Novel mechanisms of insulin receptor inhibition in liver by nPKCs and MARCH1

 描述(申请人提供)：2型糖尿病(T2D)的流行是21世纪决定性的医学挑战之一，据估计，2000年出生的美国人中有三分之一在一生中患上糖尿病。T2D的中心始发病理是胰岛素 在包括肝脏在内的几个组织中存在耐药性。正常肝脏通过糖异生和糖原分解来维持血糖。糖尿病患者肝脏葡萄糖产生的胰岛素抑制严重受损，凸显了正常肝脏胰岛素反应性的重要性。所有已知的胰岛素的生理作用都发生在胰岛素受体(InsR)的下游，InsR信号的损伤已经在多种胰岛素抵抗模型中得到证实。我们建议研究与正常生理和胰岛素抵抗相关的两种独立的InsR调节模式。首先，我们建议表征一个特定的InsR苏氨酸磷酸化事件，我们假设该事件与脂质诱导的肝细胞胰岛素抵抗有关。简而言之，肝脏胰岛素抵抗与肝细胞脂质堆积密切相关。这种联系的一个潜在的中介是通过新的蛋白激酶C(NPKC)亚型，特别是肝细胞中的PKCE，它被脂质中间体二酰甘油(DAG)激活，并被证明削弱了胰岛素信号转导。然而，PKCE损害胰岛素信号的确切机制尚不确定。在目标1中，我们将使用质谱仪和体外激酶分析来验证候选的PKCE底物：胰岛素受体的苏氨酸1160。然后，我们将通过对苏氨酸被敲入丙氨酸的转基因小鼠进行代谢表型分析，从功能上表征这种相互作用的重要性。其次，我们建议研究两个新的胰岛素受体信号调节因子：March1和CD82的生理意义和作用机制。最近在大规模的胰岛素信号负调节因子shRNA筛选中发现，我们的数据表明E3泛素连接酶March1非常近地在胰岛素受体水平上损害胰岛素信号。初步工作表明，反义寡核苷酸阻断March1的表达在小鼠中具有胰岛素增敏作用。细胞中的机制研究表明，March1通过Tetraspanin蛋白CD82间接作用于改变InsR信号。在目标2中，我们将在这些初步数据的基础上，在体外和体内研究March1-CD82轴。利用肝脏特异的基因修饰，我们将测试March1是否需要CD82来削弱体内的胰岛素信号。同时，我们将研究这种效应的机制，假设March1-CD82轴改变InsR内吞命运或稳态质膜InsR含量。这项建议代表了一种综合的科学方法和新的学习经验，利用生理学、细胞生物学和分析化学的技术来对肝脏胰岛素抵抗这一重要问题产生新的见解。

 DESCRIPTION (provided by applicant): The epidemic of type 2 diabetes (T2D) is one of the defining medical challenges of the 21st century, with one in three Americans born in 2000 estimated to develop diabetes in their lifetime. The central initiating pathology of T2D is insulin resistance in several tissues, including liver. The normal liver maintains glycemia through gluconeogenesis and glycogenolysis. Insulin suppression of hepatic glucose production is severely impaired in diabetes, highlighting the importance of normal hepatic insulin responsiveness. All known physiological actions of insulin proceed downstream from the insulin receptor (InsR), and impairments in InsR signaling have been demonstrated in multiple models of insulin resistance. We propose to investigate two independent modes of InsR regulation with relevance to both normal physiology and insulin resistance. First, we propose to characterize a specific InsR threonine phosphorylation event that we hypothesize to be involved in lipid-induced hepatocellular insulin resistance. Briefly, hepatic insulin resistance is strongly linked t hepatocellular lipid accumulation. One potential mediator of this link is signaling through novel protein kinase C (nPKC) isoforms, especially PKCe in hepatocytes, which is activated by the lipid intermediate diacylglycerol (DAG) and has been shown to impair insulin signaling. However, the precise mechanism by which PKCe impairs insulin signaling is uncertain. In Aim 1, we will use mass spectrometry and in vitro kinase assays to validate a candidate PKCe substrate: threonine 1160 of the insulin receptor. We will then functionally characterize the importance of this interaction by performing metabolic phenotyping on a genetically modified mouse in which this threonine is knocked-in to alanine. Second, we propose to investigate the physiological significance and mechanism of action of two novel regulators of insulin receptor signaling: MARCH1 and CD82. Recently identified in a large-scale shRNA screen for negative regulators of insulin signaling, our data indicate that the E3 ubiquitin ligase MARCH1 impairs insulin signaling very proximally: at the level of the insulin receptor. Preliminary work revealed that antisense oligonucleotide blockade of MARCH1 expression is insulin-sensitizing in mice. Mechanistic studies in cells suggest that MARCH1 acts indirectly through the tetraspanin protein CD82 to alter InsR signaling. In Aim 2, we will build on these preliminary data to study the MARCH1-CD82 axis in vitro and in vivo. Using liver-specific genetic modifications, we will test whether MARCH1 requires CD82 to impair insulin signaling in vivo. In parallel, we will investigate the mechanism of this effect, hypothesizing that the MARCH1-CD82 axis alters InsR endocytic fate or steady-state plasma membrane InsR content. This proposal represents an integrated scientific approach and new learning experiences that harness techniques of physiology, cell biology, and analytical chemistry to yield novel insights into the important problem of hepatic insulin resistance.