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Targeting cell-intrinsic metabolism for antifibrotic therapy

  • Helene Gilgenkrantz
    Affiliations
    Université de Paris, INSERM, U1149, CNRS, ERL 8252, Centre de Recherche sur l'Inflammation (CRI), Laboratoire d’Excellence Inflamex, F-75018 Paris, France
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  • Ariane Mallat
    Affiliations
    Université de Paris, INSERM, U1149, CNRS, ERL 8252, Centre de Recherche sur l'Inflammation (CRI), Laboratoire d’Excellence Inflamex, F-75018 Paris, France
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  • Richard Moreau
    Affiliations
    Université de Paris, INSERM, U1149, CNRS, ERL 8252, Centre de Recherche sur l'Inflammation (CRI), Laboratoire d’Excellence Inflamex, F-75018 Paris, France
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  • Sophie Lotersztajn
    Correspondence
    Corresponding author. Address: Centre de Recherches sur l’Inflammation, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris-France. Tel.: +33 1 57 27 74 29.
    Affiliations
    Université de Paris, INSERM, U1149, CNRS, ERL 8252, Centre de Recherche sur l'Inflammation (CRI), Laboratoire d’Excellence Inflamex, F-75018 Paris, France
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Published:February 22, 2021DOI:https://doi.org/10.1016/j.jhep.2021.02.012

      Summary

      In recent years, there have been major advances in our understanding of the mechanisms underlying fibrosis progression and regression, and how coordinated interactions between parenchymal and non-parenchymal cells impact on the fibrogenic process. Recent studies have highlighted that metabolic reprogramming of parenchymal cells, immune cells (immunometabolism) and hepatic stellate cells is required to support the energetic and anabolic demands of phenotypic changes and effector functions. In this review, we summarise how targeting cell-intrinsic metabolic modifications of the main fibrogenic cell actors may impact on fibrosis progression and we discuss the antifibrogenic potential of metabolically targeted interventions.

      Keywords

      Introduction

      Liver fibrosis and its end-stage, cirrhosis, remain a growing health burden owing to the rising incidence of non-alcoholic fatty liver (NAFLD) worldwide. Liver fibrosis is the common wound healing response associated with chronic liver injury and the consequence of prolonged parenchymal cell injury and/or inflammation.
      • Lotersztajn S.
      • Julien B.
      • Teixeira-Clerc F.
      • Grenard P.
      • Mallat A.
      Hepatic fibrosis: molecular mechanisms and drug targets.
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      • Marra F.
      • Lotersztajn S.
      Pathophysiology of NASH: perspectives for a targeted treatment.
      The fibrogenic process is characterised by progressive accumulation of extracellular matrix components following activation of a heterogeneous population of fibrogenic cells mainly derived from hepatic stellate cells (HSCs) and to a lesser extent from portal fibroblasts.
      • Tsuchida T.
      • Friedman S.L.
      Mechanisms of hepatic stellate cell activation.
      ,
      • Lemoinne S.
      • Cadoret A.
      • El Mourabit H.
      • Thabut D.
      • Housset C.
      Origins and functions of liver myofibroblasts.
      Parenchymal cell death, endothelial cell dysfunction and inflammatory signals originating from innate, adaptive and innate-like immune cells, are driving forces of fibrosis progression.
      • Lotersztajn S.
      • Julien B.
      • Teixeira-Clerc F.
      • Grenard P.
      • Mallat A.
      Hepatic fibrosis: molecular mechanisms and drug targets.
      ,
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Schwabe R.F.
      • Tabas I.
      • Pajvani U.B.
      Mechanisms of fibrosis development in nonalcoholic steatohepatitis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      Major advances have been made in the understanding of the mechanisms governing the coordinated dialogue between parenchymal and non-parenchymal cells during chronic liver cell injury, and its impact on fibrosis progression and regression.
      • Lotersztajn S.
      • Julien B.
      • Teixeira-Clerc F.
      • Grenard P.
      • Mallat A.
      Hepatic fibrosis: molecular mechanisms and drug targets.
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      • Marra F.
      • Lotersztajn S.
      Pathophysiology of NASH: perspectives for a targeted treatment.
      ,
      • Schwabe R.F.
      • Tabas I.
      • Pajvani U.B.
      Mechanisms of fibrosis development in nonalcoholic steatohepatitis.
      Although these findings have led to the identification of several therapeutic targets, no antifibrotic treatment has been approved. In this context, intrinsic metabolic reprogramming of HSCs, immune cells (immunometabolism) and hepatocytes has emerged as a crucial determinant of the cell phenotype switch that accompanies chronic liver injury and is now considered a potential therapeutic target. Studies on cell-intrinsic metabolism focus on how intracellular metabolic changes will modify effector functions in a given cell. A better understanding of how reprogramming metabolism in each liver cell type may impact on liver fibrosis is essential to predict the antifibrotic efficacy of targeting candidate pathways. In this review, we summarise our current understanding of the role of the intracellular metabolic machinery on parenchymal and non-parenchymal cells during fibrosis progression and regression, as well as discussing potential therapeutic options.

      Reprogramming metabolism of HSCs drives their fibrogenic/immunoregulator functions

      In the normal liver, quiescent HSCs display adipocyte-like features characterised by the expression of lipogenic genes and transcription factors, and the presence of abundant cytoplasmic lipid droplets. Following liver injury, HSCs lose their lipid droplets and undergo a phenotypic switch to a myofibroblastic phenotype in which they display fibrogenic, contractile and angiogenic functions. Myofibroblastic HSCs also display features of non-professional immune cells and contribute to the amplification of the inflammatory response by producing reactive oxygen species, pro-inflammatory cytokines and chemokines. They also respond to proinflammatory signals by expressing chemokine receptors and pattern recognition receptors. In addition, they behave as antigen-presenting cells and phagocyte apoptotic bodies, which contributes to activation of their fibrogenic properties.
      • Tsuchida T.
      • Friedman S.L.
      Mechanisms of hepatic stellate cell activation.
      The acquisition of myofibroblastic features is energetically expensive and relies on intense metabolic reprograming through activation of glycolysis, glutaminolysis and lipogenesis (Fig. 1, Fig. 2).
      Figure thumbnail gr1
      Fig. 1Summary of the main intrinsic metabolic pathways.
      Pyruvate generated from glucose through glycolysis is either converted into lactate or is oxidised to acetyl-CoA to fuel the TCA cycle. Two major products of the TCA cycle are NADH and FADH2 that transfer electrons to the electron transport chain to support OXPHOS. Fatty acid oxidation also produces NADH and FADH2 which can be used to generate ATP. Fatty acids are synthetised from acetyl-CoA, which is derived from glycolysis or from TCA cycle-derived citrate. The pentose phosphate pathway runs in parallel to glycolysis, generating ribose-5-phosphate required for the synthesis of nucleotides and amino acids. Finally, the TCA cycle can also be fed by amino acids such as glutamine via glutaminolysis for ATP or citrate production. OXPHOS, oxidative phosphorylation; TCA, tricarboxylic acid.
      Figure thumbnail gr2
      Fig. 2Targeting intracellular metabolic pathways in hepatocytes, macrophages and hepatic stellate cells: impact on liver fibrosis.
      Hepatocyte injury and inflammatory signals originating from innate, adaptive and innate-like immune cells contribute to the phenotypic switch from quiescent to activated HSC. Among immune cells, macrophages with proinflammatory and inflammatory properties are major regulators of liver fibrogenesis. In HSC, glycolysis, glutaminolysis, de novo lipogenesis, cholesterol accumulation, modulation of nuclear receptor expression support acquisition of myofibroblastic features. Regarding autophagy conflicting data have been reported (see text). In macrophages, cytotoxic lipid accumulation drives polarization toward a pro-inflammatory phenotype as does MAGL. Autophagy and acetoacetate released by hepatocytes is oxidised by macrophages, leading to the release of antifibrogenic signals. The pro-and anti-inflammatory potential of nuclear receptors, glycolysis lipogenesis and ferroptosis are suggested by in vitro studies but have not been studied in the context of fibrosis. Hepatocyte injury, as induced by cytotoxic lipid accumulation, activates HSC. Hepatocyte-specific loss of autophagy, MBOAT7 or FBP1 or inhibition of MPC will inhibit HSC activation. FBP1, fructose 1,6-bisphosphatase 1; HSC, hepatic stellate cell; MAGL, monoacylglycerol lipase; MBOAT7, membrane-bound O-acyltransferase domain containing 7; MPC, mitochondrial pyruvate carrier.

      Glycolysis drives HSC activation

      Glycolysis involves the intracellular processing of glucose to pyruvate, through several steps (Fig. 1). Pyruvate is further converted to lactate (anaerobic glycolysis) or to acetyl CoA that will fuel the tricarboxylic acid (TCA) cycle (aerobic glycolysis). The glycolytic pathway provides intermediates that are required for the synthesis of nucleotides, fatty acids and amino acids. In addition to its role in proliferation, glycolysis also supports production of extracellular matrix, as shown in the skin or lung.
      • Zhao X.
      • Psarianos P.
      • Ghoraie L.S.
      • Yip K.
      • Goldstein D.
      • Gilbert R.
      • et al.
      Metabolic regulation of dermal fibroblasts contributes to skin extracellular matrix homeostasis and fibrosis.
      • Yin X.
      • Choudhury M.
      • Kang J.H.
      • Schaefbauer K.J.
      • Jung M.Y.
      • Andrianifahanana M.
      • et al.
      Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-beta.
      • Zhao X.
      • Kwan J.Y.Y.
      • Yip K.
      • Liu P.P.
      • Liu F.F.
      Targeting metabolic dysregulation for fibrosis therapy.
      In cultured HSCs, transition to a myofibroblast phenotype is associated with a rise in glucose transporters, particularly GLUT1, and in glycolytic enzymes such as hexokinase 2, pyruvate kinase isoform M2 (PKM2) or 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), with concomitant lactate accumulation and downregulation of gluconeogenic genes.
      • Chen Y.
      • Choi S.S.
      • Michelotti G.A.
      • Chan I.S.
      • Swiderska-Syn M.
      • Karaca G.F.
      • et al.
      Hedgehog controls hepatic stellate cell fate by regulating metabolism.
      The resulting increase in glycolysis provides ATP, required for the activation process (Fig. 2). In keeping with in vitro studies, similar increases in glycolytic genes have been observed in experimental models of fibrosis and in the livers of patients with chronic liver injury.
      • Chen Y.
      • Choi S.S.
      • Michelotti G.A.
      • Chan I.S.
      • Swiderska-Syn M.
      • Karaca G.F.
      • et al.
      Hedgehog controls hepatic stellate cell fate by regulating metabolism.
      ,
      • Mejias M.
      • Gallego J.
      • Naranjo-Suarez S.
      • Ramirez M.
      • Pell N.
      • Manzano A.
      • et al.
      CPEB4 increases expression of PFKFB3 to induce glycolysis and activate mouse and human hepatic stellate cells, promoting liver fibrosis.
      Interestingly, inhibition of hexokinase 2 (with the inhibitor 2-deoxyglucose), or knockdown/inhibition of PKM2 or PFKFB3, decreases glycolysis and reduces fibrosis following a decrease in the myofibroblastic features of HSCs.
      • Mejias M.
      • Gallego J.
      • Naranjo-Suarez S.
      • Ramirez M.
      • Pell N.
      • Manzano A.
      • et al.
      CPEB4 increases expression of PFKFB3 to induce glycolysis and activate mouse and human hepatic stellate cells, promoting liver fibrosis.
      ,
      • Zheng D.
      • Jiang Y.
      • Qu C.
      • Yuan H.
      • Hu K.
      • He L.
      • et al.
      Pyruvate kinase M2 tetramerization protects against hepatic stellate cell activation and liver fibrosis.

      Glutaminolysis supports HSC activation

      Cell intrinsic metabolic reprograming of parenchymal and non-parenchymal cells has recently emerged as a potential therapeutic approach for liver fibrosis.
      Besides glycolysis, glutamine metabolism via glutaminolysis has been identified as an additional source of ATP in HSCs that drives their transdifferentiation into myofibroblasts.
      • Du K.
      • Hyun J.
      • Premont R.T.
      • Choi S.S.
      • Michelotti G.A.
      • Swiderska-Syn M.
      • et al.
      Hedgehog-YAP signaling pathway regulates glutaminolysis to control activation of hepatic stellate cells.
      Glutaminolysis comprises a 2-step reaction, initiated by the conversion of glutamine into glutamate by glutaminase. Glutamate is further metabolised into alpha-ketoglutarate that fuels the TCA cycle and provides the ATP required for cell anabolism (Fig. 1). Myofibroblastic HSCs show an increase in expression of glutaminase in cell culture,
      • Bode J.G.
      • Peters-Regehr T.
      • Gressner A.M.
      • Haussinger D.
      De novo expression of glutamine synthetase during transformation of hepatic stellate cells into myofibroblast-like cells.
      in experimental models of chronic liver injury,
      • Du K.
      • Hyun J.
      • Premont R.T.
      • Choi S.S.
      • Michelotti G.A.
      • Swiderska-Syn M.
      • et al.
      Hedgehog-YAP signaling pathway regulates glutaminolysis to control activation of hepatic stellate cells.
      ,
      • Li J.
      • Ghazwani M.
      • Liu K.
      • Huang Y.
      • Chang N.
      • Fan J.
      • et al.
      Regulation of hepatic stellate cell proliferation and activation by glutamine metabolism.
      and in liver samples of patients with non-alcoholic steatohepatitis (NASH) and advanced fibrosis.
      • Du K.
      • Chitneni S.K.
      • Suzuki A.
      • Wang Y.
      • Henao R.
      • Hyun J.
      • et al.
      Increased glutaminolysis marks active scarring in nonalcoholic steatohepatitis progression.
      Interestingly, pharmacological inhibition of glutaminolysis via a glutaminase inhibitor (BTPES), or glutamine deprivation, is more efficient than glucose deprivation at blocking HSC proliferation and the acquisition of myofibroblastic features, while pharmacological blockade of glutaminase reduces fibrosis progression
      • Du K.
      • Hyun J.
      • Premont R.T.
      • Choi S.S.
      • Michelotti G.A.
      • Swiderska-Syn M.
      • et al.
      Hedgehog-YAP signaling pathway regulates glutaminolysis to control activation of hepatic stellate cells.
      ,
      • Li J.
      • Ghazwani M.
      • Liu K.
      • Huang Y.
      • Chang N.
      • Fan J.
      • et al.
      Regulation of hepatic stellate cell proliferation and activation by glutamine metabolism.
      (Fig. 2). These data suggest that the combined inhibition of glycolysis and glutaminolysis may have synergic antifibrogenic effects. Of note, myofibroblastic HSCs also express the metabotropic glutamate receptor 5 and a recent study has shown that following alcohol exposure, hepatocytes release glutamate with paracrine effects on neighbouring HSCs, leading to the release of steatogenic/fibrogenic mediators.
      • Choi W.M.
      • Kim H.H.
      • Kim M.H.
      • Cinar R.
      • Yi H.S.
      • Eun H.S.
      • et al.
      Glutamate signaling in hepatic stellate cells drives alcoholic steatosis.
      ,
      • Mallat A.
      • Lotersztajn S.
      Glutamate signaling in alcohol-associated fatty liver: "Pas de Deux".

      Lipid metabolism directs activation of HSCs

      Though well characterised in hepatocytes, the impact of interfering with lipid metabolism in HSCs has been less well studied. However, recent studies have highlighted the fact that reducing lipid accumulation in HSCs can restrain their activation, directly modulating fibrogenesis.

      Targeting lipogenesis

      Two recent studies have shown that inhibiting de novo lipogenesis inhibits HSC activation and limits fibrosis.
      • Bates J.
      • Vijayakumar A.
      • Ghoshal S.
      • Marchand B.
      • Yi S.
      • Kornyeyev D.
      • et al.
      Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.
      ,
      • Kim C.W.
      • Addy C.
      • Kusunoki J.
      • Anderson N.N.
      • Deja S.
      • Fu X.
      • et al.
      Acetyl CoA carboxylase inhibition reduces hepatic steatosis but elevates plasma triglycerides in mice and humans: a bedside to bench investigation.
      Lipogenesis is initiated by the conversion of acetyl-CoA into malonyl-CoA, catalysed by the rate limiting enzyme acetyl-CoA carboxylase (ACC) (Fig. 1). Pharmacological inhibition of ACC by firsocostat directly suppresses the fibrogenic properties of cultured activated HSCs, and is associated with the blockade of glycolysis and oxidative phosphorylation
      • Bates J.
      • Vijayakumar A.
      • Ghoshal S.
      • Marchand B.
      • Yi S.
      • Kornyeyev D.
      • et al.
      Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.
      (Fig. 2). Moreover, in 2 mouse models of liver fibrosis (diet-induced NAFLD and diethylnitrosamine), administration of firsocostat is associated with a decrease in liver fibrosis.
      • Bates J.
      • Vijayakumar A.
      • Ghoshal S.
      • Marchand B.
      • Yi S.
      • Kornyeyev D.
      • et al.
      Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.
      In addition, inhibition of ACC has also been identified as an interesting therapeutic target in NAFLD; indeed, genetic or pharmacological blockade of ACC reduces steatosis and hepatocyte injury in experimental models. The beneficial impact of this approach is also supported by an exploratory study with the ACC inhibitor MK-4074
      • Kim C.W.
      • Addy C.
      • Kusunoki J.
      • Anderson N.N.
      • Deja S.
      • Fu X.
      • et al.
      Acetyl CoA carboxylase inhibition reduces hepatic steatosis but elevates plasma triglycerides in mice and humans: a bedside to bench investigation.
      and a phase II randomised placebo-controlled trial of firsocostat in patients with NASH.
      • Loomba R.
      • Kayali Z.
      • Noureddin M.
      • Ruane P.
      • Lawitz E.J.
      • Bennett M.
      • et al.
      GS-0976 reduces hepatic steatosis and fibrosis markers in patients with nonalcoholic fatty liver disease.
      Both compounds were reported to decrease steatosis, while firsocostat also reduced select markers of fibrosis (Table 1).
      Table 1Selected drugs targeting cell metabolism currently evaluated in NASH clinical trials with fibrosis as an endpoint.
      TargetDrugPhaseResults [Ref.]
      Glucose metabolism
      SLGT1/2 inhibitorEmpaglifozinPhase II/IIIDecrease hepatic fat.
      LicoglifozinPhase IISignificant improvement of liver stiffness
      • Francke S.
      • Bedossa P.
      • Abdelmalek M.
      • Quentin Anstee M.
      • Bugianesi E.
      • Vlad R.
      • et al.
      A randomised, double-blind, placebo-controlled, multi-centre, dose-range, proof-of-concept, 24-week treatment study of lanifibranor in adult subjects with non-alcoholic steatohepatitis: design of the NATIVE study.
      Phase II[No significant decrease in fibrosis (Harrison S AASLD: Boston, MA, USA, 2019)]
      DapaglifozinPhase IIIExpected
      Hexokinase inhibitorPF-06835919Phase IIaRecruiting
      Lipid metabolism
      ACC1&2 inhibitorFirsocostatPhase II30% reduction of liver fat.
      Decrease in the fibrosis marker TIMP-1. No decrease in ELF test and magnetic resonance elastography
      • Loomba R.
      • Kayali Z.
      • Noureddin M.
      • Ruane P.
      • Lawitz E.J.
      • Bennett M.
      • et al.
      GS-0976 reduces hepatic steatosis and fibrosis markers in patients with nonalcoholic fatty liver disease.
      SCD1 inhibitorAramcholPhase IIbReduction in fibrosis at 600 mg without worsening of NASH [Ratziu V, AASLD, San Francisco, USA 2018]
      NCT04104321
      ArmorPhase III/IVExpected
      PPARLanifibranor (α/β/γ)Phase IIResolution of NASH with no worsening of fibrosis
      • Lefere S.
      • Puengel T.
      • Hundertmark J.
      • Penners C.
      • Frank A.K.
      • Guillot A.
      • et al.
      Differential effects of selective- and pan-PPAR agonists on experimental steatohepatitis and hepatic macrophages.
      Saroglitazar (α&γ)Phase IIImprovement of liver biochemistry and steatosis. Decrease in APRI and ELF. No clear decrease in liver stiffness [Gawrieh S AASLD: Boston MA, USA, 2019]
      Elafibranor (α&δ)Phase IIINot superior to placebo for fibrosis
      • Ratziu V.
      • Harrison S.A.
      • Francque S.
      • Bedossa P.
      • Lehert P.
      • Serfaty L.
      • et al.
      Elafibranor, an agonist of the peroxisome proliferator-activated receptor-alpha and -delta, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening.
      Seladelpar (δ)Phase IISuspended for unexpected effects
      FXR agonistTropifexorPhase IIDecrease in steatosis and in ALT. Results on liver biopsies expected
      Obeticholic AcidPhase IIIImprove fibrosis w/o NASH worsening
      • Younossi Z.M.
      • Ratziu V.
      • Loomba R.
      • Rinella M.
      • Anstee Q.M.
      • Goodman Z.
      • et al.
      Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
      Mitochondrial pyruvate carrier
      MPC inhibitorMSDC-0602KPhase IIb

      Phase III
      Reduction in glucose liver enzymes. No improvement of fibrosis
      • Harrison S.A.
      • Alkhouri N.
      • Davison B.A.
      • Sanyal A.
      • Edwards C.
      • Colca J.R.
      • et al.
      Insulin sensitizer MSDC-0602K in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase IIb study.
      Autophagy
      ASK1 inhibitorSelonsertibPhase IIINot superior to placebo
      • Dickson I.
      No anti-fibrotic effect of selonsertib in NASH.
      AMPK & PG1CaResveratrolPhase II/IIIPrevents liver damage
      • Dufour J.F.
      • Caussy C.
      • Loomba R.
      Combination therapy for non-alcoholic steatohepatitis: rationale, opportunities and challenges.
      ALT, alanine aminotransferase; APRI, aspartate aminotransferase-to-platelet ratio index; ELF, enhanced liver fibrosis; NASH, non-alcoholic steatohepatitis.
      Another key lipogenic enzyme is stearoyl-CoA desaturase 1 (SCD1), which converts lipotoxic saturated fatty acids into mono-unsaturated fatty acids (Fig. 1). Inhibitors of SCD1 have been shown to reduce steatogenesis in experimental models. In addition, as described for ACC inhibitors, global or selective inhibition of SCD-1 in HSCs reduces activation of cultured HSCs and inhibits fibrosis in experimental models
      • Lai K.K.Y.
      • Kweon S.M.
      • Chi F.
      • Hwang E.
      • Kabe Y.
      • Higashiyama R.
      • et al.
      Stearoyl-CoA desaturase promotes liver fibrosis and tumor development in mice via a Wnt positive-signaling loop by stabilization of low-density lipoprotein-receptor-related proteins 5 and 6.
      (Fig. 2). Similar findings have been obtained with pharmacological SCD1 inhibitors, i.e. A939572 or aramchol in a phase IIb clinical trial (Table 1).
      • Lai K.K.Y.
      • Kweon S.M.
      • Chi F.
      • Hwang E.
      • Kabe Y.
      • Higashiyama R.
      • et al.
      Stearoyl-CoA desaturase promotes liver fibrosis and tumor development in mice via a Wnt positive-signaling loop by stabilization of low-density lipoprotein-receptor-related proteins 5 and 6.
      ,
      • Iruarrizaga-Lejarreta M.
      • Varela-Rey M.
      • Fernandez-Ramos D.
      • Martinez-Arranz I.
      • Delgado T.C.
      • Simon J.
      • et al.
      Role of Aramchol in steatohepatitis and fibrosis in mice.
      Glycolysis, glutaminolysis and lipid metabolism control hepatic stellate cell phenotype.

      Targeting cholesterol metabolism

      Free cholesterol activates HSCs, while it has been shown that feeding mice a high-fat high-cholesterol diet leads to accumulation of free cholesterol in HSCs and exacerbates liver fibrosis. Indeed, cholesterol homeostasis is dysregulated in liver diseases, particular during NAFLD (for a review see
      • Arab J.P.
      • Arrese M.
      • Trauner M.
      Recent insights into the pathogenesis of nonalcoholic fatty liver disease.
      ), leading to diet-induced accumulation of free cholesterol in HSCs, which increases Toll-like receptor 4 signalling and results in the activation of the transforming growth factor-β (TGF-β) pathway.
      • Teratani T.
      • Tomita K.
      • Suzuki T.
      • Oshikawa T.
      • Yokoyama H.
      • Shimamura K.
      • et al.
      A high-cholesterol diet exacerbates liver fibrosis in mice via accumulation of free cholesterol in hepatic stellate cells.
      ,
      • Tomita K.
      • Teratani T.
      • Suzuki T.
      • Shimizu M.
      • Sato H.
      • Narimatsu K.
      • et al.
      Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells.
      In addition, free cholesterol is converted to cholesterol esters by acetyl-CoA acetyltransferase 1 (ACAT1) and accordingly, genetic inactivation of ACAT1 results in increased concentrations of free cholesterol in HSCs and worse fibrosis.
      • Teratani T.
      • Tomita K.
      • Suzuki T.
      • Oshikawa T.
      • Yokoyama H.
      • Shimamura K.
      • et al.
      A high-cholesterol diet exacerbates liver fibrosis in mice via accumulation of free cholesterol in hepatic stellate cells.
      ,
      • Tomita K.
      • Teratani T.
      • Suzuki T.
      • Shimizu M.
      • Sato H.
      • Narimatsu K.
      • et al.
      Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells.
      A recent study also suggests that HSCs bearing the I148M PNPLA3 variant predisposing to enhanced fibrogenesis show increased levels of free cholesterol following downregulation of ACAT1, and display enhanced fibrogenic properties.
      • Bruschi F.V.
      • Claudel T.
      • Tardelli M.
      • Starlinger P.
      • Marra F.
      • Trauner M.
      PNPLA3 I148M variant impairs liver X receptor signaling and cholesterol homeostasis in human hepatic stellate cells.
      The beneficial impact of therapeutic strategies aimed at decreasing free cholesterol accumulation in HSCs has also been demonstrated. Indeed, specific inhibition of lipoprotein lipase in HSCs, or the use of SREBP2 siRNA- or anti-miR-33a-bearing vitamin A-coupled liposomes reduces free cholesterol accumulation in activated HSCs and decreases liver fibrosis in mouse models.
      • Berod L.
      • Friedrich C.
      • Nandan A.
      • Freitag J.
      • Hagemann S.
      • Harmrolfs K.
      • et al.
      De novo fatty acid synthesis controls the fate between regulatory T and T helper 17 cells.

      Targeting succinate

      Succinate is an intermediate metabolite produced during the TCA cycle that serves as a final common catabolic pathway of carbohydrates, amino-acids and fatty acids. Succinate is formed from succinyl-CoA by succinyl-CoA synthetase; it is subsequently oxidised to fumarate by succinate dehydrogenase (SDH), which is part of the respiratory electron transport chain (Fig. 1). Normally present in mitochondria, succinate is secreted into the extracellular space and binds to the G-protein coupled receptor, GPR91 (also known as succinate receptor 1). Binding of succinate to GPR91 expressed in HSCs enhances their pro-fibrogenic properties (Fig. 2). Moreover, paracrine succinate shuttle between hepatocytes and HSCs has also been described, involving enhanced succinate release by hepatocytes in stress conditions. Interestingly, inhibition of the succinate/GPR91 pathway has also been shown to have beneficial antifibrogenic effects in experimental models of NASH.
      • Li Y.H.
      • Woo S.H.
      • Choi D.H.
      • Cho E.H.
      Succinate causes alpha-SMA production through GPR91 activation in hepatic stellate cells.
      ,
      • Li Y.H.
      • Choi D.H.
      • Lee E.H.
      • Seo S.R.
      • Lee S.
      • Cho E.H.
      Sirtuin 3 (SIRT3) regulates alpha-smooth muscle actin (alpha-SMA) production through the succinate dehydrogenase-G protein-coupled receptor 91 (GPR91) pathway in hepatic stellate cells.
      However, further studies should evaluate its impact on inflammation, since the succinate/GRP91 pathway displays anti-inflammatory effects on macrophages.
      • Keiran N.
      • Ceperuelo-Mallafre V.
      • Calvo E.
      • Hernandez-Alvarez M.I.
      • Ejarque M.
      • Nunez-Roa C.
      • et al.
      SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.

      Immunometabolic regulation of liver fibrosis

      Several studies have emphasised the crucial role of various innate, adaptive or innate-like immune cell subsets in the control of hepatic inflammation, fibrosis progression and regression.
      • Lotersztajn S.
      • Julien B.
      • Teixeira-Clerc F.
      • Grenard P.
      • Mallat A.
      Hepatic fibrosis: molecular mechanisms and drug targets.
      ,
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Schwabe R.F.
      • Tabas I.
      • Pajvani U.B.
      Mechanisms of fibrosis development in nonalcoholic steatohepatitis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      Recent studies also demonstrate the potential beneficial impact of therapeutic strategies targeting their intrinsic metabolism.

      Macrophage cell metabolism and fibrogenesis

      Among immune cells, a heterogeneous population of macrophages is a major contributor to liver fibrogenesis (Fig. 2).
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      ,
      • Lefere S.
      • Tacke F.
      Macrophages in obesity and non-alcoholic fatty liver disease: crosstalk with metabolism.
      Resident activated Kupffer cells initiate and promote several key steps in the inflammatory response to liver injury. They recruit Ly6Chi macrophages with pro-inflammatory and pro-fibrogenic properties, as well as neutrophils and IL-17-positive cells, which ultimately promote hepatocyte death and induce accumulation and survival of activated HSCs.
      • Lotersztajn S.
      • Julien B.
      • Teixeira-Clerc F.
      • Grenard P.
      • Mallat A.
      Hepatic fibrosis: molecular mechanisms and drug targets.
      ,
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Schwabe R.F.
      • Tabas I.
      • Pajvani U.B.
      Mechanisms of fibrosis development in nonalcoholic steatohepatitis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      Meanwhile, following cessation of liver injury, the recovery phase is characterised by the presence of restorative macrophages with a distinct anti-inflammatory, fibrolytic Ly6Clo phenotype that drive fibrosis resolution (Fig. 2).
      • Ramachandran P.
      • Pellicoro A.
      • Vernon M.A.
      • Boulter L.
      • Aucott R.L.
      • Ali A.
      • et al.
      Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.
      Other innate immune cells also control fibrogenesis, including dendritic and natural killer (NK) cells.
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      However, data regarding the impact of reprogramming the intrinsic metabolism of innate immune cells in liver fibrosis are mainly restricted to macrophages.
      It is well established that the macrophage switch to an inflammatory phenotype is tightly controlled by macrophage modifications in glucose and lipid metabolism.
      • O'Neill L.A.
      • Kishton R.J.
      • Rathmell J.
      A guide to immunometabolism for immunologists.
      ,
      • Caputa G.
      • Castoldi A.
      • Pearce E.J.
      Metabolic adaptations of tissue-resident immune cells.
      In particular, characteristic features of inflammatory macrophages include enhanced glycolysis, activation of the TCA cycle (used to produce citrate for fatty acid synthesis), the pentose phosphate pathway and nitric oxide production. In contrast, anti-inflammatory macrophages use fatty acid oxidation, the TCA cycle (used for oxidative phosphorylation) and the arginase pathway
      • O'Neill L.A.
      • Kishton R.J.
      • Rathmell J.
      A guide to immunometabolism for immunologists.
      ,
      • Caputa G.
      • Castoldi A.
      • Pearce E.J.
      Metabolic adaptations of tissue-resident immune cells.
      (Fig. 1). These data suggest that manipulation of metabolism in monocytes/macrophages may provide a novel anti-inflammatory and antifibrogenic approach in the liver, and mirrors recent findings in atherosclerosis and diabetes.
      • Yvan-Charvet L.
      • Ivanov S.
      Metabolic reprogramming of macrophages in atherosclerosis: is it all about cholesterol?.
      ,
      • Mann J.
      • Mann D.A.
      Transcriptional regulation of hepatic stellate cells.

      Accumulation of lipids in Kupffer cells is proinflammatory

      Alterations in lipid metabolism drive the polarisation of Kupffer cells into a pro-inflammatory phenotype. Thus, binding of saturated fatty acid or trapping of oxidised lipoproteins by scavenger receptors such as MSR1 (macrophage scavenger receptor 1), leads to the formation of foamy Kupffer cells with a pro-inflammatory phenotype.
      • Bieghs V.
      • Walenbergh S.M.
      • Hendrikx T.
      • van Gorp P.J.
      • Verheyen F.
      • Olde Damink S.W.
      • et al.
      Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation.
      ,
      • Bieghs V.
      • Wouters K.
      • van Gorp P.J.
      • Gijbels M.J.
      • de Winther M.P.
      • Binder C.J.
      • et al.
      Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice.
      In line with these data, mice lacking Msr1 show less hepatic inflammation and greater resistance to fibrosis in response to a high-fat diet.
      • Bieghs V.
      • Wouters K.
      • van Gorp P.J.
      • Gijbels M.J.
      • de Winther M.P.
      • Binder C.J.
      • et al.
      Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice.
      Finally, exposure of Kupffer cells to fatty acids leads to accumulation of the cytotoxic lipids diacylglycerol and ceramide, and shifts Kupffer cells into a pro-inflammatory state
      • Leroux A.
      • Ferrere G.
      • Godie V.
      • Cailleux F.
      • Renoud M.L.
      • Gaudin F.
      • et al.
      Toxic lipids stored by Kupffer cells correlates with their pro-inflammatory phenotype at an early stage of steatohepatitis.
      (Fig. 2).

      Targeting lipid metabolism in macrophages: A role for monoacylglycerols

      Hydrolysis of monoacylglycerols by monoacylglycerol lipase (MAGL) is the final step in cellular triglyceride breakdown. MAGL hydrolyses 2-arachydonoyl-glycerol to glycerol and fatty acids, which constitutes the major source of arachidonic acid and pro-inflammatory prostaglandins in the liver. We have demonstrated that specific inhibition of MAGL in myeloid cells results in anti-inflammatory and antifibrogenic effects in the liver, via a shift of lipid metabolism in macrophages from an arachidonic acid/prostaglandin proinflammatory phenotype towards an anti-inflammatory 2-arachydonoyl-glycerol-producing phenotype (Fig. 2).
      • Habib A.
      • Chokr D.
      • Wan J.
      • Hegde P.
      • Mabire M.
      • Siebert M.
      • et al.
      Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.
      MAGL inhibition also protects mice against cholestatic injury, inflammation and biliary fibrosis.
      • Tardelli M.
      • Bruschi F.V.
      • Claudel T.
      • Fuchs C.D.
      • Auer N.
      • Kunczer V.
      • et al.
      Lack of monoacylglycerol lipase prevents hepatic steatosis by favoring lipid storage in adipose tissue and intestinal malabsorption.
      ,
      • Tardelli M.
      • Bruschi F.V.
      • Fuchs C.D.
      • Claudel T.
      • Auer N.
      • Kunczer V.
      • et al.
      Monoacylglycerol lipase inhibition protects from liver injury in mouse models of sclerosing cholangitis.
      Finally, pharmacological inhibition of MAGL with MJN110 promotes liver fibrosis regression by decreasing the frequency of Ly6Chi and increasing the frequency of Ly6Clo macrophages. Because inhibiting MAGL also decreases hepatocyte injury,
      • Habib A.
      • Chokr D.
      • Wan J.
      • Hegde P.
      • Mabire M.
      • Siebert M.
      • et al.
      Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.
      these data suggest that reprogramming lipid metabolism using MAGL inhibitors holds promise as an antifibrogenic approach through its dual effects on macrophages and hepatocytes.
      In macrophages, targeting lipid or acetoacetate metabolism by inhibiting monoacylglycerol lipase has antifibrogenic consequences.

      Targeting acetoacetate metabolism: a two-tale hepatocyte-macrophage story

      Mitochondrial metabolism of acetyl-CoA by HMG-CoA synthase in hepatocytes produces ketone bodies in 2 forms, acetoacetate and β-hydroxybutyrate. However, hepatocytes are unable to metabolize ketones due to the lack of expression of succinyl-CoA-oxoacid transferase (SCOT), an enzyme that prepares acetoacetate for terminal oxidation in the TCA cycle. In contrast, SCOT is highly expressed in macrophages. A recent study unveiled a protective metabolic response of macrophages to liver fibrogenesis, via a hepatocyte-macrophage acetoacetate shuttle. In brief, acetoacetate released by hepatocytes is further oxidised by mitochondrial SCOT in alternatively polarised macrophages, thereby reducing hepatic fibrogenesis (Fig. 2).
      • Puchalska P.
      • Martin S.E.
      • Huang X.
      • Lengfeld J.E.
      • Daniel B.
      • Graham M.J.
      • et al.
      Hepatocyte-macrophage acetoacetate shuttle protects against tissue fibrosis.
      Indeed, locally produced or systemically administered acetoacetate attenuates the hepatic fibrogenic response induced by a high-fat diet. In contrast, SCOT-KO macrophages exhibit perturbations of glycosaminoglycan metabolism, known to be associated with expansion of extracellular matrix. Accordingly, mice lacking SCOT in macrophages are predisposed to accelerated fibrogenesis.
      • Puchalska P.
      • Martin S.E.
      • Huang X.
      • Lengfeld J.E.
      • Daniel B.
      • Graham M.J.
      • et al.
      Hepatocyte-macrophage acetoacetate shuttle protects against tissue fibrosis.

      Metabolism of adaptive immune cells and innate/innate-like lymphoid cells and liver fibrosis

      Adaptive (CD4+, T helper [Th]1, 2 and 17) and innate/innate-like (ILC2, mucosal-associated invariant T [MAIT] and γδ T) lymphoid cells are major regulators of the hepatic immune response and consequently the fibrogenic process, with positive or negative outcomes depending on their phenotype (Fig. 2).
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      In particular, IL-17 produced by Th17 and MAIT cells directly activates the fibrogenic functions of HSCs, while the antifibrogenic properties of γδ T cells and Th1 lymphocytes are related to their apoptotic effects on HSCs and IFNγ production, respectively (for extensive reviews see
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Koyama Y.
      • Brenner D.A.
      Liver inflammation and fibrosis.
      ,
      • Seki E.
      • Schwabe R.F.
      Hepatic inflammation and fibrosis: functional links and key pathways.
      ). It is well established that metabolic changes in adaptive immune cells and innate-like T cells support their functions and drive their differentiation. In particular, shifting to aerobic glycolysis, lipogenesis and arginine and glutamine metabolism is fundamental for differentiation of naïve CD4+ T cells into Th1 and Th17 cells, of γδ T into γδ T1 and γδ T17 cells, and to drive proliferation and cytokine production in ILCs.
      • Bantug G.R.
      • Galluzzi L.
      • Kroemer G.
      • Hess C.
      The spectrum of T cell metabolism in health and disease.
      Gut dysbiosis is also a characteristic feature of chronic liver injury and is a major driver of liver fibrosis. Interestingly, accumulating data demonstrate that gut-derived bacterial metabolites modify immune cell metabolism
      • Michaudel C.
      • Sokol H.
      The gut microbiota at the service of immunometabolism.
      and support their activation. This could be a factor in the activation of MAIT cells, as these innate-like lymphoid cells are activated by bacterial ligands and display fibrogenic properties.
      • Hegde P.
      • Weiss E.
      • Paradis V.
      • Wan J.
      • Mabire M.
      • Sukriti S.
      • et al.
      Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.
      ,
      • Toubal A.
      • Nel I.
      • Lotersztajn S.
      • Lehuen A.
      Mucosal-associated invariant T cells and disease.
      Data are still lacking, but whether intrinsic metabolic changes in adaptive and innate immune cells govern their differentiation toward a pro-inflammatory and pro-fibrogenic phenotype, and the resulting impact on fibrosis progression or regression, merits further investigation.

      Hepatocyte metabolism and liver fibrosis

      Omics approaches related to changes in hepatocyte metabolism have been the focus of many studies in the context of NASH, but the link between the accumulation of cytotoxic lipids in hepatocytes and the development of liver fibrosis remains elusive. Hepatocyte injury is a major driver of fibrosis, via direct effects on HSC functions and indirect effects on immune cell activation.
      • Mallat A.
      • Lotersztajn S.
      Cellular mechanisms of tissue fibrosis. 5. Novel insights into liver fibrosis.
      ,
      • Marra F.
      • Lotersztajn S.
      Pathophysiology of NASH: perspectives for a targeted treatment.
      ,
      • Schwabe R.F.
      • Tabas I.
      • Pajvani U.B.
      Mechanisms of fibrosis development in nonalcoholic steatohepatitis.
      Changes in intrinsic hepatocyte metabolism may lead to inhibition of fibrogenesis, by reducing steatosis and liver injury or through indirect modifications of the macrophage-HSC dialogue, as described above for the acetoacetate shuttle. However, in the next paragraph we will focus on preclinical studies demonstrating that metabolic reprogramming of hepatocytes can directly impact on the profibrogenic functions of HSCs. Among metabolic pathways preserving hepatocyte integrity, key proteins involved in glucose or lipid metabolism are potential antifibrogenic candidates (Fig. 2).
      Changes in glucose metabolism through hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) result in hepatocyte secretion of the non-histone nuclear protein high-mobility group protein B1 (HMGB1) which causes HSC activation
      • Li F.
      • Huangyang P.
      • Burrows M.
      • Guo K.
      • Riscal R.
      • Godfrey J.
      • et al.
      FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome.
      (Fig. 2). Future studies should address whether crosstalk between hepatocyte glucose metabolism and HSC activation could contribute to liver fibrosis in chronic liver diseases and become a target for novel therapeutic approaches.
      The accumulation of cytotoxic lipids (i.e. free fatty acids, ceramides, diacylglycerol, cholesterol and phospholipids) in hepatocytes is a hallmark of NASH;
      • Diehl A.M.
      • Day C.
      Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis.
      • Ibrahim S.H.
      • Hirsova P.
      • Gores G.J.
      Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation.
      • Chaurasia B.
      • Tippetts T.S.
      • Mayoral Monibas R.
      • Liu J.
      • Li Y.
      • Wang L.
      • et al.
      Targeting a ceramide double bond improves insulin resistance and hepatic steatosis.
      • Xia J.Y.
      • Holland W.L.
      • Kusminski C.M.
      • Sun K.
      • Sharma A.X.
      • Pearson M.J.
      • et al.
      Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.
      thus, it has been suggested that therapeutic interventions, either pharmacological or dietary (e.g. caloric restriction), may result in a reduction of liver fibrosis following a decrease in hepatocyte lipid accumulation or prevention of lipid-induced apoptosis. Another link between alterations in hepatocyte lipid metabolism and liver fibrosis was recently provided by a study showing that disturbance of phosphatidylinositol (PI) chain remodelling, following inhibition of membrane-bound O-acyltransferase domain containing 7 (MBOAT7) in hepatocytes, increases fibrosis in response to a NASH-inducing diet in mice
      • Thangapandi V.R.
      • Knittelfelder O.
      • Brosch M.
      • Patsenker E.
      • Vvedenskaya O.
      • Buch S.
      • et al.
      Loss of hepatic Mboat7 leads to liver fibrosis.
      (Fig. 2). Interestingly, the rs641738C>T MBOAT7 variant which is associated with reduced hepatic levels of MBOAT7, has been identified as a risk locus for alcohol-related cirrhosis,
      • Buch S.
      • Stickel F.
      • Trepo E.
      • Way M.
      • Herrmann A.
      • Nischalke H.D.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      NAFLD and viral hepatitis-related fibrosis.
      • Mancina R.M.
      • Dongiovanni P.
      • Petta S.
      • Pingitore P.
      • Meroni M.
      • Rametta R.
      • et al.
      The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent.
      • Thabet K.
      • Asimakopoulos A.
      • Shojaei M.
      • Romero-Gomez M.
      • Mangia A.
      • Irving W.L.
      • et al.
      MBOAT7 rs641738 increases risk of liver inflammation and transition to fibrosis in chronic hepatitis C.
      • Thabet K.
      • Chan H.L.Y.
      • Petta S.
      • Mangia A.
      • Berg T.
      • Boonstra A.
      • et al.
      The membrane-bound O-acyltransferase domain-containing 7 variant rs641738 increases inflammation and fibrosis in chronic hepatitis B.
      In hepatocytes, accumulation of cytotoxic lipids or alterations of lipid or glucose metabolism result in hepatic stellate cell activation and the development of fibrosis.
      Finally, transport of pyruvate into the mitochondrial matrix is critical for lipid and glucose metabolism. Modulating pyruvate metabolism in hepatocytes by inhibiting mitochondrial pyruvate carrier (MPC) prevents fibrosis, not only because of its anti-steatogenic properties but also by inhibiting HSC activation (Fig. 2),
      • McCommis K.S.
      • Hodges W.T.
      • Brunt E.M.
      • Nalbantoglu I.
      • McDonald W.G.
      • Holley C.
      • et al.
      Targeting the mitochondrial pyruvate carrier attenuates fibrosis in a mouse model of nonalcoholic steatohepatitis.
      although no improvement in fibrosis was observed in a clinical trial.
      • Harrison S.A.
      • Alkhouri N.
      • Davison B.A.
      • Sanyal A.
      • Edwards C.
      • Colca J.R.
      • et al.
      Insulin sensitizer MSDC-0602K in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase IIb study.

      Endothelial cells

      Loss of liver sinusoidal endothelial cell (LSEC) fenestration (i.e. capillarisation of sinusoids) is an early characteristic feature of the fibrogenic process. Activated endothelial cells contribute to HSC activation, by producing TGF-β and platelet-derived growth factor (PDGF)-BB, and secreting components of the extracellular matrix, including collagen 1. Restoration of LSEC differentiation also accounts for fibrosis regression by promoting HSC quiescence.
      • Chen Y.
      • Choi S.S.
      • Michelotti G.A.
      • Chan I.S.
      • Swiderska-Syn M.
      • Karaca G.F.
      • et al.
      Hedgehog controls hepatic stellate cell fate by regulating metabolism.
      However, data regarding the impact of intrinsic metabolic reprogramming of LSECs during liver fibrosis are scarce and limited to the description of the beneficial role of autophagy on fibrosis progression, of liver X receptors (LXRs) on the restoration of endothelial cell capillarisation and of farnesoid X receptor (FXR) on endothelial dysfunction (see below, Fig. 2 and Fig. 3).
      Figure thumbnail gr3
      Fig. 3Targeting metabolic pathways in different liver cell types: expected outcomes.
      The different outcomes resulting from the activation of cell intrinsic metabolic pathways on the cellular actors of liver fibrogenesis are depicted. ND, not determined

      Targeting common metabolic pathways in different liver cell types: Benefits and pitfalls

      As depicted in the first part of the review, metabolic reprogramming is emerging as a potential therapeutic in the field of chronic liver diseases. However, given the pleiotropic role of these metabolic targets in various liver cells, the following sections focus on some of the pathways for which studies in different liver cell types are available.

      Autophagy

      One of the main functions of autophagy is to regulate cellular metabolism and energy, through lipophagy, mitophagy, amino acid pool refuelling, or degradation of proteins involved in glucose metabolism.
      • Allaire M.
      • Rautou P.E.
      • Codogno P.
      • Lotersztajn S.
      Autophagy in liver diseases: time for translation?.
      ,
      • Gual P.
      • Gilgenkrantz H.
      • Lotersztajn S.
      Autophagy in chronic liver diseases: the two faces of Janus.
      The role of autophagy in liver fibrosis has been delineated using mice with cell-specific deletions in autophagic genes.
      • Mallat A.
      • Lodder J.
      • Teixeira-Clerc F.
      • Moreau R.
      • Codogno P.
      • Lotersztajn S.
      Autophagy: a multifaceted partner in liver fibrosis.
      Mice with Atg5 or Atg7 deletions in hepatocytes or endothelial cells show increased fibrosis either spontaneously (hepatocytes,
      • 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.
      ) or in response to a fibrogenic insult (endothelial cells
      • Hammoutene A.
      • Biquard L.
      • Lasselin J.
      • Kheloufi M.
      • Tanguy M.
      • Vion A.C.
      • et al.
      A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis.
      ) (Fig. 3). In monocytes/macrophages, a non-canonical form of autophagy (i.e. LC3-associated phagocytosis) protects against inflammation-driven liver fibrosis.
      • Lodder J.
      • Denaes T.
      • Chobert M.N.
      • Wan J.
      • El-Benna J.
      • Pawlotsky J.M.
      • et al.
      Macrophage autophagy protects against liver fibrosis in mice.
      ,
      • Wan J.
      • Weiss E.
      • Ben Mkaddem S.
      • Mabire M.
      • Choinier P.M.
      • Picq O.
      • et al.
      LC3-associated phagocytosis protects against inflammation and liver fibrosis via immunoreceptor inhibitory signaling.
      Regarding HSCs, conflicting results have been reported. Pharmacological inhibition of autophagy with 3-methyladenine or chloroquine or in vivo deletion of ATG7 in HSCs results in inhibition of their fibrogenic properties.
      • Hernandez-Gea V.
      • Ghiassi-Nejad Z.
      • Rozenfeld R.
      • Gordon R.
      • Fiel M.I.
      • Yue Z.
      • et al.
      Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.
      ,
      • Thoen L.F.
      • Guimaraes E.L.
      • Dolle L.
      • Mannaerts I.
      • Najimi M.
      • Sokal E.
      • et al.
      A role for autophagy during hepatic stellate cell activation.
      However, this view has recently been challenged, as autophagy in HSCs was shown to have an antifibrogenic effect related to its capacity to inhibit the release of fibrogenic extracellular vesicles.
      • Gao J.
      • Wei B.
      • de Assuncao T.M.
      • Liu Z.
      • Hu X.
      • Ibrahim S.
      • et al.
      Hepatic stellate cell autophagy inhibits extracellular vesicle release to attenuate liver fibrosis.
      Although further studies are required, it is anticipated that the resulting global impact of autophagy activators will result in a reduction in fibrosis. Along these lines, preclinical studies have demonstrated the antifibrogenic effects of autophagy inducers such as rapamycin or carbamazepine (Fig. 3).
      • Zhu J.
      • Wu J.
      • Frizell E.
      • Liu S.L.
      • Bashey R.
      • Rubin R.
      • et al.
      Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis.
      ,
      • Hidvegi T.
      • Ewing M.
      • Hale P.
      • Dippold C.
      • Beckett C.
      • Kemp C.
      • et al.
      An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.

      Ferroptosis

      Ferroptosis is a form of iron-dependent cell death. The execution of ferroptosis relies on modulation of intracellular metabolism, particularly lipid peroxidation, amino acid metabolism (glutamate and glutamine) and autophagy. Antifibrogenic properties of ferroptosis inhibitors have been demonstrated in the heart and lung,
      • Fang X.
      • Wang H.
      • Han D.
      • Xie E.
      • Yang X.
      • Wei J.
      • et al.
      Ferroptosis as a target for protection against cardiomyopathy.
      ,
      • Li X.
      • Duan L.
      • Yuan S.
      • Zhuang X.
      • Qiao T.
      • He J.
      Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-beta1.
      but may depend on the targeted cell type in the context of liver fibrosis. Thus, in HSCs, ferroptosis is induced following inhibition of the cysteine/glutamate antiporter xCT, leading to a reduction in fibrosis.
      • Du K.
      • Oh S.
      • Sun T.
      • Yang W.-H.
      • Chi J.
      • Diehl A.
      Inhibiting xCT/SLC7A11 induces ferroptosis of myofibroblastic hepatic stellate cells and protects against liver fibrosis.
      Accordingly, induction of HSC ferroptosis by the anti-malaria drug artemether attenuates hepatic injury and liver fibrosis.
      • Wang L.
      • Zhang Z.
      • Li M.
      • Wang F.
      • Jia Y.
      • Zhang F.
      • et al.
      P53-dependent induction of ferroptosis is required for artemether to alleviate carbon tetrachloride-induced liver fibrosis and hepatic stellate cell activation.
      In contrast, ferroptosis reprograms macrophages to a pro-inflammatory and profibrogenic phenotype
      • Handa P.
      • Thomas S.
      • Morgan-Stevenson V.
      • Maliken B.D.
      • Gochanour E.
      • Boukhar S.
      • et al.
      Iron alters macrophage polarization status and leads to steatohepatitis and fibrogenesis.
      (Fig. 3). Likewise, ferroptosis triggers inflammation in NASH, while iron chelators protect parenchymal cells from necrotic death and suppress the infiltration of inflammatory cells.
      • Tsurusaki S.
      • Tsuchiya Y.
      • Koumura T.
      • Nakasone M.
      • Sakamoto T.
      • Matsuoka M.
      • et al.
      Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis.
      From a therapeutic perspective, the cell-specific effects of inhibiting ferroptosis must be considered.

      Nuclear receptors

      LXR

      LXRs are central in lipogenesis and cholesterol metabolism.
      • Evans R.M.
      • Mangelsdorf D.J.
      Nuclear receptors, RXR, and the big bang.
      LXRα is highly expressed in hepatocytes, Kupffer cells
      • Endo-Umeda K.
      • Makishima M.
      Liver X receptors regulate cholesterol metabolism and immunity in hepatic nonparenchymal cells.
      and endothelial cells.
      • Xing Y.
      • Zhao T.
      • Gao X.
      • Wu Y.
      Liver X receptor alpha is essential for the capillarization of liver sinusoidal endothelial cells in liver injury.
      While LXRβ is predominant in HSCs, its expression decreases upon activation.
      • Beaven S.W.
      • Wroblewski K.
      • Wang J.
      • Hong C.
      • Bensinger S.
      • Tsukamoto H.
      • et al.
      Liver X receptor signaling is a determinant of stellate cell activation and susceptibility to fibrotic liver disease.
      LXR displays anti-inflammatory properties in Kupffer cells, antifibrogenic effects in HSCs, and suppresses endothelial cell capillarisation, as shown in different cell culture and mouse models of chronic liver diseases
      • Xing Y.
      • Zhao T.
      • Gao X.
      • Wu Y.
      Liver X receptor alpha is essential for the capillarization of liver sinusoidal endothelial cells in liver injury.
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      • Wang J.
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      • et al.
      Liver X receptor signaling is a determinant of stellate cell activation and susceptibility to fibrotic liver disease.
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      Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice.
      (Fig. 3). Interestingly, disrupting LXRα phosphorylation at the inhibitory Ser196 site reduces hepatic inflammation and fibrosis despite enhanced steatosis in mice fed a high-fat high-cholesterol diet.
      • Becares N.
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      • Voisin M.
      • Shrestha E.
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      • et al.
      Impaired LXRalpha phosphorylation attenuates progression of fatty liver disease.
      Moreover, the overexpression of LXRβ has recently been shown to suppress fibrosis and HSC activation, without inducing steatosis, in a mouse model of carbon tetrachloride-induced fibrosis.
      • Zhong L.
      • Ning B.
      • Du X.
      • Huang S.
      • Cai C.
      • Zhou Y.
      • et al.
      Liver X receptor b controls hepatic stellate cell activation via Hedgehog Signaling.
      However, to further consider the potential therapeutic value of LXR agonists, the specific contribution of LXRα vs. LXRβ needs to be addressed, considering the lipogenic properties of LXRα and the potential steatogenic side effects of non-selective LXRαβ-targeting molecules.
      Targeting common metabolic pathways such as autophagy, ferroptosis or nuclear receptors is an interesting therapeutic approach but may have different impacts on different parenchymal or non-parenchymal liver cell types.

