Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease

Published:November 21, 2019DOI:


      • RIPA-56 reduces hepatic inflammation and fibrosis in dietary obese mice.
      • RIPA-56 reverses steatosis and dampens body weight gain in obese mice.
      • RIPK1 regulates triglyceride content in hepatocytes by activating MLKL, which controls mitochondrial biomass and activity.
      • RIPK1 and MLKL are significantly increased in the serum of patients with NASH.
      • Targeting RIPK1/MLKL represents a promising strategy for NASH treatment.

      Background & Aims

      In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD.


      C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD.


      When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity.


      The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD.

      Lay summary

      There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

      Graphical abstract


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Younossi Z.
        • Anstee Q.M.
        • Marietti M.
        • Hardy T.
        • Henry L.
        • Eslam M.
        • et al.
        Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.
        Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20
        • Friedman S.L.
        • Neuschwander-Tetri B.A.
        • Rinella M.
        • Sanyal A.J.
        Mechanisms of NAFLD development and therapeutic strategies.
        Nat Med. 2018; 24: 908-922
        • Luedde T.
        • Kaplowitz N.
        • Schwabe R.F.
        Cell death and cell death responses in liver disease: mechanisms and clinical relevance.
        Gastroenterology. 2014; 147: 765-783.e4
        • Vanden Berghe T.
        • Linkermann A.
        • Jouan-Lanhouet S.
        • Walczak H.
        • Vandenabeele P.
        Regulated necrosis: the expanding network of non-apoptotic cell death pathways.
        Nat Rev Mol Cell Biol. 2014; 15: 135-147
        • McIlwain D.R.
        • Berger T.
        • Mak T.W.
        Caspase functions in cell death and disease.
        Cold Spring Harb Perspect Biol. 2013; 5: a008656
        • Afonso M.B.
        • Rodrigues P.M.
        • Carvalho T.
        • Caridade M.
        • Borralho P.
        • Cortez-Pinto H.
        • et al.
        Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.
        Clin Sci (Lond). 2015; 129: 721-739
        • Gautheron J.
        • Vucur M.
        • Reisinger F.
        • Cardenas D.V.
        • Roderburg C.
        • Koppe C.
        • et al.
        A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.
        EMBO Mol Med. 2014; 6: 1062-1074
        • Gautheron J.
        • Vucur M.
        • Luedde T.
        Necroptosis in nonalcoholic steatohepatitis.
        Cell Mol Gastroenterol Hepatol. 2015; 1: 264-265
        • He S.
        • Wang X.
        RIP kinases as modulators of inflammation and immunity.
        Nat Immunol. 2018; 19: 912-922
        • Mandal P.
        • Berger S.B.
        • Pillay S.
        • Moriwaki K.
        • Huang C.
        • Guo H.
        • et al.
        RIP3 induces apoptosis independent of pronecrotic kinase activity.
        Mol Cell. 2014; 56: 481-495
        • Takahashi N.
        • Duprez L.
        • Grootjans S.
        • Cauwels A.
        • Nerinckx W.
        • DuHadaway J.B.
        • et al.
        Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.
        Cell Death Dis. 2012; 3: e437
        • Ren Y.
        • Su Y.
        • Sun L.
        • He S.
        • Meng L.
        • Liao D.
        • et al.
        Discovery of a highly potent, selective, and metabolically stable inhibitor of receptor-interacting protein 1 (RIP1) for the treatment of systemic inflammatory response syndrome.
        J Med Chem. 2017; 60: 972-986
        • Li D.
        • Meng L.
        • Xu T.
        • Su Y.
        • Liu X.
        • Zhang Z.
        • et al.
        RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system.
        Elife. 2017; 6
        • Bedossa P.
        • Consortium F.P.
        Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease.
        Hepatology. 2014; 60: 565-575
        • Aoudjehane L.
        • Podevin P.
        • Scatton O.
        • Jaffray P.
        • Dusanter-Fourt I.
        • Feldmann G.
        • et al.
        Interleukin-4 induces human hepatocyte apoptosis through a Fas-independent pathway.
        FASEB J. 2007; 21: 1433-1444
        • Vercammen D.
        • Beyaert R.
        • Denecker G.
        • Goossens V.
