Investigating Polycystin-2 up-regulation as a stress response through its role in non-alcoholic fatty liver disease

项目摘要/摘要 非酒精性脂肪性肝病(NAFLD)是全球最常见的慢性肝病，其特点是 通过细胞能量机制的异常失调，包括有缺陷的自噬和线粒体 功能。抑制NAFLD模型中的普遍自噬产生了相互矛盾的结果，导致 对肝脏脂肪变性和胰岛素抵抗的预防和原因。然而，它的具体作用 有选择地降解线粒体的自噬--称为有丝分裂的过程--还没有在 NAFLD发展的背景。了解有丝分裂在NAFLD中的确切作用，以及分子 自噬介导的线粒体功能障碍背后的驱动因素将使我们更接近开发出耐久的 与这种疾病作斗争的治疗策略。该项目的主要目标是建立 NAFLD发生发展过程中异常的有丝分裂吞噬，并确定多囊蛋白-2(PC2)，一种钙离子，一种 通过调节内质网(ER)上的蛋白质，促进有丝分裂的过度激活 它与Bcl2相关基因bag2的相互作用。这将允许理解如何观察 NAFLD发病后肝细胞线粒体形态和功能的改变 会因这些蛋白质的上调而改变。我对PC2和Bag2的兴趣是基于发布的 初步数据显示，1)NAFLD患者人和小鼠肝脏中PC2和Bag2的表达增加， 2)Bag2作为PC2的相互作用伙伴促进PTEN诱导的PINK1介导的 PC2上调后的有丝分裂吞噬作用；3)PC2对胰岛素抵抗和肝脏脂肪变性的保护作用 单倍体不足的小鼠喂食高脂饮食(HFD)。该提案将研究在压力下PC2是如何上调的 以调节有丝分裂和线粒体功能，以及PC2缺失的肝细胞如何对HFD- 诱导NAFLD。这些结果将证实PC2是一种能够调节有丝分裂的应激反应蛋白 并将有助于解决失调自噬在NAFLD发生中的作用。具体机制 上调PC2和Bag2以促进有丝分裂吞噬的责任将由细胞和 生物化学方法研究导致PC2mRNA转录增加的细胞应激源，如何 PC2的增加会影响bag2的表达和亚细胞定位，以及这些蛋白是如何发挥作用的 通过成熟的PINK1途径进行丝裂原吞噬。将检查NAFLD发育过程中的有丝分裂 通过饲喂正常饮食或HFD伴或不伴肝细胞PC2表达的小鼠(Pkd2fl/fl；AlbignCre-or Pkd2fl/fl；albumCre)，这些小鼠的代谢特征将通过高胰岛素- 正常血糖钳夹和体外分析检查有丝分裂，线粒体功能，肝脏脂质滞留， 和钙信号。人类肝脏样本也将被深入研究PC2、bag2和bag2 和疾病的发展。这些研究的结果将为识别新的目标提供洞察力 开发有效的治疗方法来挽救NAFLD患者过度的线粒体碎裂和功能障碍。

PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide, is characterized by aberrant dysregulation of cellular energy mechanisms, including defective autophagy and mitochondrial function. Inhibiting general autophagy in models of NAFLD has produced contradictory results, resulting in both protection against and causation of hepatic steatosis and insulin resistance. However, the specific role of autophagy to selectively degrade mitochondria—the process known as mitophagy—has not been studied in the context of NAFLD development. Understanding the precise role of mitophagy in NAFLD, as well as the molecular drivers underlying autophagy-mediated mitochondrial dysfunction, will bring us closer to developing durable therapeutic strategies to combat this disease. The main goals of this project are to establish the function of dysregulated mitophagy in the development of NAFLD, and to determine how Polycystin-2 (PC2), a Ca2+- modulating protein on the endoplasmic reticulum (ER), works to promote hyper-activation of mitophagy through its interaction with Bcl-2 associated athanogene 2 (BAG2). This will allow for understanding of how observed mitochondrial morphological and functional alterations that occur in hepatocytes following the onset of NAFLD are altered by the up-regulation of these proteins. My interest in PC2 and BAG2 is based on published and preliminary data demonstrating 1) increased PC2 and BAG2 expression in human and murine livers with NAFLD, 2) BAG2 as an interacting partner of PC2 that promotes PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy following PC2 up-regulation, and 3) protection against insulin resistance and hepatic steatosis in PC2 haploinsufficient mice fed a high fat diet (HFD). This proposal will examine how PC2 is up-regulated under stress to modulate mitophagy and mitochondrial function, as well as how PC2-null hepatocytes respond to HFD- induced NAFLD. These results will confirm PC2 as a stress response protein capable of regulating mitophagy and will help resolve the role of dysregulated autophagy in NAFLD development. The specific mechanisms responsible for up-regulation of PC2 and BAG2 to promote mitophagy will be determined by cellular and biochemical approaches investigating the cell stressors leading to transcriptional increases in PC2 mRNA, how increased PC2 affects BAG2 expression and subcellular localization, and how these proteins work to increase mitophagy through the well-established PINK1 pathway. Mitophagy in NAFLD development will be examined through mice fed normal diet or HFD with or without hepatocyte PC2 expression (Pkd2fl/fl;AlbuminCre- or Pkd2fl/fl;AlbuminCre+), and metabolic characterization of these mice will be performed through hyperinsulinemic- euglycemic clamps and ex vivo analyses examining mitophagy, mitochondrial function, hepatic lipid retention, and calcium signaling. Human liver samples will also be studied in depth for a relationship between PC2, BAG2 and disease development. The results of these studies will provide insight into the identification of novel targets to develop effective therapies for rescuing excess mitochondrial fragmentation and dysfunction in NAFLD.