Endocytic Trafficking Pathways in Adipocytes

胰岛素通过调节GLUT4葡萄糖的数量增加脂肪和肌肉对葡萄糖的摄取 这些细胞表面的转运蛋白。在缺乏胰岛素的情况下，GLUT4主要被隔离 在细胞内通过两部分的动态保留机制，包括细胞内缓慢的胞吐作用 隔室和从质膜快速取回。胰岛素诱导可逆性募集 通过改变GLUT4在细胞内部和细胞之间的动态分布来实现细胞表面的GLUT4 浮出水面。调节质膜上的GLUT4的量是调节全身的关键 葡萄糖稳态，随着胰岛素刺激条件的增加来抵消高血糖，以及 从基底细胞的质膜中排出，防止低血糖。胰岛素不起作用 妥善管理2型糖尿病患者的GLUT4贩运。潜在的分子缺陷(S) 这种对胰岛素不敏感的情况还不得而知。对GLUT4内化的更完整理解可能会导致 寻找药物干预治疗的新靶点 2型糖尿病。虽然胞吞作用和胞吐作用在确定 GLUT4在细胞表面的各种条件下，以往的大多数研究都集中在表征 胞吐作用，因此对控制GLUT4内化的机制知之甚少。 基础条件，在胰岛素存在的情况下，在刺激后恢复到基础保留，或如何 胰岛素信号被传递给GLUT4内吞作用。此应用程序的目标是使用量化 生化和显微镜分析表征GLUT4内吞作用，其结果将显著 在分子水平上扩大我们对GLUT4易位和胰岛素作用的理解。一共有三个 这个项目的具体目标。1)使用定量荧光显微镜和定量生化 方法研究GLUT4在基础和胰岛素处理的脂肪细胞中的内化情况。这些结果 将为其他目标的研究提供概念基础。2)内部化分析 GLUT4在胰岛素信号转导受药物干扰的细胞中的动力学 处理、显性干扰突变体的表达和小干扰RNA靶向蛋白 击倒对手。这些结果将为胰岛素调节的机制提供新的信息。 内吞作用。3)在GLUT4突变体的研究中定位控制内化的结构决定因素 在基础和胰岛素刺激条件下。这些研究的结果将提供更完整的 GLUT4内化的分子描述，从而提供了必要的框架 在分子水平上理解胰岛素对内吞作用的调节。

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.