HEPATOLOGY Concise Review

Hepatic Steatosis: InnocentBystander or Guilty Party?

CHRISTOPHER P. DAY AND OLIVER F. W. JAMES

Fatty liver (steatosis) is a common histological finding inhuman liver biopsies which is most often attributed to theeffects of alcohol excess, obesity, diabetes, or drugs.1 Thedistribution of lipid can be macrovesicular, with hepatocytesbeing distended by a single vacuole displacing the nucleus orit can be microvesicular with numerous droplets surroundinga centrally placed nucleus. Widespread microvesicular steato-sis is characteristically an acute condition in which impair-ment of fatty acid β-oxidation2 reflects a more generalperturbation of mitochondrial and ribosomal function bothwithin and outside the liver.3 Regardless of the etiology,microvesicular steatosis is widely acknowledged as having apoor prognosis with death caused by both liver failure andextra-hepatic causes. Macrovesicular steatosis, by contrast, istypically associated with a more long-standing disturbance ofhepatic lipid metabolism and has, until recently, been consid-ered a benign condition.4 There is now little doubt thatmacrovesicular steatosis of both alcohol and nonalcohol-related etiologies is associated with the development of moreadvanced disease: necroinflammation (steatohepatitis), fibro-sis, and cirrhosis.5 The critical question, and the subject ofthis review, is whether steatosis per se plays any direct causalrole in disease progression or is simply an ‘‘innocent by-stander’’ to other mechanisms of inflammation and scarring.

STEATOSIS SEVERITY AND PROGRESSIONTO ADVANCED DISEASE

The first indirect evidence supporting a role for steatosis inthe pathogenesis of advanced disease comes from studies onthe natural history of alcoholic liver disease (ALD). In a largeprospective study of men with alcohol-related steatosis,Sorensen et al. demonstrated that the severity of steatosis oninitial liver biopsy predicted the development of cirrhosis ona subsequent biopsy 10 years later, independent of the level ofcontinuing alcohol intake.6 We have recently confirmed thisfinding in an 11-year follow-up study of alcoholic men andwomen by demonstrating correlations between the severity ofsteatosis and the presence of a mixed macro/microvesicularpattern of steatosis, which presumably reflects a more severeand rapid accumulation of triglyceride,1 and the subsequentdevelopment of fibrosis/cirrhosis.7 We have also shown acorrelation between the severity of steatosis and the degree of

hepatic stellate cell activation in livers from alcoholics withno evidence of alcoholic hepatitis or cirrhosis.8 Hepaticstellate cells are the principal cells involved in hepaticfibrogenesis.

Further circumstantial evidence supporting a role forsteatosis in disease progression comes from the observationthat steatosis of most nonalcoholic etiologies is also associ-ated with the development of necroinflammation (so-callednon-alcoholic steatohepatitis or NASH). The causes of NASHare, therefore, the same as those of simple steatosis andinclude obesity, noninsulin-dependent diabetes mellitus, je-juno-ileal bypass/gastroplasty surgery, parenteral nutrition,bacterial contamination of the small bowel, and drugs.1 Arecent compilation of several independent studies of NASHreveals a 21% incidence of fibrosis and a 15% incidence ofcirrhosis at index biopsy with a 43% risk of fibrosis progres-sion.5 Importantly, as in alcoholics, one of the main factorsthat correlates with steatohepatitis and fibrosis in nonalcohol-ics is the severity of steatosis.9

The correlation between steatosis severity and necroinflam-mation/fibrosis in alcoholics and nonalcoholics does notprove that fat per se is causal in the development of moreadvanced pathology. An alternative explanation is that in allforms of fatty liver, common mechanisms are responsible forboth steatosis and necroinflammation/fibrosis, with the sever-ity of the steatosis acting as a surrogate marker for theintensity of the stimulus. A wide variety of mechanisms havebeen implicated in the necroinflammation attributed toexcessive alcohol intake.10 More recently, evidence hasemerged that at least two of these play a putative role in thepathogenesis of NASH: 1) oxidative stress/lipid peroxidationand 2) endotoxin-mediated cytokine release. These observa-tions not only provide an explanation for the strikinghistological similarity between NASH and alcoholic steato-hepatitis,11 but, more importantly, they implicate steatosis asa direct contributor to inflammation and/or fibrosis. First, wewill review the evidence supporting a role for oxidative stressand endotoxin/cytokines in the development of alcoholic andNASH. Then, we will discuss how these mechanisms mightbe invoked to explain the association between steatosis andadvanced liver disease.

OXIDATIVE STRESS AND LIPID PEROXIDATION

The concept that free radical-mediated oxidative stresscontributes to the pathogenesis of alcohol-induced liverinjury has gained broad acceptance. Ethanol metabolism byseveral enzyme systems results in the formation of reactiveoxygen species and carbon-centered free radicals capable ofinitiating peroxidation of the polyunsaturated fatty acid sidechains of membrane phospholipids.12 The ethanol-induciblecytochrome P450 2E1 (CYP2E1) seems particularly impor-tant in this respect, because dietary manipulations which leadto maximal enzyme induction increase disease severity13 andCYP2E1 inhibitors ameliorate liver injury and reduce lipidperoxidation in animal models of ALD.14 The membranedisruption injures cells directly, whereas the aldehyde end-products of lipid peroxidation, malondialdehyde, and4-hydroxynonenal are important pro-inflammatory media-tors15 and may also activate hepatic stellate cells.16 Lipidperoxidation may also result in immunological injury; malon-dialdehyde is capable of forming protein adducts, which have

Abbreviations: NASH, non-alcoholic steatohepatitis.From Centre for Liver Research, Floor 4, William Leech Building Medical School,

Framlington Place, Newcastle upon Tyne, UK.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.

Copyright r 1998 by the American Association for the Study of Liver Diseases.0270-9139/98/2706-0001$3.00/0

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been found in the livers of animals fed alcohol17 and inpatients with chronic liver disease of alcoholic and nonalco-holic etiology.18 Antibodies to these adducts are measurablein the serum of ethanol-fed rats,14 suggesting that they arecapable of initiating a potentially injurious immune response.

Lipid peroxidation has also been demonstrated in nonalco-holic forms of fatty liver.19-21 The precise stimulus to lipidperoxidation in nonalcoholic models of liver disease is notentirely clear, but several potential sources of free radicals doexist. For example, animals with nonalcoholic fatty liver andpatients with NASH have increased expression of CYP2E1,22,23

which may be induced by both fatty acids and ketones24 andwhich is capable of generating free radicals from endogenousmetabolites and dietary N-nitrosamines. We have previouslysuggested that all of the risk factors for steatohepatitis/fibrosisin patients with obesity-related steatosis (rapid weight losscaused by dieting or surgery, surgical stress, alcohol intake,and diabetes) have in common an increase in the concentra-tion of fatty acids and/or ketones within the liver.1 If theincrease in fatty acid concentration is enough to saturatemitochondrial β-oxidation, then peroxisomal β-oxidationwill provide a further source of oxidative stress by generatinghydrogen peroxide, which is converted to the highly reactivehydroxyl radical in the presence of free iron.25 A role for ironin the pathogenesis of NASH is further supported by datafrom a recent study showing that the C282Y mutation in thehaemochromatosis gene, HFE, is over-represented in patientswith NASH.26 Finally, the oxidative stress may come from anexogenous source, as exemplified by a report from Brazil onworkers 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 beenreviewed elsewhere.28 Endotoxin/lipopolysaccharide is in-creased in the serum of alcoholics caused by increased gutpermeability and impaired reticuloendothelial function. Thehepatotoxicity of lipoploysaccharide is mediated through therelease of cytokines, principally tumor necrosis factor a(TNFa), the levels of which correlate with mortality andimpaired liver function in patients with alcoholic steatohepa-titis. A role for endotoxin in the pathogenesis of NASH wasfirst suggested by the high incidence of NASH and cirrhosis inpatients following jejuno-ilieal bypass surgery for obesity.The surgery itself leads to portal endotoxemia,29 and the riskof NASH in the postoperative period is reduced by antibiotictreatment.30 More recently, Diehl’s group have shown thatgenetically obese mice with severe steatosis have a muchgreater sensitivity to endotoxin than their lean controls,rapidly developing NASH after exposure to low doses oflipopolysaccharide.31 The obese mice also have impairmentin the phagocytic function of Kupffer cells which, by permit-ting chronic low-grade systemic endotoxemia, might beresponsible for the increased basal adipose tissue expressionof TNFa messenger RNA observed in obesity.32 This produc-tion of TNFa by adipose tissue has been proposed as animportant mechanism of peripheral insulin resistance inobesity,33 but clearly the increased levels of circulating TNFain obesity34 might potentially contribute to the pathogenesisof NASH in these patients.

STEATOSIS, NECROINFLAMMATION AND FIBROSIS:A CAUSAL LINK?

How might oxidative stress and endotoxin/cytokines ex-plain the association between the severity of steatosis andadvanced disease? One possibility is that these factors playindependent roles in the pathogenesis of steatosis and necro-inflammation/fibrosis. Free fatty acids, which are capable ofinitiating oxidative stress, also provide the substrate forincreased synthesis of triglyceride; indeed, any trigger ofoxidative stress, by damaging mitochondrial DNA, may leadto an increase in substrate supply through impaired mitochon-drial β-oxidation of fatty acids.35 This notion is supported byan early report that the antioxidant vitamin E may preventalcohol-induced fatty liver.36 The peroxidation end-products,malondialdehyde and 4-hydroxynonenal, may also contrib-ute to the pathogenesis of steatosis by impairing the export oftriglyceride from the liver by forming adducts with tubulin inmicrotubules.1 Theories such as these which implicate oxi-dant 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 correla-tion between steatosis severity and the risk of advanceddisease is that the presence of steatosis influences the liver’sresponse to at least some of the important triggers ofnecroinflammation and/or fibrosis. Clearly the more fat in theliver, the greater the pool of substrate available for anyoxidative stress to initiate lipid peroxidation. Recent studieshave shown a striking correlation between the degree of lipidperoxidation and the severity of steatosis in both animal andhuman models with alcohol and nonalcohol-related fattyliver.19,21,37 The increase in lipid peroxidation would generatemore potentially reactive and cytotoxic intermediates whichare capable of inducing inflammation and fibrosis at least inpart via activation of NF-kB38 and/or immunological mecha-nisms. The increased sensitivity of steatotic livers to endo-toxin-induced necroinflammation31 provides a further mech-anism by which the severity of steatosis may play a direct rolein the pathogenesis of advanced disease. Finally, a recentstudy demonstrating that hepatocytes isolated from alcoholicfatty liver have increased sensitivity to anoxic injury39 sug-gests that the severity of steatosis may also influence themagnitude of hypoxic liver damage. Centrilobular hypoxiahas long been considered an important mechanism of injuryin alcoholic liver disease.10 As one component of the in-creased oxygen consumption in alcoholic liver disease isCYP2E1 induction, the increased expression and predomi-nantly centrilobular distribution of this enzyme in NASH22

suggests that centrilobular hypoxia may also play a role innonalcoholic fatty liver. Perhaps the best example of theincreased susceptibility of fatty liver to the necroinflamma-tory triggers of oxidative stress, endotoxin and hypoxia, is thehigh incidence of delayed and primary nonfunction observedwhen severe fatty livers are used for orthotopic transplanta-tion.40 Following periods of warm and cold ischemia/hypoxia, graft reperfusion is associated with oxidative stressand the arrival of gut-derived endotoxin, all three of whichwill be poorly tolerated by the donor fatty liver for the reasonswe have outlined.

Therefore, we would suggest that the correlation betweenthe severity of steatosis and the risk of advanced liver diseaseis at least, in part, explained by steatosis increasing the

1464 DAY AND JAMES HEPATOLOGY June 1998

sensitivity of the liver to the triggers of necroinflammationand fibrosis common to both alcoholic steatohepatitis andNASH, as follows: oxidative stress, endotoxin, and possiblyhypoxia. Can this hypothesis account for the clear differencesin the incidence and prognosis of advanced liver disease inalcoholic and nonalcoholic steatosis? There is little doubtthat in alcoholics and nonalcoholics with similar degrees ofsteatosis, the alcoholics more commonly have necroinflamma-tion and fibrosis and are more likely to progress to cirrhosis.One explanation for this is that the important triggers ofinflammation/fibrosis outlined earlier are more likely to bepersistent, to be of greater magnitude, and to range morewidely in alcoholics as compared with nonalcoholics. Onemight expect chronic alcoholics with heavy daily consump-tion and fatty liver to be subject to constant and severeoxidative stress (driven by ethanol metabolism) and/or endo-toxemia (caused by increased gut permeability). In contrast,individuals with nonalcoholic fatty liver may experienceeither no episodes or intermittent and milder episodes ofoxidative damage because the causes of oxidative stress(sudden weight loss, poor diabetic control, or environmentalexposure) will only occur in some patients and may notpersist. Intermittent episodes of endotoxemia may also occa-sionally cause steatohepatitis in patients with nonalcoholicfatty liver; in this context it is interesting that jejuno-ilealbypass, which is characterized by persistent portal endotox-emia, poses the greatest risk of disease progression among allcauses of NASH.29 A second and more obvious explanationfor the higher incidence and worse prognosis of steatohepati-tis in alcoholics is that there may be many other mechanismsof necroinflammation/fibrosis operating in heavy drinkersthat are unrelated to either the presence or the severity ofsteatosis.

CONCLUSIONS

Our understanding of steatosis has advanced considerablyin recent years. Careful clinical studies have challengedprevious assertions that macrovesicular steatosis indicates anentirely benign prognosis; the studies demonstrate clearlythat fatty liver of either alcoholic or nonalcoholic etiologiescan coincide with or lead to necroinflammation and fibrosis.Experimental studies in both human beings and animalmodels have at last begun to unravel the pathogenic mecha-nisms responsible for disease progression. In doing so, thestudies have, perhaps surprisingly, implicated steatosis itselfas a direct cause of more advanced pathology. This informa-tion provides a basis for evaluating the prognosis of patientswith steatosis and a rationale for their management. Individu-als with severe steatosis, particularly those with a mixedpattern7 and early evidence of steatohepatitis, appear to be atthe greatest risk of disease progression. Management strate-gies should ideally be directed at reducing the severity ofsteatosis and at avoiding and removing the triggers ofnecroinflammation and fibrosis. Specific treatment modali-ties for ‘‘at-risk’’ patients might include antioxidants, inhibi-tors of peroxisomal β-oxidation, CYP2E1, or TNFa, andantibiotics.

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