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Serum leptin levels correlate with hepatic steatosis in chronic hepatitis C
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Inc.
Vol. 98, No. 5, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)00185-0
Serum Leptin Levels Correlate With Hepatic Steatosis in Chronic Hepatitis C Manuel Romero-Go´mez, M.D., Victor M. Castellano-Megias, M.D., Lourdes Grande, M.D., Jose´ A. Irles, M.D., Marina Cruz, M.D., Marı´a Carmen Nogales, M.D., Juan Carlos Alco´n, M.D., and Antonio Robles, M.D. Units of Hepatology, Pathology, Nutrition, Biochemistry, and Microbiology, Hospital Universitario de Valme, Sevilla, Spain
OBJECTIVES: Hepatic steatosis (HS) has been related to obesity and fibrosis in chronic hepatitis C (CHC). The aim of this study was to determine the role of leptin system in HS development. METHODS: Patients (n ⫽ 131) with biopsy-proven CHC, positive HCV RNA, and raised ALT were enrolled. Body mass index, percentage of body fat by skin fold measurement, and bioelectrical impedance analysis was calculated and serum leptin concentration measured. Intrahepatic HCV RNA, HS, necroinflammatory activity, and fibrosis were determined in liver biopsy tissue. RESULTS: HS was present in 63 patients (48.1%). Steatosis was evident in 32 of 91 patients (35.2%) infected with genotype 1 and in 22 of 27 patients (81.5%) with genotype 3a (p ⬍ 0.001). In patients infected by genotype 3a, HS correlated significantly with intrahepatic HCV RNA load (r ⫽ 0.78; p ⬍ 0.001). However, in genotype 1, HS was associated with host factors such as leptin, body mass index, percentage of body fat, and visceral obesity. Multivariate analysis showed genotype (OR ⫽ 11.54, 95% CI ⫽ 1.13– 117.14, p ⫽ 0.038), leptin levels (OR ⫽ 1.09, 95% CI ⫽ 1.03–1.17, p ⫽ 0.008) and fibrosis (OR ⫽ 9.86, 95% CI ⫽ 2.11–5.86, p ⫽ 0.03) as independent variables of HS development. CONCLUSIONS: Hepatic steatosis was related to genotype, fibrosis degree, and serum leptin levels. Genotype 3 seems to have a viral specific steatogenic effect. Leptin seems to be a link between obesity and steatosis development in CHC genotype 1–infected patients. (Am J Gastroenterol 2003;98: 1135–1141. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION Hepatocyte steatosis (HS) is a frequent histopathological feature in chronic hepatitis C virus (HCV) infection (1, 2) and has been related to fibrosis progression (3). However, a link between steatosis and fibrosis remains unclear (4, 5). Host factors such as dyslipidemia, diabetes mellitus type 2, increased body mass index (BMI) (6, 7), together with viral factors such as genotype, hepatitis G virus coinfection (8)
and HCV RNA load (9) have been associated with steatosis development in chronic hepatitis C. However, although some investigators have found steatosis more often in patients infected with genotype 3a (10, 11) others have not (2, 6). Further, a relationship between BMI and steatosis was only found in patients with genotype 1 infection (12). Leptin has been shown to be involved in peripheral insulin resistance and strongly related to body fat composition. This protein is released from adipocytes and has been detected in activated liver stellate cells (13). Leptin may have a role in the regulation of fat deposition, fibrogenesis, and inflammation (14). The aims of the present study were to verify the relationship between steatosis and fibrosis, the influence of viral genotypes on steatosis development and to investigate the role of leptin in the development of steatosis in chronic hepatitis C infection.
MATERIALS AND METHODS Patient Population Patients (n ⫽ 131) with chronic hepatitis C infection were consecutively recruited. Fully informed written consent was obtained before entry into the study, and the hospital’s ethics committee approved the protocol. Patient history of alcohol consumption was obtained. Two investigators independently solicited information regarding the number of alcoholic beverages consumed daily as well as the duration of the alcohol abuse, if any. An average daily intake of alcohol (in grams) was calculated. Patients with alcohol consumption ⬎80 g/day were excluded. Weight changes over the previous year were recorded, and patients with changes ⬎10% were excluded from the study. No patient had received interferon or other antiviral or steatosis-inducing drugs. Patients previously diagnosed as having type 2 diabetes mellitus were also excluded. Laboratory Investigations An overnight (12-h) fasting blood sample was taken for routine investigations such as ALT, AST, alkaline phospha-
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tase, ␥-glutamyl transpeptidase, cholesterol, and triglycerides. All patients had positive anti-HCV (EIA3; Abbott Laboratories, Chicago, IL), increased ALT, and positive HCV RNA in serum. HBsAg, anti-HBc, anti-HIV were tested using commercially available kits (Abbott Laboratories). All patients were negative for HBsAg and anti-HIV. Serum leptin levels were measured with a commercially available ELISA kit (Quantikine Human Leptin Immunoassay; R&D systems, Minneapolis, MN). The interassay coefficient of variation was 12%. Fasting samples of serum obtained after centrifugation were stored in aliquots at ⫺70°C until assayed. The insulin resistance index was calculated on the basis of fasting values of plasma glucose and insulin according to the homeostasis model assessment for insulin sensitivity (HOMA) model formula: Insulin