Risk factors for diabetes mellitus and early insulin resistance in chronic hepatitis C

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1 Journal of Hepatology 35 (2001) 279±283 Risk factors for diabetes mellitus and early insulin resistance in chronic hepatitis C Jean-Michel Petit 1, *, Jean-Baptiste Bour 3, Catherine Galland-Jos 1, Anne Minello 2, Bruno Verges 1, Michel Guiguet 4, Jean-Marcel Brun 1, Patrick Hillon 2 1 Service de DiabeÂtologie et Endocrinologie, CHU du Bocage, 21000, Dijon, France 2 Service d'heâpatologie, CHU du Bocage, 21000, Dijon, France 3 Laboratoire de Virologie, CHU du Bocage, 21000, Dijon, France 4 Laboratoire de Biochimie, CHU du Bocage, 21000, Dijon, France Background/Aims: Our aims were to investigate the host and viral speci c factors associated with diabetes mellitus (DM) and insulin resistance in chronic hepatitis C patients. Methods: One hundred and three hepatitis C virus (HCV)-infected were studied to assess the effects of HCV genotype, hepatic iron content, steatosis, hepatic brosis, body mass index (BMI) and family history of DM on the occurrence of DM. Insulin resistance (HOMA IR) was studied in 81 non-diabetic patients to determine the mechanism associated with insulin resistance in this subgroup. Results: Sixteen of the 123 were diabetic (13.0%). The variables predictive of DM were METAVIR brosis score 4 (OR, 13.16; P ˆ 0.012), family history of diabetes (OR, 16.2; P ˆ ), BMI (OR, 1.37; P ˆ 0.017) and age (OR, 1.09; P ˆ 0.002). In non-diabetic HCV-infected patients, HOMA-IR of METAVIR brosis score 0 and 1 patients were signi cantly different than score 2 and score 3/4 patients. Conclusions: Our ndings indicate that older age, obesity, severe liver brosis and family history of diabetes help identify those HCV patients who might have potential risk factors for development of DM. We observed that insulin resistance in non-diabetic HCV-infected patients was related to grading of liver brosis, and occurs already at an early stage in the course of HCV infection. q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C; Liver; Diabetes mellitus; Insulin resistance; Body mass index; Obesity; Cirrhosis; Steatosis; Iron histological grading 1. Introduction A link between chronic hepatitis C virus infection (HCV) and type 2 diabetes mellitus has recently been suggested [1± 9]. Several studies have reported a higher prevalence of hepatitis C (HCV) in diabetic patients (11±28%), compared with control groups [1,2]. Others have found a higher prevalence of diabetes mellitus in-patients with chronic HCV infection [3,4,7,9]. The prevalence of diabetes in HCV infection is higher than in other chronic liver diseases, Received 22 November 2000; received in revised form 26 March 2001; accepted 23 April 2001 * Corresponding author. Tel.: ; fax: address: jean-michel.petit@chu-dijon.fr (J.-M. Petit). including hepatitis B [3,7]. This higher prevalence of diabetes mellitus (DM) in HCV patients is not related exclusively to cirrhosis. Non-cirrhotic patients with chronic HCV have an increased prevalence of type 2 diabetes compared to non-cirrhotic patients with chronic hepatitis B virus [8]. The pathogenesis of diabetes in patients with HCV is not well understood but an increase in fat or iron deposition in the liver is common in patients with HCV [10,11]. Insulin resistance related to excess liver fat, or excess iron deposition may have a role and has not been investigated. It has been suggested that liver fat may contribute to insulin resistance in HCV infected patient [12]. Similarly, the relationship between iron and the development of diabetes mellitus is well known [13]. An increased of iron store, even in the /01/$20.00 q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S (01)00143-X

2 280 J.-M. Petit et al. / Journal of Hepatology 35 (2001) 279±283 range not considered to be associated with haemochromatosis, contributes to the development of NIDDM [13]. The purpose of this study was to investigate the host and viral speci c factors associated with the development of diabetes mellitus and insulin resistance in patients with chronic hepatitis C. The study comprised two components: (i) a study of 123 consecutive patients with untreated chronic HCV to assess the effects of HCV genotype, viral load, hepatic iron content, steatosis, hepatic brosis, body mass index (BMI) and family history of diabetes mellitus on the occurrence of diabetes mellitus; (ii) an investigation of insulin resistance (HOMA IR) and b-cell function (HOMA b-cell) evaluated by the HOMA model assessment in nondiabetic patients to determine the factors associated with insulin resistance in this group. 2. Patients and methods 2.1. Patients One-hundred and twenty-three untreated chronic hepatitis C patients were studied from January 1996 to December1999. All of them were hospitalised for liver biopsy. The demographic data collected at the time of liver biopsy included age, sex, alcohol use and family history of diabetes. BMI was calculated as body weight in kilograms divided by the square of height in meters (kg/m 2 ). Information regarding average alcohol intake was assessed by interview. The alcohol intake was recorded in grams per day. Patients consuming more than 60 g per day of alcohol were considered as excessive drinker. Inclusion criteria were the presence of chronic hepatitis which was con rmed by a liver biopsy Laboratory determinations Venous blood samples were taken in the morning after 12-h overnight fasting. Plasma glucose concentration was measured by a glucose oxidase method on a Vitros 750 analyser (Johnson & Johnson Clinical Diagnostics, Rochester, NY, USA). Insulin was measured by monoclonal immunoradiometric assay (CIS Bio International, Gif sur Yvette, France). Diabetes was diagnosed using the 1997 American Diabetes Association fasting criteria (impaired fasting glucose; 6.1±6.9 mmol/l and diabetes;.7 mmol/l). The index of insulin resistance was calculated on the basis of fasting values of plasma glucose and insulin, according to the homeostasis model assessment method [14]. The formulas for the HOMA model are as follows: Insulin resistance (HOMA IR) ˆ fasting insulin (mui/l) fasting glucose (mmol/)/22.5 b-cell function (HOMA b-cell) ˆ 20 fasting insulin (mui/l)/fasting glucose (mmol/l) Virology Serological testing for anti-hcv was carried out using a commercial microparticle enzyme immunoassay (Axsym HCV version 3.0, Abbott laboratories, Chicago, IL) according to the manufacturer's instructions. All patients were tested for ARN of HCV by RT-PCR (Amplicor HCV, Roche) for ARN detection followed by reverse hybridization (Inno-lipa HCVII, Innogenetics, Swigdrecht, Belgium) for genotyping Histological data Histological iron grading was performed using 0 to 4 1 score as previously described on Perl's stains [15]. Liver brosis was evaluated using the METAVIR scoring systems. Steatosis was assessed according to semiquantitative intensity (grade 0,,5%; grade 1, 5±25%; grade 2, 25±50%; grade 3,.50%) Statistics Results were expressed as means ^ SD. Comparisons between groups were made using the Student's test or the Mann±Whitney U test for continuous variables and the x 2 or Fisher exact probability test for categorical data. Comparisons between different groups were performed using analysis of variance (ANOVA). HOMA-IR and HOMA b-cell were analyzed with values log10 transformed to improve skewness before t-test analysis. Statistical correlations were determined by the non-parametric Spearman test. Probability levels less than 0.05 were considered signi cant. A multivariate analysis based on a logistic regression study was used to establish associations between patients with diabetes mellitus and a series of preselected items including epidemiologic and histological data. 3. Results 3.1. Patient demographics and laboratory evaluation. The main clinical and laboratory data are summarized in Table 1. The mean age was 44.2 ^ 12 years (range, 20±77 years); 79 (64.2%) patients were male. The mean BMI was 23.5 ^ 3.36 (range, 17.3±36.5 kg/m 2 ), 16 (13.0%) patients had diabetes mellitus. Diabetes was managed mostly by diet alone (n ˆ 8) or oral hypoglycemic agents (n ˆ 7) and in only one patient by insulin. The diagnosis of diabetes mellitus was new in four patients. The mean of duration of overt diabetes mellitus was 9.6 ^ 6.5 years in the group of patients with known disease Histological ndings Steatosis was present in 66 patients (53.6%), 43 with grade 1 (34.9%) and 23 with grade 2 (18.6%). Fibrosis grade 0±1 was present in 37 patients (30.0%), grade 2 in 60 patients (48.7%) and grade 3±4 in 25 patients (20.3%). There was a signi cant effect of steatosis on severity of brosis. Indeed, among the 57 patient without steatosis, 20 (35.0%) had grade 0±1 brosis, 32 (56.1%) had grade 2 brosis, and 5 (8.7%) had grade 3±4 brosis, when among Table 1 Clinical and epidemiological characteristics of 123 HCV patients Mean/number Range n 123 Age 44.2 ^ 12 20±77 Sex ratio M/F 79/44 BMI 23.5 ^ ±36.5 AST (U/l) 80.1 ^ 55 23±328 ALT (U/l) ^ 81 17±599 Glycemia (g/l) 1.01 ^ ±3.45 HCV genotype 1 57 (52.2%) 2 10 (9.17%) 3 37 (33.94%) Viral load (log) 5.46 ^ ±6.73

3 J.-M. Petit et al. / Journal of Hepatology 35 (2001) 279± Table 2 Clinical and epidemiological characteristics of HCV patients with and without diabetes mellitus Diabetic Non-diabetic P n Age ^ ^ Sex ratio M/F 10/6 (62.5%/37.5%) 69/38 (64.4%/35.5%) 0.90 BMI ^ ^ Alcohol abuse 4 (25%) 22 (20.5%) 0.93 Family history of diabetes 8/8 (50%/50%) 15/92 (14.0%/85.9%) AST (U/l) 97.1 ^ ^ ALT (U/l) ^ ^ Glycemia (g/l) 1.76 ^ ^ HCV genotype 1 11/15 (73.3%) 46/94 (48.9%) 2 1/15 (6.6%) 9/94 (9.5%) 3 3/15 (20%) 34/94 (36.1%) 0.33 Viral load (log) 5.55 ^ ^ the 66 patients with steatosis, 18 (27.2%) had grade 0±1 brosis, 30 (45.4%) had grade 2 brosis, and 18 (27.2%) had grade 3±4 brosis. The difference for severity of brosis between the two groups of patients with and without steatosis was signi cant (P ˆ 0:03). A signi cant correlation was found between BMI and steatosis graded by percentage of cells with fatty changes (r ˆ 0:20, P ˆ 0:03) Risk factors for diabetes The mean age of patients with diabetes (57.0 ^ 14.9 years) was higher than the one of those without diabetes (42.3 ^ 11.4 years), (P, 0:001). There were no signi cant differences between the two groups for sex distribution, excessive alcohol intake or genotype distribution (P ˆ 0:33), (Table 2). Eight of 16 (50%) diabetic patients and 15 of 107 (14%) non-diabetic patients had a family history of diabetes (P, 0:001). Liver biopsy showed signi cantly more brosis activity in HCV diabetic patients than in non-diabetic patients (Table 3). Diabetic HCV patients had more steatosis than the non-diabetic patients (P ˆ 0:04), (Table 3). There was no signi cant association between histological iron grading and prevalence of DM (Table 3) Multivariate analysis The variables predictive of DM were METAVIR brosis score 4 (OR, 13.16; 95% CI, 1.75 ± 98.8; P ˆ 0:012), family history of diabetes (OR, 16.2; 95% CI; 2.71±97.5, P ˆ 0:0023), BMI (OR, 1.37; 95% CI, 1.05±1.78; P ˆ 0:017) and age. The results of the regression model are provided in Table 4. These variables explained 43% of the variability of the occurrence of diabetes mellitus Insulin sensitivity an b-cell function HOMA-IR and b-cell function were available for 81 of the 107 non-diabetic patients. HOMA-IR was correlated to BMI (r ˆ 0:43; P, 0:001). HOMA-IR was signi cantly different in patients with METAVIR brosis score 0 and 1 than in patients with score 2 and patients with score 3 and 4 (1.18 vs. 2.55; P, 0:01 and 1.18 vs. 2.67; respectively; P, 0:01). There was no correlation between HOMA-IR and on the one hand steatosis score or iron histological grading on the other hand. Multiple linear regression was used to examine factors that were associated with HOMA-IR. Independent variables that were considered included age, BMI, gender, family history of diabetes and brosis. BMI and brosis grade 2± 4 were independently related to HOMA-IR when family history of diabetes was border signi cant (Table 5). No signi cant association was found between b-cell function and brosis, steatosis or iron histological grading. 4. Discussion In our series, diabetes mellitus was present in 13.0% of chronic hepatitis C patients. Our ndings indicate that older Table 3 Histological parameters of HCV patients with and without diabetes mellitus Diabetic patients Non-diabetic patients Fibrosis activity 0±1 5 (31.2%) 32 (30.4%) (12.5%) 58 (54.2%) 3 5 (31.2%) 13 (12.3%) 4 4 (25.0%) 3 (2.8%) Steatosis 0 4 (25.0%) 53 (49.5%) (62.5%) 33 (30.8%) 2 2 (12.5%) 21 (19.6%) Histological iron grading 0 10 (62.5%) 65 (60%) (18.7%) 31 (29.5%) 2±4 3 (18.7%) 11 (10.4%) P

4 282 J.-M. Petit et al. / Journal of Hepatology 35 (2001) 279±283 Table 4 Multivariate analysis of association between diabetes mellitus and independent predictors in HCV patients. Variable OR 95% CI Signi cance Age ± BMI ± Family history of diabetes ± METAVIR brosis score ± R age, obesity, severe liver brosis and family history of diabetes help to identify the HCV patients who might have a potential risk for developing of diabetes mellitus. The pathogenesis of diabetes in patients with hepatitis C is not well understood. It has been suggested that the physiopathology of diabetes mellitus in HCV patients may result from steatosis [12]. Liver fat deposition may contribute to insulin resistance which may in turn lead to loss of the restraining effect of insulin on hepatocyte production of glucose, leading to diabetes [16]. Steatosis occurs more frequently in chronic hepatitis C than in chronic hepatitis B [10] and may explain the increased risk for development of DM in HCV patients. However, our ndings do not support this hypothesis. Indeed, in the multivariate analysis steatosis was not associated with diabetes mellitus and there was no correlation between insulin-resistance and steatosis score. The absence of association between steatosis and diabetes mellitus may be indeed surprising. However, the correlation between steatosis and BMI and the signi cant relationship between hepatic brosis and steatosis found in our study could be the explanation for the lack of signi cance of steatosis in multivariate analysis. Diabetes mellitus frequently complicates cirrhosis. We found that 50% of patients with liver cirrhosis had diabetes, consistent with previously published results [7,12]. Liver cirrhosis impairs insulin sensitivity and maximum cellular glucose disposal [17]. Insulin resistance is a primary event complicating cirrhosis but additional Beta cell secretory defect seems necessary for the development of diabetes [18]. A recent study in non-cirrhotic HCV-infected patients with normal tolerance revealed a strong relationship between insulin sensitivity and brosis score [19]. In our study, the development of diabetes in HCV patients was correlated to the severity of the liver disease, but we did Table 5 Multiple linear regression analysis independent association of anthropometric and histological factors with HOMA-IR Variables Coef cient SE t P Age Gender BMI Family history of diabetes Liver brosis (0±1 vs. 2±4) not observe a relationship between brosis and beta cell secretion. In non-diabetic subjects, we observed that HOMA-IR of patients with grading of brosis grade 0/1 was signi cantly different than patients with grade 2 and patients with grade 3/4 (1.18 vs. 2.55; P, 0:01 and 1.18 vs. 2.67; P, 0:01; respectively). Our data demonstrate an increase of insulin resistance for the patients with META- VIR brosis score 2 compared to patients with score 0 and 1. This suggests that insulin resistance occurs already in the early stages of the course of HCV infection, before the development of liver cirrhosis. However, cirrhosis and liver damage are not the only factors associated with the occurrence of DM. Recently, Knobler et al. studied a group of patients in whom cirrhosis was carefully excluded and found that patients with HCV infection have an increased prevalence of type 2 diabetes compared with patients with chronic hepatitis B infection (33% vs. 5.6%) [8]. Other factors than cirrhosis must be found to explain the increased prevalence of DM in HCVinfected patients. Some genotypes could be preferentially associated with extrahepatic syndromes during HCV infection. Liver steatosis score is signi cantly higher in chronic hepatitis C patients infected with HCV genotype 3 than in the case of infection with other viral genotypes [20]. Previous studies found an increased frequency of genotype 1b or 2a in the diabetic HCV group [3,8]. Our data do not con rm these ndings, hepatitis C virus distribution was similar among the patients with diabetes and those without. Serum iron indices are frequently increased in patients with chronic C viral hepatitis with considerable iron deposition in the liver [11]. An association between stores of iron and diabetes or insulin resistance syndrome was recently reported [13,21,22]. Iron deposition in the liver may cause insulin resistance by interfering with the ability of insulin to suppress hepatic glucose production [22]. The present study does not support this hypothesis with the lack of signi cant association between histological iron grading and prevalence of DM or insulin resistance index. In the same way, it has been suggested that iron deposition may lead to DM by a mechanism related to the direct damage of the b-cell. The lack of correlation between HOMA b-cell and iron histological grading in our study is not consistent with this hypothesis and suggests that iron does not play an important role in the development of DM in our study. Obesity could be a potential risk factor for brosis in HCV liver disease [23]. Obesity has an important role in the pathophysiology of steatosis in chronic HCV. Moreover, recently, Hourigan et al. reported that steatosis could be another potential risk factor for brosis in HCV infection [23]. Our study con rmed this association between steatosis and brosis, suggesting that in chronic HCV infection steatosis may play a role in disease progression. On the other hand, we know that diabetes or insulin resistance can also cause fatty liver and may be contribute to the severity of the hepatic brosis. Our ndings suggest that increasing BMI

5 J.-M. Petit et al. / Journal of Hepatology 35 (2001) 279± has also a role in the pathogenesis of DM in chronic HCV independently of liver brosis. Obesity and, more speci cally, the level of intra-abdominal fat, are positively associated with insulin resistance and DM [24]. These ndings may have therapeutic implications in the management of patients with chronic HCV. It appears necessary to encourage HCV-infected overweight patients to lose weight in order to decreased the risk of development of DM and to prevent the liver damage. Family history of diabetes is shown to be an important predictor for the development of DM in HCV-infected patients. In our study, patients with a family history of diabetes had a signi cantly higher prevalence of diabetes compared to those without (50 vs. 8%; P, 0:001). These results are comparable to those of Zein et al. [12]. We cannot exclude the direct destruction of beta-cells by the hepatitis C virus. However, the lack of correlation between HCV viral load and HOMA b-cell and the absence of association between HOMA b-cell and liver brosis do not support this hypothesis. In conclusion, the cause of DM or insulin resistance in HCV infected patients is not related to excess liver fat or excess iron deposition. We observed that insulin resistance occurs already in the early stages of HCV infection, before the development of liver cirrhosis. Our ndings indicate that older age, obesity, severe liver brosis and family history of diabetes help identify those HCV patients who might have potential risk factors for development of diabetes mellitus. This subgroup of patients with HCV infection should be greatly encouraged to make a concerted effort to lose weight in order to prevent the occurrence of diabetes mellitus. References [1] Simo R, Hernandez C, Genesca J, Jardi R, Mesa J. High prevalence of hepatitis C virus infection in diabetic patients. Diabetes Care 1996;19(9):998±1000. 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