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.
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.