Nonalcoholic fatty liver disease (NAFLD) is a disorder characterized by excess accumulation of fat in hepatocytes (nonalcoholic fatty liver (NAFL)); in up to 40% of individuals, there are additional findings of portal and lobular inflammation and hepatocyte injury (which characterize nonalcoholic steatohepatitis (NASH)). A subset of patients will develop progressive fibrosis, which can progress to cirrhosis. Hepatocellular carcinoma and cardiovascular complications are life-threatening co-morbidities of both NAFL and NASH. NAFLD is closely associated with insulin resistance; obesity and metabolic syndrome are common underlying factors. As a consequence, the prevalence of NAFLD is estimated to be 10–40% in adults worldwide, and it is the most common liver disease in children and adolescents in developed countries. Mechanistic insights into fat accumulation, subsequent hepatocyte injury, the role of the immune system and fibrosis as well as the role of the gut microbiota are unfolding. Furthermore, genetic and epigenetic factors might explain the considerable interindividual variation in disease phenotype, severity and progression. To date, no effective medical interventions exist that completely reverse the disease other than lifestyle changes, dietary alterations and, possibly, bariatric surgery. However, several strategies that target pathophysiological processes such as an oversupply of fatty acids to the liver, cell injury and inflammation are currently under investigation. Diagnosis of NAFLD can be established by imaging, but detection of the lesions of NASH still depend on the gold-standard but invasive liver biopsy. Several non-invasive strategies are being evaluated to replace or complement biopsies, especially for follow-up monitoring.
Nonalcoholic fatty liver disease (NAFLD) has been accepted as a bona fide disease entity in the late twentieth century. The disorder is characterized by excess accumulation of fat (in the form of triglycerides) in hepatocytes (>5% fat content in the liver; referred to as steatosis), leading to nonalcoholic fatty liver (NAFL). In the presence of additional factors, described below, hepatocyte injury and cell death occur along with lobular and portal inflammation, and the disease is referred to as nonalcoholic steatohepatitis (NASH). With progressive collagen deposition and subsequent vascular remodelling, cirrhosis may result.
Several histological features of NAFLD and NASH are common to alcoholic fatty liver disease (AFLD), including steatosis, inflammation, hepatocyte ballooning (a type of hepatocyte injury), Mallory–Denk bodies (Mallory hyaline) and fibrosis within the lobules (Fig. 1). However, in contrast to the underlying exposure to excess alcohol that initiates and drives AFLD, patients with NAFLD are insulin resistant, usually obese and do not consume excess alcohol1. Interestingly, whereas paediatric NAFLD shares similar metabolic triggers with NAFLD in adults, different patterns of histological injury may be observed2.
Morbidity and mortality in patients with NAFLD can be related to the liver disease itself, although cardiovascular disease is the primary cause of premature death3,4. Cirrhosis and hepatocellular carcinoma (HCC) are the most common liver-related causes of morbidity associated with NAFLD, followed by systemic infection5. HCC has been shown to occur in the absence of cirrhosis, which is a concern for clinical care6.
Finally, the value of the liver biopsy as a diagnostic tool, although an invasive procedure, continues to be shown by large studies analysing the correlation between long-term outcomes with histopathological evaluation. Advanced fibrosis has been identified as the most important histological feature associated with poor outcomes in two studies5,7; inflammation is associated with the initiation and progression of fibrosis8. Both in children and in adults, NASH-related cirrhosis is predicted to become the most common indication for liver transplantation9. Several non-invasive diagnostic tools are currently being validated and show promise to replace liver biopsy and identify early fibrosis in the setting of NAFLD.
In this Primer, we describe the worldwide prevalence of NAFLD in adults and children, highlight genomic and epigenomic changes associated with disease and its progression, as well as the molecular drivers of disease, and elaborate on the advances in diagnosis and the current limitations of management options, quality-of-life (QOL) concerns and outline the avenues for progress.
The United States and Europe. NAFLD is currently the most common cause of abnormal liver function tests in western countries. According to population studies using ultrasonography or CT imaging, the prevalence of NAFLD is in the range of 20–50%10,
As imaging can only detect fatty liver but not necroinflammation or fibrosis, the prevalence of NASH and the related liver fibrosis in the population is unclear. Histological analyses suggest that 6–55% of patients with NAFLD have NASH, depending on the patient inclusion criteria and the definition of NASH17,
With ongoing necroinflammation and liver injury, patients with NASH can develop progressive liver fibrosis and, eventually, cirrhosis. Advanced fibrosis or cirrhosis due to NASH has a similar mortality than when these features are caused by chronic hepatitis C virus infection and advanced liver disease. Contributing to this risk of death is the development of HCC. The annual incidence of HCC in patients with NASH-related cirrhosis is 1–2%21. In the United States, NASH has already become the second-leading aetiology of liver disease requiring liver transplantation in adults because of advanced cirrhosis or HCC22. Importantly, the number of patients with NASH on the transplantation waiting list increased by 170% from 2004 to 2013, the largest absolute and relative increase compared with other aetiologies. Hepatic complications represent the third-most common cause of death in patients with NAFLD or NASH, after cardiovascular events and malignancies4.
Asia, Africa and the Pacific Islands. The epidemiology of NAFLD in Asia-Pacific and African countries is shaped by vast differences in lifestyle and economic growth among countries and between urban and rural areas within the same country. For example, in China, the prevalence of NAFLD is higher in coastal and urban areas (up to 30%) than in inland and rural areas (around 1%)23. In urban areas, the prevalence of NAFLD is around 15–30% in Asia-Pacific countries and 10–20% in Africa.
Compared with western countries, the increase in NAFLD prevalence over time is more marked in Asia and the Pacific areas, probably reflecting rapid lifestyle changes. Studies from Japan and China recorded a twofold increase in the prevalence of NAFLD over a decade23,24. By contrast, the reported prevalence of NAFLD in the United States was already around 30–40% in 2004 and has not increased substantially thereafter14. The incidence of NAFLD is difficult to study because ultrasonography cannot reliably quantify liver fat. Nevertheless, according to a recent study from Hong Kong using paired 1H-MRS, 13.5% of community subjects developed NAFLD in 3–5 years25. Asians and Pacific Islanders develop obesity-related complications at a lower body mass index (BMI) than Europeans and Americans because of the tendency to have central fat accumulation. As a result, approximately 15–20% of Asian patients with NAFLD do not have obesity according to current definitions.
Data on the prevalence of advanced liver disease in the general Asia-Pacific and African populations are scarce. One population study in Hong Kong using transient elastography and serum markers of fibrosis suggests that around 4% of patients with NAFLD in the community have advanced fibrosis or cirrhosis26. In high-risk groups, such as patients with T2DM, advanced fibrosis and cirrhosis can be found in 10–20% of patients.
