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

 描述（由申请人提供）：2型糖尿病（T2 D）的流行是21世纪世纪的决定性医学挑战之一，据估计，2000年出生的美国人中有三分之一在其一生中患上糖尿病。T2 D的中心起始病理是胰岛素 包括肝脏在内的几种组织中的耐药性。正常的肝脏通过糖原合成和糖原分解来维持活力。胰岛素对肝脏葡萄糖生成的抑制在糖尿病中严重受损，突出了正常肝脏胰岛素反应性的重要性。胰岛素的所有已知生理作用均在胰岛素受体（InsR）下游进行，并且InsR信号传导的损伤已在多种胰岛素抵抗模型中得到证实。我们建议调查两种独立的InsR调节模式与正常生理和胰岛素抵抗相关。首先，我们提出了一个特定的InsR苏氨酸磷酸化事件，我们假设参与脂质诱导的肝细胞胰岛素抵抗的特点。简言之，肝胰岛素抵抗与肝细胞脂质蓄积密切相关。这种联系的一个潜在介质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基甘油（DAG）激活，并已被证明损害胰岛素信号传导的信号传导。然而，PKCe损害胰岛素信号传导的确切机制尚不确定。在目标1中，我们将使用质谱和体外激酶测定来验证候选PKCe底物：胰岛素受体的苏氨酸1160。然后，我们将在功能上表征这种相互作用的重要性，通过对转基因小鼠进行代谢表型分析，其中这种苏氨酸被敲入丙氨酸。其次，我们提出了两个新的胰岛素受体信号调节剂：MARCH 1和CD 82的生理意义和作用机制的调查。最近在一项针对胰岛素信号负调节因子的大规模shRNA筛选中发现，我们的数据表明E3遍在蛋白连接酶MARCH 1非常接近地损害胰岛素信号：在胰岛素受体水平。初步研究表明，MARCH 1表达的反义寡核苷酸阻断在小鼠中具有胰岛素增敏作用。细胞中的机制研究表明，MARCH 1间接通过四跨膜蛋白CD 82改变InsR信号传导。在目标2中，我们将基于这些初步数据在体外和体内研究MARCH 1-CD 82轴。使用肝脏特异性遗传修饰，我们将测试MARCH 1是否需要CD 82来损害体内胰岛素信号传导。同时，我们将研究这种效应的机制，假设MARCH 1-CD 82轴改变InsR内吞命运或稳态质膜InsR含量。该提案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技术，对肝脏胰岛素抵抗的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.