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Hepatitis C virus directly associates with insulin resistance independent of the visceral fat area in nonobese and nondiabetic patients
 
 
  Journal of Viral Hepatitis
Volume 14 Issue 9 Page 600-607, September 2007
 
Atsushi Nakajima, Division of Gastroenterology, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
 
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.
 
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.
 
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).
 
Discussion
In this study, we limited our target population to patients with HCV infection not caused by the genotype 3, who were nonobese and nondiabetic and had no history of alcohol consumption. Multiple regression analysis showed that HCV infection was associated with IR in a dose-dependent manner, independent of the other factors considered. As the report by Allison et al. [4] of an association between HCV infection and type 2 diabetes, evidence has been accumulating of a possible association between these two conditions. Elucidation of the relationship between HCV and IR would be of great clinical relevance, because IR is known to promote liver fibrosis [24]. Earlier studies which reported increased fasting plasma insulin levels and reduced insulin sensitivity in HCV-infected subjects also included patients with moderate to severe hepatic fibrosis [25,26]. However, more recently, these findings have also been confirmed in HCV-infected patients with minimal or no liver fibrosis [27]. There is one case report of HCV being independently associated with the development of IR after liver transplantation [28]. It has also been reported that HCV infection contributes to the development of IR and increases the long-term risk of development of type 2 DM [27]. Moreover, type 2 diabetes has been reported to be associated with an increased risk of hepatocellular carcinoma [29]. Thus, the results of epidemiological studies have led to a strong suspicion of an association between HCV infection and type 2 diabetes. However, there are some difficulties in establishing a definite relationship between HCV infection and type 2 DM on the basis of epidemiological studies, as there are numerous factors in patients that can confound the verification of the existence of a definite relationship, such as obesity, ageing [14], established DM, heavy alcohol consumption, fatty liver and especially, advanced liver injury. None of the epidemiological studies conducted until now has analysed the relationship between HCV infection and the risk of IR independent of the influence of obesity, type 2 DM and alcohol consumption; therefore, the precise relationship between HCV and IR remains unclear. Moreover, the biological mechanism underlying the development of type 2 DM or IR in humans with HCV infection has not been clearly elucidated. On the other hand, animal studies using transgenic mice carrying the core gene of HCV have suggested that HCV-encoded proteins may alter insulin signalling and therefore, tyrosine phosphorylation of insulin receptor substrate (IRS)-1, to explain the impaired insulin sensitivity and glycaemia dysregulation observed in these animals [18]. Moreover, a strong association between IR and the VFA is well known in the non-HCV-infected population [12]. Therefore, we investigated the relationship between HCV infection, IR and VFA in HCV-infected patients without obesity, diabetes or history of alcohol consumption, in order to elucidate the relationship between the quantity of HCV and IR.
 
In this study, although the fasting plasma glucose levels were within normal limits in all the patients, a significant relationship was found between the quantity of HCV and the fasting plasma level of insulin, and between the quantity of HCV and the HOMA-IR, an indicator of insulin resistance (Fig. 1). Mangia et al. [30] reported that they found no association between HCV infection and DM in noncirrhotic patients and that the prevalence of DM in noncirrhotic patients was comparable with that in the general population. However, in their study glucose intolerance was evaluated based on the blood glucose levels, and only patients with levels >126 mg/dL were considered as having abnormal glucose metabolism. Because fasting glucose levels can be compensated by hyperinsulinaemia, cryptic changes in glucose metabolism should be evaluated by measuring the HOMA-IR. The HOMA model used in this study has been validated and widely used for determining the degree of IR in epidemiological studies. HOMA-IR accounts for approximately 65% of the variability in insulin sensitivity as assessed by the glucose clamp technique [31,32]. It seems to be as good a predictor of clamp-determined insulin sensitivity as the short insulin tolerance test [33].
 
Abdominal or central fat distribution was first formally recognized to be related to diabetes in 1947 by Vague [34]. Vague's anthropometric observations have since been confirmed in numerous epidemiologic studies [35]. Visceral adipose tissue is a major contributor to IR, and the association between IR and VFA is also well known [36,37]. In our study also, 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 (Fig. 2d). These results were consistent with those reported by Ruderman [36], indicating the reliability of our study. However, to our surprise, no significant correlation was observed in our HCV-infected patients between the visceral adipose tissue area and the HOMA-IR (Fig. 2c). Furthermore, we could not find any significant contribution of hepatic inflammation or fibrosis to the HOMA-IR either (Fig. 3). These results suggest that the IR in HCV-infected patients may be independent of hepatic inflammation or fibrosis.
 
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. Thus, eradication of hepatitis C virus is the ideal treatment for chronic hepatitis C. Recently, combined therapy of this disorder with peg-interferon alpha-2b and ribavirin was shown to elicit a sustained virological response, with a response rate of up to 50%, which represents a very satisfactory response rate to pharmacological treatment [39]. On the other hand, recent studies have demonstrated that IR plays a crucial role in the development of liver fibrosis [38]. Therefore, the results of our study suggest that among cases with failure of virus eradication, reducing the quantity of HCV-RNA may be beneficial for reducing liver fibrosis via promoting normalization of IR. 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.
 
In conclusion, this is the first report to demonstrate the HCV by itself may be associated with IR in a dose-dependent manner before the development of overt type 2 DM, and IR in patients with HCV infection is independent of the visceral adipose tissue area and the severity of hepatic inflammation or fibrosis. Further clinical studies are necessary to identify the exact pathogenetic roles of HCV in the progression of IR, type 2 DM and liver fibrosis.
 
 
 
 
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