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

NASH 发病机制的体内 2H/13C 代谢通量分析

• 批准号: 8946823
• 负责人: Jamey D. Young
• 金额: $ 34.74万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8946823
• 关键词: Acute; Affect; Behavior; Biochemical Pathway; Calcium; Cell Death; Chronic; Cirrhosis; Citric Acid Cycle; Clinic; Collaborations; Conscious; Data; Diagnosis; Disease; Disease Progression; Dose; Enzymes; Epidemic; FDA approved; Fatty Liver; Gluconeogenesis; Goals; Healthcare Systems; Hepatic; Hepatocyte; Hepatology; Human Characteristics; Individual; Infusion procedures; Injury; Knock-out; Lead; Lipids; Liver; Liver Dysfunction; Liver diseases; Mass Fragmentography; Melanocortin 4 Receptor; Metabolic; Metabolic Pathway; Metabolic syndrome; Metabolism; Methods; Mitochondria; Molecular; Mus; Nuclear Magnetic Resonance; Outcome; Oxidative Stress; Pathogenesis; Patients; Pharmacotherapy; Phenotype; Plasma; Population; Prevalence; Prevention; Process; Public Health; Rattus; Regulation; Research; Research Personnel; Sampling; Societies; Techniques; Testing; Tissues; Transgenic Mice; Work; base; cohort; disorder prevention; feeding; gene therapy; in vivo; innovation; insight; liver inflammation; liver injury; liver metabolism; liver transplantation; mathematical model; member; metabolomics; mouse model; non-alcoholic fatty liver; nonalcoholic steatohepatitis; novel; novel strategies; prevent; public health relevance; response; stable isotope; therapeutic target; therapy design; western diet

 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.

 描述（由申请人提供）：尽管其在西方社会日益流行，但控制非酒精性脂肪性肝病（NAFLD）进展为非酒精性脂肪性肝炎（NASH）的潜在分子机制仍然知之甚少。当前提案的总体目标是。应用体内 2H/13C 代谢流分析 (MFA) 和代谢组学分析来识别加速从 NAFLD 向 NASH 转变的肝脏表型并进行体内评估这些研究将使用黑皮质素 4 受体敲除 (MC4R-/-) 小鼠模型，该模型已被证明在西方饮食喂养后能够快速自发地发展出人类 NASH 的特征。为了分析该模型和其他转基因小鼠模型中的中间肝脏通量，最近开发了一种基于 GC-MS 的新型 MFA 方法，该方法仅需要 40 μL 血浆，但可以确定体内通量，精度相当于该方法将用于同时评估慢性或急性脂毒性治疗引起的柠檬酸循环（CAC）和糖异生通量的变化。线粒体代谢途径过度激活和氧化组织损伤导致 NASH 此项研究的基本原理是，识别区分简单脂肪变性和 NASH 的体内代谢途径改变将为 NASH 预防或治疗提供更好的针对性方法。该研究涉及三个具体目标。第一个目标是确定与西方饮食引起的肝损伤相关的代谢表型。第二个目标是确定线粒体 CAC 通量的过度激活会加速肝细胞的脂毒性。抑制线粒体代谢可以在多大程度上预防脂肪肝中的氧化应激。工作假设是，药物抑制肝脏中的线粒体代谢将剂量依赖性地减少 NAFLD 小鼠模型的氧化组织损伤。目的是确定抑制内质网钙释放可以在多大程度上逆转脂肪肝中的线粒体过度激活，其工作假设是增强内质网螯合钙的能力将使线粒体代谢正常化并降低肝细胞脂毒性。该研究采用了新开发的基于 GC-MS 的 MFA 方法，该方法比基于 NMR 的方法更快、更便宜且需要更少的样本量，预计将实现以下主要成果。其次，它将确定抑制这种转变的潜在治疗靶点。第三，它将建立适合常规小鼠肝脏表型分析的可扩展的体内 MFA 方法。识别失调的代谢途径，可以有针对性地抑制从 NAFLD 到 NASH 的进展。