白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

Isolated freshly rat islets were transferred to 24-well plates and incubated with different concentrations of glucose or resveratrol for 1 or 24 h.The results showed that resveratrol dose-dependently inhibited glucose-stimulated insulin secretion from isolated rat islets after 1 h incubation,with 10%,35%,and 80% (P<0.05 or P<0.01) decrease at the concentrations of 1,I0,and 100 μmol/L.10 μmol/L resveratrol decreased the intracellular calcium concentration by 60% (P<0.05).After incubation for 24 h,resveratrol increased palmitatesuppressed insulin secretion to 75% (P<0.01) of control.These results suggest that resveratrol acutely inhibits insulin secretion from primary pancreatic islet via regulating intracellular calcium ion concentration,and in the long run resveratrol may protect β-cells from lipotoxicity.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

分离SD大鼠胰岛接种于24孔板中,用不同浓度的葡萄糖和白藜芦醇分别培养1 h或24 h,结果表明白藜芦醇孵育大鼠胰岛1 h可呈剂苗依赖地抑制大鼠高糖刺激的胰岛素分泌,1、10和100 μmol/L白藜芦醇可以分别使胰岛素的分泌降低10%、35%(P<0.05)和80%(P<0.01).显微离子成像技术爪10μmol/L的白藜芦醇可以使高糖引起的β细胞内Ca2+浓度的升高减少60%(P<0.05).白藜芦醇可使软脂酸孵育24 h大鼠胰岛的胰岛素分泌恢复到对照组的75%(P<0.01),提示白藜芦醇短期可通过调控细胞内的Ca+浓度,而抑制原代胰岛高精刺激的胰岛素分泌,长期可改善软脂酸引起的β细胞损伤.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

Isolated freshly rat islets were transferred to 24-well plates and incubated with different concentrations of glucose or resveratrol for 1 or 24 h.The results showed that resveratrol dose-dependently inhibited glucose-stimulated insulin secretion from isolated rat islets after 1 h incubation,with 10%,35%,and 80% (P<0.05 or P<0.01) decrease at the concentrations of 1,I0,and 100 μmol/L.10 μmol/L resveratrol decreased the intracellular calcium concentration by 60% (P<0.05).After incubation for 24 h,resveratrol increased palmitatesuppressed insulin secretion to 75% (P<0.01) of control.These results suggest that resveratrol acutely inhibits insulin secretion from primary pancreatic islet via regulating intracellular calcium ion concentration,and in the long run resveratrol may protect β-cells from lipotoxicity.     

Role of insulin in glucose-stimulated insulin secretion in beta cells

Diabetes is on the increase worldwide and greater than 90% are type 2. There are two features to type 2 diabetes: muscle, fat and liver tissues are insulin resistant and beta cells lose the ability to secrete insulin. Prior to developing diabetes, however, insulin resistant individuals lose the first-phase insulin secretion response. Transgenic mice lacking insulin receptors in their beta cells have no first-phase response. Primary cultures of mouse islets pre-exposed to anti-insulin do not exhibit a first-phase insulin secretion response. That is, beta cells, like muscle, fat, and liver, are an insulin sensitive tissue and in the presence of insulin resistance (type 2 diabetes), in the absence of insulin receptors (transgenic mice lacking beta cell insulin receptors), or in the absence of constitutively secreted insulin (anti-insulin treatment), beta cells are unable to respond properly to post-prandial glucose. The purpose of this report is to review our understanding of the glucose-stimulus response and of insulin signaling, and to suggest why the latter may be necessary for the former to proceed.     

The nucleotide exchange factor SIL1 is required for glucose-stimulated insulin secretion from mouse pancreatic beta cells in vivo

Aims/hypothesis

Regulation of insulin secretion along the secretory pathway is incompletely understood. We addressed the expression of SIL1, a nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 kD (GRP78), in pancreatic beta cells and investigated whether or not SIL1 is involved in beta cell function.

Methods

SIL1 expression was analysed by immunoblotting and immunofluorescence. Metabolic and islet variables, including glucose tolerance, beta cell mass, insulin secretion, islet ultrastructure, insulin content and levels of ER stress marker proteins, were addressed in Sil1 knockout (Sil1 ^?/?) mice. Insulin, proinsulin and C-peptide release was addressed in Sil1 ^?/? islets, and SIL1 overexpression or knockdown was explored in MIN6 cells in vitro. Models of type 1 diabetes and insulin resistance were induced in Sil1 ^?/? mice by administration of streptozotocin (STZ) and a high-fat diet (HFD), respectively.

Results

We show that SIL1 is expressed in pancreatic beta cells and is required for islet insulin content, islet sizing, glucose tolerance and glucose-stimulated insulin secretion in vivo. Levels of pancreatic ER stress markers are increased in Sil1 ^?/? mice, and Sil1 ^?/? beta cell ER is ultrastructurally compromised. Isolated Sil1 ^?/? islets show lower proinsulin and insulin content and impaired glucose-stimulated insulin secretion. Modulation of SIL1 protein levels in MIN6 cells correlates with changes in insulin content and secreted insulin. Furthermore, Sil1 ^?/? mice are more susceptible to STZ-induced type 1 diabetes with increased apoptosis. Upon HFD feeding, Sil1 ^?/? mice show markedly lower insulin secretion and exacerbated glucose intolerance compared with control mice. Surprisingly, however, HFD-fed Sil1 ^?/? mice display pronounced islet hyperplasia with low amounts of insulin in total pancreas.

Conclusions/interpretation

These results reveal a novel role for the nucleotide exchange factor SIL1 in pancreatic beta cell function under physiological and disease conditions such as diabetes and the metabolic syndrome.     

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

Aims/hypothesis

Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells.

Methods

We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot.

Results

Insulin secretion was increased following chronic (≥8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases.

Conclusions/interpretation

Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.     

Effect of secretin on basal- and glucose-stimulated insulin secretion in man

Summary Plasma immunoreactive secretin and insulin concentrations were measured in fasting normal humans after intraduodenal infusions of hydrochloric acid, isotonic or hypertonic glucose. The effect of intraduodenal acidification or intravenous bolus injections of secretin on plasma insulin concentrations during infusions of glucose was also examined. The intraduodenal glucose load did not cause an increase in plasma secretin concentrations. Secretin concentrations rose after acid both in the fasting state and during infusions of glucose. A concomitant rise in insulin levels was however only observed during infusions of glucose. Intravenous injection of secretin in a dose which mimicked the response to intraduodenal acidification was without effect on the glucose-stimulated insulin release, while a 30 times higher dose caused a highly significant augmentation of the insulin release. The insulin response pattern to this high dose of secretin differed completely from that observed after intraduodenal infusion of acid. It is concluded and confirmed that the stimulating effect of secretin on insulin secretion is pharmacological and that secretin plays no significant role in the entero-insular axis.     

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 and glucose-stimulated insulin secretion in pancreatic islets of Langerhans

BACKGROUND: In rodents, the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inactive 11-dehydrocorticosterone (DHC) into active corticosterone. The mRNA and activity of 11beta-HSD1 have been shown to be present in batch-incubated pancreatic islets from the ob/ob mouse. In other tissues, 11beta-HSD1 expression has been demonstrated to be regulated by glucocorticoids. In the present study, the influence of DHC on 11beta-HSD1 levels and glucose-induced changes in insulin secretion were studied in pancreatic islets isolated from the ob/ob mouse. METHODS: Western blotting with antiserum for 11beta-HSD1 verified the presence of 11beta-HSD1 in islets from obese ob/ob and normal C57BL/6J mice. Insulin secretion was determined by perifusing islets and assaying the perifusate with ELISA. RESULTS: Islets from the ob/ob mouse contained almost twofold more 11beta-HSD1 protein than islets from the C57BL/6J mouse. When islets from ob/ob mice were cultured with 50 nM DHC, the 11beta-HSD1 levels doubled compared with islets cultured in the absence of DHC. Selective inhibition of 11beta-HSD1 attenuated DHC-induced increase in 11beta-HSD1 levels, as did an antagonist of the glucocorticoid receptor. In individually perifused ob/ob mouse islets, early and late phases of glucose-stimulated insulin secretion (GSIS) were dose-dependently inhibited by 5, 50 and 500 nM DHC. Whereas inclusion of 11beta-HSD1 inhibitors restored, addition of the glucocorticoid receptor antagonist attenuated the DHC-mediated inhibition of GSIS. CONCLUSIONS: Levels of 11beta-HSD1 in islets from ob/ob mice are positively regulated by DHC and could be lowered by a selective 11beta-HSD1 inhibitor and a glucocorticoid receptor antagonist. Increased levels of 11beta-HSD1 were associated with impaired GSIS.     

Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets

Aims/hypothesis

Type 2 diabetes is characterised by impaired glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Since erythropoietin-producing hepatoma (Eph)–ephrin bidirectional signalling fine-tunes GSIS from pancreatic beta cells, we investigated Eph receptor tyrosine kinases (RTK) as potential drug targets for selectively increasing GSIS.

Methods

Insulin secretion assays were carried out using mouse and human pancreatic islets as well as mouse insulinoma (MIN6) cells in the presence or absence of two Eph RTK inhibitors. Furthermore, the most potent inhibitor was injected into mice to evaluate its effects on glucose tolerance and plasma insulin levels.

Results

We showed that the Eph RTK inhibitors selectively increased GSIS from MIN6 cells as well as mouse and human islets. Our results also showed that the insulin secretory effects of these compounds required Eph–ephrin signalling. Finally, pharmacological inhibition of Eph receptor signalling improved glucose tolerance in mice.

Conclusions/interpretation

We showed for the first time that Eph RTKs represent targets for small molecules to selectively increase GSIS and improve glucose tolerance.     

Sodium channel beta1 regulatory subunit deficiency reduces pancreatic islet glucose-stimulated insulin and glucagon secretion

Glucose-stimulated insulin and glucagon release regulates glucose homeostasis by an excitation-secretion coupling pathway beginning with ATP-sensitive K(+) channel closure, membrane depolarization, and entry of calcium ions to stimulate exocytosis. The contribution of voltage-gated sodium channels to this release pathway is still being elucidated. We demonstrate that loss of Scn1b, a major regulatory subunit expressed with Na(v)1.7 protein in mouse pancreatic islets, reduces glucose-stimulated insulin and glucagon secretion in vitro and in vivo, resulting in severe fed and fasting hypoglycemia. This genetic mouse model is the first to demonstrate that sodium channelopathy impairs the physiological excitation-release coupling pathway for pancreatic insulin and glucagon release.     

Metabotropic glutamate and GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells

Aims/hypothesis: The neurotransmitters glutamate and γ-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA_B (GABA_BRs) receptors in this process. Methods: We studied the expression of mGluRs and GABA _BRs in rat and human islets of Langerhans and in pancreatic α-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA _B R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). Results: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA _BR1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA_BR1 a/b and GABA_BR2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6–8) specific agonist inhibited insulin release at high (10–25 mmol/l) glucose concentrations. Baclofen, a GABA_BR agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. Conclusion/interpretation: These data suggest that mGluRs and GABA_BRs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca²⁺-channels, cAMP generation and G-protein-mediated modulation of K_ATP channels. [Diabetologia (2002) 45: 242–252] Received: 18 September 2001 and in revised form: 5 November 2001     

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

The high-capacity glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase are thought to be key components of the "glucose-sensing apparatus" that regulates insulin release from the beta cells of the islets of Langerhans in response to changes in external glucose concentration. AtT-20ins cells are derived from anterior pituitary cells and are like beta cells in that they express glucokinase and have been engineered to secrete correctly processed insulin in response to analogs of cAMP, but, unlike beta cells, they fail to respond to glucose and lack GLUT-2 expression. Herein we demonstrate that stable transfection of AtT-20ins cells with the GLUT-2 cDNA confers glucose-stimulated insulin secretion and glucose regulation of insulin biosynthesis and also results in glucose potentiation of the secretory response to non-glucose secretagogues. This work represents a first step toward creation of a genetically engineered "artificial beta cell."