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MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis

  • Xiaoqin Wu
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States
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  • Kyle L. Poulsen
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States
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  • Carlos Sanz-Garcia
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States
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  • Emily Huang
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States
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  • Megan R. McMullen
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States
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  • Sanjoy Roychowdhury
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States

    Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH, United States
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  • Srinivasan Dasarathy
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States

    Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, United States

    Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH, United States
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  • Laura E. Nagy
    Correspondence
    Corresponding author. Address: Cleveland Clinic, Lerner Research Institute/NE40 9500 Euclid Ave, Cleveland, OH 44195. Tel.: 216-444-4021; Fax: 216-636-1493.
    Affiliations
    Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States

    Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, United States

    Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH, United States
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Published:March 24, 2020DOI:https://doi.org/10.1016/j.jhep.2020.03.023

      Highlights

      • MLKL-mediated signaling contributes to FFC diet-induced liver injury.
      • FFC diet or palmitic acid treatment induces MLKL expression in hepatocytes.
      • Palmitic acid drives MLKL translocation to autophagosomes independently of Rip3.
      • Mlkl, but not Rip3, regulates autophagic flux in a murine model of NAFL/NASH.
      • Pharmacologic inhibition of autophagy induces MLKL expression.

      Background & Aims

      Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy.

      Methods

      Rip3+/+, Rip3−/−, Mlkl+/+ and Mlkl−/− mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA).

      Results

      The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl−/− mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes.

      Conclusions

      Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy.

      Lay summary

      Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

      Graphical abstract

      Keywords

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