Obesity-associated Mitophagy Resistance

项目摘要/摘要 线粒体功能异常被认为是肝脏胰岛素抵抗和脂肪变性的原因。 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 随着肝脏有丝分裂比率的降低，一条调节选择性的线粒体质量控制途径 将受损的线粒体从细胞中移除。线粒体损伤的有丝分裂信号转导和靶向 依赖于线粒体膜外膜上磷酸化泛素信号基序的产生 由泛素E3连接酶Parkin和丝氨酸/苏氨酸激酶协同激活而产生 PINK1。这项提议将检验肥胖相关的肝脏代谢疾病的产生部分原因是 与PINK1-Parkin介导的信号和信号受损导致的线粒体动态平衡丧失有关 随后减少了有丝分裂吞噬作用。检验这一假设的方法包括对一部小说进行刻画 在基线和饮食诱导的肥胖期间，肝脏特异性Parkin缺失的小鼠株系，并将包括 评估肝脏线粒体氧化代谢的体内和体外方法的广度， 活性氧产生、肝脏胰岛素敏感性、有丝分裂通透率和肝脏变化 胰岛素信号和应激信号通路。这些研究将定义帕金的遗传抑制是如何- 肝脏介导的线粒体吞噬作用对线粒体生物学和肥胖相关肝脏发病机制的影响 代谢性疾病。此外，还将评估PINK1-Parkin介导的有丝分裂吞噬信号的关键方面 在瘦小和肥胖的小鼠身上，使用包括定量质谱学在内的多种方法- 基于蛋白质组学、共聚焦显微镜和诱变的体内和体外功能 使用原代和稳定的细胞系和无细胞模型进行表征。这些研究将定义如何 肥胖肝脏中的营养应激导致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，这些信号事件激活 有丝分裂途径。他们将进一步深入了解PINK1和Parkin动态在 这可能是饮食诱导的肥胖小鼠肝脏中观察到的线粒体吞噬减少的原因。 总体而言，这些研究将探索一条新的途径，即肝脏的营养压力可能有助于 肝脏脂肪变性、胰岛素抵抗和线粒体功能障碍的发病机制。这一新知识将 最终提高我们对导致这些变化的生化和分子事件的理解 疾病，这些疾病可能有助于制定有效的生活方式干预措施或有针对性的 治疗肥胖症相关的肝病。

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.