REVIEWS Management of NAFLD: a stage-based approach Mary E. Rinella1 and Arun J. Sanyal2

Abstract | NAFLD is the most prevalent form of liver disease in the USA, affecting an estimated 30% of the population. The condition is associated with increased mortality related to cardiovascular disease, malignancy and liver disease. Identification of patients who might be at increased risk of adverse outcomes is critical as it is not feasible to screen all patients with suspected NAFLD. Patients with NASH, the progressive subtype of NAFLD, should be targeted for treatment, especially if they have concomitant fibrosis because such patients are more likely than those without fibrosis to have adverse outcomes. Treatment goals in patients with NAFLD vary depending on the disease stage owing to differential risk of progression and the particularities of an individual’s comorbid disease. Lifestyle intervention is important for all patients irrespective of disease stage, but other therapies should be targeted to those most likely to benefit. In this Review, we highlight risk factors for disease progression and offer a stage-based treatment approach for patients with NAFLD.

Northwestern University Feinberg School of Medicine, Department of Internal Medicine, Division of Gastroenterology and Hepatology, 676 N. St. Clair Street, Arkes Pavillion, 14–005, Chicago, Illinois 60527, USA. 2 Virginia Commonwealth University, 1200 East Broad Street, MCV BOX 980341, Richmond, Virginia 23298–0341, USA. Correspondence to A.J.S. [email protected] 1

doi:10.1038/nrgastro.2016.3 Published online 24 Feb 2016

NAFLD has emerged as a leading cause of chronic liver disease, comprising two principal clinical-histological phenotypes: nonalcoholic fatty liver (NAFL) and NASH. NAFL and NASH progress to cirrhosis in 2–3% and 15–20% of patients, respectively, over a 10–20 year time frame1. The contribution of NAFLD to the burden of endstage liver disease and hepatocellular carcinoma (HCC) is growing and is projected to surpass hepatitis C as the principal indication for liver transplantation2. Currently, no approved therapy for NASH, the more aggressive form of the disease, is available. However, in the past few years substantial progress has been made in the management of NASH. The focus of this manuscript is to provide guidance to practicing clinicians to help them identify those patients who are likely to have progressive NAFLD and appropriately target such patients for therapies based on the best evidence available. A secondary objective is to provide the current status of therapeutics in development.

Diagnosis and staging of NAFLD Diagnosis A key issue in the management of patients with NAFLD is the identification of those who are most at risk of develop­ ing clinically meaningful outcomes in order to target them for therapy. Patients with NASH are much more likely to progress to cirrhosis than those with NAFL1, although emerging data highlight that a subset of patients with NAFL (in particular those with nonspecific inflammation) might be at risk of progression to NASH or advanced fibrosis and merit follow-up3–5. Specific recommendations on how closely such patients should be followed are not

yet available. Currently, histological analy­sis of a liver biopsy sample is the only way to distinguish patients with NASH from those with NAFL with certainty. However, the presence of multiple features of the metabolic syndrome, type 2 diabetes mellitus (T2DM), persistently elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, increasing age and BMI are all well-known risk factors associated with the presence of NASH6,7. These clinical features can serve as tools to determine which individuals should be referred for a liver biopsy to confirm the presence of NASH. In addition, one patient subgroup that seems to be at heightened risk of disease progression are those with panhypopituitarism8,9. Clinicians should not be guided solely by liver enzyme levels, as these are often within the normal range even in advanced disease10,11. Neither serological nor radiological biomarkers are sufficiently accurate to distinguish NASH from NAFL or reliably detect earlier stages of fibrosis. The best-studied biomarker is cytokeratin 18 (CK‑18, also known as keratin, type I cytoskeletal 18), a breakdown product of hepatocyte apoptosis. Plasma CK‑18 levels are able to predict NASH among those with NAFLD with an area under the receiver operating character­istic curve of 0.82 (95% CI 0.78–0.88), based on a meta-­analysis of 13 studies12. However, cut-off values have not been standardized and the test is not commercially available. Although several genes have been linked to steato­hepatitis and the risk of fibrosis progression, they do not account for a large enough proportion of the variability in the phenotype of the disease to use them to identify suitable individuals for therapy at this time13–15.

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REVIEWS Key points • NAFLD is the most common cause of liver disease, affecting 30% of the US population; however, the goals of treatment differ based on stage of disease • Lifestyle modification and excellent control of comorbid metabolic illness is important for all patients • Patients with NASH, the progressive subtype of NAFLD, should be targeted for treatment, especially if they have concomitant fibrosis because such patients are more likely to have adverse outcomes • Currently available pharmacological options include vitamin E, pioglitazone and pentoxifylline • Although bariatric surgery has not been studied prospectively specifically as a treatment for NASH, indirect evidence suggests it is effective at improving histological features of NASH, including fibrosis • Several promising drug therapies for NASH targeting a broad array of targets are in clinical trials and are anticipated to pave the way for new and more effective treatment options

histo­logical improvement, although these improvements remained substantially higher than the placebo response rates in these subgroups26,27. Until noninvasive methods are developed and validated to assess response to treatment, liver biopsy will remain necessary to determine treatment efficacy, especially for clinical trials. A post‑hoc analysis of the FLINT trial, presented only in abstract form in 2015, indicated that the FIB-4 and APRI indices were sensitive to change and a decrease in these parameters after 6 months of treatment was predict­ive of improved fibrosis after 72 weeks of treatment in those receiving obeticholic acid28.

Management of low-risk NAFLD Patients with NAFL or NASH without fibrosis are generally considered to be at low risk of liver-related outcomes (such as development of ascites, variceal bleeding, hepatic encephalopathy or liver-related death) within a 10–15 year time frame29. These patients do, however, Staging often have major comorbidities such as T2DM and an Another important part of the evaluation of an individ- increased risk of mortality as a result of both cardioual with suspected NAFLD is determining the fibrotic vascular disease and cancer30,31. Although it is therefore stage of their disease. Those patients with bridging important to optimize cardiovascular risk management fibrosis or cirrhosis have an increased risk of liver-­ and pay special attention to meeting current cancer related outcomes and death16,17. In those with cirrho- screening guidelines (such as those for breast or colo­ sis, a Model for End-stage Liver Disease (MELD) score rectal cancer) in patients with low-risk NAFLD, these >10 or a hepatic venous pressure gradient >10 mmHg recommendations apply to all individuals with NAFLD identi­fies those at greatest risk of clinical deteriora- regardless of their liver-related risk status. tion within 1–2 years18,19. Furthermore, the fibrosis-4 Management of weight and overall fitness is the (FIB-4) index, AST to platelet ratio index (APRI), and cornerstone of treatment for all patients with NAFLD. NAFLD fibrosis scores have all been associated with an Several studies have demonstrated the benefit of weight increased risk of mortality20,21. Liver biopsy remains the loss in reducing steatosis or NAFLD activity score (NAS) gold-standard method for determining NAFLD stage, on histology, with increased weight loss associated with against which all surrogate measures of NAFLD stag- more substantial improvement. Reductions in ALT level ing are compared. However, the potential for sampling and steatosis are seen at lesser degrees of weight loss, error and inter-­observer variability is inherent to liver whereas resolution of NASH or even fibrosis can occur biopsies, which typically assess 1:50,000 of the liver. with more marked or sustained weight loss, such as that Noninvasive surrogate markers are poised to replace observed after bariatric surgery32–34. liver biopsy for the diagnosis of advanced stage fibrosis and cirrhosis, as a result of ongoing advances in imaging Diet technology. Both elasto­graphy-based methods such as Several short-term studies have examined the role of Fibroscan® (Echosens, France) and magnetic resonance specific macronutrients in NAFLD. Dietary carbo­ elasto­graphy can be used, although these have not been hydrate content has been linked to heightened systemic approved for this indication22,23. Emerging data suggest inflammation and carbohydrate-restricted hypo­caloric that MRI-based technologies can be particularly accurate diets have been associated with greater reductions in the detection of advanced fibrosis23, enabling patients in hepatic steatosis than a general hypocaloric diet to be categor­ized as having either low-risk or high-risk alone35–37. High dietary cholesterol has also been associ­ NAFLD. Whether categorized by noninvasive param- ated with NAFLD. Excess dietary cholesterol promotes eters or liver biopsy, individuals with advanced‑stage hepatic de novo lipogenesis and increased levels of NASH should be p ­ rioritized for treatment24 (FIG. 1). intracellular free cholesterol that can be cytotoxic38,39. Limiting dietary cholesterol in patients with NAFLD Monitoring treatment outcomes might have the additional benefit of reducing cardioAnother key issue is how best to assess the response to vascular risk, the principal cause of death in this patient therapeutics when interventions are made. Currently, population, although this link has not been proven40. noninvasive methods to document treatment efficacy Similarly, a Mediterranean diet rich in fibre and poly­ are lacking. Normalization of ALT level combined unsaturated fatty acids has been shown to effectively with even modest weight loss increases the probabil- reduce hepatic steatosis in a 6-week crossover study of ity of histological improvement in NASH to ~90% in patients with NAFLD when compared with an isocaloric those receiving vitamin E25. Patients on vitamin E in low-fat, high-carbohydrate diet41. The beneficial effects the PIVENS trial with lesser degrees of weight loss, or of the Mediterranean diet on hepatic steatosis in this with weight gain, had a lower absolute likelihood of study were independent of patient weight loss. Caution

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REVIEWS Suspected NAFLD (Hepatic steatosis on imaging ± elevated serum ALT levels)

Exclude alternate causes of ↑ALT levels

Confirmation of NAFLD

Evaluate alcohol consumption

Risk stratification for liver-related outcomes

Low-risk profile • BMI 11 kPa*  

  

Consider liver biopsy or confirmatory testing for cirrhosis such as magnetic resonance elastography

Figure 1 | Proposed risk stratificationNature for patients with NAFLD. After suspected Reviews | Gastroenterology & Hepatology NAFLD has been confirmed, patients can be stratified by their risk of NAFLD progression, on the basis of their noninvasive risk factor profiles. If noninvasive risk factors cannot definitively determine risk profile, liver biopsy can be used to aid stratification. Presence of MELD >10 or HVPG >10 mmHg is associated with development of adverse clinical outcomes within 1–2 years in 20% of patients. *High-quality data for the use of Fibroscan® (Echosens, France) in patients with NASH are emerging and require confirmation. ‡Cut-off values reported by Angulo et al.118. ALT, alanine aminotransferase; APRI, aspartate aminotransferase to platelet ratio index; FIB-4, fibrosis-4 index; HVPG, hepatic venous pressure gradient; MELD, model for end-stage liver disease; NFS, NAFLD fibrosis score; T2DM, type 2 diabetes mellitus.

must be exercised in interpreting these data because the majority of individuals are unable to sustain a substantial change in their dietary habits. Whether short-term diet-induced improvement in steatosis translates into long-term histological improvement, especially with respect to disease progression and fibrosis, is unknown. In 2015, a prospective study of 293 patients in a community health-care setting demonstrated the effect of weight loss of varying degrees on NASH histology42. Paired liver biopsy data were available from 261 patients at baseline and after 52 weeks of monitored diet (comprising a 750 kcal per day reduction from their calculated resting energy requirements) and exercise intervention. Although only 30% of patients achieved >5% weight loss, those that did showed histological improvement. The rate of NASH resolution increased in patients with higher degrees of weight loss: 90% of those achieving >10% weight loss showed resolution of NASH after biopsy, compared with 64% resolution in those losing 7.00–9.99% of their body weight and 26% resolution in those losing 5.00–6.99%. Regression in fibrosis, defined as a reduction of at least 1 point on the fibrosis score,

was only seen in patients who achieved more dramatic weight loss: regression was seen in 45% of patients who lost >10% body weight compared with 17% of those who lost 5.00–9.99% body weight42. Although these findings are intriguing, further validation is required. As is the case with any lifestyle intervention, sustained change is the goal, which is often difficult to achieve.

Coffee Coffee intake has been associated with decreased allcause and cause-specific mortality43. Furthermore, other studies suggest coffee intake might be beneficial in reducing the risk of alcohol-induced liver injury as well as the development of fibrosis and HCC44–46. The method of coffee preparation could alter the associ­ ations between coffee intake and disease risk, with evidence suggesting filtered coffee is of most benefit to the patient47. Multiple mechanisms have been put forward to explain the hepatoprotective effects of coffee, although further research is needed to determine which specific components are responsible. Although these associ­ ations need to be more carefully studied in prospective clinical trials, in our opinion, it is reasonable to encourage patients to drink unsweetened coffee as part of lifestyle modifications to promote a healthy liver, as long as the patient does not have medical conditions that might be exacerbated by coffee intake. Exercise Exercise also has well-established benefits in improving overall cardiovascular health, the predominant cause of mortality in patients with NAFLD. These benefits, which include the amelioration of both hepatic and peripheral insulin resistance, might be independent of weight loss48,49. Controlled studies on the effects of exercise on NAFLD are lacking. The data available show that aero­bic exercise can be more effective in reducing intrahepatic fat than resistance training, and reductions in hepatic and visceral fat might be independent of weight loss48–50. The intensity and duration of exercise needed to derive benefit remains poorly defined. A retrospective study conducted in Japan and published in 2014 compared the effects of mild-intensity, moderate-intensity and high-­intensity exercise regimens in 169 patients with detectable hepatic fat. In this study, only those in the highest exercise intensity group (>250 min per week) had favourable changes in metabolic parameters and a marked reduction in hepatic fat content compared with those engaging in 35 kg/m2 in the presence of severe comorbid factors (such as hypertension, diabetes, obstructive sleep apnoea or cardiomyo­pathy)57. Notably, liver disease is not at this time considered a comorbidity of obesity in the context of bariatric surgery. Many retro­spective studies and one large prospective study with 5‑year follow‑up demonstrate that bariatric surgery

can improve or even reverse NAFLD, NASH and fibrosis34,58–60. Although such results are encouraging, the indication for surgery in these trials was obesity and not NAFLD, and therefore these data cannot be considered level 1 evidence. A study using bariatric surgery as a primary treatment for NASH is not anticipated to be designed in the near future owing to the invasive nature and high cost of the procedure. However, the available evidence suggests that NASH improves after bariatric surgery and these beneficial effects seem to extend beyond the weight loss that surgery produces (with the exception of gastric banding, where part of the stomach is not resected). Surgically induced changes in gut hormones, particularly glucagon-like peptide 1 (GLP‑1), alter insulin sensitivity and lipid metabolism that can independently improve the histological features of NASH61,62. Thus, improvement of NASH could be considered a secondary benefit for patients otherwise ­meeting criteria for bariatric surgery. Although generally safe, complications of bariatric surgery can occur and these vary depending on the procedure. Proximal gastric bypass is still considered the gold-standard bariatric surgery in many centres because it results in superior weight loss compared with other procedures. However, sleeve gastrectomy has many of the same benefits and is less invasive. Wellknown complications of bariatric surgery include stomal ulceration and stenosis, dumping, malnutrition and leak, although generally complication rates are low63,64. However, in patients with cirrhosis, there is an

