Cell. Mol. Life Sci. DOI 10.1007/s00018-016-2161-x

Cellular and Molecular Life Sciences

REVIEW

Pathogenesis of nonalcoholic steatohepatitis Wensheng Liu1 • Robert D. Baker1 • Tavleen Bhatia1 • Lixin Zhu1 • Susan S. Baker1

Received: 25 November 2015 / Revised: 19 January 2016 / Accepted: 9 February 2016 Ó Springer International Publishing 2016

Abstract Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease and a risk factor for cirrhosis and hepatocellular carcinoma. The pathological features of NASH include steatosis, hepatocyte injury, inflammation, and various degrees of fibrosis. Steatosis reflects disordered lipid metabolism. Insulin resistance and excessive fatty acid influx to the liver are two important contributing factors. Steatosis is also likely associated with lipotoxicity and cellular stresses such as oxidative stress and endoplasmic reticulum stress, which result in hepatocyte injury. Inflammation and fibrosis are frequently triggered by various signals such as proinflammatory cytokines and chemokines, released by injuried hepatocytes and activated Kupffer cells. Although much progress has been made, the pathogenesis of NASH is not fully elucidated. The purpose of this review is to discuss the current understanding of NASH pathogenesis, mainly focusing on factors contributing to steatosis, hepatocyte injury, inflammation, and fibrosis. Keywords Autophagy
Gut microbiota
Genetic predisposition
Apoptosis
Hepatic stellate cells

& Wensheng Liu [email protected] & Susan S. Baker [email protected] 1

Department of Pediatrics, Digestive Diseases and Nutrition Center, Women and Children’s Hospital of Buffalo, The State University of New York at Buffalo (SUNY Buffalo), 3435 Main Street, 422 BRB, Buffalo, NY 14214, USA

Abbreviations ChREBP Carbohydrate response element binding protein CVD Cardiovascular disease DAG Diacylglycerol DAMPs Damage-associated molecular patterns DNL De novo lipogenesis ER Endoplasmic reticulum ETC Electron transport chain FFAs Free fatty acids HCC Hepatocellular carcinoma HH Hedgehog HPCs Hepatic progenitor cells HSC Hepatic stellate cell IR Insulin resistance LPS Lipopolysaccharide NAFLD Nonalcoholic fatty liver disease NASH Nonalcoholic steatohepatitis NLRs NOD like receptors PAMPs Pathogen-associated molecular patterns PNPLA3 Patatin-like phospholipase domaincontaining 3 PPARs Peroxisome proliferator-activated receptors PRR Pattern recognition receptors PUFA Polyunsaturated fatty acids ROS Reactive oxygen species SCFAs Short-chain fatty acids SNP Single nucleotide polymorphism SREBP-1c Sterol regulatory element-binding protein 1c TGF-b Transforming growth factor b TGs Triglycerides TLRs Toll like receptors VEGF Vascular endothelial growth factor VLDL Very low density lipoprotein

123

W. Liu et al.

Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, affecting 20–30 % of the general population [1–8]. The hallmark of NAFLD is hepatic steatosis, which is characterized by fat accumulation (in the form of triglyceride) in more than 5 % of hepatocytes in the absence of excessive alcohol intake (\20 g per day for men, \10 g per day for women [9, 10]) or other secondary cause of fat deposition such as viral infections, certain medications, and genetic disorders. Nonalcoholic steatohepatitis (NASH) is a severe form of NAFLD. In addition to steatosis, the pathological features of NASH include hepatocyte injury, inflammation, and various degrees of fibrosis. The prevalence of NASH is estimated to be 2–5 % in the general population [4, 11]. Compared to simple steatosis (also known as nonalcoholic fatty liver, NAFL [12]), a less severe form of NAFLD that has been generally considered to be a benign condition, NASH is a significant risk factor for hepatic cirrhosis and hepatocellular carcinoma [1, 4, 6, 13–18]. It is estimated to account for more than 13 % of overall HCC cases in US [4, 19]. Although much progress has been made since NASH was first documented by Ludwig et al. [20], the pathogenesis of NASH is not fully elucidated. The initial theory for the NASH pathothesis, two hit hypothesis, was proposed by Day and James [21]. According to this hypothesis, simple steatosis, as a result of IR and excessive fatty acids, is the first hit of NASH. The first hit sensitizes the liver to a second hit leading to inflammation, hepatocyte damage, and fibrosis. The second hit promotes disease progression from steatosis to NASH. The second hit likely involves oxidative stress, lipid peroxidation, and mitochondrial dysfunction. This hypothesis implies that the accumulation of triglyceride, being the first hit, is necessary for the development of NASH. However, subsequent studies indicated that the accumulation of triglyceride in hepatocytes may have a protective role to prevent hepatocytes from lipotoxicity, which is induced by fatty acids and derived metabolites such as diacylglycerols, ceramides, and acylcarnitines [22, 23]. Thus, NeuschwanderTetri proposed the nontriglyceride lipotoxicity hypothesis in 2010 [23, 24]. This hypothesis stresses that nontriglyceride lipids, but not triglycerides, play an important role in the processes leading to hepatocyte injury, inflammation, and fibrosis. As more contributing factors are identified, it is evident that NASH is a multifactorial disease. Therefore, the multiple parallel hits hypothesis was proposed by Tilg and Moschen [25]. In this paradigm, NASH is the result of complex interactions among a number of parallel hits including: disrupted lipid metabolism, lipotoxicity, oxidative stress, mitochondrial dysfunction, ER stress, gut

123

derived endotoxin and ethanol, altered cytokines and adipokines, and genetic predisposition. Although fat accumulation in the liver is the common feature for both simple steatosis and NASH, steatosis may not be required for the development of NASH in some cases. Some studies showed that inflammation in NASH could occur prior to steatosis, suggesting that NASH and simple steatosis could be two distinct clinical conditions [26]. Unlike the two hit hypothesis that suggests that steatosis always precedes inflammation, the multiple parallel hits hypothesis indicates that hepatic inflammation in NASH may precede steatosis in some cases. At present, it is commonly accepted that both simple steatosis and NASH are within the spectrum of NAFLD and that a significant proportion of simple steatosis can progress to NASH [2, 16]. Indeed, recent studies of paired liver biopsies have shown that up to 44 % of steatosis may progress to NASH, indicating that steatosis may not be as benign as it has been thought to be [27, 28]. NAFLD is considered to be the hepatic component of the metabolic syndrome, as it is closely associated with metabolic factors such as obesity, insulin resistance, and type 2 diabetes. Its prevalence is increased to about 70 % in type 2 diabetes and 90 % in morbidly obese patients [1, 3, 4, 29–31]. In addition, a growing body of evidence suggests that NAFLD is also a risk factor for cardiovascular disease (CVD) independent of other metabolic factors [15, 22, 32–36]. CVD is the leading cause of death in NAFLD patients [37, 38]. Moreover, recent studies have suggested that hepatokines could be the potential mediators linking NAFLD to CVD [39–41]. Hepatokines, predominately produced by the liver and secreted into the circulation, have been shown to regulate glucose and lipid metabolism. Increased production of hepatokines such as fetuin-A (a2-HS-glycoprotein) [42, 43], fibroblast growth factor 21 (FGF21) [44–46], and selenoprotein P (SeP) [47, 48] have been observed in NAFLD patients. Furthermore, elevated serum levels of hepatokines are independently associated with CVD [48–52], suggesting that hepatokines could be the potential mechanistic link between NAFLD and CVD. In summary, steatosis is the result of disrupted lipid hemostasis. Excessive fatty acid influx to the liver and IR are two important factors contributing to steatosis. Excess lipid accumulation in the liver likely results in oxidative and ER stress, lipotoxicity, and hepatocyte injury, which in turn trigger inflammatory and wound healing responses promoting disease progression from steatosis to NASH. Moreover, NASH is a multifactorial disease which also involves multiple extrahepatic factors such as dysfunctional adipose tissue, altered gut microbiota, and genetic predisposition. The purpose of this review is to discuss the

Pathogenesis of nonalcoholic steatohepatitis

current understanding of NASH pathogenesis, mainly focusing on factors contributing to steatosis, hepatocyte injury, inflammation, and fibrosis.

Steatosis Disordered lipid metabolism Hepatic steatosis, i.e. fat accumulation in the form of lipid droplets in the cytoplasm of hepatocytes, is one of histopathological features for NASH diagnosis. Within hepatocytes steatosis reflects a disordered homeostasis of lipid metabolism when lipid inputs exceed lipids utilized. The main form of lipids stored in lipid droplets is triglycerides (TGs), which are composed of three fatty acids and one glycerol backbone via ester bonds. Dietary fats, circulating free fatty acids (FFAs) from lipolysis of adipose tissue, and de novo lipogenesis (DNL) are three lipid sources for hepatocytes, contributing to 15, 59, and 26 % of hepatic lipids in NAFLD patients, respectively [53]. Dietary fats are digested and absorbed by the enterocytes, and then released into the circulation in the form of TG-rich chylomicrons [54]. About 80 % of the TG in chylomicrons is hydrolyzed into FFAs and taken up by adipose tissue. The remaining 20 % of TG in chylomicron remnants is taken up by the liver. Thus, high fat diets may increase lipid delivery to the liver. High-fat diet is the common method to induce hepatic steatosis in animal models. Circulating FFAs are mainly derived from the lipolysis of adipose tissue. Hepatic FFAs taken up from circulation is dependent on the concentration of FFAs in the blood and transport proteins including fatty acid transport proteins (FATPs), fatty acid binding proteins (FABPs), and fatty acid translocase (FAT/CD36) [6, 55– 57]. As hyperlipidemia is often associated with NASH, FFAs taken up from the circulation are increased in NAFLD. Moreover, the hepatic expression of FFA transport proteins is also increased in NAFLD, which further contributes to enhanced FFA delivery to the liver. DNL is a metabolic pathway which synthesizes fatty acids from simple metabolic precursors (i.e., acetyl-CoA) in a series of enzymatic reactions. The DNL pathway is mainly regulated by insulin and glucose at the transcriptional level in adipose tissue and liver. Fatty acids synthesized from DNL in NAFLD are increased to 26 % of total hepatic lipids, whereas DNL contributes about 5 % of total hepatic fats in healthy individuals [4, 53, 58–60]. Taken together, compared to healthy individuals, influx of fatty acids to the liver in NAFLD patients is increased. One major function of hepatic FFAs is to provide energy for the liver through oxidation [57]. Hepatic FFAs are mainly oxidized by mitochondria through b-oxidation. In

NAFLD increased FFAs enhance b-oxidation by mitochondria until mitochondrial respiration becomes severely impaired [61–63]. In addition, increased hepatic FFAs also stimulate b-oxidation in peroxisomes and microsomal xoxidation in the endoplasmic reticulum by microsomal cytochrome P450 enzymes. Peroxisome proliferator-activated receptor a (PPAR-a) is a key transcription factor regulating the expression of genes involved in mitochondrial, peroxisomal and microsomal FFA oxidation [64]. FFAs have been shown to induce PPAR-a in NAFLD [65], indicating that the enhanced FFA oxidation could be mediated by increased PPAR-a in NAFLD. Hepatic FFAs can also be used to synthesize other lipids such as phospholipids or re-esterified to form TGs. Then TGs can be assembled into very low density lipoprotein (VLDL) particles for secretion or stored in lipid droplets. Although defective VLDL seceretion may contribute to lipid accumulation, it has been shown that VLDL secretion is increased in NAFLD [57, 66–68]. Our studies have found that, compared to normal controls, a number of lipid metabolism–related genes, which are involved in fatty acid uptake, DNL, oxidation, and VLDL secretion, showed significantly increased expression levels in livers from NASH patients [69]. Increased FFA oxidation and VLDL secretion could be a compensative mechanism in response to the overload of hepatic FFAs. In addition, a study by Fujita et al. [70] has reported that VLDL secretion in NASH patients is significantly lower than that in NAFL patients although both NAFL and NASH patients have elevated serum VLDL levels than control group, suggesting that decreased VLDL secretion may contribute to disease progression from NAFL to NASH. Taken together, when FFA oxidation and VLDL secretion are unable to utilize the overloaded FFAs, excessive FFAs will be esterified into TGs and stored in lipid droplets, resulting in hepatic steatosis. Thus, various mechanisms such as increased FFA influx to the liver, enhanced DNL, reduced FFA oxidation, and decreased VLDL secretion can lead to hepatic steatosis (Fig. 1). Insulin resistance Insulin signaling pathway is initiated by insulin binding to its receptor and mediated by the activation of phosphoinositol-3-kinase and AKT/PKB kinase, leading to multiple effects on the liver. In healthy individuals, insulin inhibits hepatic glucose production, stimulates hepatic glucose uptake, and promotes DNL in the postprandial state [15]. Under IR condition, the stimulatory effect of insulin on DNL is retained in the liver, whereas the inhibitory effect on glucose production is diminished [54, 71]. It is well established that IR is the primary factor underlying hepatic steatosis. IR is present in almost all NAFLD patients [63, 72]. The disruption of normal insulin signaling in

123

W. Liu et al. Fig. 1 Factors contributing to hepatic steatosis. ?: promoting steatosis; -: reducing steatosis

Adipose tissue IR, Lipolysis , FFAs +

Gut microbiota Ethanol , Choline +

Genetic SNPs PNPLA3 I148M +

Dietary fats

+

Liver IR

+ +

DNL

hepatocytes and increased abundance of fatty acids as well lead to disordered lipid metabolism. In insulin resistant liver, insulin is unable to inhibit glucose production, leading to an increased glucose level. Glucose stimulates DNL via the transcription factor known as the carbohydrate response element binding protein (ChREBP). On the other hand, insulin still stimulates DNL by increasing the master transcription factor of lipogenesis, sterol regulatory element-binding protein 1c (SREBP-1c). In addition, NAFLD patients present IR in adipose tissue and skeletal muscle as well, which may influence hepatic steatosis. Due to IR, the adipose tissue becomes resistant to the anti-lipolytic effect of the insulin. As a result, increased FFAs are delivered to the liver [15]. Due to IR in skeletal muscle, glucose uptake is reduced, leading to increased glucose delivery to the liver [57, 73–75]. Collectively, it is well demonstrated that IR can lead to steatosis in human and animal studies. Interestingly, studies have also shown that steatosis can lead to IR [76–81]. One underlying mechanism is mediated by diacylglycerol (DAG). In steatosis DAG level is increased. DAG activates classical (b) and novel (d and e) PKC, leading to impaired insulin signaling [77]. However, patients with mutations that cause hepatic steatosis do not present IR, suggesting that steatosis may not be able to induce IR in humans. For example, patients with familial hypobetalipoproteinemia develop hepatic steatosis due to defective secretion of VLDL particles, caused by a genetic deficiency of hepatic apolipoprotein B synthesis; however, these patients do not show insulin resistance [82]. Autophagy Autophagy is a lysosome dependent degradative process that functions to recycle cellular constituents and to maintain cellular energy homeostasis. Recently autophagy has been implicated in lipid metabolism. In hepatocytes, the degradation of lipid droplets mediated by autophagy is known as lipophagy. Dysfunctional autophagy may therefore contribute to the pathogenesis of NAFLD.

123

_ FFA TG accumulation oxidation (Steatosis) _ _ Autophagy

VLDL secretion

Studies have shown that inhibition of autophagy in hepatocytes and in mouse liver increases triglyceride storage in lipid droplets, suggesting lipophagy may prevent steatosis [83]. Moreover, it is shown that the increased TG storage is due to impaired lipolysis and not to increased TG synthesis, suggesting that autophagy plays a role in the lipolysis of lipid droplet. Conversely, lipid storage in hepatocytes is decreased when autophagy is induced by pharmacological reagents [83]. Further, in genetically manipulated mice and dietary mouse models, protein levels of ATG7, an important component in autophagy, are reduced; and levels of autophagy are decreased as well [84, 85]. Overexpression of ATG7 in the liver of ob/ob mice induces autophagy and reduces steatosis significantly. These findings support a lipolytic function of autophagy. The lipolytic function of lipophagy in liver is thought to rapidly mobilize large amounts of lipids in fasting state since the level of cytosolic lipases in hepatocytes is low compared to adipocytes. A pilot immunohistochemical study on human liver tissue has shown that the autophagy marker LC3 was decreased with an increased degree of steatosis, suggesting decreased autophagy in more severe steatosis [86]. In another study increased p62 accumulation, indicative of defective autophagy, was observed on liver biopsies in NAFLD patients [87]. These studies suggest autophagy is defective in NAFLD. Similar to lipolysis, lipophagy is inhibited by insulin under physiological conditions. It is of interest that hepatic steatosis also decreases autophagy. It is possible that defective autophagy in NAFLD is related to IR [88]. Although autophagy has been shown to play a role in lipid metabolism, its exact function in NAFLD is not fully understood. Dysfunctional adipose tissue Although hepatic steatosis is a pathological process occurring in hepatocytes, several extrahepatic factors may have significant effects, one of which is adipose tissue. Normally adipose tissue is the primary organ for lipid

Pathogenesis of nonalcoholic steatohepatitis

storage. It has been demonstrated that adipose tissue in NAFLD is dysfunctional, which contributes to NASH pathogenesis in different ways. Adipose enlargement and hypertrophy, IR, and altered secretion of adipokines have been observed in NAFLD patients. Excessive lipids stored in adipose tissue results in enlargement and hypertrophy, which in turn impair its normal functions. For example, DNL is down regulated in adipose tissue in obese animals and human subjects as well [89–91]. Excessive lipid accumulation in adipose tissue, similar to the liver, can lead to IR. Normally insulin suppresses lipolysis in adipose tissue. Lipolysis is mediated by adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoglyceride lipase (MGL). Insulin activates phosphodiesterase 3B through AKT and decreases cAMP level, which leads to reduced PKA activation [92]. Lipolysis is then inhibited due to decreased HSL phosphorylation by PKA. Insulin also suppresses lipolysis through downregulating the expression of ATGL [93–95]. Conversely, ATGL expression in adipose tissue is increased in obese patients with insulin resistance [96]. Collectively, insulin resistance in the adipose tissue results in increased lipolysis and therefore increased release of FFAs into circulation and FFA uptake in the liver (Fig. 1). Another effect of dysfunctional adipose tissue on the liver is mediated by altered adipokines. In addition to storage of excessive lipids, adipose tissue also functions as an important endocrine organ. Adipose tissue secrets a number of cytokines such as TNFa and IL-6. Among all adipokines, adiponectin and leptin are directly involved in the development of hepatic steatosis. Adiponectin has an inhibitory effect on hepatic steatosis by stimulating fatty acid oxidation in the liver. This effect is mainly mediated through activation of AMP mediated protein kinase and PPAR-a [15]. NAFLD patients have reduced adiponectin levels compared to normal controls. Therefore, low adiponectin in NAFLD enhances FFA overload in the liver [15]. Leptin has been shown to regulate food intake and energy expenditure through its effects on the central nervous system [97]. Leptin-deficient (ob/ob) mice are commonly used as an obese mouse model that also develops fatty livers. Leptin treatment decreases triglyceride levels in patients with leptin deficiency [98, 99]. However, unlike murine models, most obese and NAFLD patients have increased leptin levels, suggesting leptin resistance. Altered gut microbiota In addition to adipose tissue, gut microbiota can also affect hepatic lipid metabolism and contribute to the development of steatosis. In healthy individuals, the gut microbiota has important metabolic effects on the host energy

homeostasis. [15]. Germ-free mice have less total body fat than controls with a normal gut microbiota [100]. When they are exposed to gut microbiota harvested from conventionally raised mice, germ-free mice have a 60 % increase in body fat and develope insulin resistance within 2 weeks. The weight gain and insulin resistance are due to increased energy extraction from the undigestible food component [98]. The human gut microbiota is predominantly composed of three bacterial phyla: the gram-negative Bacteroidetes, and the gram-positive Firmicutes and Actinobacteria [101]. Recent studies in mice and humans have demonstrated that NAFLD is associated with altered composition of gut microbiota [98, 102]. Initial studies have shown that obesity and NAFLD in humans as well as rodents are associated with increased levels of Firmicutes and decreased levels of Bacteroidetes; thus, an increased Firmicutes/Bacteroidetes ratio is a potential phenotype of obesity. [102, 103]. However, some studies including ours have shown that Firmicutes are decreased in NASH [104, 105]. Moreover, recent studies [15, 106] have shown that when mice fed a high fat diet were treated with antibiotics, the hepatic TG accumulation was significantly reduced, suggesting that manipulating the gut microbiota could affect the development of steatosis [15]. The effects of gut microbiota on the liver energy metabolism could be mediated by increasing short-chain fatty acids (SCFAs) and ethanol production, and by reducing choline level. SCFAs, mainly comprised of acetate, propionate and butyrate, are the major fermentation products of gut microbes [104]. In the human colon, the concentration of SCFAs produced by gut microbiota can reach 50–100 mM [107]. These SCFAs provide a significant portion of the energy needed for our body. The SCFAs produced by the gut microbiota account for about 30 % of energy extraction from the diet [108]. In humans, increased production of SCFAs by the gut microbiota was also observed in obese people, compared to lean subjects [109–111]. Therefore the gut microbiota may contribute to the development of steatosis by increasing SCFAs delivery to the liver. By contrast, recent studies have suggested that SCFAs protect against the development of metabolic syndrome [112–114]. Further studies are needed to investigate the role of SCFAs in NAFLD. It is well known that the gut microbiota can produce alcohol [115]. In vitro studies have demonstrated that, under anaerobic conditions, 1 g (wet weight) of E. coli can produce 0.8 g of alcohol in 1 h [116]. Increased endogenous ethanol produced by bacterial enzymes has been implicated in NAFLD development. Volynets et al. [117] observed that serum alcohol concentrations were elevated in adult NAFLD patients. Our studies showed that serum alcohol concentrations in pediatric NASH patients were

123

W. Liu et al.

higher than those in healthy controls and non-NASH obese patients [105]. We further found that the composition of gut microbiota in NASH patients is significantly different from that in non-NASH obese patients in the abundance of Escherichia, a genus of the Enterobacteriaceae family. Escherichia, under anaerobic conditions, can convert sugars to a mixture of products by fermentation, including alcohol [116, 118–120]. Since the gut microbiota is the major source of endogenous alcohol, it may contribute to the development of hepatic steatosis by similar mechanisms as described for alcoholic steatosis. Choline is an essential nutrient for our body. The changes of gut microbiota in NAFLD may result in choline deficiency, as dietary choline can be converted to trimethylamine by microbial enzymes. Choline-deficient diets are commonly used to generate NASH models in rodents. Similarly, choline deficiency induced by the gut microbiota can also lead to hepatic steatosis and NASH. Recently choline deficiency has been linked to NAFLD in human [111]. It is possible that choline deficiency in humans is induced by altered gut microbiota. Taken together, altered composition of gut microbiota in NAFLD can have profound effects on the host energy metabolism and contribute to the development of steatosis (Fig. 1). Genetic predeposition Although NAFLD mainly presents with metabolic disorders, it also has a strong genetic component, as revealed by sibling studies [37, 121]. Gender and ethnicity have been shown to affect the prevalence of NAFLD. A populationbased study reported that before age 60, men had a higher rate of NAFLD than women, but the prevalence in women at older ages was higher than men. The same study also showed that the prevalence of NAFLD for hispanics was 45 %, 33 % for non-Hispanic whites, and 24 % for African Americans [37, 98]. Recent studies have identified a number of genetic variants conferring susceptibility for hepatic steatosis [81]. At present, the single nucleotide polymorphism (SNP) of rs738409 in patatin-like phospholipase domain-containing 3 (PNPLA3) gene has been demonstrated as a potent genetic factor predisposing to fatty liver. This SNP results in a missense mution of codon 148 of PNPLA3 gene from isoleucine to methionine (I148M). The PNPLA3 I148M gene variant has been identified in independent genome-wide association studies [122, 123] and confirmed by several subsequent studies [25, 81, 102, 124]. The differences in the prevalence of NAFLD among different races can be explained by the prevalence of PNPLA3 I148M mutation among ethnicities [37, 77, 81, 98, 125]. In humans PNPLA3, also known as adiponutrin, is expressed in hepatocytes and hepatic stellate cells in the

123

liver and in adipocytes as well [6, 126]. PNPLA3 is a membrane protein localized in the endoplasmic reticulum and at the surface of lipid droplets [124]. However, the exact function of PNPLA3 is not known since PNPLA3 knockout mice develop neither fatty liver nor liver injury [127]. In vitro studies suggest that PNPLA3 may function as a downstream target gene of SREBP-1c and mediate lipid accumulation [128]. Purified PNPLA3 possesses phospholipase, TG lipase and acylglycerol transacylase activity [126]. Its functions are implicated in lipid droplet remodelling and VLDL secretion. The I148M variant results in a loss of these enzymatic activities, leading to impaired lipid droplet remodelling and VLDL secretion. Thus PNPLA3 I148M variant increases the susceptibility to hepatic steatosis. Recently the TM6SF2 E167K variant is identified as another genetic factor increasing the susceptibility to hepatic steatosis in a genome-wide association study [129, 130]. The TM6SF2 E167K variant is also related to reduced VLDL secretion. It is of interest that both PNPLA3 I148M and TM6SF2 E167K (SNP rs58542926) variants are associated with the spectrum of NAFLD, including fibrosis and NASH [124, 130, 131]. A number of other genetic variants in various genes have been associated with the susceptibility of NAFLD through genome-wide association studies or candidate gene studies [37, 81, 98, 102, 132]. However, only some SNPs are independently validated, including GCKR rs780094, PEMT rs7946, SOD2 rs4880, KLF6 rs3750861, and ATGR1 rs3772622 [131]. Further studies are needed to validate these candidate genetic modifiers and reveal their underlying mechanisms.

