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  Hepatitis C Virus and Liver Steatosis:
Is It the Virus? Yes It Is, but Not Always

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The mechanisms by which the hepatitis C virus (HCV) causes chronic, progressive liver damage are unknown. The lack of correlation between intrahepatic HCV RNA level and necroinflammation inchronic hepatitis C suggests that the HCV-associated liver damage is mostly immunomediated. None the less, some histopathologic features, such as liver steatosis, are suggestive of a cytopathic effect. Even when the most common causes of steatosis are excluded, fatty metamorphosis is present in about 30% of chronic hepatitis C patients, a finding that is diagnostically relevant.2Several lines of evidence indicate that the association between HCV and steatosis is not spurious and that HCV is indeed the culprit. The association with the genotype ,first reported in 1997,3raised the possibility of a direct effect of specific viral sequences on the pathogenesis of lipid accumulation. This observation, somehow unnoticed for some time, was subsequently confirmed by several groups.4-7Another piece of evidence came from the observation that the intrahepatic level of the negative strand HCV RNA4correlated with the severity of steatosis. Finally, anecdotal reports showed that response to antiviral treatment, whether temporary8or sustained,4was associated with the disappearance of fat from the liver and that a relapse of HCV infection and disease was par-alleled by a steatosis recurrence.8These observations ledus to identify hepatocyte steatosis as the morphologic correlate of a cytopathic effect caused by HCV.4The Virus and Hepatocyte Steatosis In vitrostudies9and the transgenic mouse model10have both shown that the HCV core protein can induce steatosis by itself. Electron microscopy9and indirect immunofluorescence combined with oil red O lipid stain-ing11have shown that, upon transfection of several cellslines, the HCV core protein colocalizes with cytoplasmicglobular lipid structures. The component of lipid drop lets interacting with the core protein is still a matter of debate. Although some studies have identified an interaction withthe apoAII,9,12a major component of high-density li-poprotein (HDL), the observation that chronic hepatitis C patients have decreased serum levels of-lipoproteins5rather suggests an interference with the assembly of very low­density lipoprotein (VLDL). Serum levels of apoB (a component of VLDL) are in fact decreased in chronic hepatitis C and inversely correlated with the steatosiss core, whereas a successful antiviral treatment results in return to normal levels.5An interference with the VLDL assembly is also consistent with the observation that thecore protein reduces the activity of the microsomal tri-glycerides transfer protein (MTP).13Although a direct interaction with the MTP is unlikely, as it would require the secretion of the core protein into the endoplasmicreticulum lumen, which has not been unequivocally re-ported, the MTP inhibition may still be mediated by un-known cofactors. However, mice made transgenic with the HCV core protein have normal apoB levels,13at variance with chronic hepatitis C patients,5rendering this whole scenario somewhat speculative. Recently, the core protein expression was reported to cause steatosis14 in the transgenic mice viamitochondrial toxicity and production of reactive oxygen species.15This is an attractive model, as it may explain some clinical consequences of chronic HCV infection, such as fibrosis and oncogenesis. As expected, things are not so simple, since a recent work reported that also the nonstructural protein 5A, together with the core protein, may contribute to fat accumulation in a human hepatoma cell line16viaan interaction with apoA1. Thus, the fine details of the interaction between HCV and lipids remain unclear. Hepatocytes are at the crossroad of several pathways of lipid metabolism, and HCV may interfere with one or several of them simultaneously. Genotype 3 and Hepatic Steatosis Some recent observations suggest that the pathogenesis of the mild steatosis seen in most HCV-infected patients may be metabolic, since its severity correlates with the body mass index.6In contrast, only the steatosis of mod-Abbreviations: HCV, hepatitis C virus; HDL, high-density lipoprotein; VLDL, very low density lipoprotien; MTP, microsomal triglycerides transfer protein.
