Regulatory Mechanisms of GLUT4 Exocytosis

描述（由申请人提供）：随着胰岛素抵抗和2型糖尿病在全球范围内的流行，迫切需要了解胰岛素如何在分子水平上维持血糖稳态。胰岛素的主要功能是促进葡萄糖摄取到肌肉和脂肪组织中，这是由葡萄糖转运蛋白GLUT 4介导的过程。在胰岛素刺激后，GLUT 4通过受调节的胞吐作用从细胞内储存囊泡重新定位到细胞表面。GLUT 4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。SNARE或融合调节因子的功能丧失突变消除了胰岛素触发的GLUT 4胞吐作用，并破坏了血糖稳态。此外，GLUT 4囊泡融合蛋白的失衡与肥胖相关的胰岛素抵抗有关。虽然SNARE和融合调节因子的生理重要性是清楚的，但它们如何协同作用以介导和调节GLUT 4囊泡融合仍然知之甚少。本提案的总体目标是使用新颖和互补的方法回答这一关键问题。我们将首先定义的分子机制和功能相互作用的囊泡融合蛋白使用一种新的重组融合系统。我们将使用重组蛋白和从小鼠脂肪细胞分离的天然蛋白。然后，我们将在3 T3-L1脂肪细胞和从基因敲除小鼠分离的脂肪细胞中表征GLUT 4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是否以及如何在胰岛素抵抗中受损。如果成功完成，这项研究将大大拓宽我们对GLUT 4胞吐调控机制的认识。这些发现也将揭示与葡萄糖失衡相关的疾病，如胰岛素抵抗和2型糖尿病，并将促进开发新的策略， 治疗干预

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.