Mechanisms of Rab27 regulation of insulin secretion

描述(申请人提供)：胰腺细胞功能异常可对葡萄糖稳态产生深远影响，胰岛素分泌不足，再加上靶组织对胰岛素活动的明显抵抗，导致2型糖尿病。这项建议的首要目标是了解调节细胞胰岛素分泌的分子机制，以便开发更好的促进胰岛素分泌的治疗干预措施。这项研究的重点是了解GTPase Rab27A对含有胰岛素的分泌颗粒的胞吐作用施加如此有效的调节的分子机制。特定RAB的GTP/GDP循环的速度和位置对其行动施加了精确的时间和空间调节。然而，Rab27A的GTP/GDP循环的调控程度，调控其周期速率的葡萄糖诱导的信号通路，以及Rab27A-GTP促进胰岛素分泌的机制仍不清楚。我们假设Rab27A的核苷酸状态受到严格控制，并且GSIS在特定可识别的分泌颗粒群上启动Rab27的GTP/GDP循环。Rab27-GTP的增加对通过与SLp4a的相互作用推动颗粒招募/对接很重要，而对接的颗粒上的GTP水解被认为是促进融合的启动。这项拟议的研究有三个具体目标。首先，我们将确定Rab27A GTP/GDP循环的空间和时间特性，以及它们与培养小鼠GSIS细胞分泌动力学的功能关系。其次，我们将确定Rab27A促进胰岛素分泌的调控胞吐途径中的主要位点(S)，并确定其作用机制，并表征特定的Rab27A效应器的作用机制。最后，我们将确定调控Rab27A GTP/GDP循环速率的信号通路。通过对Rab27A的信号中间调节，精确定位分泌途径中的作用部位。实验将使用从缺乏Rab27A蛋白表达的灰鼠模型和对照C3H/He小鼠分离和培养的小鼠细胞。这些实验结合了光学(FRET、TIRF-FRET)、电生理学(膜片钳、CM监测)和生化/药理学方法来严格定义Rab27A的功能机制。与公共卫生相关：胰岛细胞分泌的胰岛素对维持血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年全球将有2.5亿人受到影响，当细胞释放胰岛素的能力超过了调节葡萄糖和脂肪代谢的激素的需求时，就会发生2型糖尿病。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胰岛素分泌反应迟缓和迟钝，以及失去紧密的刺激-分泌偶联。因此，有必要全面了解胰岛素分泌背后的分子和机制细节，以最终开发糖尿病的治疗方法。

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.