Hepatitis C Genotype-4 Infection: Role of Insulin Resistance in Hepatocellular Carcinoma

Virology Journal 2011, 8:496 doi:10.1186/1743-422X-8-496Published: 1 November 2011

Chronic Hepatitis C Genotype-4 Infection: Role of Insulin Resistance in Hepatocellular Carcinoma

The complete article is available as a provisional PDF

Amal A Mohamed, Samah A Loutfy, James D Craik, Abdel Gawad M Hashem and Ibrahem SiamAbstract (provisional)

Abstract

Background
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma (HCC) and different HCV genotypes show characteristic variations in their pathological properties. Insulin resistance (IR) occurs early in HCV infection and may synergize with viral hepatitis in HCC development. Egypt has the highest reported rates of HCV infection (predominantly genotype 4) in the world; this study investigated effects of HCV genotype-4 (HCV-4) on prevalence of insulin resistance in chronic hepatitis C (CHC) and HCC in Egyptian patients.

Methods
Fifty CHC patients, 50 HCC patients and 20 normal subjects were studied. IR was estimated using HOMA-IR index and HCV-4 load determined using real-time polymerase chain reaction. Hepatitis B virus was excluded by enzyme-linked immunosorbent assay. Standard laboratory and histopathological investigations were undertaken to characterize liver function and for grading and staging of CHC; HCC staging was undertaken using intraoperative samples.

Results
HCC patients showed higher IR frequency but without significant difference from CHC (52% vs 40%, p=0.23). Multivariate logistic regression analysis showed HOMA-IR index and International Normalization Ratio independently associated with fibrosis in CHC; in HCC, HbA1c, cholesterol and bilirubin were independently associated with fibrosis. Fasting insulin and cholesterol levels were independently associated with obesity in both CHC and HCC groups. Moderate and high viral load was associated with high HOMA-IR in CHC and HCC (p<0.001).

Conclusions
IR is induced by HCV-4 irrespective of severity of liver disease. IR starts early in infection and facilitates progression of hepatic fibrosis and HCC development.

Introduction

Persistent Hepatitis C virus (HCV) infection is widespread; it affects millionsof people worldwide and induces a range of chronic liver disease[1]. Chronic HCV infection causes progressive hepatic fibrosis and cirrhosis in up to 20%of patients and approximately 10%-20% of cirrhotic patients may go on todevelop hepatocellular carcinoma (HCC) within five years[2].

HCC is the most frequent cause of death in patients infected with HCV, and epidemiological trends suggest that the mortality rate is rising[3].Understanding the risk factors for HCC development in patients infected with HCV is thus of great importance for refinement of treatment strategies and healthcare delivery. HCV has a high mutation rate and six major genotypes have been characterized, each with a distinctive geographical distribution and pathological properties[1].

Egypt has the highest country wide prevalence of HCV in the world; about 12 to 15% of the total population are infected[4],with HCV Genotype-4 (HCV-4) accounting for the overwhelming majority ofHCV infections.

HCV has been identified as a cause of metabolic syndrome, a complex that includes dyslipidemia, diabetes and insulin resistance (IR). IR is a key featureof this syndrome and a variety of potential molecular pathways by which HCV may contribute to IR have been suggested[5]. Patients infected with HCV have significantly higher IR than healthy controls matched for age, sex and body mass index[6].

Recent studies have found that HCV-associated IR may cause; (i) hepatic steatosis, (ii) resistance to anti-viral treatment, (iii) hepatic fibrosis and esophageal varices, (iv) hepato carcinogenesis and proliferation of HCC and(v) extrahepatic manifestations[7,8]. IR has emerged as a risk factor for a wide variety of cancers, including endometrial and breast (especially after menopause), colon, and rectal, esophageal, kidney, pancreatic, biliary, ovarian
and cervical cancers [9]. In chronic HCV infection, IR can favor fibrosis
progression directly and act indirectly by inducing steatosis in a genotype dependent
manner[10].

It has been reported that IR can increase the risk of developing HCC in
patients with chronic HCV infection[11]. A multiplicity of viral and host
factors may play a crucial role in facilitating the onset of IR in patients with
chronic hepatitis C (CHC) that may ultimately end with HCC
development[5]. Given high levels of endemic CHC infection in Egypt, and
that IR is a potentially modifiable factor, a better understanding of
correlations of IR with HCC among Egyptian patients infected with HCV-4 is
urgently needed.

The focus of the study was to investigate: (i) the prevalence of IR among CHC
and HCC patients and its possible role in HCC development in the context of
high prevalence of HCV-4 infection and (ii) impact of other factors such as
host characteristics (age, gender, etc.), viral parameters (viral load, viral
genotype), and other variables including obesity and dyslipidemia on IR and
hepatic carcinogenesis.

Patients and Methods
Patients.
This prospective study was conducted with 120 participants divided into
three groups. The first group comprised 50 patients with chronic hepatitis C
genotype- 4 (CHC). The second group comprised 50 hepatocellular carcinoma
(HCC) patients diagnosed and treated at Ain Shams University Specialized
Hospital, Cairo, between February 2009 and February, 2010. A third group
included 20 apparently healthy participants who had donated blood at the
National Cancer Institute, Cairo University.

The Ethical Committee of Ain Shams University Specialized Hospital
approved the study protocol, which was prepared in accordance with the
ethical guidelines of the 1975 Declaration of Helsinki and later revisions.
Written consent was obtained from all participants prior to enrollment in the
study and all were mentally and physically capable of answering a
questionnaire.

Inclusion criteria: adult patients of both sexes (20-70 years old), diagnosis
within the previous 6 months, positive for HCV RNA in serum (by RT-PCR
assay), with evidence of chronic hepatitis supported by liver biopsy. The
control group was made from adults negative for HCV RNA. Patients were
not receiving hepatitis treatment at the time of sampling.

The presence of HBV infection or co-infection was excluded by serum ELISA
for anti-HBc and HBsAg. In addition to investigations needed to fulfill the
selection criteria, all individuals included in this study were subjected to the
following:
A. Medical History:
Full history was taken with special reference to risk factors for liver diseases
such as previous HCV exposure in surgical wards, blood transfusions, dental
therapy, needlestick injury, history of HCV in the spouse and i.v. injection.
B. Physical Examination:
Complete medical examination with particular focus upon the manifestations
of hepatitis such as jaundice, hepatomegaly, and tenderness in the right

hypochondrium. BMI was calculated as body weight in kilograms divided by
the square of height in meters (kg/m2). Abdominal ultrasonography was
performed for all patients.
C. Histopathological investigations:
Liver biopsy specimens were formalin-fixed and paraffin embedded then
sectioned and stained (hematoxylin and eosin) for routine histopathological
examination. Grading and staging of chronic hepatitis was performed
according to Modified Knodell’s Score[12]. Liver tissues from HCC cases were
collected intraoperatively. These samples were graded using World Health
Organization (WHO) classification criteria[13] and staging was determined
according to the American Joint Committee on Cancer[13].
D. Laboratory investigations:
Venous blood samples were taken in the morning after 12-h overnight fast.
Plasma glucose, HbA1c, serum alanine aminotransferase (ALT), aspartate
aminotransferase (AST), gammaglutamyl transferase (GGT), albumin (Alb),
total bilirubin levels (Bil), cholesterol (Chol), and triglycerides (TG) were
measured by using Synchron CX4 clinical system (standard clinical laboratory
methods) at the Clinical Laboratory Department, Ain Shams Specialized
Hospital. Serum insulin levels and α-fetoprotein levels were estimated by
serological techniques (Axyam System, Abbott Laboratories).
Platelet count (Plt) and Prothrombin Time (PT) measurements were
performed for all patients; normal PT was 12 seconds (100% concentration
and International Normalization Ratio (INR) of 1).

