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Purpose: We tested the hypothesis that a critical biologic determinant of T2D disparities is based on ethnicity-related differences in pancreatic islet function. Methods: Utilizing donor tissues from the NIDDK-funded Human Pancreas Analysis Program (HPAP) , we examined both insulin and glucagon secretion from nondiabetic and diabetic Caucasian and African American donor islets. Initially, a physiological amino acid mixture was used to stimulate glucagon secretion, followed by low and high glucose to stimulate insulin secretion and suppress glucagon secretion. During the high glucose step, IBMX (0.1 mM) is added to potentiate secretion of both hormones. Insulin and glucagon concentration in perifusates and islet extracts was measured by radioimmunoassay or ELISA. Statistical comparisons were drawn by repeated measures ANOVA. Results: Comparing nondiabetic islets, we observed overlapping insulin secretion profiles between both ethnicities; however, glucagon secretion was distinctly greater in both nondiabetic African American (+111%) islets compared to nondiabetic Caucasian islets, under all interventions. In T2D, the reduction in insulin secretion was significantly greater in African American T2D donors (high glucose -76%; IBMX -76%) compared to Caucasian T2D donors (high glucose -47%; IBMX -50%) . More strikingly, glucagon secretion in T2D donors was markedly different based on ethnicity. Whereas Caucasian T2D islets exhibited a similar baseline of glucagon secretion compared to Caucasian controls, there was a marked reduction of glucagon secretion overall in African American T2D donors (-70%) compared to their corresponding controls. The largest detected difference was in IBMX-potentiation of glucagon secretion, which is higher in Caucasian T2D donors (+103%) but significantly decreased in African American T2D donors (-76%) . Preliminary analysis of hormone content in a subset of the donor islet preparations revealed no differences according to ethnicity. Conclusions: We propose that ethnicity-related differences in nondiabetic islet function may contribute to the enhanced risk of T2D. Disclosure N. M. Doliba: None. M. Brissova: None. K. H. Kaestner: None. D. A. Stoffers: Other Relationship; Biomarin, Correlagen. J. Roman: None. A. V. Rozo: None. W. Qin: None. C. Liu: None. A. Naji: None. M. R. Rickels: Advisory Panel; Sernova, Corp., Vertex Pharmaceuticals Incorporated, Zealand Pharma A/S, Consultant; L-Nutra Inc. M. A. Atkinson: None. A. C. Powers: None. Funding This work is supported by 3UC4-112217-01S1 and U01-DK123594-02, UC4-DK-112217, UC4-DK-112232, and U01-DK-123716.

Insulin deficiency is central to diabetes and diabetes-related cardiac dysfunction. GPCRs are known modulators of insulin secretion and a main pharmacological target in various tissues, including the heart. GPCR kinase 2 (GRK2) phosphorylates activated GPCRs, targeting receptors for recycling or degradation. Notably, we and others have shown that GRK2 can also localize to the cardiac mitochondria where it participates in substrate utilization, particularly in response to cellular stress. GRK2 is downregulated in the pancreas of diabetogenic mice, and we have shown that pancreatic loss of GRK2 impairs insulin secretion in normal and high fat diet. Mice with pancreatic-specific GRK2 KO showed glucose intolerance (AUC WT 8691 vs. KO 14766 mg/dl*min, n=22/group, p Disclosure J. W. Snyder: None. S. K. Montgomery: None. N. M. Doliba: None. J. Roman: None. Y. Tian: None. P. Y. Sato: None. W. L. Holland: None. R. Choi: None. Funding National Institutes of Health (1R56HL149887) ; University of Pennsylvania Diabetes Research Center Pilot and Feasibility Grant (P30-DK019525) ; American Heart Association Scientist Development Grant (17SDG33660407)

