HEPATOLOGY, June 1998, p. 1463-1466, Vol. 27, No. 6
HEPATOLOGY Concise Review
Hepatic Steatosis: Innocent Bystander or Guilty Party?

Christopher P. Day and Oliver F. W. James

From Centre for Liver Research, Floor 4, William Leech Building Medical School, Framlington Place, Newcastle upon Tyne, UK.

INTRODUCTION

Fatty liver (steatosis) is a common histological finding in human liver biopsies which is most often attributed to the effects of alcohol excess, obesity, diabetes, or drugs.¹ The distribution of lipid can be macrovesicular, with hepatocytes being distended by a single vacuole displacing the nucleus or it can be microvesicular with numerous droplets surrounding a centrally placed nucleus. Widespread microvesicular steatosis is characteristically an acute condition in which impairment of fatty acid -oxidation² reflects a more general perturbation of mitochondrial and ribosomal function both within and outside the liver.³Regardless of the etiology, microvesicular steatosis is widely acknowledged as having a poor prognosis with death caused by both liver failure and extra-hepatic causes. Macrovesicular steatosis, by contrast, is typically associated with a more long-standing disturbance of hepatic lipid metabolism and has, until recently, been considered a benign condition.⁴ There is now little doubt that macrovesicular steatosis of both alcohol and nonalcohol-related etiologies is associated with the development of more advanced disease: necroinflammation (steatohepatitis), fibrosis, and cirrhosis.⁵ The critical question, and the subject of this review, is whether steatosis per se plays any direct causal role in disease progression or is simply an “innocent bystander” to other mechanisms of inflammation and scarring.

STEATOSIS SEVERITY AND PROGRESSION TO ADVANCED DISEASE

The first indirect evidence supporting a role for steatosis in the pathogenesis of advanced disease comes from studies on the natural history of alcoholic liver disease (ALD). In a large prospective study of men with alcohol-related steatosis, Sorensen et al. demonstrated that the severity of steatosis on initial liver biopsy predicted the development of cirrhosis on a subsequent biopsy 10 years later, independent of the level of continuing alcohol intake.⁶ We have recently confirmed this finding in an 11-year follow-up study of alcoholic men and women by demonstrating correlations between the severity of steatosis and the presence of a mixed macro/microvesicular pattern of steatosis, which presumably reflects a more severe and rapid accumulation of triglyceride,¹ and the subsequent development of fibrosis/cirrhosis.⁷ We have also shown a correlation between the severity of steatosis and the degree of hepatic stellate cell activation in livers from alcoholics with no evidence of alcoholic hepatitis or cirrhosis.⁸ Hepatic stellate cells are the principal cells involved in hepatic fibrogenesis.

Further circumstantial evidence supporting a role for steatosis in disease progression comes from the observation that steatosis of most nonalcoholic etiologies is also associated with the development of necroinflammation (so-called non-alcoholic steatohepatitis or NASH). The causes of NASH are, therefore, the same as those of simple steatosis and include obesity, noninsulin-dependent diabetes mellitus, jejuno-ileal bypass/gastroplasty surgery, parenteral nutrition, bacterial contamination of the small bowel, and drugs.¹ A recent compilation of several independent studies of NASH reveals a 21% incidence of fibrosis and a 15% incidence of cirrhosis at index biopsy with a 43% risk of fibrosis progression.⁵ Importantly, as in alcoholics, one of the main factors that correlates with steatohepatitis and fibrosis in nonalcoholics is the severity of steatosis.⁹

The correlation between steatosis severity and necroinflammation/fibrosis in alcoholics and nonalcoholics does not prove that fat per se is causal in the development of more advanced pathology. An alternative explanation is that in all forms of fatty liver, common mechanisms are responsible for both steatosis and necroinflammation/fibrosis, with the severity of the steatosis acting as a surrogate marker for the intensity of the stimulus. A wide variety of mechanisms have been implicated in the necroinflammation attributed to excessive alcohol intake.¹⁰ More recently, evidence has emerged that at least two of these play a putative role in the pathogenesis of NASH: 1) oxidative stress/lipid peroxidation and 2) endotoxin-mediated cytokine release. These observations not only provide an explanation for the striking histological similarity between NASH and alcoholic steatohepatitis,¹¹ but, more importantly, they implicate steatosis as a direct contributor to inflammation and/or fibrosis. First, we will review the evidence supporting a role for oxidative stress and endotoxin/cytokines in the development of alcoholic and NASH. Then, we will discuss how these mechanisms might be invoked to explain the association between steatosis and advanced liver disease.

