Therapeutic approaches to non-alcoholic fatty liver disease: past achievements and future challenges

Hong Kong, China

Therapeutic approaches to non-alcoholic fatty liver disease: past achievements and future challenges

Jia Xiao, Rui Guo, Man Lung Fung, Emily C Liong and George L Tipoe

Hong Kong, China

BACKGROUND:Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver injury and mortality in Western countries and China. However, as to date, there is no direct and effective therapy for this disease. The aim of this review is to analyze the key progress and challenges of main current therapeutic approaches in NAFLD.

DATA SOURCE:We carried out a PubMed search of Englishlanguage articles relevant to NAFLD therapy.

RESULTS:There are two major therapeutic strategies for NAFLD treatment: (1) lifestyle interventions (including weight reduction, dietary modif i cation and physical exercise) and (2) pharmaceutical therapies. Lifestyle interventions, particularly chronic and moderate intensity exercise, are the most effective and recognized clinical therapies for NAFLD. For pharmaceutical therapies, although their effects and mechanisms have been extensively investigated in laboratory studies, they still need further tests and investigations in clinical human trials.

CONCLUSION:Future advancement of NAFLD therapy should focus on the mechanistic studies on cell based and animal models and human clinical trials of exercise, as well as the combination of lifestyle intervention and pharmaceutical therapy specif i cally targeting main signaling pathways related to lipid metabolism, oxidative stress and inf l ammation.

(Hepatobiliary Pancreat Dis Int 2013;12:125-135)

non-alcoholic fatty liver disease;therapy; pharmaceuticals; exercise

Introduction

In the past decades, non-alcoholic fatty liver disease (NAFLD) has become one of the leading chronic liver diseases worldwide. The estimated prevalence of NAFLD in adult population is around 15%-30%, which increases with age.[1-3]In the aff l uent regions of China, the community prevalence of NAFLD is approximately 15%.[4]Since NAFLD is a risk factor of cirrhosis and even hepatocellular carcinoma, developing effective therapies with minimal side-effects against NAFLD is vital for controlling the progression of this disease to advance to end-stage liver disease.[5]In the past, the term "NAFLD" includes a wide spectrum of chronic liver diseases, which ranges from simple steatosis (fat accumulation in the liver without inf l ammation and/or fi brosis) to nonalcoholic steatohepatitis (NASH; fat deposition with inf l ammation and/or fi brosis) and cirrhosis.[6]This view has been recently challenged by a multiple parallel hit hypothesis of NAFLD, in which steatosis and NASH are discrete entities rather than two points of the NAFLD spectrum, not only from a set of histological criteria but also from a pathophysiological standpoint.[7,8]To date, the detailed pathogenic mechanism of NAFLD including steatosis and NASH is not fully characterized, although it is regarded as a hepatic manifestation of the metabolic syndrome[9]or sometimes it occurs in the absence of metabolic syndrome.[10]Thus, uncontrolled NAFLD-induced injury may further inf l uence other body systems, such as the cardiovascular system, kidneys and pancreas, to cause hyperlipidemia, hypertension, type 2 diabetes, pancreatitis, and atherosclerosis.[11-15]Based on conventional models of "two-hit" and "multi-hit" hypothesis of NAFLD, in which the dysregulated lipid metabolism and insulin resistance are considered as the "f i rst hit" of the liver and the following "second hit" or "multi-hit" likely involves oxidative stress, lipid peroxidation, increased inf l ammatory responses, induced hepatic fi brosis and apoptosis,[16,17]recent "multiple parallel hits" model emphasizes the multifactorial pathogenesis of NAFLD and the necessity to treat NAFLD with diabetes-like and multimodal strategy.[7]Excessive free fatty acids (FFAs), either produced from excessivede novohepatic lipogenesis or from overloaded consumption of carbohydrate, play a central role in inducing lipotoxicity and following insulin resistance.[18]Impaired insulin signaling system is responsible for the occurrence of lipid peroxidation and oxidative stress which cause downstream necroinf l ammation and apoptosis through transcription factors- and kinase-dependent pathways.[19]Therefore, therapies targeted for aforementioned key events during NAFLD development are tested to retard or ameliorate the chronic liver injury at different levels. Currently, there are two major categories of NAFLD therapy namely: lifestyle interventions and pharmaceutical therapies.

Lifestyle interventions

Weight reduction

Nowadays it is clear that obesity is associated with the increased risk of NAFLD. It has become a major health burden in the present century. In the USA, more than one-fourth people are obese and three-fourth of them have fatty liver disease.[20]For patients with body mass index (BMI) >35 kg/m2, bariatric surgery is the only effective therapy for morbid obesity.[21]The phenotype of obesity is connected with increased intra-abdominal fat (e.g. visceral adipose). Since visceral adipose holds more lipolytic capability than subcutaneous adipose on a per-unit-mass basis, FFAs released from visceral fat may get into the portal vein and caused a "f i rst-pass" effect on the liver, indicating that the liver is exposed more FFAs than the peripheral tissues, especially in patients with visceral obesity.[22]Also, the increased concentration of FFAs is taken into account as a major aspect in the development of insulin resistance. Thus, the development of NAFLD is expected to be initiated by the increased obesity index and generation of insulin resistance, which is why the maintenance or reduction of weight is a key factor in the prevention of NAFLD progression. It is also an essential therapy for obese patients who have metabolic disease with features of NAFLD. Although weight loss decreases intra-hepatic fat content and improves metabolic function in young and middle-aged obese adults, the effects of weight reduction are controversial in the elderly.[23]Generally speaking, increase in life expectancy from intentional weight loss could be achieved mainly in elderly patients with diabetes.[24]However, since reduction of BMI with age frequently involves loss of muscle rather than fat tissue, waist circumference is a better indicator for elderly patients with obesity than BMI.[25]In addition, it is noted that sometimes weight loss in elderly patients is unintentional, which is probably from pathological reasons such as cancers and cardiovascular diseases.[26]Thus, the therapeutic benef i t of weight loss in NAFLD needs further investigations, particularly in different ethnic groups and ages.

