Potentiation Of Glucose-stimulated Insulin Secretion Research Articles

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in β-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the β-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the β cell. We hypothesized β-cell-specific EP3 knockout (EP3 βKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 βKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 βKO mice as compared with wild-type controls, with no effect of β-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 βKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of β-cell replication and survival, revealing severe β-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating β-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of β-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed β-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.

Kisspeptin Modulates Mitochondrial Metabolic State and Potentiates Glucose Stimulated Insulin Secretion in INS 832/3 Cells and Pseudo-Islets

Glucagon Like Peptide-1 (GLP-1) and Kisspeptin (KP) receptors are co-expressed on pancreatic β-cells, and activation of both receptors potentiates glucose-stimulated insulin secretion (GSIS). Due to their roles in enhancing GSIS, GLP-1 and KP represent targets for controlling blood glucose in patients with diabetes. Unlike GLP-1, KP has a low affinity for receptor activation (EC50 GLP-1 ~20 nM vs KP ~1 μM) making it difficult to use as a therapeutic agent. GLP-1 has been shown to potentiate insulin secretion via cAMP dependent pathways. Conversely, previous studies in isolated rat islets of Langerhans indicated that KP potentiation of GSIS was not dependent on cAMP and may be related to changes in beta cell metabolism (Schwetz TA, et al., Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10. PLoS ONE 9: e113020, 2014). In the current studies, we evaluated KP signaling in comparison to GLP-1, in order to determine if there are unique components to target in the KP signaling pathway. The effects of GLP-1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were compared in insulinoma INS 832/3 cells. As previously described, KP had no effect on insulin secretion in the absence of glucose. In the presence of stimulatory glucose (15.5 mM), KP10 (10 μM) doubled insulin secretion (GSIS without KP10= 471.3 +/- 83.8, GSIS with KP10 = 865.0 +/- 23.8 pgm/min; +/- SEM) with an EC50 of ~5-10 μM. At saturating doses, KP10 (100 μm) and GLP-1 (100 nM) had similar effects on GSIS. Importantly, in combination, they had an additive effect (GLP-1 = 612.5 +/- 25.0, GLP-1 + KP-10 = 739.8 +/- 70.0 pgm/min) consistent with activation of independent downstream signaling pathways. GLP-1 increased cAMP production (165.0 +/- 17.4% over control), whereas KP-10 had no effect. Neither GLP-1 nor KP-10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. Conversely, the addition of KP-10 enhanced glucose-stimulated oxygen consumption (% change over baseline O_2 consumption= 221.2 +/- 44.2%) from INS 832/3 pseudo-islets (Hart et al. Insulinoma-derived pseudo-islets for diabetes research. Am. J. Physio. 321, C247-C256, 2021) which was correlated with a sustained increase in mitochondrial NADH fluorescence. Our findings indicate that KP modulation of mitochondrial metabolic state is related to its ability to potentiate GSIS. Moreover, the KP induced potentiation is synergistic with that promoted by GLP-1 indicating the potential for targeting the KP activated metabolic pathway to further enhance GLP-1 therapeutic effectiveness. Funded by the Juvenile Diabetes Research Foundation (2-SRA-2018-685-S-B), and Procyon Technologies LLC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The selective serotonin reuptake inhibitor fluoxetine has direct effects on beta cells, promoting insulin secretion and increasing beta-cell mass.

AimThis study investigated whether therapeutically relevant concentrations of fluoxetine, which have been shown to reduce plasma glucose and glycated haemoglobin independent of changes in food intake and body weight, regulate beta‐cell function and improve glucose homeostasis.MethodsCell viability, insulin secretion, beta‐cell proliferation and apoptosis were assessed after exposure of MIN6 beta cells or isolated mouse and human islets to 0.1, 1 or 10 μmol/L fluoxetine. The effect of fluoxetine (10 mg/kg body weight) administration on glucose homeostasis and islet function was also examined in ob/ob mice.ResultsExposure of MIN6 cells and mouse islets to 0.1 and 1 μmol/L fluoxetine for 72 hours did not compromise cell viability but 10 μmol/L fluoxetine significantly increased Trypan blue uptake. The dose of 1 μmol/L fluoxetine significantly increased beta‐cell proliferation and protected islet cells from cytokine‐induced apoptosis. In addition, 1 μmol/L fluoxetine induced rapid and reversible potentiation of glucose‐stimulated insulin secretion from islets isolated from mice, and from lean and obese human donors. Finally, intraperitoneal administration of fluoxetine to ob/ob mice over 14 days improved glucose tolerance and resulted in significant increases in beta‐cell proliferation and enhanced insulin secretory capacity.ConclusionsThese data are consistent with a role for fluoxetine in regulating glucose homeostasis through direct effects on beta cells. Fluoxetine thus demonstrates promise as a preferential antidepressant for patients with concomitant occurrence of depression and diabetes.

