Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Division of Cardiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Center for Metabolism and Mitochondrial Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Mol Metab. 2020 Nov;41:101051. doi: 10.1016/j.molmet.2020.101051. Epub 2020 Jul 10.
PARKIN is an E3 ubiquitin ligase that regulates mitochondrial quality control through a process called mitophagy. Recent human and rodent studies suggest that loss of hepatic mitophagy may occur during the pathogenesis of obesity-associated fatty liver and contribute to changes in mitochondrial metabolism associated with this disease. Whole-body Prkn knockout mice are paradoxically protected against diet-induced hepatic steatosis; however, liver-specific effects of Prkn deficiency cannot be discerned in this model due to pleotropic effects of germline Prkn deletion on energy balance and subsequent protection against diet-induced obesity. We therefore generated the first liver-specific Prkn knockout mouse strain (LKO) to directly address the role of hepatic Prkn.
Littermate control (WT) and LKO mice were fed regular chow (RC) or high-fat diet (HFD) and changes in body weight and composition were measured over time. Liver mitochondrial content was assessed using multiple, complementary techniques, and mitochondrial respiratory capacity was assessed using Oroboros OK platform. Liver fat was measured biochemically and assessed histologically, while global changes in hepatic gene expression were measured by RNA-seq. Whole-body and tissue-specific insulin resistance were assessed by hyperinsulinemic-euglycemic clamp with isotopic tracers.
Liver-specific deletion of Prkn had no effect on body weight or adiposity during RC or HFD feeding; however, hepatic steatosis was increased by 45% in HFD-fed LKO compared with WT mice (P < 0.05). While there were no differences in mitochondrial content between genotypes on either diet, mitochondrial respiratory capacity and efficiency in the liver were significantly reduced in LKO mice. Gene enrichment analyses from liver RNA-seq results suggested significant changes in pathways related to lipid metabolism and fibrosis in HFD-fed Prkn knockout mice. Finally, whole-body insulin sensitivity was reduced by 35% in HFD-fed LKO mice (P < 0.05), which was primarily due to increased hepatic insulin resistance (60% of whole-body effect; P = 0.11).
These data demonstrate that PARKIN contributes to mitochondrial homeostasis in the liver and plays a protective role against the pathogenesis of hepatic steatosis and insulin resistance.
PARKIN 是一种 E3 泛素连接酶，通过一种称为自噬的过程来调节线粒体质量控制。最近的人类和啮齿动物研究表明，肥胖相关脂肪肝发病过程中可能会发生肝线粒体自噬丧失，并导致与该疾病相关的线粒体代谢变化。全身性 Prkn 基因敲除小鼠对饮食诱导的肝脂肪变性具有反常的保护作用；然而，由于生殖系 Prkn 缺失对能量平衡的多效作用以及随后对饮食诱导肥胖的保护作用，这种模型无法辨别肝特异性 Prkn 缺乏的作用。因此，我们生成了首个肝特异性 Prkn 基因敲除小鼠品系（LKO），以直接研究肝 Prkn 的作用。
同窝对照（WT）和 LKO 小鼠分别用普通饲料（RC）或高脂肪饮食（HFD）喂养，并随时间测量体重和体成分的变化。使用多种互补技术评估肝线粒体含量，使用 Oroboros OK 平台评估线粒体呼吸能力。通过生化方法和组织学评估肝脂肪，通过 RNA-seq 测量肝内基因表达的整体变化。通过使用同位素示踪剂的高胰岛素-正葡萄糖钳夹术评估全身和组织特异性胰岛素抵抗。
RC 或 HFD 喂养期间，肝特异性 Prkn 缺失对体重或肥胖没有影响；然而，与 WT 小鼠相比，HFD 喂养的 LKO 小鼠肝脂肪变性增加了 45%（P<0.05）。两种饮食下，基因型之间的线粒体含量没有差异，但 LKO 小鼠的肝线粒体呼吸能力和效率显著降低。肝 RNA-seq 结果的基因富集分析表明，HFD 喂养的 Prkn 基因敲除小鼠中与脂质代谢和纤维化相关的途径发生了显著变化。最后，HFD 喂养的 LKO 小鼠的全身胰岛素敏感性降低了 35%（P<0.05），这主要是由于肝胰岛素抵抗增加（全身效应的 60%；P=0.11）。
这些数据表明，PARKIN 有助于肝脏中线粒体的动态平衡，并在肝脂肪变性和胰岛素抵抗的发病机制中发挥保护作用。
