Insulin resistance is associated with liver fibrosis in non-diabetic chronic hepatitis C patients

Journal of Hepatology 42 (2005) 41–46 www.elsevier.com/locate/jhep

Insulin resistance is associated with liver fibrosis in non-diabetic chronic hepatitis C patients Alba Muzzi1,13, Gioacchino Leandro2, Laura Rubbia-Brandt3, Richard James4, Olivia Keiser5, Raffaele Malinverni6, Jean-Franc¸ois Dufour7, Beat Helbling8, Antoine Hadengue1, Jean-Jacques Gonvers9, Beat Mu¨llhaupt10, Andreas Cerny11, Mario U. Mondelli12, Francesco Negro1,3,*, For the Swiss Hepatitis C Cohort Study (SCCS) 1

Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland 2 Division of Gastroenterology, IRCCS Saverio de Bellis, Castellana Grotte, Italy 3 Division of Clinical Pathology, University Hospital, Geneva, Switzerland 4 Division of Diabetology, University Hospital, Geneva, Switzerland 5 SCCS Data Center, University Hospital of Lausanne, Lausanne, Switzerland 6 Division of Internal Medicine, Hoˆpital De Cadolle, Neuchaˆtel, Lugano, Switzerland 7 Division of Gastroenterology, University Hospital of Berne, Berne, Switzerland 8 Division of Gastroenterology, Canton Hospital, St Gallen, Switzerland 9 Division of Gastroenterology, University Hospital of Lausanne, Lausanne, Switzerland 10 Division of Gastroenterology, University Hospital of Zu¨rich, Zu¨rich, Switzerland 11 Division of Internal Medicine, Ospedale Civico, Lugano, Switzerland 12 Laboratori di Ricerca Area Infettivologica, IRCSS Policlinico San Matteo and University of Pavia, Italy 13 Department of Infectious Diseases, IRCSS Policlinico San Matteo and University of Pavia, Italy

Background/Aims: Liver steatosis is a frequent finding in chronic hepatitis C. An association has been suggested between steatosis and fibrosis progression rate, but the pathogenetic mechanisms linking fatty infiltration and collagen deposition are unknown. Methods: We measured the levels of insulin resistance (as HOMA score) and leptin in 221 non-diabetic chronic hepatitis C patients, to assess their impact on liver steatosis and fibrosis, relative to other factors, using a multivariable logistic regression. Results: When all 221 patients were considered, steatosis was associated with excessive alcohol intake, genotype 3, and serum HCV RNA level, whereas fibrosis was associated with HOMA score and age. In 152 patients infected with genotype non-3, steatosis was associated with alcohol abuse and HCV RNA level, and fibrosis with HOMA score and age. In the 69 patients with genotype 3, steatosis and fibrosis were associated with each other. The association between fibrosis and HOMA score held also when 22 obese patients were excluded from the analysis. Levels of insulin resistance were not correlated with the presence of steatosis. Conclusions: Thus, insulin resistance (but not leptin) may play a role in fibrogenesis in chronic hepatitis C patients infected with genotype non-3. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Insulin resistance; Hepatitis C virus; Diabetes; Obesity; Liver fibrogenesis; Leptin

Received 17 May 2004; received in revised form 31 August 2004; accepted 6 September 2004; available online 21 October 2004 * Corresponding author. Address: Divisions of Gastroenterology, Hepatology and Clinical Pathology, Hoˆpital Cantonal Universitaire, 24 rue Micheli-duCrest, CH-1211 Gene`ve 14, Switzerland. Tel.: C41 22 3729340; fax: C41 22 3729366. E-mail address: [email protected] (F. Negro). 0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.09.022

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1. Introduction Chronic infection with the hepatitis C virus (HCV) is a major cause of progressive liver damage, whose long-term sequels include cirrhosis and primary hepatocellular carcinoma [1]. However, the rate of histological and clinical progression of chronic hepatitis C is variable, depending on the presence of several cofactors. These include age at infection [2–6], gender [2,3,5,6], prolonged alcohol abuse [2,5,7] and co-infections with the hepatitis B virus [8] or the immunodeficiency virus [9]. Recent studies have shown that also liver steatosis may influence fibrogenesis in chronic hepatitis C [10–13], although some uncertainty exists as to whether this effect is significant in all or only some subgroups of patients [14–17]. The steatosis observed in chronic hepatitis C may result from at least two different mechanisms. In many patients with genotype 3, HCV seems to play a direct role in the pathogenesis of steatosis [11,12,16,18–21], whereas patients infected with other genotypes seem to suffer mostly from a metabolic steatosis [12] related to BMI and nonresponding to antiviral therapy [16,20,21]. Thus, the conflicting findings concerning the correlation between steatosis and fibrosis raise the issue whether this association may depend on the mechanism underlying the fat accumulation [14–16]. Possibly, the different proportion of patients with viral or, respectively, metabolic steatosis may translate into a different contribution to the liver disease progression in the population under study, and result in a different level of association—if any—between steatosis and fibrosis. Recent studies have pointed out two additional factors, the insulin resistance syndrome and the leptin/leptin receptor system, capable of influencing the degree of both steatosis and fibrosis in chronic hepatitis C [22–29], although the conclusions of these studies are not univocal. It is not clear whether insulin resistance and leptin may provide a pathogenetic link between steatosis and fibrosis in all chronic hepatitis C patients or only a subgroup of them, and this as a function of the pathogenesis of steatosis (i.e. viral vs. metabolic). In an attempt to address this issue, we measured the degree of insulin resistance and the serum level of leptin in 221 non-diabetic patients with chronic hepatitis C. These were analysed by a multivariable logistic regression to identify which of them may be independently associated with the presence of liver steatosis and fibrosis. Patients were first considered all together, and then divided according to HCV genotype (3 vs. non-3), assuming that the steatosis in each of these groups might be due to a different pathogenesis (i.e. viral vs. metabolic, respectively). 2. Patient and methods 2.1. Study population A total of 221 patients with chronic hepatitis C, fulfilling the criteria specified below, were included at seven centers throughout Switzerland, all

participating in the Swiss Hepatitis C Cohort Study (SCCS), which is aimed at prospectively collecting a cohort of anti-HCV-positive patients across Switzerland. According to the SCCS basic protocol, clinical, and laboratory data are collected once a year, together with a sample of whole blood, from all enrolled patients. Data are then entered in a centralized database, while plasma and peripheral blood mononuclear cells are stored in appropriate repositories to form a basis for further studies. This study was approved by the Ethical Committees of the SCCS participating centers and conducted in conformity with the Helsinki declaration. All patients consented to participate. Inclusion criteria were: (1) availability of a liver biopsy performed before antiviral therapy, (2) availability of a plasma sample, collected within 1 year from the liver biopsy, (3) completeness of medical records, (4) detectable HCV RNA in serum, (5) HCV genotype 1 to 4. Patients with fasting glucose level O6.2 mmol/l, under antidiabetic or immunosuppressive regimens, with concomitant infection with HBV and/or HIV, undergoing dialysis, with clinically overt hypo- or hyperthyroidism, who had received organ transplantation or with autoimmune or genetic liver disease were excluded from the study.

2.2. Plasma assays Assays were carried out on plasma samples collected by veinpuncture after overnight fasting and stored at K80 8C until use. HCV RNA was measured by quantitative PCR (Amplicor Monitor, Roche Diagnostics, Rotkreuz, Switzerland). HCV genotyping was performed with a secondgeneration reverse-hybridization line probe assay (Inno-Lipa HCV II, Innogenetics, Zwijndrecht, Belgium). Insulin and leptin levels were measured on stored samples by commercial RIAs (LINCO Research Inc., St Charles. Missouri, USA). Insulin resistance was determined by the homeostasis model assessment (HOMA) method proposed by Matthews et al. [30].

2.3. Liver histology A liver biopsy was obtained from all patients for diagnostic purposes. Liver specimens were formalin-fixed and paraffin-embedded for histological evaluation. Histological diagnoses were established according to internationally accepted criteria. All histological samples, collected at the eight different centers participating into the SCCS study, were centralized and scored blindly in terms of clinical status by a reference pathologist of the SCCS (L.R.-B.). H&E-stained sections were evaluated according to a scoring system that includes the semi-quantitative assessment of liver disease grading and staging [31]. Macrovesicular steatosis was graded as absent or minimal (less than 1% of hepatocytes) (score 0), mild (!30% hepatocytes involved) (score 1), moderate (between 30 and 60% of hepatocytes involved) (score 2) or severe (O60% of hepatocytes involved) (score 3) [32].

2.4. Statistical analysis Univariate analysis was made by chi-square test for frequencies and by Mann–Whitney rank-sum test for means. For multivariable analysis, when steatosis was used as dependent variable (i.e. absence vs. presence of steatosis, independently of its severity), we considered as possibly independent variables: gender, age, HCV genotype, ongoing alcohol abuse (O40 g/day at the time of liver biopsy or within the 6 months preceding liver biopsy, based on self-reporting), body mass index (BMI), HOMA score, plasma leptin level, liver disease activity and liver fibrosis scores (both according to the Metavir scoring system) [31]. When fibrosis was used as dependent variable (i.e. absent or present, irrespectively of the score), we considered as independent variables: gender, age, HCV genotype, past alcohol abuse (O40 g/day for O5 years, based on selfreporting), BMI, HOMA score, plasma leptin level, Metavir activity score and steatosis score. Maximum likelihood method was used for entering or removing variables at each step. Bio Medical Data Processing (BMDP—Dynamic version 7.0, Los Angeles, CA) was used for calculations.

A. Muzzi et al. / Journal of Hepatology 42 (2005) 41–46

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Table 1 Characteristics of 221 patients considered in the logistic regression analyses

Male sex (%) Age (meanGSD) Ongoing alcohol abuse (%) Past alcohol abuse (%) BMI (meanGSD) Presence of liver fibrosis (%) Presence of liver steatosis (%) Log10 serum HCV RNA (meanGSD) Glucose (mmol/l) (meanGSD) Insulin (mU/ml) (meanGSD) HOMA score (meanGSD) Leptin (ng/ml) (meanGSD)

All (nZ221)

Genotype non-3 (nZ152)

Genotype 3 (nZ69)

P*

145 (65.6) 47.0G11.8 19 (8.6) 55 (24.9) 24.1G3.7 181 (81.9) 100 (45.2) 5.88G6.35 4.0G1.2 17.1G17.0 3.4G3.9 10.6G28.8

104 (68.4) 49.3G12.5 11 (7.2) 32 (21.0) 24.3G3.5 120 (78.9) 56 (36.8) 5.84G6.4 4.0G1.3 17.9G17.8 3.6G4.2 11.5G32.3

41 (59.4) 42.0G8.2 8 (11.6) 22 (31.9) 23.7G4.0 61 (88.4) 44 (63.8) 5.96G7.3 3.9G1.0 15.4G15.1 2.9G3.0 8.6G18.9

NS !0.001 NS NS NS NS !.001 NS NS NS NS NS

*Type non-3 vs. type 3.

3. Results The baseline characteristics of the study population are shown in Table 1. When considered all patients together, liver fibrosis was observed in 181 (81.9%) and steatosis in 100 patients (45.2%). Patients with genotype 3 were younger (P!0.001) and had more frequently steatosis (P!0.001) than patients infected with other genotypes; the two groups were otherwise comparable. 3.1. Univariate analysis Tables 2 and 3 list the factors associated with steatosis and, respectively, fibrosis, as identified by univariate analysis. Patients with steatosis, independently of its severity, presented more frequently an excess alcohol intake at the time of liver biopsy (PZ0.006), were more frequently infected with HCV genotype 3 (P!0.001), and had higher Metavir fibrosis scores (P!0.001). HOMA scores were comparable in the two groups, i.e. with or without steatosis. Patients with fibrosis were older (P!0.001), had higher levels of fasting glucose (P!0.001), higher levels of fasting insulinemia (PZ0.027), a higher HOMA score (PZ0.013), and had higher Metavir activity score (P!0.001) and more steatosis (PZ0.022) than patients without fibrosis (Table 3). There were no differences as to the plasma leptin levels in the various groups of patients, irrespectively of the presence/absence of steatosis and/or fibrosis. 3.2. Multivariate analysis Tables 4 and 5 show the results of the multivariable logistic regression analysis performed on all 221 patients. Factors independently associated with steatosis were an ongoing excess alcohol intake at the time of liver biopsy, presence of genotype 3, and a high serum HCV RNA level (Table 4). When patients were divided according to HCV

genotype, an ongoing alcohol abuse and serum HCV-RNA were both independently associated with steatosis in the 152 patients infected with a non-3 genotype, whereas the association held only for the fibrosis stage among the 69 patients with genotype 3 infection. Factors independently associated with the presence of liver fibrosis were HOMA score and age (Table 5). Both factors were still significantly associated with fibrosis when only patients with genotype non-3 were considered. Table 2 Univariate analysis of the factors associated with liver steatosis in 221 non-diabetic chronic hepatitis C patients Variable

Steatosis No (nZ119)

Male sex (%) 75 (63.0) Mean age (GSD) 47.7G12.7 HCV genotype 3 (%) 24 (20.2) 5.6G5.8 Log10 serum HCV RNA (meanGSD) Ongoing alcohol 4 (3.4) abuse (%) Mean BMI (GSD) 24.1G3.3 Metavir activity score (%) A0 16 (13.4) A1 75 (63.0) A2 21 (17.6) A3 7 (5.9) Fibrosis score (%) F0 29 (24.4) F1 30 (25.2) F2 12 (10.1) F3 5 (4.2) F4 43 (36.1) Glucose (mmol/l) 4.1G1.2 (meanGSD) Insulin (mU/ml) 16.7G16.6 (meanGSD) HOMA score 3.4G4.0 (meanGSD) Leptin (ng/ml) 11.8G29.6 (meanGSD)

P Yes (nZ102) 70 (68.6) 46.2G10.7 45 (44.1) 6.1G6.5

NS NS !0.001 NS

15 (14.7)

0.006

24.2G4.1

NS

9 (8.8) 57 (55.9) 29 (28.4) 7 (6.9)

NS

11 (10.8) 25 (24.5) 17 (16.7) 20 (19.6) 29 (28.4) 3.9G1.1

!0.001

NS

17.6G17.6

NS

3.3G3.8

NS

9.3G27.8

NS

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A. Muzzi et al. / Journal of Hepatology 42 (2005) 41–46

Table 3 Univariate analysis of the factors associated with liver fibrosis in 221 non-diabetic chronic hepatitis C patients Variable

Fibrosis No (nZ40)

Male sex (%) Mean age (GSD) HCV genotype 3 (%) Log10 serum HCV RNA Past alcohol abuse (%) Mean BMI (GSD) Metavir activity score (%) A0 A1 A2 A3 Steatosis score (%) 0 1 2 3 Glucose (mmol/l) (meanGSD) Insulin (mU/ml) (meanGSD) HOMA score (meanGSD) Leptin (ng/ml) (meanGSD)

P Yes (nZ 181)

24 (60.0) 39.7G9.5 8 (20) 5.6G5.7 8 (20.0) 23.9G3.1

121 (66.9) 48.6G11.7 61 (33.7) 5.9G6.4 46 (25.4) 24.2G3.8

NS !0.001 NS NS NS NS

16 (40.0) 22 (55.0) 2 (5.0) 0

9 (5.0) 110 (60.8) 48 (26.5) 14 (7.7)

!0.001

29 (72.5) 11 (27.5) 0 0 3.4G1.2 11.7G16.1 2.0G3.3 6.4G9.2

90 (49.7) 61 (33.7) 18 (9.9) 12 (6.6) 4.1G1.2 18.3G17.1 3.7G4.0 11.6G31.4

0.022

!0.001 0.027 0.013 NS

Among patients with genotype 3 infection, steatosis was the only factor independently associated with fibrosis. We also calculated the factors associated with fibrosis in a subgroup of 142 patients in whom both the age at infection and the chronic hepatitis C duration could be estimated, based on widely accepted criteria [16]. In this subgroup, the only factor independently associated with liver fibrosis was patient’s age at the time of liver biopsy (OR 1.09, 95% CI 1.02-1.16). 3.3. Multivariate analysis in non-obese patients We performed an additional multivariate analysis excluding 22 obese patients, as defined by BMI R30. Factors independently associated with steatosis were the same as identified by the multivariate on all 221 patients, independently of the stratification by genotype Table 4 Multivariate analysis of the factors associated with liver steatosis in 221 non-diabetic chronic hepatitis C patients OR All 221 patients Ongoing alcohol abuse HCV genotype 3 Serum HCV RNA 152 Patients with genotype non-3 Ongoing alcohol abuse Serum HCV RNA 69 Patients with genotype 3 Metavir fibrosis score

Table 5 Multivariate analysis of the factors associated with liver fibrosis in 221 non-diabetic chronic hepatitis C patients OR All 221 patients Age HOMA score 152 Patients with genotype non-3 Age HOMA score 69 Patients with genotype 3 Steatosis score

2.12–32.8 1.38–4.84 1.11–1.70

19.1 1.30

2.25–162 1.01–1.67

28.5

2.81–289

1.08 1.57

1.03–1.12 1.04–2.39

1.07 1.63

1.03–1.12 1.02–2.60

11.5

1.12–119

(data not shown). Factors independently associated with the presence of liver fibrosis were Metavir activity score, steatosis, and HOMA score (Table 6). The liver activity score was also independently associated with fibrosis even when patients were divided according to viral genotype. Furthermore, among the 64 patients with genotype 3 infection, fibrosis was independently associated also with steatosis. We also performed an additional multivariate analysis where patients were divided in three subgroups according to BMI, i.e. patients with BMI !25 (nZ139), those with a BMI R25 but !30 (nZ62), and obese patients, i.e. having a BMI R30 (nZ21). According to this analysis, HCV RNA level was independently associated with steatosis (OR 1.23, 95% CI 1.001–1.52), whereas the two factors independently associated with the presence of liver fibrosis were age at the time of liver biopsy (OR 1.08, 95% CI 1.03–1.13) and HOMA score (OR 1.53, 95% CI 1.001–2.34).

4. Discussion Several studies have analysed the relationship between steatosis and fibrosis in chronic hepatitis C patients in various univariate and multivariate analyses. Most factors were independently associated with either one or both: HCV genotype 3 is associated with presence and severity of steatosis, and age (or duration of disease) is associated with fibrosis. Moreover, fibrosis and steatosis are often associated with each other, although in some studies a genotype-specific Table 6 Multivariate analysis of the factors associated with liver fibrosis in 200 non-diabetic, non-obese chronic hepatitis C patients

95% CI

8.34 2.58 1.37

95% CI

OR All 200 patients Metavir activity score Steatosis HOMA score 136 patients with genotype non-3 Metavir activity score 64 Patients with genotype 3 Metavir activity score Steatosis score

