Role of Rab GAPs AS160 and Tbc1d1 in GLUT4 translocation and glucose homeostasis

描述（由申请人提供）：主要疾病、肥胖症、代谢综合征和糖尿病与葡萄糖稳态受损相关。维持葡萄糖稳态的关键是控制葡萄糖转运蛋白GLUT 4在肌肉和脂肪细胞中的亚细胞分布。在禁食条件下，GLUT 4主要定位于细胞内囊泡（GLUT 4囊泡），并且很少GLUT 4位于细胞表面。这限制了肌肉和脂肪细胞对葡萄糖的摄取，并确保大脑有足够的葡萄糖供应。胰岛素在几分钟内刺激GLUT 4囊泡与质膜的移动、对接和融合（GLUT 4易位）。细胞表面GLUT 4的增加促进葡萄糖进入肌肉和脂肪细胞，从而使餐后循环葡萄糖水平正常化。GLUT 4易位也是至关重要的葡萄糖摄取增加响应运动。GLUT 4在禁食条件下保留以及胰岛素和运动将GLUT 4释放到体内原代脂肪和肌肉细胞的细胞表面的分子机制尚不清楚。在培养的脂肪和肌肉细胞中的研究将GLUT 4保留和释放的作用归因于两种Rab GT3激活蛋白（Rab GAP），AS 160和Tbc 1d 1。AS 160和Tbc 1d 1基因敲除小鼠的最新结果表明，脂肪细胞中的葡萄糖摄取受AS 160调节，骨骼肌中的葡萄糖摄取受AS 160和Tbc 1d 1调节。我们的假设是，AS 160和Tbc 1d 1各自单独控制GLUT 4的细胞内滞留和释放到质膜，因此葡萄糖摄取，在细胞中，只有一个两个Rab GAP主要表达（脂肪细胞和特定的骨骼肌）。在发现两个Rab GAP的细胞（大多数骨骼肌）中，我们建议它们以协作方式执行或响应不同的刺激来调节GLUT 4的保留和释放，从而调节葡萄糖摄取。然后，AS 160和Tbc 1d 1的细胞类型和信号特异性作用差异调节全身葡萄糖稳态。使用单个AS 160和Tbc 1d 1基因敲除小鼠和双AS 160和Tbc 1d 1基因敲除小鼠，以及从这些小鼠中分离的脂肪细胞和骨骼肌，我们将首先确定两个Rab GAP中的哪一个调节空腹条件下脂肪细胞和不同骨骼肌中的葡萄糖摄取和GLUT 4亚细胞分布，以及对胰岛素和运动的响应。然后，我们将研究这两个Rab GAP是否控制GLUT 4保留和释放的相同或不同步骤，并定义它们执行功能的机制。对于这些研究，将通过全内反射荧光和共聚焦显微镜在活细胞中监测GLUT 4运输。最后，我们将确定AS 160和Tbc 1d 1在空腹条件下以及对胰岛素和运动的反应中在全身葡萄糖稳态中的作用。小鼠的代谢分析将包括胰岛素敏感性和高胰岛素-正常血糖钳夹的测量。这项研究将阐明有助于维持葡萄糖稳态的基本机制，并为验证AS 160和Tbc 1d 1作为改善葡萄糖稳态的药物靶点提供关键信息。

DESCRIPTION (provided by applicant): Major diseases, obesity, the metabolic syndrome, and diabetes, are associated with impaired glucose homeo- stasis. Key to the maintenance of glucose homeostasis is the control of the subcellular distribution of the glucose transporter GLUT4 in muscle and fat cells. Under fasting conditions GLUT4 localizes mostly to intra- cellular vesicles (GLUT4 vesicles) and few GLUT4 are at the cell surface. This limits glucose uptake into muscle and fat cells and assures adequate glucose supply to the brain. Insulin stimulates, within minutes, movement, docking and fusion of GLUT4 vesicles with the plasma membrane (GLUT4 translocation). The increase of GLUT4 at the cell surface facilitates glucose disposal into muscle and fat cells, thereby normalizing circulating glucose levels after a meal. GLUT4 translocation is also crucial to increased glucose uptake in response to exercise. The molecular mechanism by which GLUT4 is retained under fasting conditions and by which insulin and exercise release GLUT4 to the cell surface in vivo in primary fat and muscle cells is unknown. Studies in cultured fat and muscle cells attribute a role in GLUT4 retention and release to the two Rab GTPase activating proteins (Rab GAPs), AS160 and Tbc1d1. Recent results with AS160 and Tbc1d1 knockout mice suggest that glucose uptake is regulated by AS160 in adipocytes, and by AS160 and Tbc1d1 in skeletal muscles. Our hypothesis is that AS160 and Tbc1d1 each alone control GLUT4 intracellular retention and release to the plasma membrane, and thus glucose uptake, in cells where only one of the two Rab GAPs is predominantly expressed (adipocytes and specific skeletal muscles). In cells in which both Rab GAPs are found (most skeletal muscles), we propose that they perform in a collaborative fashion or respond to different stimuli to regulate GLUT4 retention and release, and thus glucose uptake. The cell type- and signal-specific roles of AS160 and Tbc1d1 then differentially regulate whole body glucose homeostasis. Using single AS160 and Tbc1d1 knockout mice, and double AS160 and Tbc1d1 knockout mice, and adipocytes and skeletal muscles isolated from these mice, we will first determine which of the two Rab GAPs regulate glucose uptake and GLUT4 subcellular distribution in adipocytes and different skeletal muscles under fasting conditions and in response to insulin and exercise. We will then investigate whether the two Rab GAPs control the same or different steps in GLUT4 retention and release, and define mechanisms by which they perform their functions. For these studies GLUT4 trafficking will be monitored in live cells by total internal reflection fluorescence and confocal microscopy. Finally, we will establish the roles of AS160 and Tbc1d1 in whole body glucose homeostasis under fasting conditions and in response to insulin and exercise. Metabolic analysis of the mice will include measurements of insulin sensitivity and hyperinsulinemic-euglycemic clamps. This research will elucidate fundamental mechanisms that contribute to the maintenance of glucose homeostasis and provide crucial information towards validation of AS160 and Tbc1d1 as drug targets to improve glucose homeostasis.