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Augmenter of liver regeneration: Mitochondrial function and steatohepatitis

  • Alok Kumar Verma
    Affiliations
    Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

    Cincinnati VA Medical Center, Cincinnati, Ohio, USA
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  • Akanksha Sharma
    Affiliations
    Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

    Cincinnati VA Medical Center, Cincinnati, Ohio, USA
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  • Nithyananthan Subramaniyam
    Affiliations
    Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
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  • Chandrashekhar R. Gandhi
    Correspondence
    Corresponding author. Address: Department of Pediatrics, Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, 3333 Burnett Avenue, Cincinnati, Ohio, 45229, USA, Tel.: +(513)-517-1090; fax: +(513)-558-8677.
    Affiliations
    Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

    Cincinnati VA Medical Center, Cincinnati, Ohio, USA

    Department of Surgery, University of Cincinnati, Cincinnati, Ohio, USA
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      Summary

      Augmenter of liver regeneration (ALR), a ubiquitous fundamental life protein, is expressed more abundantly in the liver than other organs. Expression of ALR is highest in hepatocytes, which also constitutively secrete it. ALR gene transcription is regulated by NRF2, FOXA2, SP1, HNF4α, EGR-1 and AP1/AP4. ALR’s FAD-linked sulfhydryl oxidase activity is essential for protein folding in the mitochondrial intermembrane space. ALR’s functions also include cytochrome c reductase and protein Fe/S maturation activities. ALR depletion from hepatocytes leads to increased oxidative stress, impaired ATP synthesis and apoptosis/necrosis. Loss of ALR’s functions due to homozygous mutation causes severe mitochondrial defects and congenital progressive multiorgan failure, suggesting that individuals with one functional ALR allele might be susceptible to disorders involving compromised mitochondrial function. Genetic ablation of ALR from hepatocytes induces structural and functional mitochondrial abnormalities, dysregulation of lipid homeostasis and development of steatohepatitis. High-fat diet-fed ALR-deficient mice develop non-alcoholic steatohepatitis (NASH) and fibrosis, while hepatic and serum levels of ALR are lower than normal in human NASH and NASH-cirrhosis. Thus, ALR deficiency may be a critical predisposing factor in the pathogenesis and progression of NASH.

      Keywords

      Introduction

      The liver has a remarkable ability to restore its mass after major partial resection.
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      • Anderson R.M.
      Experimental pathology of liver: restoration of the liver of white rat following partial surgical removal.
      During attempts to discover growth factors responsible for this phenomenon, augmenter of liver regeneration (ALR) was identified in the extracts of regenerating livers.
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      Preparation and partial characterization of hepatic regenerative stimulator substance (SS) from rat liver.
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      Stimulation of hepatic regeneration after partial hepatectomy by infusion of a cytosol extract from regenerating dog liver.
      It was postulated that hepatocyte-stimulatory or regeneration-augmenting activity was present in the hyperplastic and regenerating livers.
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      Preparation and partial characterization of hepatic regenerative stimulator substance (SS) from rat liver.
      ,
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      • Ihara I.
      • Starzl T.E.
      Augmenter of liver regeneration: its place in the universe of hepatic growth factors.
      However, ALR’s function in physiology was suggested by subsequent research that observed equivalent expression of the protein in the hepatocytes of weanling and resting adult livers.
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      A fresh look at augmenter of liver regeneration in rats.
      Expression of ALR in non-parenchymal hepatic stellate cells, Kupffer cells and endothelial cells is much lower than in hepatocytes.
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      A fresh look at augmenter of liver regeneration in rats.
      Cholangiocytes also express ALR.
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      Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.
      Although most of the ALR-related research has been limited to its function in the liver, the presence of ALR in the heart, brain, lung, kidney, skeletal muscle, spleen and testes
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      • Sakai H.
      • Seki T.
      • et al.
      Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.
      • Giorda R.
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      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      • Klissenbauer M.
      • Winters S.
      • Heinlein U.A.
      • Lisowsky T.
      Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.
      suggests that it has important roles in the physiology and pathophysiology of other organs as well.
      Hepatocytes produce and secrete ALR constitutively,
      • Gandhi C.R.
      • Kuddus R.
      • Subbotin V.M.
      • Prelich J.
      • Murase N.
      • Rao A.S.
      • et al.
      A fresh look at augmenter of liver regeneration in rats.
      ,
      • Vodovotz Y.
      • Prelich J.
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      • Zamora R.
      • Murase N.
      • et al.
      Augmenter of liver regeneration (ALR) is a novel biomarker of hepatocellular stress/inflammation: in vitro, in vivo and in silico studies.
      and intracellular ALR is found in the mitochondria, nucleus and cytosol.
      • Gandhi C.R.
      Augmenter of liver regeneration.
      ,
      • Ibrahim S.
      • Weiss T.S.
      Augmenter of liver regeneration: essential for growth and beyond.
      A critical role of ALR in mitochondria is evident as its loss in vitro and in vivo causes lipid accumulation (steatosis), ATP depletion, oxidative stress, mitochondrial degeneration, and death of hepatocytes.
      • Thirunavukkarasu C.
      • Wang L.F.
      • Harvey S.A.
      • Watkins S.C.
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      • Prelich J.
      • et al.
      Augmenter of liver regeneration: an important intracellular survival factor for hepatocytes.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ALR also inhibits mitochondrial membrane permeability transition, thus protecting cells from injury.
      • Wu Y.
      • Zhang J.
      • Dong L.
      • Li W.
      • Jia J.
      • An W.
      Hepatic stimulator substance mitigates hepatic cell injury through suppression of the mitochondrial permeability transition.
      Interestingly, hepatic steatosis of various aetiologies is associated with downregulation of ALR expression.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ,
      • Kumar S.
      • Wang J.
      • Rani R.
      • Gandhi C.R.
      Hepatic deficiency of augmenter of liver regeneration exacerbates alcohol-induced liver injury and promotes fibrosis in mice.
      ,
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      Non-alcoholic fatty liver disease (NAFLD) has become a major clinical challenge of recent times. NAFLD begins with simple steatosis that can progress to the more aggressive form, non-alcoholic steatohepatitis (NASH) in 10-30% of affected individuals.
      • Loomba R.
      • Sanyal A.J.
      The global NAFLD epidemic.
      A significant number of patients with NASH develop cirrhosis and some may progress to hepatocellular carcinoma (HCC) with or without cirrhosis.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      ,
      • Younossi Z.M.
      Non-alcoholic fatty liver disease - a global public health perspective.
      Since disordered mitochondrial function is a critical component of NAFLD pathophysiology,
      • Pessayre D.
      • Berson A.
      • Fromenty B.
      • Mansouri A.
      Mitochondria in steatohepatitis.
      • Mansouri A.
      • Gattolliat C.H.
      • Asselah T.
      Mitochondrial dysfunction and signaling in chronic liver diseases.
      • Serviddio G.
      • Bellanti F.
      • Vendemiale G.
      • Altomare E.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis.
      • Lee J.
      • Park J.S.
      • Roh Y.S.
      Molecular insights into the role of mitochondria in non-alcoholic fatty liver disease.
      it is apparent that ALR deficiency may play an important role in its pathogenesis and progression. In this review, we discuss the current understanding of the regulation of ALR expression and function, and we review the possible mechanisms by which ALR deficiency might contribute to aggressive NAFLD.
      ALR is an evolutionally conserved protein that is abundantly expressed in hepatocytes.

      Augmenter of liver regeneration gene and isoforms

      Although known as augmenter of liver regeneration, hepatopoietin and hepatic stimulatory substance, these names are rather misnomers since ALR is present ubiquitously (in all major organs) and demonstrates functions other than liver cell proliferation.
      • Klissenbauer M.
      • Winters S.
      • Heinlein U.A.
      • Lisowsky T.
      Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.
      ,
      • Gandhi C.R.
      Augmenter of liver regeneration.
      ,
      • Ibrahim S.
      • Weiss T.S.
      Augmenter of liver regeneration: essential for growth and beyond.
      ,
      • Nalesnik M.
      • Gandhi C.R.
      Starzl. Augmenter of liver regeneration: a fundamental life protein.
      However, the highest expression of ALR is found in the liver and testes.
      • Hagiya M.
      • Francavilla A.
      • Polimeno L.
      • Ihara I.
      • Sakai H.
      • Seki T.
      • et al.
      Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.
      • Giorda R.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      • Klissenbauer M.
      • Winters S.
      • Heinlein U.A.
      • Lisowsky T.
      Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.
      ,
      • Lisowsky T.
      • Weinstat-Saslow D.L.
      • Barton N.
      • Reeders S.T.
      • Schneider M.C.
      A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.
      The ALR gene is mapped to chromosome 16 (human), 17 (mouse) or 10 (rat).
      • Hagiya M.
      • Francavilla A.
      • Polimeno L.
      • Ihara I.
      • Sakai H.
      • Seki T.
      • et al.
      Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.
      ,
      • Giorda R.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      ,
      • Lisowsky T.
      • Weinstat-Saslow D.L.
      • Barton N.
      • Reeders S.T.
      • Schneider M.C.
      A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.
      The highly conserved ALR gene and protein sequences in humans, mice and rats, and the presence of homologous proteins in yeast,
      • Lisowsky T.
      • Weinstat-Saslow D.L.
      • Barton N.
      • Reeders S.T.
      • Schneider M.C.
      A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.
      insects,
      • Klebes A.
      • Sustar A.
      • Kechris K.
      • Li H.
      • Schubiger G.
      • Kornberg T.B.
      Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes.
      and viruses
      • Senkevich T.G.
      • White C.L.
      • Koonin E.V.
      • Moss B.
      A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation.
      indicate that the gene is evolutionally conserved. Homology between the rat, mouse and human ALR and the yeast scERV1 protein is shown in Fig. 1.
      Figure thumbnail gr1
      Fig. 1Sequence homology between mouse, rat, human ALR and Saccharomyces cerevisiae scERV1 protein.
      Sequence alignment was performed using Blast 2 sequences program (https://blast.ncbi.nlm.nih.gov/). (A) Amino acid length, and sequence similarity in ALR gene among mouse, rat, human and yeast. (B) Amino acid sequence alignment between rat, mouse and human ALR, and scERV1.
      The mammalian ALR gene is named GFER (growth factor Erv1-like) because of its structural and functional similarities with the scERV1 (essential for respiration and vegetative growth-1) protein expressed by the yeast Saccharomyces cerevisiae.
      • Hagiya M.
      • Francavilla A.
      • Polimeno L.
      • Ihara I.
      • Sakai H.
      • Seki T.
      • et al.
      Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution.
      ,
      • Giorda R.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      ,
      • Lisowsky T.
      • Weinstat-Saslow D.L.
      • Barton N.
      • Reeders S.T.
      • Schneider M.C.
      A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.
      ,
      • Lisowsky T.
      Dual function of a new nuclear gene for oxidative phosphorylation and vegetative growth in yeast.
      The human ALR is a single copy gene mapped to chromosome 16 in the polycystic kidney disease locus.
      • Lisowsky T.
      • Weinstat-Saslow D.L.
      • Barton N.
      • Reeders S.T.
      • Schneider M.C.
      A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.
      Two major forms of ALR, short (∼15 kDa; s-ALR) and long (∼22 kDa; l-ALR), have been identified.
      • Gandhi C.R.
      • Kuddus R.
      • Subbotin V.M.
      • Prelich J.
      • Murase N.
      • Rao A.S.
      • et al.
      A fresh look at augmenter of liver regeneration in rats.
      ,
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ,
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      ,
      • Dayoub R.
      • Wagner H.
      • Bataille F.
      • Stöltzing O.
      • Spruss T.
      • Buechler C.
      • et al.
      Liver regeneration associated protein (ALR) exhibits antimetastatic potential in hepatocellular carcinoma.
      ,
      • Polimeno L.
      • Pesetti B.
      • Annoscia E.
      • Giorgio F.
      • Francavilla R.
      • Lisowsky T.
      • et al.
      Alrp, a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats.
      However, the presence of multiple ATG codons in the ALR gene suggests the possibility of variants generated by alternative splicing. The entire mouse ALR gene is contained in a 6.7-kb HindIII fragment, comprising 3 exons and 2 introns.
      • Giorda R.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      Exon 1 contains a 5' untranslated sequence, ATG initiation codon and 6 amino acid-coding nucleotide sequence of s-ALR or 73 amino acid-coding sequence of l-ALR, which is followed by a 400 bp intron; exon 2 contains a 66 amino acid-coding sequence, followed by the second 480 bp intron; the third exon contains the remaining portion of the amino acid-coding sequence and the entire 3' untranslated sequence.
      • Giorda R.
      • Hagiya M.
      • Seki T.
      • Shimonishi M.
      • Sakai H.
      • Michaelson J.
      • et al.
      Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.
      The N-terminal domain of l-ALR contains the mitochondrial leader sequence (absent in s-ALR), whereas the C-terminal domain is responsible for the flavin adenine dinucleotide (FAD)-linked functional activity.

      ALR gene expression regulation

      The ALR gene has a TATA-less promotor with features of housekeeping genes, oncogenes, growth factors and transcription factors.
      • Zhao Y.
      • Tang F.
      • Cheng J.
      • Li L.
      • Xing G.
      • Zhu Y.
      • et al.
      An initiator and its flanking elements function as a core promoter driving transcription of the Hepatopoietin gene.
      Analysis of the ALR promoter identified positive regulatory elements between -416 and -608 nucleotide (nt), negative regulatory elements between -236 and -416 nt, and minimal core promoter activity between −22 and +27 nt. A “CTGGAGGC” sequence within the initiator (Inr)-like element and 2 other tandem flanking repeats comprise the core promoter that controls transcriptional initiation and the constitutive expression of the ALR gene. Activator protein 1/4 (AP1/AP4) is proposed to be responsible for basal ALR promoter activity.
      • Dong L.Y.
      • Wang X.N.
      • Song Z.G.
      • Guo D.
      • Zhao Y.Y.
      • An W.
      Identification of human hepatic stimulator substance gene promoter and demonstration of dual regulation of AP1/AP4 cis-acting element in different cell lines.
      ALR gene expression is positively regulated by nuclear factor erythroid 2-related factor 2 (NRF2), forkhead box A2 (FOXA2 also known as hepatocyte nuclear factor 3β [HNF3β]), HNF4α, early growth response protein-1 (EGR-1) and specificity protein 1 (SP1). An antioxidant response element (ARE) is located at −27/−19 nt from the initial ATG codon in the proximal promoter region. Upregulation of ALR expression by oxidative stress (due to an increase in nuclear NRF2 and its binding to ARE) suggests that ALR is an ARE-regulated gene.
      • Dayoub R.
      • Vogel A.
      • Schuett J.
      • Lupke M.
      • Spieker S.M.
      • Kettern N.
      • et al.
      Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration.
      FOXA2 binds at +276/+282 nt in the intronic promoter and its binding is amplified by the IL-6 response element at +265/+271 nt.
      • Dayoub R.
      • Groitl P.
      • Dobner T.
      • Bosserhoff A.K.
      • Schlitt H.J.
      • Weiss T.S.
      Foxa2 (HNF-3beta) regulates expression of hepatotrophic factor ALR in liver cells.
      SP1 binds at −152/−145 nt and its overexpression markedly elevates ALR expression.
      • Guo D.
      • Dong L.Y.
      • Wu Y.
      • Yang L.
      • An W.
      Down-regulation of hepatic nuclear factor 4alpha on expression of human hepatic stimulator substance via its action on the proximal promoter in HepG2 cells.
      The ALR promoter also contains 2 potential bile acid-binding response elements, and bile acids suppress ALR promoter activity induced by FOXA2, HNF4α (binding site at +421/+432 nt) and EGR-1c (binding site at +304/+314 nt) via activation of small heterodimer partner (SHP).
      • Ibrahim S.
      • Dayoub R.
      • Melter M.
      • Weiss T.S.
      Bile acids down-regulate the expression of augmenter of liver regeneration (ALR) via SHP/HNF4α1 and independent of Egr-1.
      ALR loss causes oxidative stress, ATP depletion, mitochondrial degeneration and death of hepatocytes.
      Interestingly, binding of HNF4α at -209/-204 nt negatively regulates ALR promoter activity.
      • Guo D.
      • Dong L.Y.
      • Wu Y.
      • Yang L.
      • An W.
      Down-regulation of hepatic nuclear factor 4alpha on expression of human hepatic stimulator substance via its action on the proximal promoter in HepG2 cells.
      CCAAT/enhancer binding protein-β (C/EBPβ) is another negative regulator of ALR gene transcription. In HepG2 cells, electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis revealed a C/EBPβ-binding site at −292/−279 nt, and the epidermal growth factor (EGF) was found to downregulate ALR expression via C/EBPβ.
      • Dong L.Y.
      • Sun G.
      • Jiang L.
      • Shao L.
      • Hu Y.
      • Jiang Y.
      • et al.
      Epidermal growth factor down-regulates the expression of human hepatic stimulator substance via CCAAT/enhancer-binding protein beta in HepG2 cells.
      It is apparent that altered ALR expression due to variable activation of these transcription factors may influence pathophysiological changes during disease progression.

      ALR’s role in cell viability and growth

      Hepatic ALR is transiently decreased following 70% (but not 40%) hepatectomy in normal rats, with a corresponding increase in its serum concentration.
      • Gandhi C.R.
      • Kuddus R.
      • Subbotin V.M.
      • Prelich J.
      • Murase N.
      • Rao A.S.
      • et al.
      A fresh look at augmenter of liver regeneration in rats.
      ALR administration augments liver regeneration after 40% but not 70% hepatectomy.
      • Francavilla A.
      • Hagiya M.
      • Porter K.A.
      • Polimeno L.
      • Ihara I.
      • Starzl T.E.
      Augmenter of liver regeneration: its place in the universe of hepatic growth factors.
      ,
      • Gandhi C.R.
      • Murase N.
      • Starzl T.E.
      Cholera toxin-sensitive GTP-binding protein-coupled activation of augmenter of liver regeneration (ALR) receptor and its function in rat Kupffer cells.
      This suggested that ALR released after 70% hepatectomy stimulates synthesis of growth mediators. Thus, the augmenting effect of exogenous ALR is proposed to be due to ALR-induced synthesis of tumour necrosis factor (TNF)α and IL-6 by Kupffer cells,
      • Gandhi C.R.
      • Murase N.
      • Starzl T.E.
      Cholera toxin-sensitive GTP-binding protein-coupled activation of augmenter of liver regeneration (ALR) receptor and its function in rat Kupffer cells.
      which prime hepatocyte regeneration.
      • Webber E.M.
      • Bruix J.
      • Godowski P.J.
      • Fausto N.
      In vivo response of hepatocytes to growth factors requires an initial priming stimulus.
      Furthermore, ALR reduced oxidative stress, autophagy and apoptosis, and at the same time increased oxidative phosphorylation and mitochondrial expression of ATPase 6/8, ND1 subunit and mitochondrial transcription factor A (TFAM) in partially hepatectomised rats.
      • Polimeno L.
      • Pesetti B.
      • Annoscia E.
      • Giorgio F.
      • Francavilla R.
      • Lisowsky T.
      • et al.
      Alrp, a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats.
      ,
      • Polimeno L.
      • Capuano F.
      • Marangi L.C.
      • Margiotta M.
      • Lisowsky T.
      • Ierardi E.
      • et al.
      The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria.
      Intracellular ALR was found to be essential for the survival of murine hepatocytes
      • Thirunavukkarasu C.
      • Wang L.F.
      • Harvey S.A.
      • Watkins S.C.
      • Chaillet J.R.
      • Prelich J.
      • et al.
      Augmenter of liver regeneration: an important intracellular survival factor for hepatocytes.
      and human hepatoma HepG2 cells.
      • Cao Y.
      • Fu Y.L.
      • Yu M.
      • Yue P.B.
      • Ge C.H.
      • Xu W.X.
      • et al.
      Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress.
      Increasing ALR expression either through activation of transcription factors (e.g., NRF2) or via plasmid transfection was found to be pro-proliferative and anti-apoptotic.
      • Dayoub R.
      • Vogel A.
      • Schuett J.
      • Lupke M.
      • Spieker S.M.
      • Kettern N.
      • et al.
      Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration.
      The in vivo relevance of these findings is demonstrated by robust apoptosis of hepatocytes upon genetic ablation of ALR expression.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      Early studies showed that partially purified as well as cloned ALR protects the liver from portacaval shunt-induced atrophy.
      • Francavilla A.
      • Hagiya M.
      • Porter K.A.
      • Polimeno L.
      • Ihara I.
      • Starzl T.E.
      Augmenter of liver regeneration: its place in the universe of hepatic growth factors.
      However, increased hepatic synthesis of ALR and powerful mitogens, such as hepatocyte growth factor (HGF) and TGFα (transforming growth factor α), after portacaval shunt in rats indicated that first pass of gastrointestinal-derived growth factors is critical for liver cell size maintenance and function.
      • Gandhi C.R.
      • Murase N.
      • Subbotin V.M.
      • Uemura T.
      • Nalesnik M.
      • Demetris A.J.
      • et al.
      Portacaval shunt causes apoptosis and liver atrophy despite increases in endogenous levels of major hepatic growth factors.
      ALR also protects the liver from galacosamine-,
      • Francavilla A.
      • Hagiya M.
      • Porter K.A.
      • Polimeno L.
      • Ihara I.
      • Starzl T.E.
      Augmenter of liver regeneration: its place in the universe of hepatic growth factors.
      carbon tetrachloride (CCl4)-,
      • Mei M.H.
      • An W.
      • Zhang B.H.
      • Shao Q.
      • Gong D.Z.
      Hepatic stimulator substance protects against acute liver failure induced by carbon tetrachloride poisoning in mice.
      H2O2-,
      • Wu Y.
      • Chen L.
      • Yu H.
      • Liu H.
      • An W.
      Transfection of hepatic stimulator substance gene desensitizes hepatoma cells to H2O2-induced cell apoptosis via preservation of mitochondria.
      ethanol-,
      • Liatsos G.D.
      • Mykoniatis M.G.
      • Margeli A.
      • Liakos A.A.
      • Theocharis S.E.
      Effect of acute ethanol exposure on hepatic stimulator substance (HSS) levels during liver regeneration: protective function of HSS.
      and acetaminophen-mediated
      • Hu T.
      • Sun H.
      • Deng W.Y.
      • Huang W.Q.
      • Liu Q.
      Augmenter of liver regeneration protects against acetaminophen-induced acute liver injury in mice by promoting autophagy.
      acute injury. Transplantation of liver epithelial progenitor cells overexpressing ALR was shown to mitigate CCl4-induced liver damage and mortality, whereas ALR silencing had the opposite effects.
      • Dong Y.
      • Kong W.
      • An W.
      Downregulation of augmenter of liver regeneration impairs the therapeutic efficacy of liver epithelial progenitor cells against acute liver injury by enhancing mitochondrial fission.
      ALR was also shown to protect human hepatocytes in primary culture from pro-apoptotic agents.
      • Ilowski M.
      • Kleespies A.
      • de Toni E.
      • Donabauer B.
      • Jauch K.W.
      • Hengstler J.G.
      • et al.
      Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis.
      Accumulation of toxic bile acids during cholestasis causes oxidative stress and death of hepatocytes. Reduced hepatic ALR levels in human cholestatic liver disease (presumably via bile acid-induced SHP activation and suppression of ALR promoter activity),
      • Ibrahim S.
      • Dayoub R.
      • Melter M.
      • Weiss T.S.
      Bile acids down-regulate the expression of augmenter of liver regeneration (ALR) via SHP/HNF4α1 and independent of Egr-1.
      and inhibition of glycochenodeoxycholic acid-induced apoptosis in s-ALR-transfected HepG2 cells
      • Ibrahim S.
      • Dayoub R.
      • Krautbauer S.
      • Liebisch G.
      • Wege A.K.
      • Melter M.
      • et al.
      Bile acid-induced apoptosis and bile acid synthesis are reduced by over-expression of Augmenter of Liver Regeneration (ALR) in a STAT3-dependent mechanism.
      indicate the importance of this cytosolic form in cell survival. In contrast, glycochenodeoxycholic acid-mediated apoptosis is not affected by l-ALR-transfected Huh7 cells.
      • Denk G.U.
      • Wimmer R.
      • Vennegeerts T.
      • Pusl T.
      • Thasler W.
      • Rust C.
      Glycochenodeoxycholate-induced apoptosis is not reduced by augmenter of liver regeneration in the human hepatoma cell line HuH-7.
      Interestingly, ALR levels are increased in primary biliary cholangitis, sclerosing cholangitis and cholangiocarcinoma.
      • Thasler W.E.
      • Schlott T.
      • Thelen P.
      • Hellerbrand C.
      • Bataille F.
      • Lichtenauer M.
      • et al.
      Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.
      Mechanisms underlying the variable expression of the ALR gene and their implications in progression of cholestatic liver disease remain to be elucidated.
      The ALR receptor has been identified in rat hepatocytes, and binding of s-ALR promotes DNA synthesis with similar potency as the powerful hepatocyte growth factors HGF, TNFα and EGF.
      • Wang G.
      • Yang X.
      • Zhang Y.
      • Wang Q.
      • Chen H.
      • Wei H.
      • et al.
      Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1.
      The direct effect of s-ALR on hepatocytes is reported to be mediated by EGFR phosphorylation and subsequent activation of AP1,
      • Li Y.
      • Li M.
      • Xing G.
      • Hu Z.
      • Wang Q.
      • Dong C.
      • et al.
      Stimulation of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepatopoietin.
      as well as polyamine synthesis via c-Myc activation.
      • Thasler W.E.
      • Dayoub R.
      • Mühlbauer M.
      • Hellerbrand C.
      • Singer T.
      • Gräbe A.
      • et al.
      Repression of cytochrome P450 activity in human hepatocytes in vitro by a novel hepatotrophic factor, augmenter of liver regeneration.
      Binding of l-ALR to Jun activation domain-binding protein 1 (JAB1) also promotes AP1 transcriptional activity.
      • Lu C.
      • Li Y.
      • Zhao Y.
      • Xing G.
      • Tang F.
      • Wang Q.
      • et al.
      Intracrine hepatopoietin potentiates AP-1 activity through JAB1 independent of MAPK pathway.
      ,
      • Wang Y.
      • Lu C.
      • Wei H.
      • Wang N.
      • Chen X.
      • Zhang L.
      • et al.
      Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity.
      These findings suggest that both ALR forms can stimulate liver regeneration.
      ALR is essential for proper folding of mitochondrial imported proteins, electron transport chain activity and iron/sulphur maturation of cytosolic proteins.

      Importance of ALR in mitochondrial integrity and function

      Mutations in the scERV1 gene or depletion of scERV1 protein caused loss of the inner mitochondrial membrane and eventually the entire organelle, indicating the critical importance of ERV1 in mitochondrial integrity, survival and function.
      • Lisowsky T.
      ERV1 is involved in the cell-division cycle and the maintenance of mitochondrial genomes in Saccharomyces cerevisiae.
      ,
      • Becher D.
      • Kricke J.
      • Stein G.
      • Lisowsky T.
      A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution.
      Most of the mitochondrial proteins are synthesised in the cytosol and transported into the mitochondria as precursors, aided by the TOM (translocase of the outer membrane) complex. Appropriate oxidative folding of several of these proteins is essential for their functions and is catalysed by the Mia40 (mitochondrial intermembrane space import and assembly protein 40 kDA)/ALR-sulfhydryl relay system
      • Mesecke N.
      • Terziyska N.
      • Kozany C.
      • Baumann F.
      • Neupert W.
      • Hell K.
      • et al.
      A disulfide relay system in the intermembrane space of mitochondria that mediates protein import.
      • Tokatlidis K.
      A disulfide relay system in mitochondria.
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • SanjuánSzklarz L.K.
      • Schulze-Specking A.
      • et al.
      Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.
      • Herrmann J.M.
      • Riemer J.
      Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space.
      (Fig. 2). Mia40, with its redox-active cysteine-proline-cysteine disulphide bond, oxidises cysteine residues of the imported polypeptides; these stably folded proteins are prevented from transport through the outer membrane.
      • Lu H.
      • Allen S.
      • Wardleworth L.
      • Savory P.
      • Tokatlidis K.
      Functional TIM10 chaperone assembly is redox-regulated in vivo.
      ,
      • Terziyska N.
      • Grumbt B.
      • Kozany C.
      • Hell K.
      Structural and functional roles of the conserved cysteine residues of the redox-regulated import receptor Mia40 in the intermembrane space of mitochondria.
      Reduced Mia40 is re-oxidised by ALR (about 70% of Mia40 is in an oxidised state) and can then introduce disulphide bonds into the newly imported polypeptides.
      • Riemer J.
      • Bulleid N.
      • Herrmann J.M.
      Disulfide formation in the ER and mitochondria: two solutions to a common process.
      Two essential redox-active cysteine-x-x-cysteine pairs in ALR shuttle electrons from Mia40 to FAD. ALR can be directly re-oxidised by oxygen in vitro in a reaction yielding H2O2. In vivo, ALR is re-oxidised by passing its electrons through FAD to cytochrome c of the respiratory chain; these electrons are then accepted by molecular oxygen to produce water, thus preventing generation of H2O2 in the intermembrane space.
      • Farrell S.R.
      • Thorpe C.
      Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity.
      • Bihlmaier K.
      • Mesecke N.
      • Terziyska N.
      • Bien M.
      • Hell K.
      • Herrmann J.M.
      The disulfide relay system of mitochondria is connected to the respiratory chain.
      • Dabir D.V.
      • Leverich E.P.
      • Kim S.K.
      • Tsai F.D.
      • Hirasawa M.
      • Knaff D.B.
      • et al.
      A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1.
      Figure thumbnail gr2
      Fig. 2ALR/Mia40 mitochondrial disulphide relay system.
      Newly synthesised proteins in the cytosolic compartment are translocated into mitochondrial intermembrane space through the TOM channel. The imported proteins are oxidised by Mia40 allowing for their proper folding. Following oxidation by sulfhydryl oxidase activity of Erv1/ALR, Mia40 re-enters the cycle to introduce a disulphide bond in incoming proteins. ALR is subsequently re-oxidised by donating electrons to cytochrome c for the reaction involving conversion of oxygen to water by cytochrome c oxidase. ALR, augmenter of liver regeneration; Mia40, mitochondrial intermembrane space import and assembly protein 40 kDA; TOM, translocase of the outer membrane.
      The presence of ALR in excess of Mia40
      • Erdogan A.J.
      • Ali M.
      • Habich M.
      • Salscheider S.L.
      • Schu L.
      • Petrungaro C.
      • et al.
      The mitochondrial oxidoreductase CHCHD4 is present in a semi-oxidized state in vivo.
      indicates that its role in mitochondria may extend beyond Mia40 reoxidation. Indeed, mitochondrial ALR plays an essential role in iron homeostasis by catalysing Fe/S maturation of cytosolic proteins.
      • Lange H.
      • Lisowsky T.
      • Gerber J.
      • Mühlenhoff U.
      • Kispal G.
      • Lill R.
      An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.
      Mechanistically, the Mia40/ALR pathway has been shown to facilitate import of ABCB8 (ATP-binding cassette-B8), an inner mitochondrial membrane protein necessary for cytoplasmic Fe/S cluster maturation.
      • Chang H.C.
      • Shapiro J.S.
      • Jiang X.
      • Senyei G.
      • Sato T.
      • Geier J.
      • et al.
      Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8.
      The pathophysiological implication of ALR as a regulator of iron homeostasis is evidenced by excessive iron accumulation in the liver of hepatocyte-specific ALR-deficient mice upon alcohol consumption.
      • Kumar S.
      • Wang J.
      • Rani R.
      • Gandhi C.R.
      Hepatic deficiency of augmenter of liver regeneration exacerbates alcohol-induced liver injury and promotes fibrosis in mice.
      Clinically, the importance of ALR in the mitochondrial sulfhydryl relay system is exemplified by mitochondriopathy, decreased activity of respiratory complexes I, II and IV, congenital cataract, muscular hypotonia and developmental delay observed in patients with a rare (R194H) mutation in the ALR gene.
      • Di Fonzo A.
      • Ronchi D.
      • Lodi T.
      • Fassone E.
      • Tigano M.
      • Lamperti C.
      • et al.
      The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.
      ,
      • Nambot S.
      • Gavrilov D.
      • Thevenon J.
      • Bruel A.L.
      • Bainbridge M.
      • Rio M.
      • et al.
      Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.
      The mutation reduces stability of the ALR protein, as well as the expression and activity of cytochrome oxidase. The R194H mutation also causes defective accumulation of Mia40 in mitochondria suggesting that ALR regulates Mia40 localisation.
      • Sztolsztener M.E.
      • Brewinska A.
      • Guiard B.
      • Chacinska A.
      Disulfide bond formation: sulfhydryl oxidase ALR controls mitochondrial biogenesis of human MIA40.
      ALR maintains lipid homeostasis by regulating expression of lipogenic and lipolytic enzymes.
      Another mechanism by which ALR might be important in mitochondrial function is by regulating TFAM, a highly conserved nuclear-encoded DNA-binding protein.
      • Garstka H.L.
      • Schmitt W.E.
      • Schultz J.
      • Sogl B.
      • Silakowski B.
      • Pérez-Martos A.
      • et al.
      Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA.
      • Pastukh V.
      • Shokolenko I.
      • Wang B.
      • Wilson G.
      • Alexeyev M.
      Human mitochondrial transcription factor A possesses multiple subcellular targeting signals.
      • Campbell C.T.
      • Kolesar J.E.
      • Kaufman B.A.
      Mitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number.
      TFAM is critical for stabilisation and transcription of mitochondrial DNA (mtDNA).
      • Takamatsu C.
      • Umeda S.
      • Ohsato T.
      • Ohno T.
      • Abe Y.
      • Fukuoh A.
      • et al.
      Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein.
      ,
      • Scarpulla R.C.
      Transcriptional paradigms in mammalian mitochondrial biogenesis and function.
      Administration of recombinant ALR to normal rats time-dependently increases TFAM expression.
      • Polimeno L.
      • Capuano F.
      • Marangi L.C.
      • Margiotta M.
      • Lisowsky T.
      • Ierardi E.
      • et al.
      The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria.
      Genetic ablation of ALR from hepatocytes reduces TFAM expression and ATP content, which recover as ALR levels increase.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      In line with this observation, ALR-knockdown in mice reduces expression of TFAM as well as PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α), impairs mitochondrial biogenesis, and delays liver regeneration after partial hepatectomy.
      • Han L.H.
      • Dong L.Y.
      • Yu H.
      • Sun G.Y.
      • Wu Y.
      • Gao J.
      • et al.
      Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice.
      In a rat model of obstructive jaundice, administration of human ALR led to increased expression of TFAM and nuclear respiratory factor 1, mtDNA damage repair and improvement of mitochondrial functions.
      • Tang C.
      • Lin H.
      • Wu Q.
      • Zhang Y.
      • Bie P.
      • Yang J.
      Recombinant human augmenter of liver regeneration protects hepatocyte mitochondrial DNA in rats with obstructive jaundice.
      Mitochondrial fission (via phosphorylation of dynamin-related protein 1) upon knockdown of ALR from liver epithelial progenitor cells also demonstrates its importance in mitochondrial survival.
      • Dong Y.
      • Kong W.
      • An W.
      Downregulation of augmenter of liver regeneration impairs the therapeutic efficacy of liver epithelial progenitor cells against acute liver injury by enhancing mitochondrial fission.
      The mitochondrial electron transport chain is the site of ATP and reactive oxygen species (ROS) generation, as few electrons interact directly with oxygen to form superoxide anion (O2.–) and H2O2.
      • Murphy M.P.
      How mitochondria produce reactive oxygen species.
      ROS damage mtDNA, interact with lipids and proteins to form peroxidation products and cause cell death. ALR depletion in vivo and in vitro increases oxidative stress and causes mtDNA damage.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      Forced ALR deficiency-induced oxidative stress and lipid accumulation in hepatocytes is reversed by ALR treatment.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      Of note, the Mia40/ALR-sulfhydryl relay system was shown to introduce functional protein folding in superoxide dismutase 1.
      • Gross D.P.
      • Burgard C.A.
      • Reddehase S.
      • Leitch J.M.
      • Culotta V.C.
      • Hell K.
      Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system.
      • Klöppel C.
      • Suzuki Y.
      • Kojer K.
      • Petrungaro C.
      • Longen S.
      • Fiedler S.
      • et al.
      Mia40-dependent oxidation of cysteines in domain I of Ccs1 controls its distribution between mitochondria and the cytosol.
      • Reddehase S.
      • Grumbt B.
      • Neupert W.
      • Hell K.
      The disulfide relay system of mitochondria is required for the biogenesis of mitochondrial Ccs1 and Sod1.
      Increased ALR expression also imparts protection against irradiation-induced mitochondrial and cellular damage by increasing mitochondrial membrane potential, inhibiting cytochrome c release and preventing ATP loss.
      • Cao Y.
      • Fu Y.L.
      • Yu M.
      • Yue P.B.
      • Ge C.H.
      • Xu W.X.
      • et al.
      Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress.

      Role of ALR in NAFLD

      Considering the importance of ALR in mitochondrial biogenesis, survival and function, a deficiency of, or an abnormality in, the ALR protein might be a predisposing condition in the development of NASH, the aggressive form of NAFLD. NASH is characterised by steatosis, hepatocyte ballooning and injury, inflammation, and pericellular fibrosis.
      • Estes C.
      • Razavi H.
      • Loomba R.
      • Younossi Z.
      • Sanyal A.J.
      Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease.
      While the majority of patients with NAFLD have simple steatosis (NAFL), up to 30% develop NASH and may progress to cirrhosis, a fertile environment for HCC.
      • Loomba R.
      • Sanyal A.J.
      The global NAFLD epidemic.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      • Younossi Z.M.
      Non-alcoholic fatty liver disease - a global public health perspective.
      Obesity, sedentary lifestyle, type 2 diabetes mellitus/insulin resistance, altered gut microbiota, and genetic and environmental factors are all considered as contributors to NAFLD/NASH and NASH-induced cirrhosis.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      ,
      • Buzzetti E.
      • Pinzani M.
      • Tsochatzis E.A.
      The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD).
      Despite extensive clinical and experimental research, there is still a significant knowledge gap regarding the pathogenesis and progression of NAFLD, for which no pharmacological treatment has been approved.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      ,
      • Oseini A.M.
      • Sanyal A.J.
      Therapies in non-alcoholic steatohepatitis (NASH).
      Genetic ablation of Alr in mice leads to robust steatosis and cell death followed by “lean” NASH-like progression to hepatocellular carcinoma.
      Ultrastructural mitochondrial lesions, increased production of ROS and lipid peroxidation (due to decreased activity of respiratory chain complexes), decreased fatty acid β-oxidation, and reduced ability to resynthesise ATP have all been observed in patients with NASH.
      • Pessayre D.
      • Berson A.
      • Fromenty B.
      • Mansouri A.
      Mitochondria in steatohepatitis.
      • Mansouri A.
      • Gattolliat C.H.
      • Asselah T.
      Mitochondrial dysfunction and signaling in chronic liver diseases.
      • Serviddio G.
      • Bellanti F.
      • Vendemiale G.
      • Altomare E.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis.
      • Lee J.
      • Park J.S.
      • Roh Y.S.
      Molecular insights into the role of mitochondria in non-alcoholic fatty liver disease.
      However, mechanisms underlying deterioration of mitochondrial structure and function are not completely understood. It should be noted that hepatic mitochondria of obese individuals with or without NAFL exhibit an increased respiration rate, but this adaptation is lost in those with NASH who exhibit a significantly reduced rate of respiration in association with increased oxidative stress and DNA damage.
      • 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.
      ,
      • Dewidar B.
      • Kahl S.
      • Pafili K.
      • Roden M.
      Metabolic liver disease in diabetes - from mechanisms to clinical trials.
      It will be important to elucidate whether this transition occurs as patients progress from NAFL to NASH, or if patients who progress to NASH are distinct from those with NAFL who do not develop NASH.
      Hepatocyte-specific Alr-knockout (Alr-H-KO) mice were generated to investigate ALR’s role in liver physiology. Alr-H-KO mice showed severe mitochondriopathy (degenerating or enlarged mitochondria with loss of cristae, and defect at complex II of the electron transport chain), and ATP depletion due to significant loss of ALR between 1 and 2 weeks postpartum.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      There was robust mixed macro- and microvesicular steatosis, excessive liver cell death and pericellular fibrosis.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      These pathologies led to strong ductular reaction, regeneration of hepatocytes from the surviving cells, as well as from cells of the biliary compartment at 4 weeks, and regression of steatosis. It was observed that the surviving/regenerating cells expressed ALR, albeit at significantly lower magnitude than cells in the wild-type (WT) mouse liver, but there was continued inflammation and modest fibrosis. Eventually, nearly 70% of Alr-H-KO mice developed liver tumours (60% being HCC) by 1 year when their ALR expression was similar to the WT mice.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      In this regard, hepatic ALR levels were found to be greater than normal in HCC.
      • Thasler W.E.
      • Schlott T.
      • Thelen P.
      • Hellerbrand C.
      • Bataille F.
      • Lichtenauer M.
      • et al.
      Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.
      Interestingly, hepatic ALR levels, measured via western blot analysis or ELISA (pg/mg protein or DNA), are lower than normal in NASH-induced cirrhosis.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      ,
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      Since cells from the biliary compartment seem to transform into ALR-expressing hepatocytes following pronounced apoptosis at 2 weeks postpartum, they are the likely source of malignancy. Although NAFLD is generally associated with central obesity, patients with a normal body mass index developing “lean” NASH have been identified.
      • Loomba R.
      • Sanyal A.J.
      The global NAFLD epidemic.
      Furthermore, based on routine cancer screening, 35–50% of HCC cases occurring in patients with NASH arise in the absence of cirrhosis.
      • Mittal S.
      • El-Serag H.B.
      • Sada Y.H.
      • Kanwal F.
      • Duan Z.
      • Temple S.
      • et al.
      Hepatocellular carcinoma in the absence of cirrhosis in United States veterans is associated with nonalcoholic fatty liver disease.
      ,
      • Dyson J.
      • Jaques B.
      • Chattopadyhay D.
      • Lochan R.
      • Graham J.
      • Das D.
      • et al.
      Hepatocellular cancer: the impact of obesity, type 2 diabetes and a multidisciplinary team.
      Alr-H-KO mice could be used to study lean NASH and NASH-induced HCC in the absence of cirrhosis, since they do not become overweight/obese or develop cirrhosis.
      Hepatic ALR expression is lower than normal in NAFL,
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      NASH and NASH-cirrhosis.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ,
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      Our analysis showed physiological serum ALR concentrations in the 32-380 pg/ml range (n = 27), indicating interindividual variability; ALR concentrations were significantly lower in NASH (0-336 pg/ml; p <0.05 vs. Control; n = 25) and NASH-cirrhosis (24-380 pg/ml; p <0.05 vs. Control; n = 22). In contrast, serum ALR increases in acute, chronic and fulminant hepatitis due to hepatitis virus A, B or C infection.
      • Tanigawa K.
      • Sakaida I.
      • Masuhara M.
      • Hagiya M.
      • Okita K.
      Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases.
      ,
      • Wang N.
      • Sun H.
      • Tang L.
      • Deng J.
      • Luo Y.
      • Guo H.
      • et al.
      Establishment and primary clinical application of competitive inhibition for measurement of augmenter of liver regeneration.
      Because the liver is the primary source of circulating ALR,
      • Gandhi C.R.
      Augmenter of liver regeneration.
      the increase may be due to ALR released from injured/dying hepatocytes.
      • Vodovotz Y.
      • Prelich J.
      • Lagoa C.
      • Barclay D.
      • Zamora R.
      • Murase N.
      • et al.
      Augmenter of liver regeneration (ALR) is a novel biomarker of hepatocellular stress/inflammation: in vitro, in vivo and in silico studies.
      However, normal serum ALR concentrations reported in these studies ranged from 3.0±1.55 pg/ml
      • Tanigawa K.
      • Sakaida I.
      • Masuhara M.
      • Hagiya M.
      • Okita K.
      Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases.
      to 3.77±1.55 ng/ml.
      • Wang N.
      • Sun H.
      • Tang L.
      • Deng J.
      • Luo Y.
      • Guo H.
      • et al.
      Establishment and primary clinical application of competitive inhibition for measurement of augmenter of liver regeneration.
      Discrepancies in these values may be due to differential specificity of the ALR antibodies used and differences in assay procedures. Thus, a more comprehensive investigation is required to ascertain the serum ALR range in NAFL, NASH and NASH-cirrhosis. Such research might lead to determination of serum ALR concentrations predictive or diagnostic of NASH. Similar or greater hepatic ALR levels in some patients with NASH-cirrhosis compared to healthy individuals
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      indicate heterogeneity in the disease. More intense staining for ALR was also observed in some regenerating liver cell nodules of patients with NASH-cirrhosis.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      The nuclear localisation of ALR suggests that it may also have a nuclear function. Although binding of ALR to JAB1 increases AP1-activity,
      • Dayoub R.
      • Wagner H.
      • Bataille F.
      • Stöltzing O.
      • Spruss T.
      • Buechler C.
      • et al.
      Liver regeneration associated protein (ALR) exhibits antimetastatic potential in hepatocellular carcinoma.
      ,
      • Lu C.
      • Li Y.
      • Zhao Y.
      • Xing G.
      • Tang F.
      • Wang Q.
      • et al.
      Intracrine hepatopoietin potentiates AP-1 activity through JAB1 independent of MAPK pathway.
      ,
      • Wang Y.
      • Lu C.
      • Wei H.
      • Wang N.
      • Chen X.
      • Zhang L.
      • et al.
      Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity.
      how it regulates the expression of proteins involved in lipid homeostasis is unclear. The loss of ALR in vivo and in vitro reduces the expression of carnitine palmitoyl transferase a (CPT1a), sterol regulatory element-binding protein (SREBP)1c, peroxisome proliferator-activated receptor-α (PPARα), peroxisomal membrane protein 70 (PMP70) and acyl-CoA oxidase 1 (ACOX1).
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      ,
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      Forced overexpression of ALR causes upregulation of CPT1a in steatotic hepatocytes.
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      ,
      • Xiao W.
      • Ren M.
      • Zhang C.
      • Li S.
      • An W.
      Amelioration of nonalcoholic fatty liver disease by hepatic stimulator substance via preservation of carnitine palmitoyl transferase-1 activity.
      The mechanism of ALR depletion-induced dysregulated lipid homeostasis appears to involve altered expression of several microRNAs (miRNAs) with binding sequences on mRNAs encoding CPT1a, SREBP1c, PPARα, PMP70 and ACOX1.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      In fact, expression of several miRNAs implicated in human NAFL and NASH are similarly altered in Alr-H-KO mice between 1 (beginning of steatosis) and 4 (NASH-like phenotype) weeks postpartum.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      Increased miR-540 (miR-6801 in humans) was found to affect the expression of CPT1a, SREBP1c, PPARα, PMP70 and ACOX1, and treatment with anti-miR-540 or recombinant ALR between 1 and 2 weeks mitigated steatosis and pericellular fibrosis in Alr-H-KO mice.
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      Although mitochondrial injury-related oxidative stress might be a causal factor for the altered expression of miRNAs and mRNAs, whether ALR influences the binding and activity of nuclear transcription factors responsible for the expression of miRNAs remains to be determined.
      Several single nucleotide polymorphisms found in the human ALR gene cause developmental delay, progressive multiorgan pathologies and death.
      The GFER (ALR) gene contains several single nucleotide polymorphisms, some of which are pathogenic (https://www.ncbi.nlm.nih.gov/snp/) (Table 1). It is noteworthy that children receiving the hypofunctional GFER allele (R194H mutation; rs121908192) from both healthy heterozygous parents develop severe mitochondriopathy, progressive myopathy and partial combined respiratory chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay.
      • Di Fonzo A.
      • Ronchi D.
      • Lodi T.
      • Fassone E.
      • Tigano M.
      • Lamperti C.
      • et al.
      The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.
      Other investigations found that homozygous or heterozygous mutations in GFER (rs121908192; rs1555486560; rs1597063051; rs1597063303; rs771809901) in the same patient induced similar congenital progressive multiorgan pathologies.
      • Nambot S.
      • Gavrilov D.
      • Thevenon J.
      • Bruel A.L.
      • Bainbridge M.
      • Rio M.
      • et al.
      Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.
      ,
      • Calderwood L.
      • Holm I.A.
      • Teot L.A.
      • Anselm I.
      Adrenal insufficiency in mitochondrial disease: a rare case of GFER-related mitochondrial encephalomyopathy and review of the literature.
      Di Fonzo performed ultrasound in only 1 patient and did not report liver involvement.
      • Di Fonzo A.
      • Ronchi D.
      • Lodi T.
      • Fassone E.
      • Tigano M.
      • Lamperti C.
      • et al.
      The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.
      However, ultrasound evaluation for steatosis is only accurate when >25% of the liver is affected, and biopsy is essential to diagnose steatohepatitis. Importantly, liver biopsy of a patient with compound heterozygous mutation (frameshift variants c.219delC [p.(Cys74Alafs∗76)] and [c.259-25_259-24delCA]) showed mitochondrial damage, and centrolobular and portal fibrosclerosis, and electron microscopy revealed a number of pleiomorphic mitochondria containing paracrystalline inclusions.
      • Nambot S.
      • Gavrilov D.
      • Thevenon J.
      • Bruel A.L.
      • Bainbridge M.
      • Rio M.
      • et al.
      Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.
      Very little is known about these mutations in acute and chronic liver diseases. Because subnormal ALR levels are observed in human NAFL, NASH and NASH-induced cirrhosis,
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      ,
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ,
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      it is likely that individuals with inherent ALR deficiency or dysfunction might be predisposed to develop aggressive NASH. Alternatively, steatosis-induced downregulation of ALR might be an important contributing factor to the severity of disease progression.
      ALR deficiency promotes experimental high-fat diet-induced NAFLD/NASH, and its levels are reduced in human NASH and NASH-cirrhosis.
      Table 1Pathogenic SNPs in the GFER gene.
      PositiondbSNPVariant type:Gene: consequence/protein changeClinical significanceClinVar accession
      chr16:1985991 (GRCh38.p13)rs121908192 [Homo sapiens]Variant type: SNVGFER: missense variant

      NP_005253.3:p.Arg194His
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delayRCV000009228.8
      Exon -3alleles: G>AR (Arg) > H (His)

      R [CGC] > H [CAC]
      Inborn genetic diseasesRCV000624237.1
      Disease name: NDRCV000199876.1
      chr16:1985996 (GRCh38.p13)rs370475970 [Homo sapiens]Variant type: SNVGFER: missense variantMitochondrial diseasesRCV000508691.1
      Exon -3alleles: C>TNP_005253.3:p.Arg196Cys

      R [CGC] > C [TGC]
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delayRCV000709773.1
      chr16:1985976 (GRCh38.p13)

      Exon -3
      rs373135339

      [Homo sapiens]
      Variant type: SNV alleles: C>G,TGFER: stop gained missense variant

      NP_005253.3:p.Ser189Ter

      S [TCA] > ∗ [TGA]

      NP_005253.3:p.Ser189Leu

      S [TCA] > L [TTA]
      Inborn genetic diseasesRCV000622535.1
      chr16:1984861 (GRCh38.p13)rs771809901 [Homo sapiens]Variant type: SNVGFER: stop gained

      NP_005253.3:p.Gln125Ter
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delayRCV001254645.1
      Exon 2alleles: C>TQ (Gln) > ∗ (Ter)

      Q [CAG] > ∗ [TAG]
      Disease name: NDRCV000199819.1
      chr16:1984415-1984417 (GRCh38.p13)

      Exon 1
      rs863224028 [Homo sapiens]

      rs1288218335 and rs747241374 have been merged into rs863224028
      Variant type: indel alleles: C>-(delC)GFER: frameshift variantMitochondrial diseasesRCV000508880.1
      NP_005253.3:p.Arg67fs

      R (Arg) > G (Gly)
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delay (pathogenic/likely pathogenic)RCV001270124.2
      Disease name: ND (likely pathogenic)RCV000200750.1
      chr16:1984433-1984435 (GRCh38.p13)rs1555486560 [Homo sapiens]Variant type: indel alleles: delGGFER: frameshift variant

      NP_005253.3:p.Ala73fs

      A (Ala) > P (Pro)
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delayRCV000679993.2
      Exon 1A [GCC] > P [CC]Disease name: ND (likely pathogenic)RCV000676337.1
      chr16: 1985985 (GRCh38)

      chr16: 2035986 (GRCh37)

      Exon 3
      Accession: SCV001760360.1 [Homo sapiens]

      Submitted: (Jul 15, 2021)
      Variant type: SNV alleles: A>GGFER: missense

      NP_005253.3:p.Asp192Gly

      D192G

      A>G
      Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delayRCV001542783.1
      chr16:1984436-1984437 (GRCh38.p13)

      Exon 1
      rs1597063051 [Homo sapiens]Variant type: deletionGFER: frameshift variant

      NP_005253.3:p.Cys74fs

      A (Ala) > A (Ala)

      A [GCC] > A [GC]
      Myopathy, mitochondrial progressive, with congenital cataract and developmental delayRCV000824904.2
      dbSNP, single nucleotide polymorphism database; Indel, insertion and deletion; ND, not determined; SNV, single nucleotide variation.
      Hepatocyte-specific Alr-heterozygous (Alr-H-HET) mice, which demonstrate normal development and function were investigated to examine whether ALR deficiency is a risk factor for NAFLD/NASH.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      De novo lipogenesis and reduced mitochondrial β-oxidation contribute to steatosis in NAFLD.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      The basal expression of SREBP1c, acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and SREBP2 was found to be somewhat higher in Alr-H-HET compared to WT mice.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      A high-fat high-carbohydrate (HF/HC) diet induced greater obesity and hepatic steatosis (increased de novo lipogenesis and depressed lipolysis) in Alr-H-HET mice than in WT mice, with relevant changes in the expression of related enzymes (SREBP1c, ACACA, and FASN).
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      HF/HC-fed Alr-H-HET mice had greater hepatic levels of free fatty acids, triglycerides and cholesterol than their WT counterparts.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      A study published concurrently also reported increased SREBP2 expression and cholesterol accumulation in global Alr-HET mice.
      • Wang X.
      • Dong L.Y.
      • Gai Q.J.
      • Ai W.L.
      • Wu Y.
      • Xiao W.C.
      • et al.
      Lack of augmenter of liver regeneration disrupts cholesterol homeostasis of liver in mice by inhibiting the AMPK pathway.
      The authors went on to show that inactivation of AMPK (AMP-activated protein kinase) leads to increased cholesterol in high-fat diet-fed Alr-HET mice. Accumulation of free cholesterol induces endoplasmic reticulum stress and mitochondrial injury by inhibiting glutathione transport. Furthermore, mitochondrial accumulation of cholesterol causes JNK activation and subsequent apoptosis/necrosis. Thus, based on the cholesterol accumulation observed in liver biopsies of patients with NASH, which correlates with severity of NASH and NASH-fibrosis, it is proposed that increased accumulation of cholesterol is a major contributor to ongoing lipotoxicity in experimental and human NASH.
      • Arguello G.
      • Balboa E.
      • Arrese M.
      • Zanlungo S.
      Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.
      ,
      • Ioannou G.N.
      The role of cholesterol in the pathogenesis of NASH.
      These findings provide further support for the impact of ALR deficiency on NASH development, and are supported by the greater magnitude of reduction in hepatic ALR in HF/HC diet-fed Alr-H-HET mice than WT mice.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      Furthermore, overexpression of ALR via plasmid transfection mitigated high-fat diet-induced hepatic steatosis by increasing CPT1a activity.
      • Xiao W.
      • Ren M.
      • Zhang C.
      • Li S.
      • An W.
      Amelioration of nonalcoholic fatty liver disease by hepatic stimulator substance via preservation of carnitine palmitoyl transferase-1 activity.
      Also, forced downregulation of ALR increased lipid accumulation and lipotoxicity in primary hepatocytes,
      • Kumar S.
      • Rani R.
      • Karns R.
      • Gandhi C.R.
      Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.
      and exogenous ALR reduced these effects.
      • Weiss T.S.
      • Lupke M.
      • Ibrahim S.
      • Buechler C.
      • Lorenz J.
      • Ruemmele P.
      • et al.
      Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.
      HF/HC-fed Alr-H-HET mice had increased inflammation in the liver (greater incidence of TNFα-, IL-6- and IL17-producing cells and lower incidence of FoxP3+ immunosuppressive regulatory T cells) and in white adipose tissue. This model is relevant to human NASH since both female and male mice developed hepatocyte injury, inflammation, stellate cell activation, and fibrosis. Alr-H-KO mice with underlying modest inflammation and fibrosis are resistant to HF/HC-induced obesity and hepatic steatosis but progressed to cirrhosis.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      These findings are akin to human NASH-induced cirrhosis in which steatosis is absent or minimal.
      • Kumar S.
      • Verma A.K.
      • Rani R.
      • Sharma A.
      • Wang J.
      • Shah S.A.
      • et al.
      Hepatic deficiency of augmenter of liver regeneration predisposes to nonalcoholic steatohepatitis and fibrosis.
      ,
      • Caldwell S.H.
      • Oelsner D.H.
      • Iezzoni J.C.
      • Hespenheide E.E.
      • Battle E.H.
      • Driscoll C.J.
      Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease.
      The importance of ALR deficiency in steatohepatitis is exemplified by lower hepatic ALR in human alcohol-related cirrhosis and the occurrence of alcohol-induced cirrhosis within 4 weeks in mice fed the Lieber de Carli diet (while WT mice fed the same diet showed only modest steatosis at the same timepoint).
      • Kumar S.
      • Wang J.
      • Rani R.
      • Gandhi C.R.
      Hepatic deficiency of augmenter of liver regeneration exacerbates alcohol-induced liver injury and promotes fibrosis in mice.
      A similar predisposition to NAFLD or alcohol-related liver disease is likely in humans with 1 functional ALR allele.
      • Di Fonzo A.
      • Ronchi D.
      • Lodi T.
      • Fassone E.
      • Tigano M.
      • Lamperti C.
      • et al.
      The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.
      ,
      • Nambot S.
      • Gavrilov D.
      • Thevenon J.
      • Bruel A.L.
      • Bainbridge M.
      • Rio M.
      • et al.
      Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.
      Paracrystalline inclusions, loss of cristae, and multilamellar membranes in mitochondria of patients with homozygous (R194H) mutations,
      • Di Fonzo A.
      • Ronchi D.
      • Lodi T.
      • Fassone E.
      • Tigano M.
      • Lamperti C.
      • et al.
      The mitochondrial disulfide relay system protein GFER is mutated in autosomal-recessive myopathy with cataract and combined respiratory-chain deficiency.
      ,
      • Nambot S.
      • Gavrilov D.
      • Thevenon J.
      • Bruel A.L.
      • Bainbridge M.
      • Rio M.
      • et al.
      Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.
      abnormalities also recognised in NAFLD,
      • Caldwell S.H.
      • Chang C.Y.
      • Nakamoto R.K.
      • Krugner-Higby L.
      Mitochondria in nonalcoholic fatty liver disease.
      all raise the possibility that ALR dysfunction might be critically involved in mitochondriopathy in NASH. Fig. 3 summarises putative pathways of NAFLD involving deficiency or dysfunction of ALR.
      Figure thumbnail gr3
      Fig. 3Putative mechanisms of NAFLD due to ALR deficiency.
      (1) High-fat Western-style diet causes obesity, insulin resistance and hepatic steatosis. FFAs and inflammatory cytokines released from adipose tissue further contribute to steatosis, which downregulates ALR expression. Inherent deficiency or dysfunction of ALR may also predispose the liver to abnormal lipid homeostasis causing increased steatosis. (2) Decreased hepatic ALR may cause downregulation of TFAM and PGC1α expression resulting in reduced mtDNA stability, transcription and biogenesis. ALR deficiency reduces the activity of the Mia40/ALR disulphide relay system causing disrupted oxidative folding of imported proteins leading to inhibition of mitochondrial function. (3) ALR deficiency increases miR-540, which disrupts mitochondrial and peroxisomal lipid homeostasis by inhibiting expression of CPT1a, PPARα, ACOS-1 and PMP70, thus increasing steatosis. (4) ALR deficiency disrupts mitochondrial respiratory chain activity causing increased ROS generation, reduced ATP synthesis, lipid peroxidation and mtDNA damage. This and FFA toxicity cause hepatocyte injury and consequent release of ROS and DAMPs, which promote activation of hepatic stellate cells to a fibrogenic phenotype and fibrosis development. (5) Reduced ALR in steatohepatitis may also cause dysregulation of iron homeostasis through reduced expression of GLRX5 and hepcidin leading to iron accumulation and toxicity. Dashed red arrows indicate that the mechanisms of those pathways have not been elucidated. ACOX-1, acyl-coenzyme A oxidase 1; ALR, augmenter of liver regeneration; CPT1a, carnitine palmitoyl transferase I; DAMPs, damage-associated molecular patterns; FFAs, free fatty acids; GLRX5, glutaredoxin-5; HSC, hepatic stellate cell; mt, mitochondrial; PMP70, peroxisomal membrane protein 70; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; PPARα, peroxisome proliferator-activated receptor-α; ROS, reactive oxygen species; TFAM, mitochondrial transcription factor A; TG, triglyceride.

      ALR in alcohol-induced liver injury

      Mitochondrial dysfunction is also implicated in alcohol-induced liver injury, another common cause of chronic liver disease worldwide.
      • Mansouri A.
      • Gattolliat C.H.
      • Asselah T.
      Mitochondrial dysfunction and signaling in chronic liver diseases.
      ,
      • Louvet A.
      • Mathurin P.
      Alcoholic liver disease: mechanisms of injury and targeted treatment.
      Alcohol-related liver disease (ALD) also includes a spectrum of conditions such as simple steatosis, hepatitis, fibrosis, cirrhosis, and HCC.
      • Gao B.
      • Bataller R.
      Alcoholic liver disease: pathogenesis and new therapeutic targets.
      Although heavy drinkers are prone to develop hepatic steatosis, about 20-40% progress to alcohol-related steatohepatitis and 8-20% advance to cirrhosis.
      • Ohashi K.
      • Pimienta M.
      • Seki E.
      Alcoholic liver disease: a current molecular and clinical perspective.
      It is postulated that genetic and environmental factors are drivers of ALD from simple steatosis to fibrosis and cirrhosis.
      • Louvet A.
      • Mathurin P.
      Alcoholic liver disease: mechanisms of injury and targeted treatment.
      ,
      • Purohit V.
      • Russo D.
      • Coates P.M.
      Role of fatty liver, dietary fatty acid supplements, and obesity in the progression of alcoholic liver disease: introduction and summary of the symposium.
      Like humans, most animal models are resistant to more aggressive ALD, and steatosis is readily reversed upon termination of alcohol ingestion. The Lieber Di Carli liquid alcohol diet, which has been used extensively to study ALD in mice, caused steatosis in control mice, but promoted aggressive liver disease leading to cirrhosis, accompanied by reduced expression of alcohol dehydrogenases-1 and aldehyde dehydrogenases-1, in ALR-deficient mice.
      • Kumar S.
      • Wang J.
      • Rani R.
      • Gandhi C.R.
      Hepatic deficiency of augmenter of liver regeneration exacerbates alcohol-induced liver injury and promotes fibrosis in mice.
      There was also significant mitochondrial damage and iron accumulation (lower glutaredoxin-5 and hepcidin expression) in alcohol-fed ALR-deficient mice. The clinical significance of these findings is indicated by significantly lower hepatic ALR expression in patients with alcohol-related cirrhosis.
      • Gandhi C.R.
      • Chaillet J.R.
      • Nalesnik M.A.
      • Kumar S.
      • Dangi A.
      • Demetris A.J.
      • et al.
      Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.
      ,
      • Kumar S.
      • Wang J.
      • Rani R.
      • Gandhi C.R.
      Hepatic deficiency of augmenter of liver regeneration exacerbates alcohol-induced liver injury and promotes fibrosis in mice.
      However, Alr-H-KO mice already have underlying modest oxidative stress, inflammation and fibrosis that are accelerated/augmented by alcohol, as demonstrated by further increases in oxidative stress, robust lipid peroxidation and mtDNA damage. Such underlying conditions in humans are likely a prerequisite for aggressive ALD. ALR was also shown to protect mice from alcohol-induced acute liver injury by promoting autophagy through repression of mTOR (mammalian target of rapamycin).
      • Liu L.
      • Xie P.
      • Li W.
      • Wu Y.
      • An W.
      Augmenter of liver regeneration protects against ethanol-induced acute liver injury by promoting autophagy.
      Furthermore, overexpression of ALR improved mitochondrial membrane potential and increased ATP levels in alcohol-treated HepG2 cells.
      • Liu L.
      • Xie P.
      • Li W.
      • Wu Y.
      • An W.
      Augmenter of liver regeneration protects against ethanol-induced acute liver injury by promoting autophagy.
      NRF2, which upregulates ALR during oxidative stress, is protective against alcohol-induced hepatic and pancreatic damage.
      • Sun J.
      • Fu J.
      • Zhong Y.
      • Li L.
      • Chen C.
      • Wang X.
      • et al.
      NRF2 mitigates acute alcohol-induced hepatic and pancreatic injury in mice.
      ALR deficiency or dysfunction may be an important risk factor for NASH.

      Conclusions and future prospects

      Despite being an evolutionally conserved fundamental life protein with varied functions, understanding of the role of ALR in physiology and pathophysiology has been inadequate. ALR is critically important for mitochondrial biogenesis, protein folding (Mia40-sulfhydryl relay system), and respiratory chain activity, disruption of which is implicated in the pathogenesis and progression of both NAFLD and ALD. In vivo and in vitro studies of ALR-knockdown and hepatocyte-specific ALR deficiency have provided crucial evidence of ALR’s role in lipid homeostasis and in promoting the expression of several genes, including those involved in alcohol and iron metabolism. The clinical significance of ALR in NASH and ASH is inferred from its lower hepatic concentration in human NASH- and ASH-cirrhosis. Because of its lack of a DNA-binding sequence, ALR may not be directly involved in gene transcription but may act as a promoter or suppressor of certain transcription factors. In this regard, ALR immunoprecipitates with TFAM (unpublished observation), and deficiency of ALR downregulates TFAM expression. Several pathogenic single nucleotide polymorphisms are found to cause severe mitochondrial damage and progressive multiorgan disease in humans. Liver biopsy of 1 patient with mutations in both ALR alleles showed hepatic mitochondrial damage and fibrosis. Thus, humans with heterozygous mutations in the ALR gene could be predisposed to chronic liver diseases such as NASH and ASH. Future investigations to further delineate mechanisms by which ALR deficiency or dysfunction promotes NAFLD or ALD progression will be important.

      Abbreviations

      ACACA, acetyl-CoA carboxylase alpha; ACOX1, acyl-CoA oxidase 1; ALD, alcohol-related liver disease; ALR, augmenter of liver regeneration; CCl4, carbon tetrachloride; C/EBPβ, CCAAT/enhancer binding proteins; CPT1a, carnitine palmitoyl transferase I a; Egr-1, Early growth response protein-1; ERV1, essential for respiration and vegetative growth-1; ESLD, end-stage liver disease; FAD, flavin adenine dinucleotide; FASN, fatty acid synthase; FoxA2, forkhead box A2; HCC, hepatocellular carcinoma; HF/HC, high-fat high carbohydrate; HNF4α, hepatocyte nuclear factor 4 alpha; l-ALR, long form ALR; IMS, intermembrane space; JAB1, Jun activation domain-binding protein 1; Mia40, mitochondrial IMS import and assembly protein 40 kDa; miRNA, microRNA; mtDNA, mitochondrial DNA; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NRF2, nuclear factor erythroid 2-related factor 2; nt, nucleotide; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1-alpha; PMP70, peroxisomal membrane protein 70; PPARα, peroxisome proliferator-activated receptor alpha; rALR, recombinant ALR; ROS, reactive oxygen species; s-ALR, short form ALR, scERV1, Saccharomyces cerevisiae essential for respiration and vegetative growth-1; SHP, small heterodimer partner; SREBP, sterol regulatory element-binding protein; TFAM, mitochondrial transcription factor A.

      Financial support

      Department of Defense grant #W81XWH2010477 .

      Authors’ contributions

      All authors contributed to the writing of this manuscript.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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