The CB1 Antagonist Rimonabant Decreases Insulin Hypersecretion in Rat Pancreatic Islets

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose‐stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL‐treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL‐treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL‐exposed islets.     

Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.     

A low-protein diet during pregnancy alters glucose metabolism and insulin secretion

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low-protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l(-1) of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l(-1) of glucose in LPP and CP groups, respectively. The glucose dose-response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration-response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an "inverted U-shape" dose-response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that β-cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events.     

高糖通过下调FoxO1磷酸化诱导β细胞去分化

胰岛β细胞发生去分化现象是导致其功能减退的机制之一。已有研究证明,FoxO1与β细胞去分化密切相关。然而,高糖是否可通过FoxO1诱导β细胞发生去分化目前尚未见报告。本研究通过不同浓度高糖干预MIN6细胞,采用葡萄糖刺激胰岛素分泌试验（GSIS）检测β细胞功能|实时荧光定量PCR及蛋白免疫印迹、免疫荧光方法检测高糖干预后β细胞内祖细胞标志基因、β细胞标志基因及FoxO1的表达变化。结果显示,不同浓度高糖干预β细胞后,当浓度达到35 mmol/L时,β细胞祖细胞标志基因表达明显增加。且在该浓度时,检测到β细胞标志基因表达明显降低,MIN6细胞葡萄糖刺激胰岛素分泌功能减退,磷酸化FoxO1表达减少。上述结果提示,高糖可诱导胰岛β细胞去分化的发生,其机制可能是通过FoxO1介导。     

Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (KITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.     

Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus

GSIS is often measured in the sheep fetus by a square-wave hyperglycemic clamp, but maximal β-cell responsiveness and effects of fetal number and sex difference have not been fully evaluated. We determined the dose-response curve for GSIS in fetal sheep (0.9 of gestation) by increasing plasma glucose from euglycemia in a stepwise fashion. The glucose-insulin response was best fit by curvilinear third-order polynomial equations for singletons (y = 0.018x(3) - 0.26x(2) + 1.2x - 0.64) and twins (y = -0.012x(3) + 0.043x(2) + 0.40x - 0.16). In singles, maximal insulin secretion was achieved at 3.4 ± 0.2 mmol/l glucose but began to plateau after 2.4 ± 0.2 mmol/l glucose (90% of maximum), whereas the maximum for twins was reached at 4.8 ± 0.4 mmol/l glucose. In twin (n = 18) and singleton (n = 49) fetuses, GSIS was determined with a square-wave hyperglycemic clamp >2.4 mmol/l glucose. Twins had a lower basal glucose concentration, and plasma insulin concentrations were 59 (P < 0.01) and 43% (P < 0.05) lower in twins than singletons during the euglycemic and hyperglycemic periods, respectively. The basal glucose/insulin ratio was approximately doubled in twins vs. singles (P < 0.001), indicating greater insulin sensitivity. In a separate cohort of fetuses, twins (n = 8) had lower body weight (P < 0.05) and β-cell mass (P < 0.01) than singleton fetuses (n = 7) as a result of smaller pancreata (P < 0.01) and a positive correlation (P < 0.05) between insulin immunopositive area and fetal weight (P < 0.05). No effects of sex difference on GSIS or β-cell mass were observed. These findings indicate that insulin secretion is less responsive to physiological glucose concentrations in twins, due in part to less β-cell mass.     

Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca²⁺ to glucose and depolarization but to a lesser degree suggesting that altered Ca²⁺ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.     

Beneficial effect of pretreatment of islets with fibronectin on glucose tolerance after islet transplantation.