resistance (HOMA IR) ⫽ fasting insulin (mUI/L) ⫻ fasting glucose (mmol/L) ⫼ 22.5 Body Composition Measurements Height and weight were determined at baseline, and body mass index (BMI) was calculated as weight (in kg) ⫼ height (in m2). In a nutrition laboratory, skin fold thickness was measured at the triceps, biceps, and subscapular and suprailiac regions using Holtain callipers (Dietosystem, Milan, Italy). Percentage of body fat mass was calculated using the equations of Durnin and Womersley (15). Waist circumference measurement was used to estimate visceral obesity. Visceral obesity was considered when waist measurement was ⬎102 cm in men and ⬎88 cm in women. Bioelectrical impedance analysis (BIA) was performed with the patient supine, arms relaxed but not touching the body, in a fasting state, not smoking, and with an empty bladder. Fat mass and percentage body fat was calculated using Tanita-305 (Tanita, Tokyo, Japan). Leptin adjusted for body fat mass was expressed as leptin level divided by fat mass (and expressed as ng/ml/kg). Liver Histology Methods Percutaneous liver biopsy under ultrasonographic control was performed. A portion of the biopsy material was used in histological diagnosis. All liver biopsy specimens were adequate for diagnosis. The average length was 16 mm showing nine portal tracts. We assessed grading and staging separately. The stage was defined according to the Scheuer fibrosis score (16) in which F0 ⫽ absence, F1 ⫽ enlarged portal tracts, F2 ⫽ periportal or portoportal septa, F3 ⫽ fibrosis with architectural distortion, and F4 ⫽ cirrhosis. Necroinflammatory activity was determined by combining scores for portal inflammation (0 – 4) and lobular necrosis (0 – 4). Steatosis was quantified as the percentage of hepatocytes that contained fat droplets, from 0 (absent) to 100% (all hepatocytes containing fat droplets). To classify the patients, grading of steatosis was assigned as grade 0 if there was no steatosis, grade 1 when ⬍25% of hepatocytes contained fat droplets, grade 2 when between 25% and 50%
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showed steatosis, and grade 3 when ⬎50% of hepatocytes showed fat storage. Patients showing steatosis combined with hepatocyte ballooning, neutrophil infiltrate, and either Mallory hyaline or sinusoidal fibrosis were considered as having steatohepatitis and were excluded from this study. Two pathologists with no knowledge of the patient’s clinical data performed parallel histopathology assessments. When a discrepancy between pathologists’ findings occurred, the specimen was re-evaluated in parallel by the two pathologists so as to achieve a consensus. In 48 patients, the other portion of the biopsy material was rapidly frozen and used in the quantification of intrahepatic HCV levels. Virology Methods HCV genotyping was with INNO-LIPA HCV II kits (Innogenetics, Zwijnaarden, Belgium), which were used according to the manufacturer’s instructions. Amplicor-HCVMonitor (Perkin-Elmer, Norwalk, CT) was used to quantify the HCV RNA levels in serum. The level of detection was 1000 copies/ml and all patients were HCV RNA positive. Semiquantification of HCV RNA in liver was by adding 1 g of total liver RNA to 50 l of specimen diluent to which 0.2 l of internal standard had previously been mixed. A 50-l polymerase chain reaction master mix was added and the rest of the procedure was identical to that of the serum-based assay. Amplicor-HCV-Monitor (PerkinElmer) was also used to quantify the HCV RNA levels in the liver, with a level of detection of 1000 copies/g total RNA. Statistical Analysis Hepatic steatosis was assessed as categorical (absent or present) or continuous variable from 0% to 100%. Comparisons between groups with and without steatosis according genotypes were made using the Mann-Whitney U test or the Student’s t test for continuous variables and the 2 or Fisher’s exact probability test for categorical data. The Spearman coefficient was used to correlate numerical variables that were nonnormally distributed. All values are presented as means ⫾ SD. A p value of ⬍0.05 was considered to be statistically significant. Multiple linear regression has been used in the multivariate analysis of factors associated with leptin levels, including age, sex, hepatic steatosis, body mass index, percentage of body fat, insulin resistance, fibrosis, and waist measurement. A multivariate analysis based on a backward LR logistic regression was used to establish association between hepatocyte steatosis and host (and viral) factors including sex, age, alcohol consumption, genotype, body mass index, visceral obesity, leptin level, HCV RNA load, and fibrosis.
RESULTS Patient Demographic Data The mean age of the patients was 38 yr (⫾11 yr) with an estimated duration of infection of 16 ⫾ 9 yr. The study
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Leptin and Steatosis in HCV
Table 1. Multiple Linear Regression Analysis (R2 ⫽ 0.59) With Serum Leptin Levels as Dependent Variable in Patients With Chronic Hepatitis C Variable
Coefficient
SE
t
p
Age (yr) Sex Insulin resistance Fibrosis Steatosis % BF % BMI Waist (cm)
⫺0.028 0.596 0.03 0.035 0.186 ⫺0.297 0.773 ⫺0.035
0.13 4.95 0.77 1.44 0.07 0.29 0.43 0.14
⫺0.29 3.66 0.28 0.41 2.28 ⫺1.52 4.97 ⫺0.27
0.77 0.001 0.77 0.68 0.026 0.13 0.001 0.78
Body mass index, sex, and hepatocyte steatosis were independently related to leptin levels. BF % ⫽ percentage of body fat mass; Steatosis % ⫽ percentage of hepatocyte steatosis.
