Adaptive Regulation in 3T3-L1 Adipocytes

描述(申请人提供)：大多数细胞依靠葡萄糖作为其能量生产和碳储存的主要底物。此外，葡萄糖还在调节自身新陈代谢所涉及的蛋白质的表达方面发挥作用。这些包括参与葡萄糖运输的蛋白质，这通常是葡萄糖新陈代谢的限速步骤。动物研究表明，脂肪组织的基础葡萄糖摄取与GLUT1的表达和活性直接相关，GLUT1是构成葡萄糖的转运体。我们特别关注葡萄糖本身对GLUT1的调节。为了做到这一点，我们使用了3T3-L1脂肪细胞，它可以在受控环境中提供更长时间的特征。在缺乏葡萄糖的细胞中，我们已经证明转运活动增加了20倍，这不是一个微不足道的变化。与这一观察相关的是，在循环血糖的生理极限上观察到了4倍的变化。由于GLUT1的表达不会改变，这导致了GLUT1被激活的假设是对葡萄糖剥夺的反应。我们现在知道，质膜中GLUT1池的一部分驻留在脂筏中。这一比例的增加是对剥夺的反应。新的数据表明，从葡萄糖缺乏的细胞分离的脂筏中重组的GLUT1显示出比从对照细胞中观察到的更高的“内在活性”。有趣的是，我们能够证明GLUT1和口蛋白之间的特定相互作用，口蛋白是一种几乎只存在于脂筏组分中的蛋白质。缺糖会增加这种相互作用，从而推断在脂筏中，胃动素和GLUT1相互作用。综上所述，这些数据表明，脂筏环境在GLUT1功能中发挥作用。这些新颖的观察结果为我们理解依赖营养的葡萄糖转运控制提供了新的方向。在具体目标1中，我们重点研究了GLUT1对脂筏的靶向作用。在具体目标2中，我们探讨了脂类在血管运输功能中的作用。在特定的目标3中，我们使用一种可能调节GLUT1的新的蛋白质组学方法来鉴定脂筏特有的蛋白质。最后，在特定的目的4中，我们使用胃蛋白基因敲除小鼠来确定胃蛋白在GLUT1靶向和功能上的作用。

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.