Endocytic Trafficking Pathways in Adipocytes

描述（由申请人提供）：胰岛素通过调节这些细胞表面的GLUT4葡萄糖转运蛋白的数量来增加脂肪和肌肉对葡萄糖的摄取。在缺乏胰岛素的情况下，GLUT4主要通过两部分的动态保留机制被隔离在细胞内，该机制涉及细胞内室的缓慢胞外分泌和从质膜的快速回收。胰岛素通过改变GLUT4在细胞内部和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GLUT4的数量对调节全身葡萄糖稳态至关重要，胰岛素刺激条件的增加可以抵消高血糖，基底细胞被排除在质膜外可以防止低血糖。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。对GLUT4内化的更全面了解可能会为2型糖尿病药物干预的发展找到新的靶点。尽管在所有条件下，内吞作用和胞吐作用都是决定细胞表面GLUT4数量的重要因素，但过去的大多数研究都集中在胞吐作用的特征上，因此对基础条件下、胰岛素存在下、刺激后返回基础滞留时控制GLUT4内化的机制，或胰岛素信号如何传递到GLUT4内吞作用的了解相对较少。本应用程序的目的是使用定量生化和显微镜分析来表征GLUT4的内吞作用，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。这个项目有三个具体目标。1)利用定量荧光显微镜和定量生化方法表征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)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突变体的研究中找出控制基础和胰岛素刺激条件下内化的结构决定因素。这些研究的结果将提供更完整的GLUT4内化的分子描述，从而为在分子水平上理解胰岛素对内吞作用的调节提供必要的框架。

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.