Involvement of P2Y signaling in the restoration of glucose-induced insulin exocytosis in pancreatic β cells exposed to glucotoxicity.

Purinergic P2Y receptors, by binding adenosine triphosphate (ATP), are known for enhancing glucose-stimulated insulin secretion (GSIS) in pancreatic β cells. However, the impact of these receptors in the actin dynamics and insulin granule exocytosis in these cells is not established, neither in normal nor in glucotoxic environment. In this study, we investigate the involvement of P2Y receptors on the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β cells exposed to normal or glucotoxic environment and their role in GSIS. Our results show that the activation of P2Y purinergic receptors by ATP or its agonist increase the insulin granules exocytosis and the reorganization of the subcortical actin network and participate in the potentiation of GSIS. In addition, their activation in INS-1832/13 β-cells, with impaired insulin secretion following exposure to elevated glucose levels, restores GSIS competence through the distal steps of insulin exocytosis. These results are confirmed ex vivo by perifusion experiments on islets from type 2 diabetic (T2D) Goto-Kakizaki (GK) rats. Indeed, the P2Y receptor agonist restores the altered GSIS, which is normally lost in this T2D animal model. Moreover, we observed an improvement of the glucose tolerance, following the acute intraperitoneal injection of the P2Y agonist concomitantly with glucose, in diabetic GK rats. All these data provide new insights into the unprecedented therapeutic role of P2Y purinergic receptors in the pathophysiology of T2D.

Chronic Fetal Leucine Infusion Does Not Potentiate Glucose-Stimulated Insulin Secretion or Affect Pancreatic Islet Development in Late-Gestation Growth-Restricted Fetal Sheep

Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic β-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising β-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, β-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50-100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising β-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and β-cell consequences in IUGR fetuses.

Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice

ObjectiveMaintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, β, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. MethodsGlucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, β, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. ResultsAcute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and β cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. ConclusionsInhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.

Acute D-Serine Co-Agonism of β-Cell NMDA Receptors Potentiates Glucose-Stimulated Insulin Secretion and Excitatory β-Cell Membrane Activity.

Insulin-secreting pancreatic β-cells express proteins characteristic of D-serine regulated synapses, but the acute effect of D-serine co-agonism on its presumptive β-cell target, N-methyl D-aspartate receptors (NMDARs), is unclear. We used multiple models to evaluate glucose homeostasis and insulin secretion in mice with a systemic increase in D-serine (intraperitoneal injection or DAAO mutants without D-serine catabolism) or tissue-specific loss of Grin1-encoded GluN1, the D-serine binding NMDAR subunit. We also investigated the effects of D-serine ± NMDA on glucose-stimulated insulin secretion (GSIS) and β-cell depolarizing membrane oscillations, using perforated patch electrophysiology, in β-cell-containing primary isolated mouse islets. In vivo models of elevated D-serine correlated to improved blood glucose and insulin levels. In vitro, D-serine potentiated GSIS and β-cell membrane excitation, dependent on NMDAR activating conditions including GluN1 expression (co-agonist target), simultaneous NMDA (agonist), and elevated glucose (depolarization). Pancreatic GluN1-loss females were glucose intolerant and GSIS was depressed in islets from younger, but not older, βGrin1 KO mice. Thus, D-serine is capable of acute antidiabetic effects in mice and potentiates insulin secretion through excitatory β-cell NMDAR co-agonism but strain-dependent shifts in potency and age/sex-specific Grin1-loss phenotypes suggest that context is critical to the interpretation of data on the role of D-serine and NMDARs in β-cell function.

Dose-dependent efficacy of the glucose-dependent insulinotropic polypeptide (GIP) receptor antagonist GIP(3-30)NH2 on GIP actions in humans.

The glucose-dependent insulinotropic polypeptide (GIP) fragment GIP(3-30)NH2 is a selective, competitive GIP receptor antagonist, and doses of 800 to 1200 pmol/kg/min inhibit GIP-induced potentiation of glucose-stimulated insulin secretion by >80% in humans. We evaluated the effects of GIP(3-30)NH2 across a wider dose range in eight healthy men undergoing six separate and randomized 10-mmol/L hyperglycaemic clamps (A-F) with concomitant intravenous infusion of GIP (1.5 pmol/kg/min; A-E) or saline (F). Clamps A to E involved double-blinded, infusions of saline (A) and GIP(3-30)NH2 at four rates: 2 (B), 20 (C), 200 (D) and 2000 pmol/kg/min (E), respectively. Mean plasma concentrations of glucose (A-F) and GIP (A-E) were similar. GIP-induced potentiation of glucose-stimulated insulin secretion was reduced by 44 ± 10% and 84 ± 10% during clamps D and E, respectively. Correspondingly, the amounts of glucose required to maintain the clamp during D and E were not different from F. GIP-induced suppression of bone resorption and increase in heart rate were lowered by clamps D and E. In conclusion, GIP(3-30)NH2 provides extensive, dose-dependent inhibition of the GIP receptor in humans, with most pronounced effects of the doses 200 to 2000 pmol/kg/min within the tested range.

GIP and GLP-1 Potentiate Sulfonylurea-Induced Insulin Secretion in Hepatocyte Nuclear Factor 1α Mutation Carriers.

Sulfonylureas (SUs) provide an efficacious first-line treatment in patients with hepatocyte nuclear factor 1α (HNF1A) diabetes, but SUs have limitations due to risk of hypoglycemia. Treatment based on the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1) is characterized by their glucose-dependent insulinotropic actions without risk of hypoglycemia. The effect of SUs together with GIP or GLP-1, respectively, on insulin and glucagon secretion in patients with HNF1A diabetes is currently unknown. To investigate this, 10 HNF1A mutation carriers and 10 control subjects without diabetes were recruited for a double-blinded, placebo-controlled, crossover study including 6 experimental days in a randomized order involving 2-h euglycemic-hyperglycemic clamps with coadministration of: 1) SU (glimepiride 1 mg) or placebo, combined with 2) infusions of GIP (1.5 pmol/kg/min), GLP-1 (0.5 pmol/kg/min), or saline (NaCl). In HNF1A mutation carriers, we observed: 1) hypoinsulinemia, 2) insulinotropic effects of both GIP and GLP-1, 3) additive to supra-additive effects on insulin secretion when combining SU+GIP and SU+GLP-1, respectively, and 4) increased fasting and arginine-induced glucagon levels compared with control subjects without diabetes. Our study suggests that a combination of SU and incretin-based treatment may be efficacious in patients with HNF1A diabetes via potentiation of glucose-stimulated insulin secretion.

Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research Foundation

Myricetin prevents thapsigargin-induced CDK5-P66Shc signalosome mediated pancreatic β-cell dysfunction

Chronic endoplasmic reticulum (ER) stress has deleterious effects on pancreatic β-cell function and survival in type 2 diabetes (T2D). Cyclin-dependent kinase 5 (CDK5) plays a critical role in β-cell failure under diabetic milieu conditions. However, little information is available on CDK5's ability to impair the function of β-cells via a chemical ER stress inducer thapsigargin.Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. Therefore, we examined the effect of CDK5 on thapsigargin-induced β-cell apoptosis, and explored the relationship between myricetin and CDK5. Exposure of beta cells with thapsigargin, induced a Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Activated CDK5 induced antiapoptotic protein myeloid cell leukemia sequence 1 (Mcl-1) degradation which was associated with p66Shc serine 36 phosphorylation, causing beta cell apoptosis via mitochondrial dysfunction. Exposure of beta cells to myricetin resulted in an acute inhibition of Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Myricetin inhibited CDK5 activation by directly binding to its ATP-binding pocket. Treatment with myricetin also enhanced the stability of Mcl-1 after thapsigargin treatment. Inhibition of CDK5 with myricetin or roscovitine, a CDK5 inhibitor attenuates thapsigargin induced p66Shc serine 36 phosphorylation and also reduced mitochondrial dysfunction by decreasing mitochondrial ROS and caspase-3 activation. In addition, myricetin was observed to enhance PDX-1 and insulin mRNA expression and potentiate glucose stimulated insulin secretion (GSIS). Taken together, these findings indicate that thapsigargin-induced early molecular events lead to CDK5-p66Shc signalosome contributes to thapsigargin-induced pancreatic β-cell dysfunction. Myricetin blocked thapsigargin induced CDK5-p66Shc signalosome formation and prevented pancreatic beta cell dysfunction. In this study, we demonstrated for the first time that thapsigargin initiated CDK5-p66Shc signalosome mediates the pancreatic beta cell dysfunction and myricetin protects the pancreatic beta cells through the inhibition of CDK5-p66Shc signalosome.

