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. 2020 Nov:41:101051.
doi: 10.1016/j.molmet.2020.101051. Epub 2020 Jul 10.

Liver-specific Prkn knockout mice are more susceptible to diet-induced hepatic steatosis and insulin resistance

Lia R Edmunds  1 , Bingxian Xie  1 , Amanda M Mills  1 , Brydie R Huckestein  1 , Ramya Undamatla  1 , Anjana Murali  1 , Martha M Pangburn  1 , James Martin  2 , Ian Sipula  1 , Brett A Kaufman  2 , Iain Scott  2 , Michael J Jurczak  3
Affiliations

Liver-specific Prkn knockout mice are more susceptible to diet-induced hepatic steatosis and insulin resistance

Lia R Edmunds et al. Mol Metab. 2020 Nov.

Abstract

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 O2K 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.

Keywords: Bioenergetics; Hepatic steatosis; Insulin resistance; Mitochondria; Mitophagy; Parkin.

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Figures

Figure 1
Figure 1
Hepatic steatosis is increased in high-fat diet fed liver-specific Prkn knockout mice. A. Targeting strategy for loxP insertions flanking exon seven of Prkn. B. PCR confirmation of 5′ and 3′ loxP insertions by CRISPR-Cas9 genetic editing of C57BL6J zygotes. C. Western blots confirming tissue-specific deletion of PARKIN after backcrossing Prknfl/fl mice to Albumin-Cre mice. D. Changes in body weight over nine weeks during regular chow (RC) and high-fat diet (HFD) studies. E. Changes in body weight in percent relative to baseline over nine weeks during RC and HFD studies. F. Final body weights at the end of RC and HFD studies. G. Final body fat measured by 1H-NMR. H. Liver triglyceride levels. Data are the mean ± S.E.M. for n = 8–12 mice per group. The effect of genotype was compared by Student's t-test, where ∗P < 0.05.
Figure 2
Figure 2
NAFLD activity score is increased in high-fat diet fed liver-specific Prkn knockout mice. A. Representative images of H&E stained sections. B. Histological scores for steatosis ranging from 0 to 3 made in whole-value increments, where 0 = 0–5%, 1 = 6–33%, 2 = 34–66%, and 3 = 67–100% of hepatocytes positive for steatosis; C. hepatocyte ballooning ranging from 0 to 2, where 0 = none present, 1 = few, and 2 = many/prominent; and D. lobular inflammation ranging from 0 to 3, where 0 = no foci, 1 = more than 2 foci per 200X field, 2 = 2-4 foci per 200X field and 3 = greater than 4 foci per 200X field. E. Composite NAFLD activity score (NAS) reflecting sum of scores in B-D. Data are the mean ± S.E.M. for n = 5–6 mice per group. The effect of genotype was compared by Student's t-test, where ∗P < 0.05.
Figure 3
Figure 3
Liver-specific deletion of Prkn exacerbates changes in gene expression associated with NAFLD progression. A. Volcano plot of RNA-seq data comparing RC-fed LKO with WT mice. B. Gene annotation summary for enriched pathways detected by KEGG and GO analyses. C. Volcano plot of RNA-seq data comparing HFD-fed LKO with WT mice. D. Gene annotation summary for enriched pathways detected by KEGG and E. GO analyses. Abbreviations: BP = biological process; CC = cellular component; MF = molecular function. Data in A and C represent the log2 fold change in gene expression of LKO compared with WT, as well as the negative log of the adjusted P-value for the entire RNAseq dataset. F. Quantitative PCR for the indicated target genes for RC-fed mice. G-H. Quantitative PCR for the indicated target genes organized by pathway (G: fibrosis; H. metabolism) for HFD-fed mice. Data in F–H are expressed as fold-change in expression relative to WT following calculation of relative expression using a housekeeping gene (Actin, Gapdh, B2m) and individual primer efficiencies determined at the time of study. Data are the mean ± S.E.M. for n = 3 mice per group and were compared by Student's t-test, where ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Figure 4
Figure 4
Deletion of hepatic Prkn does not affect markers of mitochondrial mass. A. Western blot from liver lysates for electron transport chain respiratory complex proteins CI–CIV and the ATP synthase (CV), as well as HSP60 loading control. Lower blot shows an extended exposure for CIV marker MTCO1, which appeared faintly in upper blot. B. Liver mitochondrial DNA content measured by PCR. C. Citrate synthase activity measured in liver lysates. D. Western blots from liver lysates for markers of mitochondrial mass. Data are the mean ± S.E.M. for n = 5–6 mice per group.
Figure 5
Figure 5
Hepatic mitochondrial respiratory capacity is impaired in liver-specific Prkn knockout mice. A. Respiratory control ratio calculated as the ratio of state 3 to state 4° (ADP-stimulated respiration in the presence of pyruvate, malate, and glutamate, following oligomycin treatment). B. Coupling control ratio calculated as the ratio of basal or routine respiration (sample in the presence of pyruvate, malate, and glutamate) to state 3, as defined in A. C. Flux control ratio for NADH-linked or complex I supported respiration relative to maximal electron transport chain capacity, calculated as the ratio of state 3 to the uncoupled state (FCCP). D. Respiratory capacity committed to oxidative phosphorylation (OXPHOS), calculated as the difference in oxygen consumption or flux (JO2) in the presence of pyruvate, malate, and glutamate following oligomycin treatment. E. Respiratory capacity committed to leak respiration, calculated as the difference in JO2 in the presence of oligomycin following antimycin A treatment. F. Maximal respiratory capacity of the electron transport system (ETS), determined as the max JO2 following FCCP titration. Data are the mean ± S.E.M. for n = 9–11 mice per group. The effect of genotype was compared by Student's t-test, where ∗P < 0.05 and ∗∗P < 0.01.
Figure 6
Figure 6
Insulin sensitivity is impaired in liver-specific Prkn knockout mice. A. Fasting plasma glucose levels following a six-hour morning fast. B. Fasting or basal rates of endogenous or hepatic glucose production (EGP). C. Fasting or basal and clamped levels of plasma insulin. D. Plasma glucose levels during the hyperinsulinemic infusion. E. Glucose infusion rate (GIR) required to maintain euglycemia during the hyperinsulinemic infusion. F. The average GIR during the last 40 min or steady-state portion of the clamp. G. Whole-body glucose uptake during the clamp. H. Insulin-stimulated or clamped rates of EGP. I. Western blots of post-clamp liver for indicated target proteins. J. Quantification of data in I where AU = arbitrary units. Data are the mean ± S.E.M. for n = 6 mice per group. The effect of genotype was compared by Student's t-test, where ∗P < 0.05.
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