Regulation of AS160 and TBC1D1 by insulin and exercise in human skeletal muscle

描述（由申请人提供）：2型糖尿病是当今美国日益严重的健康问题，由胰岛素信号受损（即胰岛素作用降低）引起，主要发生在骨骼肌。胰岛素与其受体结合启动一系列磷酸化事件，导致细胞内的葡萄糖转运囊泡（GLUT-4）转运到表面并促进葡萄糖摄取。胰岛素信号传导中任何一个步骤的缺陷都可能损害GLUT-4易位并导致胰岛素抵抗。骨骼肌的独特之处在于，除了胰岛素信号外，运动（即重复肌肉收缩）也促进骨骼肌葡萄糖的摄取。这种作用与胰岛素无关，并为葡萄糖进入胰岛素抵抗的骨骼肌提供了另一种途径。大量的研究表明，这两种途径在一定程度上是不同的，但在AS160蛋白和新发现的同源物TBC1D1上汇合。动物研究表明，胰岛素和运动均可增加AS160和TBC1D1的磷酸化。然而，由于缺乏磷蛋白特异性抗体，关于人类胰岛素或运动对这些蛋白的位点特异性调节的信息很少。我们的实验室在使用HPLC- esi -MS/MS研究其他重要蛋白质（如IRS-1）的磷酸化时，避免了对磷酸基特异性抗体的需要。这些质谱技术可以适应并应用于信号蛋白如AS160和TBC1D1，以更好地了解它们在体内的翻译后调控。因此，本建议的总体目标有三个方面：利用质谱技术鉴定人类骨骼肌中AS160和TBC1D1上已知和新的磷酸化位点评估胰岛素和运动对这些磷酸化位点的调节。评估胰岛素抵抗在改变AS160和TBC1D1位点特异性磷酸化中的作用。长期的科学目标是利用从本研究中获得的信息来更好地了解胰岛素和运动如何促进骨骼肌葡萄糖摄取，并找到胰岛素抵抗骨骼肌的缺陷，这些缺陷可以通过药物或运动来靶向逆转胰岛素抵抗。

DESCRIPTION (provided by applicant): Type 2 diabetes is a huge a growing health problem in the United States today and is caused by impaired insulin signaling (i.e. decreased insulin action), primarily in skeletal muscle. Insulin binding to its receptor initiates a series of phosphorylation events that cause glucose transport vesicles (GLUT-4) that reside inside the cell to translocate to the surface and facilitate glucose uptake. Defects at any of steps in insulin signaling can impair GLUT-4 translocation and lead to insulin resistance. Skeletal muscle is unique in that in addition to insulin signaling, exercise (i.e. repeating muscle contractions) also promotes skeletal muscle glucose uptake. This effect is independent of insulin and provides an alternate means of entry for glucose into skeletal muscle that is insulin resistant. A vast amount of research has demonstrated that these 2 pathways are distinct, to a point, but converge at the protein AS160 and the newly identified homologue, TBC1D1. Animal studies demonstrate that both insulin and exercise increase phosphorylation of AS160 and TBC1D1. However, due to lack of phosphosite-specific antibodies, there is little information available on the site-specific regulation of these proteins by insulin or exercise in humans. Our laboratory has circumvented the need for phosphosite-specific antibodies when studying the phosphorylation of other important proteins (e.g. IRS-1) by using HPLC- electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). These mass spectrometry techniques can be adapted and applied to signaling proteins such as AS160 and TBC1D1 to better understand their post-translational regulation in vivo. Therefore, the overall objective of this proposal is three fold: 1. to identify known and novel phosphorylation sites on AS160 and TBC1D1 in human skeletal muscle using mass spectrometry based techniques 2. to assess the regulation of these phosphorylation sites by insulin and exercise and 3. to assess the effect of insulin resistance in altering site-specific phosphorylation of AS160 and TBC1D1. The long-term scientific objective is utilize the information gained from this study to better understand how insulin and exercise promote skeletal muscle glucose uptake and to find defects in insulin-resistant skeletal muscle that can be targeted by pharmaceuticals or exercise to reverse insulin resistance. PUBLIC HEALTH RELEVANCE: Decreasing the incidence of type 2 diabetes requires a full understanding of the underlying cause of this disease (i.e. diminished insulin action). This study will increase our understanding of how insulin and exercise affect signaling proteins that regulate glucose uptake and how these signals may be altered in insulin resistant individuals. This information may help us to target important regulation sites and improve insulin action.