Objective 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 . Methods 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 O 2 K 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. Results 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). Conclusions 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.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN accelerates the onset of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, including steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD. Mitophagy is a mitochondrial quality control mechanism that allows for selective removal of damaged mitochondria from the cell. Whether changes in mitophagy occur during and contribute to NAFLD is unknown. PARKIN is a ubiquitin E3 ligase that regulates mitophagy by generating a ubiquitin signaling motif on the outer membrane of damaged mitochondria, marking them for degradation. We undertook the studies described to determine effects of genetic inhibition of mitophagy via conditional deletion of PARKIN in liver on progression of NAFLD. Twelve-week old male liver-specific PARKIN knockout (LKO) and wild type (WT) mice were fed a diet previously demonstrated to induce NAFLD that consisted of high-fat (45%), cholesterol (0.15%) and high-fructose corn syrup drinking water (42g/L) for 8 weeks. There was no difference in body weight or composition between groups. Plasma ALT and AST levels were increased 2-fold (p=0.18) and 1.7-fold (p&lt;0.05), respectively, in LKO compared with WT mice, while plasma cholesterol and insulin levels were not different and plasma fatty acid levels were reduced by 27% (p&lt;0.05). NAFLD activity score was increased 2-fold (p&lt;0.05) in LKO mice reflecting an increase in steatosis, ballooning and inflammation. Gene expression markers of NAFLD progression including markers of inflammation (Ccl2, Cd68, Il-1b, ll-6) and fibrosis (Col1a1, Col3a1) were significantly increased in LKO mice. Consistent with these mouse data, we found PARKIN protein expression to be reduced in liver biopsies from patients with NASH compared with healthy controls. These data suggest that loss of PARKIN-mediated mitophagy may contribute to the progression of NAFLD. Disclosure R. Undamatla: None. L.R. Edmunds: None. B. Xie: None. A. Mills: None. I.J. Sipula: None. S.P. Monga: None. M.J. Jurczak: None. Funding National Institutes of Health (DK114012)

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