Regulation Of Glucose-stimulated Insulin Secretion Research Articles

(Re-)Viewing Role of Intracellular Glucose Beyond Extracellular Regulation of Glucose-Stimulated Insulin Secretion by Pancreatic Cells.

For glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells in animals, it is believed that ATP generated from glucose metabolism is primarily responsible. However, this ignores two well-established aspects in literature: (a) intracellular ATP generation from other sources resulting in an overall pool of ATP, regardless of the original source, and (b) that intracellular glucose transport is 10- to 100-fold higher than intracellular glucose phosphorylation in β-cells. The latter especially provides an earlier unaddressed, but highly appealing, observation pertaining to (at least transient) the presence of intracellular glucose molecules. Could these intracellular glucose molecules be responsible for the specificity of GSIS to glucose (instead of the widely believed ATP production from its metabolism)? In this work, we provide a comprehensive compilation of literature on glucose and GSIS using various cellular systems - all studies focus only on the extracellular role of glucose in GSIS. Further, we carried out a comprehensive analysis of differential gene expression in Mouse Insulinoma 6 (MIN6) cells, exposed to low and high extracellular glucose concentrations (EGC), from the existing whole transcriptome data. The expression of other genes involved in glycolysis, Krebs cycle, and electron transport chain was found to be unaffected by EGC, except Gapdh, Atp6v0a4, and Cox20. Remarkably, 3 upregulated genes (Atp6v0a4, Cacnb4, Kif11) in high EGC were identified to have an association with cellular secretion. Using glucose as a possible ligand for the 3 proteins, computational investigations were carried out (that will require future 'wet validation', both in vitro and in vivo, e.g., using primary islets and animal models). The glucose-affinity/binding scores (in kcal/mol) obtained were also compared with glucose binding scores for positive controls (GCK and GLUT2), along with negative controls (RPA1, KU70-80, POLA1, ACAA1A, POLR1A). The binding affinity scores of glucose molecules for the 3 proteins were found to be closer to positive controls. Therefore, we report the glucose binding ability of 3 secretion-related proteins and a possible direct role of intracellular glucose molecules in GSIS.

Absence of Slc39a14/Zip14 in mouse pancreatic beta cells results in hyperinsulinemia.

Zinc is an essential component of the insulin protein complex synthesized in β cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human β cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic β cell-specific knockout of Slc39a14 (β-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using β-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in β cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused β-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in β cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human β cells besides SLC30A8. Within the β cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.

Tau suppresses microtubule-regulated pancreatic insulin secretion.

Tau protein is implicated in the pathogenesis of Alzheimer's disease (AD) and other tauopathies, but its physiological function is in debate. Mostly explored in the brain, tau is also expressed in the pancreas. We further explored the mechanism of tau's involvement in the regulation of glucose-stimulated insulin secretion (GSIS) in islet β-cells, and established a potential relationship between type 2 diabetes mellitus (T2DM) and AD. We demonstrate that pancreatic tau is crucial for insulin secretion regulation and glucose homeostasis. Tau levels were found to be elevated in β-islet cells of patients with T2DM, and loss of tau enhanced insulin secretion in cell lines, drosophila, and mice. Pharmacological or genetic suppression of tau in the db/db diabetic mouse model normalized glucose levels by promoting insulin secretion and was recapitulated by pharmacological inhibition of microtubule assembly. Clinical studies further showed that serum tau protein was positively correlated with blood glucose levels in healthy controls, which was lost in AD. These findings present tau as a common therapeutic target between AD and T2DM.

EP3 signaling is decoupled from the regulation of glucose-stimulated insulin secretion in β-cells compensating for obesity and insulin resistance

ABSTRACT Of the β-cell signaling pathways altered by obesity and insulin resistance, some are adaptive while others contribute to β-cell failure. Two critical second messengers are Ca2+ and cAMP, which control the timing and amplitude of insulin secretion. Previous work has shown the importance of the cAMP-inhibitory Prostaglandin EP3 receptor (EP3) in mediating the β-cell dysfunction of type 2 diabetes (T2D). Here, we used three groups of C57BL/6J mice as a model of the progression from metabolic health to T2D: wildtype, normoglycemic Leptin Ob (NGOB), and hyperglycemic Leptin Ob (HGOB). Robust increases in β-cell cAMP and insulin secretion were observed in NGOB islets as compared to wildtype controls; an effect lost in HGOB islets, which exhibited reduced β-cell cAMP and insulin secretion despite increased glucose-dependent Ca2+ influx. An EP3 antagonist had no effect on β-cell cAMP or Ca2+ oscillations, demonstrating agonist-independent EP3 signaling. Finally, using sulprostone to hyperactivate EP3 signaling, we found EP3-dependent suppression of β-cell cAMP and Ca2+ duty cycle effectively reduces insulin secretion in HGOB islets, while having no impact insulin secretion on NGOB islets, despite similar and robust effects on cAMP levels and Ca2+ duty cycle. Finally, increased cAMP levels in NGOB islets are consistent with increased recruitment of the small G protein, Rap1GAP, to the plasma membrane, sequestering the EP3 effector, Gɑz, from inhibition of adenylyl cyclase. Taken together, these results suggest that rewiring of EP3 receptor-dependent cAMP signaling contributes to the progressive changes in β cell function observed in the Leptin Ob model of diabetes.

