Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH): treatment

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1 Best Practice & Research Clinical Gastroenterology Vol. 18, No. 6, pp , 2004 doi: /j.bpg available online at 6 Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH): treatment Elisabetta Bugianesi 1 MD Department of Gastroenterology, San Giovanni Battista Hospital, University of Turin, Corso Bramante 88, I Turin, Italy Rebecca Marzocchi 2 MD Nicola Villanova 3 MD Giulio Marchesini* MD Department of Internal Medicine and Gastroenterology, Alma Mater Studiorum University of Bologna, Metabolic Unit-S. Orsola-Malpighi Hospital, Via Massarenti 9, I Bologna, Italy Non-alcoholic fatty liver disease is now recognized as a cause of potentially progressive liver damage, posing patients at risk of advanced liver failure. Unfortunately, the natural history of disease is only partly known, the disease is slowly progressive and therapeutic outcomes are difficult to define. These factors have limited therapeutic trials to pilot studies, and very few randomized-controlled studies are available. The concept that insulin-resistance, coupled with oxidative stress, may be the underlying mechanism responsible for fat accumulation and disease progression points to insulin-sensitizing agents (metformin, thiazolidinediones) as the most promising drugs. They proved effective in reducing enzyme levels in the short period, but very limited information is available on liver histology, not to say progression to liver cell failure. Large, long-term, placebo-controlled randomized studies are eagerly awaited. Outside controlled studies, nutritional counselling and physical exercise aimed at moderate weight loss remain the basis of any therapeutic intervention. Key words: fatty liver; treatment; insulin-sensitizing agents; anti-oxidants; lifestyle. * Corresponding author. Tel.: C ; Fax: C address: ebugianesi@yahoo.it (E. Bugianesi), rmarzocchi@hotmail.com (R. Marzocchi), villanova@ orsola-malpighi.med.unibo.it (N. Villanova), giulio.marchesini@unibo.it (G. Marchesini). 1 Tel.: C ; Fax: C Tel.: C ; Fax: C Tel.: C ; Fax: C /$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

2 1106 E. Bugianesi et al. In the last 5 years very few topics have attracted the attention of physicians caring for patients with liver disease more than non-alcoholic fatty liver (NAFLD). In spite of this growing interest, the current best treatment for the disease is unknown, and very few randomized controlled trials (RCT) have so far been published. NAFLD constitutes a special challenge for physicians for several reasons: (a) the exact prevalence of disease is unknown, but the association with highlyprevalent conditions (obesity, type 2 diabetes, dyslipidaemia) suggests that a very high number of subjects may be at risk 1 ; (b) any distinction between non-progressive (fatty liver) and progressive disease (non-alcoholic steatohepatitis NASH) is only based on liver histology 2, which is also of help to define severity 3 ; (c) surrogate markers (mainly, alanine aminotransferase) are not universally accepted, and a large body of evidence indicates that progressive liver disease may also be present in subjects with normal enzyme levels 4 ; (d) disease progression is slow. Comparison with chronic liver disease of different aetiology indicates that NAFLD is only second to primary biliary cirrhosis 5 ; (e) young NAFLD patients are generally asymptomatic 6, and invasive procedures are not easy to propose and to accept; (f) old NAFLD patients may have multiple metabolic defects, and their final prognosis is more severely regulated by the cardiovascular complications of the metabolic syndrome than by liver disease. 