      FXR

      FXR is the main regulator of bile acid synthesis expressed in hepatocytes, Kupffer cells, endothelial cells and to a far lesser extent in HSCs.
      • Zhang S.
      • Wang J.
      • Liu Q.
      • Harnish D.C.
      Farnesoid X receptor agonist WAY-362450 attenuates liver inflammation and fibrosis in murine model of non-alcoholic steatohepatitis.
      • Fiorucci S.
      • Antonelli E.
      • Rizzo G.
      • Renga B.
      • Mencarelli A.
      • Riccardi L.
      • et al.
      The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis.
      • Verbeke L.
      • Mannaerts I.
      • Schierwagen R.
      • Govaere O.
      • Klein S.
      • Vander Elst I.
      • et al.
      FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis.
      • Fickert P.
      • Fuchsbichler A.
      • Moustafa T.
      • Wagner M.
      • Zollner G.
      • Halilbasic E.
      • et al.
      Farnesoid X receptor critically determines the fibrotic response in mice but is expressed to a low extent in human hepatic stellate cells and periductal myofibroblasts.
      In hepatocytes, FXR plays an important role in lipid metabolism, inducing genes involved in lipoprotein metabolism and repressing hepatic genes involved in the synthesis of triglycerides, but increasing cholesterol.
      • Evans R.M.
      • Mangelsdorf D.J.
      Nuclear receptors, RXR, and the big bang.
      Experimental models have also shown that FXR displays anti-inflammatory and antifibrogenic properties related to its effects on hepatocytes, macrophages and endothelial dysfunction, but most likely unrelated to direct effects on HSCs
      • Zhang S.
      • Wang J.
      • Liu Q.
      • Harnish D.C.
      Farnesoid X receptor agonist WAY-362450 attenuates liver inflammation and fibrosis in murine model of non-alcoholic steatohepatitis.
      • Fiorucci S.
      • Antonelli E.
      • Rizzo G.
      • Renga B.
      • Mencarelli A.
      • Riccardi L.
      • et al.
      The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis.
      • Verbeke L.
      • Mannaerts I.
      • Schierwagen R.
      • Govaere O.
      • Klein S.
      • Vander Elst I.
      • et al.
      FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis.
      • Fickert P.
      • Fuchsbichler A.
      • Moustafa T.
      • Wagner M.
      • Zollner G.
      • Halilbasic E.
      • et al.
      Farnesoid X receptor critically determines the fibrotic response in mice but is expressed to a low extent in human hepatic stellate cells and periductal myofibroblasts.
      • McMahan R.H.
      • Wang X.X.
      • Cheng L.L.
      • Krisko T.
      • Smith M.
      • El Kasmi K.
      • et al.
      Bile acid receptor activation modulates hepatic monocyte activity and improves nonalcoholic fatty liver disease.
      • Schwabl P.
      • Hambruch E.
      • Seeland B.A.
      • Hayden H.
      • Wagner M.
      • Garnys L.
      • et al.
      The FXR agonist PX20606 ameliorates portal hypertension by targeting vascular remodelling and sinusoidal dysfunction.
      (Fig. 3). Accordingly, obeticholic acid, an FXR agonist, which has been approved as a second-line treatment for primary biliary cholangitis, has undergone clinical development in patients with NASH (see clinical perspectives).

      PPARs

      Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors (α, β/δ and γ) regulating lipid and glucose metabolism.
      • Francque S.
      • Szabo G.
      • Abdelmalek M.F.
      • Byrne C.D.
      • Cusi K.
      • Dufour J.F.
      • et al.
      Nonalcoholic steatohepatitis: the role of peroxisome proliferator-activated receptors.
      The PPARα isoform is predominantly found in hepatocytes and to a lesser extent in Kupffer cells and endothelial cells. The PPARβ/δ isoform is expressed in all liver cells, whereas PPARγ is expressed in macrophages and quiescent HSCs (Fig. 2). Activation of PPARα results in increased fatty acid oxidation in hepatocytes, shifts the macrophage phenotype toward an anti-inflammatory state and improves endothelial cell function.
      • Rodriguez-Vilarrupla A.
      • Lavina B.
      • Garcia-Caldero H.
      • Russo L.
      • Rosado E.
      • Roglans N.
      • et al.
      PPARalpha activation improves endothelial dysfunction and reduces fibrosis and portal pressure in cirrhotic rats.
      In animal models, activation of PPARα by Wy-14,643 normalises histological changes by preventing intrahepatic lipid accumulation, liver inflammation, and fibrosis
      • Ip E.
      • Farrell G.
      • Hall P.
      • Robertson G.
      • Leclercq I.
      Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice.
      (Fig. 3). The role of PPARβ/δ is more complex, since it increases de novo lipogenesis and protects against lipotoxicity, displays anti-inflammatory properties in Kupffer cells but increases activation of HSCs (Fig. 3). Nevertheless, the beneficial hepatoprotective and antifibrogenic effect of a PPARβ/δ agonist (GW501516) has not been confirmed in clinical trials with seladelpar (Table 1). The PPARγ isoform displays anti-inflammatory properties in macrophages by downregulating inflammatory gene expression, and it is essential for maintaining the quiescent phenotype of HSCs
      • Yavrom S.
      • Chen L.
      • Xiong S.
      • Wang J.
      • Rippe R.A.
      • Tsukamoto H.
      Peroxisome proliferator-activated receptor gamma suppresses proximal alpha1(I) collagen promoter via inhibition of p300-facilitated NF-I binding to DNA in hepatic stellate cells.
      (Fig. 3). Interestingly, in response to PPARγ overexpression or stimulation with PPARγ agonists, such as thioglitazone, activated HSCs can revert to a quiescent phenotype.
      • Liu X.
      • Xu J.
      • Rosenthal S.
      • Zhang L.J.
      • McCubbin R.
      • Meshgin N.
      • et al.
      Identification of lineage-specific transcription factors that prevent activation of hepatic stellate cells and promote fibrosis resolution.
      Although encouraging results were obtained in preclinical models with pioglitazone or rosiglitazone,
      • Galli A.
      • Crabb D.W.
      • Ceni E.
      • Salzano R.
      • Mello T.
      • Svegliati-Baroni G.
      • et al.
      Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro.
      no amelioration of liver fibrosis has been obtained in humans, following 1- or 2-year treatment with rosiglitazone.
      • Ratziu V.
      • Giral P.
      • Jacqueminet S.
      • Charlotte F.
      • Hartemann-Heurtier A.
      • Serfaty L.
      • et al.
      Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebo-controlled fatty liver improvement with rosiglitazone therapy (FLIRT) trial.
      ,
      • Ratziu V.
      • Charlotte F.
      • Bernhardt C.
      • Giral P.
      • Halbron M.
      • Lenaour G.
      • et al.
      Long-term efficacy of rosiglitazone in nonalcoholic steatohepatitis: results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension trial.
      Combination strategies using pan-PPAR agonists have been developed, based on encouraging results obtained in animal models. In particular, the pan-PPARα/δ/γ agonist lanifibranor improves all features of steatohepatitis, i.e. steatosis, inflammation and fibrosis in experimental models of NASH (choline-deficient, amino acid-defined high-fat diet) and of chronic toxic injury (chronic carbon tetrachloride administration).
      • Lefere S.
      • Puengel T.
      • Hundertmark J.
      • Penners C.
      • Frank A.K.
      • Guillot A.
      • et al.
      Differential effects of selective- and pan-PPAR agonists on experimental steatohepatitis and hepatic macrophages.
      ,
      • Francke S.
      • Bedossa P.
      • Abdelmalek M.
      • Quentin Anstee M.
      • Bugianesi E.
      • Vlad R.
      • et al.
      A randomised, double-blind, placebo-controlled, multi-centre, dose-range, proof-of-concept, 24-week treatment study of lanifibranor in adult subjects with non-alcoholic steatohepatitis: design of the NATIVE study.

      Targeting cell intrinsic metabolism for antifibrotic therapy – clinical perspectives

