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

Rab GAP AS160 和 Tbc1d1 在 GLUT4 易位和葡萄糖稳态中的作用

• 批准号: 8410100
• 负责人: SUSANNA R KELLER
• 金额: $ 33.25万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8410100
• 关键词: Address; Adipocytes; Blood Glucose; Brain; Cell Fractionation; Cell membrane; Cell surface; Cells; Confocal Microscopy; Data; Defect; Development; Diabetes Mellitus; Disease; Docking; Dominant-Negative Mutation; Drug Targeting; Euglycemic Clamping; Exercise; Fasting; Fatty acid glycerol esters; Fluorescence Microscopy; GLUT4 gene; GTPase-Activating Proteins; Glucose; Glucose Clamp; Glucose Transporter; Hybrids; Infection; Insulin; Knock-out; Knockout Mice; Life; Maintenance; Measurement; Measures; Metabolic; Metabolic syndrome; Methods; Molecular; Monitor; Movement; Mus; Muscle; Muscle Cells; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Plasma; Play; Proteins; Regulation; Research; Role; Signal Transduction; Skeletal Muscle; Stimulus; Testing; Tissues; Validation; Vesicle; basal insulin; blood glucose regulation; cell type; extensor digitorum; feeding; glucose disposal; glucose tolerance; glucose uptake; improved; in vivo; insulin sensitivity; insulin tolerance; novel; prevent; public health relevance; rab GTP-Binding Proteins; response; tibialis anterior muscle; trafficking

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.

描述（由申请人提供）：主要疾病，肥胖，代谢综合征和糖尿病与葡萄糖同源性受损有关。维持葡萄糖稳态的关键是控制肌肉和脂肪细胞中葡萄糖转运蛋白glut4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），而GLUT4很少在细胞表面。这限制了葡萄糖吸收到肌肉和脂肪细胞中，并确保对大脑的葡萄糖供应足够。胰岛素在几分钟之内刺激GLUT4囊泡与质膜的运动，对接和融合（GLUT4易位）。细胞表面GLUT4的增加促进了葡萄糖处理成肌肉和脂肪细胞，从而使餐后循环葡萄糖水平正常化。 GLUT4易位对于响应运动而增加的葡萄糖摄取也至关重要。在禁食条件下保留GLUT4的分子机制，胰岛素和运动在原代脂肪和肌肉细胞中的体内释放到细胞表面的glut4尚不清楚。培养的脂肪和肌肉细胞的研究将在GLUT4保留中的作用归因于两个RAB GTPase激活蛋白（RAB GAPS），AS160和TBC1D1。 AS160和TBC1D1敲除小鼠的最新结果表明，脂肪细胞中的AS160调节葡萄糖的摄取，骨骼肌中的AS160和TBC1D1受到调节。我们的假设是，AS160和TBC1D1单独控制GLUT4细胞内保留并释放到质膜，因此在两个Rab间隙中只有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），我们建议它们以协作的方式进行或对不同的刺激做出反应以调节GLUT4保留和释放，从而调节葡萄糖的吸收。然后，AS160和TBC1D1的细胞类型和信号特异性作用差异调节了全身葡萄糖稳态。 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.然后，我们将研究两个RAB间隙是否控制GLUT4保留和释放中相同或不同的步骤，并定义它们执行其功能的机制。对于这些研究，将通过总内反射荧光和共聚焦显微镜在活细胞中监测GLUT4运输。最后，我们将在禁食条件下以及对胰岛素和运动的反应下，确定AS160和TBC1D1在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夹。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AS160和TBC1D1提供至关重要的信息，以改善葡萄糖稳态。