Pathogenesis of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of disease ranging from hepatocellular steatosis through steatohepatitis to fibrosis and irreversible cirrhosis. The prevalence of NAFLD has risen rapidly in parallel with the dramatic rise in obesity and diabetes,1,2 and is rapidly becoming the most common cause of liver disease in Western countries.3 Indeed, NAFLD is now recognized to be the aetiology in many cases previously labelled as cryptogenic cirrhosis.4 In Western populations, estimates of NAFLD prevalence vary between 20 and 30%,5,6 rising up to 90% in morbidly obese individuals.7 The more severe, and clinically significant form of NAFLD, non-alcoholic steatohepatitis (NASH) is less common, affecting an estimated 2–3% of the general population,8 and up to 37% of the morbidly obese.7 Of particular concern, and with significant implications for future disease burden, is the increasing prevalence of NAFLD in children and young adults. Studies have reported a 3% prevalence of NAFLD in the general paediatric population, rising to 53% in obese children.9,10 NAFLD has a strong association with type 2 diabetes, with steatosis present in 70% of type 2 diabetics screened with ultrasound,11 and thus it is now recognized to represent the hepatic manifestation of the metabolic syndrome. NAFLD occurs in all ethnic groups although it appears to have a lower prevalence in African-Americans compared with Hispanic and European Americans. This difference remains even after controlling for obesity and insulin resistance (IR)5,12 and may be related to ethnic differences in lipid homeostasis.5 There are no laboratory, imaging or histological findings which can accurately distinguish between NAFLD and alcohol-induced steatosis or steatohepatitis, and the diagnosis can therefore only be made in the absence of a history of significant alcohol intake. Other specific causes of steatosis need to be considered and include metabolic disorders e.g. lipodystrophy and abetalipoproteinaemia, nutritional causes such as rapid weight loss, jejuno-ileal bypass and total parenteral nutrition, and drug-induced. Commonly implicated agents include glucocorticoids, methotrexate, amiodarone, synthetic oestrogens, tamoxifen, diltiazem and highly active anti-retroviral drugs.13–15 Steatosis also commonly occurs in association with hepatitis C, particularly genotype 3, and has an increased prevalence in women with polycystic ovary syndrome, when it is usually associated with IR.16 In the great majority of patients NAFLD develops in association with features of IR and the metabolic syndrome. The metabolic syndrome comprises a cluster of clinical and biochemical features, namely IR, glucose intolerance or diabetes, central obesity, hypertension and dyslipidaemia and is associated with significant cardiovascular morbidity and mortality.17–19 Whilst simple steatosis in the absence of significant fibrosis is considered to be a relatively benign condition,20 the presence of fibrosis predicts both disease progression and liver-related complications over a subsequent 10-year period.21 Decreased survival in this sub-group is due to predominantly cardiovascular causes, although there is a significant increase in liver-related deaths.21 NASH also carries an increased risk of hepatocellular carcinoma (HCC)21 and thus the observation of increased incidence of HCC in type 2 diabetics22 is likely to be due to their high prevalence of NASH.21 In a recent US study, NASH was found to account for at least 13% of overall cases of HCC.23 There are as yet few proven therapies available for patients with NASH, and current strategies are directed towards improving aspects of the metabolic syndrome. Ultimately when such measures fail, liver transplantation remains the only option for patients with end-stage cirrhosis. Although the pathogenesis of NAFLD/NASH is not yet fully understood, much progress has been made in recent years in elucidating the mechanisms of progression from steatosis to more advanced liver inflammation and fibrosis. In this review, we discuss the current understanding of NAFLD pathogenesis, and anticipate that such knowledge will eventually translate into the development of novel treatment strategies for this increasingly important disease. Initial theories for the pathogenesis of NASH were based on a ‘2-hit hypothesis’ (Figure 1a). The ‘first hit’, hepatic triglyceride accumulation, or steatosis, increases susceptibility of the liver to injury mediated by ‘second hits’, such as inflammatory cytokines/adipokines, mitochondrial dysfunction and oxidative stress, which in turn lead to steatohepatitis and/or fibrosis.24,25 However, there is increasing recognition of the role that free fatty acids (FFA) play in directly promoting liver injury, which has led to modification of this theory (Figure 1b). In obesity and IR there is an increased influx of FFA to the liver. These FFA either undergo β-oxidation or are esterified with glycerol to form triglycerides, leading to hepatic fat accumulation. There is now substantial evidence that FFA can directly cause toxicity by increasing oxidative stress and by activation of inflammatory pathways,26 therefore hepatic triglyceride accumulation may be a protective mechanism by preventing the toxic effects of unesterified FFA.27 Additionally, a further component, or ‘third-hit’ has been added to reflect inadequate hepatocyte proliferation (Figure 1c).28 In the healthy liver, cell death stimulates replication of mature hepatocytes which replace the dead cells and reconstitute normal tissue function.28 However oxidative stress, a central feature of NAFLD pathogenesis, inhibits the replication of mature hepatocytes which results in expansion of the hepatic progenitor cell (oval cell) population.29 These cells can differentiate into hepatocyte-like cells, and both oval cell and intermediate hepatocyte-like cell numbers are strongly correlated with fibrosis stage, suggesting that cumulative hepatocyte loss promotes both accumulation of progenitor cells and their differentiation towards hepatocytes.29 Activation of these cells has also been implicated in hepatocellular carcinogenesis.29 In chronic liver injury, the development of fibrosis/cirrhosis is dependent on the efficacy of hepatocyte regeneration, and therefore cell death with impaired proliferation of hepatocyte progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.28 (a) The traditional 2-hit hypothesis: steatosis represents the ‘first hit’, which then sensitises the liver to injury mediated by ‘second hits’, such as inflammatory cytokines, adipokines, oxidative stress and mitochondrial dysfunction, leading to steatohepatitis and fibrosis. The presence of high levels of oxidative stress reduces the ability of mature hepatocytes to proliferate, resulting in reduced endogenous liver repair. (b) Modified 2-hit hypothesis: the accumulation of FFA alone has been suggested to be sufficient to induce liver damage, without recourse for a second hit. Indeed, rather than being harmful, triglyceride accumulation in the form of steatosis may actually be protective by preventing FFA-induced inflammation and oxidative stress. (c) The 3-hit hypothesis: oxidative stress reduces the ability of mature hepatocytes to proliferate, resulting in the recruitment of other pathways of liver regeneration, such as HPCs. These cells have the capability of differentiating into both cholangiocytes and hepatocytes and contributing to liver repair. It has been suggested that an inability to mount such a ductular response, as is seen in patients transplanted for NASH who have denervated livers, may be responsible for a more progressive pattern of liver damage. Thus, impaired proliferation of hepatocyte progenitors represents the proposed ‘third hit’ in NAFLD pathogenesis.28 NAFLD is characterized by the accumulation of triglycerides, which are formed from the esterification of FFA and glycerol within the hepatocyte. FFAs arise in the liver from three distinct sources; lipolysis (the hydrolysis of FFA and glycerol from triglyceride) within adipose tissue, dietary sources, and de novo lipogenesis (DNL).30 In contrast, FFA may be utilized either through β-oxidation, re-esterification to triglycerides and storage as lipid droplets, or packaged and exported as very low density lipoprotein (VLDL). Hence hepatic fat accumulation can occur as a result of increased fat synthesis, increased fat delivery, decreased fat export, and/or decreased fat oxidation (Figure 2).30 Mechanisms of hepatic fat accumulation. To establish the relative contribution of lipid accumulation in patients with NAFLD, Donnelly et al. used a multiple-stable-isotope method, demonstrating that approximately 60% of liver triglyceride content derived from FFA influx from adipose tissue, 26% from DNL, and 15% from diet.31 This contrasts with healthy individuals in whom DNL contributes <5% of hepatic triglyceride formation.32,33 Triglyceride can also be exported from the liver in VLDL particles, which are formed by the incorporation of triglyceride into apolipoprotein B (apoB) by microsomal transfer protein (MTP).34 Aberrant alterations of MTP/apoB synthesis and secretion have been proposed as potential mechanisms underpinning the pathogenesis of NAFLD leading to a decreased capacity for lipid export.35,36 In healthy individuals, binding of insulin to its receptor leads to phosphorylation of several substrates including insulin receptor substrates (IRS)-1, -2, -3 and -4, which propagate the insulin signal.30,37 Insulin stimulation of IRS-1 and -2 leads to activation of intracellular PI3K (phosphoinositide 3-kinase) and AKT/PKB (protein kinase B) pathways, which are intimately involved in mediating the metabolic effects of insulin.30 Ultimately, AKT/PKB activation results in translocation of glucose transporter, GLUT4, containing vesicles to the plasma membrane, thus facilitating glucose uptake. In addition, the expression of key lipogenic genes is increased, with a concomitant decrease in gluconeogenic gene expression via its regulation of forkhead (FOXO) transcription factor activity. Insulin has a potent action to suppress adipose tissue lipolysis. However, in situations of IR, such as NAFLD, this suppression is impaired resulting in an increased efflux of FFA from adipose tissue.38 The hyperinsulinaemia associated with IR leads to: (i) up-regulation of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c), which is a key transcriptional regulator of genes involved in DNL,15 and (ii) Inhibition of β-oxidation of FFA thus further promoting hepatic lipid accumulation.30 Many of the abnormalities reported in NAFLD interfere with the insulin signalling cascade, and thus contribute to IR. These include FFAs, tumour necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), ceramide, jun N-terminal kinase 1 (JNK1), SOCS (suppressors of cytokine signalling) and cytochrome CYP2E1.39,40 Increased lipid metabolites such as diacylglycerol (DAG) have been implicated in a protein kinase Cε (PKCε) dependent mechanism, to interfere with insulin signalling through inhibition of insulin receptor activity and modulation of IRS-2 phosphorylation.30 Similar processes occur in skeletal muscle cells, leading to a more generalized state of IR. The presence of steatosis is tightly associated with chronic hepatic inflammation,41 an effect in part mediated by activation of the Iκκ-β/NF-κB signalling pathway. In murine models of high-fat diet (HFD)-induced steatosis, increased NF-κB activity is associated with elevated hepatic expression of inflammatory cytokines such as TNF-α, interleukin-6 (IL-6) and interleukin 1-beta (IL-1β), and activation of Kupffer cells.41 Liver-specific NF-κB inhibition prevents HFD-induced inflammatory gene expression, whereas HFD-induced hyperglycaemia and IR can be reproduced by selective over-expression of constitutively active Iκκ-β in hepatocytes.41 The Iκκ-β/NF-κB pathway in hepatocytes can also be activated directly by FFA, providing a further mechanism by which central obesity with consequent increased hepatic FFA supply can contribute to inflammation (Figure 3).26 Furthermore, the conversion of FFA to hepatic triglyceride may serve as a protective measure to prevent direct hepatic lipotoxicity. This is endorsed by a murine model of NAFLD, where inhibition of DGAT2, the enzyme that catalyzes the final step in triglyceride synthesis, resulted in improvement of hepatic steatosis and IR but exacerbation of injury and fibrosis.27,42 Proposed pathogenesis of NASH. The likelihood of progression to advanced NASH/cirrhosis results from a complex interplay between genetic predisposition and the mechanisms described earlier. Both serum and hepatic levels of TNF-α are elevated in patients with NASH,43,44 and levels correlate with histological severity.45 In addition to its proinflammatory effects, TNF-α promotes IR.46 Conversely, inhibition of TNF-α signalling improves IR and histological parameters of NASH.47–49 Similarly, serum IL-6 levels are also elevated in both animal and human models of IR and NAFLD,43,50,51 and levels correlate with increasing liver inflammation and fibrosis.52 The key role of hepatocyte cytokine production in the progression of steatosis to NASH is supported by studies demonstrating that cytokines can replicate all of the histological features associated with NASH, including neutrophil chemotaxis, hepatocyte apoptosis/necrosis, Mallory body formation and stellate cell activation.25 Additionally, data suggests that inflammation and NF-κB activation can promote carcinogenesis,53 and that the chronic inflammatory state associated with hepatic steatosis may also play a key role in HCC development.25 Adipose tissue is not just an inert site of energy storage, but an actively secreting endocrine organ. The functional role of adipocyte-derived cytokines (adipokines), is now increasingly recognized, with leptin and adiponectin amongst the most well described. Leptin is a 16 kDa hormone produced mainly by mature adipocytes whose actions include the regulation of energy intake and expenditure,54 regulation of the immune system,55,56 and promotion of inflammation and fibrogenesis.56,57 Higher leptin levels are observed in obese patients and those with NAFLD,54,58–60 which are commonly regarded as states of leptin resistance.58 It remains plausible that leptin may have a functional role to play in the pathogenesis of NAFLD. In contrast to leptin, secretion and circulating levels of adiponectin are inversely proportional to body fat and are reduced in patients with is and increases insulin and the of adiponectin improves as well as the biochemical and histological parameters of NAFLD in a murine the effects of TNF-α, which adiponectin The of adiponectin in NAFLD is supported by studies that serum adiponectin levels can to distinguish NASH from simple Other adipose tissue derived found in in NAFLD include TNF-α, and all of which the lipogenic effects of but their role in the pathogenesis of NAFLD remains to be (Figure The role of oxidative stress and mitochondrial dysfunction in NASH is with more advanced disease with of oxidative β-oxidation within the normal liver in the but in the of this can as a result of increased FFA rise to induce oxidative stress, with subsequent activation of inflammatory and also mitochondrial damage. mitochondrial and a in mitochondrial activity have been observed in human studies of expression and activity of the hepatic microsomal fatty enzyme cytochrome has been observed in human and animal models of NASH and represents a potent of over-expression of activity is associated with oxidative stress, IR and hepatic fat Other mechanisms implicated in NASH pathogenesis include stress and stress can be by a of including hyperinsulinaemia and and can result in activation of pathways leading to IR, and mitochondrial stress is to be important in alcohol-induced steatohepatitis and further of its role in NASH is is also for a role of in the pathogenesis of NASH. results in production of and of both of which can TNF-α production in Kupffer cells and thus induce hepatic and increased have been found more in patients with NASH when compared with This has led to the that this may the of NASH and liver fibrosis as a of bypass This is further supported by evidence that of with and can hepatic inflammation in both and from both and endogenous are a cause of NAFLD. with syndrome, who have increased circulating levels a metabolic of central obesity, IR and a significant of these patients will also hepatic The mechanisms by which promote hepatic fat accumulation include inhibition of fatty β-oxidation and promotion of hepatocyte However most patients with NAFLD have normal circulating suggesting that mechanisms are the metabolic This has led to in enzyme which play a key role in and consequent to and the type 1 to the active and thus increases levels and Inhibition of has been to lower body weight and lipid levels and glucose in animal and increase hepatic insulin in In the and are responsible for the of to its Increased hepatic activity has been in patients with and which may represent a mechanism to decrease in an to prevent development or progression of NAFLD. In animal both and inhibition of activity has been to increase susceptibility to development of IR and fatty Hence of hepatic by modulation of and the may represent a potential for preventing the development and progression of NAFLD. and its more advanced form represents the final common pathway of all chronic liver including NASH. fibrosis results in liver and hypertension with its associated complications of and as well as an increased risk of The pathogenesis of fibrosis is not within the of this and is well There are aspects of liver fibrosis and which are relatively specific to NASH and are In most of liver injury by replication of mature the presence of such as NASH or is associated with high levels of oxidative stress which the ability of these mature hepatocytes to In this other pathways of liver regeneration, hepatic progenitor cells are are cells which in the of and which on proliferation form a complex of and cholangiocytes as a ductular This was used to the of cells at the of the and the and to proliferation of progenitor cell activation and of intermediate these cells have the capability of differentiating into both cholangiocytes and hepatocytes and contributing to liver repair. inability to mount such a ductular response, as is seen in patients transplanted for NASH who have denervated livers, may be responsible for a more progressive pattern of liver damage. to in the the interplay of the ductular and fibrosis in NAFLD. Initial a association between the expansion of and the ductular in liver of NASH. The of ductular in turn strongly correlated with the of suggesting that may be responsible for a progressive mechanisms for this include the secretion of cytokines and by the ductular as well as direct of the cholangiocytes to This is in that it a for the of fibrosis in NAFLD which is a key feature of progressive disease. recent in the model of murine NAFLD has suggested that liver fibrosis the proliferation of suggesting that fibrosis not occur as a result of The of the presence of the histological findings is less on further the of an more complex between fibrosis and in which both processes can other Indeed, the of a progenitor cell with of may not only as a pathway for to from the into the but also for the survival of these Although hepatic steatosis is common in patients with obesity and IR only a progress to NASH and suggesting an important interplay between genetic predisposition and in genes related to lipid IR, oxidative stress, and may all increase susceptibility to NASH studies have which fibrosis development in other liver particularly chronic hepatitis Studies in NASH have in the and genes to be associated with advanced hepatic fibrosis in obese In addition, in the type 1 receptor are associated with an increased risk of NAFLD and studies are to more genes which will be not only as to the pathogenesis and of the but also may represent novel treatment an increasing understanding of the mechanisms of NAFLD pathogenesis, there are few therapies are directed towards improving the metabolic parameters which contribute to disease pathogenesis, such as weight loss and IR and improving In addition to current therapies utilized for patients with NAFLD include insulin e.g. and the weight loss e.g. and and of for morbidly obese transplantation remains the only treatment option for end-stage cirrhosis. The need for specific for NAFLD has this a of with particular on which can or prevent the more advanced and clinically of NASH. The presence of fibrosis predicts likelihood of liver related complications and therefore therapies which can prevent or fibrosis are important in NASH include such as C, and and Whilst of these has yet evidence of further are may represent a novel option for the progression of NAFLD. In patients such as have been to increase insulin suppress and increase in association with weight and in animal models reduced IR, of oxidative stress and hepatic fibrosis is the of hepatic which predominantly arise from activation of hepatic stellate cells that in the of the nuclear activated receptor Studies have that by such as the and leads to reduced results with these agents have been in patients with NAFLD with in both liver and However including and weight are of also to be on of has been to promote survival and liver and thus the effects of and receptor are likely to include clinical studies of such as which have been to suppress fibrosis in have to that dietary of improves histological fibrosis in of cytokines are intimately involved in the and of of which represents potential for include factor factor tissue factor factor factor and which also their effects through with studies in animal models of liver fibrosis have effects kinase such as which is for in chronic and Other potential to include and modulation of the liver and NAFLD now represents of the causes of liver disease in the Western and the rising levels of obesity, and metabolic syndrome will that it remains a cause of morbidity and Although simple steatosis carries a relatively benign a significant of patients will progress to NASH and with risk of The traditional of NAFLD pathogenesis has been several in most patients NAFLD to with lipid accumulation, or steatosis, which is in turn by obesity and IR. to steatohepatitis and fibrosis on such as FFAs, inflammatory cytokines and adipokines, oxidative stress and mitochondrial dysfunction in a complex interplay with genetic treatment strategies for NASH on improving of the metabolic syndrome, such as obesity and IR, with no agents yet However, modulation of of the mechanisms involved in NASH pathogenesis to prevent the development of fibrosis and its associated This and the significant that to be made in understanding of the pathogenesis of NASH, are to the development of novel strategies for this increasingly important is a in and is therefore from the of