SREBP1 is required for the induction by glucose of pancreatic beta-cell genes involved in glucose sensing.
ABSTRACT: Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K(+) channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1(-/-)) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1(-/-) mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.
Project description:Glucose metabolism stimulates cell division control protein 42 homolog (Cdc42)-p21-activated kinase (Pak1) activity and initiates filamentous actin (F-actin) cytoskeleton remodeling in pancreatic β-cells so that cytoplasmic secretory granules can translocate to the plasma membrane where insulin exocytosis occurs. Since glucose metabolism also generates cAMP in β-cells, the cross talk of cAMP signaling with Cdc42-Pak1 activation might be of fundamental importance to glucose-stimulated insulin secretion (GSIS). Previously, the type-2 isoform of cAMP-regulated guanine nucleotide exchange factor 2 (Epac2) was established to mediate a potentiation of GSIS by cAMP-elevating agents. Here we report that nondiabetic human islets and INS-1 832/13 β-cells treated with the selective Epac activator 8-pCPT-2'-O-Me-cAMP-AM exhibited Cdc42-Pak1 activation at 1 mmol/L glucose and that the magnitude of this effect was equivalent to that which was measured during stimulation with 20 mmol/L glucose in the absence of 8-pCPT-2'-O-Me-cAMP-AM. Conversely, the cAMP antagonist Rp-8-Br-cAMPS-pAB prevented glucose-stimulated Cdc42-Pak1 activation, thereby blocking GSIS while also increasing cellular F-actin content. Although islets from donors with type 2 diabetes had profound defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion, these defects were rescued by the Epac activator so that GSIS was restored. Collectively, these findings indicate an unexpected role for cAMP as a permissive or direct metabolic coupling factor in support of GSIS that is Epac2 and Cdc42-Pak1 regulated.
Project description:AIM/HYPOTHESIS:Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl(2), a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. METHODS:Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive ?-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. RESULTS:As observed for Mt1a mRNA levels, ?-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 µM ZnCl(2) to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late ?-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. CONCLUSION:ZnCl(2) reduces mitochondrial oxidative stress and improves rat ?-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.
Project description:Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAs(III), MAs(III) or DMAs(III) inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs(III) and DMAs(III) were more potent than iAs(III) as GSIS inhibitors with estimated IC(50)?0.1 ?M. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAs(III), MAs(III) or DMAs(III) could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic ?-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes.
Project description:Intrauterine growth-restricted (IUGR) fetuses experience prolonged hypoxemia, hypoglycemia, and elevated norepinephrine (NE) concentrations, resulting in hypoinsulinemia and ?-cell dysfunction. Previously, we showed that acute adrenergic blockade revealed enhanced insulin secretion responsiveness in the IUGR fetus. To determine whether chronic exposure to NE alone enhances ?-cell responsiveness afterward, we continuously infused NE into fetal sheep for 7 days and, after terminating the infusion, evaluated glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-induced insulin secretion (GPAIS). During treatment, NE-infused fetuses had greater (P < 0.05) plasma NE concentrations and exhibited hyperglycemia (P < 0.01) and hypoinsulinemia (P < 0.01) compared with controls. GSIS during the NE infusion was also reduced (P < 0.05) compared with pretreatment values. GSIS and GPAIS were approximately fourfold greater (P < 0.01) in NE fetuses 3 h after the 7 days that NE infusion was discontinued compared with age-matched controls or pretreatment GSIS and GPAIS values of NE fetuses. In isolated pancreatic islets from NE fetuses, mRNA concentrations of adrenergic receptor isoforms (?1D, ?2A, ?2C, and ?1), G protein subunit-?i-2, and uncoupling protein 2 were lower (P < 0.05) compared with controls, but ?-cell regulatory genes were not different. Our findings indicate that chronic exposure to elevated NE persistently suppresses insulin secretion. After removal, NE fetuses demonstrated a compensatory enhancement in insulin secretion that was associated with adrenergic desensitization and greater stimulus-secretion coupling in pancreatic islets.
Project description:Glucose-stimulated insulin secretion (GSIS) is essential in keeping blood glucose levels within normal range. GSIS is impaired in type 2 diabetes, and its recovery is crucial in treatment of the disease. We find here that sphingosine kinase 1-interacting protein (SKIP, also called Sphkap) is highly expressed in pancreatic ?-cells but not in ?-cells. Intraperitoneal glucose tolerance test showed that plasma glucose levels were decreased and insulin levels were increased in SKIP<sup>-/-</sup> mice compared to SKIP<sup>+/+</sup> mice, but exendin-4-enhanced insulin secretion was masked. GSIS was amplified more in SKIP<sup>-/-</sup> but exendin-4-enhanced insulin secretion was masked compared to that in SKIP<sup>+/+</sup> islets. The ATP and cAMP content were similarly increased in SKIP<sup>+/+</sup> and SKIP<sup>-/-</sup> islets; depolarization-evoked, PKA and cAMP-mediated insulin secretion were not affected. Inhibition of PDE activity equally augmented GSIS in SKIP<sup>+/+</sup> and SKIP<sup>-/-</sup> islets. These results indicate that SKIP modulates GSIS by a pathway distinct from that of cAMP-, PDE- and sphingosine kinase-dependent pathways.
Project description:Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100?nmol/L) for 24?h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24?h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.
Project description:Glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into ?-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in ?-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated ?-cells. Conventional PCR assays and immunostaining confirmed that ?-cells express RyR2, the cardiac RyR isoform. Extended incubation of ?-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated ?-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to ?-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.
Project description:Stevia rebaudiana (Bert.) Bertoni besides being a source of noncaloric sweeteners is also an important source of bioactive molecules. Many plant extracts, mostly obtained with ethyl acetate solvent, are rich in polyphenol compounds that present insulinotropic effects. To investigate whether the nonsweetener fraction, which is rich in phenolic compounds isolated from Stevia rebaudiana with the solvent ethyl acetate (EAF), has an insulinotropic effect, including interference at the terminals of the autonomic nervous system of the pancreatic islets of rats. Pancreatic islets were isolated from Wistar rats and incubated with EAF and inhibitory or stimulatory substances of insulin secretion, including cholinergic and adrenergic agonists and antagonists. EAF potentiates glucose-stimulated insulin secretion (GSIS) only in the presence of high glucose and calcium-dependent concentrations. EAF increased muscarinic insulinotropic effects in pancreatic islets, interfering with the muscarinic receptor subfamily M3. Adrenergic inhibitory effects on GSIS were attenuated in the presence of EAF, which interfered with the adrenergic ?2 receptor. Results suggest that EAF isolated from stevia leaves is a potential therapy for treating type 2 diabetes mellitus by stimulating insulin secretion only in high glucose concentrations, enhancing parasympathetic signal transduction and inhibiting sympathetic signal transduction in beta cells.
Project description:SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of ?-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic ? cells.
Project description:Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.