Insulin resistance (IR) was calculated on the basis of fasting levels of plasma
glucose and serum insulin, according to the homeostasis model assessment
(HOMA) method. Calculation for the HOMA model followed a standard
protocol; insulin resistance (HOMA-IR) = fasting glucose (mg/dL) × fasting
insulin (μIU/mL)/405 [14]. The HOMA-IR index has seen widespread use,
with various cut-off values for insulin resistance. In many studies of
Caucasian populations a cut-off value of 2.5 has been applied; other studies
have used higher cut-off values, for example 3.0[15]. Genetic variation with

respect to different ethnic groups will influence choice of cut-off value
(discussed by Esteghamati[16]); for this study we used a cut-off value of 4.0
[17].
E. Viral Markers
ELISA assays: Sera of all patients and controls were tested for HBsAg, anti-
HBc and anti-HCV antibodies by ELISA, using third generation kits
(DiaSorin, Italy) according to the manufacturer’s instructions.
Polymerase chain reaction (PCR) for hepatitis C virus: RT–PCR was
performed as previously described[18] and 10μl samples of the amplicons
were analyzed by electrophoresis (1.2% agarose gel, ethidium bromide
staining).

HCV genotyping:
HCV genotype was determined using INNO-LiPAII and III versant Kit
(Innogenetics, Ghent, Belgium) according to manufacturer’s directions
Quantitation of HCV-RNA in serum:
HCV-RNA was quantitated in all patients' serum samples using Real Time
PCR (RT-PCR)[15] (primers and RT-PCR reagents from Stratagene, Qiagen,
USA). Low viremia was defined as viral load lower than 100 x103 IU/L,
moderate viremia as viral load 100-1000 x 103 IU/L, and high viremia when
viral load more then 1000 x103 IU/L[19].

Statistical analysis
Data was analyzed using SPSSwin statistical package version 15 (SPSS Inc.,
Chicago, IL). Chi-square test (Fisher’s exact test) was used to examine
relationships between qualitative variables. For quantitative data, comparison
between two groups was undertaken using Mann-Whitney test and
comparison between 3 groups with ANOVA test or Kruskal-Wallis test
followed post-Hoc "Schefe test". Spearman-rho method was used to test
correlation between numerical variables. Multivariate analysis was performed
using multiple linear regression model using forward method for the
significant factors affecting fibrosis on univariate analysis. Multivariate

analysis (logistic regression) was performed to find the predictors for HCC
development All tests are two-tailed; a p-value<0.05 was considered
significant.

Results
Baseline characteristics (Table 1): Ages of CHC patients (median 57 years;
range 34-70 yrs) and HCC patients (median 60 years; range 35-76 yrs) were
closely comparable, while control group participants were younger (median
35 years; range 19-57). Preponderance of males was observed with both CHC
and HCC groups; 1:1.5 and 1:1.6 respectively. There was no significant
difference between CHC and HCC patients regarding age or sex (p=0.14 and
0.838, respectively). No significant difference was observed in BMI
distribution between the study groups (p=0.908).

Biochemical, viral and pathological factors:
HCC patients showed significantly higher median values of INR and HbA1c,
than CHC patients (p<0.0001, p=0.05, respectively), but significantly lower
median values for albumin and platelets (p<0.001). HCC patients also showed
significantly higher median values of AST, Bil, GGT and α-fetoprotein when
compared to CHC patients (p<0.001 for all). No significant differences were
found between HCC and CHC patients regarding median values of ALT,
glucose, fasting insulin, HOMA-IR index values, cholesterol and triglycerides
(p= 0.09, =0.81, = 0.178,=0.643,= 0.954, =0.999).
Frequency of high value for HOMA-IR index (>4) and presence of
dyslipidemia were not significantly different between CHC and HCC patients
(Table 2).

HCC patients showed significantly higher frequency of presentation with
moderate viral load than CHC patients (70% vs 42%, p<0.001) and higher
grades of fibrosis (IV and V: 45/50 (90%) for HCC vs 15/50 (30%), for the
CHC group (p<0.001).

The normal control group showed significant difference when compared to
the HCC or CHC groups (p<0.001) for all parameters studied, except BMI.

Factors associated with development of fibrosis
Multiple linear regression analysis showed that HOMA-IR and INR are the
only independent predictors of fibrosis in CHC patients after adjustment (of
age, AST, total bilirubin, albumin, glucose, α-fetoprotein, platelets, GGT,
fasting insulin, HbA1c, cholesterol and triglycerides). In HCC patients,
HbA1c, triglycerides and total bilirubin are independent predictors of fibrosis
after adjustment (of age, AST, INR, albumin, glucose, α-fetoprotein, platelets,
GGT, fasting insulin, HOMA-IR and cholesterol) Table 3. Correlation between
HOMA-IR index and grades of fibrosis in CHC and HCC groups are shown in
Figures 1 and 2, respectively.

