Endocytic Trafficking Pathways in Adipocytes

描述（由申请方提供）：胰岛素通过调节脂肪和肌肉细胞表面GLUT 4葡萄糖转运蛋白的数量来增加这些细胞对葡萄糖的摄取。在不存在胰岛素的情况下，GLUT 4主要通过双向动态保留机制在细胞内隔离，该机制涉及从细胞内隔室缓慢胞吐和从质膜快速回收。胰岛素通过改变GLUT4在细胞内部和细胞表面之间的动态分布，诱导GLUT4向细胞表面的可逆募集。调节质膜上GLUT 4的量对于调节全身葡萄糖稳态是至关重要的，胰岛素刺激条件的增加抵消高血糖症，并从质膜排除基底细胞以防止低血糖症。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性的分子缺陷尚不清楚。更全面地了解GLUT4内化可能会导致识别新的目标，用于开发治疗2型糖尿病的药物干预。虽然胞吞和胞吐作用在所有条件下确定细胞表面上GLUT 4的量都很重要，但过去的大多数研究都集中在表征胞吐作用，因此对基础条件下在胰岛素存在下控制GLUT 4内化的机制知之甚少，在刺激后恢复到基础保留，或胰岛素信号如何传递到GLUT4内吞作用。本申请的目的是使用定量生物化学和显微镜分析来表征GLUT4内吞作用，其结果将显着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)使用定量荧光显微镜和定量生化方法来表征基础和胰岛素处理的脂肪细胞中GLUT 4的内化。这些结果将为其他目标的研究提供概念基础。2)分析胰岛素信号转导受到药物治疗、显性干扰突变体表达和小干扰RNA靶向蛋白敲低干扰的细胞中GLUT 4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突变体研究中绘制在基础和胰岛素刺激条件下控制内化的结构决定因素。这些研究的结果将提供一个更完整的GLUT4内化的分子描述，从而提供必要的框架，了解胰岛素调节内吞作用在分子水平上。

DESCRIPTION (provided by applicant): Insulin increases glucose uptake into fat and muscle by modulating the number of GLUT4 glucose transporters on the surface of these cells. In the absence of insulin, GLUT4 is predominantly sequestered intracellularly by a bipartite, dynamic retention mechanism that involves slow exocytosis from intracellular compartments and rapid retrieval from the plasma membrane. Insulin induces a reversible recruitment of GLUT4 to the cell surface by altering the dynamic distribution of GLUT4 between the interior and cell surface. Regulation of the amount of GLUT4 on the plasma membrane is critical for regulation of whole body glucose homeostasis, with the increase in insulin-stimulated conditions counteracting hyperglycemia, and the exclusion from the plasma membrane of basal cells protecting against hypoglycemia. 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. A more complete understanding of GLUT4 internalization may lead to the identification of novel targets for the development of pharmacologic intervention in the treatment of Type 2 diabetes. Although both endocytosis and exocytosis are important in determining the amount of GLUT4 on the cell surface in all conditions, the majority of past studies have focused on characterizing exocytosis, and therefore relatively little is know about the mechanisms that control GLUT4 internalization in basal conditions, in the presence of insulin, in the return to basal retention following stimulation, or how the insulin signal is transmitted to GLUT4 endocytosis. The objective of this application is to use quantitative biochemical and microscopy assays to characterize GLUT4 endocytosis, the results of which will significantly extend our understanding of GLUT4 translocation and insulin action at a molecular level. There are three specific aims in this project. 1) To 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) To analyze the internalization kinetics of GLUT4 in cells in which insulin-signal transduction has been perturbed by the pharmacologic treatments, expression of dominant-interfering mutants and by small interfering RNA targeted protein knockdown. These results will provide novel information on the mechanisms of insulin regulation of endocytosis. 3) To 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 GLUT4 internalization, and thereby provide the necessary framework for understanding insulin-regulation of endocytosis at a molecular level.