7 In the present review, we shall review published studies on NAFLD treatment. Special focus will be given to those published in extenso. Abstracts will only be considered when they provide data on specific treatments not covered by published studies, or when they conflict with published evidence. EVIDENCE FROM PUBLISHED STUDIES ON NAFLD TREATMENT The various treatments tested on NAFLD cases are based on different pathogenic considerations, as summarized in Table 1. The two figures indicate the effects of treatments on aminotransferase levels in studies where no control group is reported (Figure 1) or comparison between the effects of experimental treatment and control treatment (Figure 2). The various treatments are not mutually exclusive, and several authors combined pharmacological interventions with lifestyle changes to increase the effectiveness of experimental treatments. Lifestyle changes A lot of studies include lifestyle changes as first-line treatment in NAFLD, and most patients receive nutritional counselling or are prescribed a formal diet and exercise to reduce body weight. Only a few studies, however, specifically tested the effects of diet and exercise, and the control group was usually limited to subjects not compliant to lifestyle changes. Reduced food intake leads to weight loss, and exercise prevents the reduction in muscle mass, which invariably accompanies the loss of fat mass. In addition, exercise

3 Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: treatment 1107 Reference Table 1. Therapeutic trials in non-alcoholic fatty liver disease. Study type Experimental therapy Control treatment No. of cases Time Effects on biopsy Lifestyle changes Andersen 8 Case series Diet mo Variable Vajro 9 Case series Diet, exercise 9 30 mo Improved Ueno 10 Open label Diet, exercise No 25 3 mo Improved treatment Franzese 11 Case series Diet, exercise 58 6 mo n.d. Hickman 12 Case series Diet, exercise mo Improved Cytoprotective agents Laurin 13 Open label UDCA Clofibrate mo Improved Obinata 14 Case series Taurine mo n.d. Vajro 15 Open label UDCA Diet 31 6 mo n.d. Lindor 16 RCT UDCA Placebo mo No difference Insulin-sensitizers Coyle 17 Case series Metformin mo Improved Marchesini 18 Open label Metformin No 20 4 mo n.d. treatment Caldwell 19 Case series Troglitazone 10 %6 mo Variable Neuschwander- Case series Rosiglitazone wk Improved Tetri 20 Uygun 21 Open label Metformin Diet 36 6 mo No difference Promrat 22 Case series Pioglitazone wk Improved Lipid-lowering agents Laurin 13 Open label Clofibrate UDCA mo No change Basaranoglu 23 Open label Gemfibrozil Diet 46 4 wk n.d. Kiyici 24 Open label Atorvastatin UDCA 44 6 mo n.d. Anti-oxidants Lavine 25 Open label Vitamin E mo n.d. Miglio 26 Open label Betaine, Diet mo Improved Nicotinamide Abdelmalek 27 Case series Betaine 8 12 mo Improved Hasegawa 28 Open label Vitamin E Diet mo n.d. Kugelmas 29 Open label Vitamin E Diet, 16 3 mo n.d. exercise Vajro 30 RCT Vtamin E Placebo 28 5 mo n.d. Yoneda 31 Open label Vitamin E Diet mo Improved Various treatments Facchini 32 Case series Phlebotomy 17 As n.d. necessary Harrison 33 Case series Orlistat 3 6 mo Improved n.d., not done. enhances muscle insulin sensitivity and alters substrate use in skeletal muscle. The initial target of weight loss should be 10% of baseline weight. More rapid weight loss (O1.6 kg/wk) might be a cause of NAFLD in subjects without previous evidence of steatosis, or promote histological exacerbation of NASH and precipitate liver failure.

4 1108 E. Bugianesi et al Effect of Treatment (9) (22) (20) (19) (25) (27) (32) (33) (13) 20 (12) 10 0 (13) 10 Lifestyle Glitazones UDCA Betaine Vitamin E Clofibrate Orlistat Phlebothomy Figure 1. Effects of experimental treatment on alanine aminotransferase in NAFLD studies where no control group is reported. Data are reported as percent decrease in enzyme values or as percent of patients with normal aminotransferase values at the end of the treatment period. The size of circles is proportional to the number of cases (logarithmic transformation). The numbers in brackets refer to bibliography. This paradoxical effect is due to massive fatty acid mobilization from visceral stores, reaching the liver through the portal vein. After a few anecdotic reports, Andersen et al 8 published a case series of 41 obese patients with NAFLD entering a weight reducing program. After a median weight loss of 34 kg during a 4 to 23-month treatment, a significant decrease in fatty infiltration of the liver was recorded. In general, a significant improvement in liver biochemistries was noted regardless of the histological changes. There is general agreement that lifestyle changes reduce aminotransferase levels, but very few data are available on histology. Ueno et al 10 treated 15 obese NAFLD patients according to a protocol of restricted diet (25 kcal/kg ideal body weight) and exercise for 3 months. The initial exercise goal was 3000 steps of brisk walking per day, but the target was increased by 500 steps every fourth day up to steps, followed by jogging (20 minutes twice a day). By the end of the study period, BMI was reduced on average by 3 kg/m 2, accompanied by a marked decrease in aminotransferases, albumin, cholinesterase, total cholesterol and blood glucose. At liver biopsy, steatosis was reduced, whereas the score of necroinflammation did not change significantly. No clinical and histological changes were observed in a control group of 10 subjects who did not accept the program. Most focus on lifestyle changes was given to the treatment of obese NAFLD children. A weight loss of 500 g/week seems the optimum goal to improve liver enzymes and histology in the Italian paediatric setting. 9,11 Temporary changes in lifestyle may be relatively easy to attain in the short period, but extremely difficult to maintain in the long-term; the levels of exercise proposed by the Japanese group 10 are very difficult to achieve and maintain also within specific

5 Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: treatment Control Treatment (16) (24)* (29) (26) (23) (15) (30) (28) (21) (18) (31) (10) Experimental Treatment Figure 2. Effects of experimental treatment, compared with control treatment, on alanine aminotransferase in NAFLD studies. Data are reported as percent decrease in enzyme values or as percent of patients with normal aminotransferase values at the end of the treatment period. The volume of marks is proportional to the total number of cases (logarithmic transformation). Different marks are used to indicate the different classes of intervention: open squares, Lifestyle changes; open circles, insulin-sensitising agents; open triangles, vitamin E; closed squares, ursodeoxycholic acid (UDCA); closed circles, cytoprotective agents; closed triangles, lipid lowering agents. The numbers is brackets refer to bibliography. No studies are clearly in the left upper part of the figure, indicating a detrimental effect of the experimental treatment, compared with control (either no treatment or lifestyle intervention). *Controlled against UDCA, that proved ineffective in the only placebo-controlled, double-blind study. 16 programs of cognitive behavioural therapy. Participation to behavioural therapy requires motivation. Only a few patients with metabolic diseases are in the preparation stage (actively considering changing) 34 when tested according to the five motivational stages of changes 35, and it is difficult to move them into permanent action. The efforts in this area are however likely to produce significant results. Hickman et al 12 have recently reported that lifestyle changes producing a modest weight loss are followed by a sustained improvement in aminotrasferase levels in liver disease of both viral and metabolic origin, as well as a marked improvement in quality of life. Cytoprotective agents Ursodeoxycholic acid (UDCA), a cytoprotective agent preventing membrane injury, has proved useful in patients with a wide range of chronic liver diseases. 36 The drug is safe, with low drug interactions, also in long-term therapy. The potential benefits of UDCA in NAFLD were first tested in an open-label pilot study against clofibrate. After one year of continuous UDCA therapy, Laurin et al 13 reported a significant improvement in alkaline phosphatase, aminotransferases, gamma-gt and in the grade

