Autophagy in nonalcoholic steatohepatitis

Abstract

Autophagy is a critical pathway for the degradation of intracellular components by lysosomes. Established functions for both macroautophagy and chaperone-mediated autophagy in hepatic lipid metabolism, insulin sensitivity and cellular injury suggest a number of potential mechanistic roles for autophagy in nonalcoholic steatohepatitis (NASH). Decreased autophagic function in particular may promote the initial development of hepatic steatosis and progression of steatosis to liver injury. Additional functions of autophagy in immune responses and carcinogenesis may also contribute to the development of NASH and its complications. The impairment in autophagy that occurs with cellular lipid accumulation, obesity and aging may therefore have an important impact on this disease, and agents to augment hepatic autophagy have therapeutic potential in NASH.

Autophagy

Autophagy is a critical intracellular pathway that targets cell constituents to the lysosome for degradation. Three types of autophagy are known: macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy (Figure 1) [1,2]. Macroautophagy involves the engulfment of a portion of cytosol by a double membrane structure termed an autophagosome. The autophagosome fuses to a lysosome, whose enzymes degrade the cellular constituents sequestered in the autophagosome [1]. In CMA, soluble proteins containing a specific pentapeptide motif are bound to a chaperone protein for translocation to the lysosome, where binding to the lysosome-associated membrane protein type 2A (LAMP-2A) receptor leads to protein internalization and degradation [2]. After degradation of cellular components in the lysosome, the breakdown products are released into the cytosol for reuse or metabolism into energy. Macroautophagy and CMA are constitutively active in cells and increase with certain cellular stresses, the classic one being nutrient deprivation. However, basal levels of autophagy are also functionally important [3]. These two forms of autophagy differ in that macroautophagy, but not CMA, can degrade cellular organelles and protein aggregates. The third form of autophagy, microautophagy, is not inducible. Little is known about the regulation or function of this form of autophagy, which involves the lysosomal internalization of cellular constituents by an invagination of the lysosomal membrane.

Figure 1. The three types of autophagy.

In macroautophagy, a double membrane of unclear origin begins to form a phagophore around cytosolic components, such as mitochondria, lipid droplets and proteins. These cellular elements become completely enclosed within an autophagosome, which translocates to a lysosome full of hydrolases. Fusion of these two structures into an autophagolysosome leads to the enzymatic degradation of the cellular components. In chaperone-mediated autophagy, cytosolic proteins containing a pentapeptide motif bind to the chaperone Hsc70. This complex binds to the LAMP-2A receptor on the lysosome for internalization and degradation. Microautophagy involves the uptake of cellular components within an invagination of the lysosomal membrane for enzymatic degradation.

LAMP-2A: Lysosome-associated membrane protein type 2A.

Autophagy performs two general functions; as a catabolic process to generate substrates necessary to maintain cellular energy homeostasis in times of limited nutrients, and as a mechanism for the removal of worn-out or damaged cellular constituents [4–6]. Recent studies have suggested new functions for autophagy in the regulation of cellular processes such as lipid metabolism, insulin sensitivity and immune responsiveness. These functions, together with the previously established involvement of autophagy in cell death pathways, suggest multiple possible mechanisms by which autophagy may be involved in the lipid accumulation and hepatocellular injury that underlie nonalcoholic steatohepatitis (NASH). This article details the potential roles of autophagy in the pathophysiology of NASH.

Autophagy regulates hepatocyte lipid metabolism through lipophagy

Abnormal lipid metabolism and the over-accumulation of triglycerides (TGs) stored in lipid droplets characterize nonalcoholic fatty liver disease (NAFLD) [7,8]. Excessive hepatic lipid accumulation or steatosis may be an initial distinct phase of NAFLD. Alternatively, steatosis may set the stage for hepatocytes to progress to the liver injury that underlies NASH through secondary insults from cofactors such as oxidants and cytokines [9–11]. Although peripheral insulin resistance that increases the flow of free fatty acids (FFAs) to the liver is clearly critical to the development of steatosis, the mechanisms of lipid accumulation in response to this lipid challenge remain unclear.

Until recently, macroautophagy was known as a homeostatic cell pathway responsible for the recycling of long-lived proteins and organelles [4–6]. Lysosomes contain lipases to degrade organelle-associated lipids and exogenous lipoproteins, but intracellular lipids were not previously known to be a substrate for autophagic degradation. However, regulatory and functional similarities between lipolysis and macroautophagy suggested an inter-relationship between autophagy and the process of lipid breakdown [12]. Specifically, both pathways are downregulated by insulin in times of adequate nutrient supply and upregulated by glucagon during nutrient deprivation [4]. We therefore examined the potential role of macroautophagy in the regulation of hepatic lipid stores.

An inhibition of macroautophagy by genetic knockdown of the autophagy gene atg5, or pharmacological inhibition with 3-methyladenine in cultured hepatocytes challenged with a lipid load from fatty acid supplementation or culture in methionineand choline-deficient medium, significantly increased cellular TG content [13]. Excessive TGs and cholesterol were retained in lipid droplets because of a decreased rate of lipolysis and the resultant reduction in fatty acid β-oxidation in cells with an inhibition of macroautophagy. Lipid movement through the autophagic pathway was confirmed by fluorescence microscopy demonstrating colocalization of lipid with autophagosomes and lysosomes, electron microscopic evidence of lipid in autophagic vacuoles and immunogold staining demonstrating an association of the autophagosome-associated protein microtubule-associated protein light chain 3 (LC3) with lipid droplets.

In vivo studies of mouse livers confirmed LC3 association with lipid droplets and the presence of lipid in autophagosomes and lysosomes, which increased when starvation stimulated macroautophagy and provided the liver with a lipid challenge in the form of increased serum FFAs [13]. The numbers of autophagic vacuoles containing lipid alone, or together with other cellular constituents, increased with starvation, indicating that the autophagic pathway selectively targeted lipids for breakdown in response to this physiological stimulus. Mice with a hepatocyte-specific atg7 knockout to inhibit macroautophagy had marked increases in hepatic TGs and cholesterol. Together, these findings identified an essential function for macroautophagy in the regulation of hepatic lipid stores. Subsequently, an inhibition of macroautophagy has been reported to increase steatosis in models of alcohol-induced hepatic injury as well [14]. Therefore, macroautophagy is a central mechanism for the breakdown of lipid droplet-stored TGs and cholesterol through a process termed lipophagy [13]. It remains possible that autophagy regulates cellular lipid content through additional mechanisms, such as the degradation of factors that mediate lipid metabolism, as has been reported for apoprotein-B [15].

With aging, both macroautophagy and CMA decline [16] in parallel with increased lipid accumulation in various organs, including the liver [17]. In addition to autophagy regulating lipid content, hepatocyte lipid accumulation was demonstrated to decrease hepatic autophagic function. High-fat diet (HFD)-fed mice had an impairment in hepatic autophagic function, as demonstrated by decreased mobilization of lipid into the autophagic compartment [13]. Thus, not only may the decrease in macroautophagy that occurs in aging promote hepatic steatosis, but also, excessive lipid accumulation in hepatocytes may be a mechanism for the decrease in autophagic function with age. Lipid accumulation alters membrane structure, and a resultant decrease in fusion efficiency between autophagosomes and lysosomes may explain the inhibitory effect of lipid accumulation induced by a HFD on macroautophagy [18]. The ability of excessive cellular lipid accumulation to impair autophagy provides another mechanism for the progression of simple steatosis to NASH and its complications. Therapeutic efforts to lower hepatic lipid stores may be beneficial by raising autophagic function, which is needed to prevent the initiation of liver injury or the development of NASH complications, such as hepatocellular carcinoma (HCC).

Modulation of insulin resistance by macroautophagy

Insulin resistance is strongly associated with the development of the metabolic syndrome and its hepatic manifestation NASH. Peripheral insulin resistance enhances lipolysis in adipose tissue [19,20], leading to increased serum levels of FFAs [21], which travel to the liver where they are taken up and stored as TGs [22,23]. The direct effects of increased intracellular FFA levels and lipid content can promote hepatic insulin resistance, which further aggravates systemic insulin resistance through increased hepatic glucose production [24].

In addition to the connection between cellular lipid metabolism and autophagic function [13], changes in autophagy have been recently linked to hyperinsulinemia and insulin resistance [25,26]. Confirmatory of the previously discussed findings, investigations in hyperinsulinemic, HFD-fed mice demonstrated reduced hepatic autophagic function, as reflected by decreases in LC3-II and increased levels of p62, a protein degraded in part by macroautophagy [26]. In addition, hepatic expression of autophagy genes, including atg5 and atg7, was decreased. These changes were attributed to the effects of hyperinsulinemia, although cellular lipid accumulation may also have contributed. Findings that hyperinsulinemia decreases autophagic function are not surprising, as insulin is known to mediate the suppression of autophagy. However, that effect is believed to be mediated through the mTOR signaling pathway and not at the level of atg expression. Liu et al. also demonstrated that inhibition of FOXO1-mediated transcription by insulin mediated the suppression of autophagy in hepatoma cells, suggesting that this may also be the mechanism in vivo [26]. This possibility needs to be confirmed by experiments in mice with genetically altered levels of FOXO1, although FOXO1 has been previously demonstrated to promote autophagy in other cell types through divergent mechanisms [26–28]. The relevance of decreased FOXO1 signaling to human NAFLD is unclear, as FOXO1 has been reported to be upregulated in this disease [29]. The finding that both cellular lipid accumulation and hyperinsulinemia impair hepatic autophagic function suggests another mechanism by which these metabolic abnormalities may promote the progression of steatosis to NASH. The loss of autophagic function induced by these factors may promote cellular injury and death, as well as carcinogenesis, in this disease.

Studies in ob/ob mice and HFD-fed mice have also indicated that defects in autophagy in these mice contribute to insulin resistance [25]. Macroautophagy was impaired in ob/ob mice, as demonstrated by decreased levels of autophagy genes, increased p62 and decreased numbers of autophagosomes/autophagolysosomes seen by electron microscopy. Autophagy was functionally impaired, as demonstrated by a failure of the liver to upregulate macroautophagy with starvation. The suppression of autophagy in vitro and in vivo led to decreased insulin signaling, along with an induction of endoplasmic reticulum (ER) stress. Overexpression of Atg7 in the liver to restore autophagic function reversed ER stress and improved insulin sensitivity. The findings suggest that defective macroautophagy may contribute to hepatic insulin resistance in NAFLD through the generation of ER stress. Experiments to examine the effects of a direct inhibition of ER stress in the setting of impaired autophagy on insulin signaling need to be performed before the mechanism of this effect can be conclusively attributed to ER stress. The studies of lipid accumulation and autophagy suggest the existence of a critical circular relationship between NAFLD and autophagy: steatosis and hyperinsulinemia resulting from insulin resistance decrease autophagic function, and this reduction in macroautophagy acts to further promote lipid accumulation and insulin resistance (Figure 2).

Figure 2. Inter-relationships among cellular changes in lipid content or insulin sensitivity and levels of macroautophagy.

In nonalcoholic fatty liver disease, both steatosis and hyperinsulinemia may act to inhibit levels of macroautophagy. Cellular lipid accumulation can inhibit macroautophagy by impeding the fusion of autophagosomes to lysosomes. The mechanism of the inhibitory effect of hyperinsulinemia on autophagy may be through decreased activation of FOXO1. The resultant reduction in macroautophagy may then accelerate steatosis by impairing lipolytic breakdown of lipids stored in lipid droplets. The decrease in macroautophagy may also exacerbate insulin resistance by causing increased ER stress, which worsens hepatic insulin resistance.

ER: Endoplasmic reticulum.

Autophagy regulates hepatocellular injury & death

Nonalcoholic steatohepatitis is characterized by the presence of hepatocyte injury, inflammation and fibrosis, in addition to steatosis. According to the ‘two-hit’ hypothesis, after the onset of hepatic steatosis, additional factors distinct from those that led to lipid accumulation trigger hepatocellular injury and death, leading to progression to NASH. The mechanisms of cellular injury remain unclear, but may involve factors such as oxidative stress [30] and inflammatory cytokines [10]. Autophagy is known to regulate cell death pathways, including those of oxidants and TNF, and may therefore modulate the progression to hepatocellular injury in the setting of steatosis. Controversy has existed over whether autophagy functions to initiate or prevent cell death. Autophagy has been characterized as a type of cell death along with apoptosis and necrosis [31]. By contrast, many investigations have defined protective functions for autophagy that lead to cell survival from death stimuli [32–35]. Functions of autophagy that may protect against cell death include the removal of damaged organelles or proteins that contribute to cellular dysfunction [6,36] and the supply of substrates that maintain energy homeostasis. In addition, significant crosstalk exists between autophagy and apoptosis, and components of the autophagic pathway may also directly function in the regulation of apoptosis [37,38]. Current evidence clearly indicates that autophagy is predominantly a pathway that mediates cell survival, although it can also promote cell death under certain conditions.

A critical role for macroautophagic removal of mitochondria, or mitophagy, has been demonstrated, largely through studies performed in hepatocytes and liver [39,40]. NASH is characterized by the accumulation of abnormal mitochondria, suggesting that damage to this organelle, along with a possible defect in the removal of altered mitochondria, may play a role in this disease [41]. Impaired degradation of damaged mitochondria, particularly ones in which mitochondrial death pathway activation has occurred, may lead to oxidative stress or the release of mitochondrial factors that trigger apoptosis [42]. Knockdown of atg5 to inhibit macroautophagy sensitized hepatocytes to death from menadione-induced oxidative stress associated with ATP depletion and mitochondrial cytochrome c release, suggesting that macroautophagy was critical to remove damaged mitochondria and maintain mitochondrial ATP levels in response to oxidative stress [35]. However, loss of macroautophagy also led to overactivation of the JNK/c-Jun signaling pathway that mediated death from menadione upstream of ATP depletion and mitochondrial effects. Defective autophagy may therefore promote overactivation of the JNK/c-Jun signaling pathway in response to the oxidative stress that occurs in NASH. Increased JNK signaling from oxidative stress and decreased autophagy may promote disease, as JNK overactivation mediates NASH development and progression [43]. How macroautophagy could selectively regulate JNK signaling is not clear, and the mechanism of this effect remains to be determined. In these studies, CMA was also critical for hepatocyte resistance to oxidative stress. Knockdown of the CMA receptor LAMP-2A sensitized hepatocytes to death from menadione through a mechanism distinct from that mediated by macroautophagy [35]. CMA has been demonstrated to remove oxidized proteins that accumulate in the liver during aging, and CMA restoration improves hepatic function in aged mice [44]. CMA may similarly function in acute oxidative stress to limit hepatocellular injury by removing oxidized proteins that otherwise induce cellular damage. Further studies are needed to determine whether macroautophagy and/or CMA specifically modulate cell injury and death from oxidative stress resulting from mitochondrial dysfunction or other death stimuli in in vitro and in vivo models of hepatic steatosis.

Macroautophagy protects against other death stimuli that may mediate hepatocyte injury in NASH, although these studies have been performed in nonhepatic models. Potentially relevant to hepatocyte cell death in NASH is the ability of macroautophagy to protect against death receptor-mediated apoptosis from TNF and Fas, two factors implicated in hepatocellular injury in NASH [45,46]. Knockout of atg5 in mouse fibroblasts sensitized these cells to death from both Fas and TNF [47]. As previously discussed, ER stress is associated with insulin resistance and NASH [48]. Macroautophagy has been reported to protect against cell death from ER stress [49]. Macroautophagy may therefore regulate hepatocellular resistance to death receptor- and ER stress-mediated injury in NASH, but this speculation needs to be further examined in hepatocyte models.

Immune modulation by autophagy

Inflammation is a histological hallmark of NASH, and experimental evidence points to a critical role for the innate immune response in organ-specific manifestations of the metabolic syndrome, such as NAFLD [50]. In addition to the role of inflammation in the liver, adipose tissue inflammation serves as a source of proinflammatory cytokines that fuel the hepatic disease process. Autophagy has recently emerged as an important regulatory pathway of the innate immune response, particularly through effects on pathogen recognition receptors such as the Toll-like receptors (TLRs) [51]. In the liver, TLR ligands relevant to NASH pathogenesis include saturated fatty acids and lipopolysaccharide (LPS) or endotoxin. Studies of Atg16L1, an autophagy gene associated with susceptibility to inflammatory bowel disease, have demonstrated that in intestinal inflammation this factor that mediates autophagosome formation also regulates LPS-induced intestinal inflammation. Macrophages lacking Atg16L1 produced increased amounts of the proinflammatory cytokines IL-1β and IL-18 in response to LPS stimulation [52]. Mice deficient in hematopoietic cell Atg16L1 had increased susceptibility to dextran sulfate-induced inflammatory colitis mediated by these two cytokines [52]. LPS is a TLR4 ligand, and both LPS and TLR4 have been implicated in the pathogenesis of NASH [53,54]. Defects in macrophage macroautophagy may therefore amplify hepatic steatosis and liver injury. However, it is not known whether the defects in autophagy reported in fatty livers exist just in hepatocytes or extend to Kupffer cells as well. Interestingly, evidence exists that TLR signaling stimulates the autophagic response [55], suggesting that the role for TLR signaling in NASH may be complex, with both beneficial and detrimental effects.

Potential link to fibrogenesis

In addition to hepatocytes and macrophages, another cell type in which autophagy may function in NASH pathogenesis is the hepatic stellate cell. NASH is characterized by inflammation and the development of hepatic fibrosis that can progress to cirrhosis. Critical to fibrosis development is the activation of hepatic stellate cells, which leads to their production of excessive extracellular matrix [56]. Quiescent hepatic stellate cells store lipid primarily in the form of vitamin A. This lipid is lost during activation, but whether lipid depletion is mechanistically involved in the transdifferentiation process is unclear. It is interesting to speculate that autophagy may function in hepatic stellate cells to mediate this transdifferentiation process. One mechanism of autophagic involvement could be through the regulation of stellate cell lipid stores. Increased autophagy may be required for lipid removal, which is necessary to allow activation to occur or to provide critical energy stores. A second possibility is that autophagy has a direct effect on the transdifferentiation process, as previously demonstrated in adipose tissue in which an inhibition of macroautophagy alters transdifferentiation between white and brown adipocytes [57]. Investigations of hepatic stellate cell-specific autophagy gene knockouts/knockdowns in models of NASH can examine these possibilities.

Autophagy & NASH-associated hepatocellular cancer

Poorly defined factors lead to the development of HCC as a late complication of NASH [58]. Owing to the critical function of autophagy as a cell survival pathway, there is considerable interest in the role of autophagy in human carcinogenesis [59]. Controversy exists over whether autophagy promotes or inhibits cancer development. Autophagy may mediate cancer cell survival under metabolic stress, or may alternatively serve a tumor suppressor function through mechanisms such as by limiting genomic instability [60]. Supporting an anti-neoplastic role for autophagy is the fact that many cancers have defects in autophagy. In the liver, loss of the autophagy gene beclin 1 has been linked to HCC development. Mice with a knockout of beclin 1 develop spontaneous HCCs [61,62]. Loss of beclin 1 is frequent in human HCC and correlates with a poor prognosis [63]. Whether other defects in autophagy exist in HCC remains to be determined. The ability of hepatocellular lipid accumulation and hyperinsulinemia to suppress hepatic autophagic function suggests that these factors may promote HCC formation in NASH. Studies to examine the loss of critical autophagy genes in human NASH and the relationship of these events to the development of HCC must address this question.

Autophagy as a therapeutic target in NASH

Autophagy has many potentially beneficial functions that could prevent NASH development and progression (Box 1). As previously discussed, steatosis, hyperinsulinemia and other effects of aging may all contribute to an impairment in hepatic autophagic function that has a pathophysiological role in the promotion of NASH. The potential involvement of autophagy in many disease states, particularly in neurodegenerative diseases characterized by abnormal protein aggregates, is spurring interest in the development of pharmacological agents to augment autophagic function [64]. The use of such drugs to increase hepatic autophagy may offer a new therapeutic approach to NASH. Recently carbamazepine, a commonly used anticonvulsant in humans, has been demonstrated to be beneficial in the treatment of a murine model of α1-antitrypsin deficiency. The drug reduced hepatic levels of the mutant α1-antitrypsin and the amount of fibrosis [65]. Whether agents that stimulate autophagic function are effective in removing hepatic lipid or preventing cellular injury is unproven. Although carbamazepine rarely causes idiosyncratic hepatotoxicity [66], it or other novel inducers of autophagy may warrant clinical trials in NAFLD. A potential concern with such therapy is that the resultant generation of potentially toxic intracellular FFAs from autophagy-induced lipid droplet breakdown may actually increase liver injury [67]. However, an inhibition of lipid droplet formation has been demonstrated to reduce liver injury as well as steatosis [68]. A therapeutic manipulation of autophagic pathways in patients with NASH may therefore prove useful to both decrease the severity and progression of the disease and prevent NASH complications, such as HCC, which may be directly promoted by decreased autophagy.

Box 1. Potential beneficial effects of a therapeutic increase in hepatocyte autophagic function.

• Decrease triglyceride and cholesterol accumulation

• Improve insulin signaling

• Prevent cellular injury from oxidative stress

• Block TNF and Fas death receptor-mediated liver injury

• Reduce endoplasmic reticulum stress and the resultant cellular damage and insulin resistance

• Prevent hepatocellular carcinoma development

Expert commentary

Nonalcoholic fatty liver disease is the most prevalent liver disease in the USA and in most of the developed world. The pathogenesis of NAFLD and its treatment remain unclear. Recent investigations into the pathway of autophagy have delineated many functions for this degradative pathway that could potentially impact on the development and progression of NAFLD. Studies specific to hepatocytes and the liver have demonstrated critical roles for autophagy in the regulation of hepatocellular lipid accumulation and liver injury from oxidative stress. An impairment in autophagy may in part provide the ‘two hits’ that are needed for NASH development, promoting both the initial lipid accumulation and the progression to cellular injury. Findings from nonhepatic models suggest other possible functions for autophagy in immune and fibrotic responses and carcinogenesis that may also play a role in NASH. The impairment in autophagy that occurs with aging may explain, in part, the increased incidence of NAFLD with aging. The ability of excessive hepatocellular lipid accumulation to impair hepatic autophagy also suggests that steatosis may be a mechanism for the defect in autophagy that occurs in the liver with aging.

Five-year view

Functions for autophagy in hepatic steatosis and injury have only been recently described, and much work remains to be done to directly link this lysosomal pathway mechanistically to NASH. Defects in autophagy have been associated with the occurrence of hepatic steatosis. Future studies will have to demonstrate that physiological defects in autophagy directly promote disease development and progression. Whether an impairment in autophagy results in human NASH will also have to be examined. However, a determination of autophagic function in human livers is difficult, as presently only steady-state assays that measure the numbers of autophagosomes/autolysosomes, such as electron microscopy, can be applied to biopsies, and these methods do not distinguish between increased or decreased autophagy, either of which may elevate autophagosome number. Most studies have strictly addressed the role of macroautophagy in the liver, and future studies will also focus on the unique contributions of CMA to NASH. Additional investigations will examine the function of autophagy in hepatic cell types other than hepatocytes. Specifically, does autophagic function in macrophages and stellate cells mediate the manifestations of NASH? Eventually trials of pharmacological agents that augment autophagic function will need to be performed, initially in animals and then in humans, should a role for defects in autophagy be established in this disorder.