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能量产生和碳储存的主要底物。此外，葡萄糖在调节参与其自身代谢的蛋白质的表达中起作用。这些包括参与葡萄糖转运的蛋白质，这通常是其代谢中的限速步骤。动物研究表明，脂肪组织中的基础葡萄糖摄取与"组成型"葡萄糖转运蛋白GLUT 1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，其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，我们已经证明转运活性增加了20倍，这不是一个微不足道的变化。与该观察结果相关的是，在循环葡萄糖的生理极值上观察到4倍变化。由于GLUT1表达没有变化，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GLUT 1库的一部分驻留在脂筏中。这一比例随着贫困程度的增加而增加。新的数据表明，GLUT1重组从脂筏分离葡萄糖剥夺细胞表现出更高的“内在活性”比观察到的控制细胞。有趣的是，我们能够证明GLUT1和气孔蛋白，一种几乎完全存在于脂筏组分中的蛋白质之间的特定相互作用。葡萄糖剥夺增加了这种相互作用，这推断，气孔蛋白和GLUT1相互作用的脂筏。总之，这些数据表明脂筏的环境在GLUT 1功能中起作用。这些新的观察结果为我们理解葡萄糖转运的营养依赖性控制提供了新的方向。在具体目标1中，我们重点关注GLUT 1靶向脂筏。在具体目标2中，我们探讨了脂筏转运功能的作用。在具体目标3中，我们使用一种新的蛋白质组学方法鉴定了脂筏特有的蛋白质，该蛋白质可能调节GLUT 1。最后，在具体目标4中，我们使用stomatin敲除小鼠来确定stomatin对GLUT 1靶向和功能的作用。

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.