population comprised 84 men and 47 women. Related risk factors were drug abuse (n ⫽ 42), blood transfusion (n ⫽ 31), tattooing (n ⫽ 8), surgery (n ⫽ 33), health worker occupation (n ⫽ 3), and unknown etiology (n ⫽ 14). The median alcohol intake was 19 g/day with 37 of the 131 patients consuming ⬎40 g/day; 34 of 37 (91.8%) were men. The mean BMI was 26.8 ⫾ 3.6 kg/m2. Genotype 1b was found in 78 (59.5%) patients, genotype 1a in 13 (9.9%), genotype 2a in four (2.2%), genotype 3a in 27 (20.6%), and genotype 4a in two patients. In seven patients (5.3%) we were unable to establish an unequivocal genotype. Histological Evaluation Steatosis was detected in 63 of 131 patients (48.1%), grade 1 in 39 (29.7%), grade 2 in 16 (12.2%), and grade 3 in eight (6.1%) patients. Stage of fibrosis was F0 in four (3.1%), F1 in 58 (44.2%), F2 in 47 (35.8%), F3 in 12 (9.2%), and F4 in 10 (7.6%) patients. The grade of portal inflammation was P1 in 15 (11.5%), P2 in 70 (53.4%), P3 in 40 (30.5%), and P4 in six (4.6%) cases, and lobular necrosis was L0 in two (1.5%), L1 in 63 (48.1%), L2 in 63 (48.1%), and L3 in three (2.3%). Body Composition and Leptin Levels Serum leptin levels were higher in women than in men (24.3 ⫾ 18.7 vs 8.4 ⫾ 11.1 ng/ml; p ⬍ 0.0001), even when
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adjusted for fat mass (0.28 ⫾ 0.20 vs 0.79 ⫾ 0.43 ng/ml/kg). However, no differences were observed with respect to genotype (genotype 1, 14.9 ⫾ 16.8; genotype 3a, 11.8 ⫾ 13.6 ng/ml; p ⫽ ns). The leptin levels correlated with BMI (r ⫽ 0.49; p ⬍ 0.01), percentage of body fat (r ⫽ 0.77, p ⬍ 0.001), percentage of hepatocyte steatosis (r ⫽ 0.31, p ⬍ 0.005), age (r ⫽ 0.37, p ⬍ 0.01), insulin resistance (r ⫽ 0.29, p ⫽ 0.004) and fibrosis stage (r ⫽ 0.22, p ⬍ 0.05), but not with ALT and AST levels or with HCV RNA burden. In multivariate analysis (multiple linear regression), the percentage of hepatocyte steatosis, sex, and body mass index were independently related to serum leptin levels (Table 1). Leptin concentrations when adjusted for fat mass did not alter these findings, and these data were not analyzed further. Factors Associated With Hepatic Steatosis Steatosis was detected more frequently in patients infected by genotype 3 (22 of 27; [81.5%] than by genotype 1 (32 of 91, 35.2%; p ⬍ 0.001). Thus, the influence of body composition and leptin levels on the development of hepatic steatosis was analyzed segregated by genotypes. BMI, percentage of body fat, leptin levels, and visceral obesity were significantly associated with the presence of steatosis in patients infected by genotype 1 but not in patients infected by genotype 3, whereas cholesterol, triglycerides, and insulin resistance were not related to steatosis (Table 2). Furthermore, leptin levels correlated with percentage hepatocyte steatosis in patients infected by genotype 1 (r ⫽ 0.66, p ⬍ 0.01) but not in genotype 3 (r ⫽ 0.29, p ⫽ ns) (Fig. 1), whereas in patients infected with genotype 3, steatosis correlated with intrahepatic HCV RNA (r ⫽ 0.78, p ⬍ 0.001) (Fig. 2). The degree of steatosis was greater in patients with a daily average alcohol consumption ⬎40 g/day than in nondrinkers or those consuming ⬍40 g/day. This difference did not quite reach statistical significance (16 ⫾ 25% vs 10 ⫾ 17%, p ⫽ 0.08), probably because heavy drinkers had been excluded from the study. As alcohol could be a confounding variable, an analysis excluding individuals con-
Table 2. Relationship Between Hepatocyte Steatosis, Leptin Levels, and Body Composition Segregated With Respect to Genotypes Genotype 1 (n ⫽ 91)
Genotype 3 (n ⫽ 27)
Variable
Steatosis (n ⫽ 32)
None (n ⫽ 59)
p
BMI (kg/m2) BF(%) (BIA) Skinfold (%) Leptin (ng/ml), men Leptin (ng/ml), women HOMA IR Cholesterol Triglycerides Visceral obesity (%) Alcohol intake ⬎40 g/day
29.8 ⫾ 7.4 34 ⫾ 9.9 34.3 ⫾ 9.7 14.2 ⫾ 16.0 37.6 ⫾ 19.7 2.73 ⫾ 1.76 171 ⫾ 43 115 ⫾ 87 15 (46.9%) 9 (28.1%)
25.7 ⫾ 3.6 28.1 ⫾ 7.9 27.9 ⫾ 8.0 5.5 ⫾ 4.6 16.3 ⫾ 14.3 1.84 ⫾ 2.44 158 ⫾ 36 108 ⫾ 58 14 (23.7%) 15 (25.4%)
⬍0.001 ⬍0.01 ⬍0.01 ⬍0.01 ⬍0.005 ⫽0.087 ns ns ⬍0.05 NS
Steatosis (n ⫽ 22) 26.4 ⫾ 4.6 26.8 ⫾ 8.2 25.4 ⫾ 5.5 11.61 ⫾ 16.2 16.1 ⫾ 15.9 2.16 ⫾ 2.10 153 ⫾ 44 104 ⫾ 74 4 (14.8%) 10 (45.4%)
None (n ⫽ 5)
p
26.3 ⫾ 11.3 33.6 ⫾ 8.05 29.7 ⫾ 7.4 4.9 ⫾ 1.3 16.0 ⫾ 2.3 1.19 ⫾ 0.59 175 ⫾ 25 95 ⫾ 47 0 (0%) 0 (0%)
NS NS NS NS NS ns ns ns NS NS
Visceral obesity was considered to be present when waist measurement was ⬎102 cm in men and ⬎88 cm in women (reference values of Spanish Nutrition Association). Steatosis was strongly related to body composition in patients infected with genotype 1 but not in patients infected with genotype 3. BIA ⫽ bioelectrical impedance analysis; BF% ⫽ percentage body fat.
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Figure 1. Correlation between serum leptin concentrations and percentage hepatocyte steatosis in chronic hepatitis C patients segregated with respect to genotype.
suming ⬎40 g/day was performed, and the associations were observed to remain unchanged. Steatosis correlated with liver HCV RNA in genotype 3 (r ⫽ 0.75, p ⫽ 0.01) and with leptin in genotype 1 (r ⫽ 0.64, p ⬍ 0.001). To analyze the interaction between host and viral factors implicated in steatosis development, we performed a multivariate analysis (logistic regression backward LR). The independent variables predictive of presence of steatosis were genotype 3 (OR ⫽ 11.54, 95% CI ⫽ 1.13 – 117.14, p ⫽ 0.038), higher leptin levels (OR ⫽ 1.09, 95% CI ⫽ 1.03 – 1.17, p ⫽ 0.008), and advanced fibrosis stage (OR ⫽ 9.86, 95% CI ⫽ 2.11– 45.86, p ⫽ 0.03) (Table 3).
