Serum adiponectin is increased in advancing liver fibrosis and declines with reduction in fibrosis in chronic hepatitis B

Journal of Hepatology 47 (2007) 191–202 www.elsevier.com/locate/jhep

Serum adiponectin is increased in advancing liver fibrosis and declines with reduction in fibrosis in chronic hepatitis Bq Chee-Kin Hui1,2,*, Hai-Ying Zhang1, Nikki P. Lee3, Weng Chan1, Yui-Hung Yueng1, Kar-Wai Leung1, Lei Lu1, Nancy Leung4, Chung-Mau Lo3, Sheung-Tat Fan3, John M. Luk3, Aimin Xu1, Karen S. Lam1, Yok-Lam Kwong1, George K.K. Lau1,2, for The Hong Kong Liver Fibrosis Study Group 1

Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China 2 Research Centre of Infection and Immunity, The University of Hong Kong, Hong Kong SAR, China 3 Department of Surgery, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China 4 Department of Medicine, Alice Ho Miu Ling Nethersole Hospital, Hong Kong SAR, China

Background/Aims: Despite the possible role of adiponectin in the pathogenesis of liver cirrhosis, few data have been collected from patients in different stages of liver fibrosis. We studied the role of adiponectin in 2 chronic hepatitis B (CHB)-patient cohorts. Methods: Serum adiponectin was quantified by enzyme-linked immunosorbent assay. One-hundred liver biopsy specimens from CHB patients with different stages of fibrosis and 38 paired liver biopsies from hepatitis B e antigen-positive patients randomized to lamivudine (n = 15), pegylated interferon alfa-2a (n = 15) or pegylated interferon alfa-2a plus lamivudine (n = 8) therapy for 48 weeks were assessed. Results: Serum adiponectin was detected at levels ranging over fourfold magnitude with advancing fibrosis stage and correlated positively with fibrosis stage [r = 0.45, p < 0.001]. CHB patients with stage 0–1 fibrosis had higher composition of high molecular weight (HMW) form of adiponectin when compared with CHB patients with liver cirrhosis [mean ± SEM 51.2 ± 2.1% vs. 40.9 ± 1.7%, respectively, p = 0.001]. After antiviral therapy, patients with fibrosis reduction had marked decline in serum adiponectin level and increase in HMW form of adiponectin [mean ± SEM 43.5 ± 1.2% vs. 37.0 ± 3.0%, respectively, p = 0.04]. Conclusions: Serum adiponectin may have a role in fibrosis progression in CHB infection. A marked decline in serum adiponectin after antiviral therapy is associated with fibrosis reduction.  2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Adiponectin; Chronic hepatitis B; Liver cirrhosis; Pegylated interferon alfa-2a; Lamivudine; Sustained virological response; Adiponectin oligomeric form

Received 14 December 2006; received in revised form 16 January 2007; accepted 28 February 2007; available online 12 April 2007 q The authors who have taken part in this study declared that they did not receive funding from the manufacturers to carry out their research. They received funding from the Hong Kong Liver Foundation which enabled them to carry out their study. * Corresponding author. Tel.: +852 28184300; fax: +852 28184030. E-mail address: [email protected] (C.-K. Hui). Abbreviations: CHB, chronic hepatitis B; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; anti-HBe, hepatitis B e antibody; HMW, high molecular weight; MMW, middle molecular weight; LMW, low molecular weight; ALT, alanine aminotransaminase; HAI, histology activity index. 0168-8278/$32.00  2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2007.02.023

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1. Introduction Recently, adiponectin, a physiologically active polypeptide secreted by adipose tissues, has been the focus of research interest. It has been shown to demonstrate anti-diabetic, anti-obesity, anti-atherogenic and anti-inflammatory effects, making it a promising candidate for the treatment of obesity-mediated metabolic syndrome [1,2]. Adiponectin also has direct effects on hepatocytes via AdipoR2 receptor and anti-inflammatory properties mediated by its antagonism against tumor necrosis factora. This led to the postulation on the potential hepatoprotective role of adiponectin against liver fibrosis and cirrhosis. Initial studies involved mice model of alcoholic and non-alcoholic fatty liver diseases [3]. In these mice models, circulating levels of adiponectin were shown to decline following the consumption of high-fat ethanol-containing diet. However, the delivery of adiponectin could effectively alleviate both alcohol- and obesity-associated liver abnormality [3]. Hypoadiponectinemia was found to be associated with severe histologic necroinflammation in patients with non-alcoholic steatohepatitis (NASH) [4]. Further evidence on the protective role of adiponectin came from adiponectin knockout mice which were more susceptible to carbon tetrachloride-induced liver fibrosis, while liver fibrosis in knockout and in wild-type mice was attenuated by adenoviral vector-delivered adiponectin [5]. Much of the optimism on the possible role of adiponectin or its agonist as a candidate for treatment of liver cirrhosis in humans was dampened with the reports of two cohort studies on patients with liver cirrhosis with diverse etiology [4,6]. These studies found that elevated adiponectin levels in patients with liver cirrhosis correlated positively with the severity of cirrhosis and negatively with hepatic protein synthesis. The authors suggested that adiponectin might be used as a marker for liver cell injury and that the proposed use of adiponectin for treating insulin resistance and diabetes be limited to patients without liver disease [4,6]. However, more recent publications reported no such association in hepatitis C virus (HCV)or NASH-related liver cirrhosis, suggesting that the relationship between adiponectin level and liver cirrhosis may be disease-specific [7,8]. In order to further elucidate the association between adiponectin and fibrosis progression, we studied two chronic hepatitis B (CHB) infected patient cohorts, namely patients with different fibrosis stages and patients undergoing therapy in a multi-centre phase III trial on pegylated interferon alfa-2a [9].

ment of CHB infection at the Department of Medicine and Surgery, Queen Mary Hospital, The University of Hong Kong, from March 2000 to December 2003, were retrospectively included into the study. Thirty-four of these 100 patients (34.0%) had liver cirrhosis while 66 of the 100 patients (66.0%) did not. Another 38 consecutive treatment-naı¨ve HBsAg and hepatitis B e antigen (HBeAg) positive CHB patients who were recruited into a phase III multi-centre study on pegylated interferon alfa-2a at the Department of Medicine, Queen Mary Hospital, The University of Hong Kong, from March 2000 to December 2003, were retrospectively analyzed in order to determine the effect of anti-HBV therapy on adiponectin levels [9]. These 38 patients were randomized to receive lamivudine (GlaxoSmithKline, North Carolina, USA) 100 mg daily (n = 15), subcutaneous pegylated interferon alfa-2a (Roche, Basle, Switzerland) 180 lg weekly (n = 8) or combination subcutaneous pegylated interferon alfa-2a (Roche, Basle, Switzerland) 180 lg weekly plus lamivudine (GlaxoSmithKline, North Carolina, USA) 100 mg daily (n = 15) for 48 weeks. According to protocol, all 38 patients underwent liver biopsy at baseline. After completion of 48 weeks therapy, they were followed up for a further 6 months and underwent repeat liver biopsy [9]. All CHB patients included into this study had been HBsAg plus HBeAg positive for more than 6 months prior to the initial percutaneous liver biopsy. They were all negative for HCV antibody, hepatitis D antibody and human immunodeficiency virus. None of the patients had evidence of hepatocellular carcinoma according to previously published criteria [10,11]. All patients included had an alcohol intensity of less than 10 gm/day quantified as previously described [12]. None of the 138 patients had diabetes mellitus according to criteria defined by the World Health Organization [13]. Consecutive patients with histologically proven liver cirrhosis due to HCV (n = 34), autoimmune hepatitis (n = 34), alcoholic liver disease (n = 34) and primary biliary cirrhosis (n = 34) were included as control to investigate the possible disease-specific variation on adiponectin levels. This study was approved by the local Institutional Review Board and informed consent obtained from all patients.

2.2. Liver biopsy A percutaneous liver biopsy with a length of 20 mm or more was performed on all patients. Fibrosis stage, using the Ishak fibrosis score, and disease activity grade, using the modified histology activity index (HAI), were established by a group of pathologists blinded to the patients’ characteristics [9,14]. Five millimeter of the liver biopsy was stored in RNA later (Qiagen, Valencia, CA, USA) and frozen at 80 C until mRNA extraction.

2.3. Enzyme-linked immunosorbent assay (ELISA) for human adiponectin in serum Quantification of human serum adiponectin was performed as described previously [15].

2.4. Serum gel filtration for adiponectin

2. Materials and methods

Gel filtration was used to separate three oligomeric forms of adiponectin, namely the high molecular weight (HMW) (45.5–49 min), middle molecular weight (MMW) (52.5–56.5 min), and low molecular weight (LMW) (58.5–62 min), in patients with stage 0–1 fibrosis (n = 28) and in those with liver cirrhosis (n = 34) as previously described [15]. Gel filtration was also performed on patients receiving antiviral therapy in order to determine if improvement in fibrosis stage was associated with a change in HMW and MMW oligomeric form of adiponectin.

2.1. Patients

2.5. Immunohistochemistry

One hundred consecutive treatment naı¨ve hepatitis B surface antigen (HBsAg) positive patients who underwent liver biopsy for assess-

Immunohistochemistry was performed as described previously in patients with stage 0–1 fibrosis and liver cirrhosis [16]. The mouse

C.-K. Hui et al. / Journal of Hepatology 47 (2007) 191–202 monoclonal antibody used in these experiments was produced as previously described [15]. The specificity of the immunoreaction of adiponectin was tested by omitting the primary antibody and confirmed by the presence of immunostaining in sections of adult adipose tissues in which adiponectin was highly expressed. Moreover, pre-adsorption experiments were performed after pre-incubating the antiserum with soluble recombinant human ACRP30 (Alexis Biochemicals, San Diego, CA, USA).

2.6. mRNA expression analysis of adiponectin in frozen liver tissue Total RNA was extracted from fresh frozen liver biopsy tissues of patients with stage 0–1 fibrosis and liver cirrhosis using Trizol reagent (Life Technologies Inc., Gaithersburg; MD, USA) according to the manufacturer’s instruction. Total RNA (5 lg) was reverse transcribed (Promega, Madison, WI, USA) and then subjected to polymerase chain reaction (PCR) for 35 cycles at 94, 58 and 72 C for 45, 45 and 45 s, respectively, using intron-spanning specific primers for human adiponectin gene (GenBank Accession No. NM004797) (sense: 5 0 -CTT CTT GAA GAG GCT GAC CT-3 0 ) to yield a product size of 350 bp. PCRs were also performed in RNA samples before reverse transcription to exclude DNA contamination. The housekeeping GADPH gene was used as control in all RT-PCR experiments to assess cDNA quality. The PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. A positive control cDNA and ‘‘no template’’ negative control were amplified in parallel in each PCR assay as positive and negative controls, respectively.

2.7. Virological assessment HBsAg, hepatitis B surface antibody (anti-HBs), HBeAg and hepatitis B e antibody (anti-HBe) were tested by commercially available enzyme immunoassays (Abbott Laboratories, Chicago, IL, USA). Serum HBV DNA levels were quantified with an in-house real time PCR assay as described previously [17]. The linear range of this assay was 102–109 copies/ml. HBV was genotyped by analysis of restriction fragment-length polymorphism of an S region or a pre-S region amplicon as previously described [18].

2.8. Definition of endpoints Liver cirrhosis was defined as either fibrosis stage 5 or 6 on liver biopsy. Sustained virological response, assessed at 6 months after the end of therapy, was defined as HBeAg seroconversion and suppression of HBV DNA to less than 104 copies/ml with normalization of serum alanine aminotransaminase (ALT) levels as previously described [19]. HBeAg seroconversion was defined as loss of HBeAg with the development of anti-HBe for three consecutive readings 8 weeks apart [19]. In the 38 patients treated with anti-HBV therapy, improvement in liver fibrosis was defined as at least a 1-point reduction in fibrosis stage on liver biopsy at 6 months after the end of therapy, while progression of fibrosis was defined as at least a 1-point increase in fibrosis stage on liver biopsy at 6 months after the end of therapy. Improvement in modified HAI score was defined as at least a 2-point reduction in modified HAI score on liver biopsy at 6 months after the end of therapy. Biochemical response was defined as normalization of serum ALT levels at 6 months after the end of therapy.

2.9. Statistical analysis All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 12.5 for windows; SPSS Inc., Chicago, IL, USA). The chi-square test was used to analyze relationships between categorical variables. The Kruskal–Wallis test and ANOVA test were used to compare values between different groups, and the

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Student’s t test was used to analyze single specific differences of biological interest. The Student’s t test was also used to analyze paired values in the same patients at different time points. The degree of association between serum adiponectin and variables was assessed using Spearman’s correlation. Continuous variables were expressed in mean ± standard error of mean (SEM). The primary aims of this study were to determine the association of adiponectin in CHB-related liver cirrhosis and to determine if improvement in fibrosis stage after antiviral therapy is associated with a decline in serum adiponectin level. Factors significant in the univariate analysis were subsequently incorporated in a logistic regression analysis to identify significant independent risk factors for liver cirrhosis. All statistical analyses were performed on an intention-to-treat basis. Statistical significance was defined as p < 0.05 (two-tailed).

3. Results 3.1. Increase in serum adiponectin was associated with liver cirrhosis in patients with CHB The baseline characteristics of the 100 CHB patients studied are shown in Table 1. The plasma level of adiponectin was higher in CHB patients with liver cirrhosis when compared with those with stage 0–1, stage 2, and stage 3 fibrosis (mean ± SEM 20.74 ± 3.50 vs. 8.28 ± 0.89 vs. 12.02 ± 1.75 vs. 14.88 ± 1.53 lg/ml, respectively, p < 0.001 by ANOVA). There was a significant positive correlation between serum adiponectin and fibrosis stage (r = 0.45, p < 0.001) (Fig. 1a).

Table 1 Baseline characteristics of the 100 CHB patients included into the first part of study Characteristics

HBV total group (n = 100)

Age, years Sex, M:F Serum alanine aminotransaminase, U/L Serum albumin, g/dl Prothrombin time, seconds Serum HBV DNA, log10 copies/ml Modified histology activity index score Fibrosis stage Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 or 6 (liver cirrhosis) HBV genotype B C Body mass index, kg/m2 Serum adiponectin, lg/ml HBeAg Positive Negative Anti-HBe Positive Negative

57 ± 24 67:33 124 ± 13 42 ± 9 10.2 ± 2.6 8.37 ± 0.30 9±1 2 26 22 16 0 34 45 55 23.1 ± 0.7 13.53 ± 1.33 36 64 64 36

Continuous variables are expressed in mean ± standard error of mean.

Serum Adiponectin Level (μg/ml)

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C.-K. Hui et al. / Journal of Hepatology 47 (2007) 191–202 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

albumin, prothrombin time, or body mass index and serum adiponectin level (all p = NS).

R = 0.45, P<0.001

3.2. Serum adiponectin was independently associated with liver cirrhosis in CHB Patients

Stage 0-1 Fibrosis Stage 2 Fibrosis

Stage 3 Fibrosis

Liver Cirrhosis

Serum Adiponectin Level (μg/ml)

R = 0.25, p = 0.03

80 70 60 50 40

The baseline demographics between CHB patients with and without liver cirrhosis are shown in Table 2. On multiple regression analysis, serum adiponectin (p = 0.02, adjusted hazards ratio 1.07, 95% confidence interval 1.00–1.14) was independently associated with liver cirrhosis. There was a trend that modified HAI score was also independently associated with liver cirrhosis in CHB patients (p = 0.05, adjusted hazards ratio 0.19, 95% confidence interval 0.05–0.73). There was no significant difference in the baseline demographics of cirrhotic CHB patients with high (higher than 20.74 lg/ml) or low serum adiponectin (lower than 20.74 lg/ml) levels (Table 3).

30 20

3.3. Serum adiponectin level was comparable in patients with different etiology of liver cirrhosis

10 0 0

2

4

6

8

10

12

14

16

18

Modified HAI Score

Fig. 1. (a) Serum adiponectin level in different stages of HBV-related liver fibrosis patients. Serum collected from patients with different stages of CHB-related liver fibrosis was quantified for the levels of adiponectin using ELISA. Each point represented a single patient and the bars indicating the mean values; (b) graph showing correlation between modified HAI score and serum adiponectin level.

There was also a significant positive correlation between serum adiponectin and modified HAI score (r = 0.25, p = 0.03) (Fig. 1b). There was no association between HBV genotype, serum HBV DNA, sex, age, serum alanine aminotransaminase (ALT) levels, serum

The baseline demographics of 136 patients with chronic HCV infection, autoimmune hepatitis, alcoholic hepatitis and primary biliary cirrhosis were compared with those of CHB-related liver cirrhosis (all p = NS by ANOVA) (Table 4). There was no difference in the serum adiponectin level between the 5 groups (p = NS by ANOVA) (Table 4). 3.4. MMW oligomeric form of adiponectin was increased in patients with CHB-related liver cirrhosis For CHB patients with stage 0–1 fibrosis, the distribution of HMW, MMW, and LMW oligomeric form

Table 2 Baseline demographics of the 100 patients with and without CHB-related liver cirrhosis Characteristics Age, years Sex; M:F Serum alanine aminotransaminase, U/L Serum albumin, g/dl Prothrombin time, seconds Serum HBV DNA, log10 copies/ml Modified HAI score HBV genotype B C Body mass index, kg/m2 Serum adiponectin, lg/ml HBeAg Positive Negative Anti-HBe Positive Negative

Liver cirrhosis (n = 34)

No liver cirrhotics (n = 66)

p-Value

45 ± 1 24:10 79 ± 13 38 ± 6 13.3 ± 2.1 5.91 ± 0.80 12.9 ± 0.6

39 ± 2 43:23 135 ± 15 42 ± 4 11.8 ± 2.7 7.52 ± 0.34 7.1 ± 0.4

0.25 0.58 0.02 0.21 0.31 0.10 <0.001 0.90

15 19 22.1 ± 0.4 21.32 ± 3.20

30 36 23.4 ± 0.6 9.38 ± 0.70

14 30

22 34

30 14

34 22

0.54 <0.001 0.44

0.44

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Table 3 Demographics of CHB-related cirrhotics patients with serum adiponectin higher than 20.74 lg/ml or lower than 20.74 lg/ml Characteristics

Liver cirrhosis with Padiponectin 20.74 lg/ml (n = 11)

Liver cirrhotics with adiponectin <20.74 lg/ml (n = 23)

p-Value

Age, years Sex; M:F Serum alanine aminotransaminase, U/L Serum albumin, g/dl Prothrombin time, seconds Serum HBV DNA, log10 copies/ml Modified HAI score HBV genotype B C Body mass index, kg/m2 HBeAg Positive Negative Anti-HBe Positive Negative

42 ± 2 7:4 82 ± 28 36 ± 8 12.6 ± 2.7 6.03 ± 0.65 13.7 ± 0.7

45 ± 3 15:8 72 ± 14 39 ± 4 11.8 ± 2.5 5.76 ± 0.76 12.0 ± 1.1

0.45 1.00 0.72 0.52 0.69 0.43 0.32 0.76

5 6 21.3 ± 0.5

10 15 23.5 ± 0.6

6 5

8 15

5 6

15 8

0.83 0.27

0.27

of adiponectin was 51.2%, 38.5%, and 10.3%, respectively (Table 5). On the other hand, the distribution of the HMW, MMW and LMW oligomeric form of adiponectin in CHB patients with liver cirrhosis was 40.9%, 48.7%, and 10.4%, respectively (Table 5). CHB patients with stage 0–1 fibrosis had higher percentage composition of HMW form of adiponectin (p = 0.001) and a lower percentage composition of MMW form of adiponectin (p = 0.001) when compared with CHB patients with liver cirrhosis (Table 5 and Fig. 2). However, no significant difference was found in the LMW forms of adiponectin between these two CHB patient groups (p = NS) (Table 5 and Fig. 2). The ratio of HMW to MMW (HMW/MMW) was calculated in these CHB patient groups to illustrate the difference in the oligomeric form distributions of adiponectin (p = 0.001). A significant reduction in HMW/MMW was found in CHB patients with liver cirrhosis (Table 4), due to the increased level of MMW forms of adiponectin. Furthermore, the HMW oligomeric form of adiponectin was higher in female than in male CHB patients

(48.7 ± 2.7% vs. 41.7 ± 2.1%, respectively, p = 0.03). However, there was no significant difference in the MMW (47.4 ± 1.7% vs. 44.9 ± 1.3%, respectively, p = NS) and LMW (10.4 ± 0.6 vs. 10.1 ± 0.2%, respectively, p = NS) forms of adiponectin between females and males with CHB. 3.5. Hepatic mRNA expression of adiponectin The mRNA expression of adiponectin in the liver tissues of the 28 CHB patients with stage 0 or 1 fibrosis and 34 patients with CHB-related liver cirrhosis was analyzed by RT-PCR. None of the CHB patients had adiponectin mRNA detectable in liver biopsies even after 35 cycles (Fig. 3a). 3.6. Immunohistochemistry Immunohistochemistry was used to compare the expression and localization of adiponectin in liver tissues of the 34 CHB patients with liver cirrhosis and 28

Table 4 Baseline demographics of the 34 patients with chronic hepatitis B-related liver cirrhosis and 136 patients with non-chronic hepatitis B-related liver cirrhosis Characteristics

Age, years Sex; M:F Serum alanine aminotransaminase, U/L Serum albumin, g/dl Body mass index, kg/m2 Serum adiponectin, lg/ml

CHB-related liver cirrhosis (n = 34)

Chronic HCV-related liver cirrhosis (n = 34)

Autoimmune hepatitis-related liver cirrhosis (n = 34)

Alcoholic liver cirrhosis (n = 34)

Primary biliary cirrhosis-related liver cirrhosis (n = 34)

p-Value

45 ± 1 24:10 79 ± 13

46 ± 3 26:8 79 ± 15

41 ± 2 19:15 45 ± 7

39 ± 3 26:8 56 ± 9

48 ± 5 20:14 37 ± 13

0.62 0.21 0.28

38 ± 6 22.1 ± 0.4 21.32 ± 3.20

40 ± 9 24.1 ± 0.3 19.21 ± 1.03

37 ± 8 24.6 ± 0.7 18.64 ± 2.71

35 ± 3 18.2 ± 1.1 17.22 ± 2.18

37 ± 2 20.3 ± 0.4 17.93 ± 2.45

0.52 0.48 0.38

CHB, chronic hepatitis B; HCV, hepatitis C virus.

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Table 5 Mean ± standard deviation of mean percentages of different oligomeric forms of adiponectin in patients with low fibrosis and cirrhosis Patients

Mean percentages of oligomeric forms of adiponectin HMW (%)

MMW (%)

LMW (%)

Stage 0–1 fibrosis Cirrhosis p-Value

51.2 ± 2.1 40.9 ± 1.7 0.001

38.5 ± 1.7 48.7 ± 1.8 0.001

10.3 ± 0.5 10.4 ± 0.4 0.913

Mean ratios of HMW/MMW 1.4 ± 0.1 0.9 ± 0.1 0.001

HMW, high molecular weight oligomeric form of adiponectin; MMW, middle molecular weight oligomeric form of adiponectin; LMW, low molecular weight oligomeric form of adiponectin.

CHB patients with stage 0–1 fibrosis. Enhanced levels of adiponectin were detected in cirrhotic livers, when compared to stage 0–1 fibrosis stage (Fig. 3b). In accordance with the serum adiponectin levels in the 34 CHB patients with liver cirrhosis, cirrhotic patients with higher serum adiponectin levels had more intense adiponectin immunoreactivity (data not shown). 3.7. Anti-HBV therapy leads to reduction of serum adiponectin level The characteristics of the 38 CHB patients randomized to receive lamivudine monotherapy, pegylated interferon alfa-2a monotherapy or combination pegylated interferon alfa-2a plus lamivudine therapy are shown in Table 6. There was no difference in the baseline characteristics among the three different treatment groups (all p = NS by ANOVA). At 6 months after the end of therapy, antiviral therapy resulted in significant reduction of serum adiponectin level when compared with baseline (mean ± SEM 10.17 ± 1.09 lg/ ml vs. 8.48 ± 0.76 lg/ml, respectively, p = 0.01). The mean ± SEM decrease in serum adiponectin level at 6 months after the end of therapy was 1.72 ± 1.00 lg/ ml. The reduction in serum adiponectin level at 6 months after the end of therapy was comparable in the

% of Each Oligomeric Form of Adiponectin

P = 0.001

P = 0.001

60 50 40 30 20 10 0 HMW

MMW Stage 0-1 Fibrosis

LMW Liver Cirrhosis

Fig. 2. Percentage of each oligomeric complex per total serum adiponectin in patients with stage 0–1 fibrosis and patients with liver cirrhosis. HMW, high molecular weight oligomeric form; MMW, middle molecular weight oligomeric form; LMW, low molecular weight oligomeric form.

lamivudine group (mean ± SEM 1.14 ± 1.85 lg/ml), the pegylated interferon alfa-2a group (mean ± SEM 2.24 ± 1.46 lg/ml) or the combination pegylated interferon alfa-2a plus lamivudine group (mean ± SEM 2.23 ± 1.46 lg/ml) (p = NS by ANOVA). 3.8. Improvement in fibrosis stage is associated with a higher reduction in serum adiponectin level On repeat liver biopsy at 6 months after the end of therapy, 14 of the 38 patients (36.8%) had an improvement in liver fibrosis, 11 of the 38 patients (28.9%) had progression of fibrosis while the remaining 13 patients of the 38 patients (34.2%) had unchanged fibrosis stage. Those with an improvement in fibrosis stage had a higher reduction in serum adiponectin level at 6 months after the end of therapy when compared with those who developed progression of fibrosis and those with unchanged fibrosis stage at 6 months after the end of therapy (mean ± SEM 3.08 ± 1.34 lg/ml vs. 0.11 ± 1.34 vs. 1.75 ± 2.29 lg/ml, respectively, p = 0.03 by ANOVA) (Fig. 4a and b). 3.9. MMW oligomeric form of adiponectin was reduced in patients’ improvement in fibrosis stage In patients who demonstrated an improvement in fibrosis stage, there was an increase in the HMW form of adiponectin (mean ± SEM 43.5 ± 1.2% vs. 37.0 ± 3.0%, respectively, p = 0.04) with a corresponding decrease in the MMW form of adiponectin (mean ± SEM 45.9 ± 1.6% vs. 52.9 ± 2.9%, respectively, p = 0.04) at 6 months after the end of therapy when compared with baseline. There was also a trend that patients with an improvement in fibrosis stage had a significant increase in the ratio of HMW to MMW (HMW/MMW) at 6 months after the end of therapy when compared with baseline (mean ± SEM 0.97 ± 0.07 vs. 0.71 ± 0.09, respectively, p = 0.05) (Fig. 4c). 3.10. Sustained virological responders had a higher reduction in serum adiponectin level Fifteen of the 38 patients (39.8%) had sustained virological response at 6 months after the end of therapy – 6

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Fig. 3. (a) RT-PCR analysis for adiponectin mRNA was performed on liver tissues of patients with stage 0–1 fibrosis and those with liver cirrhosis. The integrity of the mRNA was documented by RT-PCR of GADPH. PC, positive control; NC; negative control, P1–P5; patients with liver cirrhosis; P6– P10, patients with stage 0–1 fibrosis. (b) The levels and localizations of adiponectin in liver tissues from patients with CHB-related liver cirrhosis and those with stage 0–1 fibrosis. Immunohistochemistry was performed to show the localizations and levels of adiponectin in patients with low fibrosis and cirrhosis. Brown immunoprecipitates represented the localizations of adiponectin. The areas within the open rectangles in the 200· were further enlarged (400·) as shown underneath each photograph of low fibrosis or cirrhosis. Magnifications are indicated in the micrographs. Table 6 Baseline characteristics of patients randomized to receive either lamivudine monotherapy, pegylated interferon alfa-2a monotherapy or pegylated interferon alfa-2a plus lamivudine combination therapy

Age, years Sex, M:F Serum alanine aminotransaminase, U/L HBV DNA, log10 copies/ml Modified histology activity index score Fibrosis stage Genotype B C Baseline serum adiponectin level, lg/ml

LAM (n = 15)a

Peg (n = 15)a

Peg+LAM (n = 8)a

39 ± 2 13:2 113 ± 17 8.59 ± 0.42 9±1 3 ± 0.4

40 ± 3 11:4 153 ± 34 8.85 ± 0.35 8±1 2 ± 0.3

41 ± 5 7:1 207 ± 61 8.93 ± 0.91 10 ± 1 3 ± 0.4

5:10

7:8

3:5

10.82 ± 2.04

9.65 ± 1.54

11.92 ± 1.95

LAM, lamivudine monotherapy; Peg, Pegylated interferon alfa-2a monotherapy; Peg+LAM, pegylated interferon alfa-2a plus lamivudine combination therapy. Continuous variables are expressed in mean ± standard error of mean. a All p = NS when the 3 groups are compared with each other (by ANOVA).

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from the lamivudine group, 6 from the pegylated interferon alfa-2a group and another 3 from the combination pegylated interferon alfa-2a plus lamivudine group. Although statistically insignificant, progression of fibrosis on repeat liver biopsy was less likely to occur in patients with sustained virological response than those

Change in Serum Adiponectin Level at 6 Months After The End of Therapy (µg/ml)

a

without sustained virological response [3 of 15 patients (20.0%) vs. 10 of 23 patients (43.5%), respectively, p = 0.33]. The reduction in serum adiponectin level at 6 months after the end of therapy was higher in patients with sustained virological response than those without sustained virological response (mean ± SEM 3.79 ± 1.92

P = 0.03 when the 3 groups were compared 2

1

0

-1

-2

-3

-4

-5 Reduction in Fibrosis Stage

Progression in Fibrosis Stage

No Change in Fibrosis Stage

b Serum Adiponectin Level (μg/ml)

30

Reduction in Fibrosis Stage

No Change in Fibrosis Stage

30

25

25

20

20

15

15

10

10

5

5

0

0 Baseline

6 Months After End of Therapy

Baseline

6 Months After End of Therapy

Serum Adiponectin Level (μg/ml)

30

Progression in Fibrosis Stage 25 20 15 10 5 0 Baseline

6 Months After End of Therapy

Fig. 4. (a) Change in serum adiponectin level between baseline and 6 months after the end of therapy in patients who had reduction in liver fibrosis, progression of liver fibrosis at 6 months after the end of therapy on longitudinal liver biopsy. (b) Serial analysis of serum adiponectin in those with improvement in fibrosis stage, progression of fibrosis stage or no change in fibrosis stage at 6 months after the end of therapy. (c) Change in the percentage of each oligomeric complex per total serum adiponectin at 6 months after the end of therapy when compared with baseline in patients with improvement in fibrosis stage. (d) Change in serum adiponectin level between baseline and at 6 months after the end of therapy in patients with and without sustained virological response. SVR, sustained virological response. (e) Serial analysis of serum adiponectin in those with and without sustained virological response at 6 months after the end of therapy. (f) Serial analysis of serum adiponectin in those with and without improvement in modified HAI score. (g) Serial analysis of serum adiponectin in those with and without biochemical response at 6 months after the end of therapy.

C.-K. Hui et al. / Journal of Hepatology 47 (2007) 191–202

c

d 60

P=0.04

50 40 30 20 10 0 HMW

MMW

LMW

Serum Adiponectin Level (μg/ml)

At 6 Months After The End of Therapy

e

Changein Serum Adiponectin Level at 6 Months After The End of Therapy (μg/ml)

% of Each Oligomeric Form of Adiponectin

P=0.04

30

Baseline

25

25

20

20

15

15

10

10

5

5

0

30

0 -1 -2 -3 -4 -5 -6

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Non-Sustained Virological Responders

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No 2-Point Improvement in Modified HAI Score

Serum Adiponectin Level (μg/ml)

30 25 25 20

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Serum Adiponectin Level (μg/ml)

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g

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f

P=0.03 2

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Sustained Virological Responders

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30

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30

Biochemical Responders

25

25

20

20

15

15

10

10

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Fig. 4 (continued)

Baseline

6 Months After End of Therapy

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lg/ml vs. 0.34 ± 1.02 lg/ml, respectively, p = 0.03) (Fig. 4d and e). 3.11. Improvement in modified HAI score was associated with higher reduction in serum adiponectin level In the 38 patients randomized to receive lamivudine monotherapy, pegylated interferon alfa-2a monotherapy or combination pegylated interferon alfa-2a plus lamivudine therapy, serum adiponectin at baseline was positively correlated with modified HAI score (r = 0.32, p = 0.04). There was also a trend that the modified HAI score at 6 months after the end of therapy correlated with serum adiponectin (r = 0.05, p = 0.08). Twenty-three patients (60.5%) had improvement in modified HAI score at 6 months after the end of therapy (lamivudine, n = 9, pegylated interferon alfa-2a, n = 9, combined pegylated interferon alfa-2a plus lamivudine, n = 5). The reduction in serum adiponectin level at 6 months after the end of therapy was higher in patients with improvement in modified HAI score when compared with those without an improvement in modified HAI score (mean ± SEM 1.87 ± 0.77 lg/ml vs. 1.62 ± 1.40 lg/ml, respectively, p = 0.02) (Fig. 4f). 3.12. Biochemical response was associated with a higher decline in serum adiponectin level Sixteen of the 38 patients (42.1%) developed biochemical response at 6 months after the end of therapy. Those with biochemical response also had a higher reduction in serum adiponectin level (mean ± SEM 2.31 ± 0.97 lg/ml vs. 0.92 ± 1.02 lg/ml, respectively, p = 0.04) (Fig. 4g).

4. Discussion Adiponectin is a secretory adipocytokine which acts as a coordinator in energy metabolism and insulin sensitivity [20]. Its level in plasma alters according to different clinical conditions, such as obesity and diabetes [21]. These observations suggested that adiponectin might be pathogenetically involved in these abnormalities. In the liver, adiponectin is found to correlate with the incidence of liver cirrhosis of different etiologies, such as alcoholism and NASH [22]. The highest level of plasma adiponectin was detected in NASH-induced liver cirrhosis [22]. In general, the level of adiponectin correlates with the severity of liver cirrhosis [4,22,23]. In this study, we reported for the first time that serum adiponectin level was independently associated with liver cirrhosis and there was no evidence of disease-specific effects of HBV as our cohort of CHB patients with liver cirrhosis had comparable serum

adiponectin level with the 4 control groups with nonCHB-related liver cirrhosis. Although hepatic adiponectin mRNA was not detectable in liver biopsy tissues of either CHB patients with stage 0–1 fibrosis, or, in cirrhotic liver, immunohistochemical staining demonstrated increased hepatic adiponectin expression in cirrhotic liver tissues, particularly, in the hepatocytes and not in the hepatocytes of CHB patients with stage 0–1 fibrosis. Furthermore, we also found a correlation between serum adiponectin level and adiponectin staining on immunohistochemistry in CHB patients with liver cirrhosis (data not shown). We did not compare CHB patients with stage 0–1 fibrosis because these patients only had minimal adiponectin staining on immunohistochemistry. Since there is no evidence to suggest that adiponectin is expressed in ‘‘normal’’ liver, and there have been no published human studies demonstrating substantial hepatic adiponectin mRNA expression, the increase in hepatic adiponectin in cirrhotic liver tissues on immunohistochemistry may be the result of uptake of the protein from the circulation [7]. As biliary excretion has previously been shown to be involved in the clearance of adiponectin, the increase in serum adiponectin level in patients with liver cirrhosis can be partly explained by the impaired biliary secretion in cirrhotic liver [4]. This leads to the accumulation of adiponectin in the circulation and its deposition in cirrhotic livers [4]. Next, we examined whether oligomeric forms of adiponectin might influence the role played by adiponectin in liver cirrhosis. Three oligomeric forms, namely the HMW, MMW, and LMW, of adiponectin have been previously identified [20]. These oligomeric forms of adiponectin have been shown to demonstrate sexual dimorphism, with males having a reduced level of HMW forms due to testosterone suppression, when compared with females [15,24,25]. In this study, we demonstrated that a selective increase in the MMW form of adiponectin was associated with liver cirrhosis when compared to stage 0–1 fibrosis. This suggests that the development of liver cirrhosis may result from an unbalanced ratio of HMW to MMW forms of adiponectin as reflected in the circulation, a condition similar to the development of vascular diseases that are associated with obesity in humans [24]. This may be explained by the fact that different oligomeric forms of adiponectin have distinct binding partners or downstream signaling pathways. For instance, the HMW and MMW forms of adiponectin bind to platelet-derived growth factor BB whereas the three oligomeric forms exhibited differential binding affinities with fibroblast growth factor and heparin-binding epidermal growth factor-like growth factor [26]. Additionally, HMW and MMW, but not the LMW, forms of adiponectin have the ability to activate nuclear factor-jB signaling pathway in vitro [27]. Collectively,

C.-K. Hui et al. / Journal of Hepatology 47 (2007) 191–202

these observations illustrate that alteration in the oligomeric complex distribution can result in different disease manifestation, which is likely due to the modulation of different downstream components and/or signaling pathways. On the other hand, clinical trials with nucleoside/ nucleotide analogues and immunomodulators have demonstrated an improvement in liver histology with antiviral therapy [28–31]. This improvement in fibrosis is more marked in those with virological response and HBeAg seroconversion [9,29,31]. However, short-term therapy with nucleoside/nucleotide analogues is usually followed by viral recrudescence in most CHB patients and deterioration in fibrosis stage following drug cessation [32]. Here, we demonstrated that antiviral therapy might result in a decline in serum adiponectin level. More importantly, the decrease in serum adiponectin level was higher in those who achie‘ved sustained virological response. There was also a parallel improvement in fibrosis stage or improvement in liver fibrosis among those with the highest decline in serum adiponectin level after antiviral therapy while those with progression of fibrosis stage had the least decline in serum adiponectin level. This provides evidence that adiponectin is being depleted in those with improvement in fibrosis stage. A decrease in the MMW oligomeric form of adiponectin in association with an increase in the HMW oligomeric form of adiponectin is associated with an improvement in fibrosis stage. Thus, serum adiponectin may not only be employed as a marker of liver fibrosis in CHB, but also as a candidate for therapy in CHBrelated liver cirrhosis. In addition to antiviral therapy, further improvement in fibrosis stage may be facilitated by an adiponectin antagonist that reduces serum adiponectin level, thereby correcting the imbalance ratio between HMW and MMW oligomeric forms of adiponectin. Further studies on the application of adiponectin as a target for reverting CHB-related liver fibrosis in a larger cohort and cohorts from European and American CHB patients would be worthwhile. In conclusion, adiponectin may have a direct role in the progression of fibrosis stage in CHB infection. Response to antiviral therapy can reduce serum adiponectin level and result in an improvement in fibrosis stage, especially those with significant decline together with restoration of HMW to MMW oligomeric form ratio of adiponectin. The potential therapeutic value of adiponectin inhibition in the prevention and reversion of liver fibrosis in CHB needs to be further investigated. Acknowledgements This work was funded by grants from The Hong Kong Liver Foundation (awarded to GKKL) and generous donation from Mr. Heung Chit Kau and family (to GKKL). The Hong Kong Liver Fibrosis Study

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Group includes the following persons: Chee-Kin Hui, George K Lau, Chung-Mau Lo, Sheung-Tat Fan, Ronnie TP Poon, Tony Shek (Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China); Nancy Leung (AHML Nethersole Hospital, Hong Kong SAR, China); Jak-Yiu Lai, Sik-To Lai, Hung Yao (Princess Margaret Hospital, Hong Kong SAR, China); Polly Lam (Queen Elizabeth Hospital, Hong Kong SAR, China); Wai-Ki Lee (Tuen Mun Hospital, Hong Kong SAR, China); Tang-Tat Fung (Kwong Wah Hospital, Hong Kong SAR, China); Lawrence Lai (Caritas Medical Centre, Hong Kong SAR, China) and Wai-Man Wong (St. Paul’s Hospital, Hong Kong SAR, China). References [1] Matsuzawa Y, Funahashi T, Kihara S, Shimomura I. Adiponectin and metabolic syndrome. Arterioscler Thromb Vasc Biol 2004;24:29–33. [2] Tschritter O, Fritsche A, Thamer C, Haap M, Shirkavand F, Rahe S, et al. Plasma adiponectin concentrations predict insulin sensitivity of both glucose and lipid metabolism. Diabetes 2003;52:239–243. [3] Xu A, Wang Y, Keshaw H, Xu LY, Lam KS, Cooper GJ. The fatderived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 2003;112:91–100. [4] Tacke F, Wustefeld T, Horn R, Luedde T, Srinivas Rao A, Manns MP, et al. High adiponectin in chronic liver disease and cholestasis suggests biliary route of adiponectin excretion in vivo. J Hepatol 2005;42:666–673. [5] Kamada Y, Tamura S, Kiso S, Matsumoto H, Saji Y, Yoshida Y, et al. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology 2003;125:1796–1807. [6] Tietge UJ, Boker KH, Manns MP, Bahr MJ. Elevated circulating adiponectin levels in liver cirrhosis are associated with reduced liver function and altered hepatic hemodynamics. Am J Physiol Endocrinol Metab 2004;287:E82–E89. [7] Jonsson JR, Moschen AR, Hickman IJ, Ricardson MM, Kaser S, Clouston AD, et al. Adiponectin and its receptors in patients with chronic hepatitis C. J Hepatol 2005;43:929–936. [8] Liu CJ, Chen PJ, Jeng YM, Huang WL, Yang WS, Lai MY, et al. Serum adiponectin correlates with viral characteristics but not histologic features in patients with chronic hepatitis C. J Hepatol 2005;43:235–242. [9] Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat T, Cooksley G, et al. Peginterferon alfa-2a (40 kDa) (PEGASYS) monotherapy and in combination with lamivudine is more effective than lamivudine monotherapy in HBeAg positive chronic hepatitis B: results from a large, multinational study. N Eng J Med 2005;352:2682–2695. [10] Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, et al. EASL panel of experts on HCC. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001;35:421–430. [11] Hui CK, Lie A, Au WY, Leung YH, Ma SY, Cheung WW, et al. A long-term follow-up study on hepatitis B surface antigen positive patients undergoing allogeneic hematopoietic stem cell transplantation. Blood 2005;106:464–469. [12] Hui CK, Lau E, Monto A, Kim M, Luk JM, Poon RT, et al. Natural history of patients with recurrent chronic hepatitis C virus and occult hepatitis B co-infection after liver transplantation. Am J Transplant 2006;6:1600–1608.

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