295-LB: Pancreatic Peroxisomes Are Required for Physiological Regulation of Insulin Secretion and Maintenance of Normal Glucose Tolerance in Male Mice

The regulation of glucose-stimulated insulin secretion (GSIS) is central to maintenance of glucose disposal. The role of lipid storage and lipid oxidation to regulate pancreatic islet β-cell insulin secretion is not completely understood. Peroxisomes are enriched in insulin-producing β-cells, but their contribution to β-cell function and maturity is lacking. Therefore, we generated a novel model of peroxisomal dysfunction to test the hypothesis that peroxisomal lipid metabolism is required to prevent a hyperlipidemic environment that interferes with β-cell function and maturity. Pex5 plays a critical role in importing the majority of peroxisomal proteins into the peroxisomal matrix; thus, deletion of the Pex5 gene creates a cellular environment with reductions in function of this organelle. In this study, we bred Pex5 mice on a C57BL/6J background containing ‘floxed’ alleles to Pdx1-cre mice to generate offspring with a pancreas-wide deficiency in Pex5 expression and abundance. Glucose tolerance was impaired by 12 weeks in male mice (n=20; AUC p<0.05). Surprisingly, GSIS was elevated in Pex5Pdx1-/- mice compared to littermate controls both in vivo (n=15; AUC p<0.05) and ex vivo in isolated islets as measured by perifusion analysis (n=12; AUC p<0.01). We further found that a subset of β-cells from Pex5Pdx1-/- mice expressed the de-differentiation marker Aldh1a3, while a subset of β-cells showed reductions in MafA and Nkx6.1 staining compared to controls. Perilipin-1 staining showed increased presence of lipid droplets in pancreatic tissue of Pex5Pdx1-/- mice versus controls. Thus, pancreatic deletion of peroxisomal function results in increased lipid storage in pancreatic tissue that is correlated with exaggerated insulin secretion. Collectively, these data are in support of a model whereby peroxisomal function supports β-cell insulin secretion and maintenance of the fully differentiated state. Disclosure S. Burke: None. S. Ghosh: None. D. Burk: None. R. Noland: None. J. Collier: None.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in β-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in β-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled β-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects β-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

301-OR: Modeling the Type 2 Diabetes–Associated ABCC8 Arg1420 His Mutation using iPSC–Derived Pancreatic Islet-Like Cells

Genetic studies in American Indians from Arizona previously identified a population specific Arg1420 His mutation in ABCC8 which causes hyperinsulinemic hypoglycemia in infancy. The His allele is carried by 3% of this population and associates with increased birthweight, suggesting increased insulin secretion in-utero, and doubles the risk of type 2 diabetes, a disease characterized by reduced insulin secretion. To model the contrary effects of this mutation, we used gene-edited induced pluripotent stem cell (iPSC) derived pancreatic islet-like cells. An iPS cell line created from an American Indian heterozygous for the mutation was gene-edited using CRISPR-CAS9 to generate iPSCs homozygous for the risk (His) and the non-risk (Arg) allele. Multiple iPSC lines for each genotype were differentiated towards pancreatic islet-like cells using a seven-stage differentiation protocol and the effect of the mutation on islet function were assessed. Higher insulin secretion was observed from the His-allele carrying islet-like cells when stimulated with low glucose (2mM) after 1 and 2 weeks of maturation, consistent with a clinical phenotype of hyperinsulinemia in His-allele carriers. Stimulation with 20mM glucose resulted in increased insulin secretion after 2 weeks of maturation only in the Arg-allele (non-risk) carrying islets while no increase was seen in the His-allele carrying islets, consistent with its role in T2D risk. Treatment with diazoxide significantly reduced insulin secretion with no difference between the genotypes. Single cell RNA sequencing of islet-like cells cultured for an extended time (4 weeks) identified many differentially expressed genes in cells with the mutant vs. reference allele that could potentially reveal the mechanism of the increased future T2D risk including G6PC2, a known negative regulator of glucose-stimulated insulin secretion, in beta-like cells and CRH, which has been linked to increased T2D risk, in alpha-like cells. Disclosure A.K.Nair: None. M.Traurig: None. J.Sutherland: Stock/Shareholder; Bristol-Myers Squibb Company, Johnson & Johnson, Pfizer Inc. R.G.Nelson: None. C.Bogardus: None. L.Baier: None. Funding NIH Intramural

