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

 描述(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(T2D)发展的基础。超过2600万美国成年人患有T2D。胰岛素信号通路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不清楚，这严重限制了有效的预防和治疗T2D的策略。大多数关于胰岛素抵抗中的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(PPP1R12)调节蛋白磷酸酶1催化亚基(PP1c)的活性和特异性，并将PP1c锚定在底物上。然而，在骨骼肌胰岛素抵抗的背景下，PPP1R12控制的去磷酸化事件和功能知之甚少。我们假设在肥胖的非糖尿病和/或T2D受试者的骨骼肌中，PPP1R12的蛋白丰度低于瘦身对照组，并且涉及PPP1R12的蛋白质-蛋白质相互作用异常，PPP1R12丰度降低有助于胰岛素抵抗和T2D的发展。我们的假设是基于现有的文献，并基于我们的新的初步发现揭示：1)。在T2D骨骼肌中，PPP1R12蛋白丰度较低(6倍于LEAN，P&lt；0.05)；在肥胖和/或T2D受试者中，骨骼肌中多个PPP1R12相互作用伙伴改变了与PPP1R12的关联；PPP1R12在L6细胞中的敲除导致多个蛋白质上100多个位点的磷酸化增加；4)。PPP1R12基因敲除导致葡萄糖摄取减少。为了验证我们的假设，我们建议：1.确定瘦身健康对照组、肥胖非糖尿病对照组和肥胖T2D受试者骨骼肌中PPP1R12蛋白丰度和蛋白相互作用伙伴。我们将进行高胰岛素-正常血糖钳夹来评估胰岛素敏感性，并获得两个骨骼肌活组织标本，一个在钳前，另一个在钳的末端。PPP1R12的丰度和相互作用伙伴将使用蛋白质组学和蛋白质印迹技术在肌肉样本中进行量化。2.确定PPP1R12的敲除或过表达如何影响瘦健康对照组、肥胖的非糖尿病对照组和肥胖的T2D受试者的初级骨骼肌细胞的胰岛素作用。我们将从这些受试者的活检组织中培养原代骨骼肌细胞，并在这些细胞中敲除或过表达PPP1R12。磷酸蛋白质组学和经典的胰岛素作用测量将被用来识别由PPP1R12调节的新的磷酸化和信号事件。我们的总体目标是利用临床研究(直接测量人类病理生理学)、体外细胞研究(用于因果机制)和尖端蛋白质组学(用于细胞信号的全球分析和无偏见的发现)相结合，解决关于磷酸酶在胰岛素抵抗和T2D中作用的知识差距。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，有助于设计更好的T2D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.