Cdc42-regulated insulin granule exocytosis

Cdc42调节的胰岛素颗粒胞吐作用

基本信息

批准号：
8037742
负责人：
Debbie C Thurmond
金额：
$ 31.21万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8037742
关键词：
Actin-Binding Protein Actins Active Sites Adverse effects Attenuated B-insulin Beta Cell Binding Binding Proteins Biochemical Calcium Channel Caveolins Cell Fractionation Cell membrane Cell surface Complex Coupled Cytoplasmic Granules Data Diabetes Mellitus Dissociation Event Exocytosis F-Actin Failure Family Future Gelsolin Glucose Goals Guanine Guanosine Triphosphate Phosphohydrolases Human Hyperglycemia Hypoglycemia Imagery Injection of therapeutic agent Insulin Investigation Islet Cell Islets of Langerhans Knowledge Lead Learning Link Mediating Microscopy Molecular Mutation Names Pancreas Patients Phase Phosphorylation Physiological Play Principal Investigator Proteins Publishing Regulation Regulatory Pathway Research Role SNAP receptor Signal Transduction Simulate Site Small Interfering RNA Structure of beta Cell of islet Sulfonylurea Compounds Testing Therapeutic Therapeutic Intervention Vesicle Work base blood glucose regulation cdc42 GTP-Binding Protein diabetic diabetic patient expectation filamin fodrin improved inhibitor/antagonist insulin granule insulin secretion islet meter novel novel therapeutics prevent programs protein function response rho src-Family Kinases syntaxin syntaxin 4 syntaxin-2 target SNARE proteins therapeutic target trafficking vesicular SNARE proteins voltage

项目摘要

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.

描述（由申请人提供）：当前对2型糖尿病患者的高血糖治疗导致组成症和非调节的胰岛素递送，并且这种未能模拟内源性胰岛素分泌的失败会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细胞使用的机制来释放胰岛素，然后设计方法在患者中概括该测量机制。 β细胞中丝状肌动蛋白（F-肌动蛋白）屏障后数千种胰岛素颗粒，已知F-肌动蛋白重塑可将颗粒动员至细胞表面的T-Snare蛋白，但在重塑和颗粒动员中涉及的机制是很大程度上尚不清楚的，并且未知。此处介绍的已发表和初步工作表明，肌动蛋白重塑的关键在于小的Rho家族GTPase蛋白Cdc42的葡萄糖特异性激活，而Cdc42对于从小岛的二相胰岛素释放至关重要。新数据还表明，需要CDC42鸟嘌呤分离抑制剂（GDI）以保持CDC42不活跃，并且其突变/耗竭会导致不适当的本构胰岛素分泌。 Cdc42-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CDC42激活与T-Snare蛋白与F-肌动蛋白之间的相互作用耦合。 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.提出的研究的中心假设是，Cdc42通过选择性重塑F-肌动蛋白将胰岛素释放的第二阶段特别激活，以协调胰岛素释放的第二阶段，以动员并将靶向颗粒靶向质膜上的SNARE位点，以使其胞吐作用。这将在三个特定目的中进行测试：1）阐明胰腺β细胞中葡萄糖调节Cdc42的激活方式； 2）确定CDC42-V-SNARE相互作用如何调节胰岛素胞吐作用； 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胞吐作用的调节中的功能作用。通过使用“救援”策略的胰岛β细胞中的siRNA介导的敲低的敲低，并通过使用显微镜和生物化学亚细胞亚细胞分离分析的cdc42相互作用可视化Cdc42相互作用的空间变化，从而实现了研究。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细胞中肌动蛋白重塑的长期目标的进展，以概括受调节的胰岛素分泌并防止β细胞衰竭。