Second-phase Glucose-stimulated Insulin Secretion Research Articles

Munc18c Depletion Selectively Impairs the Sustained Phase of Insulin Release

OBJECTIVEThe Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4–mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mechanism(s) using the islet β-cell as a model system.RESEARCH DESIGN AND METHODSPerifusion analyses of isolated Munc18c- (−/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization.RESULTSMunc18c (−/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (−/+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2–Syntaxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion.CONCLUSIONSCollectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion.

CAPS1 and CAPS2 Regulate Stability and Recruitment of Insulin Granules in Mouse Pancreatic β Cells

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

Insulin constitutively secreted by beta-cells is necessary for glucose-stimulated insulin secretion.

Four hypotheses have been posited on the role of insulin in glucose-stimulated insulin secretion; available evidence has supported insulin as being 1) essential, 2) a positive modulator, 3) a negative modulator, or 4) not necessary. Because circulating insulin levels in mice, before or after intraperitoneal glucose injection, are sufficient to elicit insulin responses in insulin-sensitive tissues, it is likely that beta-cell insulin receptors are continuously exposed to stimulating concentrations of insulin. To determine whether constitutively secreted insulin is necessary for glucose-stimulated insulin secretion, CD1 male mouse islets were incubated for 30 min at 4 degrees C in the absence (control) or presence of anti-insulin (1 micro g/ml) or anti-IgG (1 micro g/ml). Then islets were exposed to 3, 11, or 25 mmol/l glucose or to 20 mmol/l arginine. Nontreated islets exhibited first- and second-phase glucose-stimulated insulin secretion. Control and anti-IgG-treated islets, after a 5-min lag phase, increased their insulin secretion in 25 mmol/l glucose. Anti-insulin-treated islets secreted insulin at a basal rate in 3 or 25 mmol/l glucose buffers. Insulin secretion stimulated by 20 mmol/l arginine was the same in islets pretreated with either antibody and showed no lag phase. Taken together, these data suggest that constitutively secreted insulin is required and sufficient for beta-cells to maintain sensitivity to glucose.

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex.

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.

Inhibition of nitric oxide generation: Normalization of in vitro insulin secretion in mice with multiple low-dose streptozotocin and in mice injected with mononuclear splenocytes from diabetic syngeneic donors

We studied the effect on in vitro glucose-induced insulin secretion of in vivo administration of l- N G-monomethyl-arginine ( l-NMMA), a competitive inhibitor of nitric oxide (NO) synthase, to mice injected with multiple low-dose streptozotocin (mld-SZ). In addition, the effect of l-NMMA treatment on the capacity of mononuclear spleen cells (MS) from mld-SZ mice to transfer alterations in insulin secretion from normal syngeneic receptors was also investigated. We also studied the effect of in vivo treatment with l-NMMA on anti-β-cell cellular immune aggression (CIA) by coculturing MS from mld-SZ mice with rat dispersed islet cells. Our results show that mld-SZ mice treated with 0.25 mg l-NMMA/g body weight had normoglycemia, first- and second-phase glucose-stimulated insulin secretion similar to those obtained in nondiabetic mice—effects not observed with a lower dose of l-NMMA (0.17 mg/g body weight)—and a diminished anti-β-cell CIA. We also demonstrate that mice injected with MS from syngeneic donors treated with mld-SZ plus 0.25 mg l-NMMA/g had normal levels for first-phase glucose-stimulated insulin secretion and an absence of CIA. Taken together, these findings seem to indicate that prevention of in vivo NO production may block the onset of diabetes in mld-SZ mice, and that l-NMMA administration to diabetic donor mice prevents inhibition of first-phase insulin secretion and CIA in the transferred recipient mice. Although a nonimmunological mechanism or mechanisms of diabetes prevention by l-NMMA cannot be excluded, these results suggest that l-NMMA treatment could also be acting on T-cell-dependent immune reactions.