Beta-Cell Adaptation to Pregnancy - Role of Calcium Dynamics.

During pregnancy, the mother develops insulin resistance to shunt nutrients to the growing fetus. As a result, the maternal islets of Langerhans undergo several changes to increase insulin secretion in order to maintain glucose homeostasis and prevent the development of gestational diabetes. These changes include an increase in β-cell proliferation and β-cell mass, upregulation of insulin synthesis and insulin content, enhanced cell-to-cell communication, and a lowering of the glucose threshold for insulin secretion, all of which resulting in an increase in glucose-stimulated insulin secretion. Emerging data suggests that a change in intracellular calcium dynamics occurs in the β-cell during pregnancy as part of the adaptive process. Influx of calcium into β-cells is crucial in the regulation of glucose-stimulated insulin secretion. Calcium fluxes into and out of the cytosol, endoplasmic reticulum, and mitochondria are also important in controlling β-cell function and survival. Here, we review calcium dynamics in islets in response to pregnancy-induced changes in hormones and signaling molecules, and how these changes may enhance insulin secretion to stave off gestational diabetes.

Mechanistic Investigation of GHS-R Mediated Glucose-Stimulated Insulin Secretion in Pancreatic Islets.

Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr−/− mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr−/− mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr−/− mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate—glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr−/− mice, but the ATP/ADP ratio in Ghsr−/− islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr−/− islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr−/− mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.

Plasma triacylglycerols are biomarkers of β-cell function in mice and humans

ObjectivesTo find plasma biomarkers prognostic of type 2 diabetes, which could also inform on pancreatic β-cell deregulations or defects in the function of insulin target tissues. MethodsWe conducted a systems biology approach to characterize the plasma lipidomes of C57Bl/6J, DBA/2J, and BALB/cJ mice under different nutritional conditions, as well as their pancreatic islet and liver transcriptomes. We searched for correlations between plasma lipids and tissue gene expression modules. ResultsWe identified strong correlation between plasma triacylglycerols (TAGs) and islet gene modules that comprise key regulators of glucose- and lipid-regulated insulin secretion and of the insulin signaling pathway, the two top hits were Gck and Abhd6 for negative and positive correlations, respectively. Correlations were also found between sphingomyelins and islet gene modules that overlapped in part with the gene modules correlated with TAGs. In the liver, the gene module most strongly correlated with plasma TAGs was enriched in mRNAs encoding fatty acid and carnitine transporters as well as multiple enzymes of the β-oxidation pathway. In humans, plasma TAGs also correlated with the expression of several of the same key regulators of insulin secretion and the insulin signaling pathway identified in mice. This cross-species comparative analysis further led to the identification of PITPNC1 as a candidate regulator of glucose-stimulated insulin secretion. ConclusionTAGs emerge as biomarkers of a liver-to-β-cell axis that links hepatic β-oxidation to β-cell functional mass and insulin secretion.

Rheb1 promotes glucose-stimulated insulin secretion in human and mouse β-cells by upregulating GLUT expression

Reduced β-cell mass and impaired β-cell function are primary causes of all types of diabetes. However, the intrinsic molecular mechanism that regulates β-cell growth and function remains elusive. Here, we demonstrate that the small GTPase Rheb1 is a critical regulator of glucose-stimulated insulin secretion (GSIS) in β-cells. Rheb1 was highly expressed in mouse and human islets. In addition, β-cell-specific knockout of Rheb1 reduced the β-cell size and mass by suppressing β-cell proliferation and increasing β-cell apoptosis. However, tamoxifen-induced deletion of Rheb1 in β-cells had no significant effect on β-cell mass and size but significantly impaired GSIS. Rheb1 facilitates GSIS in human or mouse islets by upregulating GLUT1 or GLUT2 expression, respectively, in a mTORC1 signaling pathway-dependent manner. Our findings reveal a critical role of Rheb1 in regulating GSIS in β-cells and identified a new target for the therapeutic treatment of diabetes mellitus.

Paired box 6 programs essential exocytotic genes in the regulation of glucose-stimulated insulin secretion and glucose homeostasis.

The paired box 6 (PAX6) transcription factor is crucial for normal pancreatic islet development and function. Heterozygous mutations of PAX6 are associated with impaired insulin secretion and early-onset diabetes mellitus in humans. However, the molecular mechanism of PAX6 in controlling insulin secretion in human beta cells and its pathophysiological role in type 2 diabetes (T2D) remain ambiguous. We investigated the molecular pathway of PAX6 in the regulation of insulin secretion and the potential therapeutic value of PAX6 in T2D by using human pancreatic beta cell line EndoC-βH1, the db/db mouse model, and primary human pancreatic islets. Through loss- and gain-of-function approaches, we uncovered a mechanism by which PAX6 modulates glucose-stimulated insulin secretion (GSIS) through a cAMP response element-binding protein (CREB)/Munc18-1/2 pathway. Moreover, under diabetic conditions, beta cells and pancreatic islets displayed dampened PAX6/CREB/Munc18-1/2 pathway activity and impaired GSIS, which were reversed by PAX6 replenishment. Adeno-associated virus-mediated PAX6 overexpression in db/db mouse pancreatic beta cells led to a sustained amelioration of glycemic perturbation in vivo but did not affect insulin resistance. Our study highlights the pathophysiological role of PAX6 in T2D-associated beta cell dysfunction in humans and suggests the potential of PAX6 gene transfer in preserving and restoring beta cell function.

Deconstructing the origins of sexual dimorphism in sensory modulation of pancreatic β cells

The regulation of glucose-stimulated insulin secretion and glucose excursion has a sensory component that operates in a sex-dependent manner. ObjectiveHere, we aim to dissect the basis of the sexually dimorphic interaction between sensory neurons and pancreatic β cells and its overall impact on insulin release and glucose homeostasis. MethodsWe used viral retrograde tracing techniques, surgical and chemodenervation models, and primary cell-based co-culture systems to uncover the biology underlying sex differences in sensory modulation of pancreatic β-cell activity. ResultsRetrograde transsynaptic labeling revealed a sex difference in the density of sensory innervation in the pancreas. The number of sensory neurons emanating from the dorsal root and nodose ganglia that project in the pancreas is higher in male than in female mice. Immunostaining and confocal laser scanning microscopy confirmed the higher abundance of peri-islet sensory axonal tracts in the male pancreas. Capsaicin-induced sensory chemodenervation concomitantly enhanced glucose-stimulated insulin secretion and glucose clearance in male mice. These metabolic benefits were blunted when mice were orchidectomized prior to the ablation of sensory nerves. Interestingly, orchidectomy also lowered the density of peri-islet sensory neurons. In female mice, capsaicin treatment did not affect glucose-induced insulin secretion nor glucose excursion and ovariectomy did not modify these outcomes. Interestingly, same- and opposite-sex sensory-islet co-culture paradigms unmasked the existence of potential gonadal hormone-independent mechanisms mediating the male-female difference in sensory modulation of islet β-cell activity. ConclusionTaken together, these data suggest that the sex-biased nature of the sensory control of islet β-cell activity is a result of a combination of neurodevelopmental inputs, sex hormone-dependent mechanisms and the potential action of somatic molecules encoded by the sex chromosome complement.

Targeting Pyruvate Kinase PEPs Up Insulin Secretion and Improves Glucose Homeostasis

The consensus model of glucose-stimulated insulin secretion (GSIS) holds that ATP generation by oxidative phosphorylation directly regulates KATP channel activity and thus insulin granule release, a concept inconsistent with bioenergetic principles. Here, Lewandowski etal. (2020) and Abulizi etal. (2020) report that regulation of GSIS is much more complex as different sources of ATP generation are essential to control this process, which can be targeted invivo and additionally modulate hepatic glucose production. These findings establish an important new conceptual framework of GSIS and invivo glucose homeostasis.

Secreted PDZD2 exerts an insulinotropic effect on INS-1E cells by a PKA-dependent mechanism

Secreted PDZD2 (sPDZD2) is a signaling molecule generated upon proteolytic processing of the multi-PDZ-containing protein PDZD2. Previous analysis of gene-trap mice deficient in the synthesis of full-length PDZD2, but not the secreted form, revealed a role of PDZD2 in the regulation of glucose-stimulated insulin secretion. Here, using the pancreatic INS-1E β cells as in vitro model, we showed that depletion of PDZD2/sPDZD2 by RNA interference suppressed the expression of β-cell genes Ins1, Glut2 and MafA whereas treatment with recombinant sPDZD2 rescued the suppressive effect. Similar to GLP-1, sPDZD2 stimulated intracellular cAMP levels, activated β-cell gene expression in a PKA-dependent manner and induced the phosphorylation and nuclear localization of PDX1. Depletion of PDX1 inhibited the sPDZD2 insulinotropic effect, which could also be demonstrated in mouse islets. In summary, our findings are consistent with sPDZD2 serving a signaling function in regulating β-cell gene expression.

321-OR: Deletion of 14-3-3ζ in Pancreatic ß-Cells Increase Mitochondrial Activity and Insulin Secretion

Pancreatic β-cells continuously sense blood glucose levels and secrete insulin to maintain normoglycemia. In β-cells, ATP generated by glycolysis promotes the closure of ATP-sensitive K+ channels, thereby increasing intracellular calcium and ultimately insulin secretion. 14-3-3 proteins, and in particular 14-3-3ζ, have been found to regulate ATP synthase and mitochondrial respiration, suggesting that members of this family of scaffold proteins in β-cells may influence glucose-stimulated insulin secretion (GSIS) and glucose homeostasis. To date, we have identified critical contributions of 14-3-3 proteins to GSIS. In mouse and human islets, pan-inhibition of 14-3-3 proteins with cell-permeable inhibitors potentiated ex-vivo GSIS. This was associated with increased mitochondrial function, as oxygen consumption and ATP synthesis rates were significantly enhanced. Moreover, increased ATP production was confirmed with luciferase-based assays. Of the seven isoforms, we previously reported critical metabolic roles of 14-3-3ζ in glucose homeostasis, and to understand its role in β-cells, β-cell-specific knockout mice (Ins1CreThor:14-3-3ζfl/fl, β-KO) were generated. When compared to control mice, no differences in body weights or glucose and insulin tolerance were observed, but β-KO mice displayed significantly enhanced insulin secretion following i.p. glucose (2 g/kg), and ex vivo islet perifusion studies revealed enhanced 2nd-phase secretion of GSIS from β-KO islets. Similar to pan-14-3-3 inhibition, increased mitochondrial activity and ATP synthesis were detected in islets of β-KO mice. In summary, these results demonstrate critical functions of 14-3-3ζ and its related proteins in mitochondrial activity in β-cells and the regulation of GSIS. These data also suggest that 14-3-3ζ inhibition may represent a promising target to enhance pancreatic β-cell function in the context of diabetes. Disclosure Y. Mugabo: None. M. Galipeau: None. J. Tan: None. E. Fadzeyeva: None. R. Grygorczyk: None. G.E. Lim: None. Funding Canadian Institutes of Health Research; Canada Research Chairs Program (PJT-153144)

2078-P: APPL2 Deficiency Diminishes Glucose-Stimulated Insulin Secretion in Pancreatic Beta Cells

In response to elevated postprandial blood glucose, remodeling of F-actin enables exocytosis of insulin granules from pancreatic β cells. Dysregulation of F-actin remodeling contributes to defective glucose-stimulated insulin secretion (GSIS) in β cells and subsequent type 2 diabetes. The adaptor protein APPL1 potentiates GSIS and prevents β cell loss in diabetes, but the role of its close homolog APPL2 in β cell function remains unknown. Here we demonstrated that APPL2 controls GSIS by Rac1-mediated F-actin depolymerization via interaction with Rac GTPase activating protein 1 (RacGAP1). β-cell specific deletion of APPL2 induced defective GSIS and glucose intolerance in mice. Ex vivo study revealed a dramatic reduction in both first- and second-phase GSIS in islets, accompanied by a dysregulation of F-actin remodeling. Consistently, knockdown of APPL2 caused impaired glucose-induced F-actin remodeling and insulin secretion in INS-1E β cells. The defects were mainly due to the inactivation of Rac1- the key player controlling insulin granule secretion via F-actin remodeling. Ectopic expression of constitutively active Rac1 mutant rescued the defective GSIS and F-actin remodeling in APPL2 deficient β cells. Furthermore, we identified RacGAP1 as an interacting partner of APPL2. RacGAP1 interacted with and regulate activity of Rac1, and overexpression of RacGAP1 significantly suppressed glucose-induced F-actin depolymerization, whereas co-expression with APPL2 antagonized this suppressive effect. In addition, the defects in APPL2 knockdown β cells were largely reversed by simultaneous downregulation of RacGAP1 or co-expression of constitutively active Rac1 mutant. In summary, our study suggest that APPL2 potentiates GSIS by antagonizing the inhibitory effect of RacGAP1 on Rac1 activation and F-actin remodeling and insulin secretion in β cells, highlighting the importance of APPL2 in the regulation of GSIS in pancreatic β cells and glucose homeostasis. Disclosure H. Lin: None. Funding National Natural Science Foundation of China (81970675)

Proinflammatory Cytokine Interleukin 1β Disrupts β-cell Circadian Clock Function and Regulation of Insulin Secretion.

Intrinsic β-cell circadian clocks are important regulators of insulin secretion and overall glucose homeostasis. Whether the circadian clock in β-cells is perturbed following exposure to prodiabetogenic stressors such as proinflammatory cytokines, and whether these perturbations are featured during the development of diabetes, remains unknown. To address this, we examined the effects of cytokine-mediated inflammation common to the pathophysiology of diabetes, on the physiological and molecular regulation of the β-cell circadian clock. Specifically, we provide evidence that the key diabetogenic cytokine IL-1β disrupts functionality of the β-cell circadian clock and impairs circadian regulation of glucose-stimulated insulin secretion. The deleterious effects of IL-1β on the circadian clock were attributed to impaired expression of key circadian transcription factor Bmal1, and its regulator, the NAD-dependent deacetylase, Sirtuin 1 (SIRT1). Moreover, we also identified that Type 2 diabetes in humans is associated with reduced immunoreactivity of β-cell BMAL1 and SIRT1, suggestive of a potential causative link between islet inflammation, circadian clock disruption, and β-cell failure. These data suggest that the circadian clock in β-cells is perturbed following exposure to proinflammatory stressors and highlights the potential for therapeutic targeting of the circadian system for treatment for β-cell failure in diabetes.

2179-P: Overexpression Screening Identifies Mitochondrial Carbonic Anhydrase 5b as an Important Regulator of Glucose-Stimulated Insulin Secretion in Beta Cells

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiates insulin secretion in a concentration-dependent manner has unveiled. To identify candidate genes important for glucose-stimulated insulin secretion (GSIS), we have conducted screening of differentially expressed genes identified by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, a platform containing zeocin resistant gene flanked with a wild type and mutated flippase recognition target sites was integrated in the genome of MIN6 cells expressing Tet3G. The modified MIN6 cells have been used to generate stable transfectants expressing mRNA or shRNA of genes of interest by means of the recombinease-mediated cassette exchange method. Using this system, more than ten stable MIN6 cell transformants can be gnenerated at a time. By creating approximately 100 overexpressing transfectants, we have identified mitochondrial carbonic anhydrase 5b (Car5b) as an important regulator of GSIS. Car5b is a one of 15 members of carbonic anhydrases, which catalyze the interconversion of carbon dioxide and water to bicarbonate and protons, and modulate various intracellular processes, such as urea formation, HCO3- supply for carboxylation and regulation of intracellular acid-base balance. Car5b overexpression in MIN6 cells increased 14CO2 formation from H14CO3- (37 ± 14% increases, p < 0.05) in isolated mitochondria and resulted in 32 ± 12% increases (P < 0.05) in GSIS at 20 mM, while shCar5b expression reduced Car5b protein levels to 31.4 ± 4.4% (p < 0.05), 14CO2 formation to 68.7 ± 8.1% (p < 0.05), and GSIS at 20 mM to 77 ± 5% (p < 0.05). In addition, we found that Car5b expressions in ob/ob mice were 3.7 ± 0.4-fold higher (p < 0.01) and tended to be higher (p = 0.073) in HFD-mice. These data indicated that mitochondrial carbonic anhydrase Car5b plays a role in GSIS in pancreatic β-cells. Disclosure M. Kosuda: None. S. Yamaguchi: None. A. Nagasawa: None. M. Yamana: None. M. Koike: None. H. Nishioka: None. H. Ishihara: Research Support; Self; Daiichi Sankyo Company, Limited, Lilly Diabetes. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

New roles for dopamine D2 and D3 receptors in pancreatic beta cell insulin secretion.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) plays important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic β-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic β-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in β-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA’s inhibition of GSIS. Our results show that uptake of L-DOPA is essential for establishing intracellular DA stores in β-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which β-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, β-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.