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

描述(申请人提供)：主要疾病，肥胖，代谢综合征和糖尿病，与受损的葡萄糖稳态有关。维持葡萄糖动态平衡的关键是控制葡萄糖转运蛋白GLUT4在肌肉和脂肪细胞中的亚细胞分布。在禁食条件下，GLUT4主要定位于细胞内小泡(GLUT4小泡)，少数位于细胞表面。这限制了肌肉和脂肪细胞对葡萄糖的吸收，并确保大脑有足够的葡萄糖供应。胰岛素在几分钟内刺激GLUT4囊泡与质膜的移动、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，从而使餐后循环中的血糖水平正常化。GLUT4易位也是运动增加葡萄糖摄取的关键。在原代脂肪和肌肉细胞中，GLUT4在禁食条件下被保留以及胰岛素和运动在体内将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 GTP酶激活蛋白(Rab Gap)，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分别调节葡萄糖的摄取。我们的假设是，AS160和Tbc1d1各自单独控制GLUT4在细胞内的滞留和释放到质膜，从而控制葡萄糖的摄取，在只有两个Rab间隙中的一个主要表达的细胞中(脂肪细胞和特定的骨骼肌)。在发现两个Rab间隙的细胞中(大多数骨骼肌)，我们认为它们以协作的方式执行，或对不同的刺激做出反应，以调节GLUT4的保留和释放，从而调节葡萄糖的摄取。AS160和Tbc1d1的细胞类型和信号特异性作用随后以不同的方式调节全身葡萄糖稳态。利用单个AS160和Tbc1d1基因敲除小鼠，以及双AS160和Tbc1d1基因敲除小鼠，以及从这些小鼠分离的脂肪细胞和骨骼肌，我们将首先确定两个Rab缺口中的哪一个调节禁食条件下以及对胰岛素和运动的反应下脂肪细胞和不同骨骼肌中葡萄糖的摄取和GLUT4亚细胞分布。然后，我们将调查两个RAB缺口是否控制GLUT4保留和释放中的相同或不同步骤，并定义它们执行其功能的机制。在这些研究中，GLUT4在活细胞中的转运将通过全内反射、荧光和共聚焦显微镜进行监测。最后，我们将确定AS160和Tbc1d1在禁食条件下以及对胰岛素和运动的反应中在全身血糖稳态中的作用。对小鼠的代谢分析将包括测量胰岛素敏感性和高胰岛素-正常血糖钳夹。这项研究将阐明有助于维持血糖稳态的基本机制，并为AS160和Tbc1d1作为改善血糖稳态的药物靶点的验证提供关键信息。 公共卫生相关性：项目简介：包括肥胖、代谢综合征和2型糖尿病在内的主要疾病与葡萄糖稳态受损有关，这一缺陷在这些疾病的严重并发症的发展中发挥了作用。维持正常血糖稳态的关键是适当调节葡萄糖转运蛋白GLUT4在肌肉和脂肪细胞中的分布。这项拟议的研究将阐明两个新的蛋白质(AS160和Tbc1d1)在控制GLUT4在细胞内的分布和葡萄糖稳态中的功能。这项研究将阐明有助于维持血糖稳态的基本机制，并为AS160和Tbc1d1作为改善血糖稳态和预防后续并发症的药物靶点的有效性提供关键信息。

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. PUBLIC HEALTH RELEVANCE: Project Narrative Major diseases, including obesity, the metabolic syndrome, and type 2 diabetes, are associated with impaired glucose homeostasis, a defect that plays a role in the development of severe complications of these diseases. Key to the maintenance of normal glucose homeostasis is the proper regulation of the distribution of the glucose transporter GLUT4 within muscle and fat cells. The proposed research will elucidate the function of two novel proteins (AS160 and Tbc1d1) in the control of the distribution of GLUT4 within cells and in glucose homeostasis. This research will elucidate fundamental mechanisms that contribute to the maintenance of glucose homeostasis and provide crucial information towards the validation of AS160 and Tbc1d1 as drug targets to improve glucose homeostasis and prevent ensuing complications.