Mechanisms of non-alcoholic steatohepatitis

Alcohol 34 (2004) 67–79

Mechanisms of non-alcoholic steatohepatitis Craig J. McClaina,*, Sri Prakash L. Mokshagundama, Shirish S. Barvea, Zhenyuan Songa, Daniell B. Hilla, Theresa Chenb, Ion Deaciuca a

Department of Internal Medicine, University of Louisville Medical Center, 530 South Jackson Street, ACB 3rd Floor, Louisville, KY 40292, USA b Department of Pharmacology and Toxicology, Room 1319 Research Tower, University of Louisville Health Sciences Center, 500 South Preston Street, Louisville, KY 40202, USA Received 30 March 2004; received in revised form 9 July 2004; accepted 13 July 2004

Abstract In 1980, the term non-alcoholic steatohepatitis was coined to describe a new syndrome occurring in patients who usually were obese (often diabetic) females who had a liver biopsy picture consistent with alcoholic hepatitis, but who denied alcohol use. The causes of this syndrome were unknown, and there was no defined therapy. More than two decades later, this clinical syndrome is only somewhat better understood, and still there is no Food and Drug Administration–approved or even generally accepted drug therapy. Patients with primary non-alcoholic steatohepatitis typically have the insulin resistance syndrome (synonymous with the metabolic syndrome, syndrome X, and so forth), which is characterized by obesity, diabetes, hyperlipidemia, hypertension, and, in some instances, other metabolic abnormalities such as polycystic ovary disease. Secondary non-alcoholic steatohepatitis may be caused by drugs such as tamoxifen, certain industrial toxins, rapid weight loss, and so forth. The cause of non-alcoholic steatohepatitis remains elusive, but most investigators agree that a baseline of steatosis requires a second hit capable of inducing inflammation, fibrosis, or necrosis for non-alcoholic steatohepatitis to develop. Our research group has focused its efforts on the interactions of nutritional abnormalities, cytokines, oxidative stress with lipid peroxidation, and mitochondrial dysfunction in the induction of steatohepatitis, both alcoholic and non-alcoholic in origin. Research findings from other laboratories also support the role of increased cytokine activity, oxidative stress, and mitochondrial dysfunction in the pathogenesis of non-alcoholic steatohepatitis. The objectives of this article are to review the (1) definition and clinical features of non-alcoholic steatohepatitis, (2) potential mechanisms of non-alcoholic steatohepatitis, and (3) potential therapeutic interventions in non-alcoholic steatohepatitis. 쑖 2005 Elsevier Inc. All rights reserved. Keywords: Non-alcoholic steatohepatitis; Fatty liver; Cytokines; Mitochondria; Oxidative stress

1. Introduction In 1980, the term non-alcoholic steatohepatitis was coined to describe a new syndrome occurring in patients who usually were obese (often diabetic) females with a liver biopsy picture consistent with alcoholic hepatitis, but who denied alcohol use (Ludwig et al., 1980). The cause (or causes) of this syndrome was unknown, and there was no defined therapy. More than two decades later, this clinical syndrome is somewhat better understood, but there still is no Food and Drug Administration–approved or generally accepted drug therapy (Sheth et al., 1997). Patients with primary nonalcoholic steatohepatitis typically have the insulin resistance syndrome (synonymous with the metabolic syndrome, syndrome X, and so forth), which is characterized by obesity,

* Corresponding author. Tel.: ⫹1-502-562-3899; fax: ⫹1-502-562-4271. E-mail address: [email protected] (C.J. McClain). Editor: T.R. Jerrells 0741-8329/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.07.007

diabetes, hyperlipidemia, hypertension, and, in some instances, other metabolic abnormalities such as polycystic ovary disease (Luyckx et al., 2000). Secondary nonalcoholic steatohepatitis may be caused by drugs such as tamoxifen, certain industrial toxins, and rapid weight loss (Table 1). The cause of non-alcoholic steatohepatitis remains elusive, but most investigators agree that a baseline of steatosis requires a second hit capable of inducing inflammation, fibrosis, or necrosis for non-alcoholic steatohepatitis to develop (Day & James, 1998). One area of focus in our research group has been the interactions of cytokines, oxidative stress with lipid peroxidation, and mitochondrial dysfunction in the induction of steatohepatitis, both alcoholic and non-alcoholic in origin (Hill et al., 1999; Kugelmas et al., 2003; McClain et al., 1999). Research findings from other laboratories also support the role of increased cytokine activity, oxidative stress, and mitochondrial dysfunction in the pathogenesis of non-alcoholic steatohepatitis (Chitturi & Farrell, 2001; Falck-Ytter et al., 2001; Fromenty & Pessayre, 1995; Sanyal et al., 2001; Wigg et al., 2001).

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Table 1 Classification of steatohepatitis Non-alcoholic steatohepatitis (primary)

Non-alcoholic steatohepatitis (secondary)

Metabolic syndrome Central obesity Hypertriglyceridemia Low high-density lipoprotein cholesterold Hyperglycemia Hypertension Others

Nutrition Rapid weight loss Total parenteral nutrition Others

Gastrointestinal surgery Jejunoileal bypass Others

A second major focus of our research group is the role of nutrition and the use of complementary and alternative medicines in the treatment of liver disease. S-adenosylmethionine (also termed AdoMet, SAM, and SAMe; SAMe will be used in this article to denote both the natural metabolite and therapeutic agent) is an important methyl donor and antioxidant (Mato et al., 1997a; McClain et al., 2002). SAMe also is a commercially available complementary and alternative medicine agent with potential therapeutic efficacy in non-alcoholic steatohepatitis. SAMe can be produced in all cells, but the liver is the primary organ responsible for conversion of dietary methionine to SAMe. The hepatic form of methionine adenosyltransferase (MAT), the enzyme responsible for this conversion, is highly susceptible to oxidative stress, and decreased activity is observed in many forms of liver disease (Mato et al., 1997a; McClain et al., 2002). Depressed SAMe concentrations are observed in multiple forms of experimental liver injury, including non-alcoholic fatty liver disease, and SAMe therapy is an effective hepatoprotective agent in many forms of experimental liver injury. Although SAMe concentrations are low in steatohepatitis, homocysteine concentrations (a downstream product of SAMe) are elevated. Homocysteine is known to play a role in atherosclerosis and seems to play a role in hepatic steatosis (Nyga˚rd et al., 1997). Thus, correction of abnormalities in the hepatic transsulfuration pathway may be one form of therapy for non-alcoholic steatohepatitis. In this article, we review the definition and clinical features of non-alcoholic steatohepatitis, potential mechanisms of non-alcoholic steatohepatitis, and current therapeutic options (with a focus on SAMe and homocysteine). Findings of studies with human subjects will be emphasized, and a limited number of results from animal studies will be reviewed. There are a host of animal models of fatty liver that have unique attributes, and these models have been reviewed recently by Koteish and Diehl (2002). We will focus mainly on two models that relate to abnormal SAMe metabolism. The methionine-restricted choline-deficient (MCD) model is a diet-induced model that causes SAMe and choline deficiency (Chawla et al., 1998). Hepatic SAMe deficiency and non-alcoholic steatohepatitis develop in the methionine adenosyltransferase 1A (MAT1A) knockout mouse (Lu

