INSULIN STIMULATION OF GLUCOSE TRANSPORT

我们的长期目标是了解胰岛素是如何导致 脂肪和肌肉细胞的葡萄糖摄取-具体地说，为了识别， 鉴定并克隆转导信号的蛋白质 胰岛素激活受体蛋白-酪氨酸激酶与肿瘤细胞的融合 含有GLUT-4葡萄糖转运蛋白的细胞内小泡 质膜。我们克隆了一个新的低分子量GTP结合蛋白Rab3D， 它在胰岛素反应组织中高度表达，并具有许多 调节富含GLUT-4的胞吐的蛋白质的预期性质 水泡。事实上，我们已经产生了一个假定的显性负向Rab3D 突变，N135I，不能结合GTP；表达该突变，但不 野生型Rab3D在分化的3T3-L1脂肪细胞中抑制 胰岛素增加葡萄糖转运的能力。最近，我们克隆了 突触囊泡蛋白、突触素和突触缩短的同源物 (VAMP)，像Rab3D一样，在胰岛素反应中大量表达 组织，在体外脂肪形成过程中被诱导。我们还展示了 Rab3A，其表达被认为仅限于神经和神经内分泌 细胞，也在胰岛素反应组织中大量表达，并且是 在脂肪形成过程中诱导的。我们假设胰岛素的激活 受体在Rab3A或3D上诱导GTP与GDP的交换，在很大程度上 Ras被其他受体蛋白-酪氨酸激酶激活，并且 A-Rab3GTP直接诱导膜融合。我们的具体目标是：(一) Rab3D和Rab3A在GLUT4-和GLUT1-胞吐中的作用 含有囊泡和脂酶分泌。这包括世代 在Rab3A和3D中的其他显性-负突变并检查 胰岛素刺激对葡萄糖转运和血管内皮细胞转位的影响 Glut1和GLUT4在3T3-L1脂肪细胞中的表达； 检测GTPGammaS和与之相对应的合成肽的作用 Rab3对GLUT-4胞吐作用的推测结构域 通透性脂肪细胞系统和通透性脂肪细胞 表达突变型Rab3蛋白；(2)亚细胞鉴定 Rab3D、Rab3A及其脂肪细胞特异性同源物的定位 基础脂肪细胞和胰岛素刺激脂肪细胞中的突触素和突触素； (3)确定脂肪细胞特异性同源物的作用 基础和胰岛素刺激下血糖中的突触素和突触素 表达3T3-L1脂肪细胞的克隆性转运 这些蛋白的特定突变形式或过度表达野生型 蛋白质，并检测它们对胰岛素刺激的易位 GLUT4和GLUT1对细胞表面和脂酶分泌的影响；以及(4) 克隆Rab3D或Raba GDP-GTP交换蛋白或GAP蛋白 在脂肪形成过程中，使用表达克隆、杂交或 聚合酶链式反应和合适的脂肪细胞特异性全长消减表达 C DNA文库。我们将测试这些蛋白质是否是 胰岛素受体至胞吐作用的信号转导途径 葡萄糖转运蛋白。

Our long-term goal is to understand how insulin causes an increase in glucose uptake by fat and muscle cells-specifically, to identify, characterize, and clone proteins that transduce the signal from the insulin-activated receptor protein-tyrosine kinase to the fusion of intracellular vesicles containing the GLUT-4 glucose transporter with the plasma membrane. We have cloned a new low-mw GTP binding protein, Rab3D, that is highly expressed in insulin-responsive tissues, and has many of the properties expected of a protein that regulates exocytosis of GLUT-4-rich vesicles. Indeed, we have generated a presumed dominant-negative Rab3D mutation, N135I, that cannot bind GTP; expression of this mutant but not the wild-type Rab3D in differentiated 3T3-L1 adipocytes inhibits the ability of insulin to increase glucose transport. Recently, we have cloned homologs of the synaptic vesicle proteins synaptophysin and synaptobrevin (VAMP) that, like Rab3D, are abundantly expressed in insulin responsive tissues and are induced during adipogenesis in vitro. We also showed that Rab3A, whose expression was thought confined to nerve and neuroendocrine cells, is also abundantly expressed in insulin responsive tissues and is induced during adipogenesis. We hypothesize that activation of the insulin receptor induces an exchange of GTP for GDP on Rab3A or 3D, much in the way that ras is activated by other receptor protein-tyrosine kinases, and that a Rab3GTP directly induces membrane fusion. Our specific goals are: (1) Elucidating the roles of Rab3D and Rab3A in exocytosis of GLUT4- and GLUT1- containing vesicles and in secretion of adipsin. This includes generation of other dominant-negative mutations in Rab3A and 3D and examining the effects on insulin-stimulation of glucose transport and translocation of GLUT1 and GLUT4 to the cell surface in transfected 3T3-L1 adipocytes; and examining the effects of GTPgammaS and synthetic peptides corresponding to the presumed effector domain of Rab3 on exocytosis of GLUT- 4 in two permeabilized adipocyte systems and also in permeabilized adipocytes expressing mutant Rab3 proteins; (2) Determining the subcellular localization of Rab3D, Rab3A, and the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated adipocytes; (3) Determining the roles of the adipocyte-specific homologs of synaptophysin and synaptobrevin in basal and insulin-stimulated glucose transport by generating clonal lies of 3T3-l1 adipocytes that express specific mutant forms of these proteins or that overexpress the wild-type protein, and examining them for insulin-stimulation of translocation of GLUT4 and GLUT1 to the cell surface and secretion of adipsin; and (4) Cloning Rab3D or RabA GDP-GTP exchange protein or GAP proteins that are induced during adipogenesis, using expression cloning, hybridization, or PCR and a suitable full-length subtractive, adipocyte-specific expression cDNA library. We will test whether these proteins are intermediates in the signal transduction pathway from the insulin receptor to exocytosis of glucose transporters.