DISCUSSION In the present study, hepatic steatosis was associated with a higher degree of fibrosis and was influenced by HCV genotype. In patients infected with genotype 1, steatosis correlated with serum leptin levels, and in patients infected with genotype 3, steatosis was strongly related to intrahepatic HCV load. An explanation for these findings is not yet available, although a similar result has been reported (12, 17). The relationship between steatosis and intrahepatic HCV load has been observed in human as well as animal
studies. The core protein of hepatitis C has been shown to induce hepatic steatosis in transgenic mice (18), whereas, in patients with chronic hepatitis C, a close relationship between in situ level of core HCV and hepatic steatosis has been observed (19). In genotype 3–infected patients, steatosis has been related to HCV RNA load in serum (9) and in the liver (20), as was observed in the current study. Thus, at least in patients infected with genotype 3, hepatocyte steatosis emerges as a morphological feature that is induced directly by the virus. However, host factors such as BMI or body fat, usually associated with steatosis, seem to not play an important role in this group. The mechanism by which HCV induces steatosis remains unclear. The virus can increase glutathione turnover by eliciting free radical–mediated lipid peroxidation, which would affect iron metabolism within the hepatocyte and which may promote fat droplet deposition (21). On the other hand, an increase in the concentration of monounsaturated fatty acids has been found in the liver of patients with chronic hepatitis C relative to non–HCV-related liver disease (22). Thus, HCV may cause a change in lipid metabolism and promote triglyceride accumulation resulting in hepatocyte steatosis.
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Leptin and Steatosis in HCV
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Figure 2. Relationship between intrahepatic HCV load and percentage of hepatocyte steatosis segregated with respect to genotype.
In the current study, liver steatosis was related to serum leptin level and body composition in patients infected by genotype 1 but not in those with genotype 3. In a previous report, leptin was observed to be unrelated to steatosis in chronic hepatitis C (23), although in that report, the number of patients with genotype 1 infection was very small (only 12), and the authors were unable to find any relationship between leptin levels and steatosis. In the current study, a weak relationship between leptin levels and fibrosis has also been found. Indeed, leptin has previously been shown to be associated with steatosis and fibrosis in nonobese patients with nonalcoholic steatohepatitis (24). However, leptin shows a sex-based difference, and women have circulating plasma leptin concentrations that are three times higher than those in men, even when subjects are matched for body fat mass (25). Thus, patient sex needs to be considered when investigating leptin levels, and multivariate analysis is necessary to reduce the bias caused by this confounding variable. The mechanism by which leptin could promote steatosis and fibrosis remains unknown. Leptin could regulate body weight by decreasing food intake and increasing energy expenditure (26) and, as a result, would correlate with body mass index, percentage body fat (27), and insulin activity modulation (28). It has been suggested that leptin induces
insulin resistance and increases fatty acid concentrations in the liver while enhancing lipid peroxidation (29) and promoting steatosis. Of note is that a portal perfusion of leptin in rats induced hypertriglyceridemia and contributed to hepatic steatosis by increasing free fatty acids in the liver (30). On the other hand, leptin could induce the release of cytokines such as tumor necrosis factor-␣, interferon-␥, interferon-18, and tumor growth factor-1, and these could mediate liver steatosis and fibrosis (31–33). Recently, a link between HCV infection and leptin metabolism has been proposed. Bound leptin, but not the nonbound form, was found to be higher in chronic hepatitis C patients than in control subjects, and the concentrations decreased in sustained responders to antiviral therapy compared to nonresponders (34). In chronic hepatitis C, obesity has been associated with steatosis development and fibrosis progression (10 –12, 35). As such, leptin could be a link between these factors. Alcohol has been associated with hepatic steatosis but, as in a recent report (36), our data did not indicate a relationship between alcohol consumption and steatosis in patients with hepatitis C. In summary, hepatic steatosis was related to genotype, degree of fibrosis, and serum leptin levels. Genotype 3 seems to have a viral specific steatogenic effect, whereas
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Table 3. Host and Viral Factors Associated With Hepatocyte Steatosis: Univariate and Multivariate Analysis
Variable Age (yr) Sex Male Female BMI (kg/m2) BF(%) (BIA) Skinfold (%) Alcohol intake ⬎40 g/day ⬍40 g/day Leptin (ng/ml) Waist (cm) HCV RNA (IU ⫻ 103/ml) ALT (IU/ml) Fibrosis Genotype 1 Genotype 3
p
Logistic Regression (Backward LR) OR (95% CI)
40.7 ⫾ 12.1
36.7 ⫾ 9.9
⬍0.05
ns
40 23 28.6 ⫾ 6.4 30.8 ⫾ 11.2 30.2 ⫾ 10.6
44 24 25.8 ⫾ 3.4 28.1 ⫾ 8.3 27.5 ⫾ 9.0
ns ⬍0.025 ns ns
21 42 20.8 ⫾ 19.9
16 52 8.4 ⫾ 8.1
ns ⬍0.001
92.4 ⫾ 15.4 500 ⫾ 588 144 ⫾ 96 1.95 ⫾ 0.96
86.4 ⫾ 11.5 804 ⫾ 826 128 ⫾ 142 1.54 ⫾ 0.90
0.06 ns ns p ⬍ 0.01
31 22
60 5
p ⬍ 0.001
Steatosis
None
ns ns
1.09 (1.03–1.17) p ⫽ 0.008 ns 9.86 (2.11–45.86) p ⫽ 0.03 11.54 (1.13–117.14) p ⫽ 0.038
Steatosis was more often seen in genotype 3. Fibrosis stage and leptin levels were independently associated with hepatocyte steatosis. BF% ⫽ percentage body fat; BIA ⫽ bioelectrical impedance analysis.
leptin seems to be a link between obesity and steatosis in patients with chronic hepatitis C genotype 1 infection.
ACKNOWLEDGMENTS This article is dedicated to Gabriel Ro´ dena, M.D., who died in October, 2001. All those who knew him considered him an exemplary person. We will never forget him. The authors thank Dr. J. Salmeron, M.D., for useful comments and Schering-Plough for providing us with the leptin kits. Reprint requests and correspondence: Manuel Romero-Go´ mez, M.D., Hepatology Unit, Hospital Universitario de Valme, Ctra de Ca´ diz s/n, 41014 Sevilla, Spain. Received Jan. 14, 2002; accepted Aug. 26, 2002.