OXIDATIVE STRESS AND LIPID PEROXIDATION

The concept that free radical-mediated oxidative stress contributes to the pathogenesis of alcohol-induced liver injury has gained broad acceptance. Ethanol metabolism by several enzyme systems results in the formation of reactive oxygen species and carbon-centered free radicals capable of initiating peroxidation of the polyunsaturated fatty acid side chains of membrane phospholipids.¹² The ethanol-inducible cytochrome P450 2E1 (CYP2E1) seems particularly important in this respect, because dietary manipulations which lead to maximal enzyme induction increase disease severity¹³ and CYP2E1 inhibitors ameliorate liver injury and reduce lipid peroxidation in animal models of ALD.¹⁴ The membrane disruption injures cells directly, whereas the aldehyde end-products of lipid peroxidation, malondialdehyde, and 4-hydroxynonenal are important pro-inflammatory mediators¹⁵ and may also activate hepatic stellate cells.¹⁶ Lipid peroxidation may also result in immunological injury; malondialdehyde is capable of forming protein adducts, which have been found in the livers of animals fed alcohol¹⁷ and in patients with chronic liver disease of alcoholic and nonalcoholic etiology.¹⁸ Antibodies to these adducts are measurable in the serum of ethanol-fed rats,¹⁴ suggesting that they are capable of initiating a potentially injurious immune response.

Lipid peroxidation has also been demonstrated in nonalcoholic forms of fatty liver.^19-21 The precise stimulus to lipid peroxidation in nonalcoholic models of liver disease is not entirely clear, but several potential sources of free radicals do exist. For example, animals with nonalcoholic fatty liver and patients with NASH have increased expression of CYP2E1,^22,23 which may be induced by both fatty acids and ketones²⁴ and which is capable of generating free radicals from endogenous metabolites and dietary N-nitrosamines. We have previously suggested that all of the risk factors for steatohepatitis/fibrosis in patients with obesity-related steatosis (rapid weight loss caused by dieting or surgery, surgical stress, alcohol intake, and diabetes) have in common an increase in the concentration of fatty acids and/or ketones within the liver.¹ If the increase in fatty acid concentration is enough to saturate mitochondrial -oxidation, then peroxisomal -oxidation will provide a further source of oxidative stress by generating hydrogen peroxide, which is converted to the highly reactive hydroxyl radical in the presence of free iron.²⁵ A role for iron in the pathogenesis of NASH is further supported by data from a recent study showing that the C282Y mutation in the haemochromatosis gene, HFE, is over-represented in patients with NASH.²⁶ Finally, the oxidative stress may come from an exogenous source, as exemplified by a report from Brazil on workers who deveoped NASH at a petrochemical factory.²⁷

ENDOTOXIC-CYTOKINE MEDIATED INJURY

Several lines of evidence suggest a role for endotoxin-mediated cytokine release in ALD and these have been reviewed elsewhere.²⁸Endotoxin/lipopolysaccharide is increased in the serum of alcoholics caused by increased gut permeability and impaired reticuloendothelial function. The hepatotoxicity of lipoploysaccharide is mediated through the release of cytokines, principally tumor necrosis factor  (TNF), the levels of which correlate with mortality and impaired liver function in patients with alcoholic steatohepatitis. A role for endotoxin in the pathogenesis of NASH was first suggested by the high incidence of NASH and cirrhosis in patients following jejuno-ilieal bypass surgery for obesity. The surgery itself leads to portal endotoxemia,²⁹ and the risk of NASH in the postoperative period is reduced by antibiotic treatment.³⁰ More recently, Diehl’s group have shown that genetically obese mice with severe steatosis have a much greater sensitivity to endotoxin than their lean controls, rapidly developing NASH after exposure to low doses of lipopolysaccharide.³¹ The obese mice also have impairment in the phagocytic function of Kupffer cells which, by permitting chronic low-grade systemic endotoxemia, might be responsible for the increased basal adipose tissue expression of TNF messenger RNA observed in obesity.³² This production of TNF by adipose tissue has been proposed as an important mechanism of peripheral insulin resistance in obesity,³³ but clearly the increased levels of circulating TNF in obesity³⁴ might potentially contribute to the pathogenesis of NASH in these patients.

STEATOSIS, NECROINFLAMMATION AND FIBROSIS: A CAUSAL LINK?

How might oxidative stress and endotoxin/cytokines explain the association between the severity of steatosis and advanced disease? One possibility is that these factors play independent roles in the pathogenesis of steatosis and necroinflammation/fibrosis. Free fatty acids, which are capable of initiating oxidative stress, also provide the substrate for increased synthesis of triglyceride; indeed, any trigger of oxidative stress, by damaging mitochondrial DNA, may lead to an increase in substrate supply through impaired mitochondrial -oxidation of fatty acids.³⁵ This notion is supported by an early report that the antioxidant vitamin E may prevent alcohol-induced fatty liver.³⁶ The peroxidation end-products, malondialdehyde and 4-hydroxynonenal, may also contribute to the pathogenesis of steatosis by impairing the export of triglyceride from the liver by forming adducts with tubulin in microtubules.¹ Theories such as these which implicate oxidant stress as a cause of steatosis are quite plausible; however, it is much more difficult to invoke a role for endotoxin/cytokines in the pathogenesis of steatosis.

A second and more intriguing explanation for the correlation between steatosis severity and the risk of advanced disease is that the presence of steatosis influences the liver’s response to at least some of the important triggers of necroinflammation and/or fibrosis. Clearly the more fat in the liver, the greater the pool of substrate available for any oxidative stress to initiate lipid peroxidation. Recent studies have shown a striking correlation between the degree of lipid peroxidation and the severity of steatosis in both animal and human models with alcohol and nonalcohol-related fatty liver.^19,21,37 The increase in lipid peroxidation would generate more potentially reactive and cytotoxic intermediates which are capable of inducing inflammation and fibrosis at least in part via activation of NF-B³⁸ and/or immunological mechanisms. The increased sensitivity of steatotic livers to endotoxin-induced necroinflammation³¹ provides a further mechanism by which the severity of steatosis may play a direct role in the pathogenesis of advanced disease. Finally, a recent study demonstrating that hepatocytes isolated from alcoholic fatty liver have increased sensitivity to anoxic injury³⁹ suggests that the severity of steatosis may also influence the magnitude of hypoxic liver damage. Centrilobular hypoxia has long been considered an important mechanism of injury in alcoholic liver disease.¹⁰ As one component of the increased oxygen consumption in alcoholic liver disease is CYP2E1 induction, the increased expression and predominantly centrilobular distribution of this enzyme in NASH²² suggests that centrilobular hypoxia may also play a role in nonalcoholic fatty liver. Perhaps the best example of the increased susceptibility of fatty liver to the necroinflammatory triggers of oxidative stress, endotoxin and hypoxia, is the high incidence of delayed and primary nonfunction observed when severe fatty livers are used for orthotopic transplantation.⁴⁰ Following periods of warm and cold ischemia/hypoxia, graft reperfusion is associated with oxidative stress and the arrival of gut-derived endotoxin, all three of which will be poorly tolerated by the donor fatty liver for the reasons we have outlined.

Therefore, we would suggest that the correlation between the severity of steatosis and the risk of advanced liver disease is at least, in part, explained by steatosis increasing the sensitivity of the liver to the triggers of necroinflammation and fibrosis common to both alcoholic steatohepatitis and NASH, as follows: oxidative stress, endotoxin, and possibly hypoxia. Can this hypothesis account for the clear differences in the incidence and prognosis of advanced liver disease in alcoholic and nonalcoholic steatosis? There is little doubt that in alcoholics and nonalcoholics with similar degrees of steatosis, the alcoholics more commonly have necroinflammation and fibrosis and are more likely to progress to cirrhosis. One explanation for this is that the important triggers of inflammation/fibrosis outlined earlier are more likely to be persistent, to be of greater magnitude, and to range more widely in alcoholics as compared with nonalcoholics. One might expect chronic alcoholics with heavy daily consumption and fatty liver to be subject to constant and severe oxidative stress (driven by ethanol metabolism) and/or endotoxemia (caused by increased gut permeability). In contrast, individuals with nonalcoholic fatty liver may experience either no episodes or intermittent and milder episodes of oxidative damage because the causes of oxidative stress (sudden weight loss, poor diabetic control, or environmental exposure) will only occur in some patients and may not persist. Intermittent episodes of endotoxemia may also occasionally cause steatohepatitis in patients with nonalcoholic fatty liver; in this context it is interesting that jejuno-ileal bypass, which is characterized by persistent portal endotoxemia, poses the greatest risk of disease progression among all causes of NASH.²⁹ A second and more obvious explanation for the higher incidence and worse prognosis of steatohepatitis in alcoholics is that there may be many other mechanisms of necroinflammation/fibrosis operating in heavy drinkers that are unrelated to either the presence or the severity of steatosis.

CONCLUSIONS

Our understanding of steatosis has advanced considerably in recent years. Careful clinical studies have challenged previous assertions that macrovesicular steatosis indicates an entirely benign prognosis; the studies demonstrate clearly that fatty liver of either alcoholic or nonalcoholic etiologies can coincide with or lead to necroinflammation and fibrosis. Experimental studies in both human beings and animal models have at last begun to unravel the pathogenic mechanisms responsible for disease progression. In doing so, the studies have, perhaps surprisingly, implicated steatosis itself as a direct cause of more advanced pathology. This information provides a basis for evaluating the prognosis of patients with steatosis and a rationale for their management. Individuals with severe steatosis, particularly those with a mixed pattern⁷ and early evidence of steatohepatitis, appear to be at the greatest risk of disease progression. Management strategies should ideally be directed at reducing the severity of steatosis and at avoiding and removing the triggers of necroinflammation and fibrosis. Specific treatment modalities for “at-risk” patients might include antioxidants, inhibitors of peroxisomal -oxidation, CYP2E1, or TNF, and antibiotics.

Footnotes

Abbreviations: NASH, non-alcoholic steatohepatitis.

Received February 18, 1998; accepted April 2, 1998.

Address reprint requests to: Christopher P. Day, Ph.D., M.D., Centre for Liver Research, Floor 4, William Leech Building, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH UK. Fax: 44-191-222-0723.

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Copyright © 1998 by the American Association for the Study of Liver Diseases.

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