Nonalcoholic Steatohepatitis (NASH)

Walid Y. Farah, Yousef M. Ajlouni, M. Amer Khatib, Mustafa M. Shennak

ABSTRACT

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that is attracting increasing significance characterized by diffuse fatty infiltration and inflammation.The exact prevalence of NASH is unclear, but it is becoming more evident that the disease is much more common than previously thought, Although originally believed to be a benign clinical entity, NASH is now recognized as a cause of progressive fibrotic liver disease with adverse clinical sequelae.

Clinical characteristics associated with NASH include obesity, hyperlipidemia, and diabetes mellitus, all of which have been associated with underlying insulin resistance. Typically, this disease becomes evident in the fourth or fifth decade of life with an equal sex predilection.

NASH is thought to be caused, in part, by impaired insulin signaling, leading to elevated circulating insulin levels and subsequent altered lipid homeostasis. This process is likely multifactorial and includes both genetic and environmental factors. Treatment options to date are limited and are based on very small clinical trials. Current investigations are focusing on improving the underlying insulin resistance that has been associated with NASH as well as other therapies that decrease oxidative stress or improve hepatocyte survival.

INTRODUCTION

Ludwig et al. (1), in 1980, were the first to give the name nonalcoholic steatohepatitis (NASH) to a group of patients from the Mayo clinic with similar histological findings in nonalcoholics. More

encompasses a spectrum of fatty liver disease from steatosis to NASH through cirrhosis to end-stage liver disease. Previously considered to be benign, NASH has the potential to progress to

fibrosis, cirrhosis, and end-stage liver disease in some patients. Additionally, newer studies indicate that the disease may be more common than previously suspected. Investigations into the pathogenesis of the disease have provided clues to possible therapy options. A number of comprehensive reviews have been published (2–7).

Definition

The following criteria have been proposed for the diagnosis of NASH (3).

1. A liver biopsy showing moderate to gross macrovesicular fatty change with inflammation (lobular or portal) and with or without Mallory bodies, fibrosis, or cirrhosis.
2. Convincing evidence of negligible alcohol consumption (less than 40 g of ethanol per week) including a detailed history and interrogation of family members and local medical practitioners. Random blood assays for ethanol estimation should be negative. If performed, assays for the presence of desialylated transferrin in serum, a marker of alcohol consumption, should also be negative (4).
3. Absence of serologic evidence of infection with hepatitis B or hepatitis C.

Epidemiology

The exact prevalence of NASH is not known because of the indolent, often silent, nature of the disease, paucity of initial and especially sequential biopsies, and lack of complete consensus regarding histological diagnosis.

Nonalcoholic fatty liver disease affects 10 to 24 percent of the general population in various countries. The prevalence increases to 57.5 percent (5) to 74 percent (6,7) in obese persons. Nonalcoholic fatty liver disease affects 2.6 percent of children (8) and 22.5 percent (8) to 52.8 percent (9) of obese children.

Nonalcoholic fatty liver disease is a common explanation for abnormal liver-test results in blood donors, and it is the cause of asymptomatic elevation of aminotransferase levels in up to 90 percent of cases once other causes of liver disease are excluded.(10)

Risk Factors and associated conditions

The insulin resistance syndrome with obesity, diabetes mellitus type 2, and hyperlipidemia are conditions frequently associated with NASH (11,12).

The reported prevalence of obesity in several series of patients with nonalcoholic fatty liver disease varied between 30 and 100 percent, the prevalence of type 2 diabetes varied between 10 and 75 percent, and the prevalence of hyperlipidemia varied between 20 and 92 percent.(1,13) The prevalence of nonalcoholic fatty liver disease increases by a factor of 4.6 in obese people, defined as those with a body-mass index (the weight in kilograms divided by the square of the height in meters) of at least 30.(14) Regardless of bodymass index, the presence of type 2 diabetes mellitus significantly increases the risk and severity of nonalcoholic fatty liver disease. (15,16) Truncal obesity seems to be an important risk factor for nonalcoholic fatty liver disease, even in patients with a normal body mass index. (17)

A family history of steatohepatitis or cryptogenic cirrhosis has also been implicated as a risk factor for this disorder. (18)

A number of other conditions have also been associated with NASH and are listed in Table 1.

Table 1

Undelying Causes of NASH

Acquired Metabolic disorders associated with steatosis: • Diabetes mellitus • Obesity • IBD • Jejuno-ileal bypass • Starvation and cachexia • Severe anemia • Protein-calorie malnutrition • Kwashiorkor and marasmus • Serum lipid abnormalities • Sudden weight loss or weight cycling • Choline deficiency

Inborn Errors of Metabolism associated with steatosis: • Abetalipoproteinemia • Familial hepatosteatosis • Galactosemia • Glycogen storage disorder • Hereditary fructose intolerance • Homocystinuria • Hypobetalipoproteinemia • Refsum disease • Systemic carnitine deficiency • Tyrosinemia • Weber-Christian disease • Wilson disease • Schwachman’s syndrome

Drugs and toxins associated with Steatosis : Metals: Antimony, Barium salts, Borates,Carbon disulfide, Chromates,Phosphorus, Thalium compounds,Uranium compounds. Antibiotics: Azaserine,Bleomycin , Puromycin, Tetracycline Cytotoxic/Cytostatic Drugs: L-Asparaginase, Azacytidine,Azauridine,Methotrexate Other Drugs: Amiodarone,Coumadin,Dichloroethylene,Ethionine,Ethyl Bromide,Estrogens,Flectol H,Glucocorticoids, Hydrazine,Hypoglycin,Orotate,Perhexilene maleate,Safrole In many cases, no discernible cause is present

Pathogenesis:

In view of the varied nature of conditions that have been associated with NASH, this disease is speculated to be the end result of several diverse insults to the liver.

The most widely supported theory implicates insulin resistance as the key mechanism leading to hepatic steatosis, and perhaps also to steatohepatitis. Others have proposed that a "second hit," or additional oxidative injury, is required to manifest the necroinflammatory component of steatohepatitis. Hepatic iron, leptin, anti-oxidant deficiencies, and intestinal bacteria have all been suggested as potential oxidative stressors.

TRIGLYCERIDE ACCUMULATION

Hepatic steatosis is a manifestation of excessive triglyceride accumulation in the liver. This can occur from the excessive importation of free fatty acids (FFA) from adipose tissue, from diminished hepatic export of FFA (secondary to reduced synthesis or secretion of VLDL), or from impaired beta-oxidation of FFA.

INSULIN RESISTANCE

Insulin resistance has a key role in the development of hepatic steatosis and, potentially, steatohepatitis. (3,19) . Obesity and type 2 diabetes, conditions associated with peripheral insulin resistance, are frequently observed in patients with NAFLD. Insulin resistance has also been observed in patients with NASH who are not obese and those who have normal glucose tolerance (20).

Further supporting the role of insulin resistance are the observations from several pilot studies, which have demonstrated beneficial effects of glucose-sensitizing medications in patients with NAFLD. Two studies of patients with steatohepatitis reported marked improvements in both aminotransferase levels and steatosis grades following short-term therapy with troglitazone and pioglitazone, respectively (21,22). Another study of 20 patients with NASH showed significant declines in aminotransferase levels and liver volume following a four-month course of metformine (23).

Despite the association, not all patients with NASH exhibit hyperinsulinemia. This suggests that NASH may be a heterogeneous syndrome with more than one cause.

Resistance to the action of insulin results in important changes in lipid metabolism. These include enhanced peripheral lipolysis, increased triglyceride synthesis (24), and increased hepatic uptake of fatty acids.

FFAs are inducers of several cytochrome p-450 microsomal lipoxygenases, capable of producing hepatotoxic free oxygen radical species (25). Furthermore, the shift to FFA beta-oxidation, in the setting of preexisting defects in mitochondrial oxidative phosphorylation, may result in increased free radical formation, hepatocellular injury, and fibrosis (26). Electron microscopy of hepatocytes from patients with NAFLD demonstrated that significant mitochondrial structural abnormalities were present in patients with NASH, but not those with simple hepatic steatosis (26).

The molecular pathway leading to insulin resistance is complex and has not been completely elucidated. Several molecules appear to be involved in interfering with the actions of insulin on a cellular level. It have been suggested that the development of hepatocellular injury requires the presence of both insulin resistance and a second defect that results in the accumulation of damaging free oxygen radical species. Several potential oxidative stressors have been proposed to result in necroinflammation.

ANTIOXIDANTS

Lipid peroxidation and free oxygen radical species can deplete antioxidant enzymes, such as glutathione, vitamin E, beta-carotene and vitamin C, thus rendering the liver susceptible to oxidative injury. There is indirect evidence supporting the role of antioxidants in preventing oxidative liver injury. Vitamin E therapy normalized serum aminotransferase elevations in children with fatty liver disease (27). In another report, a six-month course of combination therapy with vitamin E and vitamin C resulted in significant histological improvement, both with respect to inflammation and fibrosis scores (28).

IRON

Increased hepatic iron may also have a role in the development of NASH.

Insulin resistance is associated with increased hepatic iron levels (29), and improved glycemic control is associated with improvements in serum ferritin and hepatic iron concentrations (30).

The specific mechanism by which hepatic iron may contribute to necroinflammation is unknown, but may be related to the generation of free oxygen radical species that occurs in the process of reduction of Fe 3+ to Fe 2+ (31).

LEPTIN

Leptin is a peptide produced primarily in adipose tissue. Absence of leptin is associated with massive obesity in mice (ob/ob) and in humans.

Leptin may contribute to the development of fibrosis in NASH. Leptin induces dephosphorylation of insulin-receptor substrate 1, rendering hepatocytes more insulin-resistant (32). Blood leptin levels correlate with the degree of fibrosis in patients with chronic hepatitis C (33), and leptin-deficient obese mice that are exposed to a methionine-choline-deficient diet, a necroinflammatory insult, do not develop hepatic fibrosis.

INTESTINAL MICRO-ORGANISMS

Intestinal microbes have been implicated as a potential source of hepatotoxic oxidative injury. In one report, small intestinal bacterial overgrowth was observed significantly more often in patients with NASH compared with controls (34). One proposed mechanism pertains to the production of endogenous alcohol and acetaldehyde (35).

Intestinal bacteria may also contribute to hepatic injury by means of endotoxin production. Rats injected with lipopolysaccharide develop steatohepatitis while anti-TNF antibodies can improve steatosis (36).

Diagnosis

Most patients with NAFLD have no symptoms or signs of liver disease at the time of diagnosis, although many patients report fatigue or malaise and a sensation of fullness or discomfort on the right side of the upper abdomen.

Hepatomegaly is the only physical finding in most patients. Acanthosis nigricans may be found in children with nonalcoholic fatty liver disease.(37,38)

Findings of chronic liver disease and diminished numbers of platelets suggest that advanced disease with cirrhosis is present. A high proportion of patients with cryptogenic cirrhosis share many of the clinical and demographic features of patients with nonalcoholic fatty liver disease,

(39) suggesting that their cryptogenic cirrhosis is unrecognized nonalcoholic fatty liver disease.

Serum AST and ALT are elevated in almost 90 percent of patients (40). The AST/ALT ratio is usually less than 1; this is much lower than the ratio in alcoholic hepatitis, which is usually above 2 and averaged 2.85 in one report and 2.6 in another (41,42). Alkaline phosphatase is less frequently elevated and hyperbilirubinemia is uncommon (40) .

Differential diagnosis includes most chronic liver diseases. Ultrasonography often reveals a hyperechoic texture or a bright liver because of diffuse fatty infiltration (43). However, this is a nonspecific finding and should not be used to make the diagnosis of NASH. Both CT and MRI can identify steatosis but are not sufficiently sensitive to detect inflammation or fibrosis (44).

Alcoholic and drug-related liver disease can usually be diagnosed by a careful history. The following additional causes of chronic liver disease and aminotransferase elevation should be considered and evaluated with appropriate laboratory studies obtained prior to performance of a liver biopsy. This includes the workup for chronic viral hepatitis (B, C, and D), hereditary hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, primary biliary cirrhosis and autoimmune hepatitis.

Table 2

Clinical Features of NASH

Symptoms: • Mostly asymptomatic • If questioned, > 50% report: Persistent fatigue, Malaise, Upper abdominal discomfort

Signs: • Hepatomegaly is common • Splenomegaly in some • Portal hypertension unusual

Laboratory investigations: • Increase AST, ALT (Typical) • +/- increase ALP, GGT • Increased cholesterol, triglycerides, glucose • Negative viral markers and autoantibodies • Iron studies abnormal in some

Imaging: Fatty liver

Role of a liver biopsy

In the absence of definitive clinical or laboratory evidence of the above disorders, liver biopsy is the only way to confirm or exclude the diagnosis of NASH (45). Liver biopsy also permits determination of disease severity and may provide insight into prognosis. Although it is difficult to predict the severity of underlying liver disease based upon clinical or laboratory features, in one study, age over 45, obesity, and diabetes mellitus were independent predictors of liver fibrosis (46). In another study focusing on obese patients, independent predictor of fibrosis included age 50 (Odds ratio 14.1), a body mass index 28 kg/m2 (Odds ratio 5.7), triglycerides 1.7 mmol/L (Odds ratio 5), and an alanine aminotransferase concentration 2 x normal (Odds ratio 4.6) (47). Other predictive models have been described but none has been extensively validated (48).

Table 3

Fibrosis Stages of NAFLD / NASH (Brunt et al)
Stage 1	Zone 3, Pericentral vein injury, sinusoidal or peri-cellular fibrosis.
Stage 2	Zone 3, sinusoidal fibrosis and zone 1, peri-portal fibrosis
Stage 3	Bridging between zone 3 and zone 1
Stage 4	Regenerating nodules indicating fibrosis

A liver biopsy should be performed in patients suspected of having NASH who have any of the following clinical and laboratory features:

• Stigmata of chronic liver disease
• Splenomegaly
• Cytopenia
• Abnormal iron studies
• Diabetes and/or significant obesity in an individual over the age of 45

In patients who do not have these features and are obese, schedule a liver biopsy in six months to one year. Patients are instructed to lose 5 to 7 kilograms, and are encouraged to get their diabetes and hyperlipidemia under control. The liver biopsy can be deferred if liver function tests normalize afterwards

Clinical course and prognosis

Relatively few patients have been observed prospectively to document the natural history of NASH. NASH is generally considered to be a clinically stable disorder and has a markedly better prognosis than alcoholic hepatitis. Patients with NASH do not appear to have a lower life expectancy than age- and sex-matched normal controls (49). However, NASH may be an important underlying cause of cryptogenic cirrhosis, particularly among older, diabetic women (50).

In most patients, there is little change in liver function tests throughout the course of the disease. In a sizable minority, however, histologic progression occurs and a small fraction of patients progress to end-stage liver disease. Data from three studies in which a total of 28 patients underwent repeat liver biopsy found that only one patient improved, 15 (54 percent) remained unchanged and 12 (43 percent) had histologic progression over follow-up of one to seven years (40). Another report described the outcome of 132 patients with NASH followed for up to 18 years (51).

Table 4

NON-ALCOHOLIC FATTY LIVER DISEASE 4 biopsy patterns (Matteoni et al 1999) (51)
Type 1	Fat alone	benign
Type 2	Fat + lymphocytes	benign
Type 3	Fat + ballooning of heaptocytes	some progress to cirrhosis
Type 4	Fat + fibrosis/Mallory’s hyaline	some progress to cirrhosis

Progression to cirrhosis was much more likely in patients whose initial biopsies demonstrated ballooning degeneration and Mallory hyaline or fibrosis than patients with steatosis alone (26 versus 4 percent).

With the exception of the histologic features discussed above, no clinical or laboratory features can predict progression in any given patient. The degree of obesity does not significantly alter the clinical course and the effect of weight loss is variable (52,53).

Treatment

There is no proven effective therapy for NASH. Attempts are made to modify potential risk factors such as obesity, hyperlipidemia, and poor diabetic control. Weight reduction should be gradual, as rapid weight loss has been associated with worsening of liver disease (52,53). A report suggested that weight loss should not exceed approximately 1.6 Kilograms per week in adults (54).

Other potential treatments have been described, none is used routinely for clinical practice.

Vitamin E. Oxidative stress is suggested to play a significant role in progression of steatosis to NASH. The use of antioxidants, such as vitamin E, is intriguing. Previous investigations have demonstrated that α-tocopherol inhibited production of cytokines by leukocytes, ex vivo (55). Furthermore,TGF-β gene expression is inhibited in an animal model (56). A recent human trial with _-tocopherol, in doses up to 300 mg per day, decreased TGF-_ levels and improved inflammation and fibrosis in a majority of patients (57). Data from a double-blind, randomized, prospective study using 1000 IU of vitamin E and 1000 mg of vitamin C in 42 patients was presented at the most recent Digestive Disease Week (58). Results revealed a small but significant decrease in fibrosis in patients in the vitamin group.