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Vol. 98, No. 5, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)00185-0
Serum Leptin Levels Correlate With Hepatic Steatosis in Chronic Hepatitis C Manuel Romero-Go´mez, M.D., Victor M. Castellano-Megias, M.D., Lourdes Grande, M.D., Jose´ A. Irles, M.D., Marina Cruz, M.D., Marı´a Carmen Nogales, M.D., Juan Carlos Alco´n, M.D., and Antonio Robles, M.D. Units of Hepatology, Pathology, Nutrition, Biochemistry, and Microbiology, Hospital Universitario de Valme, Sevilla, Spain
OBJECTIVES: Hepatic steatosis (HS) has been related to obesity and fibrosis in chronic hepatitis C (CHC). The aim of this study was to determine the role of leptin system in HS development. METHODS: Patients (n ⫽ 131) with biopsy-proven CHC, positive HCV RNA, and raised ALT were enrolled. Body mass index, percentage of body fat by skin fold measurement, and bioelectrical impedance analysis was calculated and serum leptin concentration measured. Intrahepatic HCV RNA, HS, necroinflammatory activity, and fibrosis were determined in liver biopsy tissue. RESULTS: HS was present in 63 patients (48.1%). Steatosis was evident in 32 of 91 patients (35.2%) infected with genotype 1 and in 22 of 27 patients (81.5%) with genotype 3a (p ⬍ 0.001). In patients infected by genotype 3a, HS correlated significantly with intrahepatic HCV RNA load (r ⫽ 0.78; p ⬍ 0.001). However, in genotype 1, HS was associated with host factors such as leptin, body mass index, percentage of body fat, and visceral obesity. Multivariate analysis showed genotype (OR ⫽ 11.54, 95% CI ⫽ 1.13– 117.14, p ⫽ 0.038), leptin levels (OR ⫽ 1.09, 95% CI ⫽ 1.03–1.17, p ⫽ 0.008) and fibrosis (OR ⫽ 9.86, 95% CI ⫽ 2.11–5.86, p ⫽ 0.03) as independent variables of HS development. CONCLUSIONS: Hepatic steatosis was related to genotype, fibrosis degree, and serum leptin levels. Genotype 3 seems to have a viral specific steatogenic effect. Leptin seems to be a link between obesity and steatosis development in CHC genotype 1–infected patients. (Am J Gastroenterol 2003;98: 1135–1141. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION Hepatocyte steatosis (HS) is a frequent histopathological feature in chronic hepatitis C virus (HCV) infection (1, 2) and has been related to fibrosis progression (3). However, a link between steatosis and fibrosis remains unclear (4, 5). Host factors such as dyslipidemia, diabetes mellitus type 2, increased body mass index (BMI) (6, 7), together with viral factors such as genotype, hepatitis G virus coinfection (8)
and HCV RNA load (9) have been associated with steatosis development in chronic hepatitis C. However, although some investigators have found steatosis more often in patients infected with genotype 3a (10, 11) others have not (2, 6). Further, a relationship between BMI and steatosis was only found in patients with genotype 1 infection (12). Leptin has been shown to be involved in peripheral insulin resistance and strongly related to body fat composition. This protein is released from adipocytes and has been detected in activated liver stellate cells (13). Leptin may have a role in the regulation of fat deposition, fibrogenesis, and inflammation (14). The aims of the present study were to verify the relationship between steatosis and fibrosis, the influence of viral genotypes on steatosis development and to investigate the role of leptin in the development of steatosis in chronic hepatitis C infection.
MATERIALS AND METHODS Patient Population Patients (n ⫽ 131) with chronic hepatitis C infection were consecutively recruited. Fully informed written consent was obtained before entry into the study, and the hospital’s ethics committee approved the protocol. Patient history of alcohol consumption was obtained. Two investigators independently solicited information regarding the number of alcoholic beverages consumed daily as well as the duration of the alcohol abuse, if any. An average daily intake of alcohol (in grams) was calculated. Patients with alcohol consumption ⬎80 g/day were excluded. Weight changes over the previous year were recorded, and patients with changes ⬎10% were excluded from the study. No patient had received interferon or other antiviral or steatosis-inducing drugs. Patients previously diagnosed as having type 2 diabetes mellitus were also excluded. Laboratory Investigations An overnight (12-h) fasting blood sample was taken for routine investigations such as ALT, AST, alkaline phospha-
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tase, ␥-glutamyl transpeptidase, cholesterol, and triglycerides. All patients had positive anti-HCV (EIA3; Abbott Laboratories, Chicago, IL), increased ALT, and positive HCV RNA in serum. HBsAg, anti-HBc, anti-HIV were tested using commercially available kits (Abbott Laboratories). All patients were negative for HBsAg and anti-HIV. Serum leptin levels were measured with a commercially available ELISA kit (Quantikine Human Leptin Immunoassay; R&D systems, Minneapolis, MN). The interassay coefficient of variation was 12%. Fasting samples of serum obtained after centrifugation were stored in aliquots at ⫺70°C until assayed. The insulin resistance index was calculated on the basis of fasting values of plasma glucose and insulin according to the homeostasis model assessment for insulin sensitivity (HOMA) model formula: Insulin resistance (HOMA IR) ⫽ fasting insulin (mUI/L) ⫻ fasting glucose (mmol/L) ⫼ 22.5 Body Composition Measurements Height and weight were determined at baseline, and body mass index (BMI) was calculated as weight (in kg) ⫼ height (in m2). In a nutrition laboratory, skin fold thickness was measured at the triceps, biceps, and subscapular and suprailiac regions using Holtain callipers (Dietosystem, Milan, Italy). Percentage of body fat mass was calculated using the equations of Durnin and Womersley (15). Waist circumference measurement was used to estimate visceral obesity. Visceral obesity was considered when waist measurement was ⬎102 cm in men and ⬎88 cm in women. Bioelectrical impedance analysis (BIA) was performed with the patient supine, arms relaxed but not touching the body, in a fasting state, not smoking, and with an empty bladder. Fat mass and percentage body fat was calculated using Tanita-305 (Tanita, Tokyo, Japan). Leptin adjusted for body fat mass was expressed as leptin level divided by fat mass (and expressed as ng/ml/kg). Liver Histology Methods Percutaneous liver biopsy under ultrasonographic control was performed. A portion of the biopsy material was used in histological diagnosis. All liver biopsy specimens were adequate for diagnosis. The average length was 16 mm showing nine portal tracts. We assessed grading and staging separately. The stage was defined according to the Scheuer fibrosis score (16) in which F0 ⫽ absence, F1 ⫽ enlarged portal tracts, F2 ⫽ periportal or portoportal septa, F3 ⫽ fibrosis with architectural distortion, and F4 ⫽ cirrhosis. Necroinflammatory activity was determined by combining scores for portal inflammation (0 – 4) and lobular necrosis (0 – 4). Steatosis was quantified as the percentage of hepatocytes that contained fat droplets, from 0 (absent) to 100% (all hepatocytes containing fat droplets). To classify the patients, grading of steatosis was assigned as grade 0 if there was no steatosis, grade 1 when ⬍25% of hepatocytes contained fat droplets, grade 2 when between 25% and 50%
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showed steatosis, and grade 3 when ⬎50% of hepatocytes showed fat storage. Patients showing steatosis combined with hepatocyte ballooning, neutrophil infiltrate, and either Mallory hyaline or sinusoidal fibrosis were considered as having steatohepatitis and were excluded from this study. Two pathologists with no knowledge of the patient’s clinical data performed parallel histopathology assessments. When a discrepancy between pathologists’ findings occurred, the specimen was re-evaluated in parallel by the two pathologists so as to achieve a consensus. In 48 patients, the other portion of the biopsy material was rapidly frozen and used in the quantification of intrahepatic HCV levels. Virology Methods HCV genotyping was with INNO-LIPA HCV II kits (Innogenetics, Zwijnaarden, Belgium), which were used according to the manufacturer’s instructions. Amplicor-HCVMonitor (Perkin-Elmer, Norwalk, CT) was used to quantify the HCV RNA levels in serum. The level of detection was 1000 copies/ml and all patients were HCV RNA positive. Semiquantification of HCV RNA in liver was by adding 1 g of total liver RNA to 50 l of specimen diluent to which 0.2 l of internal standard had previously been mixed. A 50-l polymerase chain reaction master mix was added and the rest of the procedure was identical to that of the serum-based assay. Amplicor-HCV-Monitor (PerkinElmer) was also used to quantify the HCV RNA levels in the liver, with a level of detection of 1000 copies/g total RNA. Statistical Analysis Hepatic steatosis was assessed as categorical (absent or present) or continuous variable from 0% to 100%. Comparisons between groups with and without steatosis according genotypes were made using the Mann-Whitney U test or the Student’s t test for continuous variables and the 2 or Fisher’s exact probability test for categorical data. The Spearman coefficient was used to correlate numerical variables that were nonnormally distributed. All values are presented as means ⫾ SD. A p value of ⬍0.05 was considered to be statistically significant. Multiple linear regression has been used in the multivariate analysis of factors associated with leptin levels, including age, sex, hepatic steatosis, body mass index, percentage of body fat, insulin resistance, fibrosis, and waist measurement. A multivariate analysis based on a backward LR logistic regression was used to establish association between hepatocyte steatosis and host (and viral) factors including sex, age, alcohol consumption, genotype, body mass index, visceral obesity, leptin level, HCV RNA load, and fibrosis.
RESULTS Patient Demographic Data The mean age of the patients was 38 yr (⫾11 yr) with an estimated duration of infection of 16 ⫾ 9 yr. The study
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Leptin and Steatosis in HCV
Table 1. Multiple Linear Regression Analysis (R2 ⫽ 0.59) With Serum Leptin Levels as Dependent Variable in Patients With Chronic Hepatitis C Variable
Coefficient
SE
t
p
Age (yr) Sex Insulin resistance Fibrosis Steatosis % BF % BMI Waist (cm)
⫺0.028 0.596 0.03 0.035 0.186 ⫺0.297 0.773 ⫺0.035
0.13 4.95 0.77 1.44 0.07 0.29 0.43 0.14
⫺0.29 3.66 0.28 0.41 2.28 ⫺1.52 4.97 ⫺0.27
0.77 0.001 0.77 0.68 0.026 0.13 0.001 0.78
Body mass index, sex, and hepatocyte steatosis were independently related to leptin levels. BF % ⫽ percentage of body fat mass; Steatosis % ⫽ percentage of hepatocyte steatosis.
population comprised 84 men and 47 women. Related risk factors were drug abuse (n ⫽ 42), blood transfusion (n ⫽ 31), tattooing (n ⫽ 8), surgery (n ⫽ 33), health worker occupation (n ⫽ 3), and unknown etiology (n ⫽ 14). The median alcohol intake was 19 g/day with 37 of the 131 patients consuming ⬎40 g/day; 34 of 37 (91.8%) were men. The mean BMI was 26.8 ⫾ 3.6 kg/m2. Genotype 1b was found in 78 (59.5%) patients, genotype 1a in 13 (9.9%), genotype 2a in four (2.2%), genotype 3a in 27 (20.6%), and genotype 4a in two patients. In seven patients (5.3%) we were unable to establish an unequivocal genotype. Histological Evaluation Steatosis was detected in 63 of 131 patients (48.1%), grade 1 in 39 (29.7%), grade 2 in 16 (12.2%), and grade 3 in eight (6.1%) patients. Stage of fibrosis was F0 in four (3.1%), F1 in 58 (44.2%), F2 in 47 (35.8%), F3 in 12 (9.2%), and F4 in 10 (7.6%) patients. The grade of portal inflammation was P1 in 15 (11.5%), P2 in 70 (53.4%), P3 in 40 (30.5%), and P4 in six (4.6%) cases, and lobular necrosis was L0 in two (1.5%), L1 in 63 (48.1%), L2 in 63 (48.1%), and L3 in three (2.3%). Body Composition and Leptin Levels Serum leptin levels were higher in women than in men (24.3 ⫾ 18.7 vs 8.4 ⫾ 11.1 ng/ml; p ⬍ 0.0001), even when
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adjusted for fat mass (0.28 ⫾ 0.20 vs 0.79 ⫾ 0.43 ng/ml/kg). However, no differences were observed with respect to genotype (genotype 1, 14.9 ⫾ 16.8; genotype 3a, 11.8 ⫾ 13.6 ng/ml; p ⫽ ns). The leptin levels correlated with BMI (r ⫽ 0.49; p ⬍ 0.01), percentage of body fat (r ⫽ 0.77, p ⬍ 0.001), percentage of hepatocyte steatosis (r ⫽ 0.31, p ⬍ 0.005), age (r ⫽ 0.37, p ⬍ 0.01), insulin resistance (r ⫽ 0.29, p ⫽ 0.004) and fibrosis stage (r ⫽ 0.22, p ⬍ 0.05), but not with ALT and AST levels or with HCV RNA burden. In multivariate analysis (multiple linear regression), the percentage of hepatocyte steatosis, sex, and body mass index were independently related to serum leptin levels (Table 1). Leptin concentrations when adjusted for fat mass did not alter these findings, and these data were not analyzed further. Factors Associated With Hepatic Steatosis Steatosis was detected more frequently in patients infected by genotype 3 (22 of 27; [81.5%] than by genotype 1 (32 of 91, 35.2%; p ⬍ 0.001). Thus, the influence of body composition and leptin levels on the development of hepatic steatosis was analyzed segregated by genotypes. BMI, percentage of body fat, leptin levels, and visceral obesity were significantly associated with the presence of steatosis in patients infected by genotype 1 but not in patients infected by genotype 3, whereas cholesterol, triglycerides, and insulin resistance were not related to steatosis (Table 2). Furthermore, leptin levels correlated with percentage hepatocyte steatosis in patients infected by genotype 1 (r ⫽ 0.66, p ⬍ 0.01) but not in genotype 3 (r ⫽ 0.29, p ⫽ ns) (Fig. 1), whereas in patients infected with genotype 3, steatosis correlated with intrahepatic HCV RNA (r ⫽ 0.78, p ⬍ 0.001) (Fig. 2). The degree of steatosis was greater in patients with a daily average alcohol consumption ⬎40 g/day than in nondrinkers or those consuming ⬍40 g/day. This difference did not quite reach statistical significance (16 ⫾ 25% vs 10 ⫾ 17%, p ⫽ 0.08), probably because heavy drinkers had been excluded from the study. As alcohol could be a confounding variable, an analysis excluding individuals con-
Table 2. Relationship Between Hepatocyte Steatosis, Leptin Levels, and Body Composition Segregated With Respect to Genotypes Genotype 1 (n ⫽ 91)
Genotype 3 (n ⫽ 27)
Variable
Steatosis (n ⫽ 32)
None (n ⫽ 59)
p
BMI (kg/m2) BF(%) (BIA) Skinfold (%) Leptin (ng/ml), men Leptin (ng/ml), women HOMA IR Cholesterol Triglycerides Visceral obesity (%) Alcohol intake ⬎40 g/day
29.8 ⫾ 7.4 34 ⫾ 9.9 34.3 ⫾ 9.7 14.2 ⫾ 16.0 37.6 ⫾ 19.7 2.73 ⫾ 1.76 171 ⫾ 43 115 ⫾ 87 15 (46.9%) 9 (28.1%)
25.7 ⫾ 3.6 28.1 ⫾ 7.9 27.9 ⫾ 8.0 5.5 ⫾ 4.6 16.3 ⫾ 14.3 1.84 ⫾ 2.44 158 ⫾ 36 108 ⫾ 58 14 (23.7%) 15 (25.4%)
⬍0.001 ⬍0.01 ⬍0.01 ⬍0.01 ⬍0.005 ⫽0.087 ns ns ⬍0.05 NS
Steatosis (n ⫽ 22) 26.4 ⫾ 4.6 26.8 ⫾ 8.2 25.4 ⫾ 5.5 11.61 ⫾ 16.2 16.1 ⫾ 15.9 2.16 ⫾ 2.10 153 ⫾ 44 104 ⫾ 74 4 (14.8%) 10 (45.4%)
None (n ⫽ 5)
p
26.3 ⫾ 11.3 33.6 ⫾ 8.05 29.7 ⫾ 7.4 4.9 ⫾ 1.3 16.0 ⫾ 2.3 1.19 ⫾ 0.59 175 ⫾ 25 95 ⫾ 47 0 (0%) 0 (0%)
NS NS NS NS NS ns ns ns NS NS
Visceral obesity was considered to be present when waist measurement was ⬎102 cm in men and ⬎88 cm in women (reference values of Spanish Nutrition Association). Steatosis was strongly related to body composition in patients infected with genotype 1 but not in patients infected with genotype 3. BIA ⫽ bioelectrical impedance analysis; BF% ⫽ percentage body fat.
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Figure 1. Correlation between serum leptin concentrations and percentage hepatocyte steatosis in chronic hepatitis C patients segregated with respect to genotype.
suming ⬎40 g/day was performed, and the associations were observed to remain unchanged. Steatosis correlated with liver HCV RNA in genotype 3 (r ⫽ 0.75, p ⫽ 0.01) and with leptin in genotype 1 (r ⫽ 0.64, p ⬍ 0.001). To analyze the interaction between host and viral factors implicated in steatosis development, we performed a multivariate analysis (logistic regression backward LR). The independent variables predictive of presence of steatosis were genotype 3 (OR ⫽ 11.54, 95% CI ⫽ 1.13 – 117.14, p ⫽ 0.038), higher leptin levels (OR ⫽ 1.09, 95% CI ⫽ 1.03 – 1.17, p ⫽ 0.008), and advanced fibrosis stage (OR ⫽ 9.86, 95% CI ⫽ 2.11– 45.86, p ⫽ 0.03) (Table 3).
