Adaptive Regulation in 3T3-L1 Adipocytes

描述（由申请人提供）：大多数细胞依赖葡萄糖作为其主要的底物来产生能量和储存碳。此外，葡萄糖在调节参与其自身代谢的蛋白质表达中起作用。其中包括参与葡萄糖运输的蛋白质，这通常是葡萄糖代谢的限速步骤。动物研究表明，脂肪组织的基础葡萄糖摄取与“组成性”葡萄糖转运蛋白GLUT1的表达和活性直接相关。我们特别关注葡萄糖本身对GLUT1的调节。为了实现这一点，我们使用了3T3-L1脂肪细胞，它在受控环境中提供了长时间的表征。在缺乏葡萄糖的细胞中，我们发现运输活性增加了20倍，这不是一个微不足道的变化。与这一观察相关的是，在循环葡萄糖的生理极限上观察到4倍的变化。由于GLUT1的表达没有改变，这导致了GLUT1在葡萄糖剥夺反应中被激活的假设。我们现在知道，质膜中的一部分GLUT1存在于脂筏中。这一比例在贫困中增加。新的数据表明，从葡萄糖剥夺细胞分离的脂筏中重建的GLUT1比从对照细胞中观察到的具有更高的“内在活性”。有趣的是，我们能够证明GLUT1和stomatin（一种几乎只存在于脂筏部分的蛋白质）之间的特异性相互作用。葡萄糖剥夺增加了这种相互作用，这推断气孔素和GLUT1在脂筏中相互作用。综上所述，这些数据表明脂筏的环境在GLUT1的功能中起作用。这些新颖的观察结果为我们理解葡萄糖转运的营养依赖控制提供了新的方向。在特异性目标1中，我们重点关注GLUT1对脂筏的靶向作用。在具体目标2中，我们探讨脂质在筏运输功能中的作用。在Specific Aim 3中，我们使用一种新的蛋白质组学方法鉴定了脂筏中独特的蛋白质，这种蛋白质组学方法可能调节GLUT1。最后，在Specific Aim 4中，我们使用stomatin敲除小鼠来确定stomatin在GLUT1靶向和功能中的作用。

DESCRIPTION (provided by applicant): Most cells depend on glucose as their primary substrate for energy production and carbon storage. In addition, glucose serves a role in regulating the expression of proteins involved in its own metabolism. These include the proteins involved in glucose transport, which is often the rate-limiting step in its metabolism. Animal studies suggest that basal glucose uptake in adipose tissue is directly related to the expression and activity of GLUT1, the "constitutive" glucose transporter. We have focused specifically on the regulation of GLUT1 by glucose, itself. To accomplish this, we have used 3T3-L1 adipocytes which afford characterization over extended time in a controlled environment. In cells deprived of glucose, we have shown that transport activity increases by 20-fold, not an insignificant change. The relevance to this observation is that a 4-fold change is observed over the physiological extremes of circulating glucose. Because GLUT1 expression does not change, this led to the hypothesis that GLUT1 is activated in response to glucose deprivation. We now know that a proportion of the GLUT1 pool in plasma membranes resides in lipid rafts. This percentage increases in response to deprivation. New data demonstrate that GLUT1 reconstituted from lipid rafts isolated from glucose-deprived cells exhibits higher "intrinsic activity" than that observed from control cells. Interestingly, we are able to demonstrate a specific interaction between GLUT1 and stomatin, a protein that exists almost exclusively in the lipid raft fraction. Glucose-deprivation increases this interaction, which infers that stomatin and GLUT1 interact within lipid rafts. Together, these data suggest that the environment of lipid rafts plays a role in GLUT1 function. These novel observations have led to new directions in our attempts to understand the nutrient-dependent control of glucose transport. In Specific Aim 1, we focus on the targeting of GLUT1 to lipid rafts. In Specific Aim 2, we explore the role of lipids in rafts on transport function. In Specific Aim 3, we identify proteins unique to lipid rafts using a novel proteomics approach which may regulate GLUT1. Finally in Specific Aim 4, we use the stomatin knockout mouse to determine the role of stomatin on GLUT1 targeting and function.