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

通过其在非酒精性脂肪肝疾病中的作用来研究多囊蛋白 2 的上调作为应激反应

• 批准号: 10085572
• 负责人: Allison Leigh Brill
• 金额: $ 3.08万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10085572
• 关键词: Acute; Affect; American; Autophagocytosis; Autophagosome; BCL2 gene; Biochemical; Biogenesis; Biological Assay; CRISPR/Cas technology; Calcium Signaling; Cell physiology; Cells; Chronic; Conflict (Psychology); Data; Development; Diet; Disease; Disputes; Endoplasmic Reticulum; Equilibrium; Etiology; Exhibits; Fatty Liver; Functional disorder; Genetic Transcription; Glucose Clamp; Goals; Health; Hepatic; Hepatocyte; High Fat Diet; Human; Human Cell Line; Inositol; Insulin Resistance; Kidney; Kidney Diseases; Knock-out; Knockout Mice; Lead; Link; Lipids; Literature; Liver; Luciferases; Maintenance; Measurement; Measures; Mediating; Messenger RNA; Metabolic; Mitochondria; Modeling; Molecular; Morphology; Mus; PKD2 protein; PTEN-induced putative kinase; Pathway interactions; Patients; Population; Prevalence; Process; Protein Analysis; Proteins; Publishing; Regulation; Research; Role; Sampling; Signal Transduction; Stress; Testing; Therapeutic; Time; Tissues; Up-Regulation; Work; base; biological adaptation to stress; chronic liver disease; combat; effective therapy; feeding; in vivo; insight; interest; knock-down; loss of function mutation; mitochondrial autophagy; mitochondrial dysfunction; mitochondrial metabolism; mutant; non-alcoholic fatty liver disease; novel; promoter; stressor

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.

项目总结/摘要 非酒精性脂肪性肝病（NAFLD）是世界上最常见的慢性肝病，其特征在于 细胞能量机制的异常失调，包括有缺陷的自噬和线粒体 功能在NAFLD模型中抑制一般自噬产生了矛盾的结果，导致了两种 防止和引起肝脂肪变性和胰岛素抵抗。但是，具体作用 自噬选择性地降解线粒体-称为线粒体自噬的过程-尚未在 NAFLD发展的背景。了解线粒体自噬在NAFLD中的确切作用，以及其分子机制， 自噬介导的线粒体功能障碍的潜在驱动因素，将使我们更接近于开发持久的 治疗策略来对抗这种疾病。该项目的主要目标是建立 线粒体自噬失调在NAFLD发展中的作用，并确定多囊蛋白-2（PC 2），一种Ca 2 +- 调节内质网（ER）上的蛋白质，通过以下途径促进线粒体自噬的过度激活： 与Bcl-2相关的凋亡基因2（BAG 2）相互作用。这将有助于了解如何观察到 NAFLD发作后肝细胞中发生的线粒体形态和功能改变 都被这些蛋白质的上调所改变。我对PC 2和BAG 2的兴趣是基于出版的， 初步数据表明1）在患有NAFLD的人和鼠肝脏中增加的PC 2和BAG 2表达， 2)BAG 2作为PC 2的相互作用伴侣，促进PTEN诱导的推定激酶1（PINK 1）介导的 PC 2上调后的线粒体自噬，以及3）保护PC 2中的胰岛素抵抗和肝脂肪变性 喂饲高脂肪饮食（HFD）的单倍不足小鼠。该提案将研究PC 2在压力下如何上调 调节线粒体自噬和线粒体功能，以及PC 2缺失肝细胞如何响应HFD- 诱发NAFLD。这些结果将证实PC 2是能够调节线粒体自噬的应激反应蛋白 并将有助于解决自噬失调在NAFLD发展中的作用。的具体机制 负责上调PC 2和BAG 2以促进线粒体自噬的蛋白将由细胞和 生物化学方法研究细胞应激导致PC 2 mRNA转录增加，如何 增加的PC 2影响BAG 2表达和亚细胞定位，以及这些蛋白质如何起作用以增加 线粒体自噬通过完善的PINK 1途径。将检查NAFLD发展中的线粒体自噬 通过喂食正常饮食或HFD（有或没有肝细胞PC 2表达）的小鼠（Pkd 2fl/fl;白蛋白Cre-或 Pkd 2fl/fl;白蛋白Cre+），并且这些小鼠的代谢表征将通过高胰岛素血症- 正常血糖钳夹和离体分析检查线粒体自噬，线粒体功能，肝脂质潴留， 和钙信号。还将深入研究人类肝脏样品中PC 2、BAG 2和BAG 3之间的关系。 和疾病的发展。这些研究的结果将为识别新靶点提供见解 开发有效的治疗方法来挽救NAFLD中的过度线粒体碎片化和功能障碍。