Hepatocyte injury and apoptosis Steatosis-associated cellular stresses In steatosis, FFA delivery to the liver is markedly increased. It is likely that metabolites derived from FFAs will also be increased in hepatic steatosis, in parallel to TG accumulation. It is well documented that increased hepatocyte triglyceride formation may have a protective role to prevent hepatocytes from FFA induced damage. Unlike TGs, FFAs and other derived metabolites such as diacylglycerols, ceramides, and acylcarnitines are harmful to hepatocytes. These toxic effects induced by fatty acids and derived metabolites are generally known as lipotoxicity [22, 23, 133, 134]. Listenberger et al. [135, 136] have shown that palmitic acid induces apoptosis through ceramide and reactive intermediates; whileas oleic acid inhibits palmitate-induced apoptosis by channeling palmitate into TG synthesis. However, both oleic and palmitic acids induce lipotoxicity in mouse embryonic fibroblasts with defective TG synthesis,

Pathogenesis of nonalcoholic steatohepatitis

highlighting that TG synthesis plays an important role in the protection from lipotoxicity [135]. Therefore, hepatic steatosis reflects disordered lipid metabolism and it may have serious lipotoxic effects on the liver. One important mediator of lipotoxicity is the over production of reactive oxygen species (ROS). When ROS production exceeds the antioxidant capacity, it leads to oxidative stress. Numerous studies have demonstrated that oxidative stress is elevated in NAFLD patients, as indicated by various oxidative stress markers present in liver biopsies or in the peripheral circulation [137]. Moreover, some studies have shown that the level of oxidative stress correlated with the disease severity of NAFLD [4, 137]. Further, in response to oxidative stress, a variety of anti-oxidant genes are upregulated in NAFLD, which could be a compensatory mechanism to counteract oxidative stress [137–141]. ROS are produced in hepatocytes as a result of FFA oxidation. Hepatic FFAs can be oxidized in mitochondria, peroxisomes, and microsomes. FFA b-oxidation in mitochondria provides energy (ATP) for the liver through the electron transport chain (ETC) and oxidative phosphorylation. Due to electron leak in the complexes I and III of ETC, mitochondria also become the major source of ROS production in cells. Under normal conditions, it has been estimated that about 0.2–2 % of the oxygen utilized in mitochondria generates ROS [142]. FFA b-oxidation in mitochondria initially is stimulated as FFA influx to the liver is increased. Therefore, more substrate derived electrons will be transferred to ETC, which results in dramatically increased ROS production. It has been estimated that approximately 2–4 % of oxygen consumed by the cell is used to generate ROS in NAFLD; whereas it is less than 2 % under normal conditions [143–145]. When mitochondria b-oxidation is overwhelmed and not sufficient to metabolize overload of FFAs in NAFLD, the b-oxidation by peroxisomes as well as x-oxidation by microsomes become increasingly important. FFA oxidation by both peroxisomes and microsomes generates a significant amount of ROS and contributes to oxidative stress. Despite the powerful anti-oxidant capacity in the liver, excessive FFA oxidation in the steatotic hepatocytes could cause substantial oxidative stress. In addition to oxidative stress, steatosis is also associated with endoplasmic reticulum (ER) stress [1, 146, 147]. ER is an important intracellular organelle that plays a crucial role in the synthesis, folding, and trafficking of proteins. ER stress, i.e. dysfunction in the ER, causes accumulation of unfolded proteins and triggers an unfolded protein response (UPR). One of the consequences of lipotoxicity in steatosis is the activation of UPR. It has been shown that saturated FFAs directly induce ER stress response in hepatocytes. Moreover, increased levels of ER stress have been reported in NAFLD patients [146, 147]. Glucose regulated protein GRP78 is a central regulator of

ER function and protects cells against ER stress [15]. A study has shown that serum GRP78 concentrations are decreased in NASH patients, compared to patients with simple steatosis [91], indicating increased ER stress. Cellular stress-induced hepatocyte injury and apoptosis Prolonged cellular stresses can cause serious damage, leading to cell death by necrosis and apoptosis [148]. Hepatocyte injury is also a defining lesion for NASH. Histologically, hepatocyte injury is indicated by hepatocyte ballooning that is characterized by an enlarged hyperchromatic nucleus and a foamy, pale cytoplasm [3, 9, 149, 150]. Hepatocyte ballooning is due to abnormal distribution of intermediate filaments indued by oxidative stress [150, 151]. Oxidative stress has been well documented to cause a variety of damages such as mitochondrial dysfunction. Mitochondria are the major targets of ROS effects because they are the primary organelle that produces ROS. Polyunsaturated fatty acids (PUFA) are essential components of phospholipids in mitochondrial membranes. ROS can attack and react with PUFA, leading to damage to mitochondrial membranes. ROS can also inactivate important antioxidant enzymes such as superoxide dismutase, which in turn reduces the antioxidant capacity [152, 153]. Lipid peroxidation products can inhibit the electron-transport chain of the mitochondria [154], which further increases ROS production. In addition, ROS also cause damage to mitochondrial DNA by inducing point mutations and DNA breaks. Collectively, overproduction of ROS can lead to mitochondrial dysfunction [144]. The capacity to oxidize FFAs is reduced in dysfunctional mitochondria, leading to more ROS production and starting a vicious circle [65, 155, 156]. Impaired ATP homeostasis [157], defective electron transport chain [158], and abnormal ultrastructure such as paracristalline inclusions and loss of cristae [159, 160] are common indications of mitochondrial dysfunction and have been well documented in NAFLD. Under normal conditions, injuried intracellular organelles are removed and recycled by degrative pathways such as autophagy. Although hepatocytes have powerful antioxidant resources, prolonged stress and unrepaired injuries lead to cell death by necrosis and apoptosis. [63]. It has been shown that apoptosis is the major mechanism of cell death in NASH and promotes disease progression from simple steatosis to NASH [161]. Increased levels of apoptosis are seen in NASH patients compared with controls; and hepatocyte apoptosis positively correlates with the severity of disease [2, 162]. Both the intrinsic and the extrinsic apoptosis pathways are implicated in the pathogenesis of NASH [63, 163–166]. Dysfunctional mitochondria can initiate apoptosis by cytochrome c release. Prolonged ER stress increases calcium release

123

W. Liu et al.

Steatosis, FFAs

ROS

Mitochondrial dysfunction

Oxidative stress

ER stress

Hepatocyte injury and apoptosis Fig. 2 Hepatocyte injury and apoptosis in NAFLD

from ER and leads to apoptosis [167]. Moreover, FFAs play an important role in apoptosis in NASH [161]; and apoptosis levels correlate with serum FFA levels [168]. The accumulation of FFAs in hepatocytes induces apoptosis via multiple mechanisms. For instance, excessive FFAs upregulate Fas ligands and activate Fas receptors, promote lysosomal permeabilization and release the lysosomal protease cathepsin B, and activate TLR4 receptor, all of which lead to cellular injury and apoptosis [15, 29, 169]. Taken together, increased cellular stresses can lead to cell injury and apoptosis (Fig. 2), which are the important drivers of inflammation and fibrosis in liver diseases.

Inflammation Kupffer cells as the central players in the inflammation in NASH Inflammation is mediated by a number of proinflammatory cytokines and chemokines such as TNFa, IL6, and CCL2 (monocyte chemoattractant protein 1, MCP-1). Those Fig. 3 Inflammation in NASH

FFAs and lipotoxic metabolites

soluble mediators of inflammation can be secreted by injuried hepatocytes and dysfunctional adipose tissue, which promote hepatic inflammation in NASH. In injuried hepatocytes, NF-jB pathway is activated, leading to expression of proinflammatory cytokines. The secretion of proinflammatory cytokines such as TNFa and IL6 is also increased in dysfunctional adipose tissue, as a result of increased macrophage infiltration [170]. TNFa activates two main proinflammatory signalling pathways: the JNK and NF-jB pathway [171], increasing production of proinflammatory cytokines (including TNFa) and further worsening the inflammation. Although injuried hepatocytes and dysfunctional adipose tissue may contribute to hepatic inflammation, Kupffer cells have been implicated to play a central role in the inflammation in NASH [172, 173] (Fig. 3). Kupffer cells are resident macrophages in the liver, accounting for about 10 % of total resting liver cells [15]. When Kupffer cells are depleted in mice, liver inflammation is reduced, indicating that Kupffer cells play a key role in liver inflammation [174]. Recent studies further showed that inflammation in the liver is regulated by the balance of proinflammatory M1 Kupffer cells and anti-inflammatory M2 Kupffer cells [175, 176]. Imbalanced M1/M2 phenotypic Kupffer cells have emerged as a central mechanism underlying steatohepatitis. In NASH Kupffer cells are activated by various stimulants and shift to the proinflammatory M1 phenotype. In agreement with this notion, adiponectin, an anti-inflammatory adipokine, promotes M2 Kupffer cell polarization through inhibiting TNFa expression and increasing the expression of anti-inflammatory cytokines such as IL-10 [174]. Histologically, inflammation in NASH is manifested by infiltration of inflammatory cells such as macrophages, monocytes, neutrophils, and lymphocytes [177]. Accumulating studies have demonstrated that macrophage and monocyte infiltration into the liver is primarily promoted by chemokine CCL2 since macrophages and monocytes express the receptor for CCL2, C–C chemokine receptor 2 (CCR2). CCL2 levels are increased in both the serum and

Hepatocyte injury and apoptosis DAMPs ROS

Kupffer cell activation (M1 phenotype)

123

Gut derived PAMPs

Inflamsome activation, IL-1 Inflammatory cytokines (e.g. TNF , IL6) ; Chemokines (e.g. CCL2) , recruitment of neutrophils and monocytes, etc

Pathogenesis of nonalcoholic steatohepatitis

the liver of NASH patients. Inhibition of CCL2 or knockout of CCR2 reduces macrophage infiltration in animal NASH models [174]. In NASH Kupffer cells play a major role in the recrutiment of inflammatory cells in the liver. Neutrophil infiltration is frequently observed in human NASH [178], although its pathogenic significance is not fully understood. It has been suggested that increased myeloperoxidase (MPO, a neutrophil enzyme) may cause oxidative damage to hepatocytes and contribute to the development of NASH [179, 180]. A recent study has shown that in a mouse model neutrophil infiltration is associated with inflammation induced by dietary triggers carbohydrate and cholesterol; whileas non-metabolic triggers LPS and interleukin-1b mainly induce intrahepatic accumulation of mononuclear cells, suggesting that different inducers may elicit different inflammatory responses [181]. Kupffer cells are exposed to various substances such as nutrients and gut derived bacterial products, and they function to sense and remove pathogens and danger molecules via pattern recognition receptors (PRR). PRR comprise at least two families of sensing proteins: the Tolllike receptors (TLR) and the NOD like receptors (NLR). Both NLRs and TLRs detect danger signals including: pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are pathogens originating from gut-derived microorganisms; while DAMPs include molecules endogenously released from stressed or injuried hepatocytes. TLR recognize bacterial products derivated from gut microbiota such as lipopolysaccharide (LPS, also known as endotoxin) and peptidoglycan. NLR is a component of inflammasome complexes in the cytosol. In response to danger signals, activation of inflammasome results in secretion of IL-1 beta, IL-18 or IL33. Kupffer cells are the primary sensors of PAMPs and DAMPs as well, and TLR and NLR receptors have emerged as important mediators of Kupffer cell activation [174]. Various factors activating Kupffer cells As mentioned above, gut-derived bacterial products can activate Kupffer cells and promote inflammation responses. A variety of danger signals activate Kupffer cells through different TLR or NLR receptors. For example, LPS is shown to bind to TLR4 and trigger a cascade of inflammatory signaling pathways [182, 183]. LPS levels are reported to be increased both in NASH patients and experimental models due to increased gut permeability [184]. FFAs have been shown to indirectly bind to TLR4 and activate Kupffer cells. FFAs may interact with TLR4 via fetuin-A, which is an endogenous ligand for TLR4 [185, 186]. Besides, palmitic acid can activate TLR2 likely

via an indirect mechanism as palmitic acid alone is unable to activate TLR2 [187, 188]. In addition, Kupffer cells can also be activated by diacylglycerol, ceramide, cholesterol, oxidized lipoproteins, and engulfment of apoptotic hepatocytes [174, 189]. Moreover, products of PUFA peroxidation such as 4-hydroxy-2-nonenal and malondialdehyde, which have longer half-lives than ROS, can diffuse into extracellular space and activate Kupffer cells [137, 144]. Inflammation is a required pathological feature for defining NASH and it is also the essential difference from simple steatosis. Clinically it is important to distinguish simple steatosis from NASH because their prognosis is different. Compared to simple steatosis, NASH is a significant risk factor for hepatic cirrhosis and hepatocellular carcinoma. [6, 190]. Although in some NASH cases, inflammation can occur independent of steatosis, about 20–30 % of simple steatosis can progress to NASH [2, 25, 146, 191, 192]. One mechanism could be mediated by steatosis-induced cellular stresses, which in turn leads to hepatocyte injury and apoptosis triggering inflammatory responses (Fig. 3).