Alcoholic steatohepatitis Drugs/environmental toxins Tamoxifen Amiodarone Petrochemical exposure Others

Ethanol

et al., 2001). Both models are susceptible to a second insult, and hepatic glutathione deficiency develops (Table 2).

2. Definition and clinical features of non-alcoholic steatohepatitis 2.1. Definition Non-alcoholic steatohepatitis is defined by both histologic (steatohepatitis) and clinical (non-alcoholic) criteria. Although some investigators have been more liberal and used 40 g per day of alcohol as a cut-off, most currently use 0 to 20 g per day to exclude alcohol-induced pathologic changes [reviewed in Falck-Ytter et al. (2001)]. The histologic abnormalities of non-alcoholic steatohepatitis are identical to those in alcohol-induced steatohepatitis and include steatosis (macro ⬎ micro), hepatocyte ballooning degeneration (usually zone 3), mixed lobular inflammation with scattered polys (polymorphonuclear neutrophils) and mononuclear cells, and, at times, other features, such as lipogranulomas, acidophil bodies, Mallory’s hyaline, zone 3 perisinusoidal fibrosis, and megamitochondria (Brunt, 2001). Excluded are patients with viral, autoimmune, or other metabolic liver diseases, including those such as Wilson’s disease that frequently exhibit steatosis. Many groups [see, for example, Younossi et al. (2001)] use a broader term such as nonalcoholic fatty liver disease to incorporate more of the spectrum of fatty liver disease (Falck-Ytter et al., 2001; Marchesini et al., 2001a; Younossi et al., 2001). 2.2. Clinical features The clinical features of non-alcoholic steatohepatitis are generally nonspecific. Patients are typically diagnosed in the fifth or sixth decade of life, although there is an increasing recognition of non-alcoholic steatohepatitis in children. Most patients are asymptomatic, although some complain of fatigue, right upper quadrant discomfort, or both. Many patients are diagnosed on routine physical examination (mild hepatomegaly) and laboratory study evaluation [mild increase in serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) less than five times the normal concentration]. The AST:ALT ratio is usually ⬍1 in non-alcoholic steatohepatitis, thus helping to

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Table 2 Animal models of non-alcoholic steatohepatitis Methionine-restricted choline-deficient (MCD) diet–induced modela,b

Methionine adenosyltransferase 1A (MAT1A) knockout mouse modelc

Decreased hepatic S-adenosylmethionine (SAMe)/glutathione (GSH) Macrovesicular fat Sensitivity to lipopolysaccharide hepatotoxicity Cirrhosis can develop Cancer can develop long term

Decreased hepatic SAMe, GSH Macrovesicular fat, inflammation Susceptibility to choline-deficient diet Cancer can develop long term

Adapted/summarized from a R. K. Chawla, W. H. Watson, C. E. Eastin, E. Y. Lee, J. Schmidt, and C. J. McClain, S-adenosylmethionine deficiency and TNF-α in lipopolysaccharideinduced hepatic injury, The American Journal of Physiology - Gastrointestinal and Liver Physiology 275(1 Pt 1), G125–G129, 1998, with permission of The American Physiological Society; b C. E. Eastin, C. J. McClain, E. Y. Lee, G. J. Bagby, and R. K. Chawla, Choline deficiency augments and antibody to tumor necrosis factor-alpha attenuates endotoxin-induced hepatic injury, Alcoholism: Clinical and Experimental Research 21(6), 1037–1041, 1997, with permission from Lippincott, Williams & Wilkins; and c S. C. Lu, L. Alvarez, Z.-Z. Huang, L. Chen, W. An, F. J. Corrales, M. A. Avila, G. Kanel, and J. M. Mato, Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation, Proceedings of the National Academy of Sciences of the United States of America 98(10), 5560–5565, 2001, with permission by the National Academy of Sciences. Copyright (2001) National Academy of Sciences, U.S.A.

distinguish it from alcoholic steatohepatitis. The serum albumin and bilirubin concentrations are usually within normal limits. Type II diabetes mellitus and obesity (especially visceral adiposity) are frequently associated medical conditions (Sheth et al., 1997). The natural history of steatosis relates to histologic severity (Brunt, 2001; Sheth et al., 1997). Fatty liver without inflammation has a relatively more benign course, whereas the presence of fibrosis and inflammation indicates a more ominous prognosis. Fat may decrease as cirrhosis develops in patients with non-alcoholic steatohepatitis, and non-alcoholic steatohepatitis is a major cause of cryptogenic cirrhosis. Non-alcoholic steatosis is increasingly recognized in the pediatric age group, which is of major concern for public health (Manton et al., 2000).

3. Potential mechanisms of non-alcoholic steatohepatitis 3.1. Oxidative stress Oxidative stress is postulated to play an etiologic role in multiple forms of liver disease, ranging from metabolic diseases such as Wilson’s disease, viral-induced liver disease such as hepatitis C, and drug- or toxin-induced liver injury such as with alcohol or carbon tetrachloride (Lieber, 1997). Reactive oxygen species can induce liver injury in many ways, mainly by interacting with biomolecules (e.g., lipids, proteins, and nucleic acids), altering their structure and, as a consequence, their function. An example of special relevance to non-alcoholic steatohepatitis is the inactivation of MAT in the liver by reactive oxygen (and nitrogen) species, thus leading to a decreased content of SAMe in the liver [for a review, see Mato et al. (2002)]. Another example is the inhibition of cytochrome c oxidase by 4-hydroxynonenal, a product of lipid peroxidation (Chen et al., 1998). Once initiated, oxidative stress undergoes self-perpetuation through

damaging mitochondria, which, in turn, accelerates the formation of reactive oxygen species. Oxidative stress is well documented in non-alcoholic steatohepatitis. Studies from Sanyal and co-workers (2001) have shown that immunohistochemical staining for 3-nitrotyrosine, a “footprint” for oxidative stress, was elevated in liver biopsy samples obtained from subjects with non-alcoholic fatty liver disease and significantly elevated above that observed for both normal control subjects and patients with non-alcoholic fatty liver disease in those patients with nonalcoholic steatohepatitis (Sanyal et al., 2001). Thioredoxin is an oxidative stress–inducible thiol-containing protein that has major antioxidant properties. Serum thioredoxin concentrations were significantly elevated in patients with nonalcoholic steatohepatitis compared with the concentrations in patients with simple steatosis or in healthy volunteers (Sumida et al., 2003). The potential sources for the reactive oxygen species are multiple, with hepatic cytochrome P450 2E1 (CYP2E1) and liver mitochondria being two of the most widely investigated causes. Increased CYP2E1 can occur with obesity (Morrow, 2003). Findings of studies by Emery and co-workers (2003) have revealed increased CYP2E1 in morbidly obese human subjects with non-alcoholic steatohepatitis. It is important to note that concentrations decreased significantly after gastric bypass surgery and weight loss. Similar to findings in human beings, animal models of fatty liver also have evidence of lipid peroxidation and oxidative stress. Results of studies from Farrell’s group show that, in rats fed an MCD diet, decreased concentrations of wholeliver glutathione (GSH) develop by 2 weeks of feeding of the deficient diet, and concentrations of thiobarbituric acid reactive substances (a measure of lipid peroxidation) in whole liver were increased 16-fold over control values at 2 weeks (George et al., 2003). The markers of oxidative stress preceded evidence of profibrotic-gene expression and

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fibrosis in these animals. Collagen alpha-1 gene expression was up-regulated by 5 weeks and was increased fivefold by 17 weeks. Smooth muscle actin immunostaining, along with fibrosis, was present at 12 weeks, and, in some animals, cirrhosis occurred by 17 weeks (George et al., 2003). Hepatocytes were thought to be a major source of lipid peroxidation, and mRNA for transforming growth factor-beta (TGF-β) concentrations was elevated in isolated hepatocytes. Increased CYP2E1 activity also develops in animals fed the MCD diet. Evidence for oxidative stress has been reported by other groups by using this animal model (Oliveira et al., 2002). The MAT1A knockout mouse model of hepatic steatosis also has evidence of oxidative stress, demonstrated by an increased expression of several antioxidant enzymes, such as catalase, glutathione peroxidase, and Cu2⫹/Zn2⫹–superoxide dismutase [Santamarı´a et al. (Epub 11 March 2003)]. This increased expression is regarded as a compensatory mechanism to remove the reactive oxygen species. Also, “spontaneous” oxidative stress in the liver develops in MAT1A knockout mice, as indicated by increased concentrations of malondialdehyde. 3.2. Mitochondrial dysfunction Mitochondria also are a major potential source of reactive oxygen species in non-alcoholic steatohepatitis. We focus, in this article, on three major pathways leading to potential interactions between dysregulation of fat metabolism and mitochondrial dysfunction in the hepatocyte in non-alcoholic steatohepatitis: (1) fatty acid (FFA, free fatty acids) metabolism, especially beta-oxidation; (2) increased hepatic FFA synthesis, along with an increased uptake of FFAs from the blood; and (3) increased esterification rate of FFAs in the hepatocyte, along with the increased triglyceride inflow into the liver from the periphery. 3.2.1. Mitochondrial fatty acid metabolism Research on mitochondrial fatty acid metabolism has focused on possible defects in fatty acid beta-oxidation. Because this is the major metabolic pathway of fatty acids within the mitochondrion, its impairment will tend to increase the intramitochondrial concentrations of FFAs, which, in turn, may have a number of deleterious effects on mitochondrial function. Indeed, fat accumulation in the liver has been shown to be associated with, and is likely to be induced, at least in part, by, inhibition of fatty acid beta-oxidation (Fromenty & Pessayre, 1995). However, somewhat contradictory clinical and experimental findings have been reported regarding whether or not beta-oxidation of fatty acids is impaired in the steatotic liver. In a study performed in the MAT1A knockout mouse model of non-alcoholic steatohepatitis, it was demonstrated that a key enzyme in fatty acid beta-oxidation, acyl-coenzyme A (CoA) dehydrogenase, was markedly down-regulated [Santamarı´a et al. (Epub 11 March 2003)]. Findings of clinical studies revealed a marked down-regulation of the activity of mitochondrial respiratory

complexes (I–IV), which was associated with increased concentrations of acyl-carnitines—the form in which longchain fatty acids are transported into the mitochondrial matrix—and decreased concentrations of free carnitine (Pere´z-Carreras et al., 2003). A lower activity of mitochondrial respiratory complexes would promote a slower fatty acid beta-oxidation. Furthermore, in patients with nonalcoholic steatohepatitis, a marked down-regulation of long-chain fatty acid-CoA synthetase was evident in the liver (Sreekumar et al., 2003). In contrast, fatty acid betaoxidation was not documented in patients with non-alcoholic steatohepatitis, as determined on the basis of measurements of 3-hydroxybutyrate in serum (Sanyal et al., 2001). However, the investigators only measured 3-hydroxybutyrate and did not determine its oxidized partner acetoacetate, which would have provided a more complete profile of ketones in the circulation. Even if beta-oxidation in hepatocytes were normal, an overload of these hepatocytes with FFAs from the blood would cause the capacity of the betaoxidation pathway to be surpassed, thus leading to accumulation of FFAs in the cell. Whatever the cause, an increased intracellular content of FFAs and their derivatives has several repercussions on the cell. Among such repercussions, several have been demonstrated in clinical or experimental settings: • uncoupling of oxidative phosphorylation (Wojtczak & Scho¨nfeld, 1993), which decreases the energy charge of the cell; • decrease in free CoAs and increase in acyl-CoAs [the latter can act as inhibitors of both gluconeogenesis (Sherratt, 1986) and ureagenesis (Corkey et al., 1988), resulting in hyperammonemia]; • inhibition of adenylate translocase; • activation of protein kinase C (Nesher & Boneh, 1994); • increased oxidation of fatty acid methyl end producing ω-hydroxy-fatty acids; and • long-chain dicarboxylic acids (Fromenty & Pessayre, 1997),resulting in inhibition of oxidative phosphorylation. Furthermore, uncoupling protein-2–like protein is upregulated in ob/ob mice and has been implicated in a decreased efficiency of oxidative phosphorylation in non-alcoholic steatohepatitis (Chavin et al., 1999). All these changes revolve around, and lead to, impairment of oxidative phosphorylation, which is decreased in the liver of patients with non-alcoholic steatohepatitis (Cortez-Pinto et al., 1999b). Experimental findings in patients with non-alcoholic steatohepatitis have shown that the ATP pool in the liver is replenished at a much slower rate than in control subjects after its depletion by a load of fructose (Cortez-Pinto et al., 1999a). 3.2.2. Increased hepatic fatty acid synthesis An increased rate of fatty acid synthesis, mainly from glucose, has been demonstrated in ob/ob mice. Increased tumor necrosis factor-alpha (TNF-α) may play an etiologic role because it increases fatty acid synthesis in the hepatocyte

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through activation of acetyl-CoA carboxylase. The latter is the rate-limiting step in the de novo synthesis of fatty acids. Some of the lines of evidence implicating TNF-α in abnormal fatty acid metabolism include the observations that TNF-α • increases fatty acid synthesis (also an effect of interleukin-6), which leads to increased intracellular concentrations of FFAs (Grunfeld et al., 1990); • up-regulates fatty acid translocase in the liver, which leads to an elevated uptake of FFAs from the blood (Memon et al., 1998); and • increases peripheral lipolysis (particularly in adipose tissue), which increases the FFA availability to the liver (Memon et al., 1998). 3.2.3. Increased hepatic triglyceride synthesis and deposition The final product of fat metabolism that accumulates in the liver is triglycerides. The rate of synthesis of these compounds is generally controlled by substrate availability (Zammit, 1984). This mechanism was recognized long ago and seems largely to explain the increase in FFA esterification in the cytoplasm of the hepatocyte. An increased availability of FFAs, mainly as a consequence of decreased beta-oxidation and increased uptake, induces an augmentation of triglyceride synthesis. To our knowledge, no experimental, nor clinical, study findings are available to indicate that enzymes involved in triglyceride synthesis might be up-regulated in nonalcoholic steatohepatitis.

Fig. 1. Rats were fed a methionine-restricted choline-deficient (MCD) diet or a methionine-and-choline-sufficient (MCS) normal diet for approximately 2 weeks. Some rats were injected with endotoxin [lipopolysaccharide (LPS)], and subgroups were pretreated with either anti–tumor necrosis factor-alpha antibody (Ab.TNF-α) or S-adenosylmethionine (SAMe) (Adomet). Anti–tumor necrosis factor-alpha antibody was administered just before LPS injection. SAMe was given once a day intramuscularly throughout the duration of the study. Fatty liver and marked liver injury developed in the MCD diet–fed animals in response to LPS. This liver injury was significantly attenuated with both anti–tumor necrosis factor-α antibody and SAMe pretreatment. ALT ⫽ Alanine aminotransferase. Adapted from Alcohol, 27, C. J. McClain, D. B. Hill, Z. Song, R. Chawla, W. H. Watson, T. Chen, and S. Barve, S-adenosylmethionine, cytokines, and alcoholic liver disease, pp. 185–192, fig. 1, Copyright 2002, with permission from Elsevier.

3.3. Dysregulated cytokine metabolism Abnormal cytokine metabolism is a major feature of both alcoholic and non-alcoholic steatohepatitis. Work from our laboratory first revealed dysregulated TNF-α metabolism in alcoholic hepatitis 15 years ago with the observation that cultured monocytes (which produce the overwhelming majority of systemic circulating TNF-α and are a surrogate marker for Kupffer cells) obtained from patients with alcoholic hepatitis spontaneously produced TNF-α and produced significantly more TNF-α in response to stimulation with lipopolysaccharide (McClain & Cohen, 1989). Increased serum TNF-α concentrations in alcoholic hepatitis were next reported by several groups, and values correlated with disease severity and mortality [reviewed in McClain et al. (1999)]. Compelling findings of animal studies also support the idea of a role for TNF-α in alcoholic liver disease. Agents that block TNF-α production, anti–TNF-α antibodies, and TNF-α receptor-1 knockout mice all block the development of experimental alcoholic liver disease (Honchel et al., 1992; Iimuro et al., 1997; Yin et al., 1999). The vast majority of cytokine abnormalities observed in alcoholic liver disease are also observed in non-alcoholic steatohepatitis (Tilg & Diehl, 2000). Much of what is known about the mechanism (or mechanisms) of non-alcoholic steatohepatitis comes from animal models such as the previously

described MCD nutritional model of fatty liver or genetically obese mice and rats (ob/ob mice and fa/fa rats exhibit obesity, insulin resistance, hyperglycemia, and hyperlipidemia and have fatty livers) (Tilg & Diehl, 2000). Similar to observations in rats fed alcohol chronically, ob/ob mice are much more sensitive to endotoxin hepatotoxicity (Yang et al., 1997). They also have increased TNF-α production (Tilg & Diehl, 2000; Yang et al., 1997). It is important to note that fat stores in these mice also serve as a major source of cytokine production. Similar to observations in ob/ob mice, severe steatosis develops and increased serum/hepatic TNF-α concentrations occur in rats fed an MCD diet (Chawla et al., 1998; Eastin et al., 1997). These MCD diet–fed rats are highly sensitive to endotoxin hepatotoxicity (Chawla et al., 1998; Eastin et al., 1997) (Fig. 1). Anti–TNF-α antibody markedly attenuated the lipopolysaccharide-enhanced hepatotoxicity (Eastin et al., 1997). Isolated Kupffer cells obtained from these rats also are primed to overproduce TNF-α in response to stimulation with lipopolysaccharide. Thus, rodents with genetically and nutritionally induced non-alcoholic steatohepatitis have dysregulated cytokine metabolism, similar to that seen in human non-alcoholic steatohepatitis and to that seen in

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Fig. 2. Plasma tumor necrosis factor-alpha (TNF-α) concentrations in patients with non-alcoholic steatohepatitis (NASH) (n ⫽ 16), patients with stable alcoholic cirrhosis (n ⫽ 9), and patients with acute alcoholic steatohepatitis (AH) (n ⫽ 17). Values are mean ⫾ S.E.M. *P ⬍ .05 non-alcoholic steatohepatitis versus controls and cirrhosis versus controls. **P ⬍ .05 alcoholic hepatitis versus cirrhosis, alcoholic hepatitis versus non-alcoholic steatohepatitis, and alcoholic hepatitis versus controls. Reprinted from M. Kugelmas, D. B. Hill, B. Vivian, L. Marsano, and C. J. McClain, Cytokines and NASH: a pilot study of the effects of lifestyle modification and vitamin E, Hepatology 38(2), pp. 413–419, fig. 2, Copyright 2003, by permission of American Association for the Study of Liver Diseases.

alcoholic liver disease. Study findings from our laboratory also demonstrate that obese patients with non-alcoholic steatohepatitis have increased serum TNF-α concentrations and increased monocyte production of inflammatory cytokines such as interleukin-8, similar to what is observed in alcoholic steatohepatitis (Fig. 2) (Kugelmas et al., 2003). Findings of studies by Crespo and co-workers (2001) have shown significantly increased hepatic TNF-α mRNA and increased hepatic TNF-α receptor-1 mRNA in patients with non-alcoholic steatohepatitis. Thus, human beings with nonalcoholic steatohepatitis have increased serum concentrations of TNF-α and hepatic TNF-α as one mechanism for liver injury. 3.4. Insulin resistance Several investigators have reported the association between insulin resistance or insulin resistance syndrome and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis. Marceau et al. (1999) reported the common presence of features of the insulin resistance syndrome in morbidly obese subjects undergoing gastric bypass surgery who had histologic diagnosis of non-alcoholic steatohepatitis. Marchesini et al. (1999) reported hyperinsulinemia and insulin resistance in subjects with ultrasonographic evidence of fatty liver. Insulin resistance was more closely related to hepatic fat than to body mass index, supporting the suggestion of an independent role of insulin resistance. Knobler et al. (1999) reported on 48 subjects with hepatic steatosis and noted the association with features of the insulin resistance syndrome: abdominal obesity, dyslipidemia, and glucose intolerance. Cortez-Pinto et al. (1999a) also noted the association of features of insulin resistance syndrome and nonalcoholic steatohepatitis. Direct assessment of insulin

resistance in nondiabetic subjects with fatty liver and nonalcoholic steatohepatitis by insulin clamp techniques has further confirmed the association between non-alcoholic steatohepatitis and insulin resistance. Sanyal et al. (2001) demonstrated that in nondiabetic subjects with hepatic steatosis and non-alcoholic steatohepatitis there is decreased peripheral insulin sensitivity and increased hepatic oxidative stress. They also noted increased lipid peroxidation, but they did not demonstrate any generalized defect in fatty acid beta-oxidation. Marchesini et al. (2001b) demonstrated that nondiabetic lean or mildly overweight subjects with nonalcoholic fatty liver disease have both peripheral and hepatic insulin resistance. These defects are similar to those seen in subjects with type 2 diabetes mellitus. The cumulative evidence from these studies shows that insulin resistance represents a reproducible predisposing factor for non-alcoholic steatohepatitis. Insulin resistance or insulin resistance syndrome (or both) has been correlated with the presence and severity of fibrosis in subjects with non-alcoholic fatty liver disease. Marceau and co-workers (1999) showed that, with each additional component of the metabolic syndrome (elevated waist:hip ratio, impaired glucose tolerance, hypertension, and dyslipidemia), the risk of steatosis increased exponentially from onefold to 99-fold. Bugianesi et al. (2004) evaluated 263 subjects with non-alcoholic fatty liver disease and demonstrated that insulin resistance is an independent risk factor for advanced fibrosis in patients with non-alcoholic fatty liver disease. Marchesini et al. (2003) demonstrated that, among subjects with fatty liver, the presence of the metabolic syndrome was associated with increased risk of nonalcoholic steatohepatitis and of severe fibrosis. The mechanisms that lead to the association between insulin resistance and non-alcoholic steatohepatitis are not clearly understood. Several possibilities have been explored:

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• Insulin resistance leads to fatty liver and non-alcoholic steatohepatitis. • Hepatic steatosis leads to insulin resistance. • Some common factors lead to both insulin resistance and non-alcoholic steatohepatitis. Insulin resistance favors accumulation of FFAs in the liver and predisposes to oxidative stress by stimulating microsomal lipid peroxidases. The direct effect of high insulin concentrations is to decrease mitochondrial beta-oxidation of fatty acids, a recognized mechanism of hepatic fat accumulation. Insulin is an antilipolytic hormone. Insulin resistance could contribute to hepatic steatosis by favoring peripheral lipolysis and hepatic uptake of fatty acids. Insulin also inhibits oxidation of FFAs, thus increasing toxic FFAs in the liver. Free fatty acids, in particular dicarboxylic acid, may themselves be cytotoxic. Fatty acids are both substrates and inducers of CYP2E1. Concentrations of CYP2E1 are invariably increased in the livers of patients with non-alcoholic steatohepatitis, and they are normally suppressed by insulin. Liver fat has also been postulated to cause insulin resistance. Banerji et al. (1995) showed that, in African American males with type 2 diabetes mellitus, liver fat was inversely related to insulin sensitivity. The effect of exogenous insulin in improving blood glucose control in subjects with type 2 diabetes mellitus has been shown to correlate inversely with liver fat (Ryysy et al., 2000). The effect of two insulinsensitizing agents, rosiglitazone and pioglitazone, on glycemic control in subjects with type 2 diabetes mellitus also correlates with reduction in liver fat (Bajaj et al., 2003; Mayerson et al., 2002). The newer thiazolidendione, ragaglitzar, has both peroxisome proliferator-activated receptorgamma and peroxisome proliferator-activated receptor-alpha agonist activity and has been shown in animal models to eliminate feeding-induced accumulation of triglyceride in the liver (Ye et al., 2003). Chalasani et al. (2003b) have shown that, in nondiabetic subjects with non-alcoholic steatohepatitis, insulin resistance was likely related to visceral fat mass. Two adipocyte-derived hormones, leptin and adiponectin, have been implicated in the pathogenesis of both nonalcoholic steatohepatitis and insulin resistance. In subjects with congenital lipodystrophy syndrome and low serum leptin concentrations, severe steatosis and insulin resistance have been reported (Garg & Misra, 2002). Treatment of these subjects with leptin reduced liver fat and liver enzyme abnormalities and improved insulin resistance. However, abnormalities of leptin have not been clearly linked to the more prevalent forms of non-alcoholic fatty liver disease in human beings (Chalasani et al., 2003a). Adiponectin, a protein secreted exclusively from adipose tissue can act directly on hepatic tissue and inhibit glucose production (Berg et al., 2001). Low concentrations of adiponectin have been associated with high visceral fat and have been implicated in the development of the insulin resistance syndrome. A threefold

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increase in plasma adiponectin concentration, by the insulinsensitizing agent pioglitazone given to subjects with type 2 diabetes mellitus, is strongly associated with a decrease in hepatic fat and improvement in hepatic and muscle insulin sensitivity (Bajaj et al., 2004). In a mouse model of nonalcoholic steatohepatitis, administration of adiponectin was associated with decreases in hepatic lipid contents and serum ALT concentrations (Xu et al., 2003). Furthermore, in a study of 90 morbidly obese Chinese subjects, plasma concentrations of adiponectin were inversely correlated with those of ALT (Xu et al., 2003). Further studies in human beings on the effects on adiponectin in non-alcoholic steatohepatitis are underway in several laboratories, including our own. Iron overload has been another factor that is common to both non-alcoholic steatohepatitis and insulin resistance. Iron overload has been associated with insulin resistance and increased risk of type 2 diabetes mellitus (Ferna´ndez-Real et al., 2002). Oxidative stress induced by iron overload could provide a potential explanation for development of both insulin resistance and non-alcoholic steatohepatitis. Mendler et al. (1999) demonstrated that, in subjects with unexplained hepatic iron overload, insulin resistance syndrome was a constant feature. Fargion et al. (2001) noted that, in subjects with hyperferritinemia, insulin resistance was found in 65% of subjects, and non-alcoholic steatohepatitis was associated with the presence of multiple metabolic alterations. However, Bugianesi et al. (2004) found no association between hepatic iron load and fibrosis in 263 subjects with nonalcoholic fatty liver disease. Insulin resistance–associated iron overload, characterized by high plasma ferritin concentrations, has been associated with higher hepatic iron, but its relation to non-alcoholic steatohepatitis remains unclear (Chitturi & George, 2003). As noted previously, increased TNF-α concentrations have been reported both in animal models of non-alcoholic steatohepatitis and in patients with non-alcoholic steatohepatitis. Not only is TNF-α produced in the liver, but increased TNF-α mRNA expression has also been observed in adipose tissue in animal models of obesity and diabetes. Tumor necrosis factor-alpha has been shown to play a role in insulin resistance in a variety of animal models and in vitro systems. The inhibitor of nuclear factor-kappa B kinases, the I-kappa B kinase (IKK) complex, which consists of two catalytic subunits IKK1 and IKK2 as well as a regulatory subunit, is a critical regulator of nuclear factor-kappa B–mediated immune/inflammatory responses. The IKK2 has been postulated to play a critical role in insulin resistance in obesity. Inhibitors of this kinase have been considered as promising targets for new therapies for insulin resistance. However, use of conditional disruption of IKK2 failed to prevent obesityrelated insulin resistance (Ro¨hl et al., 2004). Thus, this remains an important, but unsolved, area of potential mechanisms for insulin resistance in non-alcoholic steatohepatitis. 3.5. Altered methionine/SAMe/homocysteine metabolism SAMe is an obligatory intermediate in the conversion of methionine to cysteine in the transsulfuration pathway

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Fig. 3. Diagram of hepatic transsulfuration pathway. There is impaired conversion of methionine to S-adenosylmethionine (SAMe) because of decreased methionine adenosyltransferase 1A (MAT1A) activity. In contrast, there are increased concentrations of both S-adenosylhomocysteine (SAH) and homocysteine in many forms of fatty liver. Increased homocysteine has been implicated in the cause of fatty liver, and increased SAH seems to sensitize to tumor necrosis factor hepatotoxicity. Homocysteine and SAH can be removed either by giving betaine to regenerate homocysteine or through 5-methyltetrahydrofolate metabolism. Polymorphisms in the 5-methyltetrahydrofolate reductase gene can impair this pathway and potentially exacerbate fatty liver. Moreover, the ratio of SAMe:SAH is critical in controlling methyltransferase reactions. Thus, this metabolic pathway seems to be vital in the genesis of many types of fatty liver disease, and there are molecular targets, such as SAMe and betaine, that may provide novel therapeutic interventions.

(Mato et al., 1997a) (see transsulfuration pathway, Fig. 3). The conversion of methionine to SAMe involves transfer of the adenosyl moiety of ATP to the sulfur atom of methionine; the process is catalyzed by MAT. Methionine adenosyltransferase activity has been identified in most cell types studied. The MAT gene is considered one of a select group of genes absolutely required for the survival of an organism (Horowitz et al., 1981). In mammals, two distinct types of genes coding for MAT have been identified: one exclusively in adult liver and the other in all other tissues. Besides being a precursor for the synthesis of polyamines, choline, and GSH, SAMe is the major methylating agent for a vast number of molecules, including both high- and low-molecularweight compounds, through specific methyltransferases (Horowitz et al., 1981). These compounds include DNA, RNA, biogenic amines, phospholipids, histones, and other proteins, and their methylation may modulate cellular functions and integrity. In this process, SAMe is converted to S-adenosylhomocysteine (SAH), which is a competitive inhibitor of most methyltransferases studied. SAMe deficiency occurs in many forms of liver disease. This deficiency was first identified in alcoholic liver disease in the early 1980s when it was observed that alcoholic subjects had a delayed clearance of an oral bolus of methionine but had no detectable accumulation of any other metabolic intermediates of the transsulfuration pathway (presumably because of a blocked conversion of methionine to SAMe) (Horowitz et al., 1981). Subsequently, Mato and co-workers in his group (Duce et al., 1988; Mato et al., 1997b) confirmed this postulate and demonstrated that the functional MAT was indeed subnormal in liver biopsy samples obtained from

alcoholic subjects. Because SAMe is a precursor for GSH synthesis, SAMe deficiency may result in GSH deficiency, which is observed in many forms of liver disease (Chawla et al., 1984). In animal studies, exogenous SAMe corrected hepatic deficiencies of both SAMe and GSH. Glutathione is required for optimal expression of MAT activity in liver, and hepatic deficiency of MAT may be due, in part, to GSH deficiency. Also, hepatic MAT is sensitive to oxidative stress. Oxidation of Cys 121, the active site of the enzyme located in its flexible loop, results in loss of its activity (SanchezGongora et al., 1997). Thus, subnormal hepatic MAT activity in patients with non-alcoholic steatohepatitis could occur as a result of oxidation of this active site. Morever, as described previously, steatohepatitis with hepatic SAMe deficiency develops in the MAT1A knockout mouse (Lu et al., 2001). There is a seemingly paradoxic increase in homocysteine concentrations in patients with non-alcoholic steatohepatitis. Homocysteine has been highly correlated with the development of atherosclerosis, and increased homocysteine concentrations have also been implicated in the development of fatty liver [Boison et al. (Epub 7 May 2002); Ji & Kaplowitz (2003); Nyga˚rd et al. (1997)]. Unpublished observations from our group (C. J. McClain, D. B. Hill, and Z. Song, 2004) revealed not only elevated homocysteine concentrations, but also elevated SAH concentrations, in patients with nonalcoholic steatohepatitis. Similarly, findings from our group support the suggestion that SAH plays a major role in sensitizing the hepatocyte to TNF-α–induced apoptosis (Song et al., 2004). One way of removing homocysteine and SAH is by providing betaine for the conversion of homocysteine to methionine (Barak et al., 2003; Ji & Kaplowitz, 2003).

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The other pathway for regeneration of homocysteine to methionine involves the enzyme methylene tetrahydrofolate reductase (Fig. 3). However, there is a frequent polymorphism in this gene that leads to decreased activity and may potentially lead to more problems with fatty liver (Schwahn & Rozen, 2001). Indeed, fatty liver develops in methylene tetrahydrofolate reductase-knockout mice [Schwahn et al. (Epub 22 January 2003)]. In addition, the presence of this polymorphism has been linked to atherosclerosis and complications of diabetes such as accelerated nephropathy in human beings. Thus, abnormalities in the hepatic transsulfuration pathway on multiple levels may lead to the development of fatty liver and, potentially, to sensitization to TNF-α–induced hepatotoxicity. 4. Current therapeutic steatohepatitis

options

for

non-alcoholic

There is no one widely-accepted therapy for nonalcoholic steatohepatitis, and there is no Food and Drug Administration–approved treatment for non-alcoholic steatohepatitis. Because obesity is a major risk factor for nonalcoholic steatohepatitis (especially in the United States), lifestyle modifications with weight reduction are frequently recommended. Indeed, obesity can induce systemic oxidative stress, as assessed by urinary isoprostane concentrations [Keaney et al. (Epub 30 January 2003)]. Similarly, weight loss can reduce oxidative stress. In an early study of 39 obese patients, liver biochemical test results improved with weight loss, especially with greater than 10% loss of body weight (Palmer & Schaffner, 1990). In a protocol combining diet and an exercise regimen for 3 months, improvement in liver biochemical test results and hepatic fatty infiltration was observed (Ueno et al., 1997). Work from our laboratory has shown that exercise and weight loss over a 3-month period caused an improvement in both liver enzyme and serum hyaluronic acid concentrations (the latter being a marker of sinusoidal endothelial cell function and hepatic fibrosis) in all patients studied (Fig. 4). There are difficulties with lifestyle modification therapy, however. Unfortunately, it is difficult to get most patients to exercise and lose weight. Moreover, some patients with non-alcoholic steatohepatitis, especially those from Europe or the Orient, may not be overweight. Thus, additional measures are required. Because oxidative stress likely plays a role in the development of non-alcoholic steatohepatitis, antioxidants have been postulated as logical therapeutic interventions. Vitamin E has been the most widely studied agent. Results of in vitro studies demonstrate that vitamin E can inhibit proinflammatory cytokine production and attenuate hepatic fibrosis/ collagen production (Hill et al., 1999; Lee et al., 1995). Initial clinical enthusiasm came from a pilot protocol by Lavine (2000) in 11 obese children, results of which showed that vitamin E (doses of 400–1,200 IU per day) caused improvement in liver enzyme concentrations. This improvement occurred without weight loss, and selected patients