CAMP-PKA dependent ERK1/2 activation is necessary for vanillic acid potentiated glucose-stimulated insulin secretion in pancreatic β-cells

Vanillic acid (VA), a dietary phenolic compound is generally studied for its anti-oxidative and anti-inflammatory effects. However, the effect of VA on insulin secretion and its mechanism of action has never been explored. In this study, we report that VA augments glucose-stimulated insulin secretion (GSIS) in both insulin-secreting cell-line INS-1 and isolated rat pancreatic islets. Potentiation of GSIS is accompanied by a concurrent increase in 3′,5′-cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) in INS-1 and rat islets. The activated cAMP-PKA pathway, in turn, phosphorylates extracellular signal-regulated kinases 1/2 (ERK1/2) in INS-1 cells. Pharmacological intervention with PKA and ERK1/2 inhibitors revealed that VA potentiated GSIS is primarily dependent on PKA mediated ERK1/2 activity. These findings demonstrated that VA directly acts on insulin-secreting pancreatic β-cells to exert its insulinotropic effect thereby providing a novel role of VA in the regulation of insulin secretion.

The functional impact of G protein-coupled receptor 142 (Gpr142) on pancreatic \u03b2-cell in rodent

We have recently shown that the G protein-coupled receptor 142 (GPR142) is expressed in both rodent and human pancreatic β-cells. Herein, we investigated the cellular distribution of GPR142 within islets and the effects of selective agonists of GPR142 on glucose-stimulated insulin secretion (GSIS) in the mouse islets and INS-1832/13 cells. Double-immunostaining revealed that GPR142 immunoreactivity in islets mainly occurs in insulin-positive cells. Potentiation of GSIS by GPR142 activation was accompanied by increased cAMP content in INS-1832/13 cells. PKA/Epac inhibition markedly suppressed the effect of GPR142 activation on insulin release. Gpr142 knockdown (Gpr142-KD) in islets was accompanied by elevated release of MCP-1, IFNγ, and TNFα during culture period and abolished the modulatory effect of GPR142 activation on the GSIS. Gpr142-KD had no effect on Ffar1, Ffar2, or Ffar3 mRNA while reducing Gpr56 and increasing Tlr5 and Tlr7 mRNA expression. Gpr142-KD was associated with an increased expression of Chrebp, Txnip, RhoA, and mitochondrial Vdac1 concomitant with a reduced Pdx1, Pax6, and mitochondrial Vdac2 mRNA levels. Long-term exposure of INS-1832/13 cells to hyperglycemia reduced Gpr142 and Vdac2 while increased Chrebp, Txnip, and Vdac1 mRNA expression. GPR142 agonists or Bt2-cAMP counteracted this effect. Glucotoxicity-induced decrease of cell viability in Gpr142-KD INS-1 cells was not affected by GPR142-agonists while Bt2-cAMP prevented it. The results show the importance of Gpr142 in the maintenance of pancreatic β-cell function in rodents and that GPR142 agonists potentiate GSIS by an action, which most likely is due to increased cellular generation of second messenger molecule cAMP.

Negative Effects of Cyclic Palmitate Treatment on Glucose Responsiveness and Insulin Production in Mouse Insulinoma Min6 Cells Are Reversible.

