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

肥胖和 2 型糖尿病中的人类骨骼肌蛋白质组和磷酸化蛋白质组

• 批准号: 8012803
• 负责人: Zhengping Yi
• 金额: $ 32.4万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8012803
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affect; Antibodies; Binding; Biological Process; Biopsy; Cardiovascular Diseases; Co-Immunoprecipitations; Complex; Comprehension; Defect; Development; Disease; Gene Expression; Generations; Glycogen; Goals; Health; High Pressure Liquid Chromatography; Human; Individual; Infusion procedures; Insulin; Insulin Resistance; Insulin Signaling Pathway; Investigation; Laboratories; Lead; Mass Spectrum Analysis; Methods; Microarray Analysis; Molecular; Muscle; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Outcome; PDPK1 gene; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Physiological; Post-Translational Protein Processing; Prevention; Protein Biosynthesis; Proteins; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Relative (related person); Research; Signal Pathway; Signaling Protein; Skeletal Muscle; Stable Isotope Labeling; Stimulation of Cell Proliferation; Technology; Testing; United States; Western Blotting; Work; analog; base; diabetic; glucose transport; in vivo; innovation; insulin receptor substrate 1 protein; insulin signaling; interest; non-diabetic; novel; protein complex; protein degradation; protein protein interaction; research study; tandem mass spectrometry; tool; volunteer

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.

描述（由申请人提供）：骨骼肌中的胰岛素抵抗是肥胖、2型糖尿病和心血管疾病的常见疾病。这些疾病影响着全世界数百万人。胰岛素信号通路对骨骼肌的多种生物过程至关重要，如糖原合成和葡萄糖运输。它包括精确的、受控的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展有关，但蛋白质-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是不清楚的。本项目将利用最先进的高效液相色谱-纳米射线-串联质谱（HPLC-ESI-MS/MS）分析从瘦健康、肥胖非糖尿病和2型糖尿病志愿者的肌肉活检中分离的蛋白质，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蛋白质的新磷酸化。本研究的中心假设是，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苗条和健康状况相比存在差异。我们研究的总体目标是确定人类骨骼肌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。这项工作的结果将是发现与胰岛素抵抗相关并受胰岛素调节的新型蛋白复合物和磷酸化位点，这将加快提出解决2型糖尿病挑战性问题的假说。具体而言，我们建议：1。确定人类骨骼肌中涉及IRS-1的蛋白-蛋白相互作用如何在肥胖和2型糖尿病中发生改变，并受胰岛素调节。我们将验证人类骨骼肌中异常蛋白-蛋白复合物与胰岛素抵抗相关的假设，这些假设可能阻碍胰岛素作用的信号通路，并且这些蛋白-蛋白相互作用由胰岛素调节。我们将使用针对IRS-1的特异性抗体进行共免疫沉淀实验，IRS-1是胰岛素信号传导的主要参与者，随后通过HPLC-ESI-MS/MS鉴定和定量IRS-1相关蛋白。2. 确定在肥胖和2型糖尿病中，骨骼肌中参与胰岛素信号通路的蛋白磷酸化是如何功能失调的。我们将验证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型糖尿病相关的假设。我们将采用我们实验室开发的基于质谱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，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输注时进行量化。公共卫生相关性：我们建议利用创新的基于质谱的蛋白质组学技术来研究瘦肉健康、肥胖非糖尿病和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。