Regulatory Mechanisms of GLUT4 Exocytosis

项目摘要 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 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，这一过程由葡萄糖转运蛋白介导 GLUT 4。在胰岛素刺激下，GLUT 4从细胞内储存囊泡重新定位到细胞表面 通过调节胞吐作用。GLUT 4囊泡的胞吐作用需要SNARE蛋白作为核心 融合机器，以及一组融合调节器。SNARE或融合的功能丧失突变 调节剂消除胰岛素触发的GLUT 4胞吐作用并破坏血糖稳态。此外，委员会认为， GLUT 4囊泡融合蛋白的不平衡与肥胖相关的胰岛素抗性有关。 虽然SNARE和融合调节因子的生理学重要性是清楚的，但仍然知之甚少 它们如何协同作用以介导和调节GLUT 4囊泡融合。本提案的总体目标是 用新颖和互补的方法回答这个关键问题。我们将首先定义分子 使用新的重组融合物的囊泡融合蛋白的机制和功能相互作用 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。我们将 然后表征3 T3-L1脂肪细胞和从人脂肪细胞分离的脂肪细胞中的GLUT 4囊泡融合蛋白， 敲除小鼠最后，我们将确定囊泡融合蛋白的活性是否以及如何被调节。 胰岛素抵抗受损，使用高脂肪饮食喂养的小鼠作为模型系统。如果成功完成， 这项研究将大大拓宽我们对GLUT 4胞吐调节机制的认识。 这些发现也将有助于阐明与葡萄糖失衡相关的疾病，如胰岛素 耐药性和2型糖尿病，并将促进新的治疗策略的发展， 干预

PROJECT SUMMARY 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 high 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.