Endocytic Trafficking Pathways in Adipocytes

描述（由申请人提供）：胰岛素调节全身葡萄糖稳态，部分通过调节GLUT4葡萄糖转运体从细胞内腔室到肌肉和脂肪细胞的质膜的募集。因此，要了解胰岛素如何剧烈调节葡萄糖转运到脂肪和肌肉中，需要详细了解胰岛素调节的膜运输。胰岛素不能正确调节2型糖尿病患者的GLUT4运输。这种对胰岛素不敏感的分子缺陷尚不清楚。在过去的十年中，大多数研究都集中在表征GLUT4的细胞内和胞外运输途径上。然而，GLUT4在质膜上的表达是胞吞和胞吐的平衡；因此，快速的GLUT4内化在决定细胞表面GLUT4的数量方面具有重要作用。该项目的长期目标是更好地表征调节脂肪细胞内吞GLUT4的机制。在本申请中，我提出了对GLUT4内化的胰岛素调控进行定量、全面的分析，其结果将显著扩展我们在分子水平上对GLUT4易位和胰岛素作用的理解。该项目有三个具体目标。1)我们将使用定量荧光显微镜和定量生化方法来表征GLUT4在基础和胰岛素处理的脂肪细胞中的内化。这些结果将为其他目标的研究提供概念基础。2)我们将分析胰岛素信号转导被显性干扰突变体的表达或胰岛素信号转导途径蛋白的小干扰RNA敲低而受到干扰的细胞中GLUT4的内化动力学。这些结果将为胰岛素调节内吞作用的机制提供新的信息。3)在GLUT4突变体的研究中，我们将绘制出在基础和胰岛素刺激条件下控制内化的结构决定因素。这些研究结果将提供更完整的胰岛素调节GLUT4内化的分子描述，从而在分子水平上为理解胰岛素调节内吞作用提供必要的框架。

DESCRIPTION (provided by applicant): Insulin regulates whole body glucose homeostasis, in part, by regulating the recruitment of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane of muscle and fat cells. Thus, understanding how insulin acutely regulates glucose transport into fat and muscle requires a detailed understanding of insulin regulated-membrane trafficking. Insulin does not properly regulate GLUT4 trafficking in individuals with Type 2 diabetes. The molecular defect(s) underlying this insensitivity to insulin are not known. In the past decade, the majority of studies have focused on characterizing the intracellular and exocytic trafficking pathway of GLUT4. However, GLUT4 expression on the plasma membrane is a balance of endocytosis and exocytosis; hence, rapid GLUT4 internalization has a significant role in determining the amount of GLUT4 on the cell surface. The long-term objective of this project is to better characterize the mechanism that regulates the endocytosis of GLUT4 in adipocytes. In this application, I propose a quantitative, comprehensive analysis of insulin-regulation of GLUT4 internalization, the results of which will significantly extend our understanding of GLUT4 translocation and insulin action at a molecular level. The project has three specific aims. 1) We will use quantitative fluorescence microscopy and quantitative biochemical methods to characterize the internalization of GLUT4 in basal and insulin-treated adipocytes. These results will provide the conceptual foundation for the studies in the other aims. 2) We will analyze the internalization kinetics of GLUT4 in cells in which insulin-signal transduction has been perturbed by the expression of dominant-interfering mutants or by small interfering RNA knockdown of proteins of the insulin-signal transduction pathway. These results will provide novel information on the mechanisms of insulin regulation of endocytosis. 3) We will map, in studies of GLUT4 mutants, the structural determinants that control internalization in basal and insulin-stimulated conditions. The results of these studies will provide a more complete molecular description of insulin regulation of GLUT4 internalization, and thereby provide the necessary framework for understanding insulin-regulation of endocytosis at a molecular level.