Fuel stimulated hormone release (HR) and the oxygen consumption rates (OCR) of isolated normal pancreatic islets are positively correlated illustrating that cellular energy state is a critical determinant of stimulus-secretion coupling. We investigated the relationship between hormone release and OCR in islets from donors with T1DM and T2DM, using unique oxygen measurement technology and optimized perifusion protocols. HR and OCR were quantified in 20 human donor islet preparations obtained from the Human Islet Resource Center at the University of Pennsylvania within 3-4 days after isolation (5 each of nondiabetic, T1DM, T2DM and anti-islet AutoAntiBody positive (AAB+). A physiological amino acid mixture (4mM) was used to stimulate glucagon release, then 3 and 16.7mM glucose were added to stimulate insulin release and inhibit glucagon release. Respiration was then uncoupled with FCCP and finally blocked by NaN3. Basal OCR was increased in islets from AAB+, T1DM and T2DM donors compared to controls. Low glucose failed to increase OCR in T1DM and T2DM islets while high glucose-stimulated OCR was both decreased and delayed. Glucose stimulated insulin release much less effectively and failed to suppress glucagon release in both T1DM and T2DM islets. AAB+ islets showed normal insulin release but lacked glucose suppression of glucagon release. The observation of increased basal-OCR of islets in all diabetes related conditions and reduced glucose-stimulated-OCR in T1DM and T2DM islets unrelated to hormone release rates is striking and indicates a profound alteration of bioenergetics regulation in these conditions hitherto unrecognized. Common to this pathophysiology are an increase in relative alpha-cell mass (T1DM and T2DM) or a functional defect of these cells (AAB+) providing a plausible explanation for this phenomenology and implying impaired alpha-cell energy metabolism. Disclosure N.M. Doliba: None. A.V. Rozo: None. W. Qin: None. J. Roman: None. C. Liu: None. A. Naji: None. K.H. Kaestner: None. D.A. Stoffers: None. F. Matschinsky: None. Funding National Institutes of Health (UC4DK112217)

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+]i), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+]i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

Type 2 diabetes is characterized by elevated blood glucose levels resulting from a pancreatic β-cell secretory insufficiency combined with insulin resistance, most significantly manifested in skeletal muscle and liver (1). If untreated, diabetic complications develop that cause loss of vision, peripheral neuropathy, impaired kidney function, heart disease, and stroke. The disease has a polygenic basis because numerous genes (the latest count exceeding 20) participate in its pathogenesis, but modern lifestyle characterized by limited physical activity and excessive caloric intake are critical precipitating factors for the current epidemic of type 2 diabetes worldwide (2). It appears that available treatments, including attempts at lifestyle alterations and drug therapies including insulin, are insufficient to stem the tide. Therefore, new approaches, including the development of therapeutic agents with novel mechanisms of action, are needed. Selection of new drug targets to treat type 2 diabetes has to be guided primarily by consideration of established physiological chemistry of glucose homeostasis and of prevailing views about the pathophysiology of type 2 diabetes because the genetics of the disease that could serve as another guiding principle remain prohibitively perplexing. The glucose-phosphorylating enzyme glucokinase (GK) was identified as an outstanding drug target for developing antidiabetic medicines because it has an exceptionally high impact on glucose homeostasis because of its glucose sensor role in pancreatic β-cells and as a rate-controlling enzyme for hepatic glucose clearance and glycogen synthesis, both processes that are impaired in type 2 diabetes (3). Milestones in the 45-year history of GK research are listed in Supplementary Table 1 (Supplementary References S1–S27). In the mid-1990s, Hoffmann La-Roche scientists conducted a high-throughput screen in search of small molecules that could reverse the inhibition of GK by its regulatory protein (GKRP, see further discussion below) and identified a hit molecule that reversed GKRP inhibition by directly stimulating GK (4 …

