Mechanisms of Rab27 regulation of insulin secretion

Rab27调节胰岛素分泌的机制

• 批准号: 8228165
• 负责人: EDWARD L STUENKEL
• 金额: $ 33.74万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8228165
• 关键词: Affect; Binding; Biochemical; C3H/He Mouse; 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; Health; Hormones; Hydrolysis; 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; glucose metabolism; insulin secretion; lipid metabolism; mouse model; patch clamp; 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β细胞功能异常会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。该提案的总体目标是加深对调节β细胞胰岛素分泌的分子机制的了解，以便开发出更好的促进胰岛素分泌的治疗干预措施。该研究的重点是了解 GTPase Rab27A 对含胰岛素分泌颗粒的胞吐作用发挥如此有效调节的分子机制。特定 Rab 的 GTP/GDP 循环的速率和位置对其行为施加精确的时间和空间调节。然而，Rab27A 的 GTP/GDP 循环受到调节的程度、控制其循环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然未知。我们假设 Rab27A 的核苷酸状态受到严格控制，并且 GSIS 在特定的可识别分泌颗粒群体上启动 Rab27 的 GTP/GDP 循环。 Rab27-GTP 的增加被认为对于通过与 Slp4a 相互作用驱动颗粒招募/对接很重要，而对接颗粒上的 GTP 水解被认为可以促进融合的启动。拟议的研究分为三个具体目标。首先，我们将确定 Rab27A GTP/GDP 循环的空间和时间特性及其与培养的小鼠 ¿ 细胞 GSIS 期间分泌动态的功能关系。其次，我们将确定 Rab27A 促进胰岛素分泌的受调节胞吐途径中的主要位点，定义其作用机制，并表征特定 Rab27A 效应器的作用机制。最后，我们将定义调节 Rab27A 的 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。实验将使用从灰白小鼠模型（缺乏 Rab27A 蛋白表达）和对照 C3H/He 小鼠中分离和培养的小鼠 ¿-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 功能的机制。公众健康相关性：胰岛 β 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 型糖尿病预计到 2020 年将影响全球 2.5 亿人，当 β 细胞释放胰岛素的能力超过调节葡萄糖和脂质代谢的激素的需求时，就会发生这种疾病。在糖尿病患者中，K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延迟和减弱以及紧密刺激-分泌耦合的丧失。因此，非常有必要全面了解胰岛素分泌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。