Ursodeoxycholic acid. This drug is now considered standard for diseases such as primary biliary cirrhosis and primary sclerosing cholangitis. However, its role in the treatment of NASH is less convincing. Two small studies have demonstrated normalization of ALT but no histological improvement on repeat liver biopsy (59, 60).

Metformin. The insulin resistance observed in many patients with NASH prompted a trial of metformin, a hypoglycemic agent, in an animal model of NASH (61). Treatment was associated with histologic and biochemical improvement and reversal of hepatomegaly. The mechanism was hypothesized to be related to inhibition of hepatic expression of tumor necrosis factor. A possible benefit was also suggested in an open-label trial in humans (23).

Pioglitazone — The efficacy of pioglitazone plus vitamin E was compared to vitamin E alone in a pilot controlled trial (reported only as an abstract) involving 21 patients (62). After six months, no significant histologic changes were observed in the vitamin E group. In contrast, significant decreases in fat, cytologic ballooning, and Mallory hyaline were seen in the combination group. Mean serum ALT decreased significantly in both groups. No side effects were observed in the vitamin E group. One patient in the combination group had an increase in serum ALT to the 400 IU range, prompting withdrawal from the study.

A second study (also reported as an abstract) focused on 20 patients with NASH but without diabetes who were treated with pioglitazone (30 mg daily) for 48 weeks (63). In an interim report, the serum ALT declined in nine patients who had completed the study (normalizing in seven). End-of-treatment liver biopsies showed improvement in steatosis, Mallory bodies, and parenchymal inflammation. The main side effect of treatment was weight gain, which averaged 3.5 kg.

Probucol — Probucol (a lipid lowering agent with antioxidant properties) was associated with a significant reduction in serum aminotransferases in a pilot, randomized controlled trial (64). The effect on liver histology was not assessed. Probucol is not available in the United States.

Betaine. This drug, which functions as a methyl donor to generate lecithin, has recently been evaluated by the Mayo Clinic group in a small pilot trial (65). Ten patients with biopsy-proven NASH were treated with betaine daily for 12 months. Of the seven who completed the trial, ALT levels had normalized in three of the seven and improved by 50% in another three patients. More importantly, paired liver biopsies were available for six patients, demonstrating an improvement in steatosis, necroinflammatory activity, and fibrosis.

Summary of treatment is shown in table 5.

Table 5

Traetment of NASH

Dieting

Promising agents: • ursodeoxycolic acid • Anti-oxidants (vitamin E, glutathione pro-drugs) • betaine

Potential beneficial agents: • Statins (Lipid lowering agents) • Itazones (Insulin sensitizer) • Metformin (Insulin sensitizer) • S-adenosyl methionine (SAM) • Anti- Cytokines (Anti TNF antibodies, soluble receptors) • Fibrates (Lipid lowering agents)

Other therapeutic modalities: • Ursodeoxycholic acid • Calorie-free amino acid infusions • Metronidazole

Surgical Treatment : Surgical weight loss (gastric bypass)

Conclusion

NASH is a chronic liver disease characterized by diffuse fatty infiltration, lobular inflammation, and perisinusoidal fibrosis. It is thought to be caused by metabolic derangements in fatty acid metabolism to include insulin resistance.

The exact prevalence of NASH is variable, but it is much more common than previously thought. Although generally a benign, indolent disease, it can progress to significant liver disease in approximately 15–20% of patients. Risk factors associated with NASH include obesity, diabetes mellitus, hyperlipidemia, and hypertension, which are associated with increased insulin resistance.

The complete pathogenesis of NASH has not been elucidated, but progress is being made.

Treatment options are being studied, aimed at improving insulin resistance and mitochondrial energy homeostasis. There are limited data for a variety of treatment options to include controlled weight loss, glycemic control in diabetics, ursodeoxycholic acid, phlebotomy, antioxidants, control of hyperlipidemia, and replacement of important substrates necessary for energy homeostasis. Prospective, doubleblind, placebo-controlled trials are underway evaluating HMG-CoA reductase inhibitors, antioxidants, metformin, thiazolidinediones, and other promising agents. It is likely that treatment of this disease will need to be multifactorial, targeting therapy at improving lipid homeostasis through improving insulin resistance as well as altering the specific interplay of protein mitogen kinases and cytokines that allow for cellular inflammation, necrosis, apoptosis, and fibrogenesis to occur(66).

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