Obesity-associated Mitophagy Resistance

项目概要/摘要 线粒体功能异常被认为是导致肝脏胰岛素抵抗和脂肪变性的重要原因 与肥胖和2型糖尿病有关。最近，饮食诱导的小鼠肥胖被发现与 肝脏线粒体自噬率降低，线粒体自噬是一种线粒体质量控制途径， 从细胞中清除受损的线粒体。线粒体自噬信号传导和受损线粒体的靶向 依赖于线粒体外膜上磷酸化泛素信号基序的产生， 通过泛素E3连接酶PARKIN和丝氨酸/苏氨酸激酶的协同激活产生 PINK1.这项提议将检验肥胖相关的肝脏代谢疾病部分是由于 PINK 1-PARKIN介导的信号传导受损导致线粒体稳态丧失， 随后减少线粒体自噬。检验这一假设的方法包括描述一部小说 在基线和饮食诱导的肥胖期间具有肝脏特异性PARKIN缺失的小鼠系，并将包括 设计用于评价肝线粒体氧化代谢的体内和离体方法的广度， 活性氧产生、肝脏胰岛素敏感性、线粒体吞噬流量和肝脏胰岛素敏感性的变化。 胰岛素信号传导和应激信号传导途径。这些研究将确定PARKIN的遗传抑制是如何产生的。 肝脏介导的线粒体自噬影响线粒体生物学和肥胖相关肝脏的发病机制 代谢性疾病此外，还将评估PINK 1-PARKIN介导的线粒体自噬信号传导的关键方面 在瘦的和肥胖的小鼠中使用包括定量质谱法在内的方法的组合， 基于蛋白质组学，共聚焦显微镜和诱变，然后在体内和体外功能 使用原代和稳定细胞系以及无细胞模型进行表征。这些研究将确定如何 肥胖肝脏中的营养应激有助于线粒体蛋白的翻译后修饰， 然后干扰最近定义的磷酸化和泛素化信号事件， 线粒体自噬途径他们将进一步深入了解PINK 1和PARKIN动力学如何在 肥胖的肝脏，并可能解释减少线粒体自噬观察到的肝脏从饮食诱导的肥胖小鼠。 总的来说，这些研究将探索一种新的途径，通过这种途径，肝脏中的营养应激可能有助于 肝脂肪变性、胰岛素抵抗和线粒体功能障碍的发病机制。这些新知识将 最终提高我们对这些生物化学和分子事件的理解， 疾病，这可能有助于制定有效的生活方式干预措施或有针对性的 治疗肥胖相关肝病的药物。

Project summary/Abstract Abnormal mitochondrial function is proposed to contribute to the hepatic insulin resistance and steatosis associated with obesity and type 2 diabetes. Recently, diet-induced obesity in mice was found to be associated with reduced rates of hepatic mitophagy, a mitochondrial quality control pathway that regulates selective removal of damaged mitochondria from the cell. Mitophagy signaling and targeting of damaged mitochondria relies on generation of a phosphorylated ubiquitin signaling motif on the outer mitochondrial membrane that is produced through the coordinated activation of the ubiquitin E3 ligase PARKIN and the serine/threonine kinase PINK1. This proposal will test the hypothesis that obesity-associated liver metabolic disease arises in part due to a loss of mitochondrial homeostasis resulting from impaired PINK1-PARKIN-mediated signaling and subsequently reduced mitophagy. The approach to test this hypothesis consists of characterizing a novel mouse line with liver-specific deletion of PARKIN at baseline and during diet-induced obesity, and will include a breadth of in vivo and ex vivo approaches designed to evaluate hepatic mitochondrial oxidative metabolism, reactive oxygen species production, hepatic insulin sensitivity, rates of mitophagic flux, and changes in hepatic insulin signaling and stress signaling pathways. These studies will define how genetic inhibition of PARKIN- mediated mitophagy in liver impacts mitochondrial biology and the pathogenesis of obesity-associated liver metabolic disease. Additionally, key aspects of PINK1-PARKIN-mediated mitophagy signaling will be evaluated in lean and obese mice using a combination of approaches that includes quantitative mass spectrometry- based proteomics, confocal microscopy and mutagenesis followed by in vivo and in vitro functional characterization using primary and stable cell lines and cell free models. These studies will define how nutritional stress in the obese liver contributes to post-translational modifications of mitochondrial proteins that then interfere with recently defined phosphorylation and ubiquitination signaling events that activate the mitophagy pathway. They will provide further insight into how PINK1 and PARKIN dynamics are affected in the obese liver and may account for the reduced mitophagy observed in liver from diet-induced obese mice. Overall, these studies will explore a novel pathway by which nutritional stress in liver may contribute to the pathogenesis of hepatic steatosis, insulin resistance and mitochondrial dysfunction. This new knowledge will ultimately improve our understanding of the biochemical and molecular events that contribute to these diseases, which may lend themselves to the development of effective life style interventions or targeted therapeutics to treat obesity-associated liver disease.