Cdc42-regulated insulin granule exocytosis

摘要 目前2型糖尿病患者高血糖症的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问题，并最终导致低血糖。 β细胞衰竭因此，长期的目标是破译胰岛β细胞的作用机制， 细胞来测量胰岛素的释放，然后设计出重现这种测量机制的方法。 在病人身上。在β细胞中，数千个胰岛素颗粒存在于丝状肌动蛋白（F-actin）屏障后面 和F-肌动蛋白重塑是已知的动员颗粒的t-SNARE蛋白在细胞表面，但 涉及重塑和颗粒动员的机制在很大程度上是未知的和未经检验的。发表 初步研究表明，肌动蛋白重塑的关键在于葡萄糖特异性 小Rho家族GT3蛋白Cdc 42的激活，以及Cdc 42对于第二相胰岛素是必需的。 从胰岛中释放。新的数据还表明，Cdc 42鸟嘌呤解离抑制剂（GDI）是必需的， 保持Cdc 42失活，其突变/缺失导致不适当的组成型胰岛素分泌。的 Cdc 42-GDI复合物直接与胰岛素颗粒上的v-SNARE结合，并破坏这种结合 减弱葡萄糖但不减弱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偶联。因此，本申请的目的是 描述葡萄糖激活Cdc 42以促进细胞增殖的生理、细胞和分子机制。 第二相胰岛素释放，并确定Cdc 42如何通过Cdc 42-v-SNARE控制第二相 相互作用，并通过影响F-actin-t-SNARE协会来调节胰岛素颗粒靶向和肌动蛋白 重组。这项研究的中心假设是Cdc 42被激活， 特别是对葡萄糖的反应，以通过选择性重塑来协调胰岛素释放的第二阶段 F-肌动蛋白将颗粒动员并靶向质膜上的SNARE位点进行胞吐。这将 在三个特定目的中进行测试：1）阐明Cdc 42激活如何在胰腺β-淀粉样蛋白中受葡萄糖调节。 2）确定Cdc 42-v-SNARE相互作用如何调节胰岛素胞吐作用; 3）确定功能作用 在Cdc 42介导的肌动蛋白重塑和胰岛素胞吐调节中的肌动蛋白-t-SNARE相互作用。 研究将在胰岛β细胞中使用siRNA介导的敲低与“拯救”策略来完成， 通过证实双相胰岛素释放（胰岛灌流）的定量与空间变化的可视化， 利用显微镜和生化亚细胞分级分析cdc 42相互作用。获得知识 Cdc 42在第二阶段分泌中的功能将标志着朝着调节细胞周期的长期目标的进展。 肌动蛋白在β细胞中重塑以重现调节的胰岛素分泌并防止β细胞衰竭。项目叙事 ¿ 虽然糖尿病患者使用磺脲类药物或胰岛素注射剂来生存，但这些治疗方法不能 模拟仔细计量的胰岛素释放，否则这些胰岛素将来自他们自己的胰岛。 细胞我们的初步数据显示，胰岛素的定量释放是由一种名为 cdc 42和对这种蛋白质在胰岛素分泌中的功能的进一步研究将有望导致发现。 新的胰岛素输送治疗策略，更好地模拟健康人的自然胰岛素释放， 来改善糖尿病患者的生活。 ¿

Abstract 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 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 quantitation 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. Project¿Narrative¿ ¿ ¿ 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.¿ ¿