Pancreatic β-cell failure is characterized by compromised insulin secretion in response to glucose, which ultimately results in hyperglycemia, the clinical hallmark of type 2 diabetes mellitus (T2DM). Acute exposure to plasma free fatty acids (FFAs) potentiates glucose stimulated insulin secretion (GSIS), while chronic exposure impairs GSIS, and the latter has been associated with the mechanism of β cell failure in obesity linked T2DM. By contrast, growth hormone (GH) signaling has been linked positively to GSIS in β cells. Numerous studies have examined chronic exposure of β cells to elevated FFAs both with in vivo cohorts and in vitro models. Little attention, however, has been given to the fluctuation of plasma FFA levels due to rhythmic effects that are affected by daily diet and fat intake. Mouse insulinoma Min6 cells were exposed to cyclic/daily palmitate treatment over 2 and 3 days to assess effects on GSIS. Cyclic/daily palmitate treatment with a period of recovery negatively affected GSIS in a dose-dependent manner. Removal of palmitate after two cycles/day resulted in reversal of the effect on GSIS, which was also reflected by relative gene expression involved in insulin biosynthesis (Ins1, Ins2, Pdx1, and MafA) and GSIS (glucose 2 transporter and glucokinase). Modest positive effects on GSIS and glucokinase transcript levels were also observed when Min6 cells were cotreated with human GH and palmitate. These observations indicate that like continuous palmitate treatment, cyclic exposure to palmitate can acutely impair GSIS over 48 and 72 h. However, they also suggest that the negative effects of short periods of exposure to FFAs on β cell function remain reversible.

Activation of imidazoline receptor I2, and improved pancreatic β-cell function in human islets

AimThe impact of BL11282, an imidazoline receptor (NISCH) agonist, on potentiation of glucose-stimulated insulin secretion (GSIS) from isolated human non-diabetic (ND) and type 2 diabetic (T2D) islets was investigated. MethodsAnalysis of mRNA was performed by RNA-sequencing and qPCR. Insulin and cAMP by RIA and ELISA respectively. ResultsRNA-sequencing data revealed that NISCH is highly expressed in fat tissues, islets, liver and muscles, with eight detectable splice variants of transcripts in islets. NISCH had a positive correlation with GLP-1 (GLP1R) and GIP (GIPR) receptor transcripts. The expression of NISCH was confirmed by qPCR in human islets. NISCH and GLP1R were comparably higher expressed in mouse islets compared to human islets. GSIS was dose-dependently potentiated by BL11282 from incubated islets of ND and T2D human islet donors. The insulinotropic action of BL11282 was associated with increased cAMP. While the harmful effect of high glucose on reductive capacity of islet cells was enhanced by glibenclamide during long-term culture, it was counteracted by BL11282 or Bt2-cAMP. BL11282 also increased proliferation of INS-1 cells during long-time culture. ConclusionOur data suggest that BL11282 potentiates GSIS by an action involving cAMP/PKA system and BL11282 could be an attractive insulinotropic and β-cell protective agent.

Reversion of aging-related DHEAS decline in mouse plasma alleviates aging-related glucose tolerance impairment by potentiation of glucose-stimulated insulin secretion of acute phase

Aims/hypothesisThe latest research proposes mild age-related diabetes (MARD) as a subgroup of type 2 diabetes. While in human circulating dehydroepiandrosterone sulfate (DHEAS) decline with age is related to MARD, the role of circulating DHEAS in insulin secretion remains little known. MethodsAfter intraperitoneal administration of glucose (2 g/kg) together with DHEAS (50 μg/kg) or equivalent DMSO to young (6–8 week old) or aging (12 month old) male C57BL/6 mice, plasma DHEAS and blood glucose were measured at indicated time point. Then in vitro, we investigated DHEAS effects on GSIS of acute phase in aging mice pancreatic islets as well as in MIN6 cells. Finally we conducted pharmacological studies in MIN6 cells to examine whether exert its effects on insulin secretion by itself. ResultsWe found in vivo that aging mice had lower plasma DHEAS levels and impaired glucose tolerence compared to young mice and that the aged mice but not the young mice receiving DHEAS supplement had improved glucose tolerance as soon as 15 min after glucose injection compared to the ones with DMSO administration. These results indicate that in male mice, aging-related DHEAS decline in plasma contribute to aging-related impairment of glucose tolerence and that reversion of aging-related DHEAS deficiency in aging mice plasma could alleviate aging-related glucose tolerance impairment. Consistently, in vitro DHEAS glucose-and dose-dependently potentiated glucose-stimulated insulin secretion (GSIS) of acute phase in both aging male mice pancreatic islets and MIN6 cells. Moreover, none of steroid sulfatase (STS) inhibitor STX64 (10 nM), androgen receptor (AR) blocker flutamide (1 mM) or estrogen receptor (ER) antagonist ICI182780 (1 mM), affected DHEAS-potentiated high GSIS of acute phase indicating this potentiation exercised by DHEAS per se Conclusions/interpretation These results lead us to tentatively conclude that aging-related DHEAS decline may imply MARD development and that adequate DHEAS supplement may be a precise medicine and preventive measure for MARD.