        • Van Loo G.
        • Declercq W.
        • et al.
        Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.
        J Exp Med. 1998; 187: 1477-1485
        • Nagarajan S.R.
        • Paul-Heng M.
        • Krycer J.R.
        • Fazakerley D.J.
        • Sharland A.F.
        • Hoy A.J.
        Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism.
        Am J Physiol Endocrinol Metab. 2019; 316: E578-E589
        • Sureshbabu A.
        • Patino E.
        • Ma K.C.
        • Laursen K.
        • Finkelsztein E.J.
        • Akchurin O.
        • et al.
        RIPK3 promotes sepsis-induced acute kidney injury via mitochondrial dysfunction.
        JCI Insight. 2018; 3
        • Wang B.
        • Li J.
        • Gao H.M.
        • Xing Y.H.
        • Lin Z.
        • Li H.J.
        • et al.
        Necroptosis regulated proteins expression is an early prognostic biomarker in patient with sepsis: a prospective observational study.
        Oncotarget. 2017; 8: 84066-84073
        • Orozco S.
        • Oberst A.
        RIPK3 in cell death and inflammation: the good, the bad, and the ugly.
        Immunol Rev. 2017; 277: 102-112
        • Chenxu G.
        • Minxuan X.
        • Yuting Q.
        • Tingting G.
        • Jing F.
        • Jinxiao L.
        • et al.
        Loss of RIP3 initiates annihilation of high-fat diet initialized nonalcoholic hepatosteatosis: a mechanism involving Toll-like receptor 4 and oxidative stress.
        Free Radic Biol Med. 2019; 134: 23-41
        • Gautheron J.
        • Vucur M.
        • Schneider A.T.
        • Severi I.
        • Roderburg C.
        • Roy S.
        • et al.
        The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.
        Nat Commun. 2016; 7: 11869
        • Roychowdhury S.
        • McCullough R.L.
        • Sanz-Garcia C.
        • Saikia P.
        • Alkhouri N.
        • Matloob A.
        • et al.
        Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.
        Hepatology. 2016; 64: 1518-1533
        • Christofferson D.E.
        • Li Y.
        • Yuan J.
        Control of life-or-death decisions by RIP1 kinase.
        Annu Rev Physiol. 2014; 76: 129-150
        • Cho Y.S.
        • Challa S.
        • Moquin D.
        • Genga R.
        • Ray T.D.
        • Guildford M.
        • et al.
        Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.
        Cell. 2009; 137: 1112-1123
        • Liang Y.X.
        • Wang N.N.
        • Zhang Z.Y.
        • Juan Z.D.
        • Zhang C.
        Necrostatin-1 ameliorates peripheral nerve injury-induced neuropathic pain by inhibiting the RIP1/RIP3 pathway.
        Front Cell Neurosci. 2019; 13: 211
        • Cui H.
        • Zhu Y.
        • Yang Q.
        • Zhao W.
        • Zhang S.
        • Zhou A.
        • et al.
        Necrostatin-1 treatment inhibits osteocyte necroptosis and trabecular deterioration in ovariectomized rats.
        Sci Rep. 2016; 6: 33803
        • Liu Z.Y.
        • Wu B.
        • Guo Y.S.
        • Zhou Y.H.
        • Fu Z.G.
        • Xu B.Q.
        • et al.
        Necrostatin-1 reduces intestinal inflammation and colitis-associated tumorigenesis in mice.
        Am J Cancer Res. 2015; 5: 3174-3185
        • Wang J.N.
        • Liu M.M.
        • Wang F.
        • Wei B.
        • Yang Q.
        • Cai Y.T.
        • et al.
        RIPK1 inhibitor Cpd-71 attenuates renal dysfunction in cisplatin-treated mice via attenuating necroptosis, inflammation and oxidative stress.
        Clin Sci (Lond). 2019; 133: 1609-1627
        • Koliaki C.
        • Szendroedi J.
        • Kaul K.
        • Jelenik T.
        • Nowotny P.
        • Jankowiak F.
        • et al.
        Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis.
        Cell Metab. 2015; 21: 739-746
        • Pelusi S.
        • Valenti L.
        Hepatic fat as clinical outcome and therapeutic target for nonalcoholic fatty liver disease.
        Liver Int. 2019; 39: 250-256
        • Xu H.
        • Du X.
        • Liu G.
        • Huang S.
        • Du W.
        • Zou S.
        • et al.
        The pseudokinase MLKL regulates hepatic insulin sensitivity independently of inflammation.
        Mol Metab. 2019; 23: 14-23
        • Marchesini G.
        • Brizi M.
        • Morselli-Labate A.M.
        • Bianchi G.
        • Bugianesi E.
        • McCullough A.J.
        • et al.
        Association of nonalcoholic fatty liver disease with insulin resistance.
        Am J Med. 1999; 107: 450-455