health (DK27619, DK29867)

Sodium glucose cotransporter-2 (SGLT2) inhibitors promote urinary glucose excretion, consequently reducing blood glucose. Previously we showed that 6 weeks of Dapagliflozin (Dapa) treatment improved insulin sensitivity and prevented weight gain. In addition, Dapa promoted adipose tissue (AT) beiging, lipolysis and decreased adipocyte size primarily in the visceral (VIS) depot in a canine model of mild type 2 diabetes. Little is known about the effects of Dapa on AT inflammation and ceramide synthesis in both the subcutaneous (SC) and VIS fat depots. Twelve dogs were fed a high fat diet for 6 weeks, then received a single low dose of streptozotocin (18.5mg/kg) to induce mild type 2 diabetes. Animals were then exposed to Dapa (n=6, 1.25mg/kg) or placebo (n=6) once per day for 6 weeks, while remaining on the high fat diet. At the end of the treatment, fasting plasma parameters were measured, and SC and VIS fat biopsies were obtained for molecular studies. Dapa increased adiponectin, adiponectin receptors 1 and 2 gene expression by 90%, 74% and 69% ( In conclusion, Dapa promoted adiponectin and its receptors, decreased inflammation and enzymes involved in ceramide synthesis mainly in the VIS depot. These changes were independent of fasting glucose and insulin. Thus, for the first time we are beginning to identify mechanisms by which Dapa enhances AT function in regulating energy homeostasis and improving AT inflammation in mild type 2 diabetes canine model. Disclosure M. Kabir: None. R. N. Bergman: None. V. Gopaul: None. H. Yang: Employee; Self; AstraZeneca. C. M. Kolka: Research Support; Self; AstraZeneca. Funding National Institutes of Health

We recently reported that in rodent models of type 2 diabetes (T2D), a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) induces remission of hyperglycemia that is sustained for weeks. To clarify the peripheral mechanisms underlying this effect, we used the Zucker diabetic fatty fa/fa rat model of T2D, which, like human T2D, is characterized by progressive deterioration of pancreatic β-cell function after hyperglycemia onset. We report that although icv FGF1 injection delays the onset of β-cell dysfunction in these animals, it has no effect on either glucose-induced insulin secretion or insulin sensitivity. These observations suggest that FGF1 acts in the brain to stimulate insulin-independent glucose clearance. On the basis of our finding that icv FGF1 treatment increases hepatic glucokinase gene expression, we considered the possibility that increased hepatic glucose uptake (HGU) contributes to the insulin-independent glucose-lowering effect of icv FGF1. Consistent with this possibility, we report that icv FGF1 injection increases liver glucokinase activity by approximately twofold. We conclude that sustained remission of hyperglycemia induced by the central action of FGF1 involves both preservation of β-cell function and stimulation of HGU through increased hepatic glucokinase activity.

Apoptosis repressor with caspase recruitment domain (ARC) is an endogenous inhibitor of apoptosis signaling. We previously showed ARC is expressed in β cells and ameliorates amyloid-induced β-cell death in vitro. More recently, we found that wild type (WT) and whole-body ARC knockout (KO) mice on an FVB background fed a normal chow diet exhibit no differences in islet area, β-cell area, or α-cell area at 16 (n=5) or 35 weeks (n=7-8) of age. In the present study, we sought to determine whether ARC plays a role in maintaining glycemia in response to the challenge of high fat diet (HFD, 60% kcal from fat) feeding in vivo. At 12 weeks of age, just prior to starting 24 weeks of HFD, ARC KO mice displayed lower body weight than WT mice (0 wks: 26.2±0.6 vs. 30.6±0.8 g; p Disclosure A.T. Templin: None. C.R. Schmidt: None. M.F. Hogan: None. N. Esser: None. R.N. Kitsis: None. R.L. Hull: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc. Funding U.S. Department of Veterans Affairs (I01-BX001060, IK2-BX004659); National Institutes of Health (P30DK017047)

Selective sodium glucose cotransporter-2 inhibition promotes urinary glucose excretion, thus reducing blood glucose. Previously we showed that 6 weeks of Dapagliflozin (Dapa) treatment improved insulin sensitivity and prevented weight gain in a canine model of mild type 2 diabetes. Little is known about the effects of Dapa on adipose tissue (AT) function. Eleven dogs were fed a high fat diet for 6 weeks, then received a single low dose of streptozotocin (18.5mg/kg) to induce mild type 2 diabetes. Animals were then exposed to Dapa (n=5, 1.25mg/kg) or placebo (n=6) once per day for 6 weeks, while remaining on the high fat diet. At the end of the study, subcutaneous (SC) and visceral (VIS) fat depots were obtained for adipocytes isolation and molecular studies and fasting plasma parameters were measured. Fasting plasma glucose and insulin levels were unaffected by Dapa treatment. Finite mixture modeling linear regression was used for adipocyte distributions. Dapa decreased adipocyte size and bimodal distributions in both SC and VIS by 10% (P In conclusion, Dapa promotes AT beiging, lipolysis, adiponectin and leptin release by adipocytes and decreased adipocyte size and distributions. These changes were independent of glucose and insulin. Thus, for the first time we are beginning to identify mechanisms by which Dapa enhances AT function and improves energy homeostasis in mild type 2 diabetes canine model. Disclosure M. Kabir: None. R.N. Bergman: None. F. Piccinini: Employee; Self; Medtronic. D. Stefanovski: None. V. Gopaul: None. H. Yang: Employee; Self; AstraZeneca. C.M. Kolka: Research Support; Self; AstraZeneca. Funding AstraZeneca (D1690L00045-CSR212966)

Glucose homeostasis involves actions of both insulin and glucose per se (“glucose effectiveness, SG”). While consumption of a high fat diet typically induces insulin resistance, it is unclear whether SG is altered by fat feeding. We quantified SG from the intravenous glucose tolerance test (IVGTT; n=36) or hyperglycemic clamp at basal insulin (HGC; n=5) in dogs, tested before and after 6 weeks fat feeding (+ 6 g/kg per day). SG from IVGTTs were quantified by minimal model (SG-MINMOD). In HGCs, glucose was sequentially raised to 125, 180, and 250 mg/dl during somatostatin (SRIF) and basal insulin replacement; SG-HGC was calculated as the slope of steady state glucose infusion rate and plasma glucose. Insulin sensitivity (SI) was calculated from IVGTTs and from euglycemic clamps. Fat feeding induced weight gain (28.8±0.4 to 31.2±2.5 kg; p Disclosure M. Ader: None. C.M. Kolka: Research Support; Self; AstraZeneca. R.N. Bergman: None. Funding National Institutes of Health (DK29867, DK27619)

Although the β-cells secrete insulin, the liver, with its first-pass insulin extraction (FPE), regulates the amount of insulin allowed into circulation for action on target tissues. The metabolic clearance rate of insulin, of which FPE is the dominant component, is a major determinant of insulin sensitivity (SI). We studied the intricate relationship among FPE, SI, and fasting insulin. We used a direct method of measuring FPE, the paired portal/peripheral infusion protocol, where insulin is infused stepwise through either the portal vein or a peripheral vein in healthy young dogs (n = 12). FPE is calculated as the difference in clearance rates (slope of infusion rate vs. steady insulin plot) between the paired experiments. Significant correlations were found between FPE and clamp-assessed SI (rs = 0.74), FPE and fasting insulin (rs = −0.64), and SI and fasting insulin (rs = −0.67). We also found a wide variance in FPE (22.4–77.2%; mean ± SD 50.4 ± 19.1) that is reflected in the variability of plasma insulin (48.1 ± 30.9 pmol/L) and SI (9.4 ± 5.8 × 104 dL · kg−1 · min−1 · [pmol/L]−1). FPE could be the nexus of regulation of both plasma insulin and SI.

African Americans (AAs) tend to have higher plasma insulin concentrations than European Americans (EAs); the increased insulin concentrations have been attributed to increased secretion and/or decreased insulin clearance by liver or other tissues. This work characterizes the contributions of hepatic versus extrahepatic insulin degradation related to ethnic differences between AAs and EAs. By using a recently developed mathematical model that uses insulin and C-peptide measurements from the insulin-modified, frequently sampled intravenous glucose tolerance test (FSIGT), we estimated hepatic versus extrahepatic insulin clearance in 29 EA and 18 AA healthy women. During the first 20 min of the FSIGT, plasma insulin was approximately twice as high in AAs as in EAs. In contrast, insulin was similar in AAs and EAs after the 20–25 min intravenous insulin infusion. Hepatic insulin first-pass extraction was two-thirds lower in AAs versus EAs in the overnight-fasted state. In contrast, extrahepatic insulin clearance was not lower in AAs than in EAs. The difference in insulin degradation between AAs and EAs can be attributed totally to liver clearance. The mechanism underlying reduced insulin degradation in AAs remains to be clarified, as does the relative importance of reduced liver clearance to increased risk for type 2 diabetes.

Islet amyloid is present in more than 90% of individuals with type 2 diabetes, where it contributes to β-cell apoptosis and insufficient insulin secretion. Apoptosis repressor with caspase recruitment domain (ARC) binds and inactivates components of the intrinsic and extrinsic apoptosis pathways and was recently found to be expressed in islet β-cells. Using a human islet amyloid polypeptide transgenic mouse model of islet amyloidosis, we show ARC knockdown increases amyloid-induced β-cell apoptosis and loss, while ARC overexpression decreases amyloid-induced apoptosis, thus preserving β-cells. These effects occurred in the absence of changes in islet amyloid deposition, indicating ARC acts downstream of amyloid formation. Because islet amyloid increases c-Jun N-terminal kinase (JNK) pathway activation, we investigated whether ARC affects JNK signaling in amyloid-forming islets. We found ARC knockdown enhances JNK pathway activation, whereas ARC overexpression reduces JNK, c-Jun phosphorylation, and c-Jun target gene expression (Jun and Tnf). Immunoprecipitation of ARC from mouse islet lysates showed ARC binds JNK, suggesting interaction between JNK and ARC decreases amyloid-induced JNK phosphorylation and downstream signaling. These data indicate that ARC overexpression diminishes amyloid-induced JNK pathway activation and apoptosis in the β-cell, a strategy that may reduce β-cell loss in type 2 diabetes.

On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the “single gateway hypothesis,” which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis.

Dynamic adjustment of insulin secretion to compensate for changes of insulin sensitivity that result from alteration of nutritional or metabolic status is a fundamental aspect of glucose homeostasis. To investigate the role of the brain in this coupling process, we used cold exposure as an experimental paradigm because the sympathetic nervous system (SNS) helps to coordinate the major shifts of tissue glucose utilization needed to ensure that increased thermogenic needs are met. We found that glucose-induced insulin secretion declined by 50% in rats housed at 5°C for 28 h, and yet, glucose tolerance did not change, owing to a doubling of insulin sensitivity. These potent effects on insulin secretion and sensitivity were fully reversed by returning animals to room temperature (22°C) for 4 h or by intravenous infusion of the α-adrenergic receptor antagonist phentolamine for only 30 min. By comparison, insulin clearance was not affected by cold exposure or phentolamine infusion. These findings offer direct evidence of a key role for the brain, acting via the SNS, in the rapid, highly coordinated, and reciprocal changes of insulin secretion and insulin sensitivity that preserve glucose homeostasis in the setting of cold exposure.

African American (AA) children have lower hepatic insulin clearance than European Americans (EA), as obtained with a 25-sample intravenous glucose tolerance test (FSIGT), over 240 minutes (t=0, 2, 3, 4 5, 6, 8, 10, 12, 15, 19, 20, 21, 22, 24, 26, 28, 30, 35, 40, 50, 60, 70, 180, 240 minutes). C-peptide and insulin concentrations were analyzed with a recent model of hepatic (FEL) and extra-hepatic (CLP) insulin clearance. This model has not been tested in a reduced sampling protocol, easier to be performed in larger cohorts. With data from 203 children of both sexes and 3 ethnic groups [55 AA, 88 EA, 60 HA (Hispanic Americans), age 7-13 years, mean BMI = 19 kg/m2, basal plasma glucose = 99 mg/dL, insulin = 78 pmol/L, C-peptide = 511 pmol/L], a reduced 12-sample schedule was examined (t=0, 2, 4, 8, 19, 22, 26, 30, 40, 50, 70, 180 minutes). FEL and CLP from the reduced protocol were strongly correlated with the full one: FEL: r=0.91, p Disclosure F. Piccinini: None. B. Gower: None. J.R. Fernandez: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067426); Nutrition Obesity Research Center (DK56336); National Institutes of Health (M01RR00032)

Recent studies showed that hepatic insulin clearance is lower in African Americans (AA) than European Americans (EA). The aim of this work is to elucidate the possible genetic/epigenetic versus lifestyle causes. The frequently-sampled intravenous glucose tolerance test (FSIGT) was performed in 142 children [38 AA, 69 EA, 28 HA (Hispanic Americans), both sexes, age 7-13 years, mean BMI = 19 kg/m2, basal plasma glucose = 99 mg/dL, insulin = 78 pmol/L, C-peptide = 511 pmol/L]. Mathematical modeling provided hepatic (FEL) and extra-hepatic (CLP) insulin clearance values. Two-way analysis of covariance was done (groups: sex and ethnicity, covariates: age, Tanner stage and body fat from DXA). Data are mean (standard error). FEL was lower in AA than EA (18% (2%) vs. 31% (3%), p=0.001), but no difference was observed in HA. CLP was not different among ethnicities. Partial correlation was calculated between FEL, CLP and 1) diet composition (energy intake, % of calories from carbohydrate, fat, protein), 2) physical activity (PA: light, moderate, hard, very hard), 3) admixture scores (African, European, Amerindian). FEL was positively correlated with the European admixture score (r=0.3, p Disclosure F. Piccinini: None. B. Gower: None. J.R. Fernandez: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067426); Nutrition Obesity Research Center (DK56336); National Institutes of Health (M01RR00032)

ZGN-1061 is a second-generation methionine aminopeptidase 2 (MetAP2) inhibitor in development for treatment of T2D. Full results of a Phase 2 clinical trial of ZGN-1061 vs. placebo (PBO) administered SC for 12 weeks on change from baseline in A1C in subjects with T2D are reported elsewhere. Baseline (ITT population N=175) A1C 8.6±1.1%, BMI 37±7 kg/m2, T2D duration 8±6 y. ZGN-1061 was generally well tolerated; 93% completed the trial. LS mean±SE A1C change was 0.2±0.1% for PBO vs. ±0.4±0.1% and -0.9±0.2% for 0.9 and 1.8 mg ZGN-1061 (both p Disclosure R. Fabunmi: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. F. Piccinini: None. T. Kim: Employee; Spouse/Partner; Celgene Corporation. Employee; Self; Zafgen, Inc. D. Zhuang: Employee; Self; Zafgen, Inc. K. Taylor: Employee; Self; Zafgen, Inc. D.D. Kim: Employee; Self; Zafgen, Inc. Funding Zafgen, Inc.

We have previously demonstrated in dogs that following exposure to a high fat diet for 16 weeks, the brain and peripheral penetrating CB1R antagonist Rimonabant (RIM) promoted adipose tissue beiging and decreased adipocyte cell size. It is hoped that peripherally restricted CB1R antagonists, such as AM6545, will allow for the substantial beneficial metabolic effects of CB1R antagonism without the possible negative consequences associated with brain penetrance. Studies demonstrated beneficial metabolic effects of AM6545 in adipose tissue including increased energy expenditure and decreased adipocytes cell size. We thus compared the effects of RIM versus AM6545 on oxygen consumption and beiging in the well-characterized 3T3-L1 cell line model. Mature 3T3-L1s were treated for 4 or 48 hours with RIM, AM6545, vehicle or isoproterenol (ISO) (positive control). Baseline oxygen consumption and the proportional contribution of proton leak to maximal respiratory capacity was greater for cells exposed to RIM, ISO, and, in lesser extent, AM6545 after 48 hours, demonstrating greater mitochondrial uncoupling. Further, markers of adipose tissue beiging (UCP1, Prdm16, PGC1α) were also unregulated by approximately 2-fold (P Disclosure R.K. Paszkiewicz: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. A. Frank: None. O.O. Woolcott: None. R. de Souza Santos: None. M.S. Iyer: None. D. Clegg: None. M. Kabir: None.

There is wide variance among individuals in the fraction of insulin cleared by the liver (20% to 80%). Hepatic insulin clearance is 67% lower in African Americans than European Americans. Clearance is also lower in African American children 7–13 years of age. Lower hepatic insulin clearance will result in peripheral hyperinsulinemia: this exacerbates insulin resistance, which stresses the β-cells, possibly resulting in their ultimate failure and onset of type 2 diabetes. We hypothesize that lower insulin clearance can be a primary cause of type 2 diabetes in at-risk individuals.