Insulin Regulated Membrane Trafficking

描述（由申请方提供）：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加，并将随着肥胖的增加而继续上升。需要在分子水平上更好地理解胰岛素作用，以鉴定用于开发治疗胰岛素抵抗的疗法的分子靶点。胰岛素通过将GLUT 4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来增加葡萄糖摄取。重要的是要了解负责基础细胞内滞留的机制和诱导GLUT 4易位到质膜的胰岛素信号传导的机制。关于GLUT 4运输及其与胰岛素信号传导的交叉点，我们已经知道了相当多的信息，尽管仍有很多东西需要了解。在这个更新申请中，我提出了一个工作计划，将提供GLUT 4生物学三个领域的新信息。在目的1中，我们将分析GLUT 4在细胞质中的GLUTQI、LL基序和EVEY运输基序中突变的行为。该分析将包括胞吐和胞内运输的突变体的详细动力学研究，以及研究，以确定GLUT 4的胞内运输路线的突变体的影响。这些结果将在分子水平上提供关于GLUT 4分选的新信息。在目标2中，我们将确定RAB蛋白在GLUT 4的专门细胞内运输中的作用。用siRNA方法敲除Rab蛋白，并分析GLUT 4的行为。这些研究将重点关注Rab 10和Rab 14，这是胰岛素调节的AS 160 rab GAP的两个靶点。Rab 31在基础状态细胞内GLUT 4运输中起作用，也将进行研究。这些结果将提供新的信息的作用，rab蛋白在胰岛素调节GLUT 4运输。在目标3中，我们将表征Akt-1和Akt-2嵌合体的行为，以确定负责AKT-2在胰岛素信号传导至GLUT 4中的亚型特异性活性的结构域。在这些研究中，将使用基于AKT-2敲低脂肪细胞中胰岛素刺激的GLUT 4易位的拯救的功能测定和全内反射荧光显微镜测定，以测量质膜的募集。这些结果将提供新的信息Akt亚型特异性信号GLUT 4。拟议的研究是基于我们在过去的资助期间取得的成就。 项目叙述：胰岛素通过使含有转运葡萄糖的蛋白质（称为GLUT 4）的囊泡移动到质膜并与质膜融合，从而刺激葡萄糖转运到脂肪和肌肉细胞中，这一过程称为易位。GLUT 4的易位在胰岛素抵抗和2型糖尿病中是有缺陷的。本研究项目的目的是从分子水平详细研究易位过程，从而更好地了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.