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Lack of hepatic autophagy promotes severity of liver injury but not steatosis

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      To the Editor:
      We read with great interest the recently published paper by Baselli et al. in which they identified novel rare autophagy-related 7 (ATG7) genetic variants that were associated with the progression of non-alcoholic fatty liver disease (NAFLD) in the European population.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      The implication of autophagy in regulating lipid metabolism, lipid droplet (LD) biogenesis and NAFLD has been well documented in experimental settings.
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      • Nguyen T.B.
      • Louie S.M.
      • Daniele J.R.
      • Tran Q.
      • Dillin A.
      • Zoncu R.
      • et al.
      DGAT1-dependent lipid droplet biogenesis protects mitochondrial function during starvation-induced autophagy.
      However, the direct genetic evidence on autophagy-related genes in human diseases such as NAFLD has been lacking for a long time. Using several European NAFLD and chronic liver disease cohorts, Baselli et al. identified that loss-of-function variants of ATG7 increased the risk of cirrhosis and hepatocellular carcinoma (HCC). Notably, one low-frequency variant rs36117895 p. V471A impaired autophagic flux in cultured hepatocytes with increased LD accumulation, confirming V471A is a loss-of-function ATG7 mutant. Interestingly, despite a strong association with increased liver injury (increased aspartate aminotransferase levels) and hepatocellular ballooning, ATG7 V471A was not associated with steatosis in the liver biopsy cohort.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      The findings regarding ATG7 variants in human liver diseases are generally consistent with experimental mouse models from liver-specific Atg7 or Atg5 knockout (L-Atg7 or L-Atg5 KO) mice that show increased hepatocyte death, inflammation, fibrosis and spontaneous adenocarcinoma.
      • Takamura A.
      • Komatsu M.
      • Hara T.
      • Sakamoto A.
      • Kishi C.
      • Waguri S.
      • et al.
      Autophagy-deficient mice develop multiple liver tumors.
      ,
      • Ni H.M.
      • Woolbright B.L.
      • Williams J.
      • Copple B.
      • Cui W.
      • Luyendyk J.P.
      • et al.
      Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.
      However, L-Atg7 or L-Atg5 KO mice are resistant to physiological steatosis induced by fasting or partial hepatectomy, which is demonstrated by Oil red O staining for neutral lipids and electron microscope analysis for LD in mouse livers (Fig. 1). Decreased steatosis in L-Atg5 or L-Atg7 KO mouse livers is either due to nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2) activation or impaired autophagic degradation of nuclear receptor co-repressor 1 (NCoR1) that inhibits liver X receptor α-mediated de novo lipogenesis and LD biogenesis.
      • Li Y.
      • Chao X.
      • Yang L.
      • Lu Q.
      • Li T.
      • Ding W.X.
      • et al.
      Impaired fasting-induced adaptive lipid droplet biogenesis in liver-specific Atg5-deficient mouse liver is mediated by persistent nuclear factor-like 2 activation.
      ,
      • Takahashi S.S.
      • Sou Y.S.
      • Saito T.
      • Kuma A.
      • Yabe T.
      • Sugiura Y.
      • et al.
      Loss of autophagy impairs physiological steatosis by accumulation of NCoR1.
      Mice with liver-specific deletion of focal adhesion kinase family kinase-interacting protein of 200 kDa (also called Rb1cc1), a core subunit of the mammalian autophagy related 1 complex, are also resistant to high fat diet-induced fatty liver.
      • Ma D.
      • Molusky M.M.
      • Song J.
      • Hu C.R.
      • Fang F.
      • Rui C.
      • et al.
      Autophagy deficiency by hepatic FIP200 deletion uncouples steatosis from liver injury in NAFLD.
      Moreover, lipids generated from autophagic degradation of membranes are required for the LD biogenesis.
      • Nguyen T.B.
      • Louie S.M.
      • Daniele J.R.
      • Tran Q.
      • Dillin A.
      • Zoncu R.
      • et al.
      DGAT1-dependent lipid droplet biogenesis protects mitochondrial function during starvation-induced autophagy.
      These observations argue against the contribution of defective hepatic autophagy on the development of steatosis, the key early feature of NAFLD. The lack of association of ATG7 V471A with steatosis found in the human NAFLD cohort by Baselli et al. further supports the notion that impaired hepatic autophagy is not critical for steatosis.
      Figure thumbnail gr1
      Fig. 1L-ATG5 and L-ATG7 KO mice have impaired fasting-induced hepatic LD accumulation.
      L-ATG5 and L-ATG7 KO mice were either fed with a chow diet or fasted overnight (L-ATG5 KO) or 24 hrs (L-ATG7 KO). Liver tissues were fixed and subjected to Oil Red O staining (A, B) or EM analysis (C, D). Representative images are shown. Bar: 1 μm. EM, electron microscope; KO, knockout; LD, lipid droplet. (This figure appears in color on the web.)
      Defective hepatic autophagy impairs the removal of damaged mitochondria and protein aggregates that can lead to increased oxidative stress, hepatocyte death and genome instability resulting in liver fibrosis and tumorigenesis.
      • Takamura A.
      • Komatsu M.
      • Hara T.
      • Sakamoto A.
      • Kishi C.
      • Waguri S.
      • et al.
      Autophagy-deficient mice develop multiple liver tumors.
      ,
      • Ni H.M.
      • Woolbright B.L.
      • Williams J.
      • Copple B.
      • Cui W.
      • Luyendyk J.P.
      • et al.
      Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.
      Patients that have chronic liver diseases including NAFLD and HCC may have combined multiple gene mutations such as PNPLA3 and ATG7 that contribute to distinctive stages of liver pathogenesis. Therefore, the findings from Baselli et al.’s study seem to support the notion that loss-of-function ATG7 variants may not affect hepatic lipid accumulation, but rather promote the severity of NAFLD, cirrhosis and HCC in humans who may also have PNPLA3 or other gene variants that affect lipid metabolism. However, it is also likely that the ATG7 V471A mutant protein may have unknown scaffold functions that could regulate lipid metabolism independently of autophagy. Future work using ATG7 V471A knockin mice may help to further clarify its function in the pathogenesis of NAFLD.

      Financial support

      R37 AA020518, R01 DK102142, R01 AG072895 (WXD).

      Authors’ contributions

      HMN, SW and MK performed experiments. WXD conceived and drafted the manuscript and all authors read the manuscript. We thank Dr. Yssa Rodriguez for critical reading of the manuscript.

      Conflict of interest

      The authors disclose no conflicts.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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