Insulin Regulated Membrane Trafficking

描述（由申请人提供）：胰岛素抵抗导致能量代谢失调。在美国，胰岛素抵抗的患病率正以惊人的速度增长，并将随着肥胖的增加而继续上升。我们需要在分子水平上更好地了解胰岛素的作用，以确定胰岛素抵抗治疗方法的分子靶点。胰岛素通过将GLUT4葡萄糖转运蛋白从细胞内储存室募集到脂肪和肌肉细胞的质膜，从而增加葡萄糖的摄取。了解基底细胞内滞留的机制和诱导GLUT4转运到质膜的胰岛素信号传导机制是很重要的。关于GLUT4的转运及其与胰岛素信号传导的交叉，我们已经知道了相当多的信息，尽管还有很多需要了解。在这个更新申请中，我提出了一个工作计划，将提供关于GLUT4生物学三个领域的新信息。在目标1中，我们将分析突变的GLUT4在细胞质FQQI、LL-based和EVEY转运基序中的行为。分析将包括突变体胞外分泌和细胞内运输的详细动力学研究，以及确定突变体对GLUT4细胞内运输行程的影响的研究。这些结果将为GLUT4在分子水平上的分选提供新的信息。在目标2中，我们将确定兔蛋白在GLUT4的细胞内特化运输中的作用。用siRNA方法敲低Rab蛋白并分析GLUT4的行为。这些研究将集中于Rab10和Rab14，这是胰岛素调节的AS160 rab GAP的两个靶点。Rab31在细胞内GLUT4的基础转运中起作用，也将被研究。这些结果将为兔蛋白在胰岛素调节GLUT4运输中的作用提供新的信息。在目标3中，我们将描述Akt-1和Akt-2嵌合体的行为，以确定在胰岛素向GLUT4信号传导中Akt-2亚型特异性活性的结构域。在这些研究中，将使用一种基于胰岛素刺激的GLUT4易位在AKT-2敲除脂肪细胞中的拯救的功能测定和一种全内反射荧光显微镜测定，以测量质膜的招募。这些结果将为Akt同工异构体对GLUT4信号的特异性提供新的信息。建议的研究是基于我们在过去资助期间的成就。

DESCRIPTION (provided by applicant): Insulin resistance results in a dysregulation of energy metabolism. The prevalence of insulin resistance is increasing at an alarming rate in the Unite States and it will continue to rise in tandem with the increase in obesity. A better understanding of insulin action at a molecular level is required to identify molecular targets for the development of therapeutics for the treatment of insulin resistance. Insulin increases glucose uptake by recruiting the GLUT4 glucose transporter from intracellular storage compartments to the plasma membrane of fat and muscle cells. It is important to understand both the mechanism responsible for the basal intracellular retention and the mechanism for the insulin signaling that induces GLUT4 translocation to the plasma .membrane. A considerable amount is known about GLUT4 trafficking and its intersection with insulin signaling, although there is still much to be learned. In this renewal application I propose a work plan that will provide novel information on three areas of GLUT4 biology. In aim 1 we will analyze the behaviors of GLUT4 mutated in the cytoplasmic FQQI, LL-based and EVEY trafficking motifs. The analysis will include detailed kinetic studies of exocytosis and intracellular trafficking of the mutants as well as studies to define the impact of the mutants on the intracellular trafficking itinerary of GLUT4. These results will provide novel information on GLUT4 sorting at a molecular level. In aim 2 we will determine the roles of rab proteins in the specialized intracellular trafficking of GLUT4. Rab proteins will be knocked-down with siRNA methods and the behavior of GLUT4 analyzed. These studies will focus on Rab10 and Rab14, two targets of the insulin-regulated AS160 rab GAP. Rab31, which has a role in basal state intracellular GLUT4 traffic, will also be studied. These results will provide novel information on the roles of rab proteins in insulin regulation of GLUT4 trafficking. In aim 3 we will characterize the behaviors of Akt-1 and Akt-2 chimeras to identify the domains responsible for the isoform-specific activity of AKT-2 in insulin signaling to GLUT4. A functional assay based on rescue of insulin-stimulated GLUT4 translocation in AKT-2 knockdown adipocytes and a total internal reflection fluorescence microscopy assay, to measure recruitment to the plasma membrane, will be used in these studies. These results will provide novel information on Akt isoform specificity in signaling to GLUT4. The proposed studies are based on our accomplishments during the past funding period. Project Narrative: Insulin stimulates glucose transport into fat and muscle cells by causing vesicles containing a protein that transports glucose (called GLUT4) to move to and fuse with the plasma membrane, a process known as translocation. The translocation of GLUT4 is defective in insulin resistance and type 2 diabetes mellitus. The objective of this research project is to study the translocation process in molecular detail, which may lead to a better understanding of defects that give rise to insulin resistance and type 2 diabetes mellitus.