95% CI

8.62 3.04 1.60

3.10–24.0 1.25–7.39 1.03–2.48

7.08

2.12–23.6

73.4 31.2

2.01–2,690 1.6–610

A. Muzzi et al. / Journal of Hepatology 42 (2005) 41–46

association has been reported [14–16]. Furthermore, in obese patients, BMI may contribute to both steatosis and fibrosis. Since HCV genotype 1 is more frequent in older patients, and BMI tends to increase with age, this association is more likely in patients infected with genotype 1. Discrepancies among published studies may also be related to potential acute confounding events (such as diet- or drug-induced induced weight loss) that may affect the one time findings on liver biopsy. Should this be the case, the role of steatosis in liver fibrogenesis would probably be underestimated in some patients’ subsets. Future studies should consider also such potential confounding events preceding the liver biopsy. Understanding the pathogenesis of steatosis in chronic hepatitis C and its relationship to fibrogenesis, and hence to liver disease progression, is critical for proper management. Current data suggest that there are at least two types of steatosis: (1) virally-related steatosis, which correlates with HCV replication level, is often associated with genotype 3, and disappears upon successful antiviral therapy, and (2) metabolic steatosis, whose presence and severity correlate with BMI, and that does not respond to antiviral treatment, even in virological responders [11,12,16,18–21]. Both types of steatosis are likely to be associated with other factors, such as necro-inflammation, to induce fibrogenesis, but it is not clear whether fatty liver activates (or indirectly amplifies) the fibrogenic process per se, or whether it should be considered solely as an innocent hallmark of a real, unknown fibrogenetic factor, acting independently of the presence of steatosis. A recent elegant paper [22] has linked for the first time insulin resistance—but not steatosis—to fibrosis in patients with chronic hepatitis C. Other groups have confirmed these findings [23,24], that seem to suggest that the previously reported correlation between steatosis and fibrosis may be spurious, i.e. due to the fact that insulin resistance had never been considered in the logistic regression model. To test this hypothesis, we analysed the level of insulin resistance in a group of European, non-diabetic patients with chronic hepatitis C. Our data show that, apart from confirming previously reported correlations, the degree of insulin resistance (expressed as HOMA score) was independently associated with liver fibrosis (but not steatosis). This association (or lack thereof, as far as steatosis is concerned) held also when we divided patients according to HCV genotype or BMI. We think that these observations are noteworthy, in view of the debated role of HCV infection in the establishment of an insulin resistant state, since they hint at the presence of a factor (HCV?) other than overweight and/or obesity involved in the pathogenesis of insulin resistance. However, at variance with Hui et al. [22], we could not confirm a genotype-specific effect on the level of insulin resistance (data not shown). Although the present findings are simply correlative and do not prove causality, there is accumulating evidence compatible with the hypothesis that chronic hyperglycaemic and hyperinsulinemic states are fibrogenic in chronic

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hepatitis C, and that a factor other than overweight/obesity (possibly HCV itself) may be involved in the insulin resistance state, in agreement with mounting clinical [33–35] and experimental [36,37] data. The factor involved in the induction of insulin resistance does not seem to be steatosis, since fasting glucose and insulin levels, as well as HOMA scores, were comparable in patients with or without steatosis (Table 2). Furthermore, by multivariable analysis, HOMA scores were not associated with steatosis. Although the latter may play a role in fibrogenesis, especially in patients with genotype 3 (Tables 4 and 5), according to our data steatosis and insulin resistance may be fibrogenic independently of each other. This is also compatible with recent experimental data, showing that the expression of the HCV core protein (type 1) in the transgenic mouse is directly responsible of insulin resistance in this model and that this event precedes the appearance of steatosis in the liver [36]. Finally, we failed to find any association between leptin levels and either steatosis or fibrosis stage. The role of leptin in liver disease progression is controversial. In patients with non-alcoholic steatohepatitis circulating leptin levels do not correlate with the amount of fibrosis [38]. In chronic hepatitis C, leptin correlates with BMI and with fibrosis [27,29], as well as with TNF-a. Another study has suggested a possible relationship between leptin and steatosis [28]; our results seem to be in conflict with such findings, and more extensive analyses seem to be warranted. In conclusion, insulin resistance, but not leptin, seems to be associated with fibrosis (but not with steatosis) in nondiabetic patients with chronic hepatitis C. Whether this reflects an HCV-mediated effect due to the increasingly reported association between HCV and diabetes remains to be established. Acknowledgements This study was supported by grants 32-063428.00 and 32-63549.00 from the Swiss National Science Foundation. The authors wish to thank M. Rickenbach for the skilful management of the SCCS database, E. Bugianesi, A. Golay, R. Genta and A. Mangia for helpful advice and criticism, L. Minoli for kind support, Ch. Rossi and L. Bischoff for technical help. A.M. was a recipient of a scholarship from Italian Ministry of Health. This study was supported by grants 32-063428.00 and 32-63549.00 from the Swiss National Science Foundation. References [1] Niederau C, Lange S, Heintges T, Erhardt A, Buschkamp M, Hurter D, et al. Prognosis of chronic hepatitis C: results of a large, prospective cohort study. Hepatology 1998;28:1687–1695. [2] Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997;349: 825–832.

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