Munc18c depletion selectively impairs the sustained phase of insulin release.
Munc18c depletion selectively impairs the sustained phase of insulin release.
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MUNC18C耗竭选择性损害胰岛素释放的持续相。
DOI：
10.2337/db08-1059
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发表时间：
2009-05
期刊：
影响因子：
7.7
通讯作者：
Thurmond DC
中科院分区：
文献类型：
--
作者：
Oh E;Thurmond DC
Sec 1/Munc 18蛋白Munc 18 c与Syntaxin 4介导的胞吐事件有关，尽管其在胞吐中的目的仍然难以捉摸。鉴于Syntaxin 4在葡萄糖刺激的胰岛素分泌（GSIS）的第二阶段中起作用，我们假设Munc 18 c也是必需的，并使用胰岛β细胞作为模型系统来寻求对可能机制的深入了解。使用分离的Munc 18 c-（-/+）或Munc 18 c缺失（RNAi）小鼠胰岛的灌流分析来评估双相分泌。蛋白质相互作用研究使用从MIN 6 β细胞制备的亚细胞级分和去污剂裂解物来确定Munc 18 c在Syntaxin 4活化和含囊泡相关膜蛋白（VAMP）2的胰岛素颗粒的对接/融合中的机制作用。电子显微镜检查用于测量颗粒定位的变化。Munc 18 c（−/+）胰岛在第二阶段GSIS期间选择性地分泌约60%的胰岛素; RNAi介导的Munc 18 c消耗在野生型和Munc 18 c（−/+）胰岛中以基因剂量依赖性方式在功能上重演了这一点。Munc 18 c缺失消除了葡萄糖刺激的VAMP 2-Syntaxin 4结合以及Syntaxin 4激活，与胰岛素释放不足相关。值得注意的是，Munc 18 c耗竭导致响应于葡萄糖刺激的异常颗粒定位于质膜，这与其对分泌的第二阶段的选择性作用一致。总的来说，这些研究证明了Munc 18 c在第二阶段GSIS中的重要积极作用，并表明Munc 18 c在颗粒定位于质膜以及在囊泡对接/融合之前的步骤中触发突触融合蛋白4对VAMP 2的可及性中的新作用。
The 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. Perifusion 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. Munc18c (−/+) 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. Collectively, 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.
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