Integration of Inflammatory Signaling and the Unfolded Protein Response by Nitrosylation Signaling in Obesity

项目摘要 在肥胖的情况下，内质网（ER）应激已被确定为肥胖的一个突出特征。 在动物模型和人类中的代谢组织中。为了科普内质网应激，细胞激活未折叠的 蛋白质反应（UPR），以减轻压力。然而，普遍定期审议的失败导致长期未解决的压力， 促进肥胖诱导的胰岛素抵抗的发展。一氧化氮（NO）是一种关键的介质， 肥胖相关的炎症我们最近证明，NO介导的蛋白质修饰（S- 亚硝基化）损害肌醇需要酶-α（IRE 1 α）的RNase活性，导致未解决的ER 压力和胰岛素抵抗。虽然一氧化氮合酶（NOS）提供了一般的细胞内NO库 对于蛋白质S-亚硝基化，这些修饰的速率也受到NO基团的靶向去除的影响 通过蛋白质去亚硝基化。我们强有力的初步数据表明，肥胖损害了S- 亚硝基谷胱甘肽还原酶（GSNOR，一种主要的脱亚硝基酶），导致肝脏亚硝化应激升高。 此外，GSNOR的缺失或过表达直接调节IRE 1 α的S-亚硝基化状态， 饮食诱导肥胖（DIO）小鼠的ER功能。值得注意的是，肝脏特异性GSNOR过表达改善 肥胖相关的胰岛素抵抗然而，ER调节的分子机制 通过GSNOR介导的去亚硝基化信号传导的体内平衡是未知的。我们建议测试中央 假设肥胖减弱GSNOR依赖性蛋白质去亚硝基化，导致亚硝化活性升高， ER中的应激导致肥胖相关的肝脏胰岛素抵抗。为了验证这个假设，我们 将实现两个具体目标。在目标1中，我们将：1）确定肝脏特异性GSNOR缺失是否影响 肝脏胰岛素敏感性和全身葡萄糖稳态; 2）确定是否 GSNOR通过调节UPR直接调节肝脏胰岛素作用;和3）评估治疗潜力 通过补充谷胱甘肽增强肥胖小鼠的肝脏GSNOR活性， 烟酰胺腺嘌呤二核苷酸（NAD）代谢。在目标2中，我们将：1）描绘ER S-亚硝基化 蛋白质组和表征内源性S-亚硝基化位点的IRE 1 α; 2）解决如何GSNOR介导的 去亚硝基化信号调节IRE 1 α RNA酶活性; 3）确定GSNOR如何介导 去亚硝基化信号传导影响IRE 1 α相互作用体的形成。这里使用的方法是创新的， 结合细胞和分子生物学分析，S-亚硝基化蛋白的生化分析， 阐明ER S-亚硝基化蛋白质组，蛋白质-RNA相互作用分析，和体内小鼠代谢 侧写该项目阐明的机制将进一步了解炎症和 内质网应激通路与肥胖相关的肝脏胰岛素抵抗、血脂异常 最终导致2型糖尿病。深入了解适应不良细胞器应激反应的机制 驱动这些病理学应该加速代谢疾病的新治疗靶点的开发。

PROJECT SUMMARY In the setting of obesity, endoplasmic reticulum (ER) stress has been identified as a prominent feature in metabolic tissues in both animal models and in humans. To cope with ER stress, cells activate the unfolded protein response (UPR) to mitigate stress. However, failure of the UPR results in chronic unresolved stress, contributing to the development of obesity-induced insulin resistance. Nitric oxide (NO) is a key mediator of obesity-associated inflammation. We recently demonstrated that NO-mediated protein modification (S- nitrosylation) impairs the RNase activity of inositol-requiring enzyme-α (IRE1α), resulting in unresolved ER stress and insulin resistance. Although nitric oxide synthase (NOS) provides the general intracellular NO pools for protein S-nitrosylation, the rate of these modifications is also affected by the targeted removal of NO groups by protein denitrosylation. Our strong preliminary data demonstrate that obesity impairs the activity of S- nitrosoglutathione reductase (GSNOR, a major denitrosylase), leading to elevated nitrosative stress in the liver. Furthermore, deletion or overexpression of GSNOR directly regulates the S-nitrosylation state of IRE1α and ER function in mice with diet-induced obesity (DIO). Notably, liver-specific GSNOR overexpression ameliorated obesity-associated insulin resistance. However, the molecular mechanism that underlies the regulation of ER homeostasis by GSNOR-mediated denitrosylation signaling is unknown. We propose to test the central hypothesis that obesity attenuates GSNOR-dependent protein denitrosylation, resulting in elevated nitrosative stress in the ER that contributes to obesity-associated hepatic insulin resistance. To test this hypothesis, we will undertake 2 specific aims. In Aim 1, we will: 1) determine whether liver-specific GSNOR deletion impacts hepatic insulin sensitivity and whole body glucose homeostasis using DIO mouse model; 2) establish whether GSNOR regulates hepatic insulin action directly via modulation of UPR; and 3) assess the therapeutic potential of enhancing hepatic GSNOR activity in obese mice by glutathione supplementation and enhancing cellular nicotinamide adenine dinucleotide (NAD) metabolism. In Aim 2, we will: 1) profile the ER S-nitrosylation proteome and characterize the endogenous S-nitrosylation sites on IRE1α; 2) address how GSNOR-mediated denitrosylation signaling modulates the IRE1α RNase activity; and 3) establish how GSNOR-mediated denitrosylation signaling affects IRE1α interactome formation. The approaches used here are innovative, combining the use of cellular and molecular biological analysis, biochemical analysis for S-nitrosylated proteins, elucidating the ER S-nitrosylation proteome, protein-RNA interaction analysis, and in vivo mouse metabolic profiling. The mechanisms elucidated in this project will provide further understanding of how inflammatory and ER stress pathways are integrated in the context of obesity-associated hepatic insulin resistance, dyslipidemia and ultimately type 2 diabetes. Insight into the mechanisms by which maladaptive organelle stress responses drive these pathologies should speed development of novel therapeutic targets for metabolic diseases.