Dietary modif i cation

Current data clearly suggest that the level of energy intake is signif i cantly higher in NAFLD patients than in individuals with no evidence of fatty liver.[27]Highfructose diet in humans is related to the occurrence of obesity, metabolic syndrome and NAFLD with increased energy intake level, fat mass and blood pressure.[28]Thus, it is logical that reducing the dietary carbohydrate or energy content is benef i cial for the retardation of NAFLD progression. Low (800-1800 kCal/ day) and very low-calorie diets (<800 kCal/day) and/or carbohydrate restriction (20-50 g/day) signif i cantly reduce body weight and intrahepatic lipid content.[29]Another small retrospective cohort study of obese patients demonstrated that a 600-800 kCal/day diet for a mean of 16 months resulted in improvement of liver enzymes and hepatosplenomegaly.[30]Petersen et al[31]found that a low fat (3%) reduced calorie (daily intake 1200 kCal/day) diet effectively reduces body weight and intra-hepatic lipid content with improvement of insulin resistance in NAFLD patients. Thoma et al[32]also illustrated that a range of lifestyle modif i cations exert very positive inf l uence on reducing intrahepatic lipid contents and circulating enzyme levels, which lead to improved measures of glucose control and/or insulin sensitivity in patients with NAFLD. In the USA, more than one-fourth men and one-third women are trying to lose weight by lifestyle changes.[33]Considering the close link between obesity and NAFLD, all patients who exhibit NAFLD symptom should be encouraged to continue moderate intensity of aerobic exercise and to take low-fat diet.[34]

Physical exercise

The convincing evidence of physical exercise on NAFLD treatment has been demonstrated by the reduction of liver lipid contents. A study investigatingthe effects of exercise on humans found that 9-day exercise training program increased lipid metabolism gene expression in skeletal muscle.[35]A new study also proved that regular exercise (more than 3 times per week, at least for 30 minutes each time and for consecutive 3 months) was related to reduced risk of NAFLD and decreased liver enzymes in NAFLD patients independent of obesity in Korea.[36]Another study prescribing 3-month moderate intensity exercise for NAFLD patients observed a 50% reduction in aminotransferase levels.[37]However, evident benef i cial effects can only be seen in NAFLD individuals who perform ≥150 minutes of aerobic type exercise per week.[38]Furthermore, the mechanism for the direct hepatic benef i t of chronic exercise training remains unclear.[39]Since exercise has been shown to control the progression of fatty liver by reducing intrahepatic fat, fatty acid uptake and improving insulin sensitivity, it is proposed that exercise releases the burden of oxidative stress and inf l ammation caused by fat accumulation in the liver.[40]A very recent study found that exercise training drastically attenuates hepatic inf l ammation, fi brosis and macrophage inf i ltration during diet induced-obesity in obese mice, indicating the positive effects of exercise on both steatosis and NASH.[41]Interestingly, a recent well-designed randomized controlled trial tested the effects of lifestyle intervention using a combination of diet, exercise, and behavior modif i cation. They found that after 48 weeks of intervention, the testing group lost an average of 9.3% of their weight versus 0.2% in the intervention-free group. They also signif i cantly reduced their NAFLD activity score, indicating that combined intervention for a long-term therapy is effective for improving NAFLD histology.[42]

In addition, the fi nding that exercise requires hepatic glucagon receptor activation to lower hepatic fat content is a step toward understanding the benef i ts of regular exercise. Several experiments provided mechanistic insight into the understanding on how repeated bouts of exercise-stimulated hepatic glucagon action lower the hepatic fat content.[43-45]Post exercise fi ndings in gcgr+/+ (glucagon receptor null) mice showed lowered hepatic energy state, increased AMPK activity, and elevated expression of AMPK-α1/-α2, PPAR-α, and FGF21 when compared with control and wild-type mice.[44]AMPK, PPAR-α, and FGF21 are targets of hepatic glucagon action and key proteins involved in oxidative metabolism. Stimulation of these interrelated pathways suggested that repeated bouts of exercise-stimulated hepatic glucagon action heighten fat oxidation. Thus, consuming high-fat diet in mice provokes rapid and progressive fatty liver that is reversible by exercise in a glucagon receptor-dependent manner.

Pharmaceutical interventions

Antioxidants

Induced by lipotoxicity from excessive FFAs, the level of reactive oxygen species and reactive nitrogen species may exceed the intracellular neutralizing ability of antioxidant enzymes, leading to the formation of oxidative stress. It is now clear that oxidative stress not only directly causes liver injury, but also induces other pathological events contributing to NAFLD progression, including inf l ammation, chemoattraction, necrosis, and apoptosis.[46]Thus, several kinds of antioxidant therapy have been introduced to control the level of oxidative stress in NAFLD. A pilot study using combined therapy of lifestyle modif i cation and vitamin E found that increased levels of liver enzymes, cholesterol, and plasma hyaluronic acid were signif i cantly alleviated by the therapy. However, dysregulated level of cytokines was not re-balanced.[47]Another prospective, doubleblind, placebo-controlled trial in NAFLD patients found vitamin C and/or vitamin E treatment improved hepatic fi brosis, but not necro-inf l ammation and ALT levels.[48]Conversely, a pilot study conducted by Lavine pointed out longer administration of vitamin E (4-10 months) markedly reduced serum ALT and AST levels without affecting BMI.[49]Some reports indicated the safety of long-term use of vitamin E. A meta-analysis study showed that high-dosage vitamin E supplementation (≥400 IU/d) may increase all-causes of mortality.[50]

In addition to vitamins, herbal derivatives with antioxidant property are another promising therapy because 1) some common herbs (e.g. red grape, garlic, and green tea) are popular daily foods or food supplements that are easy to obtain; 2) adverse effect of herbal derivatives when administered in low dosage is usually very low or nil; and 3) several herbal derivatives are proven with high-eff i cacy in improving NAFLD parameters.[51]Although detailed protective mechanisms of several herbal derivatives have been elucidated in animal models, the clinical application is still on the way. Among many derivatives, resveratrol from red grape received most attentions since it shows protective effects against many diseases, including NAFLD, cardiovascular diseases, diabetes and cancer.[52]In animal studies, treatment of resveratrol attenuates NAFLD symptoms through inhibition of lipid peroxidation and inf l ammatory responses, as well as activation of LKB1/AMPK and Sirt1 pathways.[53-56]Green tea extract is also tested in different NAFLD models, includingin vitromodels, cultured cells,tissue models, andin vivomodels. Administration of green tea extract is shown to effectively ameliorate histological changes, fi brosis, oxidative stress, lipid accumulation, and inf l ammation occurred during NAFLD development.[57-60]In addition, epidemiological data have shown that consumption of green tea lowers lipid dysregulation, serum ALT level, and the risk of cardiovascular disorders.[61,62]We recently identif i ed the benef i cial effects of S-allylmercaptocysteine, a water-soluble monomer from garlic, on NAFLD rat model. Addition of S-allylmercaptocysteine during a high-fat diet induction of NAFLD in rat reduces liver injury, including necro-inf l ammation, oxidative stress, and apoptosis. It also attenuates fi brosis and reduces dysregulated of lipid metabolism by modulating the kinase- and transcription factor-dependent pathways.[63]

Insulin sensitizers

Since insulin resistance is one of the major triggering mechanisms for NAFLD progression, sensitization of insulin in patients with fatty liver disease receives increasing attention because it restores the ability of insulin to lower hyperinsulinemia and hyperlipidemia in peripheral adipocytes.[64]Metformin and thiazolidinediones (TZDs, including pioglitazone and rosiglitazone) are major classes of insulin sensitizers applied in clinical studies.[65]In early reports, rosiglitazone was demonstrated to normalize transaminases levels, inf l ammatory responses and hepatic steatosis.[66-68]However, due to its obvious side effects (e.g. increase risk of heart attack), the use of rosiglitazone has been highly restricted in the USA and banned in Europe.[69,70]In a large controlled trial, when pioglitazone was taken with high fat diet in NAFLD patients, insulin sensitivity, serum ALT, steatosis, necro-inf l ammation but not fi brosis were signif i cantly ameliorated when compared with high fat diet plus placebo control.[71]Another placebo-controlled trial called PIVENS (pioglitazone versus vitamin E versus placebo) also found the benef i cial properties of pioglitazone in improving hepatic histology, inf l ammation, and steatosis in non-diabetic NAFLD patients after 96-week administration.[72]Similar to rosiglitazone, the long-term safety and adverse effects of pioglitazone have not been well established. In addition, weight gain and edema are typical obvious side effects of pioglitazone, which in turn aggravates heart failure. So the clinical usage of this drug is still under strict supervision in many countries.[73]Another important insulin sensitizer used in NAFLD therapy is metformin, which showed anti-diabetic effects through increasing fatty acid/glucose metabolism and improving insulin signaling in the liver and adipose tissue.[74,75]At the molecular level, metformin activates the LKB1/AMPK pathway to accelerate glucose uptake through dissociation of the gluconeogenic CREB-CBPTORC2 transcriptional complex[76]and to increase lipolysis by decreasing the expressions of key lipid regulating members (e.g. acetyl-CoA carboxylase, HMG-CoA reductase and fatty acid synthase).[77]Over the past decades, a large number of clinical trials had been conducted to measure the benef i cial effects of metformin in NAFLD, in which the liver function, steatosis and insulin sensitivity improved.[78-84]Furthermore, metformin therapy exhibited protective effects on metabolic abnormalities and cardiovascular risk, making it a good choice for clinical treatment of NAFLD.[85,86]However, a recent meta-analysis study found that metformin, when treated with different doses and durations, did not show improvement on liver histology.[87]In addition, newly published guidelines argue that metformin has no signif i cant effect on liver histology and is not recommended as a specif i c treatment for liver disease in adults with NASH.[88]Another promising therapeutic target is the incretin pathway. During NAFLD progression, incretin hormones (e.g. glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1) were dysregulated through the action of dipeptidyl peptidase-4. Therefore, inhibition of dipeptidyl peptidase-4 has been shown to improve insulin resistance, steatosis and inf l ammation. It also contributes to the improvement of type 2 diabetes clinically.[89,90]

Lipid lowering drugs

Statins are inhibitors of HMG-CoA reductase in the liver. They are used to reduce the level of cholesterol which is closely associated with the development of atherosclerosis and the incidence of cardiovascular disease.[91]Regarding the anti-in fl ammatory and antifi brogenic properties of statins, they are also employed in the clinical test for NAFLD prevention and treatment in recent years.[92]The Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) 3-year prospective investigation found that statins successfully normalized liver enzymes and improved liver function in NAFLD patients. There was a 68% relative risk reduction of cardiovascular events in patients with suspected NAFLD who received statins compared with patients with suspected NAFLD who did not receive statin. Interestingly, among all patients who received statins, patients with suspected NAFLD derived signi fi cantly greater bene fi t from statins compared with those who had no biochemical evidence of NAFLD.[11,93]Clinicalprescription of statins on NAFLD patients signif i cantly attenuated hepatic steatosis and fi brosis in a followup study.[94]Combination of atorvastatin and vitamin C/E also improved the clinical outcome of NAFLD.[95]However, in another two pilot studies using members of statin class: atorvastatin and simvastatin, exhibited no or controversial statistically important improvement of steatosis, serum aminotransferases, and fi brosis in NAFLD patients.[96,97]Currently it is not clear if different members of statins possess different effects on NAFLD progression, due to their distinct lipophilicity.[98,99]Although administration of statins often increases liver enzymes (ALT and AST) and induces acute liver injury in a very small number of patients, the statins therapy is now proven safe.[100,101]Theoretical data have been collected to support the usefulness of statins in NAFLD treatment even without dyslipidemia, but further prospective studies are needed to verify this fi nding.[92]An animal study evaluating the effects of rosuvastatin treatment on high-fat diet induced mouse NAFLD found that insulin resistance, hepatic steatosis, and increased circulating levels of cholesterol were improved by rosuvastatin.[102]

RAS receptor blockers

The role of renin-angiotensin system (RAS) in NAFLD development has been extensively investigated, making blockers for angiotensin receptors to be a potential therapeutic strategy of NAFLD.[103]Telmisartan and losartan, the most common angiotensin II receptor type 1 (AT1) blockers, have been tested if they can ameliorate NAFLD symptoms clinically. In a randomized controlled trial, administration of telmisartan in hypertensive NAFLD patients improved hepatic steatosis, fi brosis, inf l ammation, and dysregulated lipid metabolism.[104]A study[105]in NASH mice further conf i rmed the fi nding and suggested additional roles of hepatic macrophage inf i ltration. In another clinical trial using combined losartan and simvastatin in hypertensive NAFLD patients, hepatic steatosis, visceral adiposity, and HOMA-IR were signif i cantly improved when compared with amlodipine plus simvastatin group.[106]In a more recent animal study, combination treatment of losartan and new oral iron chelator (deferasirox) exhibited strong suppressive effects on oxidative stress, neovascularization, and hepatic stellate cells activation in NASH rats.[107]

Other drugs

Ursodeoxycholic acid (UDCA) is a bile acid with anti-apoptotic and cytoprotective properties. Thus, it is used as a hepatoprotectant in NAFLD treatment. A 2-year placebo-controlled trial in NAFLD patients found that there was no signif i cant difference between UDCA-treated NAFLD and NAFLD patients, in terms of NAFLD symptoms.[108]Other studies[109,110]found that no signif i cant difference was observed regarding mortality or improvement in liver function tests after treatment with normal or high dose of UDCA. Similar to metformin, UDCA is not recommended for the treatment of NAFLD or NASH recently.[88]Pentoxifylline is another hepatoprotectant with anti-tumor necrosis factor effect and has been used in NAFLD treatment. A 12-month pilot study with pentoxifylline treatment obtained biochemical improvements. However, the side effect (e.g. nausea) is quite noticeable.[111]A more recent randomized placebo-controlled trial conf i rmed the benef i cial properties of pentoxifylline in reducing steatosis and fi brosis after one year administration in NAFLD patients.[112]

L-carnitine is the precursor of carnitine-palmitoyltransferase. As a popular food supplement, L-carnitine is used to control body weight because it is involved in the fatty acid transportation in the mitochondria during the breakdown of lipids for the energy production. When introduced to NAFLD patients, L-carnitine improved hepatic steatosis, lipid metabolism, fi brosis, and inf l ammation.[113]However, another study[114]demonstrated that when the same dose and treatment duration of L-carnitine was applied, it failed to exhibit a signif i cant effect on liver histology.

Since apoptosis is positively correlated with the severity of NAFLD, reduction of apoptosis provides another possible way to retard this disease. In a randomized, placebo-controlled trial, patients with diagnosed NAFLD received treatment of caspase inhibitor GS-9450 for 4 weeks. When compared with placebo-control, GS-9450 treatment improved ALT levels, but not other metabolic variables.[115]

Future challenges

Life style modif i cations (including weight reduction, dietary modif i cation, and physical exercise) and pharmaceutical treatments (including dietary supplementation) are still two major directions for the therapeutic development of NAFLD in future. The advantages and disadvantages of main current NAFLD therapies are summarized in Table. Generally, for the intervention of lifestyle, numerous clinical reports have extensively investigated the benef i cial effects of weight loss, dietary constraints and exercise on NAFLD symptoms but few studies have shown the molecular mechanisms that mediated these effects. For herbal supplementation, several recentstudies revealed the protective molecular pathways bothin vivoandin vitro, but the possible application of these derivatives in humans still faces some problems, including the control of adverse effects.[116]In addition, increasing attention is being given to the control of metabolic syndrome risk factors (e.g. obesity, insulin resistance, hyperlipidemia, hypertension, and inf l ammation), which are proven to induce NAFLD directly or indirectly. Metabolic syndrome itself is also a risk factor of NAFLD andvice versa.[117]Thus, therapies that effectively ameliorate metabolic syndrome should also possess positive effects on NAFLD treatment. Similar to NAFLD, lifestyle modif i cation is the fi rst and most effective treatment for metabolic syndrome. However, in the majority of cases, pharmaceutical approach is also needed. How to improve symptoms of NAFLD as well as metabolic disorders is another emerging clinical challenge facing us. Regarding the importance of the progression from simple steatosis to NASH, applying suitable pharmacological methods to stop the occurrence of inf l ammation (e.g. antiinf l ammation drugs) is vital for the control of end-stage NAFLD. In recent years, emerging evidence shows a close relationship between gut microbiota and the liver. Homeostasis between bacteria- and host-derived signals in the gut is essential for the maintenance of intestinal barrier function and liver health. Dysregulated gut fl ora (e.g. small intestinal bacterial overgrowth) is found in a large number of patients with chronic liver diseases and inf l ammation.[114]Therefore, therapies targeting disrupted gut-liver axis may also contribute to the retardation of NAFLD (especially NASH) clinically. Another potential therapeutic target is fructose. Recent fi ndings point out that high level of fructose in human diet may induce NAFLD through several mechanisms, including the induction of the metabolic syndrome, copper def i ciency, bacterial translocation from the gut to the liver, the formation of advanced glycation end-products and a direct dysmetabolic effect on liver enzymes. Thus, reduction of the intake of fructose-rich foods and beverages may help the control of NAFLD progression.[118]

Table.Pros and cons of main current therapeutic approaches to NAFLD

Although NAFLD is a leading cause of chronic liver disease all over the world, we cannot ignore the individual variability and the need for personalization of NAFLD health care. Recent family and inter-ethnic studies suggest that genetic/family factors may be important risk determinants for NAFLD progression. Genetic polymorphism (e.g. lipid metabolism genepnpla3) is closely related to the occurrence of steatosis, fi brosis and inf l ammation in the liver. Daly et al[119]carefully reviewed genetic polymorphisms that inf l uence the pathological events of NAFLD recently. Therefore, a complete genetic screen before the diagnosis and therapy of NAFLD in clinics seems to be necessary. In addition, several co-factors such as alcohol consumption and cigarette smoke exposure may also signif i cantly affect the incidence and severity of NAFLD. Several studies pointed out that for NAFLD patients, light to moderate alcohol consumption may have hepatic benef i ts in people with or at risk for NAFLD. However, a recent review by Liangpunsakul and Chalasani[120]clearly argued that due to the limitations of these studies (e.g. they are largely cross-sectional and used surrogate endpoints), people with NAFLD should avoid alcohol of any type or amount. So to elucidate the impact of alcohol intake on NAFLD progression is another challenging task for both hepatologists and surgeons. Unlike alcohol consumption, smoking exposure is clearly considered harmful to people with NAFLD. Yuan et al[121]reported that in mice and cultured hepatocytes, exposure to side-stream whole smoke stimulates lipid accumulation in the liver by modulating AMPK/SREBP-1c pathway. Another large multicohort study[122]found that smoking history is associated with advanced liver fi brosis in NAFLD patients partly through its inf l uence on insulin resistance. Thus, reducing the exposure to cigarette smoke, whether "f i rst-hand" or "second-hand", is very important for the control and cure of NAFLD.

Conclusions

During the past decades, therapies against NAFLD received many achievements, in both lifestyle and pharmaceutical interventions. However, we are still far from achieving great therapeutic success in managing NAFLD in clinical studies. To date, pharmaceutical therapies for NAFLD exhibited few positive outcomes in clinical trials, although animal and cellular studies found several promising advancements. Lifestyle interventions are still the most effective methods for control and reversal of NAFLD patients. It is clear that other than body weight reduction, exercise itself is an independent factor for the alleviation of steatosis. Therefore, in clinical setting, when counseling patients on lifestyle changes, regular and moderate intensity exercise should be emphasized. Future studies of pharmaceutical treatment or supplementation are needed to further enhance the therapeutic effects of exercise and other lifestyle modif i cations. Future advancement of NAFLD therapy should focus on the mechanistic studies on cell based and animal models and human clinical trials of exercise, as well as the combination of lifestyle intervention and pharmaceutical therapy specif i cally targeting main signaling pathways related to lipid metabolism, oxidative stress and inf l ammation.

Contributors:XJ and GR wrote the main body of the manuscript. TGL provided advice, revision and edited the manuscript. All authors contributed to the design, scientif i c input and interpretation of the study. XJ and TGL are the guarantors.

Funding:This work was supported by grants from the Seed Funding for Basic Research, University Research Committee, HKU, and General Research Fund, University Grant Council, Hong Kong SAR.

Ethical approval:Not needed.

Competing interest:No benef i ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

1 Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G, Bellentani S. Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos nutrition and liver study. Hepatology 2005;42:44-52.

2 Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004;40:1387-1395.

3 Schwimmer JB, Deutsch R, Kahen T, Lavine JE, Stanley C, Behling C. Prevalence of fatty liver in children and adolescents. Pediatrics 2006;118:1388-1393.

4 Fan JG, Farrell GC. Epidemiology of non-alcoholic fatty liver disease in China. J Hepatol 2009;50:204-210.

5 Stickel F, Hellerbrand C. Non-alcoholic fatty liver disease as a risk factor for hepatocellular carcinoma: mechanisms and implications. Gut 2010;59:1303-1307.

6 Kunde SS, Lazenby AJ, Clements RH, Abrams GA. Spectrum of NAFLD and diagnostic implications of the proposed new normal range for serum ALT in obese women. Hepatology 2005;42:650-656.

7 Polyzos SA, Kountouras J, Zavos C, Deretzi G. Nonalcoholic fatty liver disease: multimodal treatment options for a pathogenetically multiple-hit disease. J Clin Gastroenterol 2012;46:272-284.

8 Yilmaz Y. Review article: is non-alcoholic fatty liver disease a spectrum, or are steatosis and non-alcoholic steatohepatitis distinct conditions? Aliment Pharmacol Ther 2012;36:815-823.

9 Alisi A, Cianfarani S, Manco M, Agostoni C, Nobili V. Nonalcoholic fatty liver disease and metabolic syndrome in adolescents: pathogenetic role of genetic background and intrauterine environment. Ann Med 2012;44:29-40.

10 Yilmaz Y. NAFLD in the absence of metabolic syndrome: different epidemiology, pathogenetic mechanisms, risk factors for disease progression? Semin Liver Dis 2012;32:14-21.

11 Chatrath H, Vuppalanchi R, Chalasani N. Dyslipidemia in patients with nonalcoholic fatty liver disease. Semin Liver Dis 2012;32:22-29.

12 Bhatia LS, Curzen NP, Calder PC, Byrne CD. Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor? Eur Heart J 2012;33:1190-1200.

13 Tziomalos K, Athyros VG, Karagiannis A. Non-alcoholic fatty liver disease in type 2 diabetes: pathogenesis and treatment options. Curr Vasc Pharmacol 2012;10:162-172.

14 Targher G, Chonchol M, Zoppini G, Abaterusso C, Bonora E. Risk of chronic kidney disease in patients with non-alcoholic fatty liver disease: is there a link? J Hepatol 2011;54:1020-1029.

15 van Geenen EJ, Smits MM, Schreuder TC, van der Peet DL, Bloemena E, Mulder CJ. Nonalcoholic fatty liver disease is related to nonalcoholic fatty pancreas disease. Pancreas 2010;39:1185-1190.

16 Polyzos SA, Kountouras J, Zavos Ch. The multi-hit process and the antagonistic roles of tumor necrosis factor-alpha and adiponectin in non alcoholic fatty liver disease. Hippokratia 2009;13:127.

17 Jou J, Choi SS, Diehl AM. Mechanisms of disease progressionin nonalcoholic fatty liver disease. Semin Liver Dis 2008;28: 370-379.

18 Neuschwander-Tetri BA. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites. Hepatology 2010;52: 774-788.

19 Malaguarnera M, Di Rosa M, Nicoletti F, Malaguarnera L. Molecular mechanisms involved in NAFLD progression. J Mol Med (Berl) 2009;87:679-695.

20 Rodgers GP, Collins FS. The next generation of obesity research: no time to waste. JAMA 2012;308:1095-1096.

21 Buchwald H, Williams SE. Bariatric surgery worldwide 2003. Obes Surg 2004;14:1157-1164.

22 de Farias JM, Bom KF, Tromm CB, Luciano TF, Marques SO, Tuon T, et al. Effect of physical training on the adipose tissue of diet-induced obesity mice: interaction between reactive oxygen species and lipolysis. Horm Metab Res 2013;45:190-196.

23 Han TS, Tajar A, Lean ME. Obesity and weight management in the elderly. Br Med Bull 2011;97:169-196.

24 Lean ME, Powrie JK, Anderson AS, Garthwaite PH. Obesity, weight loss and prognosis in type 2 diabetes. Diabet Med 1990;7:228-233.

25 Visscher TL, Seidell JC, Molarius A, van der Kuip D, Hofman A, Witteman JC. A comparison of body mass index, waisthip ratio and waist circumference as predictors of all-cause mortality among the elderly: the Rotterdam study. Int J Obes Relat Metab Disord 2001;25:1730-1735.

26 Seidell JC, Visscher TL. Body weight and weight change and their health implications for the elderly. Eur J Clin Nutr 2000;54:S33-9.

27 Capristo E, Miele L, Forgione A, Vero V, Farnetti S, Mingrone G, et al. Nutritional aspects in patients with non-alcoholic steatohepatitis (NASH). Eur Rev Med Pharmacol Sci 2005;9: 265-268.

28 Raben A, Vasilaras TH, Møller AC, Astrup A. Sucrose compared with artif i cial sweeteners: different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects. Am J Clin Nutr 2002;76:721-729.

29 Bistrian BR. Dietary composition during weight-loss maintenance. JAMA 2012;308:1088.

30 Palmer M, Schaffner F. Effect of weight reduction on hepatic abnormalities in overweight patients. Gastroenterology 1990; 99:1408-1413.

31 Petersen KF, Dufour S, Befroy D, Lehrke M, Hendler RE, Shulman GI. Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycemia by moderate weight reduction in patients with type 2 diabetes. Diabetes 2005;54:603-608.

32 Thoma C, Day CP, Trenell MI. Lifestyle interventions for the treatment of non-alcoholic fatty liver disease in adults: a systematic review. J Hepatol 2012;56:255-266.

33 Tovar A, Chui K, Hyatt RR, Kuder J, Kraak VI, Choumenkovitch SF, et al. Healthy-lifestyle behaviors associated with overweight and obesity in US rural children. BMC Pediatr 2012;12:102.

34 Harrison SA, Day CP. Benef i ts of lifestyle modif i cation in NAFLD. Gut 2007;56:1760-1769.

35 Tunstall RJ, Mehan KA, Wadley GD, Collier GR, Bonen A, Hargreaves M, et al. Exercise training increases lipid metabolism gene expression in human skeletal muscle. Am J Physiol Endocrinol Metab 2002;283:E66-72.

36 Bae JC, Suh S, Park SE, Rhee EJ, Park CY, Oh KW, 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 2012;7:e46819.

37 Sreenivasa Baba C, Alexander G, Kalyani B, Pandey R, Rastogi S, Pandey A, et al. Effect of exercise and dietary modif i cation on serum aminotransferase levels in patients with nonalcoholic steatohepatitis. J Gastroenterol Hepatol 2006;21:191-198.

38 St George A, Bauman A, Johnston A, Farrell G, Chey T, George J. Independent effects of physical activity in patients with nonalcoholic fatty liver disease. Hepatology 2009;50:68-76.

39 Johnson NA, Keating SE, George J. Exercise and the liver: implications for therapy in fatty liver disorders. Semin Liver Dis 2012;32:65-79.

40 Yi CX, Al-Massadi O, Donelan E, Lehti M, Weber J, Ress C, et al. Exercise protects against high-fat diet-induced hypothalamic inf l ammation. Physiol Behav 2012;106:485-490.

41 Kawanishi N, Yano H, Mizokami T, Takahashi M, Oyanagi E, Suzuki K. Exercise training attenuates hepatic inf l ammation, fi brosis and macrophage inf i ltration during diet inducedobesity in mice. Brain Behav Immun 2012;26:931-941.

42 Promrat K, Kleiner DE, Niemeier HM, Jackvony E, Kearns M, Wands JR, et al. Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology 2010;51:121-129.

43 Ramnanan CJ, Edgerton DS, Kraft G, Cherrington AD. Physiologic action of glucagon on liver glucose metabolism. Diabetes Obes Metab 2011;13:118-125.

44 Berglund ED, Lustig DG, Baheza RA, Hasenour CM, Lee-Young RS, Donahue EP, et al. Hepatic glucagon action is essential for exercise-induced reversal of mouse fatty liver. Diabetes 2011;60:2720-2729.

45 Hoene M, Weigert C. The stress response of the liver to physical exercise. Exerc Immunol Rev 2010;16:163-183.

46 Rolo AP, Teodoro JS, Palmeira CM. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Free Radic Biol Med 2012;52:59-69.

47 Kugelmas M, Hill DB, Vivian B, Marsano L, McClain CJ. Cytokines and NASH: a pilot study of the effects of lifestyle modif i cation and vitamin E. Hepatology 2003;38:413-419.

48 Harrison SA, Torgerson S, Hayashi P, Ward J, Schenker S. Vitamin E and vitamin C treatment improves fi brosis in patients with nonalcoholic steatohepatitis. Am J Gastroenterol 2003;98:2485-2490.

49 Lavine JE. Vitamin E treatment of nonalcoholic steatohepatitis in children: a pilot study. J Pediatr 2000;136:734-738.

50 Miller ER 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005;142:37-46.

51 Straus SE. Herbal medicines--what's in the bottle? N Engl J Med 2002;347:1997-1998.

52 Catalgol B, Batirel S, Taga Y, Ozer NK. Resveratrol: French paradox revisited. Front Pharmacol 2012;3:141.

53 Shang J, Chen LL, Xiao FX, Sun H, Ding HC, Xiao H. Resveratrol improves non-alcoholic fatty liver disease by activating AMP-activated protein kinase. Acta Pharmacol Sin2008;29:698-706.

54 Bujanda L, Hijona E, Larzabal M, Beraza M, Aldazabal P, García-Urkia N, et al. Resveratrol inhibits nonalcoholic fatty liver disease in rats. BMC Gastroenterol 2008;8:40.

55 Um JH, Park SJ, Kang H, Yang S, Foretz M, McBurney MW, et al. AMP-activated protein kinase-def i cient mice are resistant to the metabolic effects of resveratrol. Diabetes 2010;59:554-563.

56 Purushotham A, Schug TT, Xu Q, Surapureddi S, Guo X, Li X. Hepatocyte-specif i c deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inf l ammation. Cell Metab 2009;9:327-338.

57 Bruno RS, Dugan CE, Smyth JA, DiNatale DA, Koo SI. Green tea extract protects leptin-def i cient, spontaneously obese mice from hepatic steatosis and injury. J Nutr 2008;138:323-331.

58 Bose M, Lambert JD, Ju J, Reuhl KR, Shapses SA, Yang CS. The major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice. J Nutr 2008;138:1677-1683.

59 Ueno T, Torimura T, Nakamura T, Sivakumar R, Nakayama H, Otabe S, et al. Epigallocatechin-3-gallate improves nonalcoholic steatohepatitis model mice expressing nuclear sterol regulatory element binding protein-1c in adipose tissue. Int J Mol Med 2009;24:17-22.

60 Park HJ, DiNatale DA, Chung MY, Park YK, Lee JY, Koo SI, et al. Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice. J Nutr Biochem 2011;22:393-400.

61 Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ 1995;310:693-696.

62 Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N, Nishino Y, et al. Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study. JAMA 2006;296:1255-1265.

63 Xiao J, Ching YP, Liong EC, Nanji AA, Fung ML, Tipoe GL. Garlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver diseasein vivoanimal model. Eur J Nutr 2012.

64 Samuel VT, Shulman GI. Mechanisms for insulin resistance: common threads and missing links. Cell 2012;148:852-871.

65 Vuppalanchi R, Chalasani N. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management. Hepatology 2009;49:306-317.

66 Marlatt GA, Larimer ME, Mail PD, Hawkins EH, Cummins LH, Blume AW, et al. Journeys of the Circle: a culturally congruent life skills intervention for adolescent Indian drinking. Alcohol Clin Exp Res 2003;27:1327-1329.

67 Tiikkainen M, Häkkinen AM, Korsheninnikova E, Nyman T, Mäkimattila S, Yki-Järvinen H. Effects of rosiglitazone and metformin on liver fat content, hepatic insulin resistance, insulin clearance, and gene expression in adipose tissue in patients with type 2 diabetes. Diabetes 2004;53:2169-2176.

68 Ratziu V, Giral P, Jacqueminet S, Charlotte F, Hartemann-Heurtier A, Serfaty L, et al. Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebocontrolled Fatty Liver Improvement with Rosiglitazone Therapy (FLIRT) Trial. Gastroenterology 2008;135:100-110.

69 Nissen SE, Wolski K. Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N Engl J Med 2007;356:2457-2471.

70 Mazza A, Fruci B, Garinis GA, Giuliano S, Malaguarnera R, Belf i ore A. The role of metformin in the management of NAFLD. Exp Diabetes Res 2012;2012:716404.

71 Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355: 2297-2307.

72 Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med 2010;362:1675-1685.

73 Shah P, Mudaliar S. Pioglitazone: side effect and safety prof i le. Expert Opin Drug Saf 2010;9:347-354.

74 Stumvoll M, Nurjhan N, Perriello G, Dailey G, Gerich JE. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. N Engl J Med 1995;333:550-554.

75 Ali S, Fonseca V. Overview of metformin: special focus on metformin extended release. Expert Opin Pharmacother 2012;13:1797-1805.

76 He L, Sabet A, Djedjos S, Miller R, Sun X, Hussain MA, et al. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell 2009;137:635-646.

77 Kohjima M, Higuchi N, Kato M, Kotoh K, Yoshimoto T, Fujino T, et al. SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease. Int J Mol Med 2008;21:507-511.

78 Uygun A, Kadayifci A, Isik AT, Ozgurtas T, Deveci S, Tuzun A, et al. Metformin in the treatment of patients with nonalcoholic steatohepatitis. Aliment Pharmacol Ther 2004;19: 537-544.

79 Nair S, Diehl AM, Wiseman M, Farr GH Jr, Perrillo RP. Metformin in the treatment of non-alcoholic steatohepatitis: a pilot open label trial. Aliment Pharmacol Ther 2004;20:23-28.

80 Schwimmer JB, Behling C, Newbury R, Deutsch R, Nievergelt C, Schork NJ, et al. Histopathology of pediatric nonalcoholic fatty liver disease. Hepatology 2005;42:641-649.

81 Nobili V, Marcellini M, Devito R, Ciampalini P, Piemonte F, Comparcola D, et al. NAFLD in children: a prospective clinical-pathological study and effect of lifestyle advice. Hepatology 2006;44:458-465.

82 Loomba R, Lutchman G, Kleiner DE, Ricks M, Feld JJ, Borg BB, et al. Clinical trial: pilot study of metformin for the treatment of non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2009;29:172-182.

83 de Oliveira CP, Stefano JT, de Siqueira ER, Silva LS, de Campos Mazo DF, Lima VM, et al. Combination of N-acetylcysteine and metformin improves histological steatosis and fi brosis in patients with non-alcoholic steatohepatitis. Hepatol Res 2008;38:159-165.

84 Garinis GA, Fruci B, Mazza A, De Siena M, Abenavoli S, Gulletta E, et al. Metformin versus dietary treatment in nonalcoholic hepatic steatosis: a randomized study. Int J Obes (Lond) 2010;34:1255-1264.

85 Landin K, Tengborn L, Smith U. Treating insulin resistance in hypertension with metformin reduces both blood pressure and metabolic risk factors. J Intern Med 1991;229:181-187.

86 Petersen JS, DiBona GF. Acute sympathoinhibitory actions of metformin in spontaneously hypertensive rats. Hypertension1996;27:619-625.

87 Musso G, Cassader M, Rosina F, Gambino R. Impact of current treatments on liver disease, glucose metabolism and cardiovascular risk in non-alcoholic fatty liver disease (NAFLD): a systematic review and meta-analysis of randomised trials. Diabetologia 2012;55:885-904.

88 Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, 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 2012;142:1592-1609.

89 Yilmaz Y, Atug O, Yonal O, Duman D, Ozdogan O, Imeryuz N, et al. Dipeptidyl peptidase IV inhibitors: therapeutic potential in nonalcoholic fatty liver disease. Med Sci Monit 2009;15:HY1-5.

90 Zarrinpar A, Loomba R. Review article: the emerging interplay among the gastrointestinal tract, bile acids and incretins in the pathogenesis of diabetes and non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2012;36:909-921.

91 Tzefos M, Olin JL. 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor use in chronic liver disease: a therapeutic controversy. J Clin Lipidol 2011;5:450-459.

92 Dima A, Marinescu AG, Dima AC. Non-alcoholic fatty liver disease and the statins treatment. Rom J Intern Med 2012;50: 19-25.

93 Athyros VG, Tziomalos K, Gossios TD, Griva T, Anagnostis P, Kargiotis K, et al. Safety and eff i cacy of long-term statin treatment for cardiovascular events in patients with coronary heart disease and abnormal liver tests in the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) Study: a post-hoc analysis. Lancet 2010;376:1916-1922.

94 Ekstedt M, Franzén LE, Mathiesen UL, Holmqvist M, Bodemar G, Kechagias S. Statins in non-alcoholic fatty liver disease and chronically elevated liver enzymes: a histopathological follow-up study. J Hepatol 2007;47:135-141.

95 Foster T, Budoff MJ, Saab S, Ahmadi N, Gordon C, Guerci AD. Atorvastatin and antioxidants for the treatment of nonalcoholic fatty liver disease: the St Francis Heart Study randomized clinical trial. Am J Gastroenterol 2011;106:71-77.

96 Hyogo H, Tazuma S, Arihiro K, Iwamoto K, Nabeshima Y, Inoue M, et al. Eff i cacy of atorvastatin for the treatment of nonalcoholic steatohepatitis with dyslipidemia. Metabolism 2008;57:1711-1718.

97 Nelson A, Torres DM, Morgan AE, Fincke C, Harrison SA. A pilot study using simvastatin in the treatment of nonalcoholic steatohepatitis: A randomized placebo-controlled trial. J Clin Gastroenterol 2009;43:990-994.

98 Hamelin BA, Turgeon J. Hydrophilicity/lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors. Trends Pharmacol Sci 1998;19:26-37.

99 Musso G, Cassader M, Gambino R. Cholesterol-lowering therapy for the treatment of nonalcoholic fatty liver disease: an update. Curr Opin Lipidol 2011;22:489-496.

100 Grundy SM. HMG-CoA reductase inhibitors for treatment of hypercholesterolemia. N Engl J Med 1988;319:24-33.

101 Chalasani N. Statins and hepatotoxicity: focus on patients with fatty liver. Hepatology 2005;41:690-695.

102 Fraulob JC, Souza-Mello V, Aguila MB, Mandarim-de-Lacerda CA. Benef i cial effects of rosuvastatin on insulin resistance, adiposity, inf l ammatory markers and nonalcoholic fatty liver disease in mice fed on a high-fat diet. Clin Sci (Lond) 2012;123:259-270.

103 Matthew Morris E, Fletcher JA, Thyfault JP, Scott Rector R. The role of angiotensin II in nonalcoholic steatohepatitis. Mol Cell Endocrinol 2012.

104 Fabbrini E, Mohammed BS, Korenblat KM, Magkos F, McCrea J, Patterson BW, et al. Effect of fenof i brate and niacin on intrahepatic triglyceride content, very low-density lipoprotein kinetics, and insulin action in obese subjects with nonalcoholic fatty liver disease. J Clin Endocrinol Metab 2010;95:2727-2735.

105 Kudo H, Yata Y, Takahara T, Kawai K, Nakayama Y, Kanayama M, et al. Telmisartan attenuates progression of steatohepatitis in mice: role of hepatic macrophage inf i ltration and effects on adipose tissue. Liver Int 2009;29:988-996.

106 Georgescu EF, Ionescu R, Niculescu M, Mogoanta L, Vancica L. Angiotensin-receptor blockers as therapy for mild-to-moderate hypertension-associated non-alcoholic steatohepatitis. World J Gastroenterol 2009;15:942-954.

107 Kaji K, Yoshiji H, Kitade M, Ikenaka Y, Noguchi R, Shirai Y, et al. Combination treatment of angiotensin II type I receptor blocker and new oral iron chelator attenuates progression of nonalcoholic steatohepatitis in rats. Am J Physiol Gastrointest Liver Physiol 2011;300:G1094-1104.

108 Lindor KD, Kowdley KV, Heathcote EJ, Harrison ME, Jorgensen R, Angulo P, et al. Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial. Hepatology 2004;39:770-778.

109 Orlando R, Azzalini L, Orando S, Lirussi F. Bile acids for non-alcoholic fatty liver disease and/or steatohepatitis. Cochrane Database Syst Rev 2007:CD005160.

110 Leuschner UF, Lindenthal B, Herrmann G, Arnold JC, Rössle M, Cordes HJ, et al. High-dose ursodeoxycholic acid therapy for nonalcoholic steatohepatitis: a doubleblind, randomized, placebo-controlled trial. Hepatology 2010;52:472-479.

111 Adams LA, Zein CO, Angulo P, Lindor KD. A pilot trial of pentoxifylline in nonalcoholic steatohepatitis. Am J Gastroenterol 2004;99:2365-2368.

112 Zein CO, Yerian LM, Gogate P, Lopez R, Kirwan JP, Feldstein AE, et al. Pentoxifylline improves nonalcoholic steatohepatitis: a randomized placebo-controlled trial. Hepatology 2011;54:1610-1619.

113 Malaguarnera M, Gargante MP, Russo C, Antic T, Vacante M, Malaguarnera M, et al. L-carnitine supplementation to diet: a new tool in treatment of nonalcoholic steatohepatitis--a randomized and controlled clinical trial. Am J Gastroenterol 2010;105:1338-1345.

114 Compare D, Coccoli P, Rocco A, Nardone OM, De Maria S, Cartenì M, et al. Gut--liver axis: the impact of gut microbiota on non alcoholic fatty liver disease. Nutr Metab Cardiovasc Dis 2012;22:471-476.

115 Ratziu V, Sheikh MY, Sanyal AJ, Lim JK, Conjeevaram H, Chalasani N, et al. A phase 2, randomized, doubleblind, placebo-controlled study of GS-9450 in subjects with nonalcoholic steatohepatitis. Hepatology 2012;55:419-428.

116 Gray S, West LM. Herbal medicines--a cautionary tale. N Z Dent J 2012;108:68-72.

117 Souza MR, Diniz Mde F, Medeiros-Filho JE, Araújo MS. Metabolic syndrome and risk factors for non-alcoholic fattyliver disease. Arq Gastroenterol 2012;49:89-96.

118 Yilmaz Y. Review article: fructose in non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2012;35:1135-1144.

119 Daly AK, Ballestri S, Carulli L, Loria P, Day CP. Genetic determinants of susceptibility and severity in nonalcoholic fatty liver disease. Expert Rev Gastroenterol Hepatol 2011;5: 253-263.

120 Liangpunsakul S, Chalasani N. What should we recommend to our patients with NAFLD regarding alcohol use? Am J Gastroenterol 2012;107:976-978.

121 Yuan H, Shyy JY, Martins-Green M. Second-hand smoke stimulates lipid accumulation in the liver by modulating AMPK and SREBP-1. J Hepatol 2009;51:535-547.

122 Zein CO, Unalp A, Colvin R, Liu YC, McCullough AJ; Nonalcoholic Steatohepatitis Clinical Research Network. Smoking and severity of hepatic fi brosis in nonalcoholic fatty liver disease. J Hepatol 2011;54:753-759.

123 Peng L, Wang J, Li F. Weight reduction for non-alcoholic fatty liver disease. Cochrane Database Syst Rev 2011:CD003619.

Received October 5, 2012

Accepted after revision December 24, 2012

AuthorAff i liations:Departments of Anatomy (Xiao J, Guo R, Liong EC and Tipoe GL), Physiology (Fung ML), and Research Centre of Heart, Brain, Hormone and Healthy Aging Centre (Fung ML and Tipoe GL), Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; Gene and Cell Engineering Research Center, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China (Xiao J)

George L Tipoe, MD, PhD, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China (Tel: 852-28199185; Fax: 852-28170857; Email: tgeorge@hkucc.hku.hk) © 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(13)60021-1