Fibrosis Liver cells involved in fibrosis Although the presence of fibrosis is not a required histological feature for defining NASH, it is an important pathological change as its presence confers a worse prognosis [15]. Fibrosis is a wound healing response characterized by an excessive deposition of collagen and other extracellular matrix (ECM) molecules resulting in formation of scar tissue. Hepatic stellate cells (HSCs) are the key cells involved in the production of excessive ECM seen in liver fibrosis. They are the perisinusoidal cells distributed throughout the liver, with a significant gamut of functions in normal as well as injured liver. In the normal liver, stellate cells participate in retinoid storage, vasoregulation, extracellular matrix homeostasis, drug detoxification and immunotolerance [193]. In the event of liver injury, HSCs undergo activation, from a quiescent, vitamin A-storing cell to a myofibroblast-like cell, with several new characteristics, such as augmented cell migration and adhesion, increased proliferation, production of chemotactic substances, contractibility, loss of normal retinoid-storing capacity and most notably, acquisition of fibrogenic potential [193–196]. In addition to HSCs, portal fibroblasts that primarily have a role in cholestatic liver injuries also provide a minor contribution to myofibroblast pool in NASH [193–195]. Furthermore, Kupffer cells play an essential role in

123

W. Liu et al.

Gut derived LPS, etc

Hepatocyte injury and apoptosis

HPCs ductular reaction

Kupffer cell activation TGF Stellate cell activation (autophagy , BAMBI )

Deposition of extracellular matrix (e.g. collagen)

Fig. 4 Fibrosis in NASH

perpetuating liver injury by activating HSCs through actions of several cytokines, especially reactive oxygen species and TGF- beta. Additionally, there has been recent evidence to suggest the role of hepatic progenitor cells (HPCs) in liver fibrosis in NAFLD [197–199]. HPCs are the stem cells forming a reserve compartment, which gets activated only when hepatocytes are continuously damaged and has the ability to differentiate into mature hepatocytes and cholangiocytes. In cases of recurrent liver insult, HPCs expand from periportal to pericentral zone giving rise to reactive ductules or ductular reaction [200]. The ductular reaction has been proven to strongly correlate with progressive portal fibrosis in NASH independent of the deposition of collagen by stellate cells [197, 200]. Furthermore, these reactive ductules are a source of factors such as Platelet-Derived Growth Factor, TGF-b, and Sonic Hedgehog, which perpetuate HSC activation [198, 200, 201] (Fig. 4). TGF-b signaling pathway Transforming growth factor TGF-b signaling pathway is known to play a major role in the activation of HSCs in liver fibrosis [202]. The TGFb consists of three isoforms TGFb1, TGFb2 and TGFb3. TGFb1 in particular is a potent modulator of cell proliferation, differentiation and fibrosis [203, 204]. The primary source of TGFb1 in the liver is thought to be Kupffer cells [202]. However, endothelial cells, hepatocytes and stellate cells also produce small amounts of this cytokine. Several studies in which TGF-b expression in liver is altered by using adenovirus or transgenic TGF-b mice have revealed a critical contribution of TGF-b to HSC activation and development of fibrosis [204, 205]. TGF-b signals through transmembrane receptors that stimulate cytoplasmic SMAD proteins. SMAD proteins are subdivided into three classes: receptor Smads (R-Smads), common mediator Smads, and inhibitory Smads. Smad7 is an inhibitory Smad, which in case of

123

increased TGFb signal forms a stable complex with the activated TGFb1 receptor and blocks the downstream pathways preventing fibrosis [206]. However, this protective response is altered in chronic liver injury as shown in experiments conducted by Tahashi et al. on rat livers. They reported that in chronically injured rat livers, Smad7 was not induced by the autocrine TGFb signal [207]. Failure of Smad7 induction on repeated injury to the liver could explain the ongoing TGFb effects and progression of liver fibrosis despite these inbuilt protective mechanisms. Recently, human BAMBI, a protein closely related to TGF-beta family type 1 receptor that lacks an intracellular kinase domain and blocks signal transduction after stimulation with ligands of the TGF-superfamily such as TGF, Bone morphogenetic proteins (BMPs) and activin (cytokines responsible for fibrosis) has been studied. Yan and colleagues further showed the complex interaction between Smad7, human BAMBI and TGFb signaling and reported that BAMBI also interacts with inhibitory Smad7, and the interaction between BAMBI and Smad7 was enhanced by TGF-b treatment [208]. Liu et al. [209] demonstrated that there is reduction of BAMBI receptors in both LPS-treated quiescent HSCs and in vivo activated HSCs from TLR4– wild-type, but not TLR4-mutant mice, strongly suggesting that TLR4-mediated down regulation of BAMBI is an integral part of HSC activation. These studies insinuate that appropriate down regulation of TGFb by BAMBI might be necessary for inhibition of advancement of fibrosis. Furthermore, the members of the integrin family can activate TGFb1 and TGFb3. Henderson and coworkers showed that loss of av integrin led to a decrease in Smad3 immunostaining, decrease in expression of TGF-b inducible genes and TGF-b activation in HSC culture reiterating that avb6 and avb8 integrins are required for the important functions of these two TGF-b isoforms [210]. In contrast, Saile et al. [211] reported that TGF-b in fact has anti-apoptotic effect on activated HSC. Based on this plasticity in response, caution is needed when ascribing general effects of TGF-b on HSCs. TGF-b signaling is regulated by interplay of multiple factors, cytokines, receptors etc. and might have several and sometimes, opposing effects in NASH. Hedgehog (HH) signaling pathway The hedgehog pathway is a highly conserved signaling pathway that was originally identified in Drosophila. This family of ligands such as sonic hedgehog (Shh), Indian hedgehog (Ihh) and desert hedgehog (Dhh), interact with the cell surface receptors PTCH1 and PTCH2 on hedgehog responsive target cells like HPCs. In HH signaling, the interaction of Shh with the cell surface receptor PTCH depresses Smo receptor (G protein coupled receptor)

Pathogenesis of nonalcoholic steatohepatitis

activity, leading to nuclear localization of glioblastoma family transcription factors (GLI 1, 2 and 3), which in turn regulates the expression of cell-specific target genes [212]. Guy et al. showed that the increase in sonic hedgehog protein in portal cells positively correlates with the stage of fibrosis and ballooning of hepatocytes. In tandem, there was an increase in doubly positive GLI 2?/K7? cells (K7? or CK7? marks a subpopulation of liver epithelial progenitors) with increased stages of fibrosis [213]. Nobili et al. [197] demonstrated that the number of hepatic progenitor cells (stained by CK7) positively correlated with the degree of fibrosis in pediatric NAFLD patients. Syn et al. then revealed that hepatic natural killer T cells drive production of hedgehog ligands and osteopontin (OPN). OPN is induced by HH pathway activation and correlates directly with fibrosis stage. They also revealed that recombinant OPN promoted fibrogenic responses in HSCs, suggesting the importance of HH pathway in liver fibrosis in NAFLD [214]. HH signaling pathway not only interacts with HPCs but also regulates epithelial mesenchymal transition (EMT) during liver fibrosis. HH stimulates quiescent HSC to become myofibroblasts. This is evidenced by the work of Choi et al. [215] who demonstrated that profibrogenic actions of Rac1 were mediated by its ability to activate hedgehog signaling pathway-dependent mechanisms that stimulated myofibroblast transition of HSCs and enhanced myofibroblast viability. Xie and coworkers also showed that when cultured HSC transitioned into myofibroblasts, they activated hedgehog signaling, underwent an EMT and increased Notch signaling. Blocking Notch signaling in myofibroblasts suppressed hedgehog activity and caused an EMT. Inhibiting the hedgehog pathway suppressed Notch signaling and also induced an EMT [216]. Thus, the Notch and Hedgehog pathways interact to control the fate of hepatic stellate cells. In addition, many other pathways are implicated in liver fibrosis in NAFLD, such as JAK/STAT/ERK signaling pathways, nuclear receptor pathways like farsenoid X receptor, and Rev-Erba. Evidence from cell culture as well as animal studies proves that adipokines such as leptin and adiponectin play a vital role in regulation of liver fibrosis as well [217]. Leptin binds to its receptor and activates the JAK2/STAT3 pathway [218, 219]. The activation of the JAK2/STAT3 pathway leads to matrix deposition through increased expression of tissue inhibitor of metalloproteinases (TIMPs) and inhibition of matrix degradation through decreased matrix metalloproteinases (MMPs) [219]. Adiponectin, on the other hand, suppresses the proliferation and migration of HSCs, hence attenuating fibrosis [220]. Also, extracellular molecules, such as lipopolysaccharide (LPS), tumor necrosis factor, interleukins, chemokines like CCL2, CCL5, CXCL10 and reactive oxygen species (ROS), can perpetuate liver

fibrosis by promotion of recruitment and migration of macrophages and HSC activation [195]. Factors promoting fibrosis Endotoxemia, LPS and intestinal microbiota Despite an efficient intestinal barrier, a small amount of bacteria and bacterial products frequently reach the portal circulation and are cleared without occurrence of significant inflammation. Diet high in fat alters the intestinal microbiota with increase in gram-negative bacteria as shown in several studies over the past decade. Also, in chronic liver diseases like NASH, there is increased intestinal permeability with increased translocation of microbial products [221, 222]. This change in intestinal microbiota in combination with increased gut leakiness leads to a rise in LPS levels in systemic and portal circulation. This upsurge in LPS acts as danger signals for the HSCs leading to their activation and finally fibrosis (Fig. 4). Number of studies have linked intestinal microbiome not only to hepatic inflammation but also fibrosis [223– 225]. Samuele De Minicis and colleagues reported more liver fibrosis in mice fed a control diet and transplanted with microbiota obtained from mice fed a high fat diet (HFD), as compared to HFD fed mice transplanted with microbiota obtained from mice fed a control diet [225]. Microflora transplantation was the vital experiment that confirmed the important and independent role of microbiome to the contribution to liver fibrosis, by eliminating the other factors like diet and lipid metabolism that can confound the results. To further study the role of microbiome and its close interaction with toll like receptors, Seki et al. [223] performed bile duct ligation (BDL) on TLR4-mutant mice and on TLR4–wild-type mice and demonstrated that TLR4– wild-type mice showed hepatic fibrosis, whereas TLR4mutant mice had a significant reduction in fibrosis, hence proving the important role of TLR4. They also reported that when they sterilized mice guts and performed BDL, there was a significant decrease in hepatic fibrosis as well as TLR4 expression, confirming that intestinal microbiome in combination with TLRs, especially TLR4, plays a role in hepatic fibrosis [226]. The exact mechanism of action of TLR4 on hepatic fibrosis is yet to be elucidated but it is likely that TLR4 plays a role in activation of HSCs via TGFb and Kupffer cells. However, there might be other pathways at play and further studies are needed to discern them. Autophagy Autophagy is the endogenous, tightly regulated cellular housekeeping process responsible for the degradation of

123

W. Liu et al.

damaged and dysfunctional cellular organelles and protein aggregates. It is well established from the overwhelming evidence in the literature that activated HSCs play a central role in liver fibrosis. As they transition from quiescent to an activated form, there is loss of the intracellular lipid droplets [196]. Hernandez-Gea et al. demonstrated increased levels of autophagy in mice after induction of liver injury with carbon tetrachloride or thioacetamide. Autophagy was then blocked. It was then observed that the loss of autophagic function in cultured mouse stellate cells and in mice following injury led to reduced fibrogenesis and matrix accumulation and an increase in intracellular lipid droplets and triglyceride content [227]. HSCs lacking autophagy remain in a quiescent state and autophagy is associated with lipid degradation required for HSC activation. It is most likely that autophagy by lipid droplet mobilization and degradation provides energy that is essential to support stellate cell activation in the face of increasing cellular energy demands during fibrogenesis. Angiogenesis Angiogenesis in liver is closely associated with progression of fibrosis in chronic liver diseases [228–230]. Both in humans and experimental models, chronic liver injury is characterized by an increase in endothelial cell number and microvessels [228, 229]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor [230]. Hepatic neovascularization and expression of VEGF is increased in rats developing liver fibrosis [231]. There is also a parallel increase of VEGF-A expression and hypoxic areas during progression of fibrosis indicating that hypoxia, angiogenesis and fibrosis are closely related. Also, hypoxia is one of the most important stimuli to switch on the transcription of pro-angiogenic genes through the action of hypoxia-inducible transcription factors [232, 233]. VEGF stimulates the proliferation, migration, and chemotaxis of HSCs and endothelin 1 and angiotensin II control the contractibility of HSCs. To complement, HSCs in turn contribute to angiogenesis through production of VEGF and angiopoietin-1 [230]. Inhibition of angiogenesis by blocking VEGF or angiopoietin-1 inhibits liver fibrosis, inferring that angiogenesis plays an essential role in liver fibrosis [234].

Conclusions Hepatic steatosis is the result of disordered lipid metabolism. Insulin resistance and excessive fatty acid influx into the liver are the major driving forces for steatosis. In addition, dysfunctional adipose tissue, altered gut microbiota, and genetic predisposition contribute to the development of steatosis. Steatosis may not be as benign as

123

it has been thought to be. A significant proportion of simple steatosis may progress to NASH although the underlying mechanisms are not fully elucidated. Steatosis is likely associated with oxidative stress and ER stress, which result in hepatocyte injury and apoptosis. Prolonged and unrepaired cell injuries promote inflammation and fibrogenesis. Kupffer cells play a central role in the processes leading to inflammation. Hepatic stellate cells are the primary cells responsible for fibrogenesis. NASH is a risk factor for cirrhosis and hepatocellular carcinoma, therefore there is an urgent need for effective treatments. Better understanding of NASH pathogenesis could be helpful for developing novel treatments for NASH.

References 1. Yoon HJ, Cha BS (2014) Pathogenesis and therapeutic approaches for non-alcoholic fatty liver disease. World J Hepatol 6:800–811 2. Levene AP, Goldin RD (2012) The epidemiology, pathogenesis and histopathology of fatty liver disease. Histopathology 61:141–152 3. Ahmed M (2015) Non-alcoholic fatty liver disease in 2015. World J Hepatol 7:1450–1459 4. Dowman JK, Tomlinson JW, Newsome PN (2010) Pathogenesis of non-alcoholic fatty liver disease. QJM 103:71–83 5. Milic S, Stimac D (2012) Nonalcoholic fatty liver disease/ steatohepatitis: epidemiology, pathogenesis, clinical presentation and treatment. Dig Dis 30:158–162 6. Bettermann K, Hohensee T, Haybaeck J (2014) Steatosis and steatohepatitis: complex disorders. Int J Mol Sci 15:9924–9944 7. Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, Landt CL, Harrison SA (2011) Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 140:124–131 8. Amarapurkar D, Kamani P, Patel N, Gupte P, Kumar P, Agal S, Baijal R, Lala S, Chaudhary D, Deshpande A (2007) Prevalence of non-alcoholic fatty liver disease: population based study. Ann Hepatol 6:161–163 9. Tiniakos DG, Vos MB, Brunt EM (2010) Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol 5:145–171 10. Younossi ZM (2008) Review article: current management of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Aliment Pharmacol Ther 28:2–12 11. Bhala N, Jouness RI, Bugianesi E (2013) Epidemiology and natural history of patients with NAFLD. Curr Pharm Des 19:5169–5176 12. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ (2012) The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 55:2005–2023 13. Argo CK, Northup PG, Al-Osaimi AM, Caldwell SH (2009) Systematic review of risk factors for fibrosis progression in nonalcoholic steatohepatitis. J Hepatol 51:371–379

Pathogenesis of nonalcoholic steatohepatitis 14. Starley BQ, Calcagno CJ, Harrison SA (2010) Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology 51:1820–1832 15. Than NN, Newsome PN (2015) A concise review of non-alcoholic fatty liver disease. Atherosclerosis 239:192–202 16. Angulo P (2010) Long-term mortality in nonalcoholic fatty liver disease: is liver histology of any prognostic significance? Hepatology 51:373–375 17. Ekstedt M, Franzen LE, Mathiesen UL, Thorelius L, Holmqvist M, Bodemar G, Kechagias S (2006) Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology 44:865–873 18. Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P (2005) The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129:113–121 19. Marrero JA, Fontana RJ, Su GL, Conjeevaram HS, Emick DM, Lok AS (2002) NAFLD may be a common underlying liver disease in patients with hepatocellular carcinoma in the United States. Hepatology 36:1349–1354 20. Ludwig J, Viggiano TR, McGill DB, Oh BJ (1980) Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 55:434–438 21. Day CP, James OF (1998) Steatohepatitis: a tale of two ‘‘hits’’? Gastroenterology 114:842–845 22. Cusi K (2012) Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology 142(711–725):e716 23. Neuschwander-Tetri BA (2010) Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites. Hepatology 52:774–788 24. Neuschwander-Tetri BA (2010) Nontriglyceride hepatic lipotoxicity: the new paradigm for the pathogenesis of NASH. Curr Gastroenterol Rep 12:49–56 25. Tilg H, Moschen AR (2010) Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52:1836–1846 26. Yilmaz Y (2012) Review article: is non-alcoholic fatty liver disease a spectrum, or are steatosis and non-alcoholic steatohepatitis distinct conditions? Aliment Pharmacol Ther 36:815–823 27. Adams LA, Ratziu V (2015) Non-alcoholic fatty liver—perhaps not so benign. J Hepatol 62:1002–1004 28. McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM (2015) Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol 62:1148–1155 29. Basaranoglu M, Kayacetin S, Yilmaz N, Kayacetin E, Tarcin O, Sonsuz A (2010) Understanding mechanisms of the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol 16:2223–2226 30. Machado M, Marques-Vidal P, Cortez-Pinto H (2006) Hepatic histology in obese patients undergoing bariatric surgery. J Hepatol 45:600–606 31. Angulo P (2002) Nonalcoholic fatty liver disease. N Engl J Med 346:1221–1231 32. Byrne CD, Targher G (2015) NAFLD: a multisystem disease. J Hepatol 62:S47–S64 33. Anstee QM, Targher G, Day CP (2013) Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 10:330–344 34. Armstrong MJ, Adams LA, Canbay A, Syn WK (2014) Extrahepatic complications of nonalcoholic fatty liver disease. Hepatology 59:1174–1197 35. Lim S, Oh TJ, Koh KK (2015) Mechanistic link between nonalcoholic fatty liver disease and cardiometabolic disorders. Int J Cardiol 201:408–414

36. Oni ET, Agatston AS, Blaha MJ, Fialkow J, Cury R, Sposito A, Erbel R, Blankstein R, Feldman T, Al-Mallah MH, Santos RD, Budoff MJ, Nasir K (2013) A systematic review: burden and severity of subclinical cardiovascular disease among those with nonalcoholic fatty liver; should we care? Atherosclerosis 230:258–267 37. Hassan K, Bhalla V, Ezz El Regal M, A-Kader HH (2014) Nonalcoholic fatty liver disease: a comprehensive review of a growing epidemic. World J Gastroenterol 20:12082–12101 38. Ong JP, Pitts A, Younossi ZM (2008) Increased overall mortality and liver-related mortality in non-alcoholic fatty liver disease. J Hepatol 49:608–612 39. Iroz A, Couty JP, Postic C (2015) Hepatokines: unlocking the multi-organ network in metabolic diseases. Diabetologia 58:1699–1703 40. Stefan N, Haring HU (2013) The role of hepatokines in metabolism. Nat Rev Endocrinol 9:144–152 41. Yoo HJ, Choi KM (2015) Hepatokines as a link between obesity and cardiovascular diseases. Diabetes Metab J 39:10–15 42. Yilmaz Y, Yonal O, Kurt R, Ari F, Oral AY, Celikel CA, Korkmaz S, Ulukaya E, Ozdogan O, Imeryuz N, Avsar E, Kalayci C (2010) Serum fetuin A/alpha2HS-glycoprotein levels in patients with non-alcoholic fatty liver disease: relation with liver fibrosis. Ann Clin Biochem 47:549–553 43. Lebensztejn DM, Bialokoz-Kalinowska I, Klusek-Oksiuta M, Tarasow E, Wojtkowska M, Kaczmarski M (2014) Serum fetuin A concentration is elevated in children with non-alcoholic fatty liver disease. Adv Med Sci 59:81–84 44. Yilmaz Y, Eren F, Yonal O, Kurt R, Aktas B, Celikel CA, Ozdogan O, Imeryuz N, Kalayci C, Avsar E (2010) Increased serum FGF21 levels in patients with nonalcoholic fatty liver disease. Eur J Clin Invest 40:887–892 45. Li H, Fang Q, Gao F, Fan J, Zhou J, Wang X, Zhang H, Pan X, Bao Y, Xiang K, Xu A, Jia W (2010) Fibroblast growth factor 21 levels are increased in nonalcoholic fatty liver disease patients and are correlated with hepatic triglyceride. J Hepatol 53:934–940 46. Dushay J, Chui PC, Gopalakrishnan GS, Varela-Rey M, Crawley M, Fisher FM, Badman MK, Martinez-Chantar ML, Maratos-Flier E (2010) Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology 139:456–463 47. Choi HY, Hwang SY, Lee CH, Hong HC, Yang SJ, Yoo HJ, Seo JA, Kim SG, Kim NH, Baik SH, Choi DS, Choi KM (2013) Increased selenoprotein p levels in subjects with visceral obesity and nonalcoholic Fatty liver disease. Diabetes Metab J 37:63–71 48. Yang SJ, Hwang SY, Choi HY, Yoo HJ, Seo JA, Kim SG, Kim NH, Baik SH, Choi DS, Choi KM (2011) Serum selenoprotein P levels in patients with type 2 diabetes and prediabetes: implications for insulin resistance, inflammation, and atherosclerosis. J Clin Endocrinol Metab 96:E1325–E1329 49. Chow WS, Xu A, Woo YC, Tso AW, Cheung SC, Fong CH, Tse HF, Chau MT, Cheung BM, Lam KS (2013) Serum fibroblast growth factor-21 levels are associated with carotid atherosclerosis independent of established cardiovascular risk factors. Arterioscler Thromb Vasc Biol 33:2454–2459 50. Shen Y, Ma X, Zhou J, Pan X, Hao Y, Zhou M, Lu Z, Gao M, Bao Y, Jia W (2013) Additive relationship between serum fibroblast growth factor 21 level and coronary artery disease. Cardiovasc Diabetol 12:124 51. Weikert C, Stefan N, Schulze MB, Pischon T, Berger K, Joost HG, Haring HU, Boeing H, Fritsche A (2008) Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke. Circulation 118:2555–2562 52. Dogru T, Genc H, Tapan S, Aslan F, Ercin CN, Ors F, Kara M, Sertoglu E, Karslioglu Y, Bagci S, Kurt I, Sonmez A (2013)

123

W. Liu et al.

53.

54. 55.

56.

57. 58.

59.

60.

61.

62.

63.

64.

65. 66.

67.

68.

69.

70.

Plasma fetuin-A is associated with endothelial dysfunction and subclinical atherosclerosis in subjects with nonalcoholic fatty liver disease. Clin Endocrinol (Oxf) 78:712–717 Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ (2005) Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 115:1343–1351 Cohen JC, Horton JD, Hobbs HH (2011) Human fatty liver disease: old questions and new insights. Science 332:1519–1523 Bradbury MW (2006) Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis. Am J Physiol Gastrointest Liver Physiol 290:G194–G198 Musso G, Gambino R, Cassader M (2009) Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD). Prog Lipid Res 48:1–26 Fabbrini E, Magkos F (2015) Hepatic steatosis as a marker of metabolic dysfunction. Nutrients 7:4995–5019 Hudgins LC, Hellerstein MK, Seidman CE, Neese RA, Tremaroli JD, Hirsch J (2000) Relationship between carbohydrateinduced hypertriglyceridemia and fatty acid synthesis in lean and obese subjects. J Lipid Res 41:595–604 Parks EJ (2002) Dietary carbohydrate’s effects on lipogenesis and the relationship of lipogenesis to blood insulin and glucose concentrations. Br J Nutr 87(Suppl 2):S247–S253 Diraison F, Beylot M (1998) Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification. Am J Physiol 274:E321–E327 Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, Luketic VA, Shiffman ML, Clore JN (2001) Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 120:1183–1192 Miele L, Grieco A, Armuzzi A, Candelli M, Forgione A, Gasbarrini A, Gasbarrini G (2003) Hepatic mitochondrial betaoxidation in patients with nonalcoholic steatohepatitis assessed by 13C-octanoate breath test. Am J Gastroenterol 98:2335–2336 Marra F, Gastaldelli A, Svegliati Baroni G, Tell G, Tiribelli C (2008) Molecular basis and mechanisms of progression of nonalcoholic steatohepatitis. Trends Mol Med 14:72–81 Reddy JK (2001) Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal beta-oxidation, PPAR alpha, and steatohepatitis. Am J Physiol Gastrointest Liver Physiol 281:G1333–G1339 Day CP (2002) Pathogenesis of steatohepatitis. Best Pract Res Clin Gastroenterol 16:663–678 Fabbrini E, Mohammed BS, Magkos F, Korenblat KM, Patterson BW, Klein S (2008) Alterations in adipose tissue and hepatic lipid kinetics in obese men and women with nonalcoholic fatty liver disease. Gastroenterology 134:424–431 Adiels M, Taskinen MR, Packard C, Caslake MJ, Soro-Paavonen A, Westerbacka J, Vehkavaara S, Hakkinen A, Olofsson SO, Yki-Jarvinen H, Boren J (2006) Overproduction of large VLDL particles is driven by increased liver fat content in man. Diabetologia 49:755–765 Musso G, Gambino R, Durazzo M, Biroli G, Carello M, Faga E, Pacini G, De Michieli F, Rabbione L, Premoli A, Cassader M, Pagano G (2005) Adipokines in NASH: postprandial lipid metabolism as a link between adiponectin and liver disease. Hepatology 42:1175–1183 Zhu L, Baker SS, Liu W, Tao MH, Patel R, Nowak NJ, Baker RD (2011) Lipid in the livers of adolescents with nonalcoholic steatohepatitis: combined effects of pathways on steatosis. Metabolism 60:1001–1011 Fujita K, Nozaki Y, Wada K, Yoneda M, Fujimoto Y, Fujitake M, Endo H, Takahashi H, Inamori M, Kobayashi N, Kirikoshi H, Kubota K, Saito S, Nakajima A (2009) Dysfunctional verylow-density lipoprotein synthesis and release is a key factor in

123

71. 72.

73.

74.

75.

76.

77.

78. 79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

nonalcoholic steatohepatitis pathogenesis. Hepatology 50:772–780 Brown MS, Goldstein JL (2008) Selective versus total insulin resistance: a pathogenic paradox. Cell Metab 7:95–96 Bugianesi E, Gastaldelli A, Vanni E, Gambino R, Cassader M, Baldi S, Ponti V, Pagano G, Ferrannini E, Rizzetto M (2005) Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: sites and mechanisms. Diabetologia 48:634–642 Fabbrini E, Magkos F, Mohammed BS, Pietka T, Abumrad NA, Patterson BW, Okunade A, Klein S (2009) Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity. Proc Natl Acad Sci USA 106:15430–15435 Korenblat KM, Fabbrini E, Mohammed BS, Klein S (2008) Liver, muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects. Gastroenterology 134:1369–1375 Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, Goto T, Westerbacka J, Sovijarvi A, Halavaara J, Yki-Jarvinen H (2002) Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab 87:3023–3028 Finck BN, Hall AM (2015) Does diacylglycerol accumulation in fatty liver disease cause hepatic insulin resistance? Biomed Res Int 2015:104132 Birkenfeld AL, Shulman GI (2014) Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology 59:713–723 Loria P, Lonardo A, Anania F (2013) Liver and diabetes. A vicious circle. Hepatol Res 43:51–64 Perry RJ, Samuel VT, Petersen KF, Shulman GI (2014) The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 510:84–91 Takamura T, Misu H, Ota T, Kaneko S (2012) Fatty liver as a consequence and cause of insulin resistance: lessons from type 2 diabetic liver. Endocr J 59:745–763 Williams KH, Shackel NA, Gorrell MD, McLennan SV, Twigg SM (2013) Diabetes and nonalcoholic fatty liver disease: a pathogenic duo. Endocr Rev 34:84–129 Tanoli T, Yue P, Yablonskiy D, Schonfeld G (2004) Fatty liver in familial hypobetalipoproteinemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity. J Lipid Res 45:941–947 Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ (2009) Autophagy regulates lipid metabolism. Nature 458:1131–1135 Yang L, Li P, Fu S, Calay ES, Hotamisligil GS (2010) Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab 11:467–478 Chen R, Wang Q, Song S, Liu F, He B, Gao X (2016) Protective role of autophagy in methionine–choline deficient diet-induced advanced nonalcoholic steatohepatitis in mice. Eur J Pharmacol 770:126–133 Kashima J, Shintani-Ishida K, Nakajima M, Maeda H, Unuma K, Uchiyama Y, Yoshida K (2014) Immunohistochemical study of the autophagy marker microtubule-associated protein 1 light chain 3 in normal and steatotic human livers. Hepatol Res 44:779–787 Fukuo Y, Yamashina S, Sonoue H, Arakawa A, Nakadera E, Aoyama T, Uchiyama A, Kon K, Ikejima K, Watanabe S (2014) Abnormality of autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease. Hepatol Res 44:1026–1036 Liu HY, Han J, Cao SY, Hong T, Zhuo D, Shi J, Liu Z, Cao W (2009) Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of FoxO1-

Pathogenesis of nonalcoholic steatohepatitis

89.

90.

91.

92.

93.

94.

95.

96.

97.

98. 99.

100.

101.

102. 103.

104. 105.

106.

dependent expression of key autophagy genes by insulin. J Biol Chem 284:31484–31492 Ferre P, Foufelle F (2010) Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 12(Suppl 2):83–92 Lim JW, Dillon J, Miller M (2014) Proteomic and genomic studies of non-alcoholic fatty liver disease—clues in the pathogenesis. World J Gastroenterol 20:8325–8340 Rodriguez-Suarez E, Duce AM, Caballeria J, Martinez Arrieta F, Fernandez E, Gomara C, Alkorta N, Ariz U, MartinezChantar ML, Lu SC, Elortza F, Mato JM (2010) Non-alcoholic fatty liver disease proteomics. Proteomics Clin Appl 4:362–371 DiPilato LM, Ahmad F, Harms M, Seale P, Manganiello V, Birnbaum MJ (2015) The role of PDE3B phosphorylation in the inhibition of lipolysis by insulin. Mol Cell Biol 35:2752–2760 Chakrabarti P, Kim JY, Singh M, Shin YK, Kim J, Kumbrink J, Wu Y, Lee MJ, Kirsch KH, Fried SK, Kandror KV (2013) Insulin inhibits lipolysis in adipocytes via the evolutionarily conserved mTORC1-Egr1-ATGL-mediated pathway. Mol Cell Biol 33:3659–3666 Chakrabarti P, English T, Shi J, Smas CM, Kandror KV (2010) Mammalian target of rapamycin complex 1 suppresses lipolysis, stimulates lipogenesis, and promotes fat storage. Diabetes 59:775–781 Chakrabarti P, Kandror KV (2011) Adipose triglyceride lipase: a new target in the regulation of lipolysis by insulin. Curr Diabetes Rev 7:270–277 Tinahones FJ, Garrido-Sanchez L, Miranda M, Garcia-Almeida JM, Macias-Gonzalez M, Ceperuelo V, Gluckmann E, RivasMarin J, Vendrell J, Garcia-Fuentes E (2010) Obesity and insulin resistance-related changes in the expression of lipogenic and lipolytic genes in morbidly obese subjects. Obes Surg 20:1559–1567 Larter CZ, Chitturi S, Heydet D, Farrell GC (2010) A fresh look at NASH pathogenesis. Part 1: the metabolic movers. J Gastroenterol Hepatol 25:672–690 Matherly SC, Puri P (2012) Mechanisms of simple hepatic steatosis: not so simple after all. Clin Liver Dis 16:505–524 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, Wagner AJ, DePaoli AM, Reitman ML, Taylor SI, Gorden P, Garg A (2002) Leptin-replacement therapy for lipodystrophy. N Engl J Med 346:570–578 Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 101:15718–15723 Ferolla SM, Armiliato GN, Couto CA, Ferrari TC (2014) The role of intestinal bacteria overgrowth in obesity-related nonalcoholic fatty liver disease. Nutrients 6:5583–5599 Duwaerts CC, Maher JJ (2014) Mechanisms of liver injury in non-alcoholic steatohepatitis. Curr Hepatol Rep 13:119–129 Wu GD, Chen J, Hoffmann C, Bittinger K, Chen YY, Keilbaugh SA, Bewtra M, Knights D, Walters WA, Knight R, Sinha R, Gilroy E, Gupta K, Baldassano R, Nessel L, Li H, Bushman FD, Lewis JD (2011) Linking long-term dietary patterns with gut microbial enterotypes. Science 334:105–108 Zhu L, Baker RD, Baker SS (2015) Gut microbiome and nonalcoholic fatty liver diseases. Pediatr Res 77:245–251 Zhu L, Baker SS, Gill C, Liu W, Alkhouri R, Baker RD, Gill SR (2013) Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology 57:601–609 Jiang C, Xie C, Li F, Zhang L, Nichols RG, Krausz KW, Cai J, Qi Y, Fang ZZ, Takahashi S, Tanaka N, Desai D, Amin SG, Albert I, Patterson AD, Gonzalez FJ (2015) Intestinal farnesoid

107.

108.

109.

110.

111.

112.

113.

114.

115. 116. 117.

118. 119. 120.

121.

122. 123.

X receptor signaling promotes nonalcoholic fatty liver disease. J Clin Invest 125:386–402 Duncan SH, Louis P, Thomson JM, Flint HJ (2009) The role of pH in determining the species composition of the human colonic microbiota. Environ Microbiol 11:2112–2122 Wostmann BS, Larkin C, Moriarty A, Bruckner-Kardoss E (1983) Dietary intake, energy metabolism, and excretory losses of adult male germfree Wistar rats. Lab Anim Sci 33:46–50 Schwiertz A, Taras D, Schafer K, Beijer S, Bos NA, Donus C, Hardt PD (2010) Microbiota and SCFA in lean and overweight healthy subjects. Obesity (Silver Spring) 18:190–195 Cho I, Yamanishi S, Cox L, Methe BA, Zavadil J, Li K, Gao Z, Mahana D, Raju K, Teitler I, Li H, Alekseyenko AV, Blaser MJ (2012) Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 488:621–626 Moschen AR, Kaser S, Tilg H (2013) Non-alcoholic steatohepatitis: a microbiota-driven disease. Trends Endocrinol Metab 24:537–545 den Besten G, Havinga R, Bleeker A, Rao S, Gerding A, van Eunen K, Groen AK, Reijngoud DJ, Bakker BM (2014) The short-chain fatty acid uptake fluxes by mice on a guar gum supplemented diet associate with amelioration of major biomarkers of the metabolic syndrome. PLoS One 9:e107392 den Besten G, Gerding A, van Dijk TH, Ciapaite J, Bleeker A, van Eunen K, Havinga R, Groen AK, Reijngoud DJ, Bakker BM (2015) Protection against the metabolic syndrome by guar gumderived short-chain fatty acids depends on peroxisome proliferator-activated receptor gamma and glucagon-like peptide-1. PLoS One 10:e0136364 den Besten G, Bleeker A, Gerding A, van Eunen K, Havinga R, van Dijk TH, Oosterveer MH, Jonker JW, Groen AK, Reijngoud DJ, Bakker BM (2015) Short-chain fatty acids protect against high-fat diet-induced obesity via a PPARgamma-dependent switch from lipogenesis to fat oxidation. Diabetes 64:2398–2408 Blomstrand R (1971) Observations of the formation of ethanol in the intestinal tract in man. Life Sci II 10:575–582 Dawes EA, Foster SM (1956) The formation of ethanol in Escherichia coli. Biochim Biophys Acta 22:253–265 Volynets V, Kuper MA, Strahl S, Maier IB, Spruss A, Wagnerberger S, Konigsrainer A, Bischoff SC, Bergheim I (2012) Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease (NAFLD). Dig Dis Sci 57:1932–1941 Paege LM, Gibbs M (1961) Anaerobic dissimilation of glucoseC14 by Escherichia coli. J Bacteriol 81:107–110 Clark DP (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol Rev 5:223–234 Brooks JB, Basta MT, el Kholy AM (1985) Studies of metabolites in diarrheal stool specimens containing Shigella species by frequency-pulsed electron capture gas-liquid chromatography. J Clin Microbiol 21:599–606 Loomba R, Schork N, Chen CH, Bettencourt R, Bhatt A, Ang B, Nguyen P, Hernandez C, Richards L, Salotti J, Lin S, Seki E, Nelson KE, Sirlin CB, Brenner D (2015) Heritability of hepatic fibrosis and steatosis based on a prospective twin study. Gastroenterology 149:1784–1793 Krawczyk M, Bonfrate L, Portincasa P (2010) Nonalcoholic fatty liver disease. Best Pract Res Clin Gastroenterol 24:695–708 Speliotes EK, Yerges-Armstrong LM, Wu J, Hernaez R, Kim LJ, Palmer CD, Gudnason V, Eiriksdottir G, Garcia ME, Launer LJ, Nalls MA, Clark JM, Mitchell BD, Shuldiner AR, Butler JL, Tomas M, Hoffmann U, Hwang SJ, Massaro JM, O’Donnell CJ, Sahani DV, Salomaa V, Schadt EE, Schwartz SM, Siscovick DS, Voight BF, Carr JJ, Feitosa MF, Harris TB, Fox CS, Smith AV, Kao WH, Hirschhorn JN, Borecki IB (2011) Genome-wide association analysis identifies variants associated with

123

W. Liu et al.

124.

125.

126.

127.

128.

129.

130.

131. 132. 133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

nonalcoholic fatty liver disease that have distinct effects on metabolic traits. PLoS Genet 7:e1001324 Dongiovanni P, Romeo S, Valenti L (2015) Genetic factors in the pathogenesis of nonalcoholic fatty liver and steatohepatitis. Biomed Res Int 2015:460190 Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC, Hobbs HH (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 40:1461–1465 Musso G, Gambino R, Cassader M (2010) Non-alcoholic fatty liver disease from pathogenesis to management: an update. Obes Rev 11:430–445 Basantani MK, Sitnick MT, Cai L, Brenner DS, Gardner NP, Li JZ, Schoiswohl G, Yang K, Kumari M, Gross RW, Zechner R, Kershaw EE (2011) Pnpla3/adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome. J Lipid Res 52:318–329 Qiao A, Liang J, Ke Y, Li C, Cui Y, Shen L, Zhang H, Cui A, Liu X, Liu C, Chen Y, Zhu Y, Guan Y, Fang F, Chang Y (2011) Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis. Hepatology 54:509–521 Goffredo M, Caprio S, Feldstein AE, D’Adamo E, Shaw MM, Pierpont B, Savoye M, Zhao H, Bale AE, Santoro N (2015) Role of the TM6SF2 rs58542926 in the pathogenesis of non-alcoholic pediatric fatty liver disease (NAFLD): a multiethnic study. Hepatology 63(1):117–125. doi:10.1002/hep.28283 Anstee QM, Day CP (2015) The genetics of nonalcoholic fatty liver disease: spotlight on PNPLA3 and TM6SF2. Semin Liver Dis 35:270–290 Anstee QM, Day CP (2013) The genetics of NAFLD. Nat Rev Gastroenterol Hepatol 10:645–655 Cheung O, Sanyal AJ (2010) Recent advances in nonalcoholic fatty liver disease. Curr Opin Gastroenterol 26:202–208 Zambo V, Simon-Szabo L, Szelenyi P, Kereszturi E, Banhegyi G, Csala M (2013) Lipotoxicity in the liver. World J Hepatol 5:550–557 Alkhouri N, Dixon LJ, Feldstein AE (2009) Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal. Expert Rev Gastroenterol Hepatol 3:445–451 Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS, Schaffer JE (2003) Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci USA 100:3077–3082 Listenberger LL, Ory DS, Schaffer JE (2001) Palmitate-induced apoptosis can occur through a ceramide-independent pathway. J Biol Chem 276:14890–14895 Liu W, Baker SS, Baker RD, Zhu L (2015) Antioxidant mechanisms in nonalcoholic fatty liver disease. Curr Drug Targets 16:1301–1314 Desai S, Baker SS, Liu W, Moya DA, Browne RW, Mastrandrea L, Baker RD, Zhu L (2014) Paraoxonase 1 and oxidative stress in paediatric non-alcoholic steatohepatitis. Liver Int 34:110–117 Liu W, Baker SS, Baker RD, Nowak NJ, Zhu L (2011) Upregulation of hemoglobin expression by oxidative stress in hepatocytes and its implication in nonalcoholic steatohepatitis. PLoS One 6:e24363 Baker SS, Baker RD, Liu W, Nowak NJ, Zhu L (2010) Role of alcohol metabolism in non-alcoholic steatohepatitis. PLoS One 5:e9570 Moya D, Baker SS, Liu W, Garrick M, Kozielski R, Baker RD, Zhu L (2014) Novel pathway for iron deficiency in pediatric non-alcoholic steatohepatitis. Clin Nutr 34(3):549–556. doi:10. 1016/j.clnu.2014.06.011 Paradies G, Paradies V, Ruggiero FM, Petrosillo G (2014) Oxidative stress, cardiolipin and mitochondrial dysfunction in

123

143.

144.

145.

146.

147.

148.

149. 150. 151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

nonalcoholic fatty liver disease. World J Gastroenterol 20:14205–14218 Takaki A, Kawai D, Yamamoto K (2014) Molecular mechanisms and new treatment strategies for non-alcoholic steatohepatitis (NASH). Int J Mol Sci 15:7352–7379 Rolo AP, Teodoro JS, Palmeira CM (2012) Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Free Radic Biol Med 52:59–69 Brandt ML, Harmon CM, Helmrath MA, Inge TH, McKay SV, Michalsky MP (2010) Morbid obesity in pediatric diabetes mellitus: surgical options and outcomes. Nat Rev Endocrinol 6:637–645 Zhang XQ, Xu CF, Yu CH, Chen WX, Li YM (2014) Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol 20:1768–1776 Passos E, Ascensao A, Martins MJ, Magalhaes J (2015) Endoplasmic reticulum stress response in non-alcoholic steatohepatitis: the possible role of physical exercise. Metabolism 64:780–792 Ashraf NU, Sheikh TA (2015) Endoplasmic reticulum stress and oxidative stress in the pathogenesis of non-alcoholic fatty liver disease. Free Radic Res 49:1405–1418 Brunt EM (2011) Non-alcoholic fatty liver disease: what’s new under the microscope? Gut 60:1152–1158 Yeh MM, Brunt EM (2014) Pathological features of fatty liver disease. Gastroenterology 147:754–764 Lackner C, Gogg-Kamerer M, Zatloukal K, Stumptner C, Brunt EM, Denk H (2008) Ballooned hepatocytes in steatohepatitis: the value of keratin immunohistochemistry for diagnosis. J Hepatol 48:821–828 Kang JH (2013) Modification and inactivation of Cu, Zn-superoxide dismutase by the lipid peroxidation product, acrolein. BMB Rep 46:555–560 Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels C, Raes M, Zachary MD, Remacle J (1990) Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Mech Ageing Dev 51:283–297 Pessayre D, Mansouri A, Fromenty B (2002) Nonalcoholic steatosis and steatohepatitis. V. Mitochondrial dysfunction in steatohepatitis. Am J Physiol Gastrointest Liver Physiol 282:G193–G199 Haque M, Sanyal AJ (2002) The metabolic abnormalities associated with non-alcoholic fatty liver disease. Best Pract Res Clin Gastroenterol 16:709–731 Koek GH, Liedorp PR, Bast A (2011) The role of oxidative stress in non-alcoholic steatohepatitis. Clin Chim Acta 412:1297–1305 Cortez-Pinto H, Chatham J, Chacko VP, Arnold C, Rashid A, Diehl AM (1999) Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study. JAMA 282:1659–1664 Perez-Carreras M, Del Hoyo P, Martin MA, Rubio JC, Martin A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA (2003) Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology 38:999–1007 Caldwell SH, Swerdlow RH, Khan EM, Iezzoni JC, Hespenheide EE, Parks JK, Parker WD Jr (1999) Mitochondrial abnormalities in non-alcoholic steatohepatitis. J Hepatol 31:430–434 Seki S, Kitada T, Yamada T, Sakaguchi H, Nakatani K, Wakasa K (2002) In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases. J Hepatol 37:56–62 Peverill W, Powell LW, Skoien R (2014) Evolving concepts in the pathogenesis of NASH: beyond steatosis and inflammation. Int J Mol Sci 15:8591–8638

Pathogenesis of nonalcoholic steatohepatitis 162. Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, Gores GJ (2003) Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125:437–443 163. Liu K, Lou J, Wen T, Yin J, Xu B, Ding W, Wang A, Liu D, Zhang C, Chen D, Li N (2013) Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM-induced autophagy or BAX. Liver Int 33:1566–1574 164. Farrell GC, Larter CZ, Hou JY, Zhang RH, Yeh MM, Williams J, dela Pena A, Francisco R, Osvath SR, Brooling J, Teoh N, Sedger LM (2009) Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression. J Gastroenterol Hepatol 24:443–452 165. Yahagi N, Shimano H, Matsuzaka T, Sekiya M, Najima Y, Okazaki S, Okazaki H, Tamura Y, Iizuka Y, Inoue N, Nakagawa Y, Takeuchi Y, Ohashi K, Harada K, Gotoda T, Nagai R, Kadowaki T, Ishibashi S, Osuga J, Yamada N (2004) p53 involvement in the pathogenesis of fatty liver disease. J Biol Chem 279:20571–20575 166. Panasiuk A, Dzieciol J, Panasiuk B, Prokopowicz D (2006) Expression of p53, Bax and Bcl-2 proteins in hepatocytes in non-alcoholic fatty liver disease. World J Gastroenterol 12:6198–6202 167. Lebeaupin C, Proics E, de Bieville CH, Rousseau D, Bonnafous S, Patouraux S, Adam G, Lavallard VJ, Rovere C, Le Thuc O, Saint-Paul MC, Anty R, Schneck AS, Iannelli A, Gugenheim J, Tran A, Gual P, Bailly-Maitre B (2015) ER stress induces NLRP3 inflammasome activation and hepatocyte death. Cell Death Dis 6:e1879 168. Bechmann LP, Gieseler RK, Sowa JP, Kahraman A, Erhard J, Wedemeyer I, Emons B, Jochum C, Feldkamp T, Gerken G, Canbay A (2010) Apoptosis is associated with CD36/fatty acid translocase upregulation in non-alcoholic steatohepatitis. Liver Int 30:850–859 169. Feldstein AE, Canbay A, Guicciardi ME, Higuchi H, Bronk SF, Gores GJ (2003) Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. J Hepatol 39:978–983 170. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796–1808 171. Cawthorn WP, Sethi JK (2008) TNF-alpha and adipocyte biology. FEBS Lett 582:117–131 172. Wenfeng Z, Yakun W, Di M, Jianping G, Chuanxin W, Chun H (2014) Kupffer cells: increasingly significant role in nonalcoholic fatty liver disease. Ann Hepatol 13:489–495 173. Baffy G (2009) Kupffer cells in non-alcoholic fatty liver disease: the emerging view. J Hepatol 51:212–223 174. Marra F, Lotersztajn S (2013) Pathophysiology of NASH: perspectives for a targeted treatment. Curr Pharm Des 19:5250–5269 175. Wan J, Benkdane M, Teixeira-Clerc F, Bonnafous S, Louvet A, Lafdil F, Pecker F, Tran A, Gual P, Mallat A, Lotersztajn S, Pavoine C (2014) M2 Kupffer cells promote M1 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease. Hepatology 59:130–142 176. Smith K (2013) Liver disease: Kupffer cells regulate the progression of ALD and NAFLD. Nat Rev Gastroenterol Hepatol 10:503 177. Harmon RC, Tiniakos DG, Argo CK (2011) Inflammation in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 5:189–200 178. Hubscher SG (2006) Histological assessment of non-alcoholic fatty liver disease. Histopathology 49:450–465 179. Rensen SS, Slaats Y, Nijhuis J, Jans A, Bieghs V, Driessen A, Malle E, Greve JW, Buurman WA (2009) Increased hepatic

180.

181.

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

192.

193. 194.

195. 196. 197.

myeloperoxidase activity in obese subjects with nonalcoholic steatohepatitis. Am J Pathol 175:1473–1482 Nijhuis J, Rensen SS, Slaats Y, van Dielen FM, Buurman WA, Greve JW (2009) Neutrophil activation in morbid obesity, chronic activation of acute inflammation. Obesity (Silver Spring) 17:2014–2018 Liang W, Lindeman JH, Menke AL, Koonen DP, Morrison M, Havekes LM, van den Hoek AM, Kleemann R (2014) Metabolically induced liver inflammation leads to NASH and differs from LPS- or IL-1beta-induced chronic inflammation. Lab Invest 94:491–502 Sutter AG, Palanisamy AP, Lench JH, Jessmore AP, Chavin KD (2015) Development of steatohepatitis in Ob/Ob mice is dependent on Toll-like receptor 4. Ann Hepatol 14:735–743 Kapil S, Duseja A, Sharma BK, Singla B, Chakraborti A, Das A, Ray P, Dhiman RK, Chawla Y (2015) Small intestinal bacterial overgrowth and toll like receptor signaling in patients with nonalcoholic fatty liver disease. J Gastroenterol Hepatol 31(1): 213–21. doi:10.1111/jgh.13058 Yuan J, Baker SS, Liu W, Alkhouri R, Baker RD, Xie J, Ji G, Zhu L (2014) Endotoxemia unrequired in the pathogenesis of pediatric nonalcoholic steatohepatitis. J Gastroenterol Hepatol 29:1292–1298 Pal D, Dasgupta S, Kundu R, Maitra S, Das G, Mukhopadhyay S, Ray S, Majumdar SS, Bhattacharya S (2012) Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med 18:1279–1285 Erridge C, Samani NJ (2009) Saturated fatty acids do not directly stimulate Toll-like receptor signaling. Arterioscler Thromb Vasc Biol 29:1944–1949 Tse E, Helbig KJ, Van der Hoek K, McCartney EM, Van der Hoek M, George J, Beard MR (2015) Fatty acids induce a proinflammatory gene expression profile in Huh-7 cells that attenuates the anti-HCV action of interferon. J Interferon Cytokine Res 35:392–400 Miura K, Yang L, van Rooijen N, Brenner DA, Ohnishi H, Seki E (2013) Toll-like receptor 2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation in mice. Hepatology 57:577–589 Duarte N, Coelho IC, Patarrao RS, Almeida JI, Penha-Goncalves C, Macedo MP (2015) How inflammation impinges on NAFLD: a role for Kupffer cells. Biomed Res Int 2015:984578 Kwanten WJ, Martinet W, Michielsen PP, Francque SM (2014) Role of autophagy in the pathophysiology of nonalcoholic fatty liver disease: a controversial issue. World J Gastroenterol 20:7325–7338 Macaluso FS, Maida M, Petta S (2015) Genetic background in nonalcoholic fatty liver disease: a comprehensive review. World J Gastroenterol 21:11088–11111 Schattenberg JM, Schuppan D (2011) Nonalcoholic steatohepatitis: the therapeutic challenge of a global epidemic. Curr Opin Lipidol 22:479–488 Puche JE, Saiman Y, Friedman SL (2013) Hepatic stellate cells and liver fibrosis. Compr Physiol 3:1473–1492 Iwaisako K, Brenner DA, Kisseleva T (2012) What’s new in liver fibrosis? The origin of myofibroblasts in liver fibrosis. J Gastroenterol Hepatol 27(Suppl 2):65–68 Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115:209–218 Friedman SL (2008) Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88:125–172 Nobili V, Carpino G, Alisi A, Franchitto A, Alpini G, De Vito R, Onori P, Alvaro D, Gaudio E (2012) Hepatic progenitor cells activation, fibrosis, and adipokines production in pediatric nonalcoholic fatty liver disease. Hepatology 56:2142–2153

123

W. Liu et al. 198. Fausto N (2004) Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39:1477–1487 199. Machado MV, Michelotti GA, Pereira TA, Xie G, Premont R, Cortez-Pinto H, Diehl AM (2015) Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease. J Hepatol 63:962–970 200. Espanol-Suner R, Carpentier R, Van Hul N, Legry V, Achouri Y, Cordi S, Jacquemin P, Lemaigre F, Leclercq IA (2012) Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice. Gastroenterology 143(1564–1575): e1567 201. Gouw AS, Clouston AD, Theise ND (2011) Ductular reactions in human liver: diversity at the interface. Hepatology 54:1853–1863 202. Dooley S, ten Dijke P (2012) TGF-beta in progression of liver disease. Cell Tissue Res 347:245–256 203. Weng HL, Ciuclan L, Liu Y, Hamzavi J, Godoy P, Gaitantzi H, Kanzler S, Heuchel R, Ueberham U, Gebhardt R, Breitkopf K, Dooley S (2007) Profibrogenic transforming growth factor-beta/ activin receptor-like kinase 5 signaling via connective tissue growth factor expression in hepatocytes. Hepatology 46:1257–1270 204. Nakamura T, Sakata R, Ueno T, Sata M, Ueno H (2000) Inhibition of transforming growth factor beta prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology 32:247–255 205. Kanzler S, Lohse AW, Keil A, Henninger J, Dienes HP, Schirmacher P, Rose-John S, Buschenfelde KH, Blessing M (1999) TGF-beta1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis. Am J Physiol 276:G1059–G1068 206. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA Jr, Wrana JL, Falb D (1997) The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell 89:1165–1173 207. Tahashi Y, Matsuzaki K, Date M, Yoshida K, Furukawa F, Sugano Y, Matsushita M, Himeno Y, Inagaki Y, Inoue K (2002) Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology 35:49–61 208. Yan X, Lin Z, Chen F, Zhao X, Chen H, Ning Y, Chen YG (2009) Human BAMBI cooperates with Smad7 to inhibit transforming growth factor-beta signaling. J Biol Chem 284:30097–30104 209. Liu C, Chen X, Yang L, Kisseleva T, Brenner DA, Seki E (2014) Transcriptional repression of the transforming growth factor beta (TGF-beta) pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) by nuclear factor kappaB (NFkappaB) p50 enhances TGF-beta signaling in hepatic stellate cells. J Biol Chem 289:7082–7091 210. Henderson NC, Arnold TD, Katamura Y, Giacomini MM, Rodriguez JD, McCarty JH, Pellicoro A, Raschperger E, Betsholtz C, Ruminski PG, Griggs DW, Prinsen MJ, Maher JJ, Iredale JP, Lacy-Hulbert A, Adams RH, Sheppard D (2013) Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19:1617–1624 211. Saile B, Matthes N, Knittel T, Ramadori G (1999) Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology 30:196–202 212. Omenetti A, Choi S, Michelotti G, Diehl AM (2011) Hedgehog signaling in the liver. J Hepatol 54:366–373 213. Guy CD, Suzuki A, Zdanowicz M, Abdelmalek MF, Burchette J, Unalp A, Diehl AM (2012) Hedgehog pathway activation

123

214.

215.

216.

217.

218.

219. 220.

221.

222.

223.

224.

225.

226.

227.

228.

parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease. Hepatology 55:1711–1721 Swiderska-Syn M, Suzuki A, Guy CD, Schwimmer JB, Abdelmalek MF, Lavine JE, Diehl AM (2013) Hedgehog pathway and pediatric nonalcoholic fatty liver disease. Hepatology 57:1814–1825 Choi SS, Witek RP, Yang L, Omenetti A, Syn WK, Moylan CA, Jung Y, Karaca GF, Teaberry VS, Pereira TA, Wang J, Ren XR, Diehl AM (2010) Activation of Rac1 promotes hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells. Hepatology 52:278–290 Xie G, Karaca G, Swiderska-Syn M, Michelotti GA, Kruger L, Chen Y, Premont RT, Choi SS, Diehl AM (2013) Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice. Hepatology 58:1801–1813 Aleffi S, Petrai I, Bertolani C, Parola M, Colombatto S, Novo E, Vizzutti F, Anania FA, Milani S, Rombouts K, Laffi G, Pinzani M, Marra F (2005) Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology 42:1339–1348 Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA (2004) Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J 18:1612–1614 Gao B (2005) Cytokines, STATs and liver disease. Cell Mol Immunol 2:92–100 Kamada Y, Tamura S, Kiso S, Matsumoto H, Saji Y, Yoshida Y, Fukui K, Maeda N, Nishizawa H, Nagaretani H, Okamoto Y, Kihara S, Miyagawa J, Shinomura Y, Funahashi T, Matsuzawa Y (2003) Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology 125:1796–1807 Seo YS, Shah VH (2012) The role of gut-liver axis in the pathogenesis of liver cirrhosis and portal hypertension. Clin Mol Hepatol 18:337–346 Keshavarzian A, Holmes EW, Patel M, Iber F, Fields JZ, Pethkar S (1999) Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. Am J Gastroenterol 94:200–207 Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF (2007) TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 13:1324–1332 Mazagova M, Wang L, Anfora AT, Wissmueller M, Lesley SA, Miyamoto Y, Eckmann L, Dhungana S, Pathmasiri W, Sumner S, Westwater C, Brenner DA, Schnabl B (2015) Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice. FASEB J 29:1043–1055 De Minicis S, Rychlicki C, Agostinelli L, Saccomanno S, Candelaresi C, Trozzi L, Mingarelli E, Facinelli B, Magi G, Palmieri C, Marzioni M, Benedetti A, Svegliati-Baroni G (2014) Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice. Hepatology 59:1738–1749 Cengiz M, Ozenirler S, Elbeg S (2015) Role of serum toll-like receptors 2 and 4 in non-alcoholic steatohepatitis and liver fibrosis. J Gastroenterol Hepatol 30:1190–1196 Hernandez-Gea V, Ghiassi-Nejad Z, Rozenfeld R, Gordon R, Fiel MI, Yue Z, Czaja MJ, Friedman SL (2012) Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues. Gastroenterology 142:938–946 Yang L, Kwon J, Popov Y, Gajdos GB, Ordog T, Brekken RA, Mukhopadhyay D, Schuppan D, Bi Y, Simonetto D, Shah VH (2014) Vascular endothelial growth factor promotes fibrosis resolution and repair in mice. Gastroenterology 146(1339–1350):e1331

Pathogenesis of nonalcoholic steatohepatitis 229. Kaur S, Anita K (2013) Angiogenesis in liver regeneration and fibrosis: ‘‘a double-edged sword’’. Hepatol Int 7:959–968 230. Fernandez M, Semela D, Bruix J, Colle I, Pinzani M, Bosch J (2009) Angiogenesis in liver disease. J Hepatol 50:604–620 231. Kitade M, Yoshiji H, Kojima H, Ikenaka Y, Noguchi R, Kaji K, Yoshii J, Yanase K, Namisaki T, Asada K, Yamazaki M, Tsujimoto T, Akahane T, Uemura M, Fukui H (2006) Leptinmediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats. Hepatology 44:983–991 232. Qu A, Taylor M, Xue X, Matsubara T, Metzger D, Chambon P, Gonzalez FJ, Shah YM (2011) Hypoxia-inducible transcription factor 2alpha promotes steatohepatitis through augmenting

lipid accumulation, inflammation, and fibrosis. Hepatology 54:472–483 233. Novo E, Cannito S, Zamara E, Valfre di Bonzo L, Caligiuri A, Cravanzola C, Compagnone A, Colombatto S, Marra F, Pinzani M, Parola M (2007) Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol 170:1942–1953 234. Taura K, De Minicis S, Seki E, Hatano E, Iwaisako K, Osterreicher CH, Kodama Y, Miura K, Ikai I, Uemoto S, Brenner DA (2008) Hepatic stellate cells secrete angiopoietin 1 that induces angiogenesis in liver fibrosis. Gastroenterology 135:1729–1738

123