Enhancing β‐cell Sensitivity to Kisspeptin via Linkage to GLP‐1

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic β‐cells, and activation of both receptors potentiate glucose stimulated insulin secretion (GSIS), although they appear to work via different downstream signaling pathways. Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 20 nM vs >1 uM) making it difficult to use as a drug without significant side effects. We developed a bivalent ligand composed of GLP‐17–36 and KP‐10 binding domains linked together, to evaluate if linking KP to a high affinity ligand would promote its activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. This feature may also provide some specificity for targeting to β‐cells which express both receptors. The effects of KP‐10, GLP‐1 and the KP‐10/GLP‐1 bivalent analog on insulin secretion from the INS 832/3 cell line were investigated. KP‐10 by itself had no effect on insulin secretion in the absence of glucose, but more than doubled GSIS (basal, 0.23 +/− 0.07; GSIS 0.60 +/− 0.02 ng/min) with an EC50 of ~25 μM. At saturating doses, KP‐10 (100 μm) and GLP‐1 (100 nM) had similar effects on GSIS, and together as monomers had an additive effect (0.79 +/− 0.07 (ng/min) consistent with activation of independent downstream signaling pathways. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers (0.64 +/− 0.01 ng/min); that is, an additive response was not obvious. This finding suggests that either the KP‐10 moiety is not binding or is not active in the bivalent ligand, or that GLP‐1 activity is diminished in the bivalent construct such that the combined effect of GLP‐1/KP‐10 is diminished when compared to the additive effect or the monomers. We have evidence from previous studies that binding of the GLP‐1 moiety within a bivalent construct is decreased relative to monomeric GLP‐11, suggesting an additive response may be present. Currently, we are determining the ability of GLP‐1/KP‐10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP‐10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epicatechin potentiation of glucose-stimulated insulin secretion in INS-1 cells is not dependent on its antioxidant activity.

Epicatechin (EC) is a monomeric flavan-3-ol. We have previously demonstrated that glucose-intolerant rats fed flavan-3-ols exhibit improved pancreatic islet function corresponding with an increase in circulating EC-derived metabolites. Thus, we speculate that EC may act as a cellular signaling molecule in vivo to modulate insulin secretion. In this study we further examined the effects of different concentrations of EC on H2O2 or hyperglycemia-induced ROS production, as well as on saturated fatty acid (SFA)-impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cell line in vitro. We showed that EC at a high concentration (30 μmol/L), but not a low concentration (0.3 μmol/L), significantly decreased H2O2 or hyperglycemia-induced ROS production in INS-1 cells. However, EC (0.3 μmol/L) significantly enhanced SFA-impaired GSIS in INS-1 cells. Addition of KN-93, a CaMKII inhibitor, blocked the effect of EC on insulin secretion and decreased CaMKII phosphorylation. Addition of GW1100, a GPR40 antagonist, significantly attenuated EC-enhanced GSIS, but only marginally affected CaMKII phosphorylation. These results demonstrate that EC at a physiological concentration promotes GSIS in SFA-impaired β-cells via activation of the CaMKII pathway and is consistent with its function as a GPR40 ligand. The findings support a role for EC as a cellular signaling molecule in vivo and further delineate the signaling pathways of EC in β-cells.

LH-21 and abnormal cannabidiol improve β-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55-/- mice. Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca2+ ]i, cAMP, apoptosis, β-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca2+ ]i in human islets and islets from both GPR55+/+ and GPR55-/- mice. LH-21 also increased insulin secretion and [Ca2+ ]i in human islets and GPR55+/+ mouse islets, but concentrations of LH-21 up to 0.1 μM were ineffective in islets from GPR55-/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55+/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased β-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55-/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. This study showed that Abn-CBD and LH-21 improve human and mouse islet β-cell function and viability. Use of islets from GPR55-/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised.