Insulin resistance and liver fibrosis progression in patients with chronic hepatitis C virus infection

Introduction 

Hepatitis C virus (HCV) infection appears to be endemic in most parts of the world, with an estimated overall prevalence of 3% [1]. It is estimated that 15% of the Egyptian population is seropositive [2] and there is high seroprevalence of hepatitis C infection among risk groups in Egyptian people [3].

Chronic HCV infection is associated with a wide spectrum of liver histological lesions, ranging from mild chronic hepatitis to cirrhosis and hepatocellular carcinoma [4]. Chronic HCV infection is associated with an increased risk for the development of type 2 diabetes mellitus (DM) [5]. Thus, type 2 diabetes is more prevalent among patients with chronic HCV compared with those with other liver diseases and the general population, irrespective of the presence or absence of liver cirrhosis [6], [7], [8].

Insulin resistance plays a primary role in the development of type 2 diabetes [9]. Insulin resistance and progressive pancreatic β-cell dysfunction have been identified as the two fundamental features in the pathogenesis of type 2 DM [10].

An Egyptian study showed that the incidence of type 2 DM increased twofold in patients who had HCV infection compared with those who did not and reported that HCV-infected persons with type 2 DM were more likely to need insulin [7].

It is important to determine whether HCV infection can predispose to the development of insulin resistance before diabetes occurs. Such a potential link is particularly cogent in light of recent data indicating that diabetes may be associated with increased hepatic fibrosis progression in patients with chronic HCV infection [11]. 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 [12]. Hyperglycaemia may play a role in the perpetuation and progression of fibrogenesis rather than in the initiation of the fibrotic process [13].

The aim of the study is to assess the insulin resistance status in non-diabetic patients with chronic hepatitis C (CHC) and the association between insulin resistance and liver fibrosis progression.

Patients and methods 

This cross-sectional descriptive study was approved by the local ethics committee of the Mansoura University Hospital. Informed consent was obtained from all subjects prior to inclusion in the study. Thirty-eight patients with CHC infection were randomly selected from all CHC patients attending the outpatient clinics of the Tropical Medicine and Internal Medicine Departments, Mansoura University Hospital, from January to August 2005. Furthermore, 12 healthy age- and sex-matched subjects were considered as a control group. The selected patients were classified into two groups: Group-1 included 23 CHC patients with elevated liver enzymes. This group consisted of 14 males and nine females with ages ranging from 19 to 49years (mean: 33.1±9.2years). Group-2 included 15 CHC patients with normal liver enzymes. This group included 12 males and three females with ages ranging from 22 to 41years (mean: 30.6±7.1years).

The inclusion criterion was patients with positive HCV RNA reverse transcription polymerase chain reaction (RT-PCR) for at least 6months. The exclusion criteria were patients with positive hepatitis B surface antigen concomitant infection, history of schistosomiasis, prior anti-HCV treatment, hepatic decompensation (e.g. ascites, jaundice, variceal bleeding or hepatic encephalopathy) or a history of alcohol intake.

Furthermore, patients with fasting blood glucose level >6.2mmol/L or under anti-diabetic or immunosuppressive regimens, undergoing dialysis, with clinically overt hypo- or hyperthyroidism or with clinically and laboratory evident autoimmune diseases were excluded from the study.

CHC patients with normal enzymes were defined as alanine transaminase (ALT) levels within the normal range on three separate occasions 2months apart within a 6-month period after presentation as defined by Martinot Peignoux et al. [14].

A liver biopsy to stage CHC was taken. Fasting plasma sample was collected within 1week of liver biopsy.

All patients studied were subjected to full history and clinical examination with stress on features of chronic liver disease. All subjects were investigated for the following factors: Complete blood picture, liver function tests, serum creatinine, HBsAg, hepatitis C antibodies, PCR for HCV, serum fasting glucose and serum fasting insulin levels.

The HOMA index of insulin sensitivity is derived from a mathematical model of the glucose–insulin homeostatic system [15]. HOMA-IR strongly predicts the development of type 2 diabetes, independent of obesity, body fat distribution and glucose tolerance status [16].

Insulin resistance is measured using HOMA-IR test calculation [10] as follows = (FI×FPG in mmol/L)/22.5. Where FI is fasting plasma insulin level (μUm/L) and FPG is fasting plasma glucose level (mmol/L).

Serum insulin was measured using an immunoradiometric assay kit (Insulin Riabead II kit; Dainabot, Tokyo, Japan). The intra- and inter-assay coefficients of variation of the assay were 2.0% and 2.1%, respectively.

Abdominal ultrasound, sigmoidoscopy and rectal mucosal biopsy (for exclusion of schistosomiasis) were carried out in all patients.

Liver biopsy analysed with connective tissue stains has long been considered the ‘gold standard’ for assessing liver histology, disease activity and liver fibrosis [17]. The cases with non-alcoholic fatty liver diseases (NAFLDs) were excluded based on histopathological findings. The Metavir score was used. It is comprised of five progressive stages: F0, normal; F1, portal fibrosis; F2, few fibrotic septae; F3, numerous septae; and F4, cirrhosis [18]. Liver biopsy was assessed by two pathologists.

Statistical analysis was done using SPSS version 10, 1999 (SPSS Inc., Chicago, IL, USA). Parametric data were analysed using Kolomgrov–Smirnov test. Quantitative data were presented in the form of mean, standard deviation and range. Student’s t-test was used for comparison of two groups. Qualitative data were presented in the form of number and percentage. Chi-square test was used for qualitative data. The variables (i.e., liver enzymes, degree of inflammation and stage of fibrosis) were analysed using simple and multiple linear regression methods. Significance was considered when the p-value was less than 0.05; a p-value more than 0.05 was considered non-significant.

Results 

The age, sex and body mass index of the HCV patients matched with the control group (Table 1) and showed no significant difference between HCV patients compared with the control group (p=0.071, 0.068 and 0.1, respectively). However, quantitative PCR for HCV was significantly higher in patients with elevated liver enzymes (p=0.001).

Table 1. Age, sex distribution and body mass index (BMI) of the studied groups.

	Groups	Age	Male (%)	Female (%)	BMI	Quantitative PCR
	Group-1 (23) CHC with elevated enzymes	33.13±9.15	14/60.9	9/39.1	28.13±2.44	177±159a
	Group-2 (15) CHC with normal enzymes	30.61±7.10	12/80	3/20	27.81±3.34	42.9±36.3
	Group-3 control	34.08±8.51	6/50	6/50	25.58±1.72
	Significance tests	One way ANOVA F test	Chi-square test		One way ANOVA F test	T=7.93
		F=2.35	X2=8.70		F=2.22	p=0.001⁎⁎
		p=0.071	p=0.068		p=0.101

Comparison among different groups using ANOVA test for numerical data and chi-square test for percentages %: percentage. CHC: chronic hepatitis C virus. BMI: body mass index. PCR: polymerase chain reaction. ∗p: significant (p<0.05).

⁎⁎ p: highly significant (p<0.01). a Significant difference between Group-1 and Group-2.

HCV patients with elevated liver enzymes showed significant higher fasting insulin, HOMA-IR and liver fibrosis grading than those with normal liver enzymes or healthy control (17.85±4.5, 3.98±1.41, 21/2 vs. 12.68±2.5, 2.69±0.76, 13/2 or 9.1±3.6, 1.92±0.74, respectively) (Table 2).

Table 2. Serum fasting glucose; fasting insulin (pmoll−1) and HOMA-IR of the studied groups (CHC with elevated enzymes; CHC with normal enzymes versus normal healthy control).

		FBG (mmoll−1)	FI (μUl−1)	HOMA-IR	Fibrosis
					+ve	−ve
	Group-1 CHC with elevated enzymes	4.9±0.75a	17.85±4.5a,c	3.98±1.41a,c	21	2
	Group-2 CHC with normal enzymes	4.6±0.51	12.68±2.5b	2.69±0.76	13	2
	Group-3 healthy control	4.1±0.42	9.1±3.6	1.92±0.74
	Group-1 versus Group-3	p=0.05⁎	p<0.001⁎⁎	p<0.001⁎⁎
	Group-2 versus Group-3	p=0.112	p<0.001⁎⁎	p=0.071
	Group-1 versus Group-2	p=0.071	p=0.009⁎⁎	p=0.09	p=0.023⁎

Comparison among different groups using ANOVA test for numerical data and chi-square test for percentages. %: percentage. NS: non-significant. CHC: chronic hepatitis C virus. FBG: fasting blood glucose. FI: fasting insulin. HOMA-IR: homeostasis model assessment-Insulin resistance. Significant differences between the groups:

a Group-1 versus Group-3. b Group-2 versus Group-3. c Group-1 versus Group-2. ⁎ p: significant (p<0.05). ⁎⁎ p: highly significant (p<0.01). F=6.21=p<0.001.

There was no significant difference in METAVIR score between CHC patients with elevated liver enzymes and those without elevated liver enzymes (p-value=0.51) (Table 3 and Fig. 1). METAVIR activity score was significantly higher in CHC patients with elevated liver enzymes than those without elevated liver enzymes (p-value=0.021) (Table 4, Fig. 2).

Table 3. METAVIR fibrosis staging in patients with chronic hepatitis C infection.

	Groups	METAVIR (staging) (F)
		F0 (%)	F1 (%)	F2 (%)	F3 (%)	F4 (%)
	CHC with elevated enzymes (N/%)	2/8.7	9/39.1	4/17.4	7/30.4	1/4.3
	CHC with normal enzymes (N/%)	2/13.3	7/46.7	3/20	3/20	–/–
	Comparison between two groups	NS (p=0.51)

Comparison among different groups using chi-square test for percentages. N: number. %: Percentage. NS: non-significant. CHC: chronic hepatitis C virus. ∗p: significant (p<0.05). ∗∗p: highly significant (p<0.01). X2=9.71=p=0.64.

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Fig. 1. METAVIR (staging) of chronic viral C hepatitis.

Table 4. METAVIR activity score in patients with chronic hepatitis C infection.

	Groups	METAVIR (activity) (A)
		A0 (no)	A1 (mild)	A2 (moderate)	A3 (severe)
	CHC with elevated enzymes (N/%)	–/–	9/39.1	8/34.8	6/26.1
	CHC with normal enzymes (N/%)	2/13.3	11/73.3	2/13.3	–/–
	Comparison between two groups	p=0.021⁎

Comparison among different groups using chi square test for percentages. N: number. %: percentage. NS: non-significant. CHC: chronic hepatitis C virus. ∗∗p: highly significant (p<0.01). X2=20.1 (p=0.012). X2=6.65 (p=0.063).

⁎ p: significant (p<0.05).

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Fig. 2. METAVIR (activity) of Chronic C Hepatitis.

In CHC patients with elevated liver enzymes, FPG, HOMA-IR and prothrombin time correlated with METAVIR activity and liver fibrosis scores (p=0.01, 0.02, p=0.02, 0.01 and p=0.01, 0.01, respectively) while viral load (PCR) correlated with METAVIR activity score only (p=0.031) (Table 5, Fig. 3). In CHC patients with normal liver enzymes, FPG and HOMA-IR correlated with METAVIR activity and liver fibrosis scores (p=0.04, 0.01 and p=0.01, 0.02, respectively) (Table 6, Fig. 4) while viral load (PCR) correlated with METAVIR activity score only (p=0.031) and prothrombin time correlated with METAVIR fibrosis score (p=0.02). There was no correlation between serum fasting insulin levels and liver fibrosis in CHC patients with elevated enzymes (p=0.01) (Table 5) and in those with normal enzymes (p=0.02) (Table 6).

Table 5. Relationship between METAVIR activity and fibrosis scores and laboratory data in patients CHC with elevated enzymes.

	Laboratory data	METAVIR (activity) (A)		METAVIR (staging) (F)
		r	p	r	p
	FBG	0.80	0.01⁎⁎	0.43	0.02⁎
	FI	0.38	0.1⁎	0.60	0.01⁎⁎
	HOMA-IR	0.49	0.02⁎	0.66	0.01⁎⁎
	PCR	0.41	0.031⁎	0.32	0.22
	ALT	0.42	0.04⁎	0.40	0.04⁎
	PT	0.87	0.01⁎⁎	0.49	0.01⁎⁎

⁎ p: significant (p<0.05). ⁎⁎ p: highly significant (p<0.01).

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Fig. 3. Correlation between METAVIR (staging) and HOMA-IR (group 1).

Table 6. Relationship between METAVIR activity and fibrosis scores and laboratory data in patients with CHC with normal enzymes.

	Laboratory data	METAVIR (activity) (A)		METAVIR (staging) (F)
		r	p	r	p
	FBG	0.53	0.04⁎	0.77	<0.01⁎⁎
	FI	0.76	0.01⁎⁎	0.61	0.02⁎
	HOMA-IR	0.76	0.01⁎⁎	0.73	0.02⁎⁎
	PCR	0.66	0.034⁎	0.07	0.92
	ALT	0.48	0.04⁎	0.21	0.43
	PT	0.08	0.91	0.50	0.02⁎

FBG: fasting blood glucose. FI: fasting insulin. HOMA-IR: homeostasis model assessment-Insulin resistance. CHC: chronic hepatitis C virus. PCR: polymerase chain reaction. ALT: alanine transferase. PT: prothrombin time.

⁎ p: significant (p<0.05). ⁎⁎ p: highly significant (p<0.01).

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Fig. 4. Correlation between METAVIR (staging) and HOMA-IR (group 2).

Discussion 

Recent data indicate that insulin resistance and diabetes may be associated with increased hepatic fibrosis progression in patients with chronic HCV infection [10].

In our study METAVIR stage F1 was the most common in CHC patients with normal enzymes (46.7%) and 39.1% in CHC patients with elevated enzymes. Puoti et al. [19] found that subjects with normal liver enzymes had moderate-to-severe degree of hepatitis.

In the present study, there was a significant correlation between FPG and liver fibrosis in CHC patients with either elevated or normal enzymes. This is consistent with the findings of Ratziu et al. [13] who demonstrated that high FPG is associated with liver fibrosis and higher fibrosis progression rate in CHC; the cause may be hyperglycaemia which results in enhanced formation and deposition of advanced glycation end products [20]. Specific receptors for these advanced glycation end products have been detected in the liver where they are restricted to hepatic stellate cells; the main cellular source of liver collagen [21].

In this study, there was a correlation between serum fasting insulin and liver fibrosis progression in CHC patients with elevated enzymes and in those with normal enzymes. This is in agreement with Fukui et al. [22] who reported that fasting serum insulin levels were significantly elevated in patients with chronic HCV infection compared with control subjects.

In this work, there is significantly higher HOMA-IR in CHC with elevated enzymes and those with normal enzymes compared with control group. This concurs with the results of Maeno et al. [23] who established that insulin resistance was elevated among HCV-infected patients compared with control group, which may be due to decreased liver carbohydrate metabolism and hypersecretion of insulin-resistant cytokines. This also was demonstrated in Egyptian patients with HCV infection who had a significantly higher level of tumour necrosis factor (TNF) alpha and ferritin, which may contribute to their insulin resistance [24]. In humans, HCV by itself may be associated with IR in a dose-dependent manner prior to the development of overt type 2 DM [25]. HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced suppressor of cytokine signalling 3 (SOCS3) promotes proteosomal degradation of insulin receptor substrates (IRS1 and IRS2) through ubiquitination, leading to insulin resistance [26].

The correlation between both FPG and HOMA-IR with theMETAVIR activity and liver fibrosis score, respectively, regardless of the liver enzymes is in agreement with the findings of Hui et al. [27] who approved that there is a correlation between insulin resistance and liver fibrosis in patients with CHC. Insulin plays a significant role in the development of fibrosis via a mechanism involving steatosis. In this regard, steatosis promotes cellular insulin resistance which, in turn, induces compensatory hyperinsulinaemia [28]. Hyperinsulinaemia has been shown to directly stimulate hepatic stellate cell proliferation and increase expression of connective tissue growth factor, a key factor in the progression of fibrosis [29]. D’Souza et al. [30] found that insulin resistance contributes to liver fibrosis in chronic HCV infection; this relationship is not genotype specific. Furthermore, they suggested that insulin resistance plays an important role in hepatic fibrosis in HCV patients. HOMA-IR remained significantly associated with fibrosis, even after adjustment for all other variables associated with fibrosis stage (age, FPG, HbA1c, AST, ALT and BMI).

Finally, elucidation of the relationship between HCV and insulin resistance is of great clinical relevance, because the latter promotes liver fibrosis [25].

In conclusion, the present data support the hypothesis that insulin resistance may increase the rate of fibrosis progression in non-diabetic patients with chronic HCV. Follow-up of hyperinsulinaemia in non-diabetic HCV-infected patients may be a biochemical indicator for progression of liver fibrosis. The use of insulin resistance to predict progression of fibrosis is interesting. We need a longitudinal study to address this issue.