Viral load and insulin resistance:
Analysis of HCV viral load with HOMA-IR showed that in the CHC group,
both moderate and high viral load were significantly associated with higher
values for HOMA-IR compared to those with lower median viral load (4.19,
5.15 vs 1.85, p<0.001) Figure 3. Similar differences were not observed in HCC
patients.

HCC development:
Multivariate logistic regression analysis showed that fibrosis was the only
independent predictor factor for risk of HCC development (OR: 21.64, 95%CI:
1.81-259.09, p=0.02), after adjustment of (age >56y, HBA1c >7%, AST >40, TBil
>1, GGT >50, INR>1) (Table 4).

Obesity:
Obesity was significantly associated with higher HOMA-IR index value in
both CHC and HCC patients (p<0.001). Obese patients (CHC and HCC)

showed significantly higher frequency of high viral load when compared to
non-obese patients (p=0.001) Figure 4.
Multiple linear regression analysis showed that fasting insulin and cholesterol
are independent predictors for obesity among CHC and HCC patients (OR:
1.663, p<0.001, OR: 1.052, p<0.001 respectively) (Table 5).
CHC and HCC with high HOMA-IR index
Table 6 shows a comparison of CHC and HCC patients demonstrating
HOMA-IR > 4. The strong similarities of these variables suggest that HCV-4,
like HCV genotypes 1 and 2, leads to fibrosis rather than the steatosis seen
with genotype 3.

Discussion
This study has provided valuable cues with respect to the potential role of
insulin resistance in the pathological consequences of infection with HCV
genotype 4 in a population notable for high rates of HCV infection and
obesity. Previous studies suggest a strong synergistic effect of metabolic
factors and viral hepatitis in HCC development in HCV-infected
patients[20,21]. In contrast to a previous report which found association of IR,
regardless of diabetes, with development of HCC[11], the present study
showed no significant difference in HOMA-IR values, diabetes and insulin
levels between CHC and HCC patients. This discrepancy might be due to
confounding factors such as:
i) High prevalence of HCV-4 infection; in high prevalence areas the incidence
of HCC is heavily influenced by virus-related characteristics that confound
other risk factors for HCC, blurring their effect[22]. This may indicate that the
effect of IR on the risk of HCC is modified by HCV infection.

ii) High prevalence of obesity among our patients without significant
difference between CHC and HCC. It has been documented that obesity is an
independent risk factor for IR in HCV and HCC[23].
iii) The number of participants in this study may not have been sufficient to
reveal small differences in prevalence in the studied population
In the present study, the frequency of conspicuous insulin resistance (HOMAIR
>4) was 40% among CHC patients. This finding is in agreement with
Harrison and co-workers[24], who reported that 30 to 70% of CHC patients
display some evidence of IR. These results have suggested the occurrence of
IR at early stage(s) of chronic HCV infection irrespective of the severity of
liver disease and thus the possible role of IR as a metabolic factor that
increases risk of HCC development[25]. It is noteworthy that there are a
variety of plausible direct effects of HCV in modulating insulin signaling and
molecular pathways involved in IR development in hepatocytes[26,27]. In
turn, hyperinsulinemia that results from IR stimulates growth of HCC and
inhibits apoptosis in diabetic patients[10,25,28].

It is very likely that a high prevalence of diabetes in our CHC group (96%)
may be a risk factor for HCC development[25,29] but this effect may be
confounded by the underlying liver disease[22]. Veldt and co-workers[25]
have reported that the 5 year risk of developing HCC is 11.4% for patients
with both diabetes mellitus and CHC with advanced fibrosis while patients
without diabetes have lower risk of HCC (occurrence of HCC in 5% after 5
years).

Multivariate analysis showed that HOMA-IR values and INR were
independently associated with fibrosis in CHC patients, but not in the HCC
patient group. This is in accord with our hypothesis that early occurrence of
IR may hasten progression of fibrosis to cirrhosis which may culminate in

HCC development. This is consistent with recent reports, including a
Japanese cohort study[30,31].

