Cdc42-regulated insulin granule exocytosis

描述（由申请人提供）：目前2型糖尿病患者的高血糖症治疗导致组成性和非调节性胰岛素递送，并且这种模拟内源性胰岛素分泌的失败导致低血糖症问题和最终的β细胞衰竭。因此，这里的长期目标是破译胰岛β细胞用于计量胰岛素释放的机制，然后设计出在患者体内重现这种计量机制的方法。在β细胞中的丝状肌动蛋白（F-actin）屏障后面存在数千个胰岛素颗粒，并且已知F-actin重构将颗粒动员到细胞表面的t-SNARE蛋白，但是涉及重构和颗粒动员的机制在很大程度上是未知的并且未经测试。发表的和初步的工作表明，肌动蛋白重塑的关键在于葡萄糖特异性激活的小Rho家族GTdR蛋白Cdc 42，Cdc 42是必不可少的第二相胰岛素从胰岛释放。新的数据还表明，Cdc 42鸟嘌呤解离抑制剂（GDI）需要保持Cdc 42失活，其突变/耗尽导致不适当的组成型胰岛素分泌。Cdc 42-GDI复合物直接与胰岛素颗粒上的v-SNARE结合，这种结合的破坏会减弱葡萄糖而不是KCl刺激的胰岛素分泌。此外，这种葡萄糖特异性Cdc 42激活与t-SNARE蛋白和F-肌动蛋白之间的相互作用相关联。因此，本申请的目的是描述葡萄糖激活Cdc 42以促进第二时相胰岛素释放的生理、细胞和分子机制，并确定Cdc 42如何通过Cdc 42-v-SNARE相互作用和通过影响F-肌动蛋白-t-SNARE缔合来调节胰岛素颗粒靶向和肌动蛋白重组来控制第二时相。这项研究的中心假设是Cdc 42在葡萄糖的作用下被特异性激活，通过选择性地重塑F-肌动蛋白来协调胰岛素释放的第二阶段，以动员和靶向质膜上的SNARE位点的颗粒进行胞吐。这将在三个特定目标中进行测试：1）阐明Cdc 42激活如何在胰腺β细胞中受葡萄糖调节; 2）确定Cdc 42-v-SNARE相互作用如何调节胰岛素胞吐; 3）确定肌动蛋白-t-SNARE相互作用在Cdc 42介导的肌动蛋白重塑和胰岛素胞吐调节中的功能作用。研究将使用siRNA介导的敲除胰岛β细胞与“救援”策略，并通过证实量化的双相胰岛素释放（胰岛灌流）与可视化的Cdc 42相互作用的空间变化，使用显微镜和生化亚细胞分级分析。了解Cdc 42在第二阶段分泌中的功能将标志着在β细胞中调节肌动蛋白重塑以重现调节的胰岛素分泌和预防β细胞衰竭的长期目标方面取得进展。

DESCRIPTION (provided by applicant): Current treatments of Type 2 diabetic patients for hyperglycemia result in constitutive and non-regulated insulin delivery, and this failure to mimic endogenous insulin secretion leads to hypoglycemia problems and eventual beta cell failure. Thus the long term goal here is to decipher the mechanisms used by pancreatic islet beta cells to meter insulin release, and then devise ways to recapitulate this metering mechanism pharmacologically in the patient. Thousands of insulin granules exist behind a filamentous actin (F-actin) barrier in the beta cell and F-actin remodeling is known to mobilize granules to the t-SNARE proteins at the cell surface, yet the mechanisms involved in remodeling and granule mobilization are largely unknown and untested. Published and preliminary work presented here suggests that the key to actin remodeling lies in the glucose-specific activation of the small Rho family GTPase protein Cdc42, and that Cdc42 is essential for second-phase insulin release from islets. New data also reveal that the Cdc42 guanine dissociation inhibitor (GDI) is required to keep Cdc42 inactive, and its mutation/depletion leads to inappropriate constitutive insulin secretion. The Cdc42-GDI complex binds directly to the v-SNARE on the insulin granules, and disruption of this binding attenuates glucose but not KCl-stimulated insulin secretion. Moreover, this glucose-specific Cdc42 activation is coupled to interactions between t-SNARE proteins and F-actin. Thus, the objective of this application is to delineate the physiologic, cellular and molecular mechanisms by which glucose activates Cdc42 to promote second-phase insulin release, and to determine how Cdc42 controls second phase through Cdc42-v-SNARE interactions and by impacting F-actin-t-SNARE associations to regulate insulin granule targeting and actin reorganization. The central hypothesis for the proposed research is that Cdc42 becomes activated specifically in response to glucose to coordinate the second phase of insulin release by selectively remodeling F-actin to mobilize and target granules towards SNARE sites at the plasma membrane for exocytosis. This will be tested in three Specific Aims: 1) Elucidate how Cdc42 activation is regulated by glucose in pancreatic beta cells; 2) Identify how Cdc42-v-SNARE interactions regulate insulin exocytosis; 3) Determine the functional role of actin-t-SNARE interactions in Cdc42-mediated actin remodeling and the regulation of insulin exocytosis. Studies will be accomplished using siRNA-mediated knockdown in islet beta cells with 'rescue' strategies, and by corroborating quantification of biphasic insulin release (islet perifusion) with visualization of spatial changes in Cdc42 interactions using microscopy and biochemical subcellular fractionation analyses. Gaining knowledge of how Cdc42 functions in second-phase secretion will mark progress towards the long-term goal of modulating actin remodeling in the beta cell to recapitulate regulated insulin secretion and prevent beta cell failure.