Insulin control of GLUT4 traffic to the plasma membrane of adipocytes

葡萄糖进入脂肪和肌肉的转运受到严格的调控，它是由质膜中葡萄糖转运蛋白GLUT4的数量决定的。在空腹状态下，当胰岛素水平较低时，GLUT4通过快速的内吞作用和缓慢的循环作用活跃地隔离在细胞内。餐后胰岛素升高主要是通过加速GLUT4的循环，导致GLUT4重新分配到PM。在餐后胰岛素升高期间，PM GLUT4水平的增加是通过GLUT4的快速循环来积极维持的。当循环胰岛素水平下降时，GLUT4 PM水平恢复到刺激前的水平。因此，脂肪和肌肉细胞对葡萄糖摄取的控制依赖于细胞内部和质膜之间GLUT4运输的调节。GLUT4向PM的重新分配受损会导致与胰岛素抵抗和2型糖尿病相关的高血糖。阐明GLUT4转运的分子机制以及胰岛素对GLUT4转运的调节将显著影响我们对胰岛素抵抗的理解，从而为未来治疗干预的发展提供基础。虽然GLUT4贩运的现象被很好地描述了，但关于这一过程的分子机制仍有许多需要了解。GLUT4主要分布在两个细胞膜上：专用于运输GLUT4的胰岛素反应小泡(IRV)和GLUT4 TGN核周小室。由于IRV在未经刺激的(基础)细胞和胰岛素刺激的细胞中将GLUT4运送到PM，因此受到了广泛的研究。在基础脂肪细胞中GLUT4在细胞内的隔离以及在动员GLUT4以支持胰岛素刺激细胞中增加的需求方面，核周室起着关键作用。控制GLUT4进入和离开核周部位的分子机制还没有详细描述。在这里，我建议解决知识上的差距。该工作计划以我的实验室过去的成就为基础，解决了GLUT4贩运领域的几个关键问题。该项目的主要目标是：确定核周室蛋白质组，从而确定在调节GLUT4运输中起作用的蛋白质以及确定除GLUT4以外的货物蛋白存储其GLUT4的蛋白质；发现并表征负责调节GLUT4通过这个核周室的流量的蛋白质机制。为了实现这些目标，我们将使用最先进的质谱学方法和一系列功能完整的细胞分析方法来表征调控向和从核周间隔转运的分子机制。

Glucose transport into fat and muscle, which is tightly regulated, is determined by the amount of the GLUT4 glucose transporter in the plasma membrane (PM). In the fasted state, when insulin levels are low, GLUT4 is actively sequestered intracellularly by rapid endocytosis and slow recycling. Elevated insulin in the postprandial state induces a redistribution of GLUT4 to the PM, predominantly by accelerating GLUT4 recycling. Increased levels of PM GLUT4 are actively maintained by rapid GLUT4 recycling during the postprandial period of elevated insulin. GLUT4 PM levels return to pre-stimulation amounts when circulating insulin levels decrease. Thus, the control of glucose uptake by fat and muscle cells is dependent upon regulation of GLUT4 trafficking between the interior and PM of cells. Compromised GLUT4 redistribution to the PM contributes to hyperglycemia associated with insulin-resistance and type 2 diabetes. Elucidating the molecular mechanism underlying GLUT4 traffic and its regulation by insulin will significantly impact our understanding of insulin resistance and thereby provide a foundation for the future development of therapeutic interventions. Although the phenomenon of GLUT4 trafficking is well described, there is much to be learned about the molecular mechanisms of the process. GLUT4 is largely distributed among 2 intracellular membrane compartments: insulin-responsive vesicles (IRVs) that are specialized for the traffic of GLUT4 and the GLUT4 TGN perinuclear compartment. IRVs have been intensively studied because they ferry GLUT4 to the PM in both unstimulated (basal) and insulin-stimulated cells. The perinuclear compartment has a pivotal role in the intracellular sequestration of GLUT4 in basal adipocytes and in the mobilization of GLUT4 to support the increased demand in insulin-stimulated cells. The molecular mechanisms controlling GLUT4 trafficking to and from the perinuclear site have not been described in detail. Here I propose to address that gap in knowledge. The workplan, building on my labs past accomplishments, addresses several key questions in the field of GLUT4 trafficking. The major objectives of the project are to: define the perinuclear compartment proteome, thereby identifying proteins that function in the regulation of GLUT4 traffic as well as identifying cargo proteins other than GLUT4 that are stored their; discover and characterize the protein machinery responsible for regulating flux of GLUT4 through this perinuclear compartment. To accomplish these objectives, we will use state-of-the-art mass spectrometry profiling methods, and a comprehensive battery of functional intact cell assays to characterize the molecular mechanism regulating trafficking to and from the perinuclear compartment.