Human Skeletal Muscle Proteome and Phosphoproteome in Obesity and Type 2 Diabetes

描述(申请人提供)：骨骼肌中的胰岛素抵抗是肥胖症、2型糖尿病和心血管疾病的常见症状。这些疾病影响着全世界数百万人。胰岛素信号通路在骨骼肌糖原合成和葡萄糖转运等多种生物学过程中起着至关重要的作用。它包括精确的、可控的蛋白质-蛋白质相互作用以及传递胰岛素信号的蛋白质磷酸化。胰岛素信号通路的缺陷与骨骼肌胰岛素抵抗的发生有关，但蛋白质相互作用和蛋白质磷酸化的确切异常在很大程度上还不清楚。本项目将利用最先进的高效液相-电喷雾串联质谱仪(HPLC-ESI-MS/MS)分析从瘦健康、肥胖的非糖尿病和2型糖尿病志愿者的肌肉活检组织中提取的蛋白质，以评估胰岛素受体底物-1(IRS-1)的相互作用伙伴，并发现和量化胰岛素信号通路中新的蛋白质磷酸化。这项研究的中心假设是，肥胖和2型糖尿病患者骨骼肌中胰岛素信号通路中的蛋白质-蛋白质相互作用和蛋白质磷酸化与瘦身和健康状态相比存在差异。我们研究的总体目标是确定人类骨骼肌中胰岛素抵抗的分子机制，并为2型糖尿病的预防和治疗提供新的靶点。这项拟议工作的结果将是发现与胰岛素抵抗相关并受胰岛素调控的新的蛋白质复合体和磷酸化位点，这将加速产生假说，以解决2型糖尿病的挑战性问题。具体地说，我们建议：1.确定在肥胖和2型糖尿病中，涉及IRS-1的蛋白质-蛋白质相互作用是如何改变的，并受到胰岛素的调节。我们将测试这样的假设，即人类骨骼肌中异常的蛋白质-蛋白质复合体与胰岛素抵抗有关，这可能会阻碍参与胰岛素作用的信号通路，并且这些蛋白质-蛋白质相互作用受到胰岛素的调节。我们将使用IRS-1的特异性抗体进行免疫共沉淀实验，IRS-1是胰岛素信号转导的主要参与者，然后通过高效液相-电喷雾质谱/质谱仪鉴定和定量IRS-1相关蛋白。2.确定骨骼肌中参与胰岛素信号转导途径的蛋白的磷酸化如何在肥胖和2型糖尿病中功能障碍。我们将检验这一假设，即人类骨骼肌中胰岛素信号通路中蛋白质的异常磷酸化模式与肥胖和2型糖尿病有关。我们将使用我们实验室开发的基于质谱学的磷酸化鉴定和定量方法来靶向PI-3激酶途径中感兴趣的蛋白质，如PI-3激酶、PDK1、AKT和AS160。新的和已知的磷酸化位点将在基础条件下和体内胰岛素注射时被量化。公共卫生相关性：我们建议利用创新的基于质谱学的蛋白质组学技术来研究瘦削健康、肥胖的非糖尿病和2型糖尿病志愿者骨骼肌中胰岛素信号通路中蛋白质-蛋白质相互作用和蛋白质磷酸化的差异。我们研究的总体目标是确定人类骨骼肌中胰岛素抵抗的分子机制，并为2型糖尿病的预防和治疗提供新的靶点。

DESCRIPTION (provided by applicant): Insulin resistance in skeletal muscle is a condition that is common to obesity, type 2 diabetes, and cardiovascular disease. These diseases affect millions of individuals worldwide. The insulin signaling pathway is crucial to a wide variety of biological processes in skeletal muscle such as glycogen synthesis and glucose transport. It involves precise, controlled protein-protein interactions as well as protein phosphorylation to relay the insulin signal. Defects in the insulin signaling pathway have been implicated in the development of skeletal muscle insulin resistance but the precise abnormalities in protein-protein interactions and protein phosphorylation are largely unclear. The present project will analyze proteins isolated from muscle biopsies of lean healthy, obese nondiabetic and type 2 diabetic volunteers utilizing state-of-the-art HPLC-nanospray- tandem mass spectrometry (HPLC-ESI-MS/MS) to assess interacting partners of insulin receptor substrate-1 (IRS-1) and to discover and quantify novel phosphorylation of proteins in the insulin signaling pathway. The central hypothesis of this investigation is that there are differences in protein-protein interactions and protein phosphorylation in the insulin signaling pathway in human skeletal muscle in obesity and type 2 diabetes as compared to lean and healthy conditions. The overall goal of our research is to identify molecular mechanisms responsible for insulin resistance in human skeletal muscle and to provide novel targets for prevention and treatment of type 2 diabetes. The outcome of this proposed work will be the discovery of novel protein complexes and phosphorylation sites associated with insulin resistance and regulated by insulin that will expedite the generation of hypotheses to tackle the challenging issues of type 2 diabetes. Specifically, we propose to: 1. Determine how protein-protein interactions involving IRS-1 in human skeletal muscle are altered in obesity and type 2 diabetes and are regulated by insulin. We will test the hypotheses that abnormal protein-protein complexes in human skeletal muscle are associated with insulin resistance that may impede signaling pathways involved in insulin action, and that these protein-protein interactions are regulated by insulin. We will perform co-immunoprecipitation experiments using a specific antibody against IRS-1, a major player in insulin signaling, followed by identification and quantification of IRS-1 associated proteins by HPLC-ESI-MS/MS. 2. Determine how phosphorylation of proteins involved in the insulin signaling pathway in skeletal muscle are dysfunctional in obesity and type 2 diabetes. We will test the hypothesis that abnormal phosphorylation patterns of proteins in the insulin signaling pathway in human skeletal muscle are associated with obesity and type 2 diabetes. We will employ the mass spectrometry based phosphorylation identification and quantification approach developed in our laboratory to target proteins of interest in the PI-3 kinase pathway, such as PI-3 kinase, PDK1, AKT and AS160. New and known phosphorylation sites will be quantified under both basal conditions and upon insulin infusion in vivo. PUBLIC HEALTH RELEVANCE: We propose to utilize innovative mass spectrometry based proteomic technology to study differences in protein-protein interactions and protein phosphorylation in the insulin signaling pathway in skeletal muscle from lean healthy, obese nondiabetic and type 2 diabetic volunteers. The overall goal of our research is to identify molecular mechanisms responsible for insulin resistance in human skeletal muscle and to provide novel targets for prevention and treatment of type 2 diabetes.