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monitored after withdrawal of vitamin E therapy experienced an increase in liver enzyme concentrations (Lavine, 2000). Findings of an important study from Japan showed that 300 mg of vitamin E per day significantly reduced TGF-β concentrations in patients with non-alcoholic steatohepatitis who had already gone through a lifestyle modification program, and some patients showed an improved hepatic histologic picture (Hasegawa et al., 2001). Further randomized studies of antioxidant therapy are planned, including studies sponsored by the National Institutes of Health. It is important to learn from previous large, multicenter antioxidant studies such as those in cardiovascular disease, of which most revealed negative results (Jialal & Devaraj, 2003). It is vital to perform dose-finding studies to document that the amount of antioxidant administered actually decreases oxidative stress, as assessed by improvement in parameters such as urinary isoprostane concentrations. Multiple differing types of antioxidants may need to be used, such as vitamin E in combination with vitamin C or with a glutathione prodrug. Anticytokine therapy has a strong rationale originating from work in alcoholic steatohepatitis. Studies from Diehl’s laboratory showed that probiotics (organisms with properties that are known or believed to exert a beneficial role on the gastrointestinal tract) successfully down-regulated TNF-α production and hepatic inflammation, as well as decreased fatty liver, in ob/ob mice (Li et al., 2003). In human alcoholic steatohepatitis, pentoxifylline, which has anti–TNF-α properties, has been used with success (Akriviadis et al., 2000). Thus, agents that block the stimulus for cytotoxic cytokines (e.g., endotoxin) or inhibit proinflammatory cytokine production are attractive potential therapeutic products. Results of both animal and human studies support the suggestion of a therapeutic role for insulin sensitization in non-alcoholic steatohepatitis. Initial compelling results in obese mice from Diehl’s group showed that metformin, an agent that improves hepatic insulin resistance, decreased hepatic steatosis and improved liver enzymes (Lin et al., 2000). Moreover, metformin decreased hepatic expression of TNF-α, a cytokine that is known to inhibit propagation of insulin-initiated signals in many cells. Results of pilot studies in human beings support the therapeutic efficacy of metformin in non-alcoholic steatohepatitis (Marchesini et al., 2001b). Similarly, small clinical trials with human beings have used the class of insulin-sensitizing agents, termed thiazolidenediones, and results have demonstrated improved biochemical, radiologic, or histologic features of non-alcoholic steatohepatitis (Caldwell et al., 2001; Neuschwander-Tetri et al., 2003; Promrat et al., 2004). Because this class of insulin-sensitizing agents has independent effects on hepatic triglyceride content and adiponectin concentrations, it is difficult to determine whether the observed benefits are related solely to improvement in insulin sensitivity. Therapy with an insulin-sensitizing agent will be one of the therapeutic areas of National Institutes of Health–funded clinical trials with non-alcoholic fatty liver disease.

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Fig. 4. Plasma hyaluronic acid (HA) concentrations in patients with non-alcoholic steatohepatitis (NASH) and a control group of normal subjects. No significant change occurred between weeks 6 and 12. Values are mean ⫾ S.E.M. (*P ⬍ .05 6 weeks versus entry, control versus entry, and 12 weeks versus entry). Reprinted from M. Kugelmas, D. B. Hill, B. Vivian, L. Marsano, and C. J. McClain, Cytokines and NASH: a pilot study of the effects of lifestyle modification and vitamin E, Hepatology 38(2), pp. 413–419, fig. 2, Copyright 2003, by permission of American Association for the Study of Liver Diseases.

There is increasing compelling evidence that alterations in the hepatic transsulfuration pathway and impaired transmethylation reactions play an important role in steatohepatitis. Indeed, both the MCD diet model and MAT1A knockout mice model result in hepatic SAMe deficiency with subsequent steatohepatitis [Chawla et al. (1998); Eastin et al. (1997); Lu et al. (2001); Santamarı´a et al. (Epub 11 March 2003)]. The MCD diet model can evolve to cirrhosis, and in both models liver cancer, in some situations, can ultimately develop (thus simulating the evolution of nonalcoholic steatohepatitis). Disease states or animal models that cause a build-up of SAH or homocysteine are also associated with hepatic steatosis [Barak et al. (2003); Ji & Kaplowitz (2003); Schwahn et al. (Epub 22 January 2003)]. Increased homocysteine is highly associated with endoplasmic reticulum stress and atherosclerosis. Elevated intracellular SAH concentrations inhibit most methyltransferase reactions (Kerr, 1972), and work from our laboratory (Song et al., 2004) has shown that increased intracellular SAH concentrations sensitize the liver to TNF-α killing. Thus, therapeutic modalities that increase SAMe concentrations or decrease elevated SAH and homocysteine concentrations may be beneficial in non-alcoholic steatohepatitis. Mato et al. (1999) used SAMe successfully in a multicenter study for alcoholic liver disease, noting decreased need for liver transplantation and improved survival in SAMe-treated subjects. Work is underway in our laboratory to perform a dose-finding study for SAMe therapy in patients with nonalcoholic steatohepatitis. In a small pilot study with the use of betaine in subjects with non-alcoholic steatohepatitis, improvement in liver enzyme concentrations was noted (Abdelmalek et al., 2001). Betaine should decrease homocysteine concentrations by stimulating its conversion to methionine (Fig. 3). Betaine should also, in theory, decrease SAH

concentrations. Indeed, betaine has been shown to decrease both homocysteine and SAH concentrations in animal models of alcoholic steatosis in conjunction with improved liver enzyme concentrations (Barak et al., 2003; Ji & Kaplowitz, 2003). Thus, lifestyle measures; complementary and alternative medicines, such as vitamin E and SAMe; and drug therapy such as metformin all hold promise in nonalcoholic steatohepatitis. However, large, well-designed, and mechanistic studies are required.

5. Future directions Although the understanding of the pathophysiology of non-alcoholic steatohepatitis is still limited, major strides have been made during the past 5 years. On the basis of decades of research in alcoholic liver disease, fundamental concepts have been translated into non-alcoholic steatohepatitis. However, many questions remain unanswered. We have selected 10 major areas that are important future directions/ areas of research: 1. Determine the role of genetic factors and polymorphisms (e.g., TNF-α and methylene tetrahydrofolate reductase polymorphisms). 2. Develop diagnostic, noninvasive markers of fibrosis. 3. Determine the role of oxidative stress in liver injury/ fibrosis and evaluate whether it is a unifying factor in this disease process. 4. Determine whether mitochondrial dysfunction is a cause or consequence of non-alcoholic steatohepatitis, and evaluate the role of mitochondrial dysfunction in disease development.

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5. Determine whether insulin resistance plays a causative role in non-alcoholic steatohepatitis. 6. Determine the role of alterations in the transmethylation/transsulfuration pathways in non-alcoholic steatohepatitis. 7. Determine the role of gut flora and cytokines in nonalcoholic steatohepatitis. 8. Determine ways of predicting who is likely to proceed to cirrhosis. 9. Determine the mechanisms of fatty infiltration in non-alcoholic steatohepatitis and whether special diets may attenuate these. 10. Determine new modes of therapy on the basis of the understanding of disease pathophysiology.

Acknowledgments This research was supported by the National Institutes of Health grants AA010762 (C.J.M.), AA010496 (C.J.M.), AA013170 (S.S.B.), AA000297 (D.B.H.), and AA014185 (D.B.H.); Kentucky Science and Engineering Foundation grants (C.J.M. and T.C.); and the Department of Veterans Affairs (C.J.M.).

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