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REVIEWS increased risk of postoperative complications, including hepatic decompensation, thus underscoring the importance of careful patient selection65. Finally, emerging endoscopic techniques might provide safer nonsurgical options for patients, although data on efficacy and outcomes are insufficient to provide any evidence-based ­recommendations at this time

Pharmacological treatment Given the low probability of adverse liver-related outcomes in those in the low-risk categories of NAFLD, most patients do not warrant pharmacological treatment; moreover, no FDA-approved therapies exist. In specific individuals with multiple clinical risk factors for NAFLD progression (for example, T2DM in the setting of NASH without fibrosis) a decision to prescribe pharmaco­logical therapy can be taken after assessment of the potential risks and benefits. The specific drugs that could be used are discussed later. Management of NASH with fibrosis Although the short-term liver-related prognosis of those with NASH and stage 1–2 fibrosis remains excellent, these patients are at risk of progression to cirrhosis3,5,29. A meta-analysis of data from studies presenting fibrosis progression data (measured by paired biopsy) found that there seem to be distinct groups of ‘rapid-­fibrosers’ and ‘slow fibrosers’ (as in, individuals whose disease can progress at different rates)29. Overall, fibrosis in patients with NASH progressed more rapidly than in patients with NAFL: progression of fibrosis by 1 stage took 7.1 years (95% CI, 4.8–14.3 years) in patients with NASH compared with 14.3 years (95% CI, 9.1–50.0 years) in those with NAFL29. Given that currently available noninvasive tools cannot accurately track modest changes in fibrosis progression, repeat biopsy should be considered after 5 years, or earlier if disease progression is suspected clinically. Patients with NASH, ­especially those with evidence of moderate fibrosis (≥stage 2) should be considered for pharmacological treatment, in addition to behavioural (diet and exercise) modification. Front-line therapy using available drugs Vitamin E. Vitamin E was originally used in NASH on the basis of its antioxidant effects66. Since the initial pilot study two phase IIb clinical trials, (the Pioglitazone, Vitamin E or placebo for Nonalcoholic Steatohepatitis (PIVENS) trial and the Treatment of Nonalcoholic fatty liver disease in Children (TONIC) trial), have demonstrated that vitamin E is superior to placebo for the improvement of NASH histology67,68. Specifically, these trials found that vitamin E treatment decreases steatosis, inflammation, cellular ballooning and promotes the reso­lution of steatohepatitis (number needed to treat 4.4). PIVENS was conducted in adults with NASH and a NAS of ≥4, whereas TONIC focused on children with NAFLD, some of whom had NASH. In both t­ rials, v­ itamin E improved liver enzyme levels and liver histology. However, in the TONIC trial, the vitamin E group did not meet the primary end point of a sustained reduction in ALT level (for >6 months between weeks 72 and 96).

Careful analysis suggested that a second phase of ALT level improvements in the control arm after 1 year could have been attributed to the adoption of a healthy lifestyle. Vitamin E has been studied mainly in patients with precirrhotic NASH without diabetes mellitus. However, ­indirect evidence suggests it is safe and effective in patients with diabetes mellitus. Data from patients in the vitamin E and placebo groups taking part in the PIVENS trial was pooled with data from patients taking vitamin E in the placebo arm of the FLINT (Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis) trial; patients were able to enter the FLINT trial if they had diabetes or if they continued to have active NASH despite being on a stable dose of vitamin E for 6 months. In this pooled analysis, vitamin E was associated with histo­logical improvements regardless of diabetic status. Patients on vitamin E randomly assigned to receive placebo had a markedly higher rate of histologic improvement than those not on ­vitamin E, regardless of diabetic status27,69. Neither the PIVENS nor the TONIC trial demonstrated an improvement in fibrosis with vitamin E treatment. This finding could be due to the inclusion of many patients with minimal or no fibrosis in these trials, or a lack of antifibrotic efficacy as neither trial was ­powered to detect an improvement in fibrosis. The principal concern surrounding vitamin E treatment has been the potential for toxicity with prolonged use. A meta-­ analysis of data from trials investigating ­vitamin E supplementation for a variety of indications at variable doses and formulations concluded that, on the basis of a regression model, at doses >800 IU per day, vitamin E was associated with a statistically significant increase in all-cause mortality, despite multiple methodological flaws70. Other studies have not supported these findings, therefore the effects of vitamin E on mortality remain inconclusive71–75. No credible signal for either increased all-cause or cardiovascular morbidity in clinical trials of vitamin E for NASH have been observed, although the trials were not designed for this purpose. Long-term use of vitamin E has been associated with an increased risk of prostate cancer (HR 1.13 (99% CI 0.95–1.35; n = 473, P = 0.06)76, although this association was not supported in a publication from the Physicians’ Health Study II, which followed up 14,641 patients over 8 years77. Although vitamin E seems to be protective against thrombotic strokes, it might increase the risk of haemorrhagic strokes78. These potential adverse consequences must be weighed against the risk of disease progression when deciding whether or not to initiate treatment. If a patient is deemed high risk, this risk could be mitigated by using 400 IU per day (rather than 800 IU per day), which has also been shown to improve NASH66. When vitamin E is used, the control of hypertension and atherogenic risk factors warrants special attention, and patients should be informed that its use is off-label. Insulin sensitizers. Several trials have evaluated the value of insulin sensitizers, most notably the thiazolidinediones, on the basis of the close link between insulin resistance and NASH79. Most trials have used pioglitazone at a

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REVIEWS dose of 30 mg per day. These studies have demonstrated that, compared with placebo, pioglitazone treatment can improve all of the histological features of NASH and lead to resolution of steatohepatitis. Although none of the individual studies demonstrated an improvement in fibrosis, meta-analysis of the trials suggests that ­pioglitazone also improves hepatic fibrosis80. Unfortunately, the use of pioglitazone is limited by its adverse effect profile, which includes weight gain, osteopenia, increased fracture risk, fluid retention, congestive heart failure and bladder cancer. Weight gain is particularly problematic because it continues beyond the duration of the treatment67,81. These adverse effects have largely relegated pioglitazone to a second-line treatment, whereby careful consideration of its risk–benefit profile is warranted before starting treatment. Pentoxifylline. Pentoxifylline has been evaluated in two pilot studies and seems to be a promising treatment for NASH82,83. As with vitamin E, pentoxifylline is inexpensive, making it especially promising as a NASH therapeutic in resource-constrained settings. Furthermore, it has a proven safety record and is generally well tolerated. As is the case with vitamin E, pioglitazone or obeticholic acid, only a subset of patients respond to pentoxi­ fylline27,67,82,83. To best understand the potential role of pentoxifylline in the treatment of NASH, larger studies using it alone or in combination with other agents will need to be done. Currently, its role in the treatment of NASH remains unclear. Omega‑3 polyunsaturated fatty acids. A strong theor­ etical rationale exists for the use of omega‑3 polyunsaturated fatty acids (n-3 PUFAs) as a treatment for NASH. Most commonly derived from fish oils, n-3 PUFAs improve insulin sensitivity, inflammation and reduce dyslipidaemia and lipogenesis84,85. Moreover, hepatic n-3‑PUFA deficiency has been documented in patients with NASH86. However, despite these attractive therapeutic properties, several trials have now been performed on n-3 PUFAs as a potential treatment for NASH and none has shown benefit87–89. Interestingly, the net effect on triglycerides in these trials is lower than expected, raising the possibility that there is a state of n-3 PUFA resistance as a result of its diversion to other metabolic pathways. If this is shown to be the case, higher doses might overcome the low resistance state. In addition to variation of dosage, studies of docosa­ hexanoic acid rather than eicosapentanoic acid might be ­warranted given their differential biological effects90,91.

Drugs in advanced development Obeticholic acid. Over the past 15 years, a large body of literature has accumulated documenting the important role that bile acids have in nutritional homeostasis92. In addition to their established effects on lipid emulsification in the gut, they bind a number of receptors that modulate multiple cellular processes. The two best-­ studied bile acid receptors are the farnesoid X receptor (FXR), a nuclear receptor present in hepatocytes and several other tissues, and the G‑protein coupled bile

acid receptor 1 (GPBAR1, also known as TGR5) a cell-­ surface receptor present in the intestine, pancreas and hepatic sinusoidal endothelium93. Activation of FXR by bile acid binding improves insulin sensitivity and diminishes lipogenesis94–96. FXR activation reduces plasma triglyceride concentrations by both decreasing lipogenesis and increasing the functionality of lipoprotein lipase as a result of its effects on apolipoprotein CII and apolipoprotein CIII97,98. Reverse cholesterol transport is upregulated by increased hepatic scavenger receptor expression, which results in a drop in HDL-cholesterol levels99. The potential effects of decreased HDL-cholesterol are unclear; theoretically, increased reverse cholesterol transport could decrease atherogenesis. The functionality of HDL, an important determinant of the effects of HDL-cholesterol on cardiovascular disease risk100, has not been studied in NASH and any changes in HDL function related to FXR agonist administration remain unknown. Obeticholic acid, a potent synthetic variant of the naturally occurring bile acid chenodeoxycholic acid, also produces a modest increase in LDL-cholesterol levels, probably related to CYP7A1 inhibition. This inhib­ition has been hypothesized to catalyze the conversion of cholesterol to bile acids, leading to the accumulation of cholesterol substrate101. In animal models of athero­sclerosis, FXR agonists are associated with a decrease in atherosclerosis despite increased LDLcholesterol levels102. The potential effects of obeticholic acid on cardiovascular risk in humans are currently unknown but are under investigation. A pilot trial in patients with T2DM and suspected NAFLD demonstrated that obeticholic acid improved insulin sensitivity as measured by euglycaemic clamp, the gold standard for assessment of insulin resistance95. The trial also found that the therapeutic effects of 25 mg per day and 50 mg per day were similar. On the basis of these results, the FLINT trial was designed to study the utility of obeticholic acid at 25 mg per day versus p ­ lacebo for active NASH in its pre-cirrhotic stages27. This trial was discontinued on the recommendation of an independent data safety monitoring board after a  ­priori thresholds for efficacy were met during an interim analy­ sis. Furthermore, patients who had not completed the full treatment course specified by the trial were taken off drug therapy without an end of treatment study-­related biopsy. For patients with available histological data, obeticholic acid use resulted in a statistically significant improvement in the NAS, as well as in its individual components. Importantly, an overall improvement in hepatic fibrosis was observed as well. Sensitivity analysis of high-risk patient subsets, such as those with T2DM or increasing levels of fibrosis, confirmed these benefits. Compared with those in the placebo group, patients treated with obeticholic acid were more likely to have complete resolution of fibrosis and less likely to progress to cirrhosis, although the study was not ­powered to specific­ally address these questions. Interestingly, despite compelling preliminary data showing improvements in insulin sensitivity, patients in the FLINT trial receiving obeticholic acid did not have improved insulin ­sensitivity compared with those in the placebo arm.

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REVIEWS Table 2 | Compounds in development for which positive phase II data are available Mechanism

Compound name

ALT

IR

Lipids

Imaging Hepatic fat steatosis

Cellular injury

Fibrosis

N/A

√

√

√

N/A

N/A

Bile and lipid metabolism Synthetic bile acid

Obeticholic acid27

√

√

↓ LDL ↑ HDL

Fatty acid or bile acid modifier

Aramchol™ (Galmed Pharmaceuticals, Israel)121

√

N/A

No effect √

N/A

PPAR‑α/δ agonist

GFT505 (REF. 108)

√

√

√

N/A

No effect* No effect

No effect*

Exenatide122

√

√

√

√

N/A

N/A

N/A

Liraglutide

√

√

↑ HDL

N/A

√

No effect

?√

Leptin therapy

Metreleptin123

√

↓ TG

√

N/A

√

√

No effect

PDE inhibition and/or TNF modulation

Pentoxifylline82,83

√

N/A

N/A

N/A

√

√

?√

Incretin mimetics GLP‑1 agonists

110

Other

*Details of the study are discussed in the text. √, positive treatment effect; ALT, alanine aminotransferase; GLP‑1, glucagon-like peptide 1; IR, insulin resistance; PDE, phosphodiesterase; PPAR‑α/δ, peroxisome proliferator-activated receptor α/δ; TG, triglycerides.

A principal concern is the observation of pruritus in 20% of patients treated with obeticholic acid in the FLINT trial. Interestingly, pruritus was highly associated with histological improvement. It remains to be determined if a lower dose of obeticholic acid, such as 10 mg per day, which might produce less pruritus would be equally effective. Pruritus was managed largely successfully in the FLINT trial by the use of topical emollients, as needed anti-histaminic agents and, occasionally, rifampin. In the FLINT trial, obeticholic acid treatment was also associated with a ~0.26 mmol/l increase in LDL‑cholesterol levels from baseline. These changes to LDL-cholesterol levels were maximal at 3 months and trended down thereafter. Interestingly, LDL-cholesterol decreased to below baseline levels without tachy­phylaxis during the study period after statins were initiated. Patients who had been on a stable dose of statins when enrolled in the trial still experienced an increase in LDLcholesterol levels. The biological basis for these findings has not been evaluated. In the context of modestly reduced triglycerides and decreased HDL-cholesterol, presumably due to increased reverse cholesterol transport, the clin­ ical implications of increased LDL-cholesterol levels are unclear and require further clarification. GFT505. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors with diverse effects on lipid metabolism and glucose homeostasis. Three isoforms have been identified in humans, each modulating pathways relevant to NAFLD: PPARα increases fatty acid oxidation; PPARγ has many effects, among them improving insulin resistance primarily via actions on adipose tissue and reducing lipogenesis in the liver; and PPARδ, which is less well understood, also modulates lipid metabolism and inflammatory signalling103,104. GFT505 is a fairly liver-specific dual PPAR‑α/δ

agonist that has been shown to improve hepatic insulin sensitivity and decrease steatosis, inflammation and fibrosis105–107. A phase IIb trial to determine its efficacy, the GOLDEN trial, has been completed; although the a priori primary end point (resolution of NASH without worsened fibrosis, compared with placebo) was not met, additional analyses accounting for baseline patient heterogeneity and severity of disease demonstrated a dose-dependent improvement in liver histology (data only presented in abstract form)108. Full presentation of this trial in a peer-reviewed format is needed before ­additional comments can be provided about this drug.

Other drugs in early development Many agents for NASH and NAFLD are in early clinical development; TABLE 2 outlines drugs in development for which phase II data are available. These trials can broadly be grouped into several categories: those that target the metabolic disturbances that drive inflammation, cellular injury and tissue remodelling that leads to cirrhosis; those that mainly target inflammation; and those that specific­ally target the fibrogenic process. Several compounds, particularly those that target fibrosis, have moved into large phase IIb trials with only preliminary preclinical data available. Importantly, several drugs with specific trial end points also have effects on other ­pathophysiological components of the disease. A small phase II trial of liraglutide (a GLP‑1 agonist) for the treatment of NASH was published in 2015109. Compared with placebo, liraglutide met the primary end point of NASH resolution without worsening fibrosis. Improvement in fibrosis was a secondary end point and, compared with placebo, those in the liraglutide arm did have substantially less fibrosis. However, several important caveats are worth noting: response to placebo was less than the ~20% seen in most NASH trials, and 36% of patients in the placebo group developed worsening

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REVIEWS Early-stage NAFLD Steatosis alone

Intermediate

Late-stage NAFLD

NASH stage 1–3 fibrosis*

NASH with cirrhosis

Lifestyle intervention such as diet and exercise changes Bariatric surgery such as gastric bypass or sleeve gastrectomy* Pharmacological therapy Current therapies Therapies in development • Vitamin E • Obeticholic • Pentoxifylline acid • Insulin • GFT505 sensitizers

Screening for HCC and oesophageal varices

Figure 2 | A stage-based approach toNature the treatment NAFLD. Lifestyle& Hepatology Reviews of | Gastroenterology modifications should be pursued throughout the disease course. Pharmacological therapy is not advised for patients with early-stage NAFLD as a result of their low risk of disease progression; however, in patients with intermediate to late stage NAFLD drugs can be a prescribed based on a risk-benefit analysis. Patients with cirrhosis should be screened for HCC and oesophageal varices. Bariatric surgery can be considered for any patient that meets selection criteria57, and can be safe and effective in carefully selected individuals with advanced fibrosis and cirrhosis, although patients with cirrhosis should only by treated in centres with extensive experience with patients with liver disease. *NASH stage 1–3 fibrosis confirmed by biopsy. HCC, hepatocellular carcinoma.

fibrosis (an unexpectedly high proportion given the short trial duration), which might have therefore overestimated the antifibrotic effect of liraglutide. It is also unclear to what degree the liraglutide-associated weight loss contributed to the histological improvement, compared with the contribution of the direct effects of liraglutide independent of weight loss. A full publication and further validation of the data in larger trials are awaited110.

Management of high-risk NAFLD In patients with advanced fibrosis (bridging fibrosis or cirrhosis), the risk of adverse clinical outcomes is more imminent. On the basis of existing evidence, the goals of therapy in patients with advanced liver disease include the reversal of the fibrogenic process that is driving the progression of liver fibrosis, correction of the underlying metabolic and inflammatory drivers of the disease and prevention or treatment of the complications of cirrhosis. Patients with cirrhosis should be screened for HCC and undergo screening for oesophageal varices as recommended by American Association for the Study of Liver Diseases practice guidelines24. 1. Matteoni, C. A. et al. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116, 1413–1419 (1999). 2. Wong, R. J., Cheung, R. & Ahmed, A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology 59, 2188–2195 (2014).

A study published in 2013 indicated that rifaximin in patients with alcoholic cirrhosis reduced the risk of all complications of cirrhosis and hepatic decompensation following an initial bout of variceal haemorrhage111. If validated in future trials, rifaximin might provide an aetiology-agnostic method to reduce the risk of ­decompensation in patients with cirrhosis. Until the past few years cirrhosis was not thought to be a reversible process. However, studies in patients infected with HBV have illustrated that fibrosis could be reversed with effective treatment of the underlying disease, even in the setting of cirrhosis112. Since these initial publications, substantial research has illustrated the dynamic nature of hepatic fibrogenesis and dissolution, leading to the development of multiple new antifibrotic compounds113. Currently, no approved therapy is available to specifically reverse advanced fibrosis and tissue remodelling in NASH. However, three major antifibrotic therapies are currently in phase IIb trials and the results of these are eagerly anticipated. The first trial involves a monoclonal antibody against lysyl oxidase, an enzyme critical for collagen crosslinking. Lysyl oxidase has also been implicated in the trafficking of stem cells to the liver and modulation of the microenvironment to promote hepatic carcino­ genesis114. The second therapy, cenciviroc, is a CCR2 and CCR5 antagonist. Although it primarily targets inflammation, inhibiting CCR2 and CCR5 also improves fibrosis and insulin sensitivity. Additionally, evidence suggests that macrophage infiltration in adipose tissue is CCR2 mediated, hence the rationale for the use of this agent in diabetes mellitus115,116. The third agent is an antibody against the profibrogenic galectin family of lectin proteins. Treatment with a galectin-binding complex carbohydrate markedly reduced fibrosis in an animal model of NASH117.

Conclusions The treatment of NAFLD and NASH is rapidly evolving, yet not all patients with NAFLD need pharmaco­ logical treatment. In order to recommend the most appropriate treatment approach, it is important for the clinician to be able to identify those patients at greatest risk of disease progression and to risk stratify them by the severity of their liver disease. The evidence base for treatment is growing and proof of short-term efficacy on the basis of surrogate histological end points for several agents is already available. Some concerns remain about the long-term safety of available agents, necessitating thoughtful consideration of the potential risks and benefits. Multiple new drugs are in development and the pathways for drug approval are being clarified. A variety of therapies will probably emerge over the next 5–7 years, permitting a stage-based approach (FIG. 2) and/or greater ­personalization of drug selection.

3. Pais, R. et al. A systematic review of follow‑up biopsies reveals disease progression in patients with nonalcoholic fatty liver. J. Hepatol. 59, 550–556 (2013). 4. Pais, R. et al. Progression from isolated steatosis to steatohepatitis and fibrosis in nonalcoholic fatty liver disease. Clin. Res. Hepatol. Gastroenterol. 35, 23–28 (2011). 5. McPherson, S. et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using

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paired biopsies: implications for prognosis and clinical management. J. Hepatol. 62, 1148–1155 (2015). 6. Ratziu, V. et al. Liver fibrosis in overweight patients. Gastroenterology 118, 1117–1123 (2000). 7. Angulo, P., Keach, J. C., Batts, K. P. & Lindor, K. D. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 30, 1356–1362 (1999).

www.nature.com/nrgastro © 2016 Macmillan Publishers Limited. All rights reserved

REVIEWS 8. Nishizawa, H. et al. Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy. Eur. J. Endocrinol. 167, 67–74 (2012). 9. Adams, L. A., Feldstein, A., Lindor, K. D. & Angulo, P. Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction. Hepatology 39, 909–914 (2004). 10. Portillo Sanchez, P. et al. High prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus and normal plasma aminotransferase levels. J. Clin. Endocrinol. Metab. http://dx.doi.org/10.1210/jc.2014-2739 (2014). 11. Mofrad, P. et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 37, 1286–1292 (2003). 12. Musso, G., Gambino, R., Cassader, M. & Pagano, G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non‑invasive tests for liver disease severity. Ann. Med. 43, 617–649 (2011). 13. Valenti, L. et al. Homozygosity for the patatin-like phospholipase‑3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease. Hepatology 51, 1209–1217 (2010). 14. Liu, Y. L. et al. TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with nonalcoholic fatty liver disease. Nat. Commun. 5, 4309 (2014). 15. Dongiovanni, P. et al. Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease. Hepatology 61, 506–514 (2015). 16. Angulo, P. et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 149, 389–397.e10 (2015). 17. Ekstedt, M. et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow‑up. Hepatology 61, 1547–1554 (2015). 18. Ripoll, C. et al. Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis. Gastroenterology 133, 481–488 (2007). 19. Ripoll, C. et al. Hepatic venous pressure gradient predicts development of hepatocellular carcinoma independently of severity of cirrhosis. J. Hepatol. 50, 923–928 (2009). 20. Angulo, P. et al. The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 45, 846–854 (2007). 21. Kim, D., Kim, W. R., Kim, H. J. & Therneau, T. M. Association between noninvasive fibrosis markers and mortality among adults with nonalcoholic fatty liver disease in the United States. Hepatology 57, 1357–1365 (2013). 22. Myers, R. P., Elkashab, M., Ma, M., Crotty, P. & Pomier-Layrargues, G. Transient elastography for the noninvasive assessment of liver fibrosis: a multicentre Canadian study. Can. J. Gastroenterol. 24, 661–670 (2010). 23. Loomba, R. et al. Magnetic resonance elastography predicts advanced fibrosis in patients with nonalcoholic fatty liver disease: a prospective study. Hepatology 60, 1920–1928 (2014). 24. Chalasani, N. et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 142, 1592–1609 (2012). 25. Hoofnagle, J. H. et al. Vitamin E and changes in serum alanine aminotransferase levels in patients with non‑alcoholic steatohepatitis. Aliment. Pharmacol. Ther. 38, 134–143 (2013). 26. Prinz, P. et al. Plasma bile acids show a positive correlation with body mass index and are negatively associated with cognitive restraint of eating in obese patients. Front. Neurosci. 9, 199 (2015). 27. Neuschwander-Tetri, B. A. et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non‑alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 385, 956–965 (2015). 28. Sanyal, A. Longitudinal changes in FIB‑4 and improvement in fibrosis stage with obeticholic acid: a secondary analysis of FLINT Trial. Presented at the American Academy for the Study of Liver Diseases 2015 Annual Meeting (2015).

29. Singh, S. et al. Fibrosis progression in nonalcoholic fatty liver versus nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin. Gastroenterol. Hepatol. 13, 643–654; e1–e9; quiz e39–e40 (2015). 30. Adams, L. A. et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129, 113–121 (2005). 31. Ekstedt, M. et al. Long-term follow‑up of patients with NAFLD and elevated liver enzymes. Hepatology 44, 865–873 (2006). 32. Wong, V. W. et al. Community-based lifestyle modification programme for non-alcoholic fatty liver disease: a randomized controlled trial. J. Hepatol. 59, 536–542 (2013). 33. Promrat, K. et al. Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology 51, 121–129 (2010). 34. Lassailly, G. et al. Bariatric surgery reduces features of non-alcoholic steatohepatitis in morbidly obese patients. Gastroenterology 149, 379–388; quiz e15–e16 (2015). 35. Browning, J. D. et al. Short-term weight loss and hepatic triglyceride reduction: evidence of a metabolic advantage with dietary carbohydrate restriction. Am. J. Clin. Nutr. 93, 1048–1052 (2011). 36. Browning, J. D. et al. Alterations in hepatic glucose and energy metabolism as a result of calorie and carbohydrate restriction. Hepatology 48, 1487–1496 (2008). 37. Oarada, M. et al. Refeeding with glucose rather than fructose elicits greater hepatic inflammatory gene expression in mice. Nutrition 31, 757–765 (2015). 38. Nakamuta, M. et al. Impact of cholesterol metabolism and the LXRα–SREBP‑1c pathway on nonalcoholic fatty liver disease. Int. J. Mol. Med. 23, 603–608 (2009). 39. Yasutake, K. et al. Nutritional investigation of nonobese patients with non-alcoholic fatty liver disease: the significance of dietary cholesterol. Scand. J. Gastroenterol. 44, 471–477 (2009). 40. Goff, D. C. Jr et al. 2013 ACC/AHA guideline on the assessment of cardiovascular risk: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J. Am. Coll. Cardiol. 63, 2935–2959 (2014). 41. Ryan, M. C. et al. The Mediterranean diet improves hepatic steatosis and insulin sensitivity in individuals with non-alcoholic fatty liver disease. J. Hepatol. 59, 138–143 (2013). 42. Vilar-Gomez, E. et al. Weight loss via lifestyle modification significantly reduces features of nonalcoholic steatohepatitis. Gastroenterology 149, 367–378 (2015). 43. Freedman, N. D., Park, Y., Abnet, C. C., Hollenbeck, A. R. & Sinha, R. Association of coffee drinking with total and cause-specific mortality. N. Engl. J. Med. 366, 1891–1904 (2012). 44. Klatsky, A. L. & Armstrong, M. A. Alcohol, smoking, coffee, and cirrhosis. Am. J. Epidemiol. 136, 1248–1257 (1992). 45. Johnson, S. et al. Coffee consumption and reduced risk of hepatocellular carcinoma: findings from the Singapore Chinese Health Study. Cancer Causes Control 22, 503–510 (2011). 46. Ruhl, C. E. & Everhart, J. E. Coffee and caffeine consumption reduce the risk of elevated serum alanine aminotransferase activity in the United States. Gastroenterology 128, 24–32 (2005). 47. Anty, R. et al. Regular coffee but not espresso drinking is protective against fibrosis in a cohort mainly composed of morbidly obese European women with NAFLD undergoing bariatric surgery. J. Hepatol. 57, 1090–1096 (2012). 48. Johnson, N. A. et al. Aerobic exercise training reduces hepatic and visceral lipids in obese individuals without weight loss. Hepatology 50, 1105–1112 (2009). 49. Bae, J. C. et al. Regular exercise is associated with a reduction in the risk of NAFLD and decreased liver enzymes in individuals with NAFLD independent of obesity in Korean adults. PLoS ONE 7, e46819 (2012). 50. Slentz, C. A. et al. Effects of aerobic versus resistance training on visceral and liver fat stores, liver enzymes, and insulin resistance by HOMA in overweight adults from STRRIDE AT/RT. Am. J. Physiol. Endocrinol. Metab. 301, E1033–E1039 (2011). 51. Oh, S. et al. Moderate to vigorous physical activity volume is an important factor for managing nonalcoholic fatty liver disease: a retrospective study. Hepatology 61, 1205–1215 (2014).

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2016 Macmillan Publishers Limited. All rights reserved

52. Stewart, K. E. et al. Readiness for behaviour change in non-alcoholic fatty liver disease: implications for multidisciplinary care models. Liver Int. 35, 936–943 (2014). 53. Cleator, J., Abbott, J., Judd, P., Sutton, C. & Wilding, J. P. Night eating syndrome: implications for severe obesity. Nutr. Diabetes 2, e44 (2012). 54. Briancon-Marjollet, A. et al. The impact of sleep disorders on glucose metabolism: endocrine and molecular mechanisms. Diabetol. Metab. Syndr. 7, 25 (2015). 55. Sjostrom, L. et al. Effects of bariatric surgery on mortality in Swedish obese subjects. N. Engl. J. Med. 357, 741–752 (2007). 56. Adams, T. D. et al. Long-term mortality after gastric bypass surgery. N. Engl. J. Med. 357, 753–761 (2007). 57. Gastrointestinal surgery for severe obesity: National Institutes of Health Consensus Development Conference Statement. Am. J. Clin. Nutr. 55, 615S–619S (1992). 58. Taitano, A. A. et al. Bariatric surgery improves histological features of nonalcoholic Fatty liver disease and liver fibrosis. J. Gastrointest. Surg. 19, 429–437 (2015). 59. Mathurin, P. et al. Prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease. Gastroenterology 137, 532–540 (2009). 60. Pillai, A. A. & Rinella, M. E. Non-alcoholic fatty liver disease: is bariatric surgery the answer? Clin. Liver Dis. 13, 689–710 (2009). 61. Osto, E. et al. Rapid and body weight-independent improvement of endothelial and high-density lipoprotein function after Roux‑en‑Y gastric bypass: role of glucagon-like peptide‑1. Circulation 131, 871–881 (2015). 62. Nergard, B. J. et al. Mucosal glucagon-like peptide‑1 and gastric inhibitory polypeptide cell numbers in the super-obese human foregut after gastric bypass. Surg. Obes. Relat. Dis. 11, 1237–1246 (2015). 63. Birkmeyer, N. J. et al. Hospital complication rates with bariatric surgery in Michigan. JAMA 304, 435–442 (2010). 64. Sanyal, A. J., Sugerman, H. J., Kellum, J. M., Engle, K. M. & Wolfe, L. Stomal complications of gastric bypass: incidence and outcome of therapy. Am. J. Gastroenterol. 87, 1165–1169 (1992). 65. Jan, A., Narwaria, M. & Mahawar, K. K. A. Systematic review of bariatric surgery in patients with liver cirrhosis. Obes. Surg. 25, 1518–1526 (2015). 66. Sanyal, A. J. et al. A pilot study of vitamin E versus vitamin E and pioglitazone for the treatment of nonalcoholic steatohepatitis. Clin. Gastroenterol. Hepatol. 2, 1107–1115 (2004). 67. Sanyal, A. J. et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N. Engl. J. Med. 362, 1675–1685 (2010). 68. Lavine, J. E. et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. JAMA 305, 1659–1668 (2011). 69. Kowdley, K. V. et al. CRN trials. Hepatology 62, 1 (2015). 70. Miller, E. R. 3rd et al. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann. Intern. Med. 142, 37–46 (2005). 71. Bjelakovic, G., Nikolova, D., Gluud, L. L., Simonetti, R. G. & Gluud, C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA 297, 842–857 (2007). 72. Gee, P. T. Unleashing the untold and misunderstood observations on vitamin E. Genes Nutr. 6, 5–16 (2011). 73. Curtis, A. J., Bullen, M., Piccenna, L. & McNeil, J. J. Vitamin E supplementation and mortality in healthy people: a meta-analysis of randomised controlled trials. Cardiovasc. Drugs Ther. 28, 563–573 (2014). 74. Jiang, S. et al. Meta-analysis: low-dose intake of vitamin E combined with other vitamins or minerals may decrease all-cause mortality. J. Nutr. Sci. Vitaminol. (Tokyo) 60, 194–205 (2014). 75. Ye, Y., Li, J. & Yuan, Z. Effect of antioxidant vitamin supplementation on cardiovascular outcomes: a meta‑analysis of randomized controlled trials. PLoS ONE 8, e56803 (2013). 76. Lippman, S. M. et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 301, 39–51 (2009).

ADVANCE ONLINE PUBLICATION | 9

REVIEWS 77. Gaziano, J. M. et al. Vitamins E and C in the prevention of prostate and total cancer in men: the Physicians’ Health Study II randomized controlled trial. JAMA 301, 52–62 (2009). 78. Schurks, M., Glynn, R. J., Rist, P. M., Tzourio, C. & Kurth, T. Effects of vitamin E on stroke subtypes: meta-analysis of randomised controlled trials. BMJ 341, c5702 (2010). 79. Choudhury, J. & Sanyal, A. J. Insulin resistance and the pathogenesis of nonalcoholic fatty liver disease. Clin. Liver Dis. 8, 575–594 (2004). 80. Mahady, S. E., Webster, A. C., Walker, S., Sanyal, A. & George, J. The role of thiazolidinediones in nonalcoholic steatohepatitis — a systematic review and meta analysis. J. Hepatol. 55, 1383–1390 (2011). 81. Neuschwander-Tetri, B. A., Brunt, E. M., Wehmeier, K. R., Oliver, D. & Bacon, B. R. Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-γ ligand rosiglitazone. Hepatology 38, 1008–1017 (2003). 82. Van Wagner, L. B. et al. Pentoxifylline for the treatment of non-alcoholic steatohepatitis: a randomized controlled trial. Ann. Hepatol. 10, 277–286 (2011). 83. Zein, C. O. et al. Pentoxifylline improves nonalcoholic steatohepatitis: a randomized placebo-controlled trial. Hepatology 54, 1610–1619 (2011). 84. Patterson, E., Wall, R., Fitzgerald, G. F., Ross, R. P. & Stanton, C. Health implications of high dietary omega‑6 polyunsaturated fatty acids. J. Nutr. Metab. 2012, 539426 (2012). 85. Pettinelli, P. et al. Enhancement in liver SREBP‑1c/ PPAR-α ratio and steatosis in obese patients: correlations with insulin resistance and n-3 long-chain polyunsaturated fatty acid depletion. Biochim. Biophys. Acta 1792, 1080–1086 (2009). 86. Puri, P. et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology 46, 1081–1090 (2007). 87. Sanyal, A. J. et al. No significant effects of ethyleicosapentanoic acid on histologic features of nonalcoholic steatohepatitis in a phase 2 trial. Gastroenterology 147, 377–384.e1 (2014). 88. Argo, C. K. et al. Effects of n-3 fish oil on metabolic and histological parameters in NASH: a double-blind, randomized, placebo-controlled trial. J. Hepatol. 62, 190–197 (2015). 89. Dasarathy, S. et al. Double-blind randomized placebocontrolled clinical trial of omega 3 fatty acids for the treatment of diabetic patients with nonalcoholic steatohepatitis. J. Clin. Gastroenterol. 49, 137–144 (2015). 90. Calder, P. C. Marine omega‑3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance. Biochim. Biophys. Acta 1851, 469–484 (2015). 91. Mullen, A., Loscher, C. E. & Roche, H. M. Antiinflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP‑1‑derived macrophages. J. Nutr. Biochem. 21, 444–450 (2010). 92. Ferrebee, C. B. & Dawson, P. A. Metabolic effects of intestinal absorption and enterohepatic cycling of bile acids. Acta Pharm. Sin. B 5, 129–134 (2015). 93. Keitel, V. et al. The G‑protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology 45, 695–704 (2007).

94. Fang, S. et al. Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance. Nat. Med. 21, 159–165 (2015). 95. Mudaliar, S. et al. Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology 145, 574–582.e1 (2013). 96. Matsukuma, K. E. et al. Coordinated control of bile acids and lipogenesis through FXR-dependent regulation of fatty acid synthase. J. Lipid Res. 47, 2754–2761 (2006). 97. Evans, M. J. et al. A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G543–G552 (2009). 98. Sirvent, A. et al. Farnesoid X receptor represses hepatic lipase gene expression. J. Lipid Res. 45, 2110–2115 (2004). 99. Zhang, Y. et al. Identification of novel pathways that control farnesoid X receptor-mediated hypocholesterolemia. J. Biol. Chem. 285, 3035–3043 (2010). 100. Rohatgi, A. et al. HDL cholesterol efflux capacity and incident cardiovascular events. N. Engl. J. Med. 371, 2383–2393 (2014). 101. Urizar, N. L. et al. A natural product that lowers cholesterol as an antagonist ligand for FXR. Science 296, 1703–1706 (2002). 102. Miyazaki-Anzai, S., Masuda, M., Levi, M., Keenan, A. L. & Miyazaki, M. Dual activation of the bile acid nuclear receptor FXR and G‑protein-coupled receptor TGR5 protects mice against atherosclerosis. PLoS ONE 9, e108270 (2014).
 103. Giby, V. G. & Ajith, T. A. Role of adipokines and peroxisome proliferator-activated receptors in nonalcoholic fatty liver disease. World J. Hepatol. 6, 570–579 (2014). 104. Bojic, L. A. & Huff, M. W. Peroxisome proliferatoractivated receptor δ: a multifaceted metabolic player. Curr. Opin. Lipidol. 24, 171–177 (2013). 105. Staels, B. et al. Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Hepatology 58, 1941–1952 (2013). 106. Cariou, B., Zair, Y., Staels, B. & Bruckert, E. Effects of the new dual PPAR α/δ agonist GFT505 on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia or impaired glucose metabolism. Diabetes Care 34, 2008–2014 (2011). 107. Cariou, B. et al. Dual peroxisome proliferator-activated receptor α/δ agonist GFT505 improves hepatic and peripheral insulin sensitivity in abdominally obese subjects. Diabetes Care 36, 2923–2930 (2013). 108. Ratziu, V. et al. An international, phase 2 randomized controlled trial of the dual PPAR alpha-delta aginist GFT505 in adult patients with NASH. Hepatology 62, 1 (2015). 109. Armstrong, M. J. et al. Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo‑controlled phase 2 study. Lancet (2015). 110. Armstrong, A. Liraglutide is effective in the histological clearance of non-alcoholic steatohepatitis in a multicentre, double-blinded, randomised, placebo‑controlled phase II trial. J. Hepatol. 62 (Suppl. 2) S187 (2015). 111. Vlachogiannakos, J. et al. Long-term administration of rifaximin improves the prognosis of patients with

10 | ADVANCE ONLINE PUBLICATION

decompensated alcoholic cirrhosis. J. Gastroenterol. Hepatol. 28, 450–455 (2013). 112. Marcellin, P. & Asselah, T. Long-term therapy for chronic hepatitis B: hepatitis B virus DNA suppression leading to cirrhosis reversal. J. Gastroenterol. Hepatol. 28, 912–923 (2013). 113. Rockey, D. C., Bell, P. D. & Hill, J. A. Fibrosis — a common pathway to organ injury and failure. N. Engl. J. Med. 372, 1138–1149 (2015). 114. Wong, C. C. et al. Lysyl oxidase-like 2 is critical to tumor microenvironment and metastatic niche formation in hepatocellular carcinoma. Hepatology 60, 1645–1658 (2014). 115. Di Prospero, N. A. et al. CCR2 antagonism in patients with type 2 diabetes mellitus: a randomized, placebocontrolled study. Diabetes Obes. Metab. 16, 1055–1064 (2014). 116. Pellicoro, A., Ramachandran, P., Iredale, J. P. & Fallowfield, J. A. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat. Rev. Immunol. 14, 181–194 (2014). 117. Traber, P. G. et al. Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease. PLoS ONE 8, e75361 (2013). 118. Angulo, P. et al. Simple noninvasive systems predict long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology. 145, 782–789 (2013). 119. Bambha, K. et al. Coffee consumption in NAFLD patients with lower insulin resistance is associated with lower risk of severe fibrosis. Liver Int. 34, 1250–1258 (2014). 120. Molloy, J. W. et al. Association of coffee and caffeine consumption with fatty liver disease, nonalcoholic steatohepatitis, and degree of hepatic fibrosis. Hepatology 55, 429–436 (2012). 121. Safadi, R. et al. The fatty acid-bile acid conjugate Aramchol reduces liver fat content in patients with nonalcoholic fatty liver disease. Clin. Gastroenterol. Hepatol. 12, 2085–2091.e2 (2014). 122. Bi, Y. et al. Effects of exenatide, insulin, and pioglitazone on liver fat content and body fat distributions in drug-naive subjects with type 2 diabetes. Acta Diabetol. 51, 865–873 (2014). 123. Safar Zadeh, E. et al. The liver diseases of lipodystrophy: the long-term effect of leptin treatment. J. Hepatol. 59, 131–137 (2013).

Author contributions

All authors contributed equally to discussion of content, writing and reviewing/editing the manuscript before submission. M.E.R. researched data for the article.

Competing interests statement

M.E.R. has served as a consultant to AbbVie, FibroGen, Genentech, Intercept, NGM Biopharmaceuticals, NuSirt, Shire, Takeda and W.L Gore & Associates. A.J.S. has stock o p t i o n s i n G e n f i t . H e i s t h e P re s i d e n t o f S a nya l Biotechnologies and has served as a consultant to AbbVie, Astra Zeneca, Boehringer Ingelhiem, Exalenz Bioscience, FibroGen, Genfit, Hemoshear, Immune Pharma, Immuron, L i l l y, N i m b u s T h e r a p e u t i c s, N i t t o D e n k o , S a l i x Pharmaceuticals, Takeda and Tobira Therapeutics. He has been an unpaid consultant to Echosens and Intercept Pharmaceuticals. His institution has received grant support from Astra Zeneca, Bristol Myers, Gilead, Merck, Novartis, Salix Pharmaceuticals and Tobira Therapeutics.

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