DISCUSSION In the present study, hepatic steatosis was associated with a higher degree of fibrosis and was influenced by HCV genotype. In patients infected with genotype 1, steatosis correlated with serum leptin levels, and in patients infected with genotype 3, steatosis was strongly related to intrahepatic HCV load. An explanation for these findings is not yet available, although a similar result has been reported (12, 17). The relationship between steatosis and intrahepatic HCV load has been observed in human as well as animal
studies. The core protein of hepatitis C has been shown to induce hepatic steatosis in transgenic mice (18), whereas, in patients with chronic hepatitis C, a close relationship between in situ level of core HCV and hepatic steatosis has been observed (19). In genotype 3–infected patients, steatosis has been related to HCV RNA load in serum (9) and in the liver (20), as was observed in the current study. Thus, at least in patients infected with genotype 3, hepatocyte steatosis emerges as a morphological feature that is induced directly by the virus. However, host factors such as BMI or body fat, usually associated with steatosis, seem to not play an important role in this group. The mechanism by which HCV induces steatosis remains unclear. The virus can increase glutathione turnover by eliciting free radical–mediated lipid peroxidation, which would affect iron metabolism within the hepatocyte and which may promote fat droplet deposition (21). On the other hand, an increase in the concentration of monounsaturated fatty acids has been found in the liver of patients with chronic hepatitis C relative to non–HCV-related liver disease (22). Thus, HCV may cause a change in lipid metabolism and promote triglyceride accumulation resulting in hepatocyte steatosis.
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Leptin and Steatosis in HCV
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Figure 2. Relationship between intrahepatic HCV load and percentage of hepatocyte steatosis segregated with respect to genotype.
In the current study, liver steatosis was related to serum leptin level and body composition in patients infected by genotype 1 but not in those with genotype 3. In a previous report, leptin was observed to be unrelated to steatosis in chronic hepatitis C (23), although in that report, the number of patients with genotype 1 infection was very small (only 12), and the authors were unable to find any relationship between leptin levels and steatosis. In the current study, a weak relationship between leptin levels and fibrosis has also been found. Indeed, leptin has previously been shown to be associated with steatosis and fibrosis in nonobese patients with nonalcoholic steatohepatitis (24). However, leptin shows a sex-based difference, and women have circulating plasma leptin concentrations that are three times higher than those in men, even when subjects are matched for body fat mass (25). Thus, patient sex needs to be considered when investigating leptin levels, and multivariate analysis is necessary to reduce the bias caused by this confounding variable. The mechanism by which leptin could promote steatosis and fibrosis remains unknown. Leptin could regulate body weight by decreasing food intake and increasing energy expenditure (26) and, as a result, would correlate with body mass index, percentage body fat (27), and insulin activity modulation (28). It has been suggested that leptin induces
insulin resistance and increases fatty acid concentrations in the liver while enhancing lipid peroxidation (29) and promoting steatosis. Of note is that a portal perfusion of leptin in rats induced hypertriglyceridemia and contributed to hepatic steatosis by increasing free fatty acids in the liver (30). On the other hand, leptin could induce the release of cytokines such as tumor necrosis factor-␣, interferon-␥, interferon-18, and tumor growth factor-1, and these could mediate liver steatosis and fibrosis (31–33). Recently, a link between HCV infection and leptin metabolism has been proposed. Bound leptin, but not the nonbound form, was found to be higher in chronic hepatitis C patients than in control subjects, and the concentrations decreased in sustained responders to antiviral therapy compared to nonresponders (34). In chronic hepatitis C, obesity has been associated with steatosis development and fibrosis progression (10 –12, 35). As such, leptin could be a link between these factors. Alcohol has been associated with hepatic steatosis but, as in a recent report (36), our data did not indicate a relationship between alcohol consumption and steatosis in patients with hepatitis C. In summary, hepatic steatosis was related to genotype, degree of fibrosis, and serum leptin levels. Genotype 3 seems to have a viral specific steatogenic effect, whereas
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Table 3. Host and Viral Factors Associated With Hepatocyte Steatosis: Univariate and Multivariate Analysis
Variable Age (yr) Sex Male Female BMI (kg/m2) BF(%) (BIA) Skinfold (%) Alcohol intake ⬎40 g/day ⬍40 g/day Leptin (ng/ml) Waist (cm) HCV RNA (IU ⫻ 103/ml) ALT (IU/ml) Fibrosis Genotype 1 Genotype 3
p
Logistic Regression (Backward LR) OR (95% CI)
40.7 ⫾ 12.1
36.7 ⫾ 9.9
⬍0.05
ns
40 23 28.6 ⫾ 6.4 30.8 ⫾ 11.2 30.2 ⫾ 10.6
44 24 25.8 ⫾ 3.4 28.1 ⫾ 8.3 27.5 ⫾ 9.0
ns ⬍0.025 ns ns
21 42 20.8 ⫾ 19.9
16 52 8.4 ⫾ 8.1
ns ⬍0.001
92.4 ⫾ 15.4 500 ⫾ 588 144 ⫾ 96 1.95 ⫾ 0.96
86.4 ⫾ 11.5 804 ⫾ 826 128 ⫾ 142 1.54 ⫾ 0.90
0.06 ns ns p ⬍ 0.01
31 22
60 5
p ⬍ 0.001
Steatosis
None
ns ns
1.09 (1.03–1.17) p ⫽ 0.008 ns 9.86 (2.11–45.86) p ⫽ 0.03 11.54 (1.13–117.14) p ⫽ 0.038
Steatosis was more often seen in genotype 3. Fibrosis stage and leptin levels were independently associated with hepatocyte steatosis. BF% ⫽ percentage body fat; BIA ⫽ bioelectrical impedance analysis.
leptin seems to be a link between obesity and steatosis in patients with chronic hepatitis C genotype 1 infection.
ACKNOWLEDGMENTS This article is dedicated to Gabriel Ro´ dena, M.D., who died in October, 2001. All those who knew him considered him an exemplary person. We will never forget him. The authors thank Dr. J. Salmeron, M.D., for useful comments and Schering-Plough for providing us with the leptin kits. Reprint requests and correspondence: Manuel Romero-Go´ mez, M.D., Hepatology Unit, Hospital Universitario de Valme, Ctra de Ca´ diz s/n, 41014 Sevilla, Spain. Received Jan. 14, 2002; accepted Aug. 26, 2002.
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