ERK Kinase Regulation of Insulin Signaling and Inflammation: Honing the Antidiabetic Effects of PPAR-gamma Activation

 说明(申请人提供)：噻唑烷二酮类(TZD)是一种有效的抗糖尿病药物，其在治疗2型糖尿病方面的使用受到副作用的限制。这项提议的目标是对一种将TZDS的积极代谢效应与其副作用分开的机制进行生化研究，从而有可能设计出更好的药物类别。PPARγ是TZDS的分子靶点，是全身胰岛素敏感性、脂肪生成、炎症和能量平衡的关键调节因子。在脂肪组织中，在高脂肪开始后不久，观察到PPARγ丝氨酸273(S273)的磷酸化 饮食喂养和进行性肥胖增加。这种磷酸化与PPARγ靶基因的失调有关，例如胰岛素增敏激素脂联素的表达减少。ERK是PPARγS273磷酸化的主要途径，γS273的磷酸化受抑制。令人惊讶的是，MEK/ERK抑制剂在肥胖小鼠中具有强大的抗糖尿病作用，表现出显著改善血糖稳态的效果。同样，缺乏促进脂肪生成能力但仍阻断S273磷酸化的PPARγ的高亲和力配体仍具有抗糖尿病作用。这两种阻止PPARγS273磷酸化的药物干预措施都像TZDS一样促进了外周代谢动态平衡的改善，同时由于它们不会引发与TZDS相关的副作用，因此似乎更安全。我们的假设是，ERK介导的PPARγ在肥胖和炎症中的磷酸化通过靶向脂肪组织的转录调控而导致糖稳态受损。我们将使用一种新型的转基因小鼠来验证这一假设，其中PPARγ在S273(S273A)上不能被磷酸化。我们的初步数据表明，阻断这种磷酸化足以改善肥胖患者的胰岛素敏感性。在本提案中，我们将使用三种方法来了解 PPARγ磷酸化在肥胖发病机制中的作用在目标1中，我们将询问PPARγS273磷酸化对葡萄糖稳态的影响以及PPARγS273A小鼠对PPARγ配体的反应能力。在目标2中，我们将研究阻断免疫系统中S273磷酸化在脂肪组织炎症和胰岛素抵抗中的相对作用。在目标3中，我们将研究野生型PPARγ或磷酸化非依赖性S273A PPARγ结合的全基因组转录调控和顺式调控元件。了解肥胖和炎症是如何通过ERK介导的磷酸化来调节PPARγ的，对于未来设计新的治疗分子将是重要的。

 DESCRIPTION (provided by applicant): The thiazolidinediones (TZDs) are powerful anti-diabetic drugs whose use in treating type 2 diabetes is limited by adverse side effects. The goal of this proposal is a biochemical investigation into a mechanism that separates the positive metabolic effects of TZDs from their side effects, making it possible to design a better class of agents. PPARγ, the molecular target of the TZDs, is a key regulator of systemic insulin sensitivity, adipogenesis, inflammation, and energy homeostasis. In adipose tissue, phosphorylation of PPARγ at serine 273 (S273) is observed shortly after the initiation of high fat diet feeding and increases with progressive obesity. This phosphorylation correlates with dysregulation of PPARγ target genes, such as decreased expression of the insulin-sensitizing hormone adiponectin. ERK is the primary kinase responsible for phosphorylating PPARγ S273, inhibitors of the MEK/ERK kinase pathway block PPARγ S273 phosphorylation. Surprisingly, MEK/ERK inhibitors had potent anti-diabetic effects in obese mice demonstrating markedly improved glucose homeostasis. Similarly, high-affinity ligands of PPARγwhich lack the capacity to promote adipogenesis but still block S273 phosphorylation retain anti-diabetic effects. These two pharmacological interventions blocking PPARγ S273 phosphorylation both promote improved peripheral metabolic homeostasis like the TZDs while also appearing safer as they do not trigger the side effects associated with TZDs. Our hypothesis is that ERK-mediated phosphorylation of PPARγ in obesity and inflammation causes altered impaired glucose homeostasis by targeting adipose tissue transcriptional regulation. We will test this hypothesis using a novel genetically modified mouse where PPARγ cannot be phosphorylated on S273 (S273A). Our preliminary data suggest that blocking this phosphorylation is sufficient to improve insulin sensitivity in obesity. In this proposal we will utilize three approaches to understand the contribution of PPARγ phosphorylation to the pathogenesis of obesity. In Aim 1, we will interrogate the effects of PPARγ S273 phosphorylation on glucose homeostasis and the ability of PPARγ S273A mice to respond to PPARγ ligands. In Aim 2, we will investigate the relative contribution of blocking S273 phosphorylation in the immune system to adipose tissue inflammation and insulin resistance. In Aim 3, we will examine the genome-wide set of mRNA transcripts regulated and cis-regulatory elements bound to either wild-type PPARγ or phosphorylation independent S273A PPARγ. Understanding how obesity and inflammation modulates PPARγ by ERK- mediated phosphorylation will be important for the future design of new therapeutic molecules.