In Vivo 2H/13C Metabolic Flux Analysis of NASH Pathogenesis

点击翻译按钮获取中文摘要

 DESCRIPTION (provided by applicant): Despite its growing prevalence in Western society, the underlying molecular mechanisms that control the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain poorly understood. The overall objective of the current proposal is to apply in vivo 2H/13C metabolic flux analysis (MFA) and metabolomics profiling to identify liver phenotypes that accelerate the transition from NAFLD to NASH and to assess in vivo responses to pharmacologic and genetic interventions designed to inhibit this transition. These studies will use a melanocortin-4 receptor knockout (MC4R-/-) mouse model that has been previously shown to rapidly and spontaneously develop characteristics of human NASH upon Western diet feeding. To enable the analysis of intermediary liver fluxes in this and other transgenic mouse models, a novel GC-MS-based MFA approach has been recently developed that requires only 40 μL of blood plasma, yet can determine in vivo fluxes with precision equivalent to state-of-the-art NMR-based approaches. This approach will be applied to simultaneously assess changes in citric acid cycle (CAC) and gluconeogenesis fluxes in response to chronic or acute lipotoxic treatments. The central hypothesis is that abnormal ER calcium release promotes overactivation of mitochondrial metabolic pathways and oxidative tissue injury leading to NASH. The rationale for this research is that identifying in vivo metabolic pathway alterations that differentiate simple steatosis from NASH will enable better targeted approaches for NASH prevention or treatment. The research involves three specific aims. The first aim is to identify metabolic phenotypes associated with Western diet-induced liver injury. The working hypothesis is that overactivation of mitochondrial CAC flux accelerates hepatocyte lipotoxicity in the context of NAFLD. The second aim is to determine the extent to which inhibition of mitochondrial metabolism can prevent oxidative stress in fatty liver. The working hypothesis is that pharmacological inhibition of mitochondrial metabolism in liver will dose-dependently reduce oxidative tissue injury in NAFLD mouse models. The third aim is to determine the extent to which inhibition of ER calcium release can reverse mitochondrial overactivation in fatty liver. The working hypothesis is that enhancing the ability of ER to sequester calcium will normalize mitochondrial metabolism and reduce hepatocyte lipotoxicity. The proposed research is exceptionally innovative because it applies a newly developed GC-MS-based MFA approach that is faster, less expensive, and requires less sample volume than NMR-based approaches. The research is expected to achieve the following key outcomes. First, it will identify metabolic phenotypes that promote the transition from simple hepatic steatosis to progressive NASH. Second, it will identify potential therapeutic targets for inhibiting this transition. Third, it will establish a scalable in vivo MFA approach that is suitabe for routine mouse liver phenotyping. This research is significant because it will identify dysregulated metabolic pathways that can be targeted to inhibit the progression from NAFLD to NASH.