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

描述（由申请人提供）：尽管骨骼肌胰岛素抵抗是代谢异常和慢性疾病（如2型糖尿病和心血管疾病）发展的主要因素，但其发病机制的分子机制仍然难以捉摸。胰岛素信号通路中磷酸化和去磷酸化事件的缺陷，如胰岛素受体底物-1 （IRS-1）丝氨酸/苏氨酸磷酸化，被认为是胰岛素抵抗的主要原因之一。大多数关于这些磷酸化事件调控的研究都集中在激酶上，并在细胞培养或动物模型中进行。我们假设人类骨骼肌中丝氨酸/苏氨酸蛋白磷酸酶2A （PP2A）复合物在胰岛素抵抗和2型糖尿病中过度激活和失调。这一假设是基于现有的文献表明，在细胞/动物模型中，PP2A在糖脂毒性和糖尿病的胁迫下过度激活，以及我们自己的以下新的初步发现：1)。PP2A的催化亚基（PP2Ac）与IRS-1在瘦肉健康参与者的骨骼肌中相互作用，并且这种相互作用在肥胖/超重和T2D肌肉中显著增加；2)胰岛素抑制瘦健康人肌肉中PP2Ac活性和Leu309羧甲基化，但2型糖尿病患者没有。为了验证我们的假设，我们将对活动进行评估

DESCRIPTION (provided by applicant): Although skeletal muscle insulin resistance is a primary contributor to the development of metabolic abnormalities and chronic diseases, such as type 2 diabetes and cardiovascular disease, the molecular mechanisms of its pathogenesis remain elusive. Defects in the phosphorylation and dephosphorylation events in the insulin signaling pathway, such as serine/threonine phosphorylation of Insulin Receptor Substrate-1 (IRS-1), are considered to be one of the main causes of insulin resistance. Most research on the regulation of these phosphorylation events has been focused on kinases and is performed in cell culture or animal models. We hypothesize that serine/threonine protein phosphatase 2A (PP2A) complexes in human skeletal muscle are over-activated and dysregulated in insulin resistance and type 2 diabetes. This hypothesis is formulated based on the existing literature suggesting hyperactivation of PP2A under the duress of glucolipotoxicity and diabetes in cell/animal models, and the following novel preliminary findings of our own: 1). The catalytic subunit of PP2A (PP2Ac) associates with IRS-1 in skeletal muscle of lean healthy participants, and this interaction is significantly increased in obese/overweight and T2D muscle; 2), Insulin suppresses PP2Ac activity and Leu309 carboxylmethylation in muscle of lean healthy participants, but not type 2 diabetic patients. To test our hypotheses, we will assess the activity (Aim 1), post-translational modifications (Aim 2), and interaction partners (Aim 3) of PP2A in skeletal muscle from lean healthy controls (LC), obese nondiabetic subjects (OC), and type 2 diabetic patients (T2D), using state-of-the-art HPLC-nanospray-tandem mass spectrometry (HPLC-ESI-MS/MS). The overall goal is to address the knowledge gap of phosphatase function and regulation in insulin resistance and T2D in humans, using a combination of clinical, biological, and proteomic approaches developed in our laboratory. For Aim 1, we will perform PP2Ac co- immunoprecipitation experiments followed by assay of PP2A activity in muscle biopsies from the 3 groups under basal and insulin infusion. For Aim 2, we will assess pTyr307 and mLeu309 in the 3 groups using Western blot analysis. In addition, we will employ the proteomics approach for phosphorylation quantification developed in our laboratory to provide a global view of PP2Ac phosphorylation in the 3 groups. For Aim 3, we will perform co-immunoprecipitation experiments using antibodies against PP2Ac followed by assessment of co-immunoprecipitated protein quantities under basal and insulin infusion in the 3 groups using the proteomics approach for protein complexes developed in our laboratory. The outcome of this research will be the discovery of abnormalities in PP2A activity, post-translational modifications, and interaction partners in skeletal muscle insulin resistance in humans, facilitating the design of new drugs to modulate PP2Ac post-translational modifications and protein-protein interactions. These drugs may restore abnormal PP2Ac activity and improve insulin signaling, resulting in the treatment and/or prevention of T2D, as well as other chronic disease.