irate to severe intensity, typically found in patients with genotype 3, may be of viral origin. 4,6 The data published by Kumar et al. in this issue of HEPATOLOGY 17 add convincing evidence that the latter is indeed the case. Kumaret al. tested the effect of HCV eradication on liver steatosis in 52 chronic hepatitis C patients. Patients with steatosis and genotype 1 infection had their score of fatty liver unchanged independently of the treatment outcome, whereas patients with genotype 3 infection saw their steatosis disappear, but only when a sustained response was reached. This further confirms that not all steatosis seen in the liver of a chronic hepatitis C patient is viral related, and that other causes may coexist. If one considers how frequent is liver steatosis in the general population( 15% according to several estimates), it is not surprising that a comparable (hence significant) proportion of patients with chronic hepatitis C may present with steatosis of etiology independent of HCV. What was beyond the scope of the study of Kumar et al. was an explanation for the pathogenesis of steatosis (whatever the etiology, viral or not), and an assessment of the relation of the clinical course of chronic hepatitis C with the presence and severity of steatosis. As a matter of fact, this study, confirming that only the steatosis associated with genotype 3 infection is viral in origin, rather complicates the issue by underscoring that observations carried out in humans may question the significance of experimental findings. In fact, all in vitro models have used HCV genotype 1­derived constructs, in contrast with studies conducted in chronic hepatitis C patients, which have linked the presence of steatosis to genotype 3.We have recently established an in vitro expression model using constructs derived from HCV genotype 3 isolates18transfected in a hepatoma derived cell line. The genotype3 core protein expression was associated with lipid drop-lets, but a similar fat accumulation was observed, albeit less severe, also in cell lines transfected with the HCV core type 1b. This suggests that, at least in vitro, the derangement of lipid metabolism induced by the HCV core protein may not be genotype specific. Whether patients infected with genotype 1 without steatosis (but levels of HCV replication comparable to those of patients with genotype 3 and massive steatosis) may express a factor protecting them from a fatty liver remains matter of speculation. Another unsolved issue is whether the steatosis is caused directly, i.e., via a molecular interaction between an HCV product and a cellular constituent, or indirectly ,i.e., via a soluble factor released by cells infected with HCV. The question is far from academic, if one considers the severity of steatosis occasionally observed in chronic hepatitis C patients.4If one accepts that a large number of hepatocytes are engulfed with lipids due to a direct effect of HCV, the obvious, necessary implication is that a corresponding proportion of cells will be infected with the virus. Therefore, those who met with skepticism the first images of HCV RNA stained in the vast majority of hepatocytes by in situhybridization19should now, just for the sake of consistency, object to a direct interaction between HCV product(s) and lipid secretory pathway(s). The Impact of Steatosis on Hepatitis C The most important question concerning the clinical significance of steatosis in hepatitis C is whether the lipid accumulation influences the liver disease progression or is solely an innocent bystander. A role of steatosis in the progression of hepatitis C is suggested by the finding that the severity of fatty accumulation correlates with the fibrosis stage.4-6,20This issue is not an easy task to address, if one considers that steatosis in chronic hepatitis C is almost invariably accompanied by some degree of necro-inflammation. Evidence from cohort studies on patients with nonalcoholic fatty liver disease shows that simple steatosis, even if associated with mild and nonspecific inflammation, mostly runs a benign, nonprogressive clinicalcourse.21,22So, one may infer that chronic hepatitis C may progress to liver fibrosis and cirrhosis independently of the presence of steatosis. The question, however, should be a different one: when in the presence of chronic inflammation, does the steatosis contribute synergistically to liver disease progression? Since cross-sectional and retrospective surveys are unlikely to address this issue, due to patients' recruitment bias, a correct answer might only come from ongoing, prospective cohort studies. What may be the pathogenic link between steatosis and fibrosis? At least three research areas that have been the recent focus of investigators may provide the clue: the lipid peroxidation via production of reactive oxygen species, the leptin/leptin receptor system, and the insulin resistance syndrome. The relative contribution of each of these potential factors to the progression of chronic hepatitis C remains to be assessed in future, prospective studies. As mentioned above, the excess reactive oxygen species produced upon expression of the HCV core protein provides a very interesting model to explain the progression of liver damage,14,15since the accumulated fat may constitute the fuel for further free radical production via an amplification process. In conclusion, although steatosis of the liver is a frequent occurrence in chronic hepatitis C, it is not always due to HCV, and therefore, may be resistant to antiviral treatment. Whatever the etiology, its contribution to liver disease progression seems accepted. Its appropriate man-agreement, however, cannot fall short of an in-depth knowledge of the possibly multifaceted mechanisms un-derlying its pathogenesis.

Acknowledgment: The author is indebted to Laura Rubbia-Brandt for continous support, and Karim  Abid, Elisabetta Bugianesi, Simona Paganin, and Roberto Genta for critically reading the manuscript. FRANCESCONEGRO, M.D.Division of Gastroenterology and Hepatology Division of Clinical Pathology University Hospital Geneva, SwitzerlandReferences1. Negro F, Krawczynski K, Quadri R, Rubbia-Brandt L, Mondelli M, ZarskiJP, Hadengue A. Detection of genomic- and minus-strand of hepatitis Cvirus RNA in the liver of chronic hepatitis C patients by strand-specificsemi-quantitative RT-PCR. HEPATOLOGY1999;29:536-542.2. Goodman ZD, Ishak KG. Histopathology of hepatitis C virus infection.Semin Liver Dis 1995;15:70-81.3. Mihm S, Fayyazi A, Hartmann H, Ramadori G. Analysis of histopatho-logical manifestations of chronic hepatitis C virus infection with respect tovirus genotype. HEPATOLOGY1997;25:735-739.4. Rubbia-Brandt L, Quadri R, Abid K, Giostra E, Male PJ, Mentha G, SpahrL, et al. Hepatocyte steatosis is a cytopathic effect of hepatitis C virusgenotype 3. J Hepatol 2000;33:106-115.5. Serfaty L, Andreani T, Giral P, Carbonell N, Chazouille res O, Poupon R.Hepatitis C virus induced hypobetalipoproteinemia: a possible mechanismfor steatosis in chronic hepatitis C. J Hepatol 2001;34:428-434.6. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, RuggieroG. Steatosis accelerates the progression of liver damage of chronic hepatitisC patients and correlates with specific HCV genotype and visceral obesity.HEPATOLOGY2001;33:1358-1364.7. Monto A, Alonzo J, Watson JJ, Grunfeld C, Wright TL. Steatosis inchronic hepatitis C: relative contributions of obesity, diabetes mellitus, andalcohol. HEPATOLOGY2002;36:729-736.8. Rubbia-Brandt L, Giostra E, Mentha G, Quadri R, Negro F. Expression ofliver steatosis in hepatitis C virus infection and pattern of response toalpha-interferon. J Hepatol 2001;35:307.9. Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura Y, EderG, et al. Hepatitis C virus core protein shows a cytoplasmic localization andassociates to cellular lipid storage droplets. Proc Natl Acad Sci U S A1997;94:1200-1205.10. Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K, Matsuura Y,Miyamura T, et al. Hepatitis C virus core protein induces hepatic steatosisin transgenic mice. J Gen Virol 1997;78:1527-1531.11. Hope RG, McLauchlan J. Sequence motifs required for lipid droplet asso-ciation and protein stability are unique to the hepatitis C virus core protein.J Gen Virol 2000;81:1913-1925.12. Sabile A, Perlemuter G, Bono F, Kohara K, Demaugre F, Kohara M,Matsuura Y, et al. Hepatitis C virus core protein binds to apolipoproteinAII and its secretion is modulated by fibrates. HEPATOLOGY1999;30:1064-1076.13. Perlemuter G, Sabile A, Letteron P, Vona G, Topilco A, Chretien Y, KoikeK, et al. Hepatitis C virus core protein inhibits microsomal triglyceridetransfer protein activity and very low density lipoprotein secretion: a modelof viral-related steatosis. FASEB J 2002;16:185-194.14. Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, Okuda M, et al.Steatosis and liver cancer in transgenic mice expressing the structural andnonstructural proteins of hepatitis C virus. Gastroenterology 2002;122:352-365.15. Okuda M, Li K, Beard MR, Showalter LA, Scholle F, Lemon SM, Wein-man SA. Mitochondrial injury, oxidative stress, and antioxidant gene ex-pression are induced by hepatitis C virus core protein. Gastroenterology2002;122:366-375.16. Shi ST, Polyak SJ, Tu H, Taylor DR, Gretch DR, Lai MM. Hepatitis Cvirus NS5A colocalizes with the core protein on lipid droplets and interactswith apolipoproteins. Virology 2002;292:198-210.17. Kumar D, Farrell GC, Fung C, George J. Hepatitis C virus genotype 3 iscytopathic to hepatocytes. Genotype-specific reversal of hepatic steatosisafter sustained response to antiviral therapy. HEPATOLOGY2002;36:1266-1272.18. Abid K, Rossi C, Latorre P, Rubbia-Brandt L, Hadengue A, Negro F. Thecore protein of HCV genotype 3a, 3h and 1b induces lipid accumulationin Huh7 cells. Proceedings of the 9thInternational Meeting on HCVand Related Viruses, San Diego, July 7-11, 2002. Abstract no. P-166,p. 143.19. Negro F, Pacchioni D, Shimizu Y, Miller RH, Bussolati G, Purcell RH,Bonino F. Detection of intrahepatic replication of hepatitis C virus RNAby in situ hybridization and comparison with histopathology. Proc NatlAcad Sci U S A 1992;89:2247-2251.20. Hourigan LF, Macdonald GA, Purdie D, Whitehall VH, Shorthouse C,Clouston A, Powell EE. Fibrosis in chronic hepatitis C correlates signifi-cantly with body mass index and steatosis. HEPATOLOGY1999;29:1215-1219.21. Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural historyof nonalcoholic fatty liver: a follow-up study. HEPATOLOGY1995;22:1714-1719.22. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCul-lough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and patho-logical severity. Gastroenterology 1999;116:1413-1419.1052NEGROHEPATOLOGY, November 2002

Steatosis Raises Fibrosis Risk In Some Hepatitis C Patients

A DGReview of :"Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients."
Journal of Hepatology

12/09/2002
By Anne MacLennan

Patients with hepatitis C virus (HCV) genotype 3 and steatosis should be recommended for early treatment.

This warning is from researchers in Sweden following a study of the impact of steatosis on fibrosis progression over time in relation to HCV genotype.

The study has confirmed the link between HCV genotype 3 and steatosis. Moreover, it suggests steatosis in patients infected with this genotype is a risk factor for progression of fibrosis.

Steatosis is common in HCV patients. Its influence on the progression of the disease, however, has not been well delineated.

In this study, Dr J Westin and colleagues from the Department of Infectious Diseases, Goteborg University, Goteborg, retrospectively analysed 98 patients who underwent dual liver biopsies prior to antiviral treatment.

Biopsy specimens were assessed for necroinflammatory activity, fibrosis and steatosis, and the patients were followed up for a median of 5.8 years.

Independent of patient gender, age, body mass index and alcohol use, the prevalence and grade of steatosis were strongly linked with HCV genotype three.

Progressive fibrosis was more prevalent in patients whose initial biopsy showed steatosis. This effect was seen mainly in patients infected with genotype-three infection.

Although low-grade steatosis was observed in patients who were overweight, high-grade steatosis was associated with genotype three, independent of body mass index.
J Hepatol 2002 Dec;37(6):837-42. "Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients."