Insulin Regulated Membrane Trafficking

描述(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在以惊人的速度增加，而且随着肥胖症的增加，它将继续上升。需要在分子水平上更好地了解胰岛素的作用，以确定治疗胰岛素抵抗的治疗方法的分子靶点。胰岛素通过将GLUT4葡萄糖转运体从细胞内的储存室招募到脂肪和肌肉细胞的质膜上来增加葡萄糖的摄取。重要的是要了解基础细胞内滞留的机制和诱导GLUT4移位到质膜的胰岛素信号机制。关于GLUT4的贩运及其与胰岛素信号的交叉，人们已经知道了相当多，尽管仍有许多需要了解。在这份续签申请中，我提出了一项工作计划，将在GLUT4生物学的三个领域提供新的信息。在目标1中，我们将分析GLUT4在细胞质FQQI、基于LL和Evey运输基序中的突变行为。该分析将包括对突变体胞吐和胞内转运的详细动力学研究，以及确定突变体对GLUT4胞内转运路线的影响的研究。这些结果将为GLUT4在分子水平上的分类提供新的信息。在目标2中，我们将确定Rab蛋白在GLUT4的专业细胞内运输中的作用。用siRNA方法敲除RAB蛋白，并分析GLUT4的行为。这些研究将集中在Rab10和Rab14上，这是胰岛素调节的AS160Rab GAP的两个靶点。在基础状态细胞内GLUT4运输中起作用的Rab31也将被研究。这些结果将为Rab蛋白在胰岛素调节GLUT4转运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，以确定负责AKT-2在胰岛素信号转导GLUT4中的异构体特异性活性的结构域。在这些研究中，将使用基于挽救AKT-2基因敲除脂肪细胞中胰岛素刺激的GLUT4易位的功能分析和全内反射荧光显微镜分析来测量质膜的募集。这些结果将为Akt异构体在GLUT4信号传递中的特异性提供新的信息。拟议的研究是基于我们在过去筹资期间取得的成就。 项目简介：胰岛素通过使含有葡萄糖运输蛋白(称为GLUT4)的囊泡移动到质膜并与质膜融合来刺激葡萄糖向脂肪和肌肉细胞的运输，这一过程称为移位。GLUT4易位在胰岛素抵抗和2型糖尿病中存在缺陷。这项研究的目的是从分子细节上研究易位过程，这可能有助于更好地理解导致胰岛素抵抗和2型糖尿病的缺陷。

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.