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16300Fw: NATAP: HCV Causes Insulin Resistance-SVR Improves Insulin Resistance

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  • alleypat
    Feb 3, 2007
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      From: nataphcv@...
      To: nataphcv@... ; nataphcvhiv@... ; natapdoctors@... ; natapindustry@...
      Sent: Thursday, February 01, 2007 9:31 AM
      Subject: NATAP: HCV Causes Insulin Resistance-SVR Improves Insulin Resistance


      NATAP http://natap.org/
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      Clearance of HCV Improves Insulin Resistance, Beta-Cell Function, and Hepatic Expression of Insulin Receptor Substrate 1 and 2

      The American Journal of Gastroenterology Feb 1, 2007 (OnlineEarly Articles).

      Takumi Kawaguchi, M.D., Ph.D.1,21Department of Digestive Disease Information and Research2Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan,
      1Department of Digestive Disease Information and Research, 2Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan

      â?oâ?¦.Chronic hepatitis C virus (HCV) infection is associated with a greater risk for the development of insulin resistanceâ?¦..In patients with HCV infection, insulin resistance is involved in progression of hepatic fibrosis (3), the development of hepatocellular carcinomaâ?¦. The present study demonstrates that clearance of HCV improves HOMA-IR, HOMA-%B, and hepatic expression of IRS1/2. These findings indicate that HCV itself is involved in the development of insulin resistanceâ?¦..Improved HOMA-IR was only seen in sustained responders and HOMA-IR remained unchanged in nonresponders, despite a decrease in BMI after antiviral therapyâ?¦.findings suggest that HCV itself causes insulin resistanceâ?¦..Interferon is known to induce insulin resistance. However, our results showed that interferon leads to a reduction in insulin resistance in sustained respondersâ?¦.Even in nonresponders or relapsers, interferon did not worsen insulin resistance. Interferon-induced insulin resistance is observed only in the early phase of treatment (27). Indeed, after 3 months of treatment, interferon-induced insulin resistance disappearsâ?¦.â?

      ABSTRACT
      OBJECTIVES: Hepatitis C virus (HCV) infection is linked to greater insulin resistance. Although HCV itself is a candidate for the development of insulin resistance, the effects of antiviral treatment on impaired glucose metabolism remain unclear. The aim of this study is to examine the effects of clearance of HCV on insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate (IRS)1/2, central molecules for insulin signaling.

      METHODS: We analyzed 89 biopsy-proven patients with chronic HCV infection. Patients received interferon-α or interferon-α plus ribavirin for 6 months and were classified into three groups at 6 months after the conclusion of antiviral therapy according to their response to antiviral therapy: sustained responders (N = 29), relapsers (N = 12), and nonresponders (N = 48). Insulin resistance and beta-cell function were assessed by the homeostasis model assessment method (HOMA-IR and HOMA-%B, respectively). Hepatic expression of IRS1/2 was evaluated by immunoblotting and immunostaining in 14 sustained responders.

      RESULTS: In nonresponders and relapsers, there were no significant changes in HOMA-IR and HOMA-%B values after antiviral therapy. On the other hand, in sustained responders, HOMA-IR values significantly decreased to 1.7 ± 0.8 from 3.1 ± 1.1 (P < 0.05) after antiviral therapy. Similarly, HOMA-%B values significantly decreased to 90.6 ± 10.0 from 113.7 ± 15.3 (P < 0.05). Immunoblotting showed a threefold increase in IRS1/2 expression after clearance of HCV. Immunostaining revealed that greater IRS1/2 expression was seen in hepatocytes.

      CONCLUSIONS: We showed that clearance of HCV improves insulin resistance, beta-cell function, and hepatic IRS1/2 expression.

      INTRODUCTION
      Chronic hepatitis C virus (HCV) infection is associated with a greater risk for the development of insulin resistance (1). Greater insulin resistance is more prevalent among patients with HCV infection compared with those with other liver diseases and with the general population (2). In patients with HCV infection, insulin resistance is involved in progression of hepatic fibrosis (3), the development of hepatocellular carcinoma (4, 5), extrahepatic manifestations (6), and prognosis (7). Thus, insulin resistance plays a crucial role in patients with HCV infection.

      Insulin resistance can be caused by many factors. In general, obesity, inflammation, and various kinds of metabolic disorders are common factors for the development of insulin resistance. Similarly, body mass index (BMI), serum tumor necrosis factor-α (TNF-α) and hepatic iron concentrations, and hepatic steatosis are reported to be possible causative factors for the development of insulin resistance in patients with HCV infection (8-11). In addition to these factors, HCV itself is also known to have a variety of biological effects (12).

      In HCV core transgenic mice, the development of insulin resistance is seen by 1 month of age, in the absence of either overt liver injury or excessive body weight gain (12, 13). Furthermore, even if liver function is restored by transplantation, postliver transplantation diabetes mellitus occurs more frequently among patients who undergo transplantation for HCV than for other conditions (14). Although precise mechanisms for HCV-associated insulin resistance have not been fully elucidated, we recently demonstrated the involvement of insulin receptor substrate 1 and 2 (IRS1/2), central molecules in insulin signaling. Downregulation of IRS1/2 is seen in livers from HCV core transgenic mice as well as in patients with HCV infection (15). Thus, HCV itself is a candidate risk factor for the development of insulin resistance.

      If HCV is a causal factor, then clearance of HCV might decrease insulin resistance just as histologic improvement of fibrosis and reduction in the risk of hepatocellular carcinoma are seen in patients with hepatitis C who have sustained response to interferon therapy (16, 17). The ability of antiviral therapy to improve glucose metabolism would support the notion that HCV causes insulin resistance in patients with HCV infection. Accordingly, we studied the effects of HCV clearance on insulin resistance, beta-cell function, and hepatic expression of IRS1/2.

      DISCUSSION
      The present study demonstrates that clearance of HCV improves HOMA-IR, HOMA-%B, and hepatic expression of IRS1/2. These findings indicate that HCV itself is involved in the development of insulin resistance.

      Insulin resistance can be caused by many factors. Obesity is a common factor for the development of insulin resistance (24). Although greater insulin resistance was seen in patients with chronic hepatitis C, BMI values were within normal limits in this study. Improved HOMA-IR was only seen in sustained responders and HOMA-IR remained unchanged in nonresponders, despite a decrease in BMI after antiviral therapy. In an epidemiologic study, Bahtiyar et al. reported that obesity is not associated with the development of insulin resistance in patients with HCV infection (25). In addition, the development of insulin resistance is seen by 1 month of age, in the absence of either overt liver injury or excessive body weight gain in HCV core transgenic mice (13) and serum HCV core protein levels are associated with HOMA-IR values in patients with chronic hepatitis C (15). Moreover, a significant increase in the incidence of diabetes was seen in subjects with high titers of HCV core compared with subjects with low titers of HCV core or anti-HCV-negative subjects at the population level during 7 yr of follow-up (26). Taken together, these findings suggest that HCV itself causes insulin resistance.

      Interferon is known to induce insulin resistance. However, our results showed that interferon leads to a reduction in insulin resistance in sustained responders. Even in nonresponders or relapsers, interferon did not worsen insulin resistance. Interferon-induced insulin resistance is observed only in the early phase of treatment (27). Indeed, after 3 months of treatment, interferon-induced insulin resistance disappears (28). Romero-Gomez et al. reported that improved insulin resistance during and after interferon therapy is correlated with a positive response to antiviral therapy (29), which is in good agreement with our findings. These finding also suggest the involvement of HCV in the development of insulin resistance.

      Pancreatic beta-cells play a crucial role in maintaining glucose homeostasis. Although HCV infects not only liver but also pancreas (30), our results demonstrated that beta-cell function, especially the ability to secrete insulin, was preserved in patients with chronic hepatitis C. Because HOMA-%B was significantly decreased after antiviral therapy in sustained responders, increase in HOMA-%B seems to be an adaptation against greater insulin resistance. On the other hand, Narita et al. reported that beta-cell function is significantly decreased in patients with HCV infection (31). Although the reasons for this discrepancy are not clear, it could be explained by following reasons: First, BMI in the previous study is higher than that in our study. Second, patients who consumed alcohol were enrolled in the previous study, while we excluded the patients who had >80 g/day of alcohol. Obesity and alcohol consumption lead to a decrease in early-phase insulin secretion (32, 33). In addition, HCV core-transgenic mice exhibited a significant increase in early-phase insulin secretion compared with control mice (13). Thus, dysfunction of beta-cells does not seem to be responsible for HCV-associated insulin resistance.

      TNF-α is a causative factor for greater insulin resistance. However, there was no significant difference in serum TNF-α level between HCV patients with insulin resistance and without insulin resistance (34). Impairment of insulin receptor can cause insulin resistance. However, there was no significant change in hepatic insulin receptor between controls and HCV core-transgenic mice (15). Recently, we identified a molecular mechanism for HCV-associated insulin resistance. HCV core downregulates hepatic expression of IRS1/2 (15). Because IRS1 and IRS2 are central molecules in intracellular insulin signaling, downregulation of these molecules should decrease downstream insulin effects such as glucose uptake, thereby contributing to insulin resistance. In this study, we first demonstrated increases in hepatic expression of IRS1/2 after antiviral therapy in sustained responders. These findings support our proposed molecular mechanism that HCV directly downregulates hepatic expression of IRS1/2.

      In conclusion, we showed that clearance of HCV improves HOMA-IR, HOMA-%B, and hepatic expression of IRS1/2. These findings indicate that HCV itself is involved in the development of insulin resistance in patients with HCV infection.

      RESULTS

      Characteristics of the Patients
      Characteristics of the patients before antiviral therapy are summarized in Table 1. There was no significant difference in age or sex distribution among the groups. In sustained responders, higher infection rates of genotype 2 (62.0%) were seen compared with nonresponders (12.5%) or relapsers (16.7%). Although HCV viral load, hepatic fibrosis, and HOMA-IR were lower in sustained responders, BMI and hepatic necroinflammatory activity were not significantly different among the groups.

      Changes in BMI, Insulin Resistance, and Beta-Cell Function After Antiviral Therapy
      Changes in BMI, insulin resistance, and beta-cell function after antiviral therapy are summarized in Figure 1. In nonresponders (N = 48), BMI significantly decreased to 21.7 ± 1.6 kg/m2 from 22.7 ± 2.3 kg/m2 (P < 0.01) at the end of follow-up. However, there were no significant changes in HOMA-IR and HOMA-%B values at the end of follow-up compared with those before antiviral therapy (HOMA-IR 4.0 ± 1.7 vs 3.6 ± 1.2, P = 0.11, HOMA-%B 120.0 ± 26.1 vs 112.4 ± 24.1, P = 0.09) (Fig. 1A). In relapsers (N = 12) no significant differences were seen in BMI (21.8 ± 1.7 kg/m2 vs 22.1 ± 1.6 kg/m2, P = 0.70), HOMA-IR values (3.7 ± 1.2 vs 3.6 ± 1.2, P = 0.69), and HOMA-%B values (121.5 ± 13.3 vs 117.4 ± 17.4, P = 0.24) at the end of follow-up compared with those before antiviral therapy (Fig. 1B). In sustained responders (N = 29), there was no significant difference in BMI at the end of follow-up (22.6 ± 1.6 kg/m2 vs 21.9 ± 1.9 kg/m2, P = 0.07). On the other hand, HOMA-IR values significantly decreased to 2.2 ± 0.7 from 3.1 ± 1.0 (P < 0.01) by the end of follow-up. Similarly, HOMA-%B values significantly decreased to 92.6 ± 14.0 from 113.7 ± 21.3 (P < 0.01) (Fig. 1C).

      Figure 1. BMI, HOMA-IR, and HOMA-%B before and after antiviral therapy in nonresponders (N = 48; A), relapsers (N = 12; B), and sustained responders (N = 29; C). Data were obtained before antiviral therapy and 6 months after its conclusion. Data are expressed as mean ± SD. *P < 0.01. N.S., not significant.



      Changes in Hepatic Expression of IRS1/2 in Sustained Responders
      Immunoblotting demonstrated a significant increase in expression of IRS1/2 after antiviral therapy in livers from sustained responders (Fig. 2A). After antiviral therapy, mean IRS1 and IRS2 intensities showed a two- and threefold increase, respectively, compared with intensities before antiviral therapy (Table 2). In immunostaining, IRS1 occurred mainly in lymphocytes (Fig. 2B, left upper panel) before antiviral therapy, but occurred in hepatocytes after antiviral therapy (Fig. 2B, right upper panel). On the other hand, IRS2 occurred in hepatocytes both before and after antiviral therapy (Fig. 2B, lower panels). After antiviral therapy, expression of IRS2 was upregulated mainly in periportal hepatocytes (Fig. 2B, right lower panel).

      MATERIALS AND METHODS
      Materials
      All reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise indicated.

      Patients
      We analyzed 89 patients with HCV infection. The diagnosis was based on elevated serum aminotransferase level, histological examination, consistent detection of anti-HCV, and HCV-RNA. Patients who coincided with other causes of liver disease such as chronic hepatitis B, autoimmune hepatitis, or alcoholic liver disease (greater than 80 g alcohol per day for at least 1-month duration prior to the onset of illness) were excluded, as were those who had been taking corticosteroids or those with a history of, or evidence of, pancreatitis or a pancreatic tumor. Clinical data collected before antiviral therapy included age, sex, and alcohol use. BMI was calculated as body weight in kilograms divided by the square of height in meters (kg/m2). Informed consent for participation in the study was obtained from each subject. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in prior approval by the Ethics Committee of the Kurume University School of Medicine. None of the subjects was institutionalized.

      Laboratory Determinations
      Venous blood samples were taken in the morning after a 12-h overnight fast. Plasma glucose, serum aspartate aminotransferase, alanine aminotransferase, albumin, total bilirubin, and immunoreactive insulin (IRI) levels were measured by using standard clinical methods (Department of Clinical Laboratory, Kurume University Hospital). Beta-cell function and insulin resistance were calculated on the basis of fasting levels of plasma glucose and IRI, according to the homeostasis model assessment (HOMA) method (18). The formulas for the HOMA model are as follows: beta-cell function (HOMA-%B) = fasting IRI (μU/mL) Ã- 360/(fasting glucose (mg/dL) - 63); insulin resistance (HOMA-IR) = fasting glucose (mg/dL) Ã- fasting IRI (μU/mL)/405. HCV genotyping was performed according to Okamoto's method (19) and genotypes were classified according to Simmonds's classification system (20). An Amplicor-HCV-Monitor 1.0 (Roche Diagnostics K.K., Tokyo, Japan) was used to quantify HCV-RNA levels.

      Liver Biopsy
      Liver tissue was obtained by percutaneous ultrasound image-guided liver biopsy. The biopsies were performed by two staff gastroenterologists using a Pro-Magâ"¢ Biopsy Needle (Medical Device Technologies Inc., Gainesville, FL), which has a biopsy specimen notch of 20.00 mm in width and 2.05 mm in diameter. More than 95% (vol/vol) of liver tissue was used for histological and immunostaining analyses. Less than 5% (vol/vol) of liver tissue was homogenized and 80 μg of protein was used for immunoblotting analysis.

      Histological Data
      For each patient, a liver biopsy specimen was fixed in 10% formalin buffer and stained with hematoxylin-eosin. Liver biopsy specimens were evaluated by a single, experienced pathologist who was unaware of the patients' clinical and laboratory data. The specimens were scored according to the METAVIR scoring system (21), which is suited for evaluation of chronic hepatitis C.

      Treatment Outcome
      All patients were treated with 3 to 10 million U of interferon-α (interferon-α2a, Nippon Roche K.K., Tokyo, Japan; interferon-α2b, Schering-Plough K.K., Osaka, Japan; or natural interferon-α, Dainippon Sumitomo Pharma Co., Osaka, Japan) by subcutaneous injection three times per week, or 6 to 10 million U of interferon-α2b plus ribavirin (600 to 1,000 mg daily, Schering-Plough Co) for 6 months. Patients were followed up until 6 months after the conclusion of antiviral therapy and classified into three groups: sustained responders (N = 29), who had undetectable serum HCV-RNA; relapsers (N = 12), who had undetectable HCV-RNA at the end of antiviral therapy but HCV-RNA relapse during follow-up; and nonresponders (N = 48), who had detectable HCV-RNA during and after treatment.

      Immunoblotting
      Liver tissue was homogenized on ice in 1 mmol/L NaHCO3 containing protease inhibitors, stored at -80°C as previously described (22, 23). Equal amounts of protein (40 μg) from liver homogenates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5% acrylamide gel. The resolved proteins were transferred electrophoretically onto polyvinylidene difluoride membranes (Amersham International, Buckinghamshire, UK). The membranes were incubated with an antihuman IRS1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or an antihuman IRS2 polyclonal antibody (Santa Cruz Biotechnology), and were subsequently incubated with an HRP-conjugated goat antirabbit IgG (Amersham International). The membranes were then incubated with chemiluminescence reagents (ECL kit, Amersham International) and immediately exposed on radiograph film. Immunoblotting intensities were determined using NIH-Image J (developed at the National Institutes of Health and available from the Internet by an anonymous FTP from http://rsb.info.nih.gov/ij/download.html) as previously described (22, 23).

      Immunohistochemistry
      In 14 sustained responders, liver biopsy was performed before and after conclusion of antiviral therapy. Paraffin-embedded liver sections from patients with HCV infection were deparaffinized and subjected to immunohistochemical staining using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with an antihuman IRS1 polyclonal antibody (Santa Cruz Biotechnology) or an antihuman IRS2 polyclonal antibody (Santa Cruz Biotechnology), and developed with 3,3'-diaminobenzidine (DAB). The primary antibodies for IRS1/2 were used at a 1:100 dilution. The specificity of IRS1/2 staining was confirmed by immunization using an excess amount of the N-terminal peptide of IRS1/2.

      Statistical Analysis
      All data are expressed as mean ± SD. The Wilcoxon's single-rank test was employed for analysis of paired samples. Statistical comparisons among multiple groups were performed by analysis of variance followed by Scheffe's post hoc test. P values < 0.05 were considered significant.



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