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，加上靶组织对其作用的明显抵抗，导致2型糖尿病。本提案的总体目标是发展对调节胰岛素分泌的分子机制的理解，从而可以开发更好的促进胰岛素分泌的治疗干预措施。本研究的重点是了解GTPase Rab27A对含胰岛素分泌颗粒胞吐作用的有效调控的分子机制。GTP/GDP循环的速率和位置对特定Rab的作用施加了精确的时间和空间调节。然而，Rab27A的GTP/GDP循环在多大程度上受到调节，葡萄糖诱导的控制其循环速率的信号通路，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启动了Rab27在特定可识别的分泌颗粒群体上的GTP/GDP循环。Rab27-GTP的增加被认为是通过与Slp4a的相互作用来驱动颗粒招募/对接的重要因素，而GTP在对接颗粒上的水解被认为是促进融合的启动。拟议的研究分为三个具体目标。首先，我们将确定小鼠培养细胞GSIS过程中Rab27A GTP/GDP循环的时空特性及其与分泌动力学的功能关系。其次，我们将确定Rab27A在受调节的胞外通路中促进胰岛素分泌的主要位点，明确其作用机制，并表征特定Rab27A效应物的作用机制。最后，我们将定义调控Rab27A GTP/GDP循环速率的信号通路。通过对Rab27A的影响，确定信号中间体调节的分泌途径中的作用位点。实验将使用从缺乏Rab27A蛋白表达的灰色小鼠模型和对照C3H/He小鼠中分离和培养的小鼠¿-细胞。实验采用光学（FRET, TIRF-FRET），电生理（膜片钳，CM-监测）和生化/药理学方法严格定义Rab27A功能的机制。公共卫生相关性：胰岛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。预计到2020年，全球将有2.5亿人罹患2型糖尿病。当细胞释放胰岛素的能力被调节葡萄糖和脂质代谢的激素需求所超过时，就会发生这种疾病。在糖尿病患者中，细胞功能障碍的早期表现包括胰岛素分泌对葡萄糖挑战的反应延迟和迟钝，以及刺激-分泌紧密耦合的丧失。因此，有必要全面了解胰岛素分泌背后的分泌途径的分子和机制细节，以最终开发糖尿病的治疗方法。