The scarcity of available islets is an obstacle for clinically successful islet transplantation. One solution might be to increase the efficacy of the limited islets. Isolated islets are exposed to a variety of cellular stressors, and disruption of the cell-matrix connections damages islets. We examined the effect of fibronectin, a major component of the extracellular matrix, on islet viability, mass and function, and also examined whether fibronectin-treated islets improved the results of islet transplantation. Islets cultured with fibronectin for 48 hours maintained higher cell viability (0.146 +/- 0.010 vs. 0.173 +/- 0.007 by MTT assay), and also had a greater insulin and DNA content (86.8 +/- 3.6 vs. 72.8 +/- 3.2 ng/islet and 35.2 +/- 1.4 vs. 30.0 +/- 1.5 ng/islet, respectively) than islets cultured without fibronectin (control). Absolute values of insulin secretion were higher in fibronectin-treated islets than in controls; however, the ratio of stimulated insulin secretion to basal secretion was not significantly different (206.9 +/- 23.3 vs. 191.7 +/- 20.2% when the insulin response to 16.7 mmol/l glucose was compared to that of 3.3 mmol/l glucose); the higher insulin secretion was thus mainly due to larger islet cell mass. The rats transplanted with fibronectin-treated islets had lower plasma glucose and higher plasma insulin levels within 2 weeks after transplantation, and had more favorable glucose tolerance 9 weeks after transplantation. These results indicate that cultivation with fibronectin might preserve islet cell viability, mass and insulin secretory function, which could improve glucose tolerance following islet transplantation.     

Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects

Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 is positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 is negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c ? 6.1). We suggest that the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.     

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.     

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.     

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.     

The effects of interleukin-1 on pancreatic beta cell function in vitro depend on the glucose concentration.

Insulin-dependent diabetes mellitus is characterized by progressive autoimmune destruction of pancreatic Beta cells mediated by ill-defined effector mechanisms. Experimental data suggest that cytokines, e.g. interleukin 1 and tumor necrosis factor, could play a fundamental role. The aim of this study was to analyze the effect of recombinant IL-1 beta (rIL-1 beta) on both islet functional capacity and morphology, using long-term cultures and various glucose concentrations. Islet cultured with 1 g/l (5.5 mmol/l) glucose maintained normal insulin- secretion and morphology for more than two months. In contrast, islets cultured with 2 g/l (11 mmol/l) glucose showed an altered insulin secretion and a shorter survival (40 days). At 11 g/l (60 mmol/l) glucose, islets died by 2 weeks of culture. rIL-1 beta exerted a cytotoxic effect on islet cells only when added to cultures containing supraphysiological glucose concentrations. But, in the presence of 1 g/l glucose, the addition of rIL-1 beta (40 ng/ml) for prolonged periods (14 days), did not alter islet function. Our results suggest that in auto-immune type I diabetes, IL-1 beta represents an aggravating factor in lesion formation more than a primary pathogenic mechanism.     

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.     

Plasma glucose regulation and insulin secretion in hypertriglyceridemic mice.

In this study, we examined glucose homeostasis and insulin secretion in transgenic mice overexpressing the human apolipoprotein CIII gene (apo CIII tg). These mice have elevated plasma levels of triglycerides, FFA and cholesterol compared to control mice. The body weight, plasma glucose, and insulin levels, glucose disappearance rates, areas under the ipGTT curve for adult (4 - 8 mo. old) and aged (20 - 24 mo. old) apo CIII tg mice and the determination of insulin during the ipGTT were not different from those of control mice. However, an additional elevation of plasma FFA by treatment with heparin for 2 - 4 h impaired the ipGTT responses in apo CIII tg mice compared to saline-treated mice. The glucose disappearance rate in heparin-treated transgenic mice was slightly lower than in heparin-treated controls. Glucose (22.2 mmol/l) stimulated insulin secretion in isolated islets to the same extent in saline-treated control and apo CIII tg mice. In islets from heparin-treated apo CIII tg mice, the insulin secretion at 2.8 and 22.2 mmol glucose/l was lower than in heparin-treated control mice. In conclusion, hypertriglyceridemia per se or a mild elevation in FFA did not affect insulin secretion or insulin resistance in adult or aged apo CIII tg mice. Nonetheless, an additional elevation of FFA induced by heparin in hypertriglyceridemic mice impaired the ipGTT by reducing insulin secretion.     

The apj receptor is expressed in pancreatic islets and its ligand, apelin, inhibits insulin secretion in mice

Apelin is the endogenous ligand of the G-protein coupled apj receptor. Apelin is expressed in the brain, the hypothalamus and the stomach and was recently shown also to be an adipokine secreted from the adipocytes. Although apelin has been suggested to be involved in the regulation of food intake, it is not known whether the peptide affects islet function and glucose homeostasis. We show here that the apj receptor is expressed in pancreatic islets and that intravenous administration of full-length apelin-36 (2 nmol/kg) inhibits the rapid insulin response to intravenous glucose (1 g/kg) by 35% in C57BL/6J mice. Thus, the acute (1-5 min) insulin response to intravenous glucose was 682+/-23 pmol/l after glucose alone (n=17) and 445+/-58 pmol/l after glucose plus apelin-36 (n=18; P=0.017). This was associated with impaired glucose elimination (the 5-20 min glucose elimination was 2.9+/-0.1%/min after glucose alone versus 2.3+/-0.2%/min after glucose plus apelin-36, P=0.008). Apelin (2 nmol/kg) also inhibited the insulin response to intravenous glucose in obese insulin resistant high-fat fed C57BL/6J mice (P=0.041). After 60 min incubation of isolated islets from normal mice, insulin secretion in the presence of 16.7 mmol/l glucose was inhibited by apelin-36 at 1 mumol/l, whereas apelin-36 did not significantly affect insulin secretion at 2.8 or 8.3 mmol/l glucose or after stimulation of insulin secretion by KCl. Islet glucose oxidation at 16.7 mmol/l was not affected by apelin-36. We conclude that the apj receptor is expressed in pancreatic islets and that apelin-36 inhibits glucose-stimulated insulin secretion both in vivo and in vitro. This may suggest that the islet beta-cells are targets for apelin-36.     

Stevioside does not cause increased basal insulin secretion or beta-cell desensitization as does the sulphonylurea, glibenclamide: studies in vitro

We have shown that stevioside (SVS) enhances insulin secretion and thus may have a potential role as antihyperglycemic agent in the treatment of type 2 diabetes mellitus. However, whether SVS stimulates basal insulin secretion (BIS) and/or cause desensitization of beta cells like sulphonylureas (SU), e.g. glibenclamide (GB), is not known. To explore and compare the effects of SVS pretreatment with those of GB and glucagon-like peptide-1 (GLP-1), we exposed isolated mouse islets to low or high glucose for 1 h after short-term (2 h) or long-term (24 h) pretreatment with SVS, GB or GLP-1, respectively. BIS at 3.3 or 5.5 mM glucose were not changed after short-term pretreatment with SVS (10(-7) M), while it increased about three folds after pretreatment with GB (10(-7) M). Glucose stimulated insulin secretion (GSIS) (16.7 mM) increased dose-dependently after long-term pretreatment with SVS at concentrations from 10(-7) to 10(-5) M. Pretreatment for 24 h with GB (10(-7) M) increased the subsequent BIS (3.3 mM glucose) (p < 0.001), but decreased GSIS (16.7 mM glucose) (p < 0.001). In contrast SVS (10(-7) M) and GLP-1 (10(-7) M) did not stimulate BIS but both enhanced the subsequent GSIS (16.7 mM glucose) (p < 0.05 and p < 0.05, respectively). While SVS pretreatment increased the intracellular insulin content, GB pretreatment decreased the insulin content. Our study suggests that SVS pretreatment does not cause a stimulation of BIS and does not desensitize beta-cells, i.e. SVS seems to have advantageous characteristics to GB as a potential treatment of type 2 diabetes.     

Intra-islet glucagon confers β-cell glucose competence for first-phase insulin secretion and favors GLP-1R stimulation by exogenous glucagon

We report that intra-islet glucagon secreted from α-cells signals through β-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9–39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on β-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a “spillover” effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the β-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9–39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test, that is, diagnostic of type 2 diabetes in the clinical setting.     

The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.