Autophagy in liver diseases

Autophagy in li Pierre-Emmanuel Rautou1,2,3,⇑, Abdellah Mansou Dominique Valla1,2,3, R 1Service d’Hépatologie, Hôpital Beaujon, Assistance Publique-Hôpitau Bichat-Beaujon CRB3, Clichy, France; 3Université Autophagy, or cellular self-digestion, is a cellular pathway cru- phagosome formation involves three major steps described in Fig. 3: initiation with the uncoordinated 51-like kinase 1 gy 2010 vol. 53 j 1123–1134 Keywords: Autophagic vacuole; Autophagosome; Hepatitis; Hepatocellular car- cinoma; LC3; Starvation; Survival. Received 29 April 2010; received in revised form 21 July 2010; accepted 22 July 2010 Review Journal of Hepatolo (ULK1) complex, nucleation with the Beclin-1-class III phosphati- dylinositol 3-kinase (PI3K) complex and elongation of the isola- tion membrane with a key role of microtubule-associated protein light chain 3 (LC3) lipidation [3,5,6]. Information on the methods for monitoring autophagy can be found elsewhere [7]. Within the past decade, numerous new techniques have been developed, allowing to understand the role of autophagy both in ⇑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. E-mail address: perautou@yahoo.fr (P.-E. Rautou). Abbreviations: a1AT, alpha-1-antitrypsin; AMPk, 50-AMP-activated protein Kina- se; ATZ, a1AT mutant Z gene; Atg, autophagy genes; Bcl, B-cell leukemia/lymp- homa; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; LC3, microtubule-associated protein light chain 3; mTOR, mammalian ta- rget of rapamycin; Nrf2, nuclear factor erythroid 2-related factor 2; PI3K, phos- phatidylinositol 3-kinase; ULK1, uncoordinated 51-like kinase 1. the Atg proteins that are required for the formation of the isola- tion membrane and the autophagosome. The process of auto- cial 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 a-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 pro- teins may be useful in a-1-antitrypsin deficiency. Following alco- hol consumption, autophagy is decreased in liver cells, likely due to a decrease in intracellular 50-AMP-activated protein kinase (AMPk) and due to an alteration in vesicle transport in hepato- cytes. 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. � 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Definition and molecular machinery of autophagy Autophagy (Greek for ‘‘self eating”) is a general term for pro- cesses by which cytoplasmic materials, including organelles, ver diseases ri2,3, Didier Lebrec1,2,3, François Durand1,2,3, ichard Moreau1,2,3 x de Paris, Clichy, France; 2INSERM, U773, Centre de Recherche Denis Diderot-Paris 7, 75018 Paris, France reach lysosomes for degradation. Three types of autophagy (mac- roautophagy, microautophagy, and chaperone-mediated autoph- agy) 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 [1]. This pathway is conserved from yeast to mammals (Fig. 1). Upon induction, a small vesicular sac called the isolation mem- brane or phagophore elongates and subsequently encloses a por- tion 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 [2]. Then, the outer membrane of the autophagosome fuses with a lysosome (to form an autolyso- some), 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 deg- radation are delivered back to the cytoplasm for recycling or energy production. Autophagy occurs at low basal levels in virtu- ally all cells to perform homeostatic functions such as protein and organelle turnover. It is rapidly upregulated through the inhibi- tion of mammalian target of rapamycin (mTOR) when cells need to generate intracellular nutrients and energy, for example, dur- ing starvation, growth factor withdrawal, or high bioenergetic demands [1,3]. Subsequently, prolonged starvation reactivates mTOR signaling that both attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolyso- somes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell [4]. The execution of autophagy involves a set of evolutionarily conserved gene products (initially identified in yeast) known as normal and in pathological conditions [1,5]. The physiological tion [21]. In this study, 61 patients who underwent liver surgery Review Autophagy and liver ischemia reperfusion and liver surgery Liver ischemia/reperfusion injury occurs during liver transplanta- tion, trauma, shock, and elective liver resection. During this pro- cess, 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 [11]. As the first known role of autophagy is its action during nutri- ent starvation, studies on autophagy and liver diseases have rap- idly focussed on liver ischemia/reperfusion [12–14]. Two types of experimental protocols have been performed reflecting two dif- ferent clinical situations: (a) hepatic ischemia induced by occlu- sion 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 [15–17]; and (b) liver transplantation with 24 h cold ischemia followed by reperfusion [18–20]. 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 [15–17]. Nevertheless, the unique study performed in patients provides interesting informa- role of autophagy in nutrient and energy metabolism in hepato- cytes has been reviewed elsewhere [8]. As detailed in Box 1, this process is particularly crucial in newborn mammals survival [9,10]. Several recent studies have demonstrated the implication of autophagy in major fields of hepatology. This review aims at providing an overview of currently available knowledge on autophagy and liver diseases. 1124 Journal of Hepatology 2010 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 pro- longed ischemia required by liver resection and another after the liver reperfusion (median: 88 min; range:57–125 min), before clo- sure of the abdomen. A unique vascular occlusion had almost no effect on autophagy, since the LC3-II rarely increased and the num- ber of cells containing autophagic vacuoles remained stable. How- ever, 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 [21], previous studies including specific groups of patients, such as young patients and patients with liver steatosis or cirrhosis obtained a clinical improvement [22,23]. This suggests that in this context, autophagy enhancement could allow for decreasing liver cell death (Table 1). Studies on liver transplantation had also apparently contra- dictory 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–trypto- phan–ketoglutarate cold-storage solution for 24 h cold preserva- tion [20], while the contrary was reported by authors using the University of Wisconsin (UW) cold-storage solution [18,19]. Elec- tron 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 solu- tion does not contain amino acids. It is well demonstrated that amino acid depletion rapidly induces autophagy [12] and that anoxia decreases autophagy protein level [16]. 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 [11]. 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 mito- chondria laden with reactive oxygen species and calcium undergo the mitochondrial permeability transition, which in turn leads to uncoupling of oxidative phosphorylation, energetic fail- ure, ATP depletion, and ultimately cell death. In case of associated nutrient depletion, autophagy is enhanced and facilitates autoph- agy of damaged mitochondria, leading to cell survival [16]. 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 PPARc 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, [15,17,20]. It is striking to notice that the two studies suggesting that inhibiting autophagy could ameliorate liver tolerance to ischemia [14,18] 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 vol. 53 j 1123–1134 mTOR Protolysosomal tubules Beclin-1 Class III PI3K AMPk Atg Lysosome Bafilomycin A1 Nutrient abundance Starvation Rapamycin Wortmanin PI3K Class I + + - - - - - - Proteinase inhibitors (pepstatin, leupeptin) - + JOURNAL OF HEPATOLOGY Isolation membrane Autophagosome Bcl-2 - could explain at least partly theworse tolerance to ischemia reper- fusion in aged patients [15,24]. Second, autophagy level decreases following partial hepatectomy suggesting a shift from the physio- logical steady state between anabolismand catabolism to the posi- tive balance which is required for the compensatory growth of the Autophagy genes knock down or knock out Fig. 1. Cellular and molecular aspects of autophagy. Note that wortmannin inhibits inhibit autophagy as represented here. Abbreviations: AMPk, adenosine monophosphate- 2; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase. 2 µm ld n m m m f A B Fig. 2. Electron micrographs showing ultrastructure of hepatocytes from a chro magnification image showing hepatocytes containing several autophagic vacuoles (origin vacuole (original magnification, 100,000�). F, fibrosis; ld, lipid droplet; m, mitochondria Journal of Hepatology 2010 Autolysosome Lysosome liver after partial hepatectomy [13]. Third, reperfusion had more effect on autophagy level than ischemia alone [16,19,25], which is in linewith the histological lesions observed [11]. Unfortunately, no study has yet specifically evaluated the autophagic pathway in liver sinusoidal endothelial cells. This is a limitation inunderstand- Amino acids both class I (inhibitory) and class III PI3K, but the overall phenotypic effect is to activated protein kinase; Atg, autophagy genes; Bcl-2, B-cell leukemia/lymphoma m 0.2 m nic hepatitis C patient. Black arrows point to autophagic vacuoles. (A) Low- al magnification, 8000�). (B) Partial view of a hepatocyte containing an autophagic ; n, nucleus. vol. 53 j 1123–1134 1125 from chronic hepatitis C patients [27–32]. Whatever the LC3 Class III PI3K Vesicle elongation and closure Review conjugation system Beclin-1 Ambra1 Atg14L Atg12 conjugation system PE PE Vps15 mTOR Initiation - Atg101 Atg13 FIP200 ULK1 Atg5 Atg12 Atg10 Atg16 Atg12 Atg5 Atg16 Atg7 LC3-I Atg4 Atg3 LC3-II ing 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 [26] (Table 1). Several studies have assessed the autophagic pathway in hepatocytes infected with HCV both in vitro and in liver biopsies Vesicle nucleation Bcl-2 /xL - Fig. 3. Basic 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 phosphati- dylinositol 3-phosphate. This activation depends on the formation of a multi- protein 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 [6]. The functional relationship between the ULK1 complex (initiation) and Beclin-1-class III PI3K complex (nucleation) complexes remains to be determined [86]. Vesicle elongation, vesicle completion. The membrane formed elongates and closes on itself to form an autophagosome. Two conjuga- tion 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 adhe- sion 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. 1126 Journal of Hepatology 2010 approach used (LC3, Atg5 or Beclin-1 immunoblotting, electron microscopy or GFP-LC3 immunofluorescence), these studies con- sistently 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) [27,28,30] and also in patients infected with HCV genotypes 1, 2, 3 and 4 [29]. However, this autophagy was inefficient. Indeed, although HCV JFH1 induced autophagosomes, it was not able to enhance autophagic protein degradation [30]. 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) [26]. 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 [27,28,30,32]. 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 [30]; (b) the absence of co-localization of lysosomes (stained with LysoTrac- ker-red) with autophagic vacuoles (GFP-LC3) in HCV-infected cells contrary to starved cells [30]; (c) the reduction in the num- ber of autophagic vacuoles following HCV elimination using interferon alpha for 14 days [31]; (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 [29]. 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 [28]. However, autophagy proteins are not needed for the trans- lation 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 ini- tial 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 cytoplas- mic transport of the incoming RNA to cellular factors or sites that are required for its translation [28]. Importantly, autophagy pro- teins are required neither for HCV entry nor for HCV secretion [28,32]. Altogether, these data explain the apparent contrast between the results of some in vitro studies reporting the impli- cation of autophagy proteins in HCV replication [30–32] and the absence of correlation between the numbe