Serine/threonine Protein Phosphatase 1 in Insulin resistance and Type 2 Diabetes

 描述（由申请人提供）：骨骼肌胰岛素抵抗是代谢综合征和2型糖尿病（T2 D）发展的基础。超过2600万美国成年人患有T2 D。胰岛素信号转导中磷酸化事件的缺陷被认为是胰岛素抵抗的主要原因之一，然而，这些缺陷的机制仍然难以捉摸，这严重限制了预防和治疗T2 D的有效策略。大多数关于胰岛素抵抗中磷酸化事件的研究都集中在激酶上。丝氨酸/苏氨酸蛋白磷酸酶1调节亚基12（PPP 1 R12）调节蛋白磷酸酶1（PP 1c）催化亚基的活性和特异性，并将PP 1c锚定在其底物上。尽管如此，很少有人知道PPP 1 R12在骨骼肌胰岛素抵抗的背景下控制的去磷酸化事件和功能。我们假设，与瘦对照相比，在肥胖非糖尿病和/或T2 D受试者的骨骼肌中，PPP 1 R12的蛋白丰度较低，并且涉及PPP 1 R12的蛋白-蛋白相互作用异常，并且较低的PPP 1 R12丰度有助于胰岛素抵抗和T2 D的发展。我们的假设是基于现有的文献，我们的新的初步发现揭示：1）。PPP 1 R12蛋白丰度在T2 D骨骼肌中较低（6倍于瘦肌，P<0.05）; 2）。与瘦对照相比，肥胖和/或T2 D受试者中骨骼肌中的多个PPP 1 R12相互作用配偶体与PPP 1 R12的相关性改变; 3）。L 6细胞中PPP 1 R12敲低导致多种蛋白质上超过100个位点的磷酸化增加; 4）。PPP 1 R12敲低导致葡萄糖摄取减少。为了验证我们的假设，我们建议：1。确定瘦健康对照、肥胖非糖尿病对照和肥胖T2 D受试者骨骼肌中PPP 1 R12蛋白丰度和蛋白相互作用伴侣。我们将进行高胰岛素-正常血糖钳夹以评估胰岛素敏感性，并获得两个骨骼肌活检样本，一个在钳夹前，另一个在钳夹结束时。PPP 1 R12丰度和相互作用伴侣将使用蛋白质组学和蛋白质印迹法在肌肉样品中定量。2.确定PPP 1 R12的敲低或过表达如何影响瘦健康对照、肥胖非糖尿病对照和肥胖T2 D受试者的原代骨骼肌细胞中的胰岛素作用。我们将从这些受试者的活检组织中培养原代骨骼肌细胞，并在这些细胞中敲低或过表达PPP 1 R12。磷酸化蛋白质组学和经典的胰岛素作用测量将用于确定PPP 1 R12调节的新型磷酸化和信号传导事件。我们的总体目标是解决磷酸酶在胰岛素抵抗和T2 D中作用的知识缺口，使用临床研究（直接测量人类病理生理学），体外细胞研究（因果机制）和尖端蛋白质组学（用于细胞信号传导的全球分析和无偏见的发现）。这项研究的结果将为胰岛素抵抗的发病机制提供新的见解，促进设计更好的T2 D预防和治疗药物。

 DESCRIPTION (provided by applicant): Skeletal muscle insulin resistance underlies the metabolic syndrome and the development of type 2 diabetes (T2D). More than 26 million US adults have T2D. Defects in phosphorylation events in insulin signaling are considered to be among the main causes of insulin resistance, however, the mechanisms for these defects remain elusive, which severely limits effective strategies to prevent and treat T2D. Most research on phosphorylation events in insulin resistance has been focused on kinases. Serine/threonine protein phosphatase 1 regulatory subunit 12 (PPP1R12) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1 (PP1c), and anchors PP1c to its substrates. Nonetheless, little is known about the dephosphorylation events and functions controlled by PPP1R12 in the context of skeletal muscle insulin resistance. We hypothesize that protein abundance of PPP1R12 is lower and protein-protein interactions involving PPP1R12 are abnormal in skeletal muscle of obese non-diabetic and/or T2D subjects compared to lean controls, and that the lower PPP1R12 abundance contributes to the development of insulin resistance and T2D. Our hypothesis is based on the existing literature, and on our novel preliminary findings revealing that: 1). PPP1R12 protein abundance was lower in T2D skeletal muscle (6-fold vs. lean, P<0.05); 2). Multiple PPP1R12 interaction partners in skeletal muscle had altered associations with PPP1R12 in obese and/or T2D subjects vs. lean controls; 3). PPP1R12 knockdown in L6 cells led to increased phosphorylation for more than 100 sites on multiple proteins; 4). PPP1R12 knockdown resulted in reduced glucose uptake. To test our hypotheses, we propose to: 1. Determine PPP1R12 protein abundance and protein interaction partners in skeletal muscle from lean healthy controls, obese non-diabetic controls, and obese T2D subjects. We will perform a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity, and obtain two skeletal muscle biopsy samples, one before the clamp and the other at the end of the clamp. PPP1R12 abundance and interaction partners will be quantified in muscle samples using proteomics and western blotting. 2. Determine how knockdown or overexpression of PPP1R12 affects insulin action in primary skeletal muscle cells from lean healthy controls, obese non-diabetic controls, and obese T2D subjects. We will culture primary skeletal muscle cells from biopsies from these subjects, and knockdown or overexpress PPP1R12 in these cells. Phosphoproteomics and classic insulin action measurements will be used to identify novel phosphorylation and signaling events regulated by PPP1R12. Our overall goal is to address the knowledge gap of phosphatase role in insulin resistance and T2D, using a combination of clinical studies (directly measure human pathophysiology), in vitro cell studies (for causal mechanisms), and cutting-edge proteomics (for global analysis of cell signaling & unbiased discovery). The outcome of this research will provide novel insights into the pathogenesis of insulin resistance, facilitating the design of better drugs for T2D prevention and treatment.