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Hepatitis C virus directly associates with insulin resistance independent of the visceral fat area in nonobese and nondiabetic patients
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Journal of Viral Hepatitis Feb 1, 2007 (OnlineEarly Articles).
M. Yoneda1 *1Division of Gastroenterology
S. Saito1 *1Division of Gastroenterology
T. Ikeda11Division of Gastroenterology,
K. Fujita11Division of Gastroenterology,
H. Mawatari11Division of Gastroenterology,
H. Kirikoshi11Division of Gastroenterology,
M. Inamori11Division of Gastroenterology,
Y. Nozaki11Division of Gastroenterology,
T. Akiyama11Division of Gastroenterology,
H. Takahashi11Division of Gastroenterology,
Y. Abe11Division of Gastroenterology,
K. Kubota11Division of Gastroenterology,
T. Iwasaki22Division of Endocrinology and Metabolism,
Y. Terauchi22Division of Endocrinology and Metabolism,
S. Togo33Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan and
A. Nakajima11Division of Gastroenterology
1Division of Gastroenterology, 2Division of Endocrinology and Metabolism, 3Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan
"....To determine the hypothesis that HCV infection may by itself predispose to IR before the onset of overt diabetes independently of the visceral adipose tissue area, we assessed the association of HOMA-IR with the quantity of HCV-RNA and the visceral adipose tissue area..... Multiple regression analysis showed that HCV infection was associated with IR in a dose-dependent manner, independent of the other factors considered.... we showed the existence of a significant correlation between the visceral adipose tissue area and the HOMA-IR in our control nondiabetic, non-HCV-infected patients.... The results of this study conducted in subjects without a history of DM or obesity suggest that HCV infection increases IR in a dose-dependent manner, independent of the visceral adipose tissue area. Based on the results of our study, we hypothesize that HCV infection by itself may cause IR..... Because IR has been reported to play a crucial role in fibrogenesis in chronic hepatitis C infection [38], the presence of chronic HCV infection in addition to a high-fat diet and lack of adequate exercise may be associated with not only an increase of the IR, but also rapid progression of liver fibrosis..... In addition to the anti-viral therapy, addition of oral hypoglycaemic agents for the improvement of IR, such as thiazolidinedione and metformin may greatly contribute to deter the progression of fibrosis...."
Abstract
Summary. Insulin resistance (IR) is known to be associated with the visceral adipose tissue area. Elucidation of the relationship between hepatitis C virus (HCV) and IR is of great clinical relevance, because IR promotes liver fibrosis. In this study, we tested the hypothesis that HCV infection by itself may promote IR. We prospectively evaluated 47 patients with chronic HCV infection who underwent liver biopsy. Patients with obesity, type 2 diabetes mellitus (DM), or a history of alcohol consumption were excluded. IR was estimated by calculation of the modified homeostasis model of insulin resistance (HOMA-IR) index. Abdominal fat distribution was determined by computed tomography. Fasting blood glucose levels were within normal range in all the patients. The results of univariate analysis revealed a significant correlation between the quantity of HCV-RNA and the HOMA-IR (r = 0.368, P = 0.0291). While a significant correlation between the visceral adipose tissue area and the HOMA-IR was also observed in the 97 control, nondiabetic, non-HCV-infected patients (r = 0.398, P < 0.0001), no such significant correlation between the visceral adipose tissue area and the HOMA-IR (r = 0.124, P = 0.496) was observed in the patients with HCV infection. Multiple regression analysis with adjustment for age, gender and visceral adipose tissue area revealed a significant correlation between the HCV-RNA and the HOMA-IR (P = 0.0446). HCV is directly associated with IR in a dose-dependent manner, independent of the visceral adipose tissue area. This is the first report to demonstrate the direct involvement of HCV and IR in patients with chronic HCV infection.
Introduction
Currently, approximately 200 million people around the world are chronically infected with the hepatitis C virus (HCV). Chronic HCV infection often leads to hepatic cirrhosis and hepatocellular carcinoma, thus posing a worldwide problem, both from the medical and the socioeconomical aspect [1,2]. Recent epidemiological studies have suggested that HCV infection is associated with an increased risk of development of type 2 diabetes mellitus (DM), and that type 2 DM is more prevalent among patients with chronic HCV infection than in patients with other liver diseases and in the general population, irrespective of whether or not hepatic cirrhosis is present [3-6].
Insulin resistance (IR) plays a primary role in the development of type 2 DM. This is supported by the results of prospective longitudinal studies showing that IR is the best predictor of the development of type 2 DM, preceding its onset by 10-20 years [7-10], and the results of cross-sectional studies showing that IR is a consistent finding in patients with type 2 DM [1,2,11]. Recently, visceral adipose tissue has drawn attention as a source of several bioactive substances, known as adipocytokines, such as adiponectin, leptin, plasminogen activator inhibitor-1 and tumour necrosis factor-α, all of which are thought to contribute to IR. It is also well known that IR is strongly associated with the visceral adipose tissue area, and furthermore, that it is a common feature in patients with obesity, type 2 DM and fatty liver [12]. In view of the strong association between HCV infection and the risk of development of DM, it is important to determine whether HCV infection can predispose to the development of IR even before overt diabetes sets in. The effect of HCV infection on IR depends on the viral genotype; patients infected with the genotype 3 virus have a lower prevalence of IR when compared with those infected with the other viral genotypes, even after adjustment for the effects of body mass index (BMI) and other confounders [13,14]. On the other hand, despite the lower prevalence of IR, subjects with HCV genotype 3 infection have more extensive hepatic steatosis [15-17]. Animal studies using transgenic mice carrying the core gene of HCV have suggested that HCV-encoded proteins might alter insulin signalling, thereby causing impaired insulin sensitivity and glycaemia dysregulation [18]. However, the pathogenesis of HCV-associated IR in humans remains to be clearly elucidated. None of the epidemiological studies conducted until now have analysed the relationship between HCV infection and the risk of IR independent of the influence of obesity, type 2 diabetes mellitus or alcohol consumption; therefore, the precise relationship between HCV and IR remains unclear in patients with HCV.
In this study, we tested the hypothesis that in Japanese nondiabetic, nonobese patients without a history of alcohol consumption having chronic HCV infection not caused by HCV genotype 3, the HCV infection by itself may promote IR independent of the visceral adipose tissue area, by determining the degree of IR in the subjects based on the fasting blood glucose and plasma insulin levels, and the homeostasis model assessment of IR (HOMA-IR) index.
Results
Patient characteristics and liver biopsy findings
The baseline characteristics of the 47 patients and the 97 nondiabetic and non-HCV-infected gender- and age-matched control subjects are shown in Table 1. The mean age of the patients was 53.0 ± 2.3 years; 29 (61.7%) were male, and the mean BMI was 23.1 ± 2.6 kg/m2. The distribution of the histopathological scoring of the inflammatory activity in the liver biopsy specimens was as follows: grade 1, 29 (61.7%) patients; grade 2, 15 (31.9%) patients; grade 3, three (6.4%) patients. The distribution of the severity of fibrosis was as follows: absent, seven (14.9%) patients; stage 1, 20 (40.4%) patients; stage 2, 11 (23.4%) patients; stage 3, nine (19.1%) patients. None of the patients had any evidence of cirrhosis. The distribution of the degree of steatosis in the patients was as follows: absent, 32 (68.1%) patients; stage 1, 8 (17.0%) patients; stage 2, seven (14.9%) patients; none of the patients in the study had stage 3 steatosis (shown in Table 2).
Insulin resistance and HCV infection
To determine the hypothesis that HCV infection may by itself predispose to IR before the onset of overt diabetes independently of the visceral adipose tissue area, we assessed the association of HOMA-IR with the quantity of HCV-RNA and the visceral adipose tissue area.
The fasting blood glucose levels were within normal range in all the patients. The results of univariate analysis revealed that while there was no significant correlation between the quantity of HCV-RNA and the fasting blood glucose (r =0.178, P = 0.3078) (Fig. 1a), a significant correlation existed between the quantity of HCV-RNA and the fasting plasma insulin level (r = 0.347, P = 0.0408) (Fig. 1b). A significant correlation was also found between the quantity of HCV-RNA and the HOMA-IR (r = 0.368, P = 0.0291) (Fig. 1c). There was no significant correlation between inflammatory activity index and the HOMA-IR (Fig. 3a), and between the fibrosis index and the HOMA-IR (Fig. 3b). These results suggested that neither hepatic inflammation nor fibrosis was responsible for the increase in IR in the patients with HCV infection in our study.
Visceral adipose tissue may be a major contributor to IR and indeed, there was a significant correlation between the visceral adipose tissue area and the HOMA-IR in the 97 nondiabetic, non-HCV-infected control subjects in this study (r = 0.398, P < 0.0001) (Fig. 2d). However, to our surprise, we found no significant correlation between the visceral adipose tissue area and the fasting plasma glucose, fasting plasma insulin or the HOMA-IR in the patients with HCV infection in our study [r = 0.227, P = 0.207 for fasting plasma glucose (Fig. 2a), r = 0.090, P = 0.6218 for fasting plasma insulin (Fig. 2b) and r = 0.124, P = 0.496 for HOMA-IR (Fig. 2c)].
Multiple regression analysis
Multiple regression analysis was used to quantify the influence of the measured variables on the HOMA-IR (Table 3). After adjustment for age, gender and visceral adipose tissue area, the quantity of HCV was still significantly correlated with the HOMA-IR (P = 0.0446).
Methods
Patients
We prospectively evaluated 47 patients with chronic HCV infection who underwent liver biopsy at Yokohama City University Hospital between January 2005 and December 2005. The study was conducted with the approval of the Ethics Committee of Yokohama City University Hospital. Hepatitis C genotyping was conducted based on the detection of anti-HCV antibodies using the line probe assay (Inno-LiPA HCV II; Innogenesis, Ghent, Belgium), a second-generation reverse hybridization technique. The serum HCV-RNA titres were examined by the Cobas Amplicor HCV monitor assay (version 2.0) (Roche, Tokyo, Japan) method, with a lower limit of quantitation of 5000 IU /mL and upper limit of quantitation of 5 000 000 IU /mL. The HCV genotype was determined using type-specific primers from the core region of the HCV genome. None of the patients had any clinical evidence of hepatic decompensation, such as hepatic encephalopathy, ascites, variceal bleeding or elevation of the serum bilirubin levels to beyond twofold of the upper limit of normal. Ninety-seven gender- and age-matched nondiabetic, nonobese, nonalcoholic and non-HCV-infected patients were evaluated as controls.
Patients with the following conditions were excluded: concurrent active hepatitis B virus infection (positive for hepatitis B surface antigen) or autoimmune hepatitis, primary biliary cirrhosis (PBC), sclerosing cholangitis, haemochromatosis, α1-antitrypsin deficiency or Wilson's disease. Patients with obesity (BMI > 25), type 2 DM, or a history of alcohol consumption (20 g/day) were also excluded from the study.
Clinical and laboratory evaluation
The 47 patients with chronic HCV infection finally enrolled in the study were interviewed to determine their current average daily alcohol intake (g/day), i.e. in the previous 6 months, and also their past history of alcohol intake (g/day), i.e. prior to the previous 6 months. A current or past history of daily alcohol intake of more than 20 g/day represented a criterion for exclusion from the study. The weight and height of the patients were measured with a calibrated scale.
Venous blood samples were drawn from the patients after overnight (12 h) fasting to determine the serum levels of albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl-transferase (GGT), plasma levels of glucose and insulin, and the platelet count. The plasma insulin level was measured by radioimmunoassay, and the other biochemical analyses were conducted in a conventional automated analyser.
Insulin resistance was calculated by the modified homeostasis model assessment of insulin resistance (HOMA-IR) method using the following formula: HOMA-IR = fasting insulin (μU/mL) X plasma glucose (mg/dL)/405. HOMA-IR, originally developed by Matthews, has since been modified [19,20]. This index has been shown to be well correlated with the results of the euglycaemic-hyperinsulinemic clamp method of assessment of IR in type 2 DM patients.
Determination of the visceral and subcutaneous fat areas
The abdominal fat distribution in the subjects was determined by computed tomography (CT) with the subjects in the supine position, in accordance with a previously described procedure [21]. The subcutaneous fatty area (SFA) and intra-abdominal visceral fatty area (VFA) were measured at the level of the umbilicus using a standardized method, in terms of the CT number. In brief, a region of interest was defined in the subcutaneous fat layer by tracing its contour on each scan, and the attenuation range for fat tissue was measured in terms of the CT number (in Hounsfield units).
Histopathology
The degree of necroinflammatory activity and fibrosis in the liver biopsy specimens were scored semiquantitatively, as described by Scheur [22], by two hepatopathologists blinded to the clinical data. The portal or periportal and lobular inflammatory activities were scored from 0 to 4. Fibrosis was scored as follows: F0, no fibrosis; F1, enlarged fibrotic portal tracts; F2, periportal or portal-portal septa, but intact hepatic architecture; F3, architectural distortion, but no obvious cirrhosis; and F4, probable or definite cirrhosis. The degree of steatosis was assessed based on the percentage of hepatocytes containing macrovesicular fat deposits; the grading was conducted as follows: grade 0, no steatosis; grade 1, <33% of hepatocytes affected; grade 2, 33-66% of hepatocytes affected; grade 3, >66% of hepatocytes affected [23].
Statistical analysis
All the data were expressed as mean ± SD, unless otherwise indicated. The relationship between any two variables was analysed by standard correlation analysis conducted using the StatView software, version 5.0 (SAS, Cary, NC, USA). The relationships between the quantity of HCV-RNA, VFA, SFA and other relevant covariates were examined by multiple regression analysis and determination of the standardized correlation coefficients. P-values <0.05 were considered to be significant.
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