6 1110 E. Bugianesi et al. of steatosis at liver biopsy. These data were partly supported by a study against atorvastatin 24, but have not been confirmed in a paediatric setting 15 and in a recently published placebo-controlled, randomized trial in adults. 16 In 166 NAFLD patients, UDCA at the dose of mg/kg/day (80 patients) was no better than placebo (86 cases) at 2-year follow-up. Serum liver enzymes were stable or improved in both groups, and changes in liver histology (107 patients had both pre- and post-treatment biopsies) were not significant between UDCA and placebo in the degree of steatosis, necroinflammation or fibrosis. This study, although negative, provides evidence that spontaneous improvements in liver enzymes can occur in NAFLD, not necessary related to decreased body weight, associated with improvements in the degree of steatosis and fibrosis. Also taurine, an amino acid largely present into chenodeoxycholic acid, has a cytoprotective action. In an open-label, pilot study, taurine improved aminotranferase levels regardless of weight changes, when administered chronically to obese children with NASH. 14 Insulin-sensitizing agents Metformin Given the strong association of insulin-resistance with NAFLD 37 and with elevated aminotransferase levels 38, any attempt to improve insulin sensitivity might be expected to ameliorate NAFLD. Metformin is a biguanide that down-regulates hepatic glucose production and diverts fatty acids from triglyceride production to mitochondrial betaoxidation. In type 2 diabetes, metformin reduces fasting glucose levels, decreases hyperinsulinaemia and improves hepatic insulin resistance. In normoglycaemic subjects metformin action does not produce hypoglycaemia. In the congenitally obese ob/ob mice, metformin reduces steatosis, possibly via reduced hepatic expression of tumour necrosis factor-alpha. 39 Marchesini et al 18 treated 14 NAFLD cases with metformin (500 mg tid) in an open label, pilot study, where six subjects who refused treatment were used as controls. All subjects received nutritional counselling, and the pretreatment evaluation also included the quantitative measurement of insulin resistance by means of the euglycaemic clamp technique and liver volume measurement by ultrasounds. Metformin significantly reduced liver volume, moderately improved insulin sensitivity, and normalized aminotransferase levels in 50% of cases. Treatment withdrawal was accompanied by a return of aminotransferase levels to pre-treatment values. No significant changes were observed in the control arm. Very recently the effects of metformin in NAFLD were compared with a lipid and calorie-restricted diet in an open label, randomized study of 6-month duration. 21 The mean changes in insulin and aminotransferase levels, and insulin sensitivity (homeostasis model assessment 40 ) were greater in the experimental arm than in the group given dietary advice alone. In particular, 59% of patients given metformin normalized alanine aminotransferase levels vs. 37% in the control arm. At post-treatment liver biopsy, no significant changes from baseline were observed in both groups, and the frequency of improvement in necroinflammatory score was 46% vs. 10% in the experimental and control group, respectively (PZ0.17). During a post-treatment follow-up of 6 12 months, no return of aminotransferase levels to pre-treatment values was observed. The drug appears to be safe also in the presence of mild liver disease, and the risk of lactic acidosis 41 has not been substantiated in the above trials. 18,21

7 Thiazolidinediones (Glitazones) The novel class of insulin sensitizing agents, thiazolidinediones, has also been tested in NAFLD. They improve insulin sensitivity in adipose tissue by acting on peroxisome proliferators-activated receptor gamma (PPAR-gamma). Caldwell et al 19 first reported a beneficial effect of troglitazone, in an observational study initiated before the withdrawal of the drug from clinical use because of idiosyncratic hepatitis. Seven of 10 patients receiving troglitazone normalized aminotransferase levels, but the biochemical response was not accompanied by a comparable histological response. Pioglitazone and rosiglitazone, second-generation thiazolidinediones, have now entered the market. They have a lower intrinsic hepatotoxicity, and no serious hepatic effects have been reported so far. A few pilot studies with pioglitazone have been published in abstract form, and recently a large study sponsored by the National Institute of Health has appeared in extenso. 22 Eighteen NASH patients received pioglitazone at the dose of 30 mg per day for 48 weeks. Treatment produced a significant decrease in serum alanine aminotransferase, which returned to normal in 72% of the patients. Also liver histology improved significantly; steatosis, necro-inflammation, and fibrosis were all reduced. Magnetic resonance imaging of the liver confirmed a marked decrease in liver fat content and liver volume. A large study with rosiglitazone is also available, and preliminary data 42 have been recently confirmed in a longer follow-up. 20 In 30 overweight or obese patients (22 with NASH), rosiglitazone at a dose of 4 mg bid for 48 weeks, improved insulin sensitivity and serum aminotransferase, in the absence of side effects. Post-treatment biopsies of 10 patients (45%) no longer met the published criteria for NASH. The only concern for the use of thiazolidinediones in patients with NAFLD/NASH is subclinical cardiac failure, a risk in hypertensive patients with the metabolic syndrome. 43 In summary, insulin-sensitising agents can be considered promising therapies for NAFLD, possibly improving even advanced NASH histology, but no controlled studies are available for this specific target. Randomised, placebo-controlled trials are needed to prove or disprove the real effectiveness of treatment in the long-term, considering its costs and benefits compared with lifestyle interventions. Lipid-lowering agents Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: treatment 1111 Several studies have consistently associated NAFLD with a disordered hepatic lipid homeostasis and hypertriglyceridemia. This forms the basis for the use of lipidlowering agents as a possible treatment for NASH. Clofibrate decreases hepatic triglyceride in experimental models of hepatic steatosis. However, in a pilot study clofibrate at the dose of 2 g/die for 1 year did not produce any significant change from baseline in either enzyme levels or histology. 13 On the contrary, gemfibrozil was significantly more effective than placebo in reducing aminotransferase levels, irrespective of baseline triglyceride levels, in a small placebo-controlled study. 23 Bezafibrate, a PPAR-alpha activator, prevented the histological progression of NASH in two patients treated with tamoxifen 44, a drug responsible for secondary NASH. Very few studies are available with the use of statins. Kiyici et al 24 in 27 hyperlipidemic NASH patients reported a significant improvement in cholesterol

8 1112 E. Bugianesi et al. and liver enzyme levels with atorvastatin (10 mg/day for 6 months), associated with increased liver density at ultrasonography. Changes in fibrosis and necroinflammation were no better than those observed with UDCA. In conclusion, the evidence in favour of a specific benefit of hypolipidemic agents in NAFLD is scanty. Antioxidants VitaminE(DL-alpha-tocopherol) is a cheap and well-tolerated, potent antioxidant, and is a particularly attractive potential treatment for NAFLD/NASH, particularly in children. In 11 obese children with NAFLD, vitamin E at a dose of IU per day for 4 10 months either improved markedly or normalized aminotransferase levels. 25 Discontinuation of therapy was followed by a return of aminotransferase to pre-treatment levels within 2 months. However, a singleblind study from the same group did not confirm any advantage of vitamin E compared with placebo in subjects compliant to a weight-reducing diet. 30 Similarly, when compared with diet alone, no significant advantage of vitamin E was reported in a small group of adults randomised to receive 800 IU of vitamin Efor3months. 29 Also betaine, a normal component of the metabolic cycle of methionine, decreases hepatic steatosis possibly via increased S-adenosylmethionine. In an open label trial 27, betaine treatment promoted a significant improvement or normalization of aminotransferase, as well as a histological improvement after 1 year of treatment. When associated with diethanolamine glucuronate (a liver detoxifying agent) and nicotinamide ascorbate (a substrate for the synthesis of phospholipids and a coenzyme for the redox reactions), betaine decreased aminotransferase levels by only 10% in 191 patients with NASH treated for 8 weeks. 26 In conclusion, although pathogenically sound considering lipid peroxidation as the culprit of progressive disease, there is no evidence that antioxidant therapy may be better than diet and lifestyle changes in the treatment and progression of hepatic disease. Lipase inhibitor Orlistat, the lipase inhibitor designed for the long-term management of obesity, at a dose of 120 mg t.i.d. decreases fat absorption on average by 30%, and increases the excretion of the unabsorbed triglycerides and cholesterol in the faeces. A very recent pilot study showed the efficacy of orlistat in reducing liver enzymes, as well as in improving the grade of steatosis, inflammation and fibrosis. 33 Interestingly, in one of the three obese patients enrolled, this improvement was maintained despite gradual weight gain over the following 2-year period. No conclusion can be drawn by this occasional case report, but orlistat is the only case where treatment stop is not followed by return of liver enzymes to pre-treatment levels. Phlebotomy There is a proven association between NASH and elevated serum ferritin, transferrin and iron concentration or transferrin saturation 45, although hyperferritinemia is more a marker of inflammation than a correlate of increased iron burden. 46 Increased hepatic iron and excessive fat accumulation may interact to

9 Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: treatment 1113 generate the oxidative stress responsible for liver injury. There is also evidence that iron accumulation may induce insulin resistance 47 that may be treated by appropriate iron removal. 48 Any iron depletion method, namely phlebotomy, is likely to reduce the inflammatory process. Facchini et al 32 induced iron depletion by quantitative phlebotomy to a level of near-iron deficiency in 39 patients with reduced glucose tolerance (17 with NAFLD). In this last group, phlebotomy produced a significant improvement of aminotransferase and glucose levels, despite normal body iron stores at the beginning of the study. The authors suggest that this type of treatment may be generally proposed in NAFLD patients, but long-term and biopsy-controlled trials are needed to prove the effectiveness and safety of the procedure in the absence of iron excess. CONCLUSIONS Only a few years ago, NAFLD was not considered a disease requiring specific treatment. Nutritional counselling or diet prescription were standard care. A better knowledge of the natural history of disease and its potential progression towards liver failure raised a lot of interest on pathogenesis and treatment. 6 A large body of evidence indicates that insulin resistance may be the primary reason for fat accumulation in the liver 49, hence the rationale for treating patients with insulin sensitising agents. The rationale for other therapeutic option is less sound. Lipidlowering drugs may indeed increase fat deposition in the liver; cytoprotective drugs and antioxidants do not remove the cause of liver cell damage. A lot of pilot studies and case series have proved that a relatively high number of patients normalize liver enzymes with a variety of drugs, but their efficacy remains low, and generally not superior to that of healthy lifestyle. Whether this is accompanied by an improvement in liver histology, remains to be determined. As recently pointed out 50, good, randomised clinical studies on standard treatment of NAFLD are not easy, due to its relatively low severity and the need of invasive procedures to confirm effectiveness. Nonetheless, any effort should be done, considering the potential long-term progression of the disease, and the emerging threat for a high number of subjects in Western countries, where the epidemic of obesity is still growing. Practice points there is no established treatment for NAFLD/NASH; the need for invasive procedures to determine the effectiveness of treatment, coupled with the relative slow progression of disease, is a severe limit to research most patients improve their aminotransferase levels by moderate weight loss, achieved by diet and/or exercise insulin-sensitising agents are the most promising treatment, able to reduce aminotransferase levels as well as to improve liver histology

10 1114 E. Bugianesi et al. Research agenda a better definition of histological criteria to predict progression is needed methods to improve adherence to lifestyle changes, the first line of any therapeutic intervention, need proper consideration and intensive research after a lot of pilot, uncontrolled studies, it is time for randomized, controlled studies with adequate sample size, long-term duration, and based on hard outcomes (histology) SUMMARY Current best treatment for non-alcoholic fatty liver disease has not yet been defined. The slow progression of disease, the lack of accurate surrogate markers, the need for liver biopsy to define stage of disease, limits the feasibility of controlled studies. Only a few years ago, NAFLD was not considered a disease requiring specific treatment. Nutritional counselling or diet prescription to reduce body weight, coupled with increased physical exercise, was standard care and remains the first line of treatment. Drug treatment may be added, to address specific pathogenic targets. Insulin sensitising agents are the most promising drugs. Pilot studies have convincingly shown that both metformin and thiazolidinediones reduce aminotransferase levels, reduce liver fat, and improve liver histology. Cytoprotective agents are less effective, and ursodeoxycholic acid was not superior to placebo in a randomised controlled trial. Antioxidants have a specific place in non-alcoholic fatty liver in children, but their effectiveness is low. Lipidlowering drugs and iron depletion may have a specific place in selected patients. It is time to move from pilot studies to randomised, controlled studies, having histology and disease progression as primary outcomes. Specific programs of cognitive-behavioural therapy, aimed at reducing body weight and permanently maintain weight loss should also be tested. Any effort should be done, considering the potential long-term progression of the disease, and the emerging threat for a high number of obese subjects in Western countries. REFERENCES *1. Neuschwander-Tetri BA & Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003; 37: American Gastroenterological Association. American Gastroenterological Association medical position statement: nonalcoholic fatty liver disease. Gastroenterology 2002; 123: Gramlich T, Kleiner DE, McCullough AJ et al. Pathologic features associated with fibrosis in nonalcoholic fatty liver disease. Hum Pathol 2004; 35: Mofrad P, Contos MJ, Haque M et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 2003; 37: Poynard T, Mathurin P, Lai CL et al. A comparison of fibrosis progression in chronic liver diseases. J Hepatol 2003; 38: *6. Falck-Ytter Y, Younossi ZM, Marchesini G & McCullough AJ. Clinical features and natural history of nonalcoholic steatosis syndromes. Semin Liver Dis 2001; 21: Isomaa B, Almgren P, Tuomi T et al. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24:

11 Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: treatment Andersen T, Gluud C, Franzmann MB & Christoffersen P. Hepatic effects of dietary weight loss in morbidly obese subjects. J Hepatol 1991; 12: Vajro P, Fontanella A, Perna C et al. Persistent hyperaminotransferasemia resolving after weight reduction in obese children. J Pediatr 1994; 125: Ueno T, Sugawara H, Sujaku K et al. Therapeutic effects of restricted diet and exercise in obese patients with fatty liver. J Hepatol 1997; 27: Franzese A, Vajro P, Argenziano A et al. Liver involvement in obese children. Ultrasonography and liver enzyme levels at diagnosis and during follow-up in an Italian population. Dig Dis Sci 1997; 42: Hickman IE, Jonsson JR, Prins JB et al. Modest weight loss and physical activity in overweight patients with chronic liver disease results in sustained improvements in alanine aminotransferase, fasting insulin, and quality of life. Gut 2004; 53: Laurin J, Lindor KD, Crippin JS et al. Ursodeoxycholic acid or clofibrate in the treatment of non-alcoholinduced steatohepatitis: a pilot study. Hepatology 1996; 23: Obinata K, Maruyama T, Hayashi M et al. Effect of taurine on the fatty liver of children with simple obesity. Adv Exp Med Biol 1996; 403: Vajro P, Franzese A, Valerio G et al. Lack of efficacy of ursodeoxycholic acid for the treatment of liver abnormalities in obese children. J Pediatr 2000; 136: *16. Lindor KD, Kowdley KV, Heathcote EJ et al. Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial. Hepatology 2004; 39: Coyle WJ, Delaney N, Yoshihashi A & Lawson P. Metformin treatment in patients with nonalcoholic steatohepatitis normalizes LFTs and improves histology. Gastroenterology 1999; 116: Marchesini G, Brizi M, Bianchi G et al. Metformin in non-alcoholic steatohepatitis. Lancet 2001; 358: Caldwell SH, Hespenheide EE, Redick JA et al. A pilot study of a thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis. Am J Gastroenterol 2001; 96: *20. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR et al. Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-gamma ligand rosiglitazone. Hepatology 2003; 38: *21. Uygun A, Kadayifci A, Isik AT et al. Metformin in the treatment of patients with non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2004; 19: *22. Promrat K, Lutchman G, Uwaifo GI et al. A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 2004; 39: Basaranoglu M, Acbay O & Sonsuz A. A controlled trial of gemfibrozil in the treatment of patients with nonalcoholic steatohepatitis. J Hepatol 1999; 31: Kiyici M, Gulten M, Gurel S et al. Ursodeoxycholic acid and atorvastatin in the treatment of nonalcoholic steatohepatitis. Can J Gastroenterol 2003; 17: Lavine JE. Vitamin E treatment of nonalcoholic steatohepatitis in children: a pilot study. J Pediatr 2000; 136: Miglio F, Rovati LC, Santoro A & Setnikar I. Efficacy and safety of oral betaine glucuronate in non-alcoholic steatohepatitis. A double-blind, randomized, parallel-group, placebo-controlled prospective clinical study. Arzneimittelforschung 2000; 50: Abdelmalek MF, Angulo P, Jorgensen RA et al. Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study. Am J Gastroenterol 2001; 96: Hasegawa T, Yoneda M, Nakamura K et al. Plasma transforming growth factor-beta1 level and efficacy of alpha-tocopherol in patients with non-alcoholic steatohepatitis: a pilot study. Aliment Pharmacol Ther 2001; 15: Kugelmas M, Hill DB, Vivian B et al. Cytokines and NASH: a pilot study of the effects of lifestyle modification and vitamin E. Hepatology 2003; 38: Vajro P, Mandato C, Franzese A et al. Vitamin E treatment in pediatric obesity-related liver disease: a randomized study. J Pediatr Gastroenterol Nutr 2004; 38: Yoneda M, Hasegawa T, Nakamura K et al. Vitamin E therapy in patients with NASH. Hepatology 2004; 39: 568 author reply Facchini FS, Hua NW & Stoohs RA. Effect of iron depletion in carbohydrate-intolerant patients with clinical evidence of nonalcoholic fatty liver disease. Gastroenterology 2002; 122: Harrison SA, Ramrakhiani S, Brunt EM et al. Orlistat in the treatment of NASH: a case series. Am J Gastroenterol 2003; 98:

12 1116 E. Bugianesi et al. 34. Vallis M, Ruggiero L, Greene G et al. Stages of change for healthy eating in diabetes: relation to demographic, eating-related, health care utilization, and psychosocial factors. Diabetes Care 2003; 26: Prochaska JO & Velicer WF. The transtheoretical model of health behavior change. Am J Health Promot 1997; 12: Lazaridis KN, Gores GJ & Lindor KD. Ursodeoxycholic acid mechanisms of action and clinical use in hepatobiliary disorders. J Hepatol 2001; 35: Marchesini G, Brizi M, Morselli-Labate AM et al. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med 1999; 107: Vozarova B, Stefan N, Lindsay RS et al. High alanine aminotransferase is associated with decreased hepatic insulin sensitivity and predicts the development of type 2 diabetes. Diabetes 2002; 51: Lin HZ, Yang SQ, Chuckaree C et al. Metformin reverses fatty liver disease in obese, leptin-deficient mice. Nat Med 2000; 6: Matthews DR, Hosker JP, Rudenski AS et al. Homeostasis model assessment: insulin resistance and beta-cell function from plasma fasting glucose and insulin concentrations in man. Diabetologia 1985; 28: Urso R & Visco-Comandini U. Metformin in non-alcoholic steatohepatitis. Lancet 2002; 359: Neuschwander-Tetri BA, Brunt EM, Wehmeier KR et al. Interim results of a pilot study demonstrating the early effects of the PPAR-gamma ligand rosiglitazone on insulin sensitivity, aminotransferases, hepatic steatosis and body weight in patients with non-alcoholic steatohepatitis. J Hepatol 2003; 38: Nesto RW, Bell D, Bonow RO et al. Thiazolidinedione use, fluid retention, and congestive heart failure: a consensus statement from the American Heart Association and American Diabetes Association. Diabetes Care 2004; 27: Saibara T, Onishi S, Ogawa Yet al. Bezafibrate for tamoxifen-induced non-alcoholic steatohepatitis. Lancet 1999; 353: Younossi ZM, Gramlich T, Bacon BR et al. Hepatic iron and nonalcoholic fatty liver disease. Hepatology 1999; 30: Bugianesi E, Manzini P, D Antico S et al. Relative contribution of iron burden, HFE mutations and insulin resistance to fibrosis in nonalcoholic fatty liver. Hepatology 2004; 39: Ferrannini E. Insulin resistance, iron, and the liver. Lancet 2000; 355: Guillygomarc h A, Mendler MH, Moirand R et al. Venesection therapy of insulin resistance-associated hepatic iron overload. J Hepatol 2001; 35: *49. Bugianesi E, Zannoni C, Vanni E et al. Non-alcoholic fatty liver and insulin resistance: a cause-effect relationship? Dig Liver Dis 2004; 36: *50. Clark JM & Brancati FL. Negative trials in nonalcoholic steatohepatitis: why they happen and what they teach us. Hepatology 2004; 39: