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Increased circulating adiponectin in males with chronic HCV hepatitis
European Journal of Internal Medicine 26 (2015) 635–639
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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Original Article
Increased circulating adiponectin in males with chronic HCV hepatitis Elena Canavesi a,1, Marianna Porzio a,1, Massimiliano Ruscica b, Raffaela Rametta a, Chiara Macchi b, Serena Pelusi a, Anna Ludovica Fracanzani a, Paola Dongiovanni a, Silvia Fargion a, Paolo Magni b, Luca Valenti a,⁎ a Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Department of Pathophysiology and Transplantation, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, Milano, Italy b Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy
a r t i c l e
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Article history: Received 23 December 2014 Received in revised form 4 May 2015 Accepted 3 August 2015 Available online 17 August 2015 Keywords: Adipokines Adiponectin Gender Hepatic fibrosis Hepatitis C virus Nonalcoholic fatty liver disease
a b s t r a c t Background: Increased levels of adiponectin, a major adipokine with insulin sensitizing properties showing a strong sexual dimorphism, have been reported in individuals with chronic HCV infection (CHC), but data are limited by small samples and lack of control for the genetic background and hepatic fibrosis. The aim of this study was to compare adiponectin levels between CHC patients and accurately matched controls. Methods: We considered 184 CHC patients, matched (1:1) for age, gender, body mass index, and Adiponectin genotype (ADIPOQ) with healthy individuals. To control for the severity of liver disease, a second control group consisting of 95 patients with histological nonalcoholic fatty liver disease (NAFLD) further matched (1:1) for severe fibrosis was exploited. ADIPOQ genotype was evaluated by Taqman assays, serum adiponectin measured by ELISA. Results: Serum adiponectin was higher in CHC patients than in healthy individuals (9.0 ± 5.0 μg/ml vs. 7.3 ± 4.0 μg/ml; p = 0.001; adjusted estimate +1.8, 1.7–2.9; p = 0.001), and than in NAFLD patients (8.3 ± 4.5 μg/ml vs. 6.0 ± 4.2 μg/ml; p b 0.001; adjusted estimate + 0.8, 0.2–1.4, p = 0.006). After stratification for sex, serum adiponectin was higher in males with CHC than in healthy individuals and NAFLD patients (p b 0.005 for both), whereas the difference was not significant in females. Conclusions: CHC is associated with increased serum adiponectin independently of age, body mass, diabetes, ADIPOQ genotype, and of severe liver fibrosis, particularly in men. © 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Background Chronic hepatitis C virus (HCV) infection (CHC) affects 170 million individuals worldwide, representing a leading cause of cirrhosis and hepatocellular carcinoma [1,2]. CHC is frequently associated with alterations in the metabolism of glucose and lipids, as HCV interferes with very low-density lipoproteins secretion from hepatocytes and impairs glucose metabolism with development of insulin resistance (IR) and Abbreviations: HCV, hepatitis C virus; CHC, chronic HCV hepatitis; IR, insulin resistance; T2DM, type 2 diabetes mellitus; NAFLD, nonalcoholic fatty liver disease; ADIPOQ, adiponectin gene; BMI, body mass index; AST, aspartate aminotransferases; ALT, alanine aminotransferases; GGT, gamma-glutamyl-transferases. ⁎ Corresponding author at: Department of Pathophysiology and Transplantation, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Pad. Granelli, via F Sforza 35, 20122 Milano, Italy. E-mail addresses: [email protected] (E. Canavesi), [email protected] (M. Porzio), [email protected] (M. Ruscica), [email protected] (R. Rametta), [email protected] (C. Macchi), [email protected] (S. Pelusi), [email protected] (A.L. Fracanzani), [email protected] (P. Dongiovanni), [email protected] (S. Fargion), [email protected] (P. Magni), [email protected] (L. Valenti). 1 These authors contributed equally to the work.
type 2 diabetes mellitus (T2DM) [3–5]. The association between HCV and metabolic alterations is clinically remarkable, since steatosis and IR are associated with faster progression of fibrosis [6,7], lower rates of response to interferon-based therapy [8,9] and increased risk of T2DM and cardiovascular disease [4,10–12]. HCV interacts with insulin signaling in the hepatocytes, leading to the development of IR in organs such as muscles or the adipose tissue [13, 14]. From this perspective, CHC shares some pathologic features with non-alcoholic fatty liver disease (NAFLD), which is related to the metabolic syndrome and results from the development of adipose tissue IR [15] and decreased adiponectin [16], increased de novo lipogenesis and altered hepatic beta-oxidation and lipoprotein export [17–19]. Adipose tissue produces a number of adipokines, which modulate insulin sensitivity. Among these, adiponectin, a molecule with insulin-sensitizing, anti-inflammatory and anti-fibrotic effects, plays a major role [20]. Based on the observation of increased circulating adiponectin in CHC patients, which was associated with adverse metabolic and clinical outcomes including hepatocellular carcinoma, altered adiponectin activity has been hypothesized to play a role in the pathogenesis of hepatic and extra-hepatic complications of this condition [21–24]. A possible state of adiponectin resistance has been hypothesized based on the observation of downregulation of the hepatic adiponectin receptor AdipoR1 in
http://dx.doi.org/10.1016/j.ejim.2015.08.001 0953-6205/© 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
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CHC patients with advanced liver fibrosis [25]. However, the sample size of available studies was limited, and increased adiponectin levels were not always confirmed, or limited to patients with severe fibrosis [23, 26], which could also interfere with biliary clearance of adiponectin [27]. Furthermore, controls matched for liver disease severity were never assessed. Therefore, the aim of this study was to evaluate whether CHC is associated with increased adiponectin levels independently of demographic and anthropometric features, the genetic background, and severe hepatic fibrosis, and secondarily to assess whether the association is influenced by sex, as adiponectin levels show a strong sexual dimorphism. Serum adiponectin is indeed a partly heritable trait, the major determinant being Adiponectin genotype (ADIPOQ) genotype [28]. To this end, CHC patients were matched to healthy subjects for age, gender, body mass index (BMI), and ADIPOQ, and further matched to patients with NAFLD for the presence of severe liver fibrosis. 2. Patients and methods 2.1. Study design From a previously described larger series of patients with CHC, NAFLD, and healthy controls with a very low probability of hepatic steatosis [29,30], we conducted a two-step nested case–control study. The inclusion criteria have previously been described in details [29]. Briefly, we selected consecutive adult patients with active CHC with available histological evaluation and without comorbidities, and with a histological diagnosis of NAFLD without coexistent liver diseases. We also considered healthy controls with normal liver enzymes and without metabolic abnormalities with a very low probability of steatosis based on the fatty liver index [31]. In a first step, we matched 1:1 CHC patients with healthy controls for age (±8 years), sex, body mass index (BMI) category (normal range: 20–25 kg/m2, overweight: 25.1–29.9 kg/m2, and obese: N 30 kg/m2), and ADIPOQ genotype (presence of rs2241766TT or rs1501299TT/TG genotypes, associated with higher adiponectin levels [29]; henceforth defined high expression genotypes). We identified 184 CHC patients (46 females, age 54 ± 13 years) and matched healthy subjects; their clinical features are presented in Table 1. In a second step, to control for the severity of liver disease, we matched 1:1 a second group of CHC patients (selected within the 184 patients identified in the first stage) for age, gender, BMI category, ADIPOQ (as reported above) and the presence of severe hepatic fibrosis
(bridging fibrosis or cirrhosis) with NAFLD patients. We identified 95 CHC patients (24 females, age 50 ± 11 years) and matched NAFLD patients; their clinical features are presented in Table 2. The study protocol was approved by the Institutional Review Board of the Fondazione Ca' Granda IRCCS Milano. Informed written consent was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki. 2.2. Clinical and laboratory data In all subjects the following information was available: lifestyle habits (smoking and daily alcohol intake, the last one confirmed by at least one family member), clinical and pharmacologic history, age, race, BMI, complete blood count, alanine-aminotransferase (ALT), gamma-glutamyltransferase (GGT), fasting glucose, total and highdensity lipoprotein (HDL) cholesterol, and triglycerides. T2DM was diagnosed according to the WHO criteria [32]. Serum adiponectin was measured on an aliquot of plasma collected after overnight fasting at the time of liver biopsy (when available) or of abdominal ultrasonography and metabolic evaluation, and stored at −80 °C. Plasma adiponectin levels (all isoforms) were measured using a commercial enzyme-linked immunosorbent assay kit with a lowest limit of sensitivity of 0.246 ng/ml (R&D Systems, Minneapolis, MN) [29,33]. ADIPOQ rs2241766, + 45 GNT and r150299, + 276 GNT polymorphisms, previously associated with the susceptibility to NAFLD were genotyped on peripheral blood DNA samples by a Taqman assay (assay on demand, Applied Biosystems, Foster City, CA) [29]. Liver biopsies were routinely processed and read by a single expert pathologist. The minimum biopsy size was 1.7 cm and the number of portal areas 10. Liver biopsies were scored the severity of fibrosis according to Ishak for CHC patients [34] and NAFLD clinical research network activity score for NAFLD patients [35]. Severe fibrosis was defined as Ishak fibrosis score F4–F6 for CHC patients or NAFLD fibrosis stage F3–F4 for NAFLD patients. 2.3. Statistical analysis For descriptive statistics, results are expressed as means ± standard deviations for continuous variables and as frequencies for categorical variables. p-Values from ANOVA or chi-square test were considered statistically significant if ≤0.05 (two-tailed). Non-normally distributed variables (e.g., GGT) were log-transformed before entering the analyses.
Table 2 Clinical features of 95 CHC and NAFLD patients. Table 1 Clinical features of 184 CHC patients and 184 healthy controls.
Age Sex (F) BMI classes Normal range Overweight Obesity ADIPOQ (high expression genotypes) T2DM Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Fasting insulin (IU/ml) Fasting glucose (mg/dl) ALT (IU/l) GGT (IU/l) Adiponectin (μg/ml)
CHC patients
Healthy controls
p
54 ± 13 46 (25)
49 ± 12 46 (25)
b0.001 1 1
90 (49) 88 (48) 6 (3) 74 (40)
90 (49) 88 (48) 6 (3) 74 (40)
30 (16) 175 ± 36 48 ± 16 106 ± 56 20 ± 9 93 ± 20 72 ± 63 58 ± 58 9.0 ± 5.0
0 194 ± 32 57 ± 13 89 ± 50 13 ± 8 70 ± 14 23 ± 9 23 ± 16 7.3 ± 4.0
1 b0.001 b0.001 b0.001 0.003 b0.001 b0.001 b0.001 b0.001 0.001
Controls were matched 1:1 for age (±10 years), sex, BMI, and ADIPOQ genotype. (): % values; BMI: body mass index; T2DM: type 2 diabetes mellitus.
Age Sex (F) BMI classes Normal range Overweight Obesity ADIPOQ (high expression genotypes) Advanced liver fibrosis T2DM Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Fasting insulin (IU/ml) Fasting glucose (mg/dl) ALT (IU/l) GGT (IU/l) Adiponectin (μg/ml)
CHC patients
NAFLD patients
50 ± 11 24 (25)
49 ± 11 24 (25)
30 (32) 54 (57) 11 (11) 41 (43)
30 (32) 54 (57) 11 (11) 41 (43)
18 (19) 9 (9) 180 ± 34 47 ± 14 107 ± 62 23 ± 11 90 ± 17 77 ± 63 54 ± 50 8.3 ± 4.5
18 (19) 27 (28) 208 ± 42 48 ± 13 128 ± 65 21 ± 19 100 ± 32 68 ± 53 104 ± 137 6.0 ± 4.2
p 0.65 1 1
1 1 0.001 b0.001 0.67 0.031 0.37 0.013 0.26 0.001 b0.001
NAFLD patients were matched 1:1 for age (±8 years), sex, BMI, ADIPOQ genotype, and presence of advanced liver fibrosis (): % values; BMI: body mass index; T2DM: type 2 diabetes mellitus.
E. Canavesi et al. / European Journal of Internal Medicine 26 (2015) 635–639
Multivariate general linear models were fitted to estimate the impact of CHC on adiponectin levels (for CHC vs. healthy subjects and vs. NAFLD). High adiponectin levels were defined as those above 7.5 μg/ml, the median value in CHC patients [29]. Variables considered in the matching procedure were entered into the final multivariate model. The study had a N 80% power (alpha = 0.05; two-tailed) to detect a 1 SD difference in adiponectin levels between CHC patients and both healthy controls and NAFLD patients (in the overall cohort, and both in males and in females). All statistical analyses were performed by the SPSS 20.0 statistical discovery software (IBM, Burbank, NJ, USA).
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Table 3 Independent predictors of serum adiponectin in CHC patients and healthy controls.
Age (years) Sex (F) BMI (kg/m2) T2DM ALT (IU/ml) ADIPOQ genotype HCV infection
Estimate
95% CI
p value
0.02 +3.39 −0.72 +0.04 −0.006 +1.09 +1.80
−0.015 to +0.58 +2.33 to +4.45 −1.56 to +0.11 −1.744 to +1.82 −0.016 to +0.004 +0.18 to +2.00 +0.74 to +2.86
0.24 b0.001 0.088 0.22 0.22 0.019 0.001
p values were determined at generalized linear model considering all the variables presented in the table. BMI: body mass index; CI: confidence interval; T2DM: type 2 diabetes mellitus.
3. Results 3.1. Adiponectin levels in CHC vs. healthy subjects The clinical characteristics and adiponectin levels in 184 CHC patients and matched healthy subjects are shown in Table 1. Despite matching for age (± 8 years) CHC patients were about 5 years older (p b 0.05), but had the same gender, BMI distribution, and genetic background. Women affected by CHC were not significantly older than those included among healthy subjects (52 ± 11 vs. 49 ± 11 years; p = 0.1). As expected, CHC had IR and higher prevalence of T2DM than healthy subjects, but lower serum cholesterol, and higher liver enzymes (p b 0.05). Serum adiponectin levels were about 22% higher in CHC patients than in matched healthy controls (Table 1; p = 0.001). As adiponectin levels display a strong sexual dimorphism, we next analyzed adiponectin levels according to gender (shown in Fig. 1A). Serum adiponectin levels were markedly higher in male CHC patients than in healthy controls (p b 0.001; p = 0.001 after adjustment for age, BMI, and ADIPOQ genotype), whereas the difference was not significant in females (p N 0.05), characterized by higher adiponectin levels. To correct for potential residual confounders (in particular age, which was significantly different between groups, but also metabolic features and hepatic inflammation), we adjusted the association of adiponectin with CHC for age, sex, BMI class, ADIPOQ genotype, presence of T2DM, and ALT levels (Table 3). CHC remained strongly associated with higher adiponectin levels (Estimate 1.8, 95% CI 0.7–2.9; p = 0.001).
A
After adjustment for sex, BMI, and T2DM, adiponectin levels were inversely correlated with fasting insulin both in CHC and healthy individuals (estimate −0.016 ± 0.05, p b 0.001, and −0.02 ± 0.01, p = 0.049, respectively).
3.2. Adiponectin levels in CHC vs. NAFLD patients The clinical features of 95 CHC and matched NAFLD patients are shown in Table 2. This second group had different clinical features because NAFLD patients were more overweight than healthy subjects. Despite the demographic and anthropometric features were superimposable between the two groups, as expected NAFLD patients had higher serum lipids (total cholesterol and triglycerides), and higher GGT and prevalence of T2DM (p b 0.05). Women affected by CHC were not significantly older than those affected by NAFLD (51 ± 7 vs. 55 ± 9 years; p = 0.1). Again, adiponectin levels were about 25% higher in CHC patients than in with subjects with NAFLD, matched for age, gender, BMI, ADIPOQ genotype, and the severity of fibrosis (p b 0.001; Table 2). We next analyzed adiponectin levels according to gender (shown in Fig. 1B). Again, serum adiponectin levels were higher in male CHC than in NAFLD patients (p b 0.001; p = 0.005 after adjustment for age, BMI, ADIPOQ genotype, and the severity of fibrosis), whereas the difference was not significant in females (p N 0.05), To correct for potential residual confounders (age, metabolic features, and hepatic inflammation), we
B
Fig. 1. Comparison of serum adiponectin levels between CHC patients and controls stratified by gender. A) Serum adiponectin levels in 184 CHC patients and in 1:1 matched healthy controls stratified by gender. B) Serum adiponectin levels in 95 CHC patients and in 1:1 matched NAFLD patients stratified by gender.
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Table 4 Independent predictors of serum adiponectin in CHC and matched NAFLD patients.
Age (years) Sex (F) BMI (kg/m2) T2DM ALT (IU/ml) ADIPOQ genotype Advanced fibrosis HCV infection
Estimate
95% CI
p value
+0.12 +0.52 −0.18 −0.62 −0.001 +0.03 +0.36 +0.81
+0.06 to +0.17 −0.13 to +1.17 0.00 to −0.36 +0.15 to −1.39 +0.001 to +0.01 −0.59 to +0.53 −0.43 to +1.15 +0.24 to +1.38
b0.001 0.11 0.052 0.11 0.77 0.92 0.37 0.006
p values were determined at generalized linear model considering all the variables presented in the table. BMI: body mass index; CI: confidence interval; T2DM: type 2 diabetes mellitus.
adjusted the association of adiponectin with CHC at a generalized model adjusted for age, sex, BMI class, ADIPOQ genotype, ALT levels, presence of T2DM, and of advanced liver fibrosis (Table 4). Chronic HCV infection remained associated with higher adiponectin levels (Estimate +0.8 95% CI 0.2–1.4; p = 0.006). After adjustment for sex, BMI, and T2DM, adiponectin levels were inversely correlated with fasting insulin both in CHC and NAFLD individuals (estimate − 0.13 ± 0.05, p = 0.006, and − 0.05 ± 0.02, p = 0.045, respectively). 3.3. Impact of viral features on adiponectin levels in CHC patients Finally, in an exploratory analysis we evaluated whether viral features influences serum adiponectin levels in 184 CHC patients described in Table 1. To this end, we analyzed whether viral genotype (G1 and G4 vs. G2 and G3) and viral load (high or low viral load, N or ≤8 × 105 IU/ml) were associated with high adiponectin levels (N or ≤7.5 μg/dl, median value). Results are shown in Table 5. Viral genotype and viral load were not associated with adiponectin levels (p N 0.4). Results did not change when treating adiponectin as a continuous variable. 4. Discussion In this study, adiponectin levels were compared between a relatively large series of well-characterized CHC patients and carefully matched controls, first healthy individuals and then patients with NAFLD. The main finding of the study is that adiponectin levels are higher in CHC independently of the presence of advanced liver fibrosis, and that the effect of CHC on adiponectin levels is more marked in males. These results are in line with previous reports of increased adiponectin in CHC [21–23,25,26, 36,37], which however did not take into account difference in body mass, the genetic background, and in particular the presence of severe liver fibrosis. Although the mechanism of fibrogenesis is different between CHC and NAFLD, also characterized by adipose tissue insulin resistance, these data suggest that the increased adiponectin in CHC patients is not due to disruption of the hepatic lobule related to advanced fibrosis. Adiponectin is the major insulin-sensitizing adipokine, mainly secreted by adipose tissue in adult life. Decreased adiponectin release is associated with obesity and IR, subclinical inflammation, T2DM, and the risk of cardiovascular disease and cancer [20]. Furthermore, adiponectin levels are lower in males and post-menopausal females, groups at higher risk of cardio-metabolic diseases [20]. Since CHC is associated with insulin resistance [13], it would be expected to be also linked to decreased
circulating adiponectin [23]. Increased adiponectin levels in the presence of IR have thus led some Authors to hypothesize a state of adiponectin resistance [25], that would account for the association of high adiponectin levels with the risk of hepatocellular carcinoma and overall mortality in CHC [26,38]. However, the inverse correlation between adiponectin and insulin levels in CHC patients does not support adiponectin resistance in this condition. A state of tissue or receptor specific adiponectin resistance cannot be ruled out, but different mechanisms may lead to increased adiponectin levels in CHC patients. Alternative explanations may be represented by a) decreased adiponectin biliary clearance due to advanced liver fibrosis [27], which however was not observed in NAFLD, and b) a feedback mechanism to counteract insulin resistance, which would be consistent with the negative correlation between adiponectin and insulin levels in CHC patients. Noteworthy, the observed differences in serum adiponectin were influenced by gender. Specifically, adiponectin levels were higher in male CHC patients than controls, both healthy subjects without liver disease and patients with NAFLD, whereas the difference was smaller and not significant in females. Therefore, altered adiponectin metabolism may be specific to male gender. Alternatively, CHC infection might exert a milder effect on serum adiponectin in females, which could not be detected because of the lower power in this subgroup. Additional studies with larger sample sizes and better characterization of the hormonal status of women will be required to definitively rule out a milder effect of HCV infection on adiponectin metabolism in women. Limitations of the present study include the cross sectional design, so that we could not causally assess the effect of HCV infection on adiponectin levels, and the impact on clinical outcomes. However, previous data indicate that successful eradication of HCV infection is associated with a decrease in serum adiponectin [22], and that increased adiponectin is associated with hepatocellular carcinoma risk and mortality [26,38]. The sample size was relatively limited, but larger than in previous studies. Furthermore, patients and controls, who did not undergo ultrasonography, but had very low probability of steatosis [31], were very well characterized and meticulously matched for confounding factors. Finally, although we could not adjust the analyses for abdominal obesity, adiponectin levels were higher in CHC patients independently of BMI and T2DM. Evaluation of the modifications of serum total adiponectin and specific isoforms in CHC patients with advanced fibrosis following viral eradication by new direct antiviral agents will be useful to gain further insight into the alterations of adiponectin metabolism in CHC patients. In conclusion, CHC is associated with increased serum adiponectin independently of age, body mass, diabetes, ADIPOQ genotype, and of severe liver fibrosis, particularly in men. Altered adiponectin metabolism may modulate metabolic disturbances and the susceptibility to cardiovascular disease and hepatocellular carcinoma in CHC patients.
Conflict of interests Authors declare that they have no conflict of interest to disclose.
Ethical statement The study protocol was approved by the Institutional Review Board of the Fondazione Ca' Granda IRCCS Milano. Informed written consent
Table 5 Adiponectin levels stratified by HCV genotype and viral load in CHC patients.
Genotype (G1–G4 vs. G2–G3) Viral load (N8 × 105 IU/ml) (): % values.
Adiponectin high (N7.5 μg/dl; n = 92)
Adiponectin low (b7.5 μg/dl; n = 92)
p value
63 (68) 39 (42)
61 (66) 35 (38)
0.75 0.47
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was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Funding The study was funded by the Ricerca Corrente Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano (Institutional Internal Funding). Author contributions EC designed the study, analyzed the data, and contributed to manuscript drafting; MP analyzed the data, and contributed to manuscript drafting; MR, RR, CM, SP, ALF, and PD collected the data, analyzed serum samples, genotyped the patients; SF and PM critically reviewed the manuscript for important intellectual content; and LV designed and supervised the study. Acknowledgements We thank all the members of the Metabolic Liver Diseases Lab for helpful comments and discussion. References [1] Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol 2013; 58:593–608. [2] Maasoumy B, Wedemeyer H. Natural history of acute and chronic hepatitis C. Best Pract Res Clin Gastroenterol 2012;26:401–12. [3] Mf Bassendine, Da Sheridan, Dj Felmlee, Bridge Sh, Gl Toms, Rd Neely. HCV and the hepatic lipid pathway as a potential treatment target. J Hepatol 2011;55:1428–40. [4] Kaddai V, Negro F. Current understanding of insulin resistance in hepatitis C. Expert Rev Gastroenterol Hepatol 2011;5:503–16. [5] Arase Y, Suzuki F, Suzuki Y, Akuta N, Kobayashi M, Kawamura Y, et al. Sustained virological response reduces incidence of onset of type 2 diabetes in chronic hepatitis C. Hepatology 2009;49:739–44. [6] Leandro G, Mangia A, Hui J, Fabris P, Rubbia-Brandt L, Colloredo G, et al. Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data. Gastroenterology 2006;130:1636–42. [7] Le Adinolfi, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001;33:1358–64. [8] Romero-Gomez M, Del Mar Viloria M, Andrade RJ, Salmeron J, Diago M, FernandezRodriguez CM, et al. Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients. Gastroenterology 2005; 128:636–41. [9] Khattab M, Emad M, Abdelaleem A, Eslam M, Atef R, Shaker Y, et al. Pioglitazone improves virological response to peginterferon alpha-2b/ribavirin combination therapy in hepatitis C genotype 4 patients with insulin resistance. Liver Int 2009;30: 447–54. [10] Aghemo A, Prati GM, Rumi MG, Soffredini R, D'ambrosio R, Orsi E, et al. Sustained virological response prevents the development of insulin resistance in patients with chronic hepatitis C. Hepatology 2012;56:1681–7. [11] Petta S, Torres D, Fazio G, Camma C, Cabibi D, Di Marco V, et al. Carotid atherosclerosis and chronic hepatitis C: a prospective study of risk associations. Hepatology 2012;55:1317–23. [12] Yc Hsu, Lin Jt, Hj Ho, Kao Yh, Huang Yt, Nw Hsiao, et al. Antiviral treatment for hepatitis C virus infection is associated with improved renal and cardiovascular outcomes in diabetic patients. Hepatology 2014;59:1293–302. [13] Bugianesi E, Salamone F, Negro F. The interaction of metabolic factors with HCV infection: does it matter? J Hepatol 2012;56(Suppl. 1):S56–65. [14] Vanni E, Abate ML, Gentilcore E, Hickman I, Gambino R, Cassader M, et al. Sites and mechanisms of insulin resistance in nonobese, nondiabetic patients with chronic hepatitis C. Hepatology 2009;50:697–706.
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[15] Bugianesi E, Gastaldelli A, Vanni E, Gambino R, Cassader M, Baldi S, et al. Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: sites and mechanisms. Diabetologia 2005;48:634–42. [16] Bugianesi E, Pagotto U, Manini R, Vanni E, Gastaldelli A, De Iasio R, et al. Plasma adiponectin in nonalcoholic fatty liver is related to hepatic insulin resistance and hepatic fat content, not to liver disease severity. J Clin Endocrinol Metab 2005;90: 3498–504. [17] Di Filippo M, Moulin P, Roy P, Samson-Bouma ME, Collardeau-Frachon S, ChebelDumont S, et al. Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia. J Hepatol 2014;61:891–902. [18] Dongiovanni P, Petta S, Maglio C, Fracanzani AL, Pipitone R, Mozzi E, et al. Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease. Hepatology 2015;61:506–14. [19] Rametta R, Mozzi E, Dongiovanni P, Motta BM, Milano M, Roviaro G, et al. Increased insulin receptor substrate 2 expression is associated with steatohepatitis and altered lipid metabolism in obese subjects. Int J Obes (Lond) 2013;37:986–92. [20] Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev 2005; 26:439–51. [21] My Sheikh, Choi J, Qadri I, Friedman JE, Sanyal AJ. Hepatitis C virus infection: molecular pathways to metabolic syndrome. Hepatology 2008;47:2127–33. [22] Ma Khattab, Eslam M, Shatat M, Abd-Aalhalim H, Yi Mousa, Samir F, et al. Changes in adipocytokines and insulin sensitivity during and after antiviral therapy for hepatitis C genotype 4. J Gastrointest Liver Dis (JGLD) 2012;21:59–65. [23] Ma Khattab, Eslam M, Mousa YI, Ela-Adawy N, Fathy S, Shatat M. Association between metabolic abnormalities and hepatitis C-related hepatocellular carcinoma. Ann Hepatol 2012;11:487–94. [24] Arano T, Nakagawa H, Tateishi R, Ikeda H, Uchino K, Enooku K, et al. Serum level of adiponectin and the risk of liver cancer development in chronic hepatitis C patients. Int J Cancer 2011;129:2226–35. [25] Corbetta S, Redaelli A, Pozzi M, Bovo G, Ratti L, Redaelli E, et al. Fibrosis is associated with adiponectin resistance in chronic hepatitis C virus infection. Eur J Clin Invest 2011;41:898–905. [26] Sumie S, Kawaguchi T, Kuromatsu R, Takata A, Nakano M, Satani M, et al. Total and high molecular weight adiponectin and hepatocellular carcinoma with HCV infection. PLos One 2011;6 (E26840). [27] Tacke F, Wustefeld T, Horn R, Luedde T, Srinivas Rao A, Manns MP. High adiponectin in chronic liver disease and cholestasis suggests biliary route of adiponectin excretion in vivo. J Hepatol 2005;42:666–73. [28] Richards JB, Waterworth D, O'rahilly S, Hivert MF, Loos RJ, Perry JR, et al. A genomewide association study reveals variants in Arl15 that influence adiponectin levels. PLoS Genet 2009;5 [E1000768]. [29] Valenti L, Rametta R, Ruscica M, Dongiovanni P, Steffani L, Motta Bm BM. The I148m Pnpla3 polymorphism influences serum adiponectin in patients with fatty liver and healthy controls. BMC Gastroenterol 2012;12:111. [30] Rametta R, Ruscica M, Dongiovanni P, Macchi C, Fracanzani AL, Steffani L. Hepatic steatosis and Pnpla3 I148m variant are associated with serum fetuin-A independently of insulin resistance. Eur J Clin Invest 2014;44:627–33. [31] Bedogni G, Bellentani S, Miglioli L, Masutti F, Passalacqua M, Castiglione A, et al. The fatty liver index: a simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol 2006;6:33. [32] Organization Wh. Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia; 2006. [33] Magni P, Ruscica M, Dozio E, Passafaro L, Steffani L, Morelli P, et al. Plasma adiponectin and leptin concentrations in professional rugby players. J Biol Regul Homeost Agents 2010;24:87–91. [34] Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696–9. [35] De Kleiner, Em Brunt, Van Natta M, Behling C, Contos MJ, Cummings OW. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21. [36] Ta Zografos, Liaskos C, Rigopoulou EI, Togousidis E, Makaritsis K, Germenis A. Adiponectin: a new independent predictor of liver steatosis and response to IFNalpha treatment in chronic hepatitis C. Am J Gastroenterol 2008;103:605–14. [37] Ma Khattab, Eslam M, Aly MM, Shatat M, Hussen A, Moussa YI, et al. Association of serum adipocytokines with insulin resistance and liver injury in patients with chronic hepatitis C genotype 4. J Clin Gastroenterol 2012;46:871–9. [38] Nakagawa H, Fujiwara N, Tateishi R, Arano T, Nakagomi R, Kondo M, et al. Impact of serum levels of Il-6 and adiponectin on all-cause. And Liver-Unrelated Mortality In Chronic Hepatitis C Patients. J Gastroenterol Hepatol: Liver-Related; 2014.
Contents lists available at ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Original Article
Increased circulating adiponectin in males with chronic HCV hepatitis Elena Canavesi a,1, Marianna Porzio a,1, Massimiliano Ruscica b, Raffaela Rametta a, Chiara Macchi b, Serena Pelusi a, Anna Ludovica Fracanzani a, Paola Dongiovanni a, Silvia Fargion a, Paolo Magni b, Luca Valenti a,⁎ a Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Department of Pathophysiology and Transplantation, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, Milano, Italy b Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy
a r t i c l e
i n f o
Article history: Received 23 December 2014 Received in revised form 4 May 2015 Accepted 3 August 2015 Available online 17 August 2015 Keywords: Adipokines Adiponectin Gender Hepatic fibrosis Hepatitis C virus Nonalcoholic fatty liver disease
a b s t r a c t Background: Increased levels of adiponectin, a major adipokine with insulin sensitizing properties showing a strong sexual dimorphism, have been reported in individuals with chronic HCV infection (CHC), but data are limited by small samples and lack of control for the genetic background and hepatic fibrosis. The aim of this study was to compare adiponectin levels between CHC patients and accurately matched controls. Methods: We considered 184 CHC patients, matched (1:1) for age, gender, body mass index, and Adiponectin genotype (ADIPOQ) with healthy individuals. To control for the severity of liver disease, a second control group consisting of 95 patients with histological nonalcoholic fatty liver disease (NAFLD) further matched (1:1) for severe fibrosis was exploited. ADIPOQ genotype was evaluated by Taqman assays, serum adiponectin measured by ELISA. Results: Serum adiponectin was higher in CHC patients than in healthy individuals (9.0 ± 5.0 μg/ml vs. 7.3 ± 4.0 μg/ml; p = 0.001; adjusted estimate +1.8, 1.7–2.9; p = 0.001), and than in NAFLD patients (8.3 ± 4.5 μg/ml vs. 6.0 ± 4.2 μg/ml; p b 0.001; adjusted estimate + 0.8, 0.2–1.4, p = 0.006). After stratification for sex, serum adiponectin was higher in males with CHC than in healthy individuals and NAFLD patients (p b 0.005 for both), whereas the difference was not significant in females. Conclusions: CHC is associated with increased serum adiponectin independently of age, body mass, diabetes, ADIPOQ genotype, and of severe liver fibrosis, particularly in men. © 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Background Chronic hepatitis C virus (HCV) infection (CHC) affects 170 million individuals worldwide, representing a leading cause of cirrhosis and hepatocellular carcinoma [1,2]. CHC is frequently associated with alterations in the metabolism of glucose and lipids, as HCV interferes with very low-density lipoproteins secretion from hepatocytes and impairs glucose metabolism with development of insulin resistance (IR) and Abbreviations: HCV, hepatitis C virus; CHC, chronic HCV hepatitis; IR, insulin resistance; T2DM, type 2 diabetes mellitus; NAFLD, nonalcoholic fatty liver disease; ADIPOQ, adiponectin gene; BMI, body mass index; AST, aspartate aminotransferases; ALT, alanine aminotransferases; GGT, gamma-glutamyl-transferases. ⁎ Corresponding author at: Department of Pathophysiology and Transplantation, Centro Malattie Metaboliche del Fegato, Università degli Studi di Milano, Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Pad. Granelli, via F Sforza 35, 20122 Milano, Italy. E-mail addresses: [email protected] (E. Canavesi), [email protected] (M. Porzio), [email protected] (M. Ruscica), [email protected] (R. Rametta), [email protected] (C. Macchi), [email protected] (S. Pelusi), [email protected] (A.L. Fracanzani), [email protected] (P. Dongiovanni), [email protected] (S. Fargion), [email protected] (P. Magni), [email protected] (L. Valenti). 1 These authors contributed equally to the work.
type 2 diabetes mellitus (T2DM) [3–5]. The association between HCV and metabolic alterations is clinically remarkable, since steatosis and IR are associated with faster progression of fibrosis [6,7], lower rates of response to interferon-based therapy [8,9] and increased risk of T2DM and cardiovascular disease [4,10–12]. HCV interacts with insulin signaling in the hepatocytes, leading to the development of IR in organs such as muscles or the adipose tissue [13, 14]. From this perspective, CHC shares some pathologic features with non-alcoholic fatty liver disease (NAFLD), which is related to the metabolic syndrome and results from the development of adipose tissue IR [15] and decreased adiponectin [16], increased de novo lipogenesis and altered hepatic beta-oxidation and lipoprotein export [17–19]. Adipose tissue produces a number of adipokines, which modulate insulin sensitivity. Among these, adiponectin, a molecule with insulin-sensitizing, anti-inflammatory and anti-fibrotic effects, plays a major role [20]. Based on the observation of increased circulating adiponectin in CHC patients, which was associated with adverse metabolic and clinical outcomes including hepatocellular carcinoma, altered adiponectin activity has been hypothesized to play a role in the pathogenesis of hepatic and extra-hepatic complications of this condition [21–24]. A possible state of adiponectin resistance has been hypothesized based on the observation of downregulation of the hepatic adiponectin receptor AdipoR1 in
http://dx.doi.org/10.1016/j.ejim.2015.08.001 0953-6205/© 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
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CHC patients with advanced liver fibrosis [25]. However, the sample size of available studies was limited, and increased adiponectin levels were not always confirmed, or limited to patients with severe fibrosis [23, 26], which could also interfere with biliary clearance of adiponectin [27]. Furthermore, controls matched for liver disease severity were never assessed. Therefore, the aim of this study was to evaluate whether CHC is associated with increased adiponectin levels independently of demographic and anthropometric features, the genetic background, and severe hepatic fibrosis, and secondarily to assess whether the association is influenced by sex, as adiponectin levels show a strong sexual dimorphism. Serum adiponectin is indeed a partly heritable trait, the major determinant being Adiponectin genotype (ADIPOQ) genotype [28]. To this end, CHC patients were matched to healthy subjects for age, gender, body mass index (BMI), and ADIPOQ, and further matched to patients with NAFLD for the presence of severe liver fibrosis. 2. Patients and methods 2.1. Study design From a previously described larger series of patients with CHC, NAFLD, and healthy controls with a very low probability of hepatic steatosis [29,30], we conducted a two-step nested case–control study. The inclusion criteria have previously been described in details [29]. Briefly, we selected consecutive adult patients with active CHC with available histological evaluation and without comorbidities, and with a histological diagnosis of NAFLD without coexistent liver diseases. We also considered healthy controls with normal liver enzymes and without metabolic abnormalities with a very low probability of steatosis based on the fatty liver index [31]. In a first step, we matched 1:1 CHC patients with healthy controls for age (±8 years), sex, body mass index (BMI) category (normal range: 20–25 kg/m2, overweight: 25.1–29.9 kg/m2, and obese: N 30 kg/m2), and ADIPOQ genotype (presence of rs2241766TT or rs1501299TT/TG genotypes, associated with higher adiponectin levels [29]; henceforth defined high expression genotypes). We identified 184 CHC patients (46 females, age 54 ± 13 years) and matched healthy subjects; their clinical features are presented in Table 1. In a second step, to control for the severity of liver disease, we matched 1:1 a second group of CHC patients (selected within the 184 patients identified in the first stage) for age, gender, BMI category, ADIPOQ (as reported above) and the presence of severe hepatic fibrosis
(bridging fibrosis or cirrhosis) with NAFLD patients. We identified 95 CHC patients (24 females, age 50 ± 11 years) and matched NAFLD patients; their clinical features are presented in Table 2. The study protocol was approved by the Institutional Review Board of the Fondazione Ca' Granda IRCCS Milano. Informed written consent was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki. 2.2. Clinical and laboratory data In all subjects the following information was available: lifestyle habits (smoking and daily alcohol intake, the last one confirmed by at least one family member), clinical and pharmacologic history, age, race, BMI, complete blood count, alanine-aminotransferase (ALT), gamma-glutamyltransferase (GGT), fasting glucose, total and highdensity lipoprotein (HDL) cholesterol, and triglycerides. T2DM was diagnosed according to the WHO criteria [32]. Serum adiponectin was measured on an aliquot of plasma collected after overnight fasting at the time of liver biopsy (when available) or of abdominal ultrasonography and metabolic evaluation, and stored at −80 °C. Plasma adiponectin levels (all isoforms) were measured using a commercial enzyme-linked immunosorbent assay kit with a lowest limit of sensitivity of 0.246 ng/ml (R&D Systems, Minneapolis, MN) [29,33]. ADIPOQ rs2241766, + 45 GNT and r150299, + 276 GNT polymorphisms, previously associated with the susceptibility to NAFLD were genotyped on peripheral blood DNA samples by a Taqman assay (assay on demand, Applied Biosystems, Foster City, CA) [29]. Liver biopsies were routinely processed and read by a single expert pathologist. The minimum biopsy size was 1.7 cm and the number of portal areas 10. Liver biopsies were scored the severity of fibrosis according to Ishak for CHC patients [34] and NAFLD clinical research network activity score for NAFLD patients [35]. Severe fibrosis was defined as Ishak fibrosis score F4–F6 for CHC patients or NAFLD fibrosis stage F3–F4 for NAFLD patients. 2.3. Statistical analysis For descriptive statistics, results are expressed as means ± standard deviations for continuous variables and as frequencies for categorical variables. p-Values from ANOVA or chi-square test were considered statistically significant if ≤0.05 (two-tailed). Non-normally distributed variables (e.g., GGT) were log-transformed before entering the analyses.
Table 2 Clinical features of 95 CHC and NAFLD patients. Table 1 Clinical features of 184 CHC patients and 184 healthy controls.
Age Sex (F) BMI classes Normal range Overweight Obesity ADIPOQ (high expression genotypes) T2DM Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Fasting insulin (IU/ml) Fasting glucose (mg/dl) ALT (IU/l) GGT (IU/l) Adiponectin (μg/ml)
CHC patients
Healthy controls
p
54 ± 13 46 (25)
49 ± 12 46 (25)
b0.001 1 1
90 (49) 88 (48) 6 (3) 74 (40)
90 (49) 88 (48) 6 (3) 74 (40)
30 (16) 175 ± 36 48 ± 16 106 ± 56 20 ± 9 93 ± 20 72 ± 63 58 ± 58 9.0 ± 5.0
0 194 ± 32 57 ± 13 89 ± 50 13 ± 8 70 ± 14 23 ± 9 23 ± 16 7.3 ± 4.0
1 b0.001 b0.001 b0.001 0.003 b0.001 b0.001 b0.001 b0.001 0.001
Controls were matched 1:1 for age (±10 years), sex, BMI, and ADIPOQ genotype. (): % values; BMI: body mass index; T2DM: type 2 diabetes mellitus.
Age Sex (F) BMI classes Normal range Overweight Obesity ADIPOQ (high expression genotypes) Advanced liver fibrosis T2DM Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Fasting insulin (IU/ml) Fasting glucose (mg/dl) ALT (IU/l) GGT (IU/l) Adiponectin (μg/ml)
CHC patients
NAFLD patients
50 ± 11 24 (25)
49 ± 11 24 (25)
30 (32) 54 (57) 11 (11) 41 (43)
30 (32) 54 (57) 11 (11) 41 (43)
18 (19) 9 (9) 180 ± 34 47 ± 14 107 ± 62 23 ± 11 90 ± 17 77 ± 63 54 ± 50 8.3 ± 4.5
18 (19) 27 (28) 208 ± 42 48 ± 13 128 ± 65 21 ± 19 100 ± 32 68 ± 53 104 ± 137 6.0 ± 4.2
p 0.65 1 1
1 1 0.001 b0.001 0.67 0.031 0.37 0.013 0.26 0.001 b0.001
NAFLD patients were matched 1:1 for age (±8 years), sex, BMI, ADIPOQ genotype, and presence of advanced liver fibrosis (): % values; BMI: body mass index; T2DM: type 2 diabetes mellitus.
E. Canavesi et al. / European Journal of Internal Medicine 26 (2015) 635–639
Multivariate general linear models were fitted to estimate the impact of CHC on adiponectin levels (for CHC vs. healthy subjects and vs. NAFLD). High adiponectin levels were defined as those above 7.5 μg/ml, the median value in CHC patients [29]. Variables considered in the matching procedure were entered into the final multivariate model. The study had a N 80% power (alpha = 0.05; two-tailed) to detect a 1 SD difference in adiponectin levels between CHC patients and both healthy controls and NAFLD patients (in the overall cohort, and both in males and in females). All statistical analyses were performed by the SPSS 20.0 statistical discovery software (IBM, Burbank, NJ, USA).
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Table 3 Independent predictors of serum adiponectin in CHC patients and healthy controls.
Age (years) Sex (F) BMI (kg/m2) T2DM ALT (IU/ml) ADIPOQ genotype HCV infection
Estimate
95% CI
p value
0.02 +3.39 −0.72 +0.04 −0.006 +1.09 +1.80
−0.015 to +0.58 +2.33 to +4.45 −1.56 to +0.11 −1.744 to +1.82 −0.016 to +0.004 +0.18 to +2.00 +0.74 to +2.86
0.24 b0.001 0.088 0.22 0.22 0.019 0.001
p values were determined at generalized linear model considering all the variables presented in the table. BMI: body mass index; CI: confidence interval; T2DM: type 2 diabetes mellitus.
3. Results 3.1. Adiponectin levels in CHC vs. healthy subjects The clinical characteristics and adiponectin levels in 184 CHC patients and matched healthy subjects are shown in Table 1. Despite matching for age (± 8 years) CHC patients were about 5 years older (p b 0.05), but had the same gender, BMI distribution, and genetic background. Women affected by CHC were not significantly older than those included among healthy subjects (52 ± 11 vs. 49 ± 11 years; p = 0.1). As expected, CHC had IR and higher prevalence of T2DM than healthy subjects, but lower serum cholesterol, and higher liver enzymes (p b 0.05). Serum adiponectin levels were about 22% higher in CHC patients than in matched healthy controls (Table 1; p = 0.001). As adiponectin levels display a strong sexual dimorphism, we next analyzed adiponectin levels according to gender (shown in Fig. 1A). Serum adiponectin levels were markedly higher in male CHC patients than in healthy controls (p b 0.001; p = 0.001 after adjustment for age, BMI, and ADIPOQ genotype), whereas the difference was not significant in females (p N 0.05), characterized by higher adiponectin levels. To correct for potential residual confounders (in particular age, which was significantly different between groups, but also metabolic features and hepatic inflammation), we adjusted the association of adiponectin with CHC for age, sex, BMI class, ADIPOQ genotype, presence of T2DM, and ALT levels (Table 3). CHC remained strongly associated with higher adiponectin levels (Estimate 1.8, 95% CI 0.7–2.9; p = 0.001).
A
After adjustment for sex, BMI, and T2DM, adiponectin levels were inversely correlated with fasting insulin both in CHC and healthy individuals (estimate −0.016 ± 0.05, p b 0.001, and −0.02 ± 0.01, p = 0.049, respectively).
3.2. Adiponectin levels in CHC vs. NAFLD patients The clinical features of 95 CHC and matched NAFLD patients are shown in Table 2. This second group had different clinical features because NAFLD patients were more overweight than healthy subjects. Despite the demographic and anthropometric features were superimposable between the two groups, as expected NAFLD patients had higher serum lipids (total cholesterol and triglycerides), and higher GGT and prevalence of T2DM (p b 0.05). Women affected by CHC were not significantly older than those affected by NAFLD (51 ± 7 vs. 55 ± 9 years; p = 0.1). Again, adiponectin levels were about 25% higher in CHC patients than in with subjects with NAFLD, matched for age, gender, BMI, ADIPOQ genotype, and the severity of fibrosis (p b 0.001; Table 2). We next analyzed adiponectin levels according to gender (shown in Fig. 1B). Again, serum adiponectin levels were higher in male CHC than in NAFLD patients (p b 0.001; p = 0.005 after adjustment for age, BMI, ADIPOQ genotype, and the severity of fibrosis), whereas the difference was not significant in females (p N 0.05), To correct for potential residual confounders (age, metabolic features, and hepatic inflammation), we
B
Fig. 1. Comparison of serum adiponectin levels between CHC patients and controls stratified by gender. A) Serum adiponectin levels in 184 CHC patients and in 1:1 matched healthy controls stratified by gender. B) Serum adiponectin levels in 95 CHC patients and in 1:1 matched NAFLD patients stratified by gender.
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Table 4 Independent predictors of serum adiponectin in CHC and matched NAFLD patients.
Age (years) Sex (F) BMI (kg/m2) T2DM ALT (IU/ml) ADIPOQ genotype Advanced fibrosis HCV infection
Estimate
95% CI
p value
+0.12 +0.52 −0.18 −0.62 −0.001 +0.03 +0.36 +0.81
+0.06 to +0.17 −0.13 to +1.17 0.00 to −0.36 +0.15 to −1.39 +0.001 to +0.01 −0.59 to +0.53 −0.43 to +1.15 +0.24 to +1.38
b0.001 0.11 0.052 0.11 0.77 0.92 0.37 0.006
p values were determined at generalized linear model considering all the variables presented in the table. BMI: body mass index; CI: confidence interval; T2DM: type 2 diabetes mellitus.
adjusted the association of adiponectin with CHC at a generalized model adjusted for age, sex, BMI class, ADIPOQ genotype, ALT levels, presence of T2DM, and of advanced liver fibrosis (Table 4). Chronic HCV infection remained associated with higher adiponectin levels (Estimate +0.8 95% CI 0.2–1.4; p = 0.006). After adjustment for sex, BMI, and T2DM, adiponectin levels were inversely correlated with fasting insulin both in CHC and NAFLD individuals (estimate − 0.13 ± 0.05, p = 0.006, and − 0.05 ± 0.02, p = 0.045, respectively). 3.3. Impact of viral features on adiponectin levels in CHC patients Finally, in an exploratory analysis we evaluated whether viral features influences serum adiponectin levels in 184 CHC patients described in Table 1. To this end, we analyzed whether viral genotype (G1 and G4 vs. G2 and G3) and viral load (high or low viral load, N or ≤8 × 105 IU/ml) were associated with high adiponectin levels (N or ≤7.5 μg/dl, median value). Results are shown in Table 5. Viral genotype and viral load were not associated with adiponectin levels (p N 0.4). Results did not change when treating adiponectin as a continuous variable. 4. Discussion In this study, adiponectin levels were compared between a relatively large series of well-characterized CHC patients and carefully matched controls, first healthy individuals and then patients with NAFLD. The main finding of the study is that adiponectin levels are higher in CHC independently of the presence of advanced liver fibrosis, and that the effect of CHC on adiponectin levels is more marked in males. These results are in line with previous reports of increased adiponectin in CHC [21–23,25,26, 36,37], which however did not take into account difference in body mass, the genetic background, and in particular the presence of severe liver fibrosis. Although the mechanism of fibrogenesis is different between CHC and NAFLD, also characterized by adipose tissue insulin resistance, these data suggest that the increased adiponectin in CHC patients is not due to disruption of the hepatic lobule related to advanced fibrosis. Adiponectin is the major insulin-sensitizing adipokine, mainly secreted by adipose tissue in adult life. Decreased adiponectin release is associated with obesity and IR, subclinical inflammation, T2DM, and the risk of cardiovascular disease and cancer [20]. Furthermore, adiponectin levels are lower in males and post-menopausal females, groups at higher risk of cardio-metabolic diseases [20]. Since CHC is associated with insulin resistance [13], it would be expected to be also linked to decreased
circulating adiponectin [23]. Increased adiponectin levels in the presence of IR have thus led some Authors to hypothesize a state of adiponectin resistance [25], that would account for the association of high adiponectin levels with the risk of hepatocellular carcinoma and overall mortality in CHC [26,38]. However, the inverse correlation between adiponectin and insulin levels in CHC patients does not support adiponectin resistance in this condition. A state of tissue or receptor specific adiponectin resistance cannot be ruled out, but different mechanisms may lead to increased adiponectin levels in CHC patients. Alternative explanations may be represented by a) decreased adiponectin biliary clearance due to advanced liver fibrosis [27], which however was not observed in NAFLD, and b) a feedback mechanism to counteract insulin resistance, which would be consistent with the negative correlation between adiponectin and insulin levels in CHC patients. Noteworthy, the observed differences in serum adiponectin were influenced by gender. Specifically, adiponectin levels were higher in male CHC patients than controls, both healthy subjects without liver disease and patients with NAFLD, whereas the difference was smaller and not significant in females. Therefore, altered adiponectin metabolism may be specific to male gender. Alternatively, CHC infection might exert a milder effect on serum adiponectin in females, which could not be detected because of the lower power in this subgroup. Additional studies with larger sample sizes and better characterization of the hormonal status of women will be required to definitively rule out a milder effect of HCV infection on adiponectin metabolism in women. Limitations of the present study include the cross sectional design, so that we could not causally assess the effect of HCV infection on adiponectin levels, and the impact on clinical outcomes. However, previous data indicate that successful eradication of HCV infection is associated with a decrease in serum adiponectin [22], and that increased adiponectin is associated with hepatocellular carcinoma risk and mortality [26,38]. The sample size was relatively limited, but larger than in previous studies. Furthermore, patients and controls, who did not undergo ultrasonography, but had very low probability of steatosis [31], were very well characterized and meticulously matched for confounding factors. Finally, although we could not adjust the analyses for abdominal obesity, adiponectin levels were higher in CHC patients independently of BMI and T2DM. Evaluation of the modifications of serum total adiponectin and specific isoforms in CHC patients with advanced fibrosis following viral eradication by new direct antiviral agents will be useful to gain further insight into the alterations of adiponectin metabolism in CHC patients. In conclusion, CHC is associated with increased serum adiponectin independently of age, body mass, diabetes, ADIPOQ genotype, and of severe liver fibrosis, particularly in men. Altered adiponectin metabolism may modulate metabolic disturbances and the susceptibility to cardiovascular disease and hepatocellular carcinoma in CHC patients.
Conflict of interests Authors declare that they have no conflict of interest to disclose.
Ethical statement The study protocol was approved by the Institutional Review Board of the Fondazione Ca' Granda IRCCS Milano. Informed written consent
Table 5 Adiponectin levels stratified by HCV genotype and viral load in CHC patients.
Genotype (G1–G4 vs. G2–G3) Viral load (N8 × 105 IU/ml) (): % values.
Adiponectin high (N7.5 μg/dl; n = 92)
Adiponectin low (b7.5 μg/dl; n = 92)
p value
63 (68) 39 (42)
61 (66) 35 (38)
0.75 0.47
E. Canavesi et al. / European Journal of Internal Medicine 26 (2015) 635–639
was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Funding The study was funded by the Ricerca Corrente Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano (Institutional Internal Funding). Author contributions EC designed the study, analyzed the data, and contributed to manuscript drafting; MP analyzed the data, and contributed to manuscript drafting; MR, RR, CM, SP, ALF, and PD collected the data, analyzed serum samples, genotyped the patients; SF and PM critically reviewed the manuscript for important intellectual content; and LV designed and supervised the study. Acknowledgements We thank all the members of the Metabolic Liver Diseases Lab for helpful comments and discussion. References [1] Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol 2013; 58:593–608. [2] Maasoumy B, Wedemeyer H. Natural history of acute and chronic hepatitis C. Best Pract Res Clin Gastroenterol 2012;26:401–12. [3] Mf Bassendine, Da Sheridan, Dj Felmlee, Bridge Sh, Gl Toms, Rd Neely. HCV and the hepatic lipid pathway as a potential treatment target. J Hepatol 2011;55:1428–40. [4] Kaddai V, Negro F. Current understanding of insulin resistance in hepatitis C. Expert Rev Gastroenterol Hepatol 2011;5:503–16. [5] Arase Y, Suzuki F, Suzuki Y, Akuta N, Kobayashi M, Kawamura Y, et al. Sustained virological response reduces incidence of onset of type 2 diabetes in chronic hepatitis C. Hepatology 2009;49:739–44. [6] Leandro G, Mangia A, Hui J, Fabris P, Rubbia-Brandt L, Colloredo G, et al. Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data. Gastroenterology 2006;130:1636–42. [7] Le Adinolfi, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001;33:1358–64. [8] Romero-Gomez M, Del Mar Viloria M, Andrade RJ, Salmeron J, Diago M, FernandezRodriguez CM, et al. Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients. Gastroenterology 2005; 128:636–41. [9] Khattab M, Emad M, Abdelaleem A, Eslam M, Atef R, Shaker Y, et al. Pioglitazone improves virological response to peginterferon alpha-2b/ribavirin combination therapy in hepatitis C genotype 4 patients with insulin resistance. Liver Int 2009;30: 447–54. [10] Aghemo A, Prati GM, Rumi MG, Soffredini R, D'ambrosio R, Orsi E, et al. Sustained virological response prevents the development of insulin resistance in patients with chronic hepatitis C. Hepatology 2012;56:1681–7. [11] Petta S, Torres D, Fazio G, Camma C, Cabibi D, Di Marco V, et al. Carotid atherosclerosis and chronic hepatitis C: a prospective study of risk associations. Hepatology 2012;55:1317–23. [12] Yc Hsu, Lin Jt, Hj Ho, Kao Yh, Huang Yt, Nw Hsiao, et al. Antiviral treatment for hepatitis C virus infection is associated with improved renal and cardiovascular outcomes in diabetic patients. Hepatology 2014;59:1293–302. [13] Bugianesi E, Salamone F, Negro F. The interaction of metabolic factors with HCV infection: does it matter? J Hepatol 2012;56(Suppl. 1):S56–65. [14] Vanni E, Abate ML, Gentilcore E, Hickman I, Gambino R, Cassader M, et al. Sites and mechanisms of insulin resistance in nonobese, nondiabetic patients with chronic hepatitis C. Hepatology 2009;50:697–706.
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