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Re: Adiponectin receptor II in non-alcoholic fatty liver disease

Dear Editor,

Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity and metabolic syndrome. The histological spectrum of NAFLD includes fatty liver to non-alcoholic steatohepatitis (NASH). Adipocyte-derived factors might play a role in the progression of the disease. Of these, adiponectin has been reported to alleviate NAFLD in an animal model [1], and its plasma level is reduced in patients with NASH [1]. Kaser et al. [2] recently reported in Gut reduced hepatic expression of adiponectin receptor II (AdipoRII), a predominant receptor for adiponectin in the liver, in patients with NASH compared with those with simple fatty liver with a similar steatosis score. However, we have obtained different results of AdipoRII expression in search for pathology-associated gene expression profiles in the liver of patients with NAFLD.

Between 2000 and 2003 at Kanazawa University Hospital, we recruited 63 consecutive patients clinically diagnosed as having NAFLD, and histologically examined their livers. All liver biopsy specimens were examined using hematoxylin-eosin and silver reticulin stain. The severity of steatosis, inflammation and fibrosis were histologically scored on a scale from 0 to 4, using previously reported criteria [3]. Patients with histologically typical livers were categorized into 3 groups: patients with normal liver (NL, n=9), patients with simple fatty liver (FL, n=8), and patients with NASH (n=8). NL was diagnosed when the steatosis score was not more than 1 without inflammation and fibrosis, FL was when the steatosis score was 3 or more with minimal inflammation and fibrosis (<=1), and NASH was when the fibrosis score was 3. Mean } SD age (years), BMI (kg/m2) and HOMA-R were 53}3, 23.6}3.1 and 1.93}1.42 in NL; 46}15, 28.3}4.2 and 4.91}3.57 in FL; and 45}15, 28.3}4.2 and 3.60}1.85 in NASH. There were no significant differences in age and sex among the groups. Mean } SD scores for steatosis, inflammation and fibrosis in each group were 0.7}0.5, 0 and 0 in NL; 3.4}0.5, 0.6}0.5 and 0.6}0.5 in FL, and 2.6}1.3, 2.8}0.7 and 3.0}0 in NASH. Patients with FL and NASH were similar in BMI, HOMA-R, steatosis score and serum levels of adiponectin (3.39}1.26 vs. 5.13}2.63 ƒΚg/ml, P=0.14).

Kaser et al. calculated the expression levels of AdipoRII as ratios to GAPDH levels. Firstly, we verified their results using real-time PCR. In our study, expression of GAPDH mRNA normalized with respect to 18S ribosomal RNA levels were up-regulated in the livers of patients with NASH compared with those with FL (54.8}11.6 vs. 11.3}2.6, mean}SE, P=0.0036). AdipoRII expression tended to be down-regulated in NASH compared with FL when standardized with GAPDH mRNA levels (8.6}1.5 vs. 36.1}14.4, P=0.0785). On the other hand, the real-time PCR method also confirmed no significant differences in AdipoRII expression levels between NASH and FL when standardized with 18S ribosomal RNA levels (46.0}12.3 vs. 26.4}7.2, P=0.1894).

To confirm the results, we made cDNA mircoarrays consisting of 10,800 human cDNAs, all of which are known to be expressed in various liver diseases (manuscript in submission). Expression levels were calculated as ratios of reference RNA, and then normalized with mean expression levels of 87 ribosomal RNAs, including 18S [3]. To find genes differentially expressed between predefined clinical groups, we used BRB-ArrayTools software (Biometric Research Branch of the National Cancer Institute, USA), the Class Comparison Tool based on univariate F-tests. The permutation distribution of the F-statistic, based on 2,000 random permutations, was also used to confirm statistical significance. Among the differentially expressed genes, AdipoRII expression was significantly up-regulated in FL (0.7, parametric P=0.000738, permutation P=0.0011) and NASH (0.8, parametric P=0.000135, permutation P=0.0001) compared with NL (0.5). Unlike in the study by Kaser et al. [2], there were no significant differences in the AdipoRII expression level between FL and NASH.

Kaser et al. [2] also presented immunohistochemistry of AdipoRII protein in the liver biopsy specimens. However, even in the representative experiment, there was little difference in the staining of AdipoRII between livers with NASH and simple steatosis. Thus, hepatic expression of AdipoRII might be unaltered between simple fatty liver and NASH, whereas it might be up-regulated during steatosis of the liver.

Therefore, before concluding the alteration in expression of AdipoRII in NASH, internal control for the mRNA expression should be carefully selected. Several groups [2,4] have reported decreased circulating levels of adiponectin in patients with NASH. Considering the possible protective effect of adiponectin against the development of NAFLD [1] and liver fibrosis [5], reduced hepatic expression of adiponectin and adipoRII might be of pathophysiological relevance in NASH. On the other hand, it might also be possible to speculate that the liver up-regulates the expression of AdipoRII to compensate for decreased circulating levels of adiponectin and to protect the liver from development of NAFLD in patients with metabolic syndrome. Further studies using animal models and a large scale clinical trial should be performed to clarify the regulation of AdipoRII expression in metabolic syndrome and NAFLD.

T Takamura, A Shimizu, N Matsuzawa, M Honda, S Kaneko
Department of Diabetes and Digestive Disease,
Kanazawa University Graduate School of Medical Science,
Kanazawa, Japan

Correspondence to:
Dr T Takamura
Department of Diabetes and Digestive Disease
Kanazawa University Graduate School of Medical Science
Kanazawa, Japan
email: tt{at}medf.m.kanazawa-u.ac.jp

References

1. Xu A, Wang Y, Keshaw H, et al. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 2003;112:91-100.

2. Kaser S, Moschen A, Cayon A, et al. Adiponectin and its receptors in non-alcoholic steatohepatitis. Gut 2005;54:117-21.

3. Takamura T, Sakurai M, Ota T, et al. Genes for systemic vascular complications are differentially expressed in the livers of type 2 diabetic patients. Diabetologia 2004;47:638-47.

4. Hui JM, Hodge A, Farrell GC, et al. Beyond insulin resistance in NASH: TNF-alpha or adiponectin? Hepatology 2004;40:46-54.