      The antifibrotic effects of strategies targeting lipid or glucose metabolism are increasingly being evaluated in patients with NASH (Table 1).
      • Taheri H.
      • Malek M.
      • Ismail-Beigi F.
      • Zamani F.
      • Sohrabi M.
      • Reza Babaei M.
      • et al.
      Effect of empagliflozin on liver steatosis and fibrosis in patients with non-alcoholic fatty liver disease without diabetes: a randomized, double-blind, placebo-controlled trial.
      • Younossi Z.M.
      • Ratziu V.
      • Loomba R.
      • Rinella M.
      • Anstee Q.M.
      • Goodman Z.
      • et al.
      Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
      • Dufour J.F.
      • Caussy C.
      • Loomba R.
      Combination therapy for non-alcoholic steatohepatitis: rationale, opportunities and challenges.
      Drugs targeting metabolism may display antifibrogenic properties by reducing hepatocyte injury. In addition, results from the aforementioned preclinical studies suggest that they may also reduce fibrosis via direct effects on HSCs and/or changes in inflammatory cell phenotype (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Cellular targets of molecules reprograming intrinsic metabolism currently under development for NASH.
      In addition to directly targeting HSC metabolism, molecules tested in clinical trials as antifibrogenic compounds may also indirectly impact on hepatocyte injury, or on inflammation via changes in macrophage or adaptive and innate-like immune cell phenotype. HSC, hepatic stellate cell; NASH, non-alcoholic steatohepatitis.
      Several phase II and III trials with these new classes of drugs are underway and interesting results have been reported for a variety of compounds in the past 2 years. Fibrosis endpoints in phase II trials may rely on non-invasive measures, such as enhanced liver fibrosis test or fibroscan. Phase III trials use the surrogate liver histology endpoints currently required for accelerated approval in NASH: i) a decrease of fibrosis stage of at least 1 point with no worsening of NASH (hepatocyte ballooning and inflammation scores), and ii) NASH resolution (hepatocyte ballooning and inflammation scores 0-1) with no worsening of fibrosis.
      Following promising phase II data, the FXR agonist obeticholic acid has been evaluated in a pivotal placebo-controlled phase III trial assessing the impact of a 10 or 25 mg dose. An interim analysis in a subgroup of 660 patients reported positive results, with improvement of fibrosis and prevention of fibrosis progression in the 25 mg group (23% vs. 12% on placebo), However, the NASH resolution endpoint was not met (12% vs. 8%).
      • Younossi Z.M.
      • Ratziu V.
      • Loomba R.
      • Rinella M.
      • Anstee Q.M.
      • Goodman Z.
      • et al.
      Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial.
      Thus, the drug was not granted accelerated approval by the FDA, pending submission of additional efficacy and safety data. Encouraging results have also been reported for the stearoyl CoA desaturase inhibitor aramchol. Preliminary data indicate that 1 year of treatment with a 600 mg dose of aramchol significantly decreases fibrosis stage without worsening NASH and a phase III/IV trial evaluating the safety and efficacy of a 300 mg dose is underway (NCT04104321) (Table 1). In contrast, other potential drugs, such as the PPARγ agonist elafibranor,
      • Ratziu V.
      • Harrison S.A.
      • Francque S.
      • Bedossa P.
      • Lehert P.
      • Serfaty L.
      • et al.
      Elafibranor, an agonist of the peroxisome proliferator-activated receptor-alpha and -delta, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening.
      have failed to demonstrate clinical efficacy.
      The antifibrotic effects of strategies targeting lipid or glucose metabolism are increasingly being evaluated in patients with NASH, with fibrosis as a surrogate primary endpoint.
      Overall, therapeutic strategies based on a single target seem to have limited antifibrogenic efficacy in NASH, hence multiple targeting is emerging as an attractive alternative.
      • Dufour J.F.
      • Caussy C.
      • Loomba R.
      Combination therapy for non-alcoholic steatohepatitis: rationale, opportunities and challenges.
      Thus, a recent study has shown that activated HSCs display limited response to obeticholic acid due to progressive FXR sumoylation during the activation process.
      • Zhou J.
      • Cui S.
      • He Q.
      • Guo Y.
      • Pan X.
      • Zhang P.
      • et al.
      SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis.
      Interestingly, combined treatment with a sumoylation inhibitor and obeticholic acid downregulated the fibrogenic phenotype in cultured HSCs and reduced experimental liver fibrosis.
      • Zhou J.
      • Cui S.
      • He Q.
      • Guo Y.
      • Pan X.
      • Zhang P.
      • et al.
      SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis.
      Whether this combined strategy proves useful in patients with NASH remains to be determined. The validity of multiple targeting has also been shown with the use of a pan-PPARα/δ/γ agonist, as initially shown in preclinical studies.
      • Lefere S.
      • Puengel T.
      • Hundertmark J.
      • Penners C.
      • Frank A.K.
      • Guillot A.
      • et al.
      Differential effects of selective- and pan-PPAR agonists on experimental steatohepatitis and hepatic macrophages.
      Interestingly, a recent press release – based on a phase IIb placebo controlled-trial – indicated that a 1,200 mg dose of lanifibranor met its fibrosis endpoints.
      • Francke S.
      • Bedossa P.
      • Abdelmalek M.
      • Quentin Anstee M.
      • Bugianesi E.
      • Vlad R.
      • et al.
      A randomised, double-blind, placebo-controlled, multi-centre, dose-range, proof-of-concept, 24-week treatment study of lanifibranor in adult subjects with non-alcoholic steatohepatitis: design of the NATIVE study.
      Future studies should also evaluate whether combining drugs targeting cell-intrinsic metabolism and compounds targeting other antifibrogenic mechanisms, such as inflammation, lead to better outcomes. Along this line, the TANDEM phase II trial will assess the efficacy of combining the FXR agonist tropifexor with the CCR2/5 antagonist cenicriviroc.
      • Dufour J.F.
      • Caussy C.
      • Loomba R.
      Combination therapy for non-alcoholic steatohepatitis: rationale, opportunities and challenges.
      ,
      • Pedrosa M.
      • Seyedkazemi S.
      • Francque S.
      • Sanyal A.
      • Rinella M.
      • Charlton M.
      • et al.
      A randomized, double-blind, multicenter, phase 2b study to evaluate the safety and efficacy of a combination of tropifexor and cenicriviroc in patients with nonalcoholic steatohepatitis and liver fibrosis: study design of the TANDEM trial.

      Towards cell-specific metabolic targeting of antifibrotic agents

      Drugs targeting metabolism in hepatocytes may display antifibrogenic properties, by reducing liver injury. However, results from aforementioned preclinical studies suggest that they may also have antifibrogenic effects by directly targeting HSCs, or indirectly by changing inflammatory cell phenotypes (Fig. 4). Nevertheless, the efficacy of strategies targeting a metabolic pathway in a given cell type may be limited by opposite effects in another liver cell type or by safety concerns due to interferences with extrahepatic metabolic pathways. Targeted delivery of antifibrotic agents to a specific liver cell type has thus emerged as an attractive option and surface markers or receptors expressed by specific liver cell types have been targeted using si-RNA, antisense oligonucleotides, non-lipid nanoparticles or vitamin A-conjugated liposomes (reviewed in
      • Poelstra K.
      • Schuppan D.
      Targeted therapy of liver fibrosis/cirrhosis and its complications.
      ). A number of protein markers can be used, such the asialoglycoprotein receptor in hepatocytes
      • Prakash T.P.
      • Graham M.J.
      • Yu J.
      • Carty R.
      • Low A.
      • Chappell A.
      • et al.
      Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.
      ,
      • Zimmermann T.S.
      • Karsten V.
      • Chan A.
      • Chiesa J.
      • Boyce M.
      • Bettencourt B.R.
      • et al.
      Clinical proof of concept for a novel hepatocyte-targeting GalNAc-siRNA conjugate.
      or the PDGFR, IGFIIR, or type VI collagen receptor for activated HSCs.
      • Bansal R.
      • Prakash J.
      • Post E.
      • Beljaars L.
      • Schuppan D.
      • Poelstra K.
      Novel engineered targeted interferon-gamma blocks hepatic fibrogenesis in mice.
      Cell-based macrophage therapies are more challenging, due to the heterogeneity of the cell population, but folate or mannose-scavenger receptors have been identified as potential targets. Nanoparticle-based medicine has also been suggested for the specific targeting of macrophages, HSCs, hepatocytes or LSECs.
      • Bai X.
      • Su G.
      • Zhai S.
      Recent advances in nanomedicine for the diagnosis and therapy of liver fibrosis.
      ,
      • Bartneck M.
      • Warzecha K.T.
      • Tacke F.
      Therapeutic targeting of liver inflammation and fibrosis by nanomedicine.
      Further studies will need to adapt these cell-based approaches to the fibrotic liver, since hepatic accumulation of extracellular matrix and closure of endothelial fenestrae may profoundly affect targeting efficiency.
      • Ergen C.
      • Niemietz P.M.
      • Heymann F.
      • Baues M.
      • Gremse F.
      • Pola R.
      • et al.
      Liver fibrosis affects the targeting properties of drug delivery systems to macrophage subsets in vivo.

      Conclusion

      Concordant preclinical studies have undoubtedly demonstrated that targeting cell-intrinsic metabolism has a beneficial effect on liver fibrosis through the combined reprogramming of hepatocyte, HSC and macrophage phenotypes. Clinical translation remains challenging, although promising results have emerged in the context of NASH-related fibrosis. Whether these metabolic approaches can also be extended to fibrosis originating from other aetiologies remains uncertain.

      Abbreviations

      ACAT1, acetyl-CoA acetyltransferase; ACC, acetyl-CoA carboxylase; BPTES, Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide; CCR2/5, C–C chemokine receptor type 2/5; FBP1, fructose 1,6-bisphosphatase 1; FXR, farnesoid X receptor; GPR91, G-protein coupled receptor 91; HMGB1, high-mobility group protein B1; HSCs, hepatic stellate cells; ILCs, innate lymphoid cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; LXR, liver X receptors; MAGL, monoacylglycerol lipase; MAIT, mucosal-associated invariant T cells; MBOAT7, membrane-bound O-acyltransferase domain containing 7; MPC, mitochondrial pyruvate carrier; MSR1, macrophage scavenger receptor 1; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NK, natural killer; PDGF, platelet-derived growth factor; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3; PKM2, pyruvate kinase isoenzyme M2; PNPLA3, patatin Like phospholipase domain containing 3; PPAR, peroxisome proliferator-activated receptor; SCD1, stearoyl-CoA desaturase 1; SCOT, succinyl-CoA-oxoacid transferase; SDH, succinate dehydrogenase; SREBP, sterol regulatory element-binding protein; TCA, tricarboxylic acid cycle; TGF-β, transforming growth factor-β.

      Financial support

      This work was supported by grants from INSERM (France), the Université de Paris , Labex Inflamex and National Research Agency to SL ( ANR-10-LABX-17 , ANR-18-CE14-0006 , ANR-19-CE14-0041 ).

      Authors’ contributions

      HG, AM RM and SL all contributed to writing.

      Conflict of interest

      The authors declare no competing financial interests.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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