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

描述（由申请人提供）：重大疾病，肥胖，代谢综合征和糖尿病，与葡萄糖稳态受损有关。葡萄糖稳态维持的关键是控制葡萄糖转运体GLUT4在肌肉和脂肪细胞中的亚细胞分布。在禁食条件下，GLUT4主要定位于细胞内囊泡（GLUT4囊泡），很少出现在细胞表面。这限制了肌肉和脂肪细胞对葡萄糖的摄取，保证了大脑有足够的葡萄糖供应。胰岛素在几分钟内刺激GLUT4囊泡与质膜的运动、对接和融合（GLUT4易位）。细胞表面GLUT4的增加有助于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。GLUT4易位对运动后葡萄糖摄取的增加也至关重要。在体内原代脂肪和肌肉细胞中，空腹条件下GLUT4保留的分子机制以及胰岛素和运动将GLUT4释放到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GLUT4的保留和释放归因于两种Rab GTPase激活蛋白（Rab GAPs） AS160和Tbc1d1。最近对AS160和Tbc1d1基因敲除小鼠的研究结果表明，脂肪细胞中的AS160和骨骼肌中的AS160和Tbc1d1调节葡萄糖摄取。我们的假设是，AS160和Tbc1d1各自单独控制GLUT4在细胞内的保留和释放到质膜，从而控制葡萄糖的摄取，在细胞中只有两种Rab gap中的一种主要表达（脂肪细胞和特定骨骼肌）。在发现这两种Rab gap的细胞（大多数骨骼肌）中，我们认为它们以协作的方式发挥作用，或对不同的刺激作出反应，以调节GLUT4的保留和释放，从而调节葡萄糖的摄取。然后AS160和Tbc1d1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AS160和Tbc1d1基因敲除小鼠，双AS160和Tbc1d1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，我们将首先确定两种Rab gap中哪一种在禁食条件下以及对胰岛素和运动的反应中调节脂肪细胞和不同骨骼肌中的葡萄糖摄取和GLUT4亚细胞分布。然后，我们将研究这两种Rab gap在GLUT4的保留和释放中是否控制相同或不同的步骤，并确定它们执行其功能的机制。在这些研究中，GLUT4的运输将通过全内反射荧光和共聚焦显微镜在活细胞中监测。最后，我们将确定AS160和Tbc1d1在空腹条件下以及胰岛素和运动反应下的全身葡萄糖稳态中的作用。小鼠的代谢分析将包括测量胰岛素敏感性和高胰岛素-血糖钳。本研究将阐明维持葡萄糖稳态的基本机制，并为验证AS160和Tbc1d1作为改善葡萄糖稳态的药物靶点提供重要信息。

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.