Cdc42-regulated insulin granule exocytosis

描述(申请人提供)：目前对2型糖尿病患者的高血糖治疗导致结构性和非调节性胰岛素分泌，这种不能模拟内源性胰岛素分泌的失败导致低血糖问题和最终的β细胞衰竭。因此，这里的长期目标是破译胰岛β细胞用来测量胰岛素释放的机制，然后设计出在患者身上从药理学上概括这种测量机制的方法。数以千计的胰岛素颗粒存在于β细胞的丝状肌动蛋白(F-actin)屏障后面，F-肌动蛋白重塑被认为是将胰岛素颗粒动员到细胞表面的t-SNARE蛋白上，然而重塑和颗粒动员的机制在很大程度上是未知的，也没有得到检验。已发表的初步工作表明，肌动蛋白重塑的关键在于小Rho家族GTPase蛋白CDC42的葡萄糖特异性激活，而CDC42是胰岛第二相胰岛素释放所必需的。新的数据还显示，cdc42鸟嘌呤解离抑制剂(GDI)是保持cdc42失活所必需的，它的突变/缺失导致不适当的结构性胰岛素分泌。CDC42-GDI复合体直接与胰岛素颗粒上的v-SNARE结合，破坏这种结合会减弱葡萄糖的分泌，但不会影响KCl刺激的胰岛素分泌。此外，这种葡萄糖特异性的CDC42激活与t-SNARE蛋白和F-肌动蛋白之间的相互作用有关。因此，本研究的目的是阐明葡萄糖激活CDC42促进第二时相胰岛素释放的生理、细胞和分子机制，并确定CDC42是如何通过CDC42-v-SNARE相互作用和影响F-肌动蛋白-t-SNARE结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的。这项研究的中心假设是，CDC42被特异性地响应于葡萄糖而被激活，通过选择性地重塑F-肌动蛋白来动员和靶向质膜上的陷阱位置来协调胰岛素释放的第二阶段，以实现胞吐作用。这将在三个特定的目标中进行测试：1)阐明葡萄糖如何调节胰岛β细胞中的CDC42激活；2)确定CDC42-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CDC42介导的肌动蛋白重塑和胰岛素分泌调节中的功能作用。这项研究将利用siRNA介导的胰岛β细胞基因敲除和“挽救”策略，并通过显微镜和生化亚细胞分级分析来证实双相胰岛素释放(胰岛灌流)的量化和对Cdc42相互作用的空间变化的可视化。了解CDC42如何在第二阶段分泌中发挥作用，将标志着朝着调节β细胞肌动蛋白重塑以概括调节胰岛素分泌和防止β细胞衰竭的长期目标取得进展。公共卫生相关性：尽管糖尿病患者使用磺脲类药物或胰岛素注射来生存，但这些治疗方法未能模拟胰岛素的仔细计量释放，否则胰岛素将来自他们自己的胰岛细胞。我们的初步数据显示，胰岛素的定量释放受一种名为CDC42的蛋白质控制，进一步研究该蛋白质在胰岛素分泌中的功能将有望发现新的胰岛素释放治疗策略，更好地模拟健康人胰腺的自然胰岛素释放，以改善糖尿病患者的生活。

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. PUBLIC HEALTH RELEVANCE: Although diabetic patients use sulfonylureas or insulin injection to survive, these treatments fail to simulate the carefully metered release of insulin that would otherwise come from their own pancreatic islet cells. Our preliminary data have revealed that the metered release of insulin is controlled by a protein named Cdc42, and further investigation of this protein's function in insulin secretion will hopefully lead to discovery of new therapeutic strategies for insulin delivery that better simulate natural insulin release from a healthy human pancreas to improve the livelihood of people with diabetes.