The glucose-phosphorylating enzyme glucokinase has structural, kinetic, and molecular genetic features that are ideal for its primary role as glucose sensor in a network of neuro/endocrine sentinel cells that maintain glucose homeostasis in many vertebrates including humans. The glucokinase-containing, insulin-producing β-cells of the pancreas take the prominent lead in this network, functioning in the aggregate as the master gland. The β-cells are also conceptualized as the prototype for all other glucose sensor cells, which determines our current understanding of many extrapancreatic glucose sensors. About 99% of the enzyme resides, however, in the hepato-parenchymal cells and serves its second role in a high-capacity process of blood glucose clearance. Two examples strikingly illustrate how pivotal a position glucokinase has in the regulation of glucose metabolism: 1) activating and inactivating mutations of the enzyme cause hypo- and hyperglycemia syndromes in humans described collectively as “glucokinase disease” and fully explained by the glucose sensor paradigm, and 2) glucokinase activator drugs (GKAs) have been discovered that bind to an allosteric site and increase the kcat and lower the glucose S0.5 of the enzyme. GKAs enhance glucose-stimulated insulin release from pancreatic islets and glucose disposition by the liver. They are now intensively explored to develop a novel treatment for diabetes. Future biophysical, molecular, genetic, and pharmacological studies hold much promise to unravel the evolving complexity of the glucokinase glucose sensor system.

Type 2 diabetic subjects manifest both disordered insulin action and abnormalities in their pancreatic islet cells. Whether the latter represents a primary defect or is a consequence of the former is unknown. To examine the β-cell mass and function of islets from type 2 diabetic patients directly, we isolated islets from pancreata of type 2 diabetic cadaveric donors (n = 14) and compared them with islets from normal donors (n = 14) matched for age, BMI, and cold ischemia time. The total recovered islet mass from type 2 diabetic pancreata was significantly less than that from nondiabetic control subjects (256,260 islet equivalents [2,588 IEq/g pancreas] versus 597,569 islet equivalents [6,037 IEq/g pancreas]). Type 2 diabetic islets were also noted to be smaller on average, and histologically, islets from diabetic patients contained a higher proportion of glucagon-producing α-cells. In vitro study of islet function from diabetic patients revealed an abnormal glucose-stimulated insulin release response in perifusion assays. In addition, in comparison with normal islets, an equivalent number of type 2 diabetic islets failed to reverse hyperglycemia when transplanted to immunodeficient diabetic mice. These results provide direct evidence for abnormalities in the islets of type 2 diabetic patients that may contribute to the pathogenesis of the disease.

In the present study, noninvasive 31 P and 23 Na + –nuclear magnetic resonance (NMR) technology and respirometry were used to compare the effect of high glucose (30 mmol/l) with the effect of the antidiabetic sulfonylurea (SU) compound glyburide (GLY) on energy metabolism, Na + flux, insulin, and cAMP release of continuously superfused β-HC9 cells encapsulated in microscopic agarose beads. Both high glucose and GLY increased oxygen consumption in β-HC9 cells (15–30%) with a maximal effect at 8 mmol/l for glucose and at 250 nmol/l for GLY. At the same time, insulin release from β-cells increased by 15- and 25-fold with high glucose or GLY, respectively. The P-creatine (PCr) level was greatly increased and inorganic phosphate (P i ) was decreased with 30 mmol/l glucose in contrast to the decreased level of PCr and increased P i with GLY. ATP levels remained unchanged during both interventions. Studies on isolated mitochondria of β-HC9 cells showed that GLY added to mitochondria oxidizing glutamine or glutamate abolished the stimulation of respiration by ADP (state 3) meanwhile leaving state 3 respiration unchanged during oxidation of other substrates. Exposure of β-HC9 cells to 5 mmol/l glucose decreased intracellular Na + levels monitored by 23 Na + -NMR spectroscopy and 30 mmol/l glucose resulted in a further decrease in cytosolic Na + . In contrast, Na + increased when 1 μmol/l GLY was added to the perfusate containing 5 mmol/l glucose. These data support the hypothesis that glucose activates the β-cell through a “push mechanism” due to substrate pressure enhancing fuel flux, energy production, and extrusion of Na + from the cells in contrast to SU receptor (SUR)-1 inhibitors, which may modify intermediary and energy metabolism secondarily through a “pull mechanism” due to higher energy demand resulting from increased ion fluxes and the exocytotic work load.