Children and adolescents
The United States and Europe. NAFLD is the most common liver disease in children and adolescents (Table 2). Similarly to adult patients, NAFLD is associated with obesity and metabolic syndrome27 and probably with increased levels of alanine aminotransferase (ALT) in the blood and PNPLA3 polymorphisms28,29 (Fig. 2). A recent analysis of the National Health and Nutrition Examination Survey, including data from >12,000 12–19-year-old Americans, showed that the prevalence of NAFLD (with NAFLD defined as a BMI in the >85th percentile and increased ALT levels of >22.1 U per litre for girls and >25.8 U per litre for boys) more than doubled over the past two decades27. The associated risk factors identified in this study confirm previously identified factors, such as age, increased BMI, male gender and Mexican-American race30. Modifiable factors were increased consumption of sugar-sweetened beverages and other lifestyle factors (such as a sedentary behaviour)27. Paediatric NAFLD prevalence has been predicted to increase even further31.
Ethnicity also has a role in NAFLD risk in children. Given the similar BMI and metabolic syndrome features, black and non-Hispanic children have a lower risk of developing liver disease than Hispanic children (reviewed in Ref. 29). Hereditability, particularly in first-degree relatives, is documented. Indeed, 59% of siblings and 78% of parents of children with fatty liver will also develop NAFLD32. Age is another striking factor; one post-mortem study in California showed that 17% of teenagers had NAFLD compared with 0.7% of 2–4-year-olds, owing to both a longer period to accumulate steatosis and an increased incidence in adolescents30.
Asia, Africa and the Pacific Islands. Given that overweight and obesity are important risk factors for NAFLD, the prevalence of NAFLD in Korea is greater in patients admitted to obesity clinics (71.9% based on ultrasonography measures and 61.8% based on serum ALT levels) than in general health clinics (8.7% based on ultrasonography measures and 5.9% based on serum ALT levels)33. In most of Asia, a significant difference is reported between urban and rural populations, with a prevalence of 16–32% in urban areas versus 9% in rural populations34. Obesity-related NAFLD was reported in 64 out of 84 Chinese children (77%) in one study35. In Australia, the prevalence of NAFLD in the paediatric population was estimated to be approximately 10% in the total population and 27.6% among children who are overweight or obese36.
Pathophysiology of NAFLD
Fatty liver disease is characterized by hepatic steatosis; there may also be varying degrees of hepatocyte injury, inflammation and, in some cases, fibrosis (Fig. 1).
The evolution of hepatic steatosis in obesity. Several factors contribute to the accumulation of hepatic lipids in the setting of obesity (Fig. 3). First, weight gain is associated with marked expansion of adipose tissue, which leads to dysfunction and eventual death of adipocytes. Dysfunction of adipose tissue results in local inflammation and the upregulation of cytokines that promote insulin resistance. Insulin resistance, in turn, compromises the ability of adipocytes to store fat, resulting in the release of free fatty acids into the circulation, which then become available for uptake by ectopic organs such as the liver37. As a consequence of obesity-related adipose tissue dysfunction, the liver is exposed to high levels of circulating free fatty acids as well as high levels of insulin, which is produced to compensate for systemic insulin resistance. Hepatocytes take up these fatty acids via the transporters fatty acid transport protein 5 (FATP5; also known as bile acyl-CoA synthetase) and CD36, which are also upregulated in obesity38,39. Fatty acid accumulation in hepatocytes prompts the synthesis of triglycerides; during this process, diacylglycerol intermediates accumulate, which impair hepatic insulin signalling by activating protein kinase Cε (PKCε)40. Hepatocyte insulin resistance fuels gluconeogenesis, promoting hyperglycaemia and prompting even more compensatory insulin production.
The liver itself can contribute to hepatic steatosis by producing lipid from carbohydrate in a process called de novo lipogenesis (DNL). The enzymes responsible for DNL are upregulated by insulin and glucose through the action of two transcription factors, sterol regulatory element-binding protein 1 (SREBP1) and carbohydrate-responsive element-binding protein (ChREBP)41. In the normal liver, DNL is not a main source of hepatic lipid, but in the setting of obesity and hyperinsulinaemia, DNL can contribute as much as 25% of total hepatic lipid stores, and is considered an important factor in the development of NAFLD42,43. Why an insulin-dependent process such as DNL would be upregulated in hepatocytes if uptake of excess circulating fatty acids has rendered them insulin resistant seems paradoxical. One explanation is that hepatocyte insulin resistance manifests downstream from the insulin receptor44; another is that DNL can also be induced by insulin-independent pathways, such as endoplasmic reticulum stress-mediated activation of SREBP1 (Ref. 45).
Nutrients from the diet also serve as substrates for hepatic triglyceride synthesis and thus participate in the development of hepatic steatosis. Dietary sugars are converted to fatty acids via DNL, and dietary fats are taken up by the liver along with adipose tissue-derived fatty acids. Roughly 40% of the lipid that accumulates in a fatty liver derives from dietary sugars and fats. Lipolysis of dysfunctional adipose tissue contributes the remaining 60%43.
The gut–liver axis in NAFLD. Interactions between the intestine and the liver are emerging as important determinants of NAFLD. Obesity, which is a key factor contributing to the development of NAFLD, is associated with alterations in the intestinal microbiota that enhance nutrient extraction from the diet46. To date, NAFLD has not yet been linked to any specific microbiotic signature, but people with NAFLD have intestinal microbiota that is clearly different from that of healthy individuals47. Intestinal dysbiosis can contribute to liver disease by weakening the intestinal barrier and enabling the translocation of bacteria or bacterial products, such as endotoxin or ethanol, into the portal circulation. Dysbiosis can also lead to the luminal degradation of beneficial nutrients, such as choline, which are important for the maintenance of hepatic lipid homeostasis11.
Mechanisms of cell death in a fatty liver. One of the hallmarks that differentiates NASH from NAFL is the presence of hepatocyte injury. Hepatocytes can be damaged by several mechanisms in the setting of NAFLD (Fig. 3). First, the demand for metabolism of excess fatty acids places strain on hepatocyte mitochondria; over time this leads to mitochondrial uncoupling, the production of reactive oxygen species (ROS) and the activation of Jun N-terminal kinase (JNK)48. Once initiated, ROS production and JNK activation continue in a feed-forward loop, which results in mitochondrial damage, impaired ATP production and cell death49,50. Excess fatty acids can also injure hepatocytes by inducing endoplasmic reticulum stress, which leads to mitochondrial dysfunction and cell death through caspase-2-mediated cleavage of BH3-interacting domain death agonist (BID)51,
Inflammation and fibrosis in fatty livers. Activation of the immune system is a key feature of NASH (Fig. 3). The classic effectors of hepatic inflammation in NASH are Kupffer cells and recruited macrophages, but natural killer T cells play an important part in macrophage recruitment58, and both natural killer T cells and T cells are emerging as contributors to progressive liver disease59,
The development of hepatic fibrosis portends a poor outcome in NASH7. The central event in hepatic fibrogenesis is the activation of hepatic stellate cells68; these cells are susceptible to stimulation by various compounds that are present in a diseased fatty liver. Like inflammatory cells, stellate cells can be activated by DAMPs from dying hepatocytes or other sources that engage TLRs on the cell surface69. Other compounds such as free cholesterol can amplify hepatic fibrosis by upregulating the expression of TLRs on stellate cells70. Oxidative stress can also activate stellate cells; advanced glycation end products, which are abundant in individuals with diabetes, induce ROS production by stellate cells by inducing the enzyme NADPH oxidase 2 (NOX2; encoded by CYBB)71. One unique consideration in NASH is the role of ballooned hepatocytes in hepatic fibrosis. Ballooned cells produce Sonic Hedgehog72, a protein that promotes tissue fibrosis73. Whether Sonic Hedgehog stimulates fibrosis through direct effects on stellate cells or indirect effects on fatty liver injury is currently under study74.
A substantial proportion of the population are at risk of progressive NAFLD (that is, progression to NASH with advanced fibrosis or cirrhosis) due to obesity and insulin resistance. However, only a minority of people with NAFLD progress to more-advanced disease, including HCC75. The reasons for this remain incompletely understood, but NAFLD is best considered a complex disease trait, the development and progression of which are attributed to subtle interindividual variations, including host genetic factors and environmental factors that interact to produce disease phenotype and determine disease progression. Although the presence of NAFLD is principally determined by environmental factors, genetic factors contribute and, crucially, determine how individuals respond to the challenge of caloric excess and consequent metabolic stressors.
Until 2008, studies looking for genetic variants involved in susceptibility to NAFLD had been restricted to case–control allelic association studies limited by the fact that they are reliant on a priori hypotheses for gene selection. Candidate genes are drawn from the limited pool of genes of which biological function is understood and considered relevant to the disease. Only a minority of genes associated with NAFLD through candidate gene analysis have been independently validated in large independent studies or through the use of transmission disequilibrium testing. This short list includes: mitochondrial superoxide dismutase 2 (SOD2)76, phosphatidylethanolamine N-methyltransferase (PEMT)77, fatty acid desaturase 1 (FADS1)78, and Kruppel-like factor 6 (KLF6)79. All of these genes were associated with progressive NAFLD rather than NAFLD. More recently, several novel genetic associations with NAFLD have been identified through genome-wide association studies (GWAS)80,
The first of these genes encodes PNPLA3 (Ref. 80). The index single-nucleotide polymorphism (SNP) in PNPLA3 (rs738409; c.444 C>G; p.I148M) is a non-synonymous cytosine to guanine nucleotide transversion mutation that results in an isoleucine to methionine amino acid change at codon 148. A gene dosage effect for PNPLA3I148M carriage is observed with a stepwise increase in 1H-MRS-quantified hepatic triglyceride content with increasing carriage of the minor allele80. The minor allele frequency also correlates with ethnic differences in susceptibility to NAFLD. Multiple histologically based studies have clearly demonstrated that the PNPLA3I148M variant is associated with the severity of steatohepatitis and fibrosis88 and more recently with the presence of HCC89. PNPLA3 might, therefore, be used as part of an HCC surveillance strategy. The PNPLA3I148M SNP has also been associated with a greater response to dietary or lifestyle modification and may therefore be a biomarker for both greater risk of progressive disease and greater benefit from intervention. The precise mechanism through which the PNPLA3I148M SNP contributes to disease remains controversial despite intensive study, although alterations in lipid droplet architecture and in retinol metabolism of the hepatic stellate cell have been associated with the PNPLA3I148M variant90,91.
The second of these genes encodes transmembrane 6 superfamily member 2 (TM6SF2) on chromosome 19, with a non-synonymous SNP (rs58542926; c.449 C>T; p.E167K) associated with 1H-MRS-quantified hepatic triglyceride content86. As with PNPLA3, candidate gene studies have shown that the TM6SF2E167K variant is also associated with progressive NAFLD92. Interestingly, evidence has been provided that carriage of the more common TM6SF2E167K major allele is associated with dyslipidaemia (that is, increased serum LDL cholesterol and triglyceride levels), increased myocardial infarction and cardiovascular disease risk, whereas minor allele carriage is protective93. Functional studies suggest that TM6SF2 regulates hepatic lipid efflux, with its deletion or mutation resulting in a reduction in lipoprotein secretion (VLDL, triglycerides and apolipoprotein B) leading to increased hepatocellular lipid droplet size and triglyceride accumulation. The TM6SF2E167K variant results in lower total cholesterol in humans and expression levels of the variant are lower than the wild-type form. These data indicate that the TM6SF2E167K variant confers a ‘loss of function’ to the protein that may be both qualitative and quantitative93.
Although these studies fail to provide any explanation for the association between the TM6SF2 variant and progressive NAFLD, they do suggest an interesting paradox — the ‘TM6SF2 Catch-22’ paradigm94. Here, on a background of insulin resistance and metabolic stress, TM6SF2 genotype acts as a determinant of metabolic syndrome-related end-organ damage and clinical outcome: protecting the liver at the expense of increased risk of atherosclerosis and cardiovascular disease or vice versa. The best evidence in support of this paradigm has recently been provided by a study in patients with NAFLD demonstrating that, although the minor threonine SNP (TM6SF2E167K) is associated with advanced disease, carriage of the major cysteine allele (TM6SF2167E) is associated with dyslipidaemia and cardiovascular disease95.
The role of epigenetic factors in the biology of NAFLD is starting to emerge and has enabled integration of information on gene expression with the external and internal metabolic information (Fig. 4). Epigenetic alterations orchestrate the reprogramming of transcriptional machinery of hepatocytes, which occurs during their adaptation to a lipotoxic environment, inflammation and oxidative stress. These epigenetic changes could at least partially explain why the degree of histological damage and/or recovery differs among individuals with an identical genetic background (for example, the presence of the PNPLA3I148M risk allele96) and in response to similar injury (that is, fat overload).
DNA methylation. DNA methylation in particular is suggested to be involved in the progression of NAFLD towards advanced (fibrotic) clinical stages (Fig. 4). Joined global expression and methylation analysis of human liver specimens demonstrated that advanced disease is associated with global deregulation (mostly hypomethylation) of methylation in multiple CpG sites97. Hypomethylation and increased expression of genes involved in tissue repair, regeneration and tumorigenesis were found, as were hypermethylated transcriptionally repressed genes that are involved in basic metabolic functions (such as lipid, urea and amino acid metabolism) or belonged to the cytochrome P450 family97. In addition, DNA hypomethylation was associated with transdifferentiation of stellate cells into myofibroblasts in cell models, a phenomenon commonly associated with fibrosis98. Abnormal methylation of the PNPLA3 promoter was associated with fibrosis severity in a large number of Japanese people99. Differences in the methylation status at specific CpG islands within the promoter of anti-fibrogenic (peroxisome proliferator-activated receptor-α (PPARA)) and PPARD) and pro-fibrogenic (transforming growth factor-β1 (TGFB1), collagen type I-α1 (COL1A1) and platelet-derived growth factor-α (PDGFA)) genes might have a prognostic value in differentiating patients with mild fibrosis from those with advanced fibrosis100.
Liver-specific epigenetic changes that affect physiological processes and lead to perturbations of normal hepatocyte and mitochondrial function not only affect the progression of NAFLD but also affect NAFLD-related metabolic phenotypes, such as insulin resistance. Targeted analysis of the methylation status of the PPARγ co-activator 1α (PPARGC1A) — a master transcriptional regulator of glucose and lipid metabolism and mitochondrial biogenesis — showed that methylation levels significantly correlated with plasma insulin levels, β-cell function and the homeostatic model assessment of insulin resistance (HOMA-IR) marker (calculated as (glucose mmol per litre × insulin mU per ml)/22.5) in humans101. A complex interaction between PPARGC1A mRNA levels and liver mitochondrial DNA (mtDNA) copy number was also reported. Interestingly, low mtDNA copy number was also directly associated with insulin resistance and NAFLD101. Methylation of mtDNA might have a role in modulating the liver phenotype beyond genomic DNA102. Specifically, methylation and transcriptional activation of the mitochondrially encoded enzyme NADH dehydrogenase subunit 6 (encoded by MT-ND6), which is involved in oxidative phosphorylation, in the liver was associated with more-advanced liver pathology in humans102.
A possible explanation for the changes in DNA methylation in NAFLD is its association with obesity, which has been suggested to be associated with reduced DNA demethylation103. Furthermore, evidence suggests that the activity of 5-hydroxymethylcytosine (5hmC) might be an additional mechanism that explains liver DNA demethylation pathways in patients with NAFLD104.
Epigenetic changes can be potentially inherited from one generation to the next. For instance, an experimental study in rodents showed that liver injury induced by the hepatotoxin carbon tetrachloride (CCl4) led to hypomethylation in the PPARG promoter and associated chromatin modifications in sperm, stem cells and/or mature sperm105. Although this experimental study suggests that the hepatic wound-healing response might be heritable, it remains unknown whether the same is true in patients with NAFLD. In addition, epigenetic alterations acquired during development may contribute to fetal metabolic programming106. Maternal protein restriction in mice led not only to fatty liver in the offspring but also to reduced lifespan; further in vitro analysis showed that these changes were associated with changes in DNA methylation107. Likewise, in utero overnutrition affects a sirtuin-mediated histone acetylase–deacetylase pathway that has an effect on the hepatic histone code of the progeny, as shown in a non-human primate experimental model of high-fat-diet-induced NAFLD108.
Non-coding RNAs. A large part (∼60%) of the cellular gene expression machinery is regulated by non-coding RNAs — RNA transcripts that do not encode proteins (Fig. 4). MicroRNAs (miRNAs) have been studied extensively in liver disease research, and evidence for their role in NAFLD is consistent. Numerous miRNAs have been implicated in NAFLD severity, liver injury and lipid metabolism, including miR-122, miR-192, miR-375 and miR-34a109,
Of particular importance in the pathophysiology of NAFLD is miR-122, the expression of which is highly enriched in the healthy liver. Its network of regulated genes are involved in cholesterol metabolism112, modulation of liver inflammation and progression of hepatocarcinogenesis113. An in vitro study showed that miR-122 regulates the production of collagen in hepatic stellate cells, suggesting a pivotal role in liver fibrogenesis114. Notably, the expression levels of miR-122 are decreased in the liver of patients with NAFLD, whereas levels are increased in the circulating (extracellular) compartment111; mouse models of diet-induced steatohepatitis confirmed these findings115. This marked downregulation of liver miR-122 observed in NAFLD seems to be of particular relevance in the pathogenesis of the disease. In fact, silencing of mir-122 is an early event during hepatocarcinogenesis as a consequence of NASH in humans116. Whether the downregulation of miR-122 in the liver is the cause or the consequence of the progression of NAFLD remains to be investigated, because of the cross-sectional nature of the above-mentioned studies.
Several studies have explored the role of circulating (extracellular) miR-122 levels as a biomarker of NASH — or disease progression — in humans, because miR-122 levels significantly correlate with histological damage (including hepatocyte ballooning and fibrosis) and increased liver enzymes (a marker for liver inflammation or injury)111. A two-stage study, exploring a large panel of circulating miRNAs at different stages of NAFLD, showed that circulating levels of miR-122 were increased 7.2-fold in patients with NASH versus healthy controls and 3.1-fold in NASH versus simple steatosis111. Although the performance of circulating miR-122 suggests that it could be a reliable biomarker of liver injury in NAFLD, the overall predictive value for liver fibrosis is very similar to currently available non-invasive measurements, including serum cytokeratin 18 levels111. Nevertheless, the research agenda is still open; for instance, preliminary data from a rat model of Roux-en-Y gastric bypass showed that circulating miR-122 levels decrease after surgery, suggesting a putative role as a biomarker of response to therapeutic interventions117. Clinical evidence has suggested that bariatric surgery was able to partially reverse epigenetic alterations in the liver associated with NAFLD. Notably, reversing differentially methylated sites in NAFLD correlates with improvement in liver histology118.
Furthermore, miR-122 released from the liver circulate in the bloodstream — like a hormone — and is capable of integrating global metabolic signalling111. A functional polymorphism in the 3′ untranslated region of a validated miR-122 target gene (that is, rs41318021 of solute carrier family 7 member 1 (SLC7A1)) was significantly associated with arterial hypertension, suggesting that miR-122 might potentially modulate the susceptibility to increased blood pressure in people who carry the risk allele of this variant111. Moreover, the expression levels of liver miR-122 significantly and negatively correlated with systolic arterial blood pressure111. Together, the reduction of miR-122 levels in NAFLD might explain in part the connection between NAFLD and cardiovascular risk.
Finally, although the role of long non-coding RNAs in the pathophysiology of NAFLD remains largely unknown, they are implicated in lipid homeostasis. For instance, a liver-enriched long non-coding RNA in mice regulates lipoprotein lipase activation through a farnesoid X receptor (FXR)-mediated pathway119.
Diagnosis, screening and prevention
Diagnosis of NAFLD
Most patients with NAFLD are asymptomatic or complain about nonspecific symptoms, such as fatigue, sleep disturbances or right upper quadrant discomfort. Hepatomegaly is the most common physical finding. More-advanced liver disease is associated with signs of portal hypertension. Features of polycystic ovary syndrome (associated with hyperandrogenism) should be sought in young women with suspected NAFLD120,121. Obstructive sleep apnoea syndrome and psoriasis can be associated with NAFLD122. Apolipoprotein B and lysosomal acid lipase partial deficiency are rare familial causes of NAFLD and should be suspected in the absence of a clear association with components of metabolic syndrome, although lysosomal acid lipase partial deficiency is usually associated with high levels of total and LDL cholesterol123,124.
As no specific marker is currently available, the diagnosis of NAFLD requires the exclusion of all the known other causes of chronic liver disease, haemochromatosis and autoimmune hepatitis and of other causes of steatosis, such as medications (Box 1), heavy alcohol consumption, viral hepatitis (especially hepatitis C genotype 3), Wilson disease, among others. To discriminate NAFLD from AFLD, a threshold for daily alcohol intake has been set at <30 g for men and <20 g for women125, but a clear differentiation between the two is difficult in those patients who consume a slightly higher amount of alcohol (up to 40 g per day in both men and women).
Another important criterion is that NAFLD is tightly associated with metabolic syndrome and insulin resistance in the liver, muscle and adipose tissues126. NAFLD should be suspected in all individuals with one or more components of the metabolic syndrome, defined as the cluster of any three of the following five features: increased waist circumference (ethnicity adjusted), increased fasting glucose or T2DM, hypertriglyceridaemia, low HDL levels (sex adjusted) and increased blood pressure127. This association does not characterize NAFLD owing to the genetic variant in PNPLA3, but PNPLA3 mutation and metabolic syndrome may be associated; that is, frequently present in the same person80.
Diagnostic workup should include evaluation of family and personal history of components of the metabolic syndrome and assessment of liver tests, including platelet counts, albumin and coagulation, fasting blood glucose, triglycerides and HDL levels. The most common modes of presentation of NAFLD are the detection of unexplained abnormal liver enzymes and/or of bright liver at ultrasonography.
Blood parameters. Liver tests can show mild (twofold to fivefold) increases in the levels of ALT, alkaline phosphatase (ALP) and γ-glutamyltranspeptidase (GGT), but levels are normal in the majority of people with NAFLD (80%)128. The ratio of ALT to aspartate aminotransferase (AST) is greater than 1 unless advanced fibrotic NAFLD is present or the patient has covert AFLD. Increased levels of GGT do not discriminate between AFLD and NAFLD, as raised GGT levels are commonly associated with metabolic disease129. Ferritin may be increased in up to 60% of patients, but is mainly a marker of subclinical inflammation, given that iron overload is uncommon in NAFLD (4–6%)130. However, high ferritin levels (twofold to threefold) have been associated with more-advanced disease125. Autoantibodies (antinuclear antibody and α-smooth muscle actin-specific antibody) are present in approximately one-third of patients at low titres (1/80 and 1/160)131 as compared to absent in healthy individuals.
A test of insulin sensitivity should be considered in all suspected patients. The HOMA-IR is a good surrogate for insulin resistance in individuals who do not have diabetes132, although there is no universal agreement on threshold defining what is abnormal. Diagnostic workup should eventually include an oral glucose tolerance test according to standard criteria133 that provides additional information about the glucose tolerance status.
Ultrasound-based techniques. A quantitative ultrasound biomarker for hepatic steatosis is the controlled attenuation parameter (CAP)134, which is a quantitative measure of the attenuation (energy loss) of the ultrasound beam as it traverses the hepatic parenchyma. The rationale is that ultrasound energy is attenuated more by fat than by non-fatty tissue; hence, greater attenuation implies greater fat content. The parameter is measured with the same device used in vibration-controlled transient elastography (VCTE, see below) and is reported in units of dB per metre134. An anatomic image is not acquired, therefore, the location where the measurements were made is not recorded. Numerous single-centre studies suggest that CAP can accurately identify patients with hepatic steatosis135,
MRI. The leading quantitative MRI biomarker for hepatic steatosis is proton density fat fraction (PDFF)138. This biomarker measures the relative proportion of mobile protons that are attributable to fat and correlates closely with biochemically determined hepatic triglyceride concentration138. In numerous single-centre and multi-centre studies, PDFF has shown high accuracy for diagnosing, grading and longitudinally monitoring steatosis in adults and children, including individuals who are morbidly obese139,
Diagnosis of NASH
The diagnosis of NAFLD can be derived from classic risk factor assessment along with biochemical measures, and ultrasound-based and magnetic resonance-based detection of hepatic steatosis, yet the most relevant challenge to the clinician is the distinction between simple fatty liver and NASH because patients with NASH are at greater risk for developing progressive fibrosis. The presence of normal liver enzymes in the majority (80%) of individuals renders the identification of patients at risk particularly challenging128. Importantly, histological severity between patients with and those without abnormal liver aminotransferases does not differ.
The actual diagnosis of NASH requires microscopic evaluation of liver tissue, obtained by liver biopsy (Fig. 1). Histological analysis shows the constellation of steatosis, hepatocyte ballooning and lobular inflammation, typically in acinar zone 3 — the microcirculatory unit through which blood exits the liver146. Fibrosis better predicts outcomes and mortality7. Semi-quantitative grading and staging systems have been developed (Box 2). These systems are primarily used to the assess the severity of lesions. Grading involves the assessment of the activity of the disease: steatosis, inflammation and hepatocyte ballooning, whereas staging involves the assessment of fibrosis location and vascular remodelling. Although the grade might be reversible with appropriate intervention, the stage is probably less reversible.
As liver disease progression has been associated with persistence or worsening of metabolic risk factors147, most predictive indices of disease severity are based on components of the metabolic syndrome along with biochemical and imaging indicators of advanced liver disease. However, the large number of patients with NAFLD and the risk of complications associated with liver biopsy have led to the refinement of non-invasive techniques to predict the histological features of NAFLD and NASH.
In clinical practice, interpretation of non-invasive markers should be performed by hepatologists according to the clinical context and considering all the other clinical and laboratory findings. The combination of two unrelated markers is recommended, as no single test has an advantage over the others in the prediction of the severity of liver disease148. Among the different strategies, algorithms that combine transient elastography and serum biomarkers are the most attractive and validated148. However, in case of unexplained discordance of non-invasive tests, a liver biopsy should be performed.
Biomarkers and scores. Screening of steatosis is usually based on ultrasonography in individual patients, but indices or biomarkers are preferred in larger-scale screening studies. The best-validated biomarkers are the Fatty Liver Index, the SteatoTest and the NAFLD liver fat score. Simple steatosis does not increase liver-related mortality, but these biomarkers can variably predict metabolic and cardiovascular outcomes or mortality149. Serum markers of fibrosis seem to have a better performance, particularly the NAFLD Fibrosis Score (NFS), Fibrosis-4 (FIB-4), BARD and commercially available panels, such as FibroTest, FibroMeter and the Enhanced Liver Fibrosis (ELF) test150 (Table 3). All of these tools have acceptable diagnostic accuracy, but only NFS and FIB-4 have been extensively validated19. All of these tests perform best at excluding severe fibrosis–cirrhosis, with negative predictive values of >90%, but are typically less accurate in the determination of less-severe fibrosis150. NFS, FIB-4, ELF and FibroTest can also predict overall, cardiovascular and liver-related mortality149.
Several non-invasive procedures have been tested for the non-invasive prediction of NASH. Plasma levels of cytokeratin 18 fragments (a marker of apoptosis) had a promising performance, but recent data have highlighted the limited accuracy151 and to date no recommendation can be issued for the non-invasive diagnosis of NASH.
Ultrasonography. Conventional ultrasonography, in which standard B-mode (grey scale) ultrasonographic images are acquired and interpreted qualitatively, is frequently used to screen for hepatic steatosis, but conventional ultrasonography is insensitive to mild steatosis, especially in individuals who are obese, and is limited by reader variability152,153.
Elastography. Imaging techniques that are collectively known as elastography have been developed to indirectly measure tissue stiffness non-invasively. The measurement involves the assessment of the propagation of shear waves within the liver, from which the hepatic stiffness is inferred. Depending on the device, results can be reported as shear wave speed in metres per second or as one of several elastic moduli (for example, Young modulus or complex shear modulus) in kPa154. These various elastography-derived parameters have emerged as the leading imaging biomarkers of hepatic fibrosis, but like serum markers, they perform best only at detecting advanced (stage 3 or stage 4) fibrosis.
Ultrasound-based elastography. The first ultrasound-based method to measure liver stiffness was VCTE155. This technology uses a non-imaging device to track shear waves that are transmitted transiently into the liver by an external vibration source; an anatomical image is not acquired and the location from which the measurements are made is not recorded.
More recently, elastography capabilities have been added to clinical ultrasound scanners154. Elastography-enabled scanners use ultrasound to track shear waves that are generated transiently in situ within the liver by an ultrasound push pulse (also known as an acoustic radiation force impulse); measurement locations are recorded on an anatomical image154,156,157 (Fig. 5b). As each manufacturer uses proprietary hardware and software, measurements made with different devices and even with different transducers (probes) on the same device do not agree157,158. Thus, any given patient should be monitored serially with a single device and using the same probe. VCTE and elastography-enabled scanners have been shown to provide high accuracy for the diagnosis of advanced fibrosis in patients with viral hepatitis infection159,160. Single-centre studies suggest these devices also accurately diagnose advanced fibrosis in patients with NAFLD160, although multi-centre validation studies have not yet been performed. None of these devices can diagnose early fibrosis accurately. The technical success rate, reliability, robustness and precision of these devices for measuring stiffness parameters in NAFLD has not been extensively studied, and their performance in detecting changes in fibrosis in the context of treatment trials is unknown157. Until such data become available, caution is advised for use of these devices in NAFLD clinical trials.
Magnetic resonance-based elastography. Magnetic resonance elastography uses a specialized MRI sequence to visualize shear waves that are delivered continuously into the liver by an external acoustic source161. The wave images are processed by a so-called inversion algorithm to generate ‘elastograms’ (Ref. 162) (Fig. 5c). These computer-generated images depict the spatial distribution of a stiffness parameter known as the magnitude of the complex shear modulus (usually referred to as ‘shear stiffness’ in the medical literature). The hepatic stiffness is recorded from representative portions of the liver. Magnetic resonance elastography is now commercially available on commercial MRI scanners, with the same hardware to generate the shear waves and the same inversion algorithm to process the wave images. Small single-centre studies in healthy volunteers suggest that measurements made with different scanners agree163. Large studies in patients with NAFLD are needed to confirm cross-platform reproducibility.
In single-centre studies, magnetic resonance elastography has been shown to diagnose advanced fibrosis in adult NAFLD with high accuracy164,
Patient monitoring should include routine biochemistry, non-invasive monitoring of fibrosis and the assessment of co-morbidities. Given that the risk of developing T2DM and cardiovascular complications is increased in patients with NAFLD122, oral glucose tolerance tests should be performed whenever glucose metabolism is abnormal (that is, a fasting glucose of 100–126 mg per dl; glycated haemoglobin (HbA1c) of 5.7–6.4%)133 and carotid doppler ultrasonography122; referral to other specialists might be required. The timing of follow-up testing is still debated, and a definite evaluation for risks of progression and costs associated with investigations has not yet been performed. In patients with simple fatty liver, a reasonable approach is a follow up in primary care setting, unless worsening of metabolic risk factors occurs. In patients with NASH and/or fibrosis, a yearly monitoring policy is advisable. If indicated, on a case-by-case basis a repeat biopsy could be performed after 5–7 years.
Current management of NAFLD
The first step in the management of NASH in most patients is the implementation of lifestyle modifications that focus on healthy eating habits and regular exercise168. Without biopsy confirmation of the presence of NASH, lifestyle modification is still appropriate to reduce cardiovascular risks in patients with NAFLD and also to treat NASH if it is present. Weight loss for patients who are overweight and obese is recommended based on the collective observations from multiple studies showing that histological improvement can occur when patients lose 5–10% of their body weight169,170. Unfortunately, meaningful lifestyle modification is difficult to achieve and difficult to sustain for most people171,172 and, therefore, alternatives are needed to treat NASH and to prevent the associated complications of the metabolic syndrome. At this time, no pharmaceutical treatments are approved for NASH, although many agents are in various stages of evaluation in clinical trials. Bariatric surgery is a good option for select patients who are obese and have NASH as it typically leads to improvement in NASH173,174.
Pathogenesis-based targets of therapy
As reviewed above, one perspective on the pathogenesis of NASH is that lipotoxic lipid species, which are derived from an excess supply of fatty acids in hepatocytes, mediate cellular injury and the resulting inflammatory and fibrotic response that results in the histological phenotype of NASH175,
If the genomic or epigenetic changes responsible for these differences can be identified and exploited, then treatments could be effective for NASH as well as the underlying insulin resistance, hypertension, hyperlipidaemia and other manifestations of the metabolic syndrome that lead to disability and early death178. Animal studies have provided some insights into these differences, but how these will be translated into treatment of human disease is yet to be shown. Examples include the divergence of rat strains into those having high and low exercise tolerance, with features of the metabolic syndrome developing in the latter179, the role of β-adrenergic stimulation of highly metabolic brown adipose tissue180, the role of serotonin in modulating metabolism181 and the role of peripheral cannabinoid signalling in modulating energy efficiency182. At this time, insights into the regulation of energy metabolism are mostly at early preclinical stages, and clinical trials have generally focused on manipulating more downstream processes, such as lipogenesis, lipolysis, inflammation, oxidative stress and fibrogenesis.
Preventing an oversupply of fatty acids in the liver
Inhibition of DNL. One approach to reducing lipotoxic liver injury is to reduce the endogenous production of fatty acids in the liver by inhibiting DNL. The transcription factor SREBP1C is a major regulator of DNL; thus, treatments that decrease SREBP1C may be beneficial. This is one potential mechanism by which the bile acid derivative obeticholic acid may work. As an activating ligand for the nuclear receptor FXR, obeticholic acid increases the expression of tyrosine-protein phosphatase non-receptor type 6 (PTPN6; also known as SHP1), which decreases the expression of SREBP1C183. A Phase II clinical trial of obeticholic acid has demonstrated histological improvement in NASH after 72 weeks (45% versus 21% in placebo-treated patients; P = 0.0002)184. However, treatment was associated with significant pruritus in 23% in the treatment group compared with 6% in the placebo group, and with an increase in total and LDL cholesterol and a decrease in HDL cholesterol in treated patients, the importance of which will be further assessed in another clinical trial.
Another approach to inhibiting DNL includes using inhibitors of key enzymes in the pathway of fatty acid synthesis, such as acetyl-CoA carboxylase, and such agents are being evaluated in clinical trials. Augmenting the incretin axis with glucagon-like peptide 1 (GLP-1) analogues such as exenatide or impairing the breakdown of endogenous GLP-1 with the dipeptidyl peptidase 4 inhibitors (gliptins) may also decrease hepatic lipogenesis by decreasing substrate availability; this approach is also being examined in clinical trials.
Reducing the burden of fatty acids delivered to the liver. Lipolysis of stored triglyceride in adipose tissue is the main source of fatty acids delivered via the circulation to the liver. Adipose tissue lipolysis is normally suppressed by insulin, but when adipose tissue is insulin resistant, it continues to release fatty acids into the blood at inappropriate times and thus contributes to the development of NASH185. The PPARγ ligands thiazolidinediones (TZDs or ‘glitazones’) have been shown to improve adipocyte insulin responsiveness and thus decrease inappropriate lipolysis. One TZD, pioglitazone, has been shown to improve adipose insulin sensitivity and improve NASH186,187. Unfortunately, this benefit comes at the expense of increased body weight in some patients because of adipocyte triglyceride retention. This and possibly other adverse effects such as osteoporosis and bladder cancer have dampened the enthusiasm for using pioglitazone for NASH and it has not gained widespread use.
Metformin is another insulin sensitizing agent but its effect is primarily in the liver. There is little evidence that hepatic insulin resistance has a role in causing NASH. A large randomized clinical trial (RCT) of metformin in children did not show any benefit on ALT levels or histology but fortunately neither did it show worsening188. Nonetheless, metformin may have an adjunctive role in the treatment of NASH because it is often associated with weight loss, and recent data indicate that it is associated with a reduced risk of several malignancies189.
Fatty acids and the carbohydrates used to make fatty acids can also be diverted to oxidative pathways and this may explain the benefit of regular exercise in improving the metabolic syndrome. Oxidative pathways can also be stimulated pharmacologically. The PPARα and PPARδ ligand GFT505 was recently evaluated in a clinical trial because it improves insulin sensitivity, which may be explained by the effect of PPARα in promoting oxidative metabolism in the liver and PPARδ in promoting oxidative metabolism in muscle190.
The mechanisms of lipotoxic injury remain uncertain, but oxidative stress has been proposed to have a role191. Oxidative stress with associated lipid peroxidation clearly occurs in NASH192, but whether it is causative or an epiphenomenon remains unknown. Two large RCTs of natural vitamin E (d-α-tocopherol) at a dose of 800 IU daily have been conducted: one in adults (PIVENS) and one in children (TONIC). The PIVENS trial demonstrated improvement in liver histology in 43% compared with a placebo response of 19% (P = 0.001)187, and similarly the TONIC trial showed resolution of NASH in 58% compared with 28% in placebo (P = 0.006)188. Neither trial showed evidence that indices of oxidative stress improved, so it remains somewhat unclear how vitamin E improves NASH. Both the PIVENS and the TONIC trials were conducted in patients without diabetes or cirrhosis, therefore, the benefits of vitamin E in patients with these commonly associated conditions remain unknown. Concern has been raised about the cardiovascular safety of high-dose vitamin E and, consequently, patients and doses should be carefully selected. The ideal patient may be someone with aggressive NASH and no diabetes or major risks for cardiovascular disease, but such patients comprise a relative minority. It should be noted that weight loss has an additive benefit in patients treated with vitamin E, so the use of vitamin E should be an adjunct to weight loss and not an alternative193. Fish oil (poly-omega-3 fatty acids) may also exert beneficial effects on lipid peroxidation, but a large trial of ethyl-eicosapentaenoic acid in patients with NASH did not show any benefit194.
Endoplasmic reticulum stress is another process known to occur in NASH that may be central to the pathogenesis of lipotoxic hepatocyte injury. Studies of agents that diminish the endoplasmic reticulum stress response have provided provocative data in animals but these have yet to be introduced into RCTs195.
The contribution of hypercholesterolaemia to the development of steatohepatitis and liver fibrosis has not been fully established, but several studies suggest that it may have a role in some patients. A morphometric study of liver biopsies demonstrated cholesterol crystals in lipid droplets in patients with steatohepatitis but not steatosis196. Statin use is safe in patients with liver disease197, and a Cochrane review concluded that statins can improve steatosis and reduce the levels of aminotransferase, but treatment trials with histological endpoints are lacking198. More recently, in a large European observational cohort with NAFLD, statin use was associated with less steatosis, NASH and fibrosis in a dose-dependent manner; interestingly, the presence of the PNPLA3I148M polymorphism prevented the protective effect of statins199. In another recent study, statin use in patients with NAFLD was associated with a reduction in liver-related deaths, liver transplantations or other liver outcomes7.
Inhibition of inflammation
The role of inflammation in contributing to hepatocyte injury versus a consequence of injury is unknown but should be clarified by the results of clinical trials examining various anti-inflammatory approaches. One clinical trial of an anti-inflammatory phosphodiesterase type 4 inhibitor was negative despite the demonstration of reduced circulating tumour necrosis factor levels in patients given the active agent200. However, like all contributors to the pathogenesis of NASH, inflammation may have an important role in only a subgroup of patients. Post-hoc analysis of RCT results to identify and then predict responders to a specific therapy will be essential for all agents.
Inhibition of apoptosis
Caspase inhibitors have been developed and are being explored in RCTs for numerous diseases including NASH based on the recognized role of hepatocyte apoptosis in the pathogenesis of NASH201. Although this might be a rational approach in acute processes, theoretical concerns about the safety of long-term inhibition of apoptosis persist with this treatment approach in NASH.
Inhibition of fibrosis
In the absence of effective therapy for NASH, being able to prevent the progression of fibrosis to cirrhosis would be advantageous. Even when effective NASH therapies are identified, there may be a role for short-term use of effective anti-fibrotic agents to accelerate the reversal of fibrosis. No anti-fibrotic agents for liver disease are available but several are being explored in RCTs, including an anti-lysyl oxidase homologue 2 (LOXL2) antibody and a galectin-binding molecule that have shown early promise.
Quality of life
QOL in patients with NAFLD is understudied, although its importance is substantial and broadens our understanding of the overall burden of the disease (Box 3). Several studies have reported QOL scores in the context of NAFLD202,
Most patients with NAFLD are overweight or obese, and even in those who are not, lifestyle changes that incorporate dietary modification and exercise are the cornerstone of therapy. Obesity has both health and psychosocial ramifications and can have a profound effect on an individual patient's QOL. In a cross-sectional study, obesity was not associated with lower QOL205; however, the effects of imposing lifestyle change were not measured. One study evaluated the effect of weight loss on HRQOL in patients with NAFLD and found that, compared with those who did not lose weight, those that did had improved HRQOL scores over baseline values, comparable to the general population204. A prospective study of intensive lifestyle intervention showed that patients who lost >7% body weight benefited not only from improvement or resolution in steatosis, necroinflammation and cellular ballooning (NASH resolution) but also improvement or resolution in fibrosis170. Importantly, no patient who lost >7% body weight had progression of fibrosis. In those that lost >10% body weight, fibrosis improved in 81%, although overall numbers were small as was baseline extent of fibrosis. Weight loss and dietary change are hard to achieve and even harder to maintain, particularly in the case of morbid obesity. Not only is behavioural change difficult in itself but also barriers — physical, social and economic (among others) — can pose a challenge to accomplishing them effectively.
Bariatric surgery for NASH
Compared with patients who are obese and are matched for co-morbidities, those that undergo bariatric surgery have decreased long-term mortality206,
However, practitioners and patients are reluctant to pursue bariatric surgery, even when patients are appropriate candidates. Although there are known complications that vary according to the procedure performed, the overall risk is low in properly selected patients213. Although carefully selected patients with established compensated (that is, stable without profound abnormalities in liver function) cirrhosis can safely undergo bariatric surgery, cirrhosis is associated with a higher risk of postoperative complications, including hepatic decompensation (that is, the development of ascites, hepatic encephalopathy or variceal bleeding)207. Therefore, careful patient selection cannot be overemphasized. Success of the intervention requires that patients remain committed to necessary lifestyle changes postoperatively to avoid regaining lost weight. More importantly, compliance with recommendations and follow-up are imperative to avoid metabolic and nutritional problems that are related to malabsorption or other consequences of bariatric surgery214.
NASH and HCC
Another consequence of liver disease in patients with NASH is HCC. The incidence of HCC is increasing in both the United States and worldwide. In western countries, approximately 20% of HCCs are found in patients with NASH-related liver disease; and even more if HCCs occurring in the setting of cryptogenic cirrhosis are included, which often represents ‘burned out’ NASH that has lost its classic histological features (Fig. 1)215,216. A recent study in the United States based on Surveillance, Epidemiology, and End Results (SEER)-Medicare registries from 2004 to 2009 found NASH to be the underlying cause of HCC in 14.1% of nearly 5,000 cases, following alcoholic liver disease in 16% and hepatitis C virus infection in 54.9%. While this represented a 9% annual increase in NASH-related HCC, NASH–HCC cases represented only 5% of liver transplantations for HCC during the same time period, probably reflecting the burden of co-morbid illness in this population. Furthermore, patients with NASH and HCC were older and had shorter survival if they did not receive liver transplantation than other aetiologies of liver disease217.
Welzel and colleagues estimated the population attributable fraction (PAF), otherwise stated as the proportion of cases that can be attributed to specific risk factors, in patients with HCC from various causes of liver disease using the SEER-Medicare databases from 1994 to 2007 (Ref. 218). Analysis of a cohort of 6,991 people showed that those with obesity and diabetes (presumably with NAFLD) had the highest PAF of HCC (36.6%) compared with other aetiologies, in descending order of magnitude: alcohol intake (23.5%), hepatitis C virus infection (22.4%), hepatitis B virus infection (6.3%) and other (3.2%). An important limitation of this study is that patients did not have a biopsy diagnosis of NAFLD or NASH. The PAF takes into account disease prevalence; thus, if NAFLD were to be eliminated, the effect on HCC would be substantial. However, the risk estimate of HCC in an individual patient with NAFLD is only in the order of 1.5–2.5 compared with 20–25 in a patient with hepatitis C virus infection. Per annum rates of HCC development in patients with NASH are lower than for patients with hepatitis C virus infection (2.6 versus 4)21. As NASH is so prevalent, it is not surprising that HCC as an indication for liver transplantation has increased 3.64-fold in patients with underlying NASH and a BMI of >30 kg per m2. In comparison, HCC due to hepatitis C virus has increased only 2.25-fold over a 10-year period from 2002–2012. Whereas diabetes and obesity are independent risk factors for HCC, the increase in NASH-related HCC persisted after controlling for these variables219. Although older age, obesity, T2DM and male sex are risk factors for the development of HCC, NASH does seem to contribute independently218,220.
Cirrhosis creates a milieu that is permissive for the development of HCC; thus, current screening recommendations focus primarily on patients with cirrhosis. However, reports of HCC occurring in the absence of cirrhosis are on the rise, with some even developing in the absence of profound fibrosis. By some estimates, 10–75% of cases of HCC may occur in non-cirrhotic NAFLD221,222. This is a serious problem because of the large number of patients affected by NAFLD and NASH at a population level. The individual risk of HCC in patients without cirrhosis who have NAFLD is even less well understood. Consequently, although some individuals at risk for HCC may not be identified at an early stage of malignancy, there is presently insufficient evidence to alter current screening guidelines. Optimal recommendations for HCC screening in patients with NAFLD may change in the future as more is learnt about individual risk in this challenging population.
Both the short-term and the long-term outlook for NAFLD will evolve as clinical and basic research continue to re-define the field (Box 4). NAFLD is an extremely complex and nuanced disease that represents the convergence of many pathways, risk factors and external influences that are not uniform in all patients. Therefore, it seems likely that NAFLD represents a constellation of various phenotypes that will require study from many angles as illustrated in this Primer. NAFLD is highly prevalent, yet only a minor subset of patients progress to advanced liver disease, including HCC. Although many patients with NAFLD have a common metabolic profile (T2DM, hypertension and obesity, among others), not all patients do. The underlying pathophysiology of NAFLD and in particular NASH is strongly linked to insulin resistance, aberrant hepatic lipid metabolism, visceral adiposity and inflammation. However, several other important modulators of disease, such as the environment and diet, can further modify triggers of chronic extrahepatic and intrahepatic immune pathways223 and the pathogenetic roles of gut dysbiosis224,
Furthermore, differences in genetic susceptibility as well as epigenetics may help to better understand at-risk populations. Therefore, a more in-depth evaluation of these differences will allow us to better understand the phenotypes of NAFLD. In turn, a more sophisticated ability to phenotype patients with NAFLD will further clarify differences in disease progression between patients and permit a more personalized approach to therapy.
Animal models of disease have offered tremendous insight into relevant mechanisms, but to date, no animal model has faithfully replicated all aspects of the human condition. However, this benchmark may not be compulsory or attainable given the heterogeneity of the human disease.
Just as the pathophysiology of NAFLD is not the same in all patients, treatment will need to be individualized. With rare exception, a commitment to lifestyle modification should be the foundation of any treatment plan, but this is not sufficient for most patients. In the morbidly obese or in those with severe co-morbid illness, bariatric surgery may have substantial benefit. Many patients will require more-intense intervention — behavioural and pharmacological.
Until recently, the landscape of clinical trials for NASH was somewhat limited. Currently there are hundreds of clinical trials in NASH. The breadth of mechanisms being targeted reflects the complexity of the disease. As the field continues to advance and patients can be better classified by constellations of risk factors, or sub-classifications using genomics or metabolomics, we will be able to offer a more personalized, mechanistically derived treatment approach to each patient.
E.B. is a member of the EPoS (Elucidating Pathways of Steatohepatitis) consortium funded by the Horizon 2020 Framework Programme of the European Union under Grant Agreement 634413.