Mechanisms of Rab27 regulation of insulin secretion

Rab27调节胰岛素分泌的机制

• 批准号: 8007206
• 负责人: EDWARD L STUENKEL
• 金额: $ 7.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-20 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007206
• 关键词: Affect; Binding; Biochemical; Cell physiology; Cells; Competence; Complex; Coupled; Coupling; Cytoplasmic Granules; Data; Diabetes Mellitus; Disease; Docking; Electric Capacitance; Event; Exocytosis; Family; Fluorescence Resonance Energy Transfer; Functional disorder; GTP Binding; Glucose; Goals; Growth and Development function; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hormones; Hydrolysis; Inbred C3H Mice; Individual; Infection; Insulin; Islets of Langerhans; Kinetics; Life; Link; Location; Maintenance; Membrane; Membrane Fusion; Molecular; Monitor; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Optics; Organelles; Pancreas; Pathway interactions; Population; Property; Proteins; Rab27a protein; Regulation; Reporting; Research; Resistance; Role; Secretory Vesicles; Signal Pathway; Signal Transduction; Site; Specificity; Staging; Stimulus; Subgroup; Techniques; Therapeutic; Therapeutic Intervention; Time; Tissues; Work; blood glucose regulation; diabetic patient; insulin secretion; lipid metabolism; mouse model; patch clamp; public health relevance; research study; response

DESCRIPTION (provided by applicant): Abnormal pancreatic ¿-cell function can have a profound impact on glucose homeostasis, with insufficient secretion of insulin, coupled with marked resistance to its actions by target tissues, resulting in type-2 diabetes. The overarching goal of this proposal is to develop an understanding of the molecular mechanisms that regulate insulin secretion from ¿-cells, such that better therapeutic interventions promoting insulin secretion can be developed. The research is specifically focused on understanding the molecular mechanism by which the GTPase Rab27A exerts such potent regulation over exocytosis of insulin-containing secretory granules. The rate and location of GTP/GDP cycling for a particular Rab imposes precise temporal and spatial regulation to its action. Yet, the extent to which GTP/GDP cycling of Rab27A is regulated, the glucose-induced signaling pathways that govern its rate of cycling, as well as the mechanism by which Rab27A-GTP facilitates secretion of insulin, remain unknown. We hypothesize that the nucleotide state of Rab27A is strictly controlled and that GSIS initiates GTP/GDP cycling of Rab27 on specific identifiable populations of secretory granules. An increase in Rab27-GTP is proposed important to drive granule recruitment/docking via interaction with Slp4a, while GTP hydrolysis on docked granules is proposed to promote priming for fusion. The proposed research is placed into three specific aims. First, we will determine the spatial and temporal properties of Rab27A GTP/GDP cycling and their functional relationships to secretory dynamics during GSIS in cultured mouse ¿-cells. Second, we will determine the primary site(s) in the regulated exocytotic pathway at which Rab27A acts to facilitate insulin secretion, define its mechanism of action, and characterize the mechanism of action of specific Rab27A effectors. Lastly, we will define the signaling pathways that regulate the rate of GTP/GDP cycling of Rab27A. Pinpoint the site of action in the secretory pathway regulated by signaling intermediate via effects on Rab27A. The experiments will use mouse ¿-cells isolated and cultured from the ashen mouse model, which lack expression of Rab27A protein, and from control C3H/He mice. The experiments incorporate optical (FRET, TIRF-FRET), electrophysiological (patch-clamp, CM- monitoring) and biochemical/ pharmacological approaches to rigorously define mechanisms of Rab27A function. PUBLIC HEALTH RELEVANCE: Insulin secreted by ¿-cells of pancreatic islets is essential for maintenance of glucose homeostasis as well as for tissue development and growth. Type-2 diabetes, a disease predicted to affect 250 million people worldwide by the year 2020, occurs when the ability of the ¿-cells to release insulin is exceeded by the requirements for the hormone to regulate glucose and lipid metabolism. In diabetic patients, early manifestations of ¿-cell dysfunction include delayed and blunted insulin secretory responses to glucose challenges and loss of tight stimulus-secretion coupling. Therefore, there is a critical necessity to understand in full molecular and mechanistic detail the secretory pathway underlying insulin secretion to ultimately develop therapeutic treatments for diabetes.

描述（由应用提供）：胰腺功能异常可能对葡萄糖稳态的影响产生深远的影响，胰岛素的分泌不足，再加上对其作用的明显耐药性，导致2型糖尿病。该提案的总体目标是对调节胰岛素分泌的分子机制有一种理解，从而可以开发出更好的治疗性干预措施来促进胰岛素分泌。这项研究专门用于理解GTPase RAB27A对含胰岛素分泌颗粒的胞吐作用的潜在调节的分子机制。特定RAB的GTP/GDP循环的速率和位置不可能精确地进行临时和空间调节其作用。然而，调节了RAB27A的GTP/GDP循环的程度，葡萄糖诱导的信号通路控制了其循环速率，以及RAB27A-GTP促进胰岛素分泌的机制仍然未知。我们假设Rab27a的核状态受到严格控制，GSIS在分泌颗粒的特定鉴定人群上启动了RAB27的GTP/GDP循环。提出，RAB27-GTP的增加对于通过与SLP4A的相互作用驱动颗粒募集/对接很重要，而对停靠颗粒上的GTP水解提议促进融合启动。拟议的研究置于三个特定目标中。首先，我们将确定RAB27A GTP/GDP循环的空间和临时特性及其在培养小鼠中GSIS期间与秘书动力学的功能关系。其次，我们将确定RAB27A作用以促进胰岛素分泌，定义其作用机理并表征特定RAB27A效应的作用机理的受调节胞吐途径中的主要部位。最后，我们将定义调节RAB27A GTP/GDP循环速率的信号通路。通过对RAB27A的影响中间信号传导调节的秘书途径中的作用部位。实验将使用从缺乏RAB27A蛋白表达的灰小鼠模型和对照C3H/HE小鼠中分离出来的小鼠�-细胞。该实验结合了光学（FRET，TIRF-FRET），电生理（贴片钳，CM-监测）和生化/药物方法，以严格定义RAB27A功能的机制。公共卫生相关性：由胰岛的细胞分泌的胰岛素对于维持葡萄糖稳态以及组织发育和生长至关重要。 2型糖尿病是一种疾病，预计到2020年将在全球范围内影响2.5亿人。在糖尿病患者中， - 细胞功能障碍的早期表现包括对葡萄糖挑战的延迟和钝性的胰岛素秘密反应以及紧密刺激 - 分泌耦合的丧失。因此，迫切需要在完整的分子和机理细节中理解胰岛素分泌的秘密途径，以最终开发糖尿病治疗治疗。