Elsevier

Journal of Hepatology

Volume 72, Issue 4, April 2020, Pages 627-635
Journal of Hepatology

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

https://doi.org/10.1016/j.jhep.2019.11.008Get rights and content

Highlights

  • 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.

Methods

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.

Results

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.

Conclusion

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.

Introduction

Non-alcoholic fatty liver disease (NAFLD) has been paralleling the worldwide increase in obesity for the last few decades. It has become the most common chronic liver disease, and now affects up to one-third of the adult population in Western countries.1 NAFLD encompasses a continuum of entities that span from plain steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis and ultimately end-stage liver disease.2 Hepatocyte cell death is a critical event in the progression of all chronic inflammatory liver diseases including NAFLD.3 Until recently, two main forms of cell death were recognized: apoptosis, which occurs in a highly controlled manner, and necrosis that is accidentally triggered. However, during the past few years, it became clear that programmed cell death was not restricted to apoptosis, and comprised other forms of regulated cell death.4 Necroptosis is one of them, combining the molecular machinery of the extrinsic apoptotic pathways with an execution similar to necrosis.4 Unlike apoptosis that requires the activation of aspartate-specific proteases known as caspases,5 necroptosis is driven by the activation of the receptor-interacting protein kinase (RIPK) 1 and 3, and the pseudo kinase mixed lineage kinase domain-like (MLKL).4 Previous studies have shown that ablation of RIPK3 protected mice from the development of steatohepatitis and fibrosis in a methionine and choline-deficient (MCD) diet model.6,7 In addition, it has been demonstrated that necroptosis was activated in the hepatocytes of patients with NASH.[6], [7], [8] Overall, compelling studies have shown that RIP kinases were pleiotropic modulators of cell death and participated in the pathogenesis of many chronic diseases.9 Therefore, there is evidence to suggest that by preventing the formation and/or activation of the necrosome, one could limit cell death and possibly disease progression in NASH.

At present, there are limited options to inhibit the necroptosis pathway. Stable inhibitors that may specifically target RIPK3 or MLKL in vivo are lacking or induce undesirable effects.10 The RIPK1 inhibitor necrostatin-1 (Nec-1) has been recognized as a potent inhibitor of necroptosis.11 However, it is poorly specific, as it also inhibits the indoleamine 2,3-dioxygenase pathway. More recently, RIPA-56 was identified as a highly specific and metabolically stable inhibitor of RIPK1.12

In the present study, we investigated whether inhibiting RIPK1 would improve features of NASH. Specifically, we tested the effect of RIPA-56 in human cells and murine models of steatosis and steatohepatitis. We also explored a hitherto undescribed interaction between the necroptosis pathway and mitochondrial respiration, as a new targetable mechanism of fat accumulation. In addition, we analyzed if the mediators of necroptosis might serve as markers of activity in NAFLD.

Section snippets

RIPA-56 feeding experiment

Six-week-old male C57BL/6J mice (Charles River Laboratories, Ecully, France) were fed a high-fat diet (HFD – 45 kcal% fat) or a normal chow diet (NCD) (Ssniff spezialdiäten GmbH, Soest, Germany) for 16 weeks (the time necessary for an inflammatory and fibrotic response to develop). The effects of a highly potent and highly specific RIPK1 kinase inhibitor (referred as to RIPA-56) were evaluated by incorporating it into HFD at 300 mg/kg dose as initially described.13 (see supplementary information

RIPK1 inhibitor reduces necro-inflammatory and fibrotic NASH features in HFD-fed mice

RIPA-56 is a highly potent, selective and metabolically stable RIPK1 inhibitor, able to prevent MLKL activation and MLKL-mediated cell death,12 as we herein confirmed using a well-established cell model of tumour necrosis factor-α (TNFα)-induced cell death, i.e., L929 cells16 (Fig. S1A-C). To explore the therapeutic potential of RIPK1 inhibition in NASH, we tested RIPA-56 in an HFD mouse model. Six-week-old male C57BL/6J mice were fed NCD or HFD for 16 weeks. HFD-fed mice received no additional

Discussion

The present study provides evidence to indicate that RIPK1 inhibition and subsequent downstream necrosome inactivation improves all histologic features of NASH including liver inflammation and fibrosis. Importantly, we show that RIPK1/MLKL axis also controls lipid homeostasis and cellular fat deposition. Therefore, the RIPK1/MLKL axis could be a valuable therapeutic target in NAFLD/NASH.

The cell commitment towards apoptosis or necroptosis is largely dependent on the interactions between caspase

Financial support

Jérémie Gautheron is funded by the Fondation pour la Recherche Médicale (FRM – ARF20170938613), the Institute of Cardiometabolism and Nutrition (ICAN – PAP17NECJG), the Société Francophone du Diabète (SFD – R19114DD) and the Mairie de Paris (Emergences – R18139DD). Amine Majdi is supported by the Ministère de l'Enseignement Supérieur et de la Recherche (MESR). Cecília M. P. Rodrigues is funded by the Fundação para a Ciência e a Tecnologia (PTDC/MED-FAR/29097/2017), and the EU H2020 Marie

Conflict of interest

The authors have no conflict of interest to declare.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors' contributions

J.G. conceived and designed the study; A.M., L.A., T.M., and J.G. performed, analyzed and interpreted most of the experiments; F.B. and T.L. provided technical assistance; L.A., F.C. and O.S. designed and performed the primary human hepatocyte experiments; V.R. provided human patients' serum samples and clinical data; T.I. analyzed MRC complex activity; M.B.A. performed Ripk3-KO animal experiments; V.R., F.C., M.L., F.F., M.M., A.C., L.F., T.A-S., J-L.D., G.C., B.F., O.S., C.P-B., C.M.P.R. and

Acknowledgements

The authors thank L. Dinard, Q. Pointout, T. Coulais and A. Guyomard from the animal facilities platform (PHEA Saint-Antoine) for their technical support, A. Munier from the cytometry platform for her help in FACS analysis, R. Morichon from the image platform for his help in microscopy analysis, and B. Solhonne and F. Merabtene for their contribution in liver tissue histomorphological analysis. We are also thankful to R. Castro and P. Rodrigues (iMed.ULisboa) for their help with Ripk3-KO animal

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