Notch signaling in Nos2 activation and steatohepatitis by obesity and alcohol

肥胖和酒精引起的 Nos2 激活和脂肪性肝炎中的 Notch 信号传导

• 批准号: 8165990
• 负责人: Jun Xu
• 金额: $ 11.11万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-20 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8165990
• 关键词: Advisory Committees; Alcohol abuse; Alcohol consumption; Alcohols; Area; Attenuated; Bone Marrow; Cells; Citric Acid Cycle; Committee Members; Coupled; DNA; Deoxyglucose; EMSA; Elements; Enzymes; Epidemic; Fatty Liver; Gene Activation; Gene Components; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Glucose; Glycolysis; Hepatic; Hypoxia; Individual; Injection of therapeutic agent; Injury; Kupffer Cells; Laboratories; Life Style; Ligands; Link; Liver; Liver diseases; Macrophage Activation; Maps; Mediating; Mentors; Metabolic; Metabolism; Mixed Function Oxygenases; Modeling; Molecular; Molecular Biology; Mus; Mutation Analysis; NF-kappa B; Nitric Oxide Synthase; Obesity; Pathway interactions; Predisposition; Procedures; Production; Public Health; Pyruvate; Radiation; Regulation; Research; Risk Factors; Role; Scheme; Severities; Signal Transduction; Site; Steatohepatitis; Testing; Transplantation; Up-Regulation; aerobic glycolysis; alpha ketoglutarate; anaerobic glycolysis; arginase; base; career development; fatty acid oxidation; feeding; gene induction; gene interaction; in vivo; inhibitor/antagonist; macrophage; metabolomics; moderate obesity; mouse model; nitrosative stress; nonalcoholic steatohepatitis; notch protein; problem drinker; promoter; secretase; small hairpin RNA; synergism

DESCRIPTION (provided by applicant): Obesity synergistically aggravates alcohol-induced steatohepatitis (SH). As obesity is a global epidemic, this synergism is an eminent public health issue and its mechanistic understanding is urgently needed. This application seeks support for my career development in a new area specifically directed to molecular and metabolic regulation of proinflammatory (M1) nitric oxide synthase gene (Nos2) activation in synergistic SH caused by obesity and alcohol. We have recently established a mouse intragastric feeding model in which moderate obesity markedly aggravates alcoholic SH. One notable change in the model is accentuated nitrosative stress with a 40-fold induction of hepatic Nos2. Analysis of hepatic macrophages (HM) from the model reveals heightened induction of M1 Nos2 gene over moderate induction of M2 arginase-1 (Arg1) gene as a plausible cause of the nitrosative stress. The expression of Notch receptors (Notch 1 and 2), ligands (Dll4, Jag1), and target gene (Hes1) are upregulated selectively in these HM. Inhibition of Notch signaling by the 3- secretase inhibitor DAPT abrogates induction of Nos2 and Notch component genes but not Arg1. ChIP analysis detects an enrichment of Notch Intracellular Domain (NICD), the effector of Notch signaling, along with HIF-11 at the proximal Nos2 promoter including HRE and CSL sites in these cells. These results suggest the pivotal role of Notch in HM Nos2 upregulation and consequent synergistic SH in the model. Fatty acid oxidation is causally linked to M2 gene induction while glycolytic pathway is associated with M1 activation. We demonstrate inhibition of anaerobic glycolysis with oxamate at the level of LDH, augments Nos2 induction but suppresses Arg1 expression, suggesting that partitioning of a glucose flux at the level of pyruvate determines the M1 vs. M2 gene regulation, with a flux into TCA cycle favoring Nos2 induction. Based on these findings, we propose a central hypothesis that heightened HM Nos2 induction mediated by Notch contributes to synergistic SH by obesity and alcohol and that this M1 activation is mediated by increased aerobic glycolysis in a Notch dependent manner. To test this hypothesis, we will aim to: 1) confirm the role of Notch in Nos2 activation in HM from the synergism model using shRNA-based silencing; 2) determine whether and how glycolytic or TCA cycle metabolites regulate Nos2 transcription using metabolomic analysis and shRNA-based manipulation of glycolytic and TCA cycle enzymes; and 3) determine in vivo the causal role of Notch 1/2 in HM M1 activation and the genesis of synergistic SH by alcohol and obesity by using Notch1/2flox/flox:Mx1-Cre chimeric mice. Through the proposed research, I will pursue a career development in metabolism-gene interaction for HM M1 gene activation in alcoholic SH under the individual and integrated guidance of the mentor, advisory committee members, and collaborators, who are experts in alcoholic and non-alcoholic SH, HM molecular biology, metabolomics and production and use of genetic chimeric mouse models. PUBLIC HEALTH RELEVANCE: Obesity and alcohol are two most common life-style risk factors of fatty liver disease, but more importantly, alcohol drinking in obese individual significantly increases both the susceptibility and severity of such liver injury. As obesity is a global epidemic, this aggravation in liver damage represents one of the most important public health issues, and for this reason, the proposed mechanistic studies of liver injury caused by obesity and alcohol, is urgently needed.

描述（由申请人提供）：肥胖协同加剧酒精引起的脂肪性肝炎（SH）。由于肥胖是一种全球流行病，这种协同作用是一个杰出的公共卫生问题，迫切需要其机械理解。该应用程序寻求支持我的职业发展，该领域专门针对促炎（M1）一氧化氮合酶基因（NOS2）激活的分子和代谢调节，这是由肥胖和酒精引起的协同SH。我们最近建立了一种小鼠胃内进食模型，其中中度肥胖显着加剧了酒精性sh。该模型中的一个显着变化是强调硝化应激，并具有40倍肝NOS2的诱导。该模型中对肝巨噬细胞（HM）的分析表明，M1 NOS2基因在中度诱导M2精氨酸酶-1（arg1）基因中的诱导增加，这是硝化应激的合理原因。在这些HM中，Notch受体（Notch 1和2），配体（DLL4，JAG1）和靶基因（Hes1）的表达在这些HM中被选择性上调。 3-泌尿酶抑制剂DAPT对NOTCH信号的抑制作用消除了NOS2和Notch成分基因的诱导，而不是ARG1。 CHIP分析检测Notch信号传导的效应子（NICD）的富集以及在NOS2启动子上的HIF-11以及这些细胞中的HRE和CSL位点的富集。这些结果表明Notch在HM NOS2上调以及随之而来的协同SH中的关键作用在模型中。脂肪酸氧化与M2基因诱导有因果关系，而糖酵解途径与M1激活有关。我们证明了在LDH水平上用Oxamate抑制厌氧性糖酵解，增强NOS2诱导但抑制了ARG1的表达，这表明在丙酮酸水平上对葡萄糖通量的分配决定了M1与M2基因调节的水平，并以flux循环为TCA循环，促进NOS2诱导。基于这些发现，我们提出了一个中心假设，即Notch介导的HM NOS2诱导增强了肥胖和酒精的协同SH，并且这种M1激活是由有氧糖糖溶解以NOTCH依赖性方式介导的。为了检验这一假设，我们将目的是：1）使用基于SHRNA的沉默来确认Notch在HM中的NOS2激活中的作用； 2）确定糖酵解或TCA循环代谢物是否使用代谢组分析以及基于SHRNA的糖酵解和TCA循环酶的操纵来调节NOS2转录； 3）在体内确定Notch 1/2在HM M1激活中的因果作用以及通过使用Notch1/2flox/Flox：MX1-CRE嵌合小鼠通过酒精和肥胖来协同SH的起源。通过拟议的研究，我将在指导者，咨询委员会成员和合作者的个人和综合指导下，在酒精SH的HM M1基因激活的代谢 - 基因互动中进行职业发展，这些人是酒精和非酒精SH，HM分子生物学，代谢性和生产，代谢性和生产以及对遗传型固定型固定型固有型固定型固定型固定型固定型固定型的HM分子生物学。 公共卫生相关性：肥胖和酒精是脂肪肝病的两个最常见的生命风格危险因素，但更重要的是，肥胖者的饮酒显着增加了这种肝损伤的敏感性和严重程度。由于肥胖是一种全球流行病，因此急性需要迫切需要肝脏损害的这种加重是最重要的公共卫生问题之一，因此，迫切需要对肥胖和酒精引起的肝损伤的机械性研究。