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Autophagy in liver diseases

  • Pierre-Emmanuel Rautou
    Correspondence
    Corresponding author. Address: Service d’Hépatologie, Hôpital Beaujon, 92 110 Clichy, France. Tel.: +33 1 40 87 55 19; fax: +33 1 40 87 44 26.
    Affiliations
    Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy, France

    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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  • Abdellah Mansouri
    Affiliations
    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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  • Didier Lebrec
    Affiliations
    Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy, France

    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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  • François Durand
    Affiliations
    Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy, France

    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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  • Dominique Valla
    Affiliations
    Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy, France

    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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  • Richard Moreau
    Affiliations
    Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy, France

    INSERM, U773, Centre de Recherche Bichat-Beaujon CRB3, Clichy, France

    Université Denis Diderot-Paris 7, 75018 Paris, France
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Open AccessPublished:August 02, 2010DOI:https://doi.org/10.1016/j.jhep.2010.07.006
      Autophagy, or cellular self-digestion, is a cellular pathway crucial for development, differentiation, survival, and homeostasis. Its implication in human diseases has been highlighted during the last decade. Recent data show that autophagy is involved in major fields of hepatology. In liver ischemia reperfusion injury, autophagy mainly has a prosurvival activity allowing the cell for coping with nutrient starvation and anoxia. During hepatitis B or C infection, autophagy is also increased but subverted by viruses for their own benefit. In hepatocellular carcinoma, the autophagy level is decreased. In this context, autophagy has an anti-tumor role and therapeutic strategies increasing autophagy, as rapamycin, have a beneficial effect in patients. Moreover, in hepatocellular carcinoma, Beclin-1 level, an autophagy protein, has a prognostic significance. In α-1-antitrypsin deficiency, the aggregation-prone ATZ protein accumulates in the endoplasmic reticulum. This activates the autophagic response which aims at degrading mutant ATZ. Some FDA-approved drugs which enhance autophagy and the disposal of aggregation-prone proteins may be useful in α-1-antitrypsin deficiency. Following alcohol consumption, autophagy is decreased in liver cells, likely due to a decrease in intracellular 5′-AMP-activated protein kinase (AMPk) and due to an alteration in vesicle transport in hepatocytes. This decrease in autophagy contributes to the formation of Mallory-Denk bodies and to liver cell death. Hepatic autophagy is defective in the liver in obesity and its upregulation improves insulin sensitivity.

      Keywords

      Abbreviations:

      α1AT (alpha-1-antitrypsin), AMPk (5′-AMP-activated protein Kinase), ATZ (α1AT mutant Z gene), Atg (autophagy genes), Bcl (B-cell leukemia/lymphoma), HCC (hepatocellular carcinoma), HBV (hepatitis B virus), HCV (hepatitis C virus), LC3 (microtubule-associated protein light chain 3), mTOR (mammalian target of rapamycin), Nrf2 (nuclear factor erythroid 2-related factor 2), PI3K (phosphatidylinositol 3-kinase), ULK1 (uncoordinated 51-like kinase 1)

      Definition and molecular machinery of autophagy

      Autophagy (Greek for “self eating”) is a general term for processes by which cytoplasmic materials, including organelles, reach lysosomes for degradation. Three types of autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy) have been identified and they differ with respect to their physiological functions and mode of cargo delivery to the lysosome.
      This review will focus on macroautophagy (hereafter referred to as autophagy), the major regulated catabolic mechanism that eukaryotic cells use to degrade long-lived proteins and organelles [
      • Mizushima N.
      • Levine B.
      • Cuervo A.M.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      ]. This pathway is conserved from yeast to mammals (Fig. 1). Upon induction, a small vesicular sac called the isolation membrane or phagophore elongates and subsequently encloses a portion of cytoplasm, which results in the formation of a double-membraned structure, the autophagosome (Fig. 2). Recent data show that the outer membrane of mitochondria participates in autophagosome biogenesis [
      • Hailey D.W.
      • Rambold A.S.
      • Satpute-Krishnan P.
      • Mitra K.
      • Sougrat R.
      • Kim P.K.
      • et al.
      Mitochondria supply membranes for autophagosome biogenesis during starvation.
      ]. Then, the outer membrane of the autophagosome fuses with a lysosome (to form an autolysosome), leading to the degradation of the enclosed materials together with the inner autophagosomal membrane. Amino acids and other small molecules that are generated by autophagic degradation are delivered back to the cytoplasm for recycling or energy production. Autophagy occurs at low basal levels in virtually all cells to perform homeostatic functions such as protein and organelle turnover. It is rapidly upregulated through the inhibition of mammalian target of rapamycin (mTOR) when cells need to generate intracellular nutrients and energy, for example, during starvation, growth factor withdrawal, or high bioenergetic demands [
      • Mizushima N.
      • Levine B.
      • Cuervo A.M.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      ,
      • Mehrpour M.
      • Esclatine A.
      • Beau I.
      • Codogno P.
      Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview.
      ]. Subsequently, prolonged starvation reactivates mTOR signaling that both attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell [
      • Yu L.
      • McPhee C.K.
      • Zheng L.
      • Mardones G.A.
      • Rong Y.
      • Peng J.
      • et al.
      Termination of autophagy and reformation of lysosomes regulated by mTOR.
      ].
      Figure thumbnail gr1
      Fig. 1Cellular and molecular aspects of autophagy. Note that wortmannin inhibits both class I (inhibitory) and class III PI3K, but the overall phenotypic effect is to inhibit autophagy as represented here. Abbreviations: AMPk, adenosine monophosphate-activated protein kinase; Atg, autophagy genes; Bcl-2, B-cell leukemia/lymphoma 2; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase.
      Figure thumbnail gr2
      Fig. 2Electron micrographs showing ultrastructure of hepatocytes from a chronic hepatitis C patient. Black arrows point to autophagic vacuoles. (A) Low-magnification image showing hepatocytes containing several autophagic vacuoles (original magnification, 8000×). (B) Partial view of a hepatocyte containing an autophagic vacuole (original magnification, 100,000×). F, fibrosis; ld, lipid droplet; m, mitochondria; n, nucleus.
      The execution of autophagy involves a set of evolutionarily conserved gene products (initially identified in yeast) known as the Atg proteins that are required for the formation of the isolation membrane and the autophagosome. The process of autophagosome formation involves three major steps described in Fig. 3: initiation with the uncoordinated 51-like kinase 1 (ULK1) complex, nucleation with the Beclin-1-class III phosphatidylinositol 3-kinase (PI3K) complex and elongation of the isolation membrane with a key role of microtubule-associated protein light chain 3 (LC3) lipidation [
      • Mehrpour M.
      • Esclatine A.
      • Beau I.
      • Codogno P.
      Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview.
      ,
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ,
      • He C.
      • Levine B.
      The Beclin 1 interactome.
      ].
      Figure thumbnail gr3
      Fig. 3Basic molecular machinery of autophagy. There are at least three steps in the formation of autophagosomes: initiation, nucleation, and elongation/closure. Autophagy is initiated by the ULK1 complex. This complex is formed by ULK1 Ser/Thr protein kinase, Atg13, and FIP200. Among the initial steps of vesicle nucleation is the activation of the class III PI3K (Vps34) to generate phosphatidylinositol 3-phosphate. This activation depends on the formation of a multiprotein complex that includes Beclin-1, Vps15, Atg14L (Atg14-like protein), and Ambra1. Beclin-1 constitutively interacts with Bcl-2 or its close homolog Bcl-XL and autophagy induction requires the dissociation of Beclin-1 from its inhibitors Bcl-2 or Bcl-XL
      [
      • He C.
      • Levine B.
      The Beclin 1 interactome.
      ]
      . The functional relationship between the ULK1 complex (initiation) and Beclin-1-class III PI3K complex (nucleation) complexes remains to be determined
      [
      • Mehrpour M.
      • Esclatine A.
      • Beau I.
      • Codogno P.
      Overview of macroautophagy regulation in mammalian cells.
      ]
      . Vesicle elongation, vesicle completion. The membrane formed elongates and closes on itself to form an autophagosome. Two conjugation systems are successively involved. The first involves the covalent conjugation of Atg12 to Atg5, with the help of Atg7 and Atg10. This conjugate is organized into a complex by associating with Atg16 to form the Atg16–Atg5–Atg12 complex. The second involves the conjugation of phosphatidylethanolamine (PE) to a LC3 by the sequential action of the Atg4, Atg7, and Atg3. This lipid conjugation leads to the conversion of the soluble form of LC3 (named LC3-I) to the autophagic vesicle-associated form (LC3-II), allowing for the closure of the autophagic vacuole. Abbreviations: Ambra1, activating molecule in Beclin-1-regulated autophagy; Atg, autophagy genes; Bcl, B-cell leukemia/lymphoma; FIP200, 200-kDa focal adhesion kinase family-interacting protein; LC3, microtubule-associated protein light chain 3; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; ULK1, uncoordinated 51-like kinase 1; Vps, vacuolar protein sorting.
      Information on the methods for monitoring autophagy can be found elsewhere [
      • Mizushima N.
      • Yoshimori T.
      • Levine B.
      Methods in mammalian autophagy research.
      ].
      Figure thumbnail fx2

      Autophagy and liver ischemia reperfusion and liver surgery

      Liver ischemia/reperfusion injury occurs during liver transplantation, trauma, shock, and elective liver resection. During this process, hypoxic organ damage is accentuated following the return of blood flow and oxygen delivery. The pathophysiology includes direct cellular damage as a result of the ischemic insult and delayed dysfunction and injury resulting from inflammatory pathway activation [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.A.
      Protective strategies against ischemic injury of the liver.
      ].
      As the first known role of autophagy is its action during nutrient starvation, studies on autophagy and liver diseases have rapidly focussed on liver ischemia/reperfusion [
      • Mortimore G.E.
      • Schworer C.M.
      Induction of autophagy by amino-acid deprivation in perfused rat liver.
      ,
      • Pfeifer U.
      Inhibited autophagic degradation of cytoplasm during compensatory growth of liver cells after partial hepatectomy.
      ,
      • Schneider P.D.
      • Gorschboth C.M.
      Limiting ischemic liver injury by interfering with lysosomal autophagy.
      ]. Two types of experimental protocols have been performed reflecting two different clinical situations: (a) hepatic ischemia induced by occlusion of the portal triad for a duration ranging from 30 to 90 min, followed or not by a reperfusion period ranging from 30 min to 3 h [
      • Shin T.
      • Kuboki S.
      • Huber N.
      • Eismann T.
      • Galloway E.
      • Schuster R.
      • et al.
      Activation of peroxisome proliferator-activated receptor-gamma during hepatic ischemia is age-dependent.
      ,
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ,
      • Cardinal J.
      • Pan P.
      • Dhupar R.
      • Ross M.
      • Nakao A.
      • Lotze M.
      • et al.
      Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury.
      ]; and (b) liver transplantation with 24 h cold ischemia followed by reperfusion [
      • Gotoh K.
      • Lu Z.
      • Morita M.
      • Shibata M.
      • Koike M.
      • Waguri S.
      • et al.
      Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation.
      ,
      • Lu Z.
      • Dono K.
      • Gotoh K.
      • Shibata M.
      • Koike M.
      • Marubashi S.
      • et al.
      Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.
      ,
      • Minor T.
      • Stegemann J.
      • Hirner A.
      • Koetting M.
      Impaired autophagic clearance after cold preservation of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning.
      ].
      Despite very similar protocols, results of studies performed in mice, assessing the impact on autophagy of portal triad occlusion, are highly controversial, some reporting an increase and others a decrease in autophagy protein level [
      • Shin T.
      • Kuboki S.
      • Huber N.
      • Eismann T.
      • Galloway E.
      • Schuster R.
      • et al.
      Activation of peroxisome proliferator-activated receptor-gamma during hepatic ischemia is age-dependent.
      ,
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ,
      • Cardinal J.
      • Pan P.
      • Dhupar R.
      • Ross M.
      • Nakao A.
      • Lotze M.
      • et al.
      Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury.
      ]. Nevertheless, the unique study performed in patients provides interesting information [
      • Domart M.C.
      • Esposti D.D.
      • Sebagh M.
      • Olaya N.
      • Harper F.
      • Pierron G.
      • et al.
      Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy.
      ]. In this study, 61 patients who underwent liver surgery with total vascular occlusion and preservation of the caval flow after receiving several courses of chemotherapy were studied. For all patients, two liver biopsies were taken, one before the prolonged ischemia required by liver resection and another after the liver reperfusion (median: 88 min; range: 57–125 min), before closure of the abdomen. A unique vascular occlusion had almost no effect on autophagy, since the LC3-II rarely increased and the number of cells containing autophagic vacuoles remained stable. However, a subgroup of patients underwent ischemic preconditioning consisting of 10 min of portal triad clamping followed by 10 min of reperfusion before the prolonged ischemia required by liver resection. In these patients, a frank increase in liver cell autophagy was observed. Even if this study failed to demonstrate a beneficial effect of such ischemic preconditioning in postresection liver injury tests or measure of patient morbidity [
      • Domart M.C.
      • Esposti D.D.
      • Sebagh M.
      • Olaya N.
      • Harper F.
      • Pierron G.
      • et al.
      Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy.
      ], previous studies including specific groups of patients, such as young patients and patients with liver steatosis or cirrhosis obtained a clinical improvement [
      • Li S.Q.
      • Liang L.J.
      • Huang J.F.
      • Li Z.
      Ischemic preconditioning protects liver from hepatectomy under hepatic inflow occlusion for hepatocellular carcinoma patients with cirrhosis.
      ,
      • Clavien P.A.
      • Selzner M.
      • Rudiger H.A.
      • Graf R.
      • Kadry Z.
      • Rousson V.
      • et al.
      A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning.
      ]. This suggests that in this context, autophagy enhancement could allow for decreasing liver cell death (Table 1).
      Table 1Level and function of autophagy in liver diseases.
      Studies on liver transplantation had also apparently contradictory results. The explanation for such discrepancies must be the solution for cold preservation used. Indeed, a decrease in autophagy was observed in a study using a histidine–tryptophan–ketoglutarate cold-storage solution for 24 h cold preservation [
      • Minor T.
      • Stegemann J.
      • Hirner A.
      • Koetting M.
      Impaired autophagic clearance after cold preservation of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning.
      ], while the contrary was reported by authors using the University of Wisconsin (UW) cold-storage solution [
      • Gotoh K.
      • Lu Z.
      • Morita M.
      • Shibata M.
      • Koike M.
      • Waguri S.
      • et al.
      Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation.
      ,
      • Lu Z.
      • Dono K.
      • Gotoh K.
      • Shibata M.
      • Koike M.
      • Marubashi S.
      • et al.
      Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.
      ]. Electron microscopy analysis of surgical biopsies performed after revascularisation of human liver grafts conserved in UW solution also disclosed the existence of numerous autophagic vacuoles (personal unpublished data). Importantly, UW cold storage solution does not contain amino acids. It is well demonstrated that amino acid depletion rapidly induces autophagy [
      • Mortimore G.E.
      • Schworer C.M.
      Induction of autophagy by amino-acid deprivation in perfused rat liver.
      ] and that anoxia decreases autophagy protein level [
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ]. This induction of autophagy due to the absence of amino acids, may explain not only the apparent discrepancy between these studies but also the protection of the liver obtained with preservation solution such as the UW solution [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.A.
      Protective strategies against ischemic injury of the liver.
      ]. Indeed, anoxia/reoxygenation induces mitochondrial dysfunction. Due to the decrease in autophagy proteins induced by anoxia/reoxygenation, autophagy fails to remove dysfunctional mitochondria, so that the mitochondria laden with reactive oxygen species and calcium undergo the mitochondrial permeability transition, which in turn leads to uncoupling of oxidative phosphorylation, energetic failure, ATP depletion, and ultimately cell death. In case of associated nutrient depletion, autophagy is enhanced and facilitates autophagy of damaged mitochondria, leading to cell survival [
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ].
      This hypothesis is supported by the beneficial effect on liver tolerance to ischemia–reperfusion of several strategies aiming at increasing autophagy in murine models: stimulation of PPARγ with rosiglitazone, infusion of nontoxic doses of cisplatin and liver graft hypothermic reconditioning by insufflation of gaseous oxygen via the caval vein during the last 90 min of preservation, [
      • Shin T.
      • Kuboki S.
      • Huber N.
      • Eismann T.
      • Galloway E.
      • Schuster R.
      • et al.
      Activation of peroxisome proliferator-activated receptor-gamma during hepatic ischemia is age-dependent.
      ,
      • Cardinal J.
      • Pan P.
      • Dhupar R.
      • Ross M.
      • Nakao A.
      • Lotze M.
      • et al.
      Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury.
      ,
      • Minor T.
      • Stegemann J.
      • Hirner A.
      • Koetting M.
      Impaired autophagic clearance after cold preservation of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning.
      ]. It is striking to notice that the two studies suggesting that inhibiting autophagy could ameliorate liver tolerance to ischemia [
      • Schneider P.D.
      • Gorschboth C.M.
      Limiting ischemic liver injury by interfering with lysosomal autophagy.
      ,
      • Gotoh K.
      • Lu Z.
      • Morita M.
      • Shibata M.
      • Koike M.
      • Waguri S.
      • et al.
      Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation.
      ] used non specific inhibitors of autophagy, such as general lysosome protease inhibitors (pepstatin and leupeptin) or PI3K inhibitors (wortmannin or LY294002), known to also have autophagy independent activities.
      Currently available studies provide additional information. First, autophagic activity declines in aged organisms which could explain at least partly the worse tolerance to ischemia reperfusion in aged patients [
      • Shin T.
      • Kuboki S.
      • Huber N.
      • Eismann T.
      • Galloway E.
      • Schuster R.
      • et al.
      Activation of peroxisome proliferator-activated receptor-gamma during hepatic ischemia is age-dependent.
      ,
      • Zhang C.
      • Cuervo A.M.
      Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function.
      ]. Second, autophagy level decreases following partial hepatectomy suggesting a shift from the physiological steady state between anabolism and catabolism to the positive balance which is required for the compensatory growth of the liver after partial hepatectomy [
      • Pfeifer U.
      Inhibited autophagic degradation of cytoplasm during compensatory growth of liver cells after partial hepatectomy.
      ]. Third, reperfusion had more effect on autophagy level than ischemia alone [
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ,
      • Lu Z.
      • Dono K.
      • Gotoh K.
      • Shibata M.
      • Koike M.
      • Marubashi S.
      • et al.
      Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.
      ,
      • Yu Q.C.
      • Lipsky M.
      • Trump B.F.
      • Marzella L.
      Response of human hepatocyte lysosomes to postmortem anoxia.
      ], which is in line with the histological lesions observed [
      • Selzner N.
      • Rudiger H.
      • Graf R.
      • Clavien P.A.
      Protective strategies against ischemic injury of the liver.
      ]. Unfortunately, no study has yet specifically evaluated the autophagic pathway in liver sinusoidal endothelial cells. This is a limitation in understanding the effect of autophagy in liver ischemia reperfusion injury since these cells are the most sensitive to ischemia and lesions to these cells are a key event in this context.

      Autophagy and viral hepatitis

      Besides the physiological function of autophagy in maintaining cellular homeostasis, autophagy is a newly recognized facet of innate and adaptative immunity. Not surprisingly, certain viruses such as hepatitis C virus (HCV) and hepatitis B virus (HBV) have developed strategies to subvert or use autophagy for their own benefit [
      • Deretic V.
      • Levine B.
      Autophagy, immunity, and microbial adaptations.
      ] (Table 1).
      Several studies have assessed the autophagic pathway in hepatocytes infected with HCV both in vitro and in liver biopsies from chronic hepatitis C patients [
      • Ait-Goughoulte M.
      • Kanda T.
      • Meyer K.
      • Ryerse J.S.
      • Ray R.B.
      • Ray R.
      Hepatitis C virus genotype 1a growth and induction of autophagy.
      ,
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ,
      • Rautou P.E.
      • Cazals-Hatem D.
      • Feldmann G.
      • Asselah T.
      • Grodet A.
      • Mansouri A.
      • et al.
      In vivo evidence of altered hepatocyte autophagic response in livers from patients with chronic Hepatitis C virus infection.
      ,
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ,
      • Mizui T.
      • Yamashina S.
      • Tanida I.
      • Takei Y.
      • Ueno T.
      • Sakamoto N.
      • et al.
      Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.
      ,
      • Tanida I.
      • Fukasawa M.
      • Ueno T.
      • Kominami E.
      • Wakita T.
      • Hanada K.
      Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles.
      ]. Whatever the approach used (LC3, Atg5 or Beclin-1 immunoblotting, electron microscopy or GFP-LC3 immunofluorescence), these studies consistently demonstrated an accumulation of autophagic vacuoles in HCV-infected hepatocytes. This increase was independent of HCV genotype since it was observed in vitro in cells harboring the HCV strain H77 (genotype 1a), Con1 (genotype 1b) and JFH1 (genotype 2a) [
      • Ait-Goughoulte M.
      • Kanda T.
      • Meyer K.
      • Ryerse J.S.
      • Ray R.B.
      • Ray R.
      Hepatitis C virus genotype 1a growth and induction of autophagy.
      ,
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ,
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ] and also in patients infected with HCV genotypes 1, 2, 3 and 4 [
      • Rautou P.E.
      • Cazals-Hatem D.
      • Feldmann G.
      • Asselah T.
      • Grodet A.
      • Mansouri A.
      • et al.
      In vivo evidence of altered hepatocyte autophagic response in livers from patients with chronic Hepatitis C virus infection.
      ]. However, this autophagy was inefficient. Indeed, although HCV JFH1 induced autophagosomes, it was not able to enhance autophagic protein degradation [
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ].
      Contrary to certain viruses such as vesicular stomatitis virus and mutant herpes simplex virus 1, that can be captured and eliminated by the autophagic pathway, HCV has evolved to avoid and subvert autophagy using three strategies (Fig. 4) [
      • Deretic V.
      • Levine B.
      Autophagy, immunity, and microbial adaptations.
      ].
      Figure thumbnail gr4
      Fig. 4HCV adaptations to evade and take advantage of autophagy. (1) HCV avoids its recognition by the autophagic machinery. (2) HCV prevents the maturation of the autophagosome into an autolysosome. (3) HCV utilizes functions or components of autophagy to enhance intracellular replication. HCV cycle. (a) HCV binding to cell surface receptors; (b) internalization; (c) fusion with endosomal membranes, allowing release of the plus-strand RNA viral genome into the cytosol; (d) translation of viral RNA; (e) HCV replication; (f) production of progeny viruses; (g) secretion. Atg, autophagy genes.
      First, HCV seems to avoid its recognition by the autophagic machinery. Indeed, both immuno-electron microscopy and immunofluorescence studies revealed no or rare co-localization of HCV proteins with autophagic vacuoles [
      • Ait-Goughoulte M.
      • Kanda T.
      • Meyer K.
      • Ryerse J.S.
      • Ray R.B.
      • Ray R.
      Hepatitis C virus genotype 1a growth and induction of autophagy.
      ,
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ,
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ,
      • Tanida I.
      • Fukasawa M.
      • Ueno T.
      • Kominami E.
      • Wakita T.
      • Hanada K.
      Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles.
      ].
      Second, HCV prevents the maturation of the autophagosome into an autolysosome, as supported by the following elements: (a) the increase in the number of autophagic vacuole without enhancement in autophagic protein degradation [
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ]; (b) the absence of co-localization of lysosomes (stained with LysoTracker-red) with autophagic vacuoles (GFP-LC3) in HCV-infected cells contrary to starved cells [
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ]; (c) the reduction in the number of autophagic vacuoles following HCV elimination using interferon alpha for 14 days [
      • Mizui T.
      • Yamashina S.
      • Tanida I.
      • Takei Y.
      • Ueno T.
      • Sakamoto N.
      • et al.
      Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.
      ]; (d) the absence of increase in the number of late autophagic vesicles in hepatocytes from chronic hepatitis C patients as compared to controls, while a strong augmentation in the number of autophagic vesicles is observed [
      • Rautou P.E.
      • Cazals-Hatem D.
      • Feldmann G.
      • Asselah T.
      • Grodet A.
      • Mansouri A.
      • et al.
      In vivo evidence of altered hepatocyte autophagic response in livers from patients with chronic Hepatitis C virus infection.
      ]. This may be related to a lack of fusion between autophagosome and lysosome.
      Third, HCV utilizes functions or components of autophagy to enhance its intracellular replication. Indeed, it has been recently shown that autophagy proteins are required for translation and/or delivery of incoming HCV RNA to the cell translation apparatus [
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ]. However, autophagy proteins are not needed for the translation of progeny HCV once replication is established since down-regulation of autophagy proteins 10 days after transduction had no effect on HCV replication. Therefore, authors hypothesized that, by remodelling endoplasmic reticulum membranes, the autophagy proteins or autophagic vesicles might provide an initial membranous support for translation of incoming RNA, prior to accumulation of viral proteins and the eventual establishment of virus-induced cellular modifications. Alternatively, autophagy proteins might contribute directly or indirectly to the cytoplasmic transport of the incoming RNA to cellular factors or sites that are required for its translation [
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ]. Importantly, autophagy proteins are required neither for HCV entry nor for HCV secretion [
      • Dreux M.
      • Gastaminza P.
      • Wieland S.F.
      • Chisari F.V.
      The autophagy machinery is required to initiate hepatitis C virus replication.
      ,
      • Tanida I.
      • Fukasawa M.
      • Ueno T.
      • Kominami E.
      • Wakita T.
      • Hanada K.
      Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles.
      ]. Altogether, these data explain the apparent contrast between the results of some in vitro studies reporting the implication of autophagy proteins in HCV replication [
      • Sir D.
      • Chen W.L.
      • Choi J.
      • Wakita T.
      • Yen T.S.
      • Ou J.H.
      Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.
      ,
      • Mizui T.
      • Yamashina S.
      • Tanida I.
      • Takei Y.
      • Ueno T.
      • Sakamoto N.
      • et al.
      Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.
      ,
      • Tanida I.
      • Fukasawa M.
      • Ueno T.
      • Kominami E.
      • Wakita T.
      • Hanada K.
      Knockdown of autophagy-related gene decreases the production of infectious hepatitis C virus particles.
      ] and the absence of correlation between the number of autophagic vacuoles or the LC3-II level and the HCV load in chronic hepatitis C patients [
      • Rautou P.E.
      • Cazals-Hatem D.
      • Feldmann G.
      • Asselah T.
      • Grodet A.
      • Mansouri A.
      • et al.
      In vivo evidence of altered hepatocyte autophagic response in livers from patients with chronic Hepatitis C virus infection.
      ]: autophagy proteins are required only for initial steps of HCV replication, but not once replication is established.
      Notably, cytosolic RNA-sensing protein kinase PKR and eIF2-α phosphorylation regulate virus- and starvation-induced autophagy [
      • Talloczy Z.
      • Jiang W.
      • HWt Virgin.
      • Leib D.A.
      • Scheuner D.
      • Kaufman R.J.
      • et al.
      Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway.
      ,
      • Talloczy Z.
      • HWt Virgin.
      • Levine B.
      PKR-dependent autophagic degradation of herpes simplex virus type 1.
      ]. It is tempting to speculate that recognition of the incoming HCV RNA by RNA-sensing molecules induces autophagy and hence, favours its initial translation. Alternatively, constitutive basal autophagic vesicle formation might be required for this initial HCV RNA translation.
      In conclusion, these data show that autophagy proteins are proviral factors for HCV.
      HBV also induces autophagosomes in liver cells, as demonstrated both in vitro in several liver derived cell lines and in vivo in the liver of transgenic mouse lines harboring low (Tg08) and high (Tg05) replication levels of the HBV DNA. Importantly, this induction was also observed in the liver of an HBV-infected patient but not of a non-infected patient [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ]. Contrary to HCV, HBV can enhance the autophagic flux, as late autophagic vacuoles could be detected in mouse hepatocytes using electron microscopy and given the existence of an extensive co-localization of lysosome-associated membrane protein 1 (LAMP1) with GFP–LC3 puncta (Fig. 5). However, without being able to provide the reason for it, no significant increase in protein degradation was observed in HBV DNA-transfected cells [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ].
      Figure thumbnail gr5
      Fig. 5HBV cycle. (a) HBV binding to cell surface receptors; (b) endocytosis; (c) transfer of the partially double stranded viral DNA to nucleus (d) repair to obtain fully double stranded and transformation into covalently closed circular DNA (cccDNA); (e) transcription; (f) the envelope proteins insert themselves as integral membrane proteins into the lipid membrane of the endoplasmic reticulum; (g) translation; (h) the pregenomic RNA (pgRNA) is packaged together with HBV polymerase and a protein kinase into core particles where it serves as a template for reverse transcription of negative-strand DNA (i); mature, viral nucleocapsids can follow two different intracellular pathways: the formation and secretion of new virions (j), or the amplification of the viral genome inside the cell nucleus (k). Implication of autophagy proteins in HBV life cycle: autophagy proteins are required mainly for HBV DNA replication (i), have a marginal effect on HBV RNA transcription and have no impact on other steps of HBV life cycle. As shown in the lower part of the picture, HBV can induce autophagic vacuole formation.
      An HBV-encoding protein, HBx, plays a crucial role in this HBV-induced autophagy [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ,
      • Tang H.
      • Da L.
      • Mao Y.
      • Li Y.
      • Li D.
      • Xu Z.
      • et al.
      Hepatitis B virus X protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression.
      ]. Indeed, transfection of Huh7.5 cells with an HBV unable to express HBx did not enhance autophagy. Moreover, expression of HBx alone was sufficient to induce autophagy; similar results were obtained in vivo in transgenic mice [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ]. This effect of HBx is due, at least partly, to its ability to bind to class III PI3K, a regulatory molecule that controls autophagy. Although conflicting, HBx may also upregulate the transcription of beclin-1, a protein that forms a complex with class III PI3K and thus sensitizes the cells to starvation-induced autophagy [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ,
      • Tang H.
      • Da L.
      • Mao Y.
      • Li Y.
      • Li D.
      • Xu Z.
      • et al.
      Hepatitis B virus X protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression.
      ]. Whether the role of HBx is confined to short nutrient starvation conditions (8 h) or also exists in normal conditions remains controversial [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ,
      • Tang H.
      • Da L.
      • Mao Y.
      • Li Y.
      • Li D.
      • Xu Z.
      • et al.
      Hepatitis B virus X protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression.
      ].
      If, in the same way as HCV, HBV subverts autophagy, the strategy applied is somewhat different (Fig. 5). Autophagy enhances HBV replication mostly at the step of viral DNA replication, slightly at the step of RNA transcription, and not at other levels [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ]. How autophagy may enhance HBV DNA replication remains unresolved.
      The question whether HBV could be engulfed in autophagic vacuoles is not fully elucidated despite the observation that HBV core/e antigens and surface antigens partially co-localized with autophagic vacuoles [
      • Sir D.
      • Tian Y.
      • Chen W.L.
      • Ann D.K.
      • Yen T.S.
      • Ou J.H.
      The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication.
      ]. Immuno-electron microscopy studies would be required to address this issue. However, as HBV seems to benefit from autophagy proteins and as the autophagic protein degradation rate is not increased, this hypothesis seems unlikely and autophagic vacuoles may rather serve as the sites for viral DNA replication and morphogenesis.

      Autophagy and acute liver injury

      Data regarding autophagy in acute liver injury are scarce. Two classic models of acute liver injury have been used: the concanavalin A and the lipopolysaccharide/d-galactosamine induced acute hepatitis [
      • Yang M.C.
      • Chang C.P.
      • Lei H.Y.
      Endothelial cells are damaged by autophagic induction before hepatocytes in Con A-induced acute hepatitis.
      ,
      • Chang C.P.
      • Yang M.C.
      • Liu H.S.
      • Lin Y.S.
      • Lei H.Y.
      Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model.
      ,
      • Chang C.P.
      • Lei H.Y.
      Autophagy induction in T cell-independent acute hepatitis induced by concanavalin A in SCID/NOD mice.
      ,
      • Wang K.
      • Damjanov I.
      • Wan Y.J.
      The protective role of pregnane X receptor in lipopolysaccharide/d-galactosamine-induced acute liver injury.
      ]. In both models, autophagy was enhanced in mice liver. However, results are discordant for what regards the suggested role of autophagy. In concanavalin A-induced acute liver injury, concanavalin A induced autophagic cell death in hepatocytes and likely also in liver endothelial cells [
      • Yang M.C.
      • Chang C.P.
      • Lei H.Y.
      Endothelial cells are damaged by autophagic induction before hepatocytes in Con A-induced acute hepatitis.
      ,
      • Chang C.P.
      • Yang M.C.
      • Liu H.S.
      • Lin Y.S.
      • Lei H.Y.
      Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model.
      ,
      • Chang C.P.
      • Lei H.Y.
      Autophagy induction in T cell-independent acute hepatitis induced by concanavalin A in SCID/NOD mice.
      ]. On the contrary, in lipopolysaccharide/d-galactosamine-induced acute liver injury, autophagy may be hepatoprotective. Indeed, lipopolysaccharide/d-galactosamine increased autophagy in the livers of both wild-type and transgenic mice deficient of pregnane X receptor (PXR). Autophagy level was sustained in the former group but decreased rapidly in the latter. In parallel, the transgenic mice displayed more severe liver injury than wild-type mice [
      • Wang K.
      • Damjanov I.
      • Wan Y.J.
      The protective role of pregnane X receptor in lipopolysaccharide/d-galactosamine-induced acute liver injury.
      ] (Table 1).
      However, the extrapolation of these data to human acute liver injury is uncertain. Indeed, electron microscopy analysis performed on liver biopsies from 5 patients with acute liver disease (2 with autoimmune hepatitis, 1 drug induced acute liver injury, 1 acute fatty liver of pregnancy and 1 Mauriac syndrome) did not show elements suggesting induction of autophagy [
      • Rautou P.E.
      • Cazals-Hatem D.
      • Moreau R.
      • Francoz C.
      • Feldmann G.
      • Lebrec D.
      • et al.
      Acute liver cell damage in patients with anorexia nervosa: a possible role of starvation-induced hepatocyte autophagy.
      ].
      Increased autophagy has only been reported in patients with acute liver insufficiency in a context of anorexia nervosa, a rare cause of liver failure. The role of autophagy in anorexia nervosa seems to be dual. As illustrated in Figure 6, initially, when body weight decreases, liver tests abnormalities are moderate, suggesting that autophagy can cope with nutrient starvation. However, when undernutrition reaches a critical level (body mass index at 13 or less), a flare in alanine aminotransferase (ALT) level occurs together with liver insufficiency. At that time, hepatocytes contain numerous autophagic vacuoles and interestingly, some liver cells present with the characteristic features of autophagic cell death, also called type 2 programmed cell death. This type of cell death could explain the contrast between the high serum alanine aminotransferase level (on average 1900 IU/L) and the absence of hepatocyte necrosis or liver cell apoptosis upon histological analysis. Indeed, while apoptosis cell plasma membrane integrity is preserved, during autophagic cell death the permeability increases, so that the aminotrasferase can be released in the blood flow [
      • Rautou P.E.
      • Cazals-Hatem D.
      • Moreau R.
      • Francoz C.
      • Feldmann G.
      • Lebrec D.
      • et al.
      Acute liver cell damage in patients with anorexia nervosa: a possible role of starvation-induced hepatocyte autophagy.
      ]. Data obtained in perfused rat liver may explain this dual action of autophagy, since authors show that the degree of amino acid deprivation determines not only the number of autophagic vacuoles but also the types of cytoplasmic elements engulfed. Autophagic vacuoles initially contain glycogen and smooth endoplasmic reticulum, and subsequently mitochondria, rough endoplasmic reticulum, and free ribosomes [
      • Mortimore G.E.
      • Schworer C.M.
      Induction of autophagy by amino-acid deprivation in perfused rat liver.
      ]. This second step may jeopardize cell survival, particularly in a context of possible hypoxia, given the low cardiac output observed in these patients [
      • Mazure N.M.
      • Pouyssegur J.
      Hypoxia-induced autophagy: cell death or cell survival?.
      ,
      • Abeliovich H.
      Mitophagy: the life-or-death dichotomy includes yeast.
      ]. Altogether, these data suggest that in acute liver injury, autophagy fights to keep cells alive under stressful “life-threatening” conditions, the autophagic cell death being the outcome of failed adaptation.
      Figure thumbnail gr6
      Fig. 6Anorexia nervosa. Course of alanine aminotransferase (ALT) level (plain line) and body weight (dotted line) in a 24-year old girl (size 1.58 m) with anorexia nervosa. Mild elevations in liver tests were observed several months before onset of acute liver insufficiency. (personal data). Abbreviation: ULN, upper limit of normal values.

      Autophagy in alpha1-antitrypsin deficiency

      The genetic disease alpha-1-antitrypsin (α1AT) deficiency is caused by homozygosity for the α1AT mutant Z gene (ATZ) and occurs 1 in 2000 births. The Z mutation confers an abnormal conformation on the nascent polypeptide, resulting in an accumulation of the mutant protein within the endoplasmic reticulum of hepatocytes rather than the appropriate, highly efficient, secretion of the wild-type protein.
      When ATZ accumulates in the ER, it can be degraded by two major mechanisms, the proteasomal and autophagic pathways. The proteasome is probably specialized for the soluble forms of ATZ that accumulates in the ER, presumably bound to multiple chaperones [
      • Perlmutter D.H.
      Autophagic disposal of the aggregation-prone protein that causes liver inflammation and carcinogenesis in alpha-1-antitrypsin deficiency.
      ]. Several lines of evidence show that the autophagic pathway is specialized for the polymerized/aggregated forms of ATZ (Table 1): (a) an increased accumulation of autophagosomes is observed in fibroblast cell lines engineered for the expression of mutant ATZ, in the liver cells of the PiZ mouse model of α1AT deficiency and in the liver cells of patients with α1AT deficiency [
      • Teckman J.H.
      • Perlmutter D.H.
      Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response.
      ]; (b) these autophagic vacuoles contain ATZ as demonstrated using both immuno-electron microscopy and immunofluorescence [
      • Teckman J.H.
      • Perlmutter D.H.
      Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response.
      ,
      • Kamimoto T.
      • Shoji S.
      • Hidvegi T.
      • Mizushima N.
      • Umebayashi K.
      • Perlmutter D.H.
      • et al.
      Intracellular inclusions containing mutant alpha1-antitrypsin Z are propagated in the absence of autophagic activity.
      ], but also frequently contain mitochondria, even when compared with liver from patients with other hepatic disorders [
      • Teckman J.H.
      • An J.K.
      • Blomenkamp K.
      • Schmidt B.
      • Perlmutter D.
      Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency.
      ]; (c) in cell lines deleted for the Atg5 gene, that is necessary for autophagy, the degradation of ATZ is retarded and the characteristic cellular inclusions of ATZ accumulate [
      • Kamimoto T.
      • Shoji S.
      • Hidvegi T.
      • Mizushima N.
      • Umebayashi K.
      • Perlmutter D.H.
      • et al.
      Intracellular inclusions containing mutant alpha1-antitrypsin Z are propagated in the absence of autophagic activity.
      ].
      This induction of autophagy is specific for the aggregation-prone properties of ATZ because it was not seen in a Saar mouse, a mouse with hepatocyte-specific inducible expression of the AT Saar variant that accumulates in the ER but does not polymerize/aggregate [
      • Kamimoto T.
      • Shoji S.
      • Hidvegi T.
      • Mizushima N.
      • Umebayashi K.
      • Perlmutter D.H.
      • et al.
      Intracellular inclusions containing mutant alpha1-antitrypsin Z are propagated in the absence of autophagic activity.
      ].
      The mechanisms by which autophagy is activated in the liver in α1AT deficiency are largely unknown. The marked upregulation of regulator of G signaling 16 (RGS16) observed both in the liver of mice that have hepatocyte-specific inducible expression of ATZ and in the liver of patients with α1AT deficiency may be implicated [
      • Hidvegi T.
      • Mirnics K.
      • Hale P.
      • Ewing M.
      • Beckett C.
      • Perlmutter D.H.
      Regulator of G Signaling 16 is a marker for the distinct endoplasmic reticulum stress state associated with aggregated mutant alpha1-antitrypsin Z in the classical form of alpha1-antitrypsin deficiency.
      ]. Indeed, RGS16 binds to and inhibits the heterotrimeric G protein Gαi3, a protein that inhibits hepatic insulin-induced autophagic activity [
      • Gohla A.
      • Klement K.
      • Nurnberg B.
      The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor.
      ]. Therefore, RGS16 might de-repress autophagy.
      The pathogenesis of liver injury in α1AT deficiency is still incompletely understood. Nevertheless, it has been shown that markers of apoptosis are more pronounced in hepatocytes with greater levels of insoluble ATZ. Furthermore, stimulation of the extrinsic apoptotic pathway with antibody to Fas, resulted in increased apoptosis almost exclusively of the globule-containing cells [
      • Lindblad D.
      • Blomenkamp K.
      • Teckman J.
      Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model.
      ]. Autophagy activation and/or mitochondrial dysfunction present in α1AT hepatocytes must therefore be responsible for this increased apoptotic hepatocellular death.
      Interestingly, recent studies have shown that carbamazepin, a well-known food and drug administration-approved drug that enhances autophagy, decreases the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing carbamazepin in α1AT deficiency patients [
      • Hidvegi T.
      • Ewing M.
      • Hale P.
      • Dippold C.
      • Kemp C.B.
      • Maurice N.
      • et al.
      An autophagy-enhancing drug promotes degradation of mutant {alpha}1-antitrypsin z and reduces hepatic fibrosis.
      ,
      • Kaushal S.
      • Annamali M.
      • Blomenkamp K.
      • Rudnick D.
      • Halloran D.
      • Brunt E.M.
      • et al.
      Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model.
      ].
      More details regarding the implication of autophagy in alpha-1-antitrypsin deficiency are provided in a recent review [
      • Perlmutter D.H.
      Autophagic disposal of the aggregation-prone protein that causes liver inflammation and carcinogenesis in alpha-1-antitrypsin deficiency.
      ].

      Autophagy in alcoholic liver disease

      Excessive alcohol consumption is the third leading preventable cause of death in the United States [
      • Lucey M.R.
      • Mathurin P.
      • Morgan T.R.
      Alcoholic hepatitis.
      ]. Chronic alcohol use may cause several types of liver injury: fatty liver (also called steatosis), hepatic fibrosis, cirrhosis, and alcoholic hepatitis.
      Several direct and indirect arguments suggest that alcohol consumption suppresses liver cell autophagy (Table 1): (a) rats chronically fed with ethanol have a reduced number of autophagic vacuoles in liver cells, as determined morphometrically [
      • Poso A.R.
      • Hirsimaki P.
      Inhibition of proteolysis in the liver by chronic ethanol feeding.
      ]; (b) chronic ethanol consumption slows down the catabolism of long-lived proteins in the rat liver [
      • Poso A.R.
      • Hirsimaki P.
      Inhibition of proteolysis in the liver by chronic ethanol feeding.
      ,
      • Donohue Jr., T.M.
      • Zetterman R.K.
      • Tuma D.J.
      Effect of chronic ethanol administration on protein catabolism in rat liver.
      ]; (c) alcohol abuse is associated with protein accumulation in the liver, as demonstrated in ethanol fed rats [
      • Baraona E.
      • Leo M.A.
      • Borowsky S.A.
      • Lieber C.S.
      Alcoholic hepatomegaly: accumulation of protein in the liver.
      ]; (d) hepatocytes from patients with alcoholic steatohepatitis contain protein aggregates called Mallory-Denk bodies. The major constituents of these cytoplasmic inclusions are cytokeratins 8 and 18, in association with other proteins including ubiquitin and p62 [
      • Harada M.
      Autophagy is involved in the elimination of intracellular inclusions, Mallory-Denk bodies, in hepatocytes.
      ,
      • Zatloukal K.
      • French S.W.
      • Stumptner C.
      • Strnad P.
      • Harada M.
      • Toivola D.M.
      • et al.
      From Mallory to Mallory-Denk bodies: what, how and why?.
      ]. These Mallory-Denk bodies may witness a decrease in autophagy level. Indeed, autophagy participates in the elimination of components of Mallory-Denk bodies, since cytokeratin 8/18 are detected in autophagic vacuoles using immuno-electron microscopy, and since stimulating the autophagic process using rapamycin decreases the number of these protein aggregates [
      • Harada M.
      Autophagy is involved in the elimination of intracellular inclusions, Mallory-Denk bodies, in hepatocytes.
      ,
      • Harada M.
      • Hanada S.
      • Toivola D.M.
      • Ghori N.
      • Omary M.B.
      Autophagy activation by rapamycin eliminates mouse Mallory-Denk bodies and blocks their proteasome inhibitor-mediated formation.
      ]; (e) loss of autophagy in transgenic mice induces the formation of protein aggregates in hepatocytes, resembling Mallory-Denk bodies [
      • Komatsu M.
      • Waguri S.
      • Ueno T.
      • Iwata J.
      • Murata S.
      • Tanida I.
      • et al.
      Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.
      ]; (f) ethanol also suppresses autophagy in cortical neuroepithelial progenitors, suggesting that this effect is not restricted to liver cells [
      • Prock T.L.
      • Miranda R.C.
      Embryonic cerebral cortical progenitors are resistant to apoptosis, but increase expression of suicide receptor DISC-complex genes and suppress autophagy following ethanol exposure.
      ].
      The mechanisms responsible for the decrease in autophagy are not clear, but two explanations can be advanced [
      • Donohue Jr, T.M.
      Autophagy and ethanol-induced liver injury.
      ] (Fig. 7). First, ethanol consumption significantly reduces adenosine monophosphate-activated protein kinase (AMPk) activity in the liver [
      • You M.
      • Matsumoto M.
      • Pacold C.M.
      • Cho W.K.
      • Crabb D.W.
      The role of AMP-activated protein kinase in the action of ethanol in the liver.
      ]. As shown in Figure 7, AMPk suppression reduces autophagy via the mTOR pathway. Second, ethanol is known to alter vesicle transport in hepatocytes. Autophagy requires the action of cytoskeletal elements, including microtubules and microfilaments. Both are necessary for autophagosome formation and fusion with other vesicular bodies, as demonstrated by blocking these processes with specific inhibitors, including nocadazole and vinblastine (microtubules), and cytochalasins (microfilaments) [
      • Aplin A.
      • Jasionowski T.
      • Tuttle D.L.
      • Lenk S.E.
      • Dunn Jr., W.A.
      Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.
      ,
      • Kochl R.
      • Hu X.W.
      • Chan E.Y.
      • Tooze S.A.
      Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes.
      ]. Disruption, by ethanol treatment, of the vesicular movement within the hepatocyte, occurs by mechanisms that are independent of the molecular motors dynein and kinesin, although there is evidence for alterations in the protein dynamin [
      • Torok N.
      • Marks D.
      • Hsiao K.
      • Oswald B.J.
      • McNiven M.A.
      Vesicle movement in rat hepatocytes is reduced by ethanol exposure: alterations in microtubule-based motor enzymes.
      ].
      Figure thumbnail gr7
      Fig. 7Ethanol may decrease autophagy level by reducing AMPk activity in the liver (a) and by disrupting vesicular movement within hepatocyte (b). This decrease in the autophagic process results in the accumulation of protein aggregates called Mallory-Denk bodies (c) and of mitochondria damaged following ethanol ingestion (d). Depolarization of the inner membrane of mitochondria may occur leading to mitochondrial permeability transition and cell death. Abbreviations: AMPk, adenosine monophosphate-activated protein kinase; CK, cytokeratin; MDB, Mallory-Denk body.
      This decline in the autophagic pathway must contribute to the pathological consequences of alcohol ingestion. First, as mentioned above, the decrease in protein catabolism likely contributes to the formation of Mallory-Denk bodies. These inclusions contain the protein p62. Recent studies have demonstrated that accumulation of p62 results in hyperactivation of the transcription factor (nuclear factor erythroid 2-related factor 2 (Nrf2) that causes liver changes such as hepatomegaly, liver cell swelling, and aminotransferase elevation [
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • et al.
      The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.
      ]. Second, ethanol causes mitochondrial damage leading, in certain cases, to depolarization of the inner membrane of mitochondria, called mitochondrial permeability transition. It is crucial that such damaged mitochondria be removed from the cell by engulfment in autophagic vacuoles. The absence of mitochondrial autophagy leads to uncoupling of oxidative phosphorylation and cell death [
      • Kim J.S.
      • Nitta T.
      • Mohuczy D.
      • O’Malley K.A.
      • Moldawer L.L.
      • Dunn Jr., W.A.
      • et al.
      Impaired autophagy: a mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.
      ,
      • Donohue Jr, T.M.
      Autophagy and ethanol-induced liver injury.
      ].

      Autophagy and non alcoholic fatty liver disease

      The implication of autophagy in hepatocyte lipid metabolism has been recently demonstrated [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ]. Besides cytosolic lipases, autophagy regulates intracellular lipid stores through a process called macrolipophagy. Portions of lipid droplets, or even whole droplets, become trapped inside the double-membrane-bound autolipophagosome vesicles and are transported to lysosomes, where they are degraded to fatty acids (Fig. 8). The presence of this alternative lipid degradative pathway in hepatocytes explains their ability to rapidly mobilize large amounts of lipids despite their low levels of cytosolic lipases in comparison with adipocytes [
      • Zechner R.
      • Madeo F.
      Cell biology: another way to get rid of fat.
      ,
      • Czaja M.J.
      Autophagy in health and disease: 2. Regulation of lipid metabolism and storage by autophagy: pathophysiological implications.
      ]. In physiological state, both lipolysis and macrolipophagy are inhibited by the hormone insulin [
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ,
      • Yin X.M.
      • Ding W.X.
      • Gao W.
      Autophagy in the liver.
      ,
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ,
      • Zechner R.
      • Madeo F.
      Cell biology: another way to get rid of fat.
      ].
      Figure thumbnail gr8
      Fig. 8Fatty liver disease and metabolic syndrome. Under physiological conditions (black arrows), autophagy functions in the basal turnover of lipids by engulfing and degrading lipid droplets. Autophagy is inhibited by the insulin amino acid-mTOR signaling pathway via both short-term and long-term regulation mechanisms. Short-term inhibition can be produced by the mTOR complex. Long-term regulation occurs via the transcription factors FoxO
      [
      • Liu H.Y.
      • Han J.
      • Cao S.Y.
      • Hong T.
      • Zhuo D.
      • Shi J.
      • et al.
      Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-dependent expression of key autophagy genes by insulin.
      ]
      , which control the transcription of autophagy genes and become inhibited by insulin-induced activation of protein kinase B. In obesity (red arrows), autophagy level is decreased in hepatocytes. Several mechanisms may account for this decline. First, obesity-induced increase in the calcium-dependent protease calpain 2 leads to down-regulation of Atg7 and then defective autophagy. Acute inhibition of calpain is able to restore Atg7 expression
      [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ]
      . Second, in obesity, the autophagy inhibitor mTOR is overactivated in the liver, presumably as the result of increased amino acid concentrations following overnutrition [
      • Codogno P.
      • Meijer A.J.
      Autophagy: a potential link between obesity and insulin resistance.
      ,
      • Newgard C.B.
      • An J.
      • Bain J.R.
      • Muehlbauer M.J.
      • Stevens R.D.
      • Lien L.F.
      • et al.
      A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.
      ]. Third, although controversial, hyperinsulinemia may also contribute to downregulating autophagy in obese mice. Indeed, inhibiting Akt which is a key molecule in the insulin pathway, increases autophagy in the liver of obese mice
      [
      • Liu H.Y.
      • Han J.
      • Cao S.Y.
      • Hong T.
      • Zhuo D.
      • Shi J.
      • et al.
      Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-dependent expression of key autophagy genes by insulin.
      ]
      . However, destruction of insulin production in β-cells by streptozotocin does not increase autophagy in the liver of obese mice
      [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ]
      , contrary to lean mice
      [
      • Liu H.Y.
      • Han J.
      • Cao S.Y.
      • Hong T.
      • Zhuo D.
      • Shi J.
      • et al.
      Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-dependent expression of key autophagy genes by insulin.
      ]
      . The reasons for these discrepancies are unclear. In obesity, defect in autophagy and its associated decrease in lysosomal degradation rate contribute to further increasing the ER stress induced by nutrient overload in an inflammatory milieu [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ,
      • Hotamisligil G.S.
      Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.
      ]. Together, autophagy decline and ER stress lead to insulin resistance
      [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ]
      . Abbreviations: Atg, autophagy genes; ld, lipid droplet; ER, endoplasmic reticulum; mTOR, mammalian target of rapamycin; PKB, protein kinase B.
      The efficiency of macrolipophagy varies with the nutritional status. In response to a short-term increase in lipid availability, in vitro studies have demonstrated that the autophagy level increases, leading to a greater breakdown of stored lipids to supply fatty acids for β-oxidation or other uses [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ]. In the same way, hepatocyte-specific atg7-knockout mice had markedly increased hepatic lipid [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ].
      In contrast, in both genetic (ob/ob mice) and dietary (high fat diet) mouse models of chronic obesity and insulin resistance, a sustained increase in lipid availability results in markedly decreased hepatic autophagy indicators [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ,
      • Czaja M.J.
      Autophagy in health and disease: 2. Regulation of lipid metabolism and storage by autophagy: pathophysiological implications.
      ,
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ,
      • Liu H.Y.
      • Han J.
      • Cao S.Y.
      • Hong T.
      • Zhuo D.
      • Shi J.
      • et al.
      Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-dependent expression of key autophagy genes by insulin.
      ,
      • Codogno P.
      • Meijer A.J.
      Autophagy: a potential link between obesity and insulin resistance.
      ]. Proposed mechanisms are summarized in Figure 8.
      This decreased autophagy level impacts on other cellular functions and particularly on ER stress. Indeed, in the liver tissue of lean mice, deficiency of autophagy, induced by suppression of Atg7, results in ER stress [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ]. This could be explained by the known role of autophagy in the degradation of unfolded proteins and in the removal of superfluous ER membranes: defect in autophagy may lead to the accumulation of unfolded proteins and thus to ER stress [
      • Bernales S.
      • Schuck S.
      • Walter P.
      ER-phagy: selective autophagy of the endoplasmic reticulum.
      ]. In obesity, ER function is compromised due to nutrient and energy surplus in an inflammatory milieu [
      • Hotamisligil G.S.
      Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.
      ]. Concomitantly, autophagy level is decreased, suggesting that failure to achieve autophagy may further impair ER function in the face of continuous energy and nutrient stress, engage organelle dysfunction and contribute to insulin resistance, a known consequence of ER stress [
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ,
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • et al.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ]. Interestingly, restoration of Atg7 expression results in significant reduction in obesity-induced ER stress in the liver of ob/ob mice, rescues the defects in insulin receptor signaling, reduces serum insulin level, improves glucose tolerance and whole body insulin sensitivity through the suppression of hepatic glucose production and enhancement of insulin-stimulated glucose disposal in the periphery, and decreases hepatic fatty acid infiltration and liver triglyceride content [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ,
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ]. However, the interest of increasing autophagy, e.g., by pharmacological means, remains uncertain given the opposite role of autophagy in adipocytes: the inhibition, rather than the stimulation, of autophagy in adipocytes gives them a brown-fat-cell-like appearance that favours fatty acid oxidation and increases insulin [
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      ,
      • Codogno P.
      • Meijer A.J.
      Autophagy: a potential link between obesity and insulin resistance.
      ].

      Autophagy and hepatocellular carcinoma

      Early, cancer has been genetically linked to autophagy malfunction. Indeed, the ATG gene beclin-1 is mono-allelically deleted in 40–75% of cases of human breast, ovarian, and prostate cancer [
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ,
      • Morselli E.
      • Galluzzi L.
      • Kepp O.
      • Vicencio J.M.
      • Criollo A.
      • Maiuri M.C.
      • et al.
      Anti- and pro-tumor functions of autophagy.
      ,
      • Mathew R.
      • Karantza-Wadsworth V.
      • White E.
      Role of autophagy in cancer.
      ]. Moreover, the regulation of autophagy overlaps closely with signaling pathways that regulate tumorigenesis.
      Studies assessing autophagy in hepatocellular carcinoma (HCC) have clearly demonstrated in vitro, in mice and in patients that, in this context, autophagy is a tumor suppressor mechanism (Table 1).
      First, mice with heterozygous disruption of beclin-1 have a high frequency of spontaneous hepatocellular carcinoma. Moreover, crossing beclin-1 +/− mice, with mice, that transgenically express the HBV large-envelope polypeptide under the transcriptional control of the mouse albumin promoter, resulted in the acceleration of the development of hepatitis B virus-induced small-cell dysplasia – an important histopathologic predictor of malignant transformation [
      • Qu X.
      • Yu J.
      • Bhagat G.
      • Furuya N.
      • Hibshoosh H.
      • Troxel A.
      • et al.
      Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.
      ].
      Second, expression of several autophagic genes (ATG5, ATG7 and BECLIN-1) and their corresponding autophagic activity is decreased in HCC cell lines compared to that in a normal hepatic cell line. Similarly, Beclin-1 mRNA and protein levels are lower in HCC tissue samples than in adjacent non-tumor tissues from the same patients [
      • Ding Z.B.
      • Shi Y.H.
      • Zhou J.
      • Qiu S.J.
      • Xu Y.
      • Dai Z.
      • et al.
      Association of autophagy defect with a malignant phenotype and poor prognosis of hepatocellular carcinoma.
      ].
      Third, the most aggressive malignant HCC cell lines and HCC tissues with recurrent disease display much lower autophagic levels than less aggressive cell lines or tissues, especially when the anti-apoptotic B-cell leukemia/lymphoma (Bcl)-xL protein is over-expressed [
      • Ding Z.B.
      • Shi Y.H.
      • Zhou J.
      • Qiu S.J.
      • Xu Y.
      • Dai Z.
      • et al.
      Association of autophagy defect with a malignant phenotype and poor prognosis of hepatocellular carcinoma.
      ]. Interestingly, in a tissue microarray study consisting of 300 HCC patients who underwent curative resection, the expression of Beclin-1 was significantly correlated with disease-free survival and overall survival only in the Bcl-xL+ patients. Multivariate analyses revealed that Beclin-1 expression was an independent predictor for disease-free survival and overall survival in Bcl-xL+ patients. In addition, there was a significant correlation between Beclin-1 expression and tumor differentiation in Bcl-xL+ but not in Bcl-xL− HCC patients. These data suggest that autophagy defect synergizes with altered apoptotic activity and facilitates tumor progression and poor prognosis of HCC [
      • Ding Z.B.
      • Shi Y.H.
      • Zhou J.
      • Qiu S.J.
      • Xu Y.
      • Dai Z.
      • et al.
      Association of autophagy defect with a malignant phenotype and poor prognosis of hepatocellular carcinoma.
      ].
      The mechanisms responsible for this low autophagy protein level are not elucidated. However, a recent study has demonstrated that HAb18G/CD147, a transmembrane glycoprotein highly expressed in HCC, contributes to this decreased autophagic level in HCC through the class I phosphatidylinositol 3-kinase–Akt pathway upregulation [
      • Gou X.
      • Ru Q.
      • Zhang H.
      • Chen Y.
      • Li L.
      • Yang H.
      • et al.
      HAb18G/CD147 inhibits starvation-induced autophagy in human hepatoma cell SMMC7721 with an involvement of Beclin 1 down-regulation.
      ]. Other oncoproteins such as the Bcl-2 family proteins may also be implicated in HCC, like in other cancers [
      • Morselli E.
      • Galluzzi L.
      • Kepp O.
      • Vicencio J.M.
      • Criollo A.
      • Maiuri M.C.
      • et al.
      Anti- and pro-tumor functions of autophagy.
      ]. Stimulation of hypoxia-inducible factors (HIFs) due to hypoxic stress within HCC may also contribute to autophagy modulation [
      • Menrad H.
      • Werno C.
      • Schmid T.
      • Copanaki E.
      • Deller T.
      • Dehne N.
      • et al.
      Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids.
      ].
      Fourth, therapeutic approaches aiming at increasing autophagy level have been successfully tested in vitro and/or in mice using molecules such as Concanavalin A, a lectin with mannose specificity [
      • Chang C.P.
      • Yang M.C.
      • Liu H.S.
      • Lin Y.S.
      • Lei H.Y.
      Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model.
      ], or cyclo-oxygenase-2 inhibitors [
      • Gao M.
      • Yeh P.Y.
      • Lu Y.S.
      • Hsu C.H.
      • Chen K.F.
      • Lee W.C.
      • et al.
      OSU-03012, a novel celecoxib derivative, induces reactive oxygen species-related autophagy in hepatocellular carcinoma.
      ,
      • Mazzanti R.
      • Platini F.
      • Bottini C.
      • Fantappie O.
      • Solazzo M.
      • Tessitore L.
      Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line.
      ]. Importantly, a large recent study has assessed the survival after liver transplantation according to the immunosuppression protocol administrated. All patients included, 2491 adult recipients of isolated liver transplantation for HCC and 12,167 for non-HCC, remained on stable maintenance immunosuppression protocols for at least 6 months post-transplant. Therapy using rapamycin, a well-known activator of autophagy, was associated with improved survivals after transplantation for HCC. Interestingly, in non-HCC patients, rapamycin showed a trend toward lower rates of survival in non-HCC recipients, confirming the specificity of its beneficial impact for cancer patients [
      • Toso C.
      • Merani S.
      • Bigam D.L.
      • Shapiro A.M.
      • Kneteman N.M.
      Sirolimus-based immunosuppression is associated with increased survival after liver transplantation for hepatocellular carcinoma.
      ].
      Given the primary prosurvival role of autophagy, this anti-tumor activity may be surprising. If the mechanisms for this tumor suppressor role of autophagy are not yet clear, the following functions of autophagy have been proposed: (a) limiting chromosomal instability therefore preventing the accumulation of oncogenic mutations; (b) restricting oxidative stress, which is also an oncogenic stimulus; and (c) reducing intratumoral necrosis and local inflammation [
      • Morselli E.
      • Galluzzi L.
      • Kepp O.
      • Vicencio J.M.
      • Criollo A.
      • Maiuri M.C.
      • et al.
      Anti- and pro-tumor functions of autophagy.
      ,
      • Mathew R.
      • Karantza-Wadsworth V.
      • White E.
      Role of autophagy in cancer.
      ].

      Funding

      PER was supported by a “poste d’accueil INSERM”. R.M. is in receipt of an Interface INSERM-AP-HP fellowship.

      Data sources and searches

      We searched MEDLINE (1966–2010) for studies on autophagy and liver diseases by using the terms autophagy, autophagosome, liver, and hepatitis. We also reviewed publications in personal reference lists and citation sections of the recovered articles. We performed the final search on July 20 2010.

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
      There is no financial disclosure.

      Acknowledgments

      The authors thank Dr. Dominique Cazals-Hatem and Pr Gérard Feldmann for helpful discussions and expertise in electron microscopy and Alain Grodet for technical assistance in electron microscopy.

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