Regulatory Mechanisms of GLUT4 Exocytosis

描述(申请人提供)：随着胰岛素抵抗和2型糖尿病在世界范围内的流行，迫切需要从分子水平上了解胰岛素如何维持血糖稳态。胰岛素的主要功能是促进肌肉和脂肪组织对葡萄糖的吸收，这一过程是由葡萄糖转运体GLUT4介导的。在胰岛素刺激下，GLUT4通过调控的胞吐作用从细胞内的储存囊重新定位到细胞表面。GLUT4囊泡的胞吐作用需要SNARE蛋白作为核心融合机制，以及一组融合调节因子。SNARs或融合调节器的功能缺失突变可消除胰岛素触发的GLUT4胞吐作用，并破坏血糖稳态。此外，GLUT4囊泡融合蛋白的失衡与肥胖相关的胰岛素抵抗有关。虽然陷阱和融合调节因子的生理重要性是显而易见的，但它们如何协同作用来调节和调节GLUT4囊泡融合仍然知之甚少。这项提案的总体目标是使用新颖和互补的办法回答这一关键问题。我们将首先使用一种新的重组融合系统来确定囊泡融合蛋白的分子机制和功能相互作用。我们将同时使用重组蛋白和从小鼠脂肪细胞中分离的天然蛋白。然后，我们将在3T3-L1脂肪细胞和从基因敲除小鼠分离的脂肪细胞中鉴定GLUT4囊泡融合蛋白。最后，我们将以高脂饮食喂养的小鼠为模型系统，确定囊泡融合蛋白的活性在胰岛素抵抗中是否以及如何受到损害。如果成功完成，这项研究将极大地拓宽我们对GLUT4胞吐调节机制的认识。这些发现还将揭示与血糖失衡有关的疾病，如胰岛素抵抗和2型糖尿病，并将促进开发新的治疗策略 治疗性干预。

DESCRIPTION (provided by applicant): As the epidemic of insulin resistance and type 2 diabetes emerges worldwide, there is an urgent need to understand how insulin maintains blood glucose homeostasis at the molecular level. A major function of insulin is to promote glucose uptake into muscle and adipose tissues, a process mediated by the glucose transporter GLUT4. Upon insulin stimulation, GLUT4 is relocated from intracellular storage vesicles to the cell surface through regulated exocytosis. The exocytosis of GLUT4 vesicles requires the SNARE proteins as the core fusion machinery, as well as a group of fusion regulators. Loss-of-function mutations of the SNAREs or fusion regulators abrogate insulin-triggered GLUT4 exocytosis and disrupt blood glucose homeostasis. Moreover, imbalances in the GLUT4 vesicle fusion proteins have been implicated in obesity-associated insulin resistance. While the physiological importance of the SNAREs and fusion regulators is clear, it remains poorly understood how they act in concert to mediate and regulate GLUT4 vesicle fusion. The overall goal of this proposal is to answer this key question using novel and complementary approaches. We will first define the molecular mechanisms and functional interactions of the vesicle fusion proteins using a novel reconstituted fusion system. We will use both recombinant proteins and native proteins isolated from mouse adipocytes. We will then characterize GLUT4 vesicle fusion proteins in 3T3-L1 adipocytes and in adipocytes isolated from knockout mice. Finally, we will determine whether and how the activities of the vesicle fusion proteins are impaired in insulin resistance, using hig fat diet-fed mice as a model system. If successfully accomplished, this research will substantially broaden our knowledge about the regulatory mechanisms of GLUT4 exocytosis. The findings will also shed light upon the diseases associated with glucose imbalances such as insulin resistance and type 2 diabetes, and will facilitate the development of novel strategies for therapeutic intervention.