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Insulin Resistance Plays a Significant Role in Liver Fibrosis in Chronic Hepatitis C and in the Response to Antiviral Therapy
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.....Our pilot study that excluded diabetic patients, suggests that insulin resistance plays an important role in hepatic fibrosis in HCV patients,..... In multivariable analyses, HOMA-IR remained significantly associated with fibrosis score, even after adjusting for all other variables associated with fibrosis stage in univariable analyses (age, fasting glucose, HbA1c, AST, ALT, BMI, and high alcohol consumption)..... higher fasting insulin levels, and higher HOMA-IR levels were all independently associated with a poorer virological response to therapy....
The American Journal of Gastroenterology
July 2005
R. D'Souza, C. A. Sabin1, and G. R. Foster
Hepatobiliary Group, Institute of Cellular and Molecular Science, Queen Mary's School of Medicine and Dentistry; and 1Department of Primary Care and Population Sciences, Royal Free and University College Medical School, London, United Kingdom
ABSTRACT
OBJECTIVES: To assess whether insulin resistance is associated with liver fibrosis in a group of patients with chronic hepatitis C virus (HCV) infection and whether there were any differences in insulin resistance between Asians and the indigenous Caucasian population. Secondly, to assess whether insulin resistance is associated with sustained virological response to antiviral therapy.
METHODS: We determined insulin resistance in 59 (30 Caucasians; 29 Asians) consecutive patients with HCV prior to starting antiviral therapy. Insulin resistance was measured using the homeostasis model assessment of insulin resistance (HOMA-IR). The relationship between insulin resistance and biochemical, virological, and histological data together with response to antiviral therapy was assessed.
RESULTS: In multivariable analyses, insulin resistance as measured using the HOMA-IR model correlated positively with the stage of fibrosis, with higher degrees of insulin resistance in those with greater degrees of fibrosis (p< 0.001). This significant relationship remained even after excluding cirrhotic patients, or after adjusting for other factors associated with fibrosis in univariable analyses. Insulin resistance was significantly higher in Asians than Caucasians (p= 0.004). Around half (55.6%) of patients completing a course of antiviral treatment had a sustained virological response. Multivariable logistic regression identified HCV genotype 3, lower fasting glucose levels, and lower aspartate transaminase (AST) levels as being associated with a higher odds of a sustained virological response. After adjusting for these variables, Asian ethnicity, higher fasting insulin levels, and higher HOMA-IR levels were all associated with a poorer virological response to therapy.
CONCLUSIONS: Insulin resistance contributes to liver fibrosis in chronic HCV infection; this relationship is not genotypic specific. Asian patients had higher insulin resistance than Caucasians. Insulin resistance is also an important predictor of sustained response to antiviral therapy.
RESULTS
Anthropometric, Biochemical, and Histological Findings by Ethnicity
The median age of the 59 patients was 47 (range: 34-60) yr; 49 (83.1%) were male, and the median BMI was 26 (23-30) kg/m2.
Approximately equal numbers of Caucasian (n = 30) and Asian (n = 29) patients were included in the study. Neither sex, age, BMI, alcohol consumption, liver function tests, fibrosis score, portal and lobular scores nor grade of steatosis differed significantly between the two groups. A similar number of Asian patients were infected with HCV genotype 1/2 and 3, whereas Caucasian patients were more likely to be infected with genotype 1/2 (p= 0.02). Both fasting insulin and insulin resistance as measured using the HOMA model were significantly higher in Asians than in Caucasians (p= 0.004 and p= 0.004, respectively). Glycosylated hemoglobin levels were also higher in Asians than in Caucasians (p= 0.03). The characteristics of those infected with genotype 1/2 or genotype 3 were similar (data not shown).
Insulin Resistance (HOMA-IR) and Anthropometric, Biochemical, and Histological Findings
There was a strong positive correlation between the patient's BMI and the level of HOMA-IR (Spearman's r = 0.54; p= 0.0001). After stratifying by ethnicity, this correlation was slightly stronger in Caucasian (0.58; p= 0.0007) than in Asian (0.49; p= 0.007) patients. In addition, the level of HOMA-IR was positively correlated with fasting insulin (Spearman's r = 0.94; p= 0.0001) and glucose (0.38; p= 0.003) levels as well as with HbA1c (0.29; p= 0.05), AST (0.33; p= 0.02), and ALT (0.33; p= 0.01). HOMA-IR levels were also higher in those with high (median 3.51; range: 0.98-5.83) alcohol consumption, compared to those with mild/moderate (2.45; 1.10-5.51), or no (2.91; 1.25-5.16) consumption (p= 0.07, Kruskal-Wallis test). In addition, levels were higher in those with portal scores of 2 (4.13; 2.30-5.23) compared to those with scores of 1 (2.81; 0.98-5.83, p= 0.03). No significant relationships were seen with age, sex, HCV genotype, lobular score, steatosis grade, or the AST:ALT ratio.
Fibrosis and Anthropometric, Biochemical, and Histological Findings
Age, BMI, fasting insulin and glucose, HOMA-IR, glycosylated hemoglobin, AST, and ALT were all positively correlated with fibrosis score in univariable analyses. Excluding cirrhotic patients (fibrosis score 5 or 6) from the analysis did not change the association between HOMA-IR and fibrosis stage (Spearman's correlation after excluding these patients: 0.68; p= 0.0001). There were no significant relationships, however, between the fibrosis score and either sex, HCV genotype, steatosis, alcohol consumption, fasting glucose, or the AST:ALT ratio. In multivariable analyses, HOMA-IR remained significantly associated with fibrosis score, even after adjusting for all other variables associated with fibrosis stage in univariable analyses (age, fasting glucose, HbA1c, AST, ALT, BMI, and high alcohol consumption). In this multivariable model, a 1-unit increase in HOMA-IR was associated with an increase in the estimated fibrosis score of 0.87 (95% CI: 0.60-1.13; p= 0.001). Older age was the only other factor independently associated with a worse fibrosis stage in this multivariable model.
Factors Associated with Sustained Virological Response
Overall, 29 of 52 patients (55.8%) who completed a full course of antiviral treatment, experienced a sustained virological response. In univariable analyses, patients with a sustained virological response were less likely to be male (p= 0.03) and had lower fasting insulin (p= 0.01), fasting glucose (p= 0.0007), HOMA-IR (p= 0.0009), AST (p= 0.01), and ALT (p= 0.003) levels but higher AST:ALT ratios (p= 0.004). In addition, those with a sustained virological response tended to have lower BMIs (p= 0.005) and were more likely to be infected with HCV gnotype 3 (p= 0.05) than those without a sustained virological response. In multivariable logistic regression analyses (Table 4), three different models gave a very similar fit to the data. In each model, HCV genotype 3 was independently associated with an increased likelihood of sustained viral response (with odds ratios ranging from 9.9 to 29.2 depending on the model chosen). In contrast, higher fasting glucose levels and higher AST levels were both independently associated with a poorer virological response to therapy. After adjusting for these three variables, Asian ethnicity, higher fasting insulin levels, and higher HOMA-IR levels were all independently associated with a poorer virological response to therapy. However, due to the strong correlations among these variables, it was not possible to tease out the independent effects of these three variables.
Because of the strong relationship between virological response and HCV genotype 3, the analyses were repeated after excluding those with genotype 3. Although the power to consider factors associated with virological response was much reduced in this subgroup, the relationships among fasting glucose, Asian ethnicity, fasting insulin and HOMA-IR levels, and virological response remained significant.
DISCUSSION
Our pilot study that excluded diabetic patients, suggests that insulin resistance plays an important role in hepatic fibrosis in HCV patients, irrespective of viral genotype. This association even remained when patients with cirrhosis were excluded. Only insulin resistance and older age were independently associated with a worse fibrosis stage in multivariable analysis. Asian HCV-infected patients had significantly higher insulin resistance than Caucasians.
In the second part of the study we assessed the effects of insulin resistance on treatment response. Patients with HCV genotype 3 were independently associated with an increased likelihood of sustained virological response. Nonresponders to antiviral therapy had significantly higher insulin resistance than responders. In multivariable regression analysis Asian ethnicity, higher fasting insulin levels, and higher HOMA-IR levels were all independently associated with a poorer virological response to therapy.
Type II DM seems to be more common in HCV infection than in other liver disorders (2-5). This could be explained by the fact that patients with cirrhosis have increased insulin resistance, and consequently, a higher incidence of DM. However, in studies where cirrhotic patients have been excluded this association still exists (4).
Insulin resistance is a consistent finding in diabetic patients (14, 15). It is the best predictor for the development of DM preceding the latter by 10-20 yr (16, 17). Insulin resistance is increased in HCV infected nondiabetic patients as compared to healthy controls (5). Our pilot study is in agreement with other studies that suggest that insulin resistance may facilitate fibrogenesis in chronic HCV (5, 12, 13). We found no genotype-specific effects on insulin resistance, which is in concordance with another study (33).
The mechanisms for insulin resistance in chronic HCV infection are only just beginning to emerge. Studies from transgenic mice that specifically express the HCV core protein at high levels in hepatocytes show that hepatic insulin resistance can be induced solely by HCV core protein (24). This resulted in increased levels of tumor necrosis factor alpha (TNF-alpha), which prevented phosphorylation of the insulin receptor-insulin substrate 1 (IRS-1) pathway (24). This defect in the insulin receptor substrate-1 tyrosine phosphorylation has been found in HCV patients' liver biopsies but not in noninfected controls (25, 27, 28). These provocative data suggest that TNF-alpha-mediated alterations in IRS-1 signaling are relevant to hepatitis-C-induced insulin resistance. Some studies have ascribed the increased insulin resistance to be secondary to the increase of iron deposits such as ferritin in HCV patients (34, 35). Insulin resistance may cause fibrogenesis by directly stimulating hepatic stellate cells to proliferate (26) or by upregulation of connective growth factor, a cytokine involved in the pathogenesis of fibrosing liver diseases (29).
Despite the small sample size and heterogeneity of the study group, our study showed that increased insulin resistance is associated with the stage of fibrosis. An alternative explanation could be that worsening hepatic fibrosis impairs insulin degradation. We feel that this possibility has been excluded for two reasons. HOMA-IR is an independent predictor of fibrosis score after exclusion of cirrhotic patients, which is known to cause insulin resistance and impaired insulin clearance. Secondly, insulin resistance is higher in chronic hepatitis C than other chronic liver diseases (5).
In this study, Asian HCV-infected patients had significantly higher insulin resistance than Caucasians. This is an expected finding on account of the fact that BMI values for the diagnosis of obesity in the Asian population are lower (>22 kg/m2) than those for Caucasians (>25 kg/m2) (23). Indeed, all of the Asians in our study were classified as obese according to this definition compared to only 62% of Caucasians. This may partly account for any ethnicity-related differences seen in insulin resistance. A recent study suggests that hyperinsulinemia may be a factor responsible for the association between BMI and fibrosis in patients with HCV (33).
Patients with HCV genotype 3 were independently associated with an increased likelihood of sustained virological response. Two pivotal studies of pegylated interferon and ribavirin combination therapy have shown that HCV genotype 2 and 3 had an increased chance of SVR compared to genotype 1 (30, 31). Our study had very few patients with genotype 2 and the numbers were too small to analyze separately.
Nonresponders to antiviral therapy had significantly higher insulin resistance than responders. In multivariable regression analysis Asian ethnicity, higher fasting insulin levels, and higher HOMA-IR levels were all independently associated with a poorer virological response to therapy. Studies have shown that patients with obesity, steatosis, and insulin resistance may have reduced response rates (13, 36). Sustained virological response rates to antiviral therapy also differ among ethnic groups (37). The Asians in our group manifest the metabolic features of the insulin resistance syndrome at lower values of BMI than Caucasians. Therefore, it is expected that insulin resistance might mediate the differences in the response rate to antiviral therapy seen here.
These factors have previously been found to be important in fibrosis progression (13), and measures aimed at weight reduction prior to antiviral therapy may improve insulin sensitivity and the chance of response. Our study is only a small pilot study and as such, the results from our study should be interpreted cautiously. In particular, it was not possible to tease out the independent effects of Asian ethnicity, fasting insulin, or HOMA-IR levels when considering factors associated with response to therapy.
In summary, we have shown that liver fibrosis is independently associated with insulin resistance in HCV patients confirming other studies and highlighting the fact that progression of liver disease in chronic hepatitis C infection shares the same mechanisms involved in the progression of nonalcoholic fatty liver disease (NAFLD) from fatty liver to cirrhosis, via nonalcoholic steatohepatitis (NASH). This article shows that insulin resistance is also an important predictor of sustained virological response to antiviral therapy. Further studies on this subject are eagerly awaited.
INTRODUCTION
Chronic infection with the hepatitis C virus (HCV) affects over 170 million individuals worldwide (1). When compared to other liver conditions, chronic HCV infection is associated with an increased risk for the development of type 2 diabetes mellitus (DM) (2-5). Among patients in the Third National Health and Nutrition Examination Survey (1988-1994), the rate of type 2 DM was three times more likely in HCV-infected individuals aged 40 yr or older than in uninfected individuals (4); studies that have excluded cirrhotic patients confirm this association (5).
A clear biological relationship between HCV infection and type 2 DM is not obvious (2). Insulin resistance plays an important role in the development of type 2 DM and is the best predictor of DM, preceding it by 10-20 yr. There is some evidence that insulin resistance can contribute to fibrosis progression in chronic hepatitis C infection, although the mechanism is still not fully understood (6, 7).
The aim of this pilot study was two-fold. Firstly, to assess whether insulin resistance using the homeostasis model assessment (HOMA-IR) is associated with increased fibrosis in a group of patients with chronic HCV infection and whether there were any differences in insulin resistance between Asians and indigenous Caucasian population. Secondly, to assess whether insulin resistance plays a part in response to antiviral therapy.
METHODS
Patients and Protocol
This was an open-label study in which consecutive patients with chronic HCV requiring therapy received a full course of treatment with pegylated interferon 2alpha plus ribavirin.
Patients with chronic HCV with a liver biopsy (LB) confirming this diagnosis who were eligible for antiviral therapy were considered for inclusion in the study. Patients were required to have had a recent liver biopsy (within the last 6 months from starting date of the study) showing mild, moderate, or severe chronic hepatitis C. If patients had evidence of cirrhosis then only patients with compensated cirrhosis were accepted for entry into the study. Entry criteria included patients aged between 18 and 70 yr who were HCV antibody and HCV RNA positive. Patients with other causes of liver disease such as hepatitis B, HIV, autoimmune liver disease, Wilsons, or hemochromatosis were excluded. All patients had to be willing to use effective contraception during the duration of the study. Exclusion criteria included patients who were diabetic, pregnant, unable to take effective contraception during the trial, had an abnormal ECG, decompensated cirrhosis (Child-Pugh score of B or C), or any contraindication to combination treatment. Patients were diagnosed with diabetes mellitus according to the American Diabetes Association classification criteria (33) as follows: they had symptoms of diabetes together with a plasma glucose concentration more than or equal to 11.1 mmol/L, fasting plasma glucose more than or equal to 7.0 mmol/L 2 h postload, or glucose of more than or equal to 11.1 mmol/l during an oral glucose tolerance test.
The study took place at The Royal London Hospitals and was approved by the local ethics committee.
Characteristics such as body mass index (BMI), alcohol intake, ethnicity, and HCV genotype were recorded. Body mass index was calculated by dividing the weight (in kg) of the patient by the square of their height (in meters2). Alcohol intake was defined as zero (nondrinkers), mild/moderate (<14 units per week for females, <21 units per week for males), or heavy (>14 units per week for females, >21 units per week for males) (32).
Fifty-nine patients in total were recruited of whom 49 were male and 10 female. Their ages varied from 34 to 60 yr. Thirty patients were Caucasians and 29 patients were Asian.
Liver Biopsy
Each patient had a liver biopsy that was fixed, paraffin-embedded, and stained with hematoxylin and eosin (H&E), reticulin, PAS, Perls', Van Gieson, and Victoria Blue. Each biopsy was reviewed by one of the three senior histopathology consultants who specialize in gastrointestinal/liver pathology and then rescored by a second independent pathologist. Disagreements were resolved by discussion to reach a consensus score. The liver biopsies were scored using the Modified Ishak scoring system with fibrosis stage scored on a scale from 0 to 6 (8). The majority of patients (86.4%) had fibrosis scores of 1-4 on liver biopsy. Portal and lobular necroinflammatory grades were also recorded using the Modified Ishak scoring system (8). Steatosis was assessed as the percentage of hepatocytes containing macrovesicular fat droplets and was graded as no s teatosis, mild (<33% of hepatocytes affected), moderate (33-66% of hepatocytes affected), or severe (>66% of hepatocytes affected) steatosis.
Laboratory Investigations
Before starting antiviral treatment, patients were required to fast for 12 h and venous blood was then drawn for fasting plasma glucose, insulin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values and glycosylated hemoglobin (HbA1c).
Fasting plasma glucose, ALT, and AST were measured using Olympus AU 640 (Olympus UK Limited). Fasting insulin was measured using the DPC Immulite 2000 analyzer, Diagnostics Products Cooporation. HbA1c was analyzed by high-pressure liquid chromatography using the H1-Auto A1c, HA-8140 A Menarini Diagnostics.
The gold standard for in vivo measurement of insulin resistance is the euglycemic hyperinsulinemic clamp technique (9). As this technique can induce severe hypoglycemia, particularly in cirrhotic patients, we used the homeostasis model assessment (HOMA). This has been validated against insulin sensitivity measured directly with the euglycemic/hyperinsulinemic clamp technique in both diabetic and nondiabetic subjects (10, 11). Using HOMA, insulin resistance is calculated using the following equation:
The duration of the therapy was determined by the genotype of the infecting virus. Patients infected with HCV genotype 2 or 3 received 24 wk of therapy; patients infected with HCV genotype 1 received 48 wk of treatment, unless they chose to stop treatment prematurely. If patients were unable to tolerate one of the antiviral treatments then the dose of the pegylated interferon or ribavirin were reduced accordingly to see if this was tolerated better, before considering withdrawing treatment. If the qualitative PCR HCV RNA assay (Cobas Amplicor TM HCV Monitor [version 2.0], Roche Diagnostics) was negative 6 months after completion of treatment then the patient was considered to have had a sustained virological response to treatment. Although all patients started treatment, 7 (11.9%) did not tolerate the treatment and withdrew from treatment during the first 12 wk. In these patients we tried first to reduce the dose of the offending antiviral agent, to make it more tolerable to the patient. Nevertheless, they still did not tolerate treatment and withdrew. This left a total of 52 (88.1%) patients who completed treatment, comprising 27 Caucasians and 25 Asians.
Statistical Methods
Comparisons of factors in different subgroups of the study population were performed using chi2 or Fisher's exact tests (for categorical variables) or Mann-Whitney U-tests (for numerical variables). Correlations between pairs of numerical variables were performed using Spearman's rank correlation methods.
Factors independently associated with the fibrosis score were identified using multivariable linear regression analyses, by treating the fibrosis score as the numerical outcome variable in the analysis. Preliminary analyses of this and other similar datasets had suggested that although the fibrosis score is not strictly numerical (it is an ordinal variable taking values from 0 to 6), this assumption would be reasonable for the purposes of our analyses. Variables considered in the multivariable regression are those shown in Table 1.
Factors associated with sustained virological response were identified using chi2 tests, Fisher's exact tests, Mann-Whitney U-tests, or Kruskal-Wallis tests as appropriate. Those factors identified as being most strongly associated with sustained virological response in these analyses were progressively included in a multivariable logistic regression model to identify factors that were independently associated with response. Factors that were not independently associated with response were excluded from these models. Because of the strong correlation between some of the variables, it was not possible to separate the effects of three factors (Asian ethnicity, fasting insulin, and HOMA-IR levels) in these models. Therefore, three separate models that include each of these variables separately are reported to allow readers to judge for themselves the relative importance of each variable.
All analyses were performed using the Statistical Analysis System (SAS, SAS Institute, Cary, NC) version 8.02. All p-values are two-sided and were considered to be significant if they were less than 0.05.
Fibrosis and Anthropometric, Biochemical, and Histological Findings
Age, BMI, fasting insulin and glucose, HOMA-IR, glycosylated hemoglobin, AST, and ALT were all positively correlated with fibrosis score in univariable analyses. Excluding cirrhotic patients (fibrosis score 5 or 6) from the analysis did not change the association between HOMA-IR and fibrosis stage (Spearman's correlation after excluding these patients: 0.68; p= 0.0001). There were no significant relationships, however, between the fibrosis score and either sex, HCV genotype, steatosis, alcohol consumption, fasting glucose, or the AST:ALT ratio. In multivariable analyses, HOMA-IR remained significantly associated with fibrosis score, even after adjusting for all other variables associated with fibrosis stage in univariable analyses (age, fasting glucose, HbA1c, AST, ALT, BMI, and high alcohol consumption). In this multivariable model, a 1-unit increase in HOMA-IR was associated with an increase in the estimated fibrosis score of 0.87 (95% CI: 0.60-1.13; p= 0.001). Older age was the only other factor independently associated with a worse fibrosis stage in this multivariable model.
Factors Associated with Sustained Virological Response
Overall, 29 of 52 patients (55.8%) who completed a full course of antiviral treatment, experienced a sustained virological response. In univariable analyses, patients with a sustained virological response were less likely to be male (p= 0.03) and had lower fasting insulin (p= 0.01), fasting glucose (p= 0.0007), HOMA-IR (p= 0.0009), AST (p= 0.01), and ALT (p= 0.003) levels but higher AST:ALT ratios (p= 0.004). In addition, those with a sustained virological response tended to have lower BMIs (p= 0.005) and were more likely to be infected with HCV gnotype 3 (p= 0.05) than those without a sustained virological response. In multivariable logistic regression analyses (Table 4), three different models gave a very similar fit to the data. In each model, HCV genotype 3 was independently associated with an increased likelihood of sustained viral response (with odds ratios ranging from 9.9 to 29.2 depending on the model chosen). In contrast, higher fasting glucose levels and higher AST levels were both independently associated with a poorer virological response to therapy. After adjusting for these three variables, Asian ethnicity, higher fasting insulin levels, and higher HOMA-IR levels were all independently associated with a poorer virological response to therapy. However, due to the strong correlations among these variables, it was not possible to tease out the independent effects of these three variables.
Because of the strong relationship between virological response and HCV genotype 3, the analyses were repeated after excluding those with genotype 3. Although the power to consider factors associated with virological response was much reduced in this subgroup, the relationships among fasting glucose, Asian ethnicity, fasting insulin and HOMA-IR levels, and virological response remained significant.
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