It has been reported that the mean IR index increases with stage of fibrosis
and may help in differentiating fibrosis stages[32]. This was observed in our
results (Figure 1) where HOMA-IR was significantly associated with high
grades of fibrosis in CHC patients (p<0.001). IR promotes fibrosis progression
in the liver of patients with HCV through development of hepatic steatosis,
hyperleptinemia, increased TNF production and reduced expression of
peroxisome proliferator activated receptors (PPAR receptors)[27,33]. High
levels of insulin and glucose could promote fibrogenesis by stimulating
release of connective tissue growth factor from hepatic stellate cells[34]. The
present study did not investigate steatosis as a possible link between IR and
fibrosis.

In our HCC patient group, HbA1c, triglycerides and total bilirubin levels
were found to be independently associated with fibrosis. This finding was not
observed in previous studies. Furthermore, multivariate analysis showed that
fibrosis was the only independent predictor for risk of HCC development
after adjustment of age and other markers of advanced liver disease (Plt, Bil,
albumin, INR AST, GGT). These results confirm a distinct role of diabetes in
HCC development[25]; in tumorigenesis, transformed cells require more
glucose[35] .

It is well established that IR is associated with abdominal obesity, elevated
blood cholesterol and hypertension[36]. In the present study, obesity was
found significantly associated with IR (higher HOMA-IR index value) in both
CHC and HCC patient groups (p<0.001 compared to non-obese patients).
Obesity may directly lead to a state of chronic inflammation with consequent
increases in the expression of signaling molecules, some of which (for
example, NFKB, and fibroblast growth factor) are thought to be involved in

carcinogenesis[37,38]. Chen and co-workers[21] have reported that although
overweight itself did not appreciably increase risk of HCC, a combination of
high BMI and diabetes showed a synergistic effect. Our results confirmed a
synergistic effect of obesity when combined with high level of insulin
resistance (HOMA-IR index) in risk of HCC development associated with
HCV-4 infection.

Multivariate analysis in the present study showed that fasting insulin and
cholesterol were independent predictors for obesity. This readily explains the
prevalence of obesity among our patients. Type 2 diabetes is a major health
problem in the Eastern Mediterranean Regional Office (EMRO) region and
many studies have established a panel of risk factors associated with IR and
type 2 diabetes; family history, body fat distribution, age, gender, smoking
and physical activity. Obesity and IR are strong cofactors of HCV-related liver
disease and predict lack of response to treatment[39].

Division of patient groups using age of >57 revealed that most of our HCC
patients were over 57 years in age and the number in this age group showed
significant difference when compared to CHC patients (p= 0.04). In univariate
analysis, IR was correlated with age in CHC patients. It has been suggested
that age is associated with a decline in mitochondrial function which could
contribute to IR[40]. However, this relationship disappeared in multivariate
analysis; this indicates that age is one of the confounding factors for
development of IR[31].

Our HCC patients showed higher frequency of moderate viral load than
patients with CHC infection; a result in accord with that reported by Hsu[33].
Our patients (CHC or HCC) showed that moderate or high viral load was
significantly associated with high levels of insulin resistance (high HOMA-IR
index) compared to those with low viral load (median 4.19, 4.80 vs 2.24,
p<0.001). Interestingly, on analysis of correlation between viral load and

HOMA-IR in our CHC and HCC groups separately, it was apparent that in
the CHC patient group, cases with moderate and high viral load were
significantly associated with higher HOMA-IR index compared to those with
lower viral load (4.19, 5.15 vs 1.85, p<0.001) while in HCC patients, moderate
and high viral loads were not significantly associated with higher HOMA-IR
values (median 4.19, 4.67 vs 3.06 p=0.70 compared to cases with lower viral
load). This observation is readily rationalized by a role of HCV infection in
production of IR before cirrhosis and HCC development. This supports the
hypothesis that a modulating effect of HCV on insulin signaling pathways
plays a role in a separate pathway involved in the process of
hepatocarcinogenesis[41].

The association of HOMA-IR with viral load is an important finding of the
study. The patient with HCV infection can present with IR due to metabolic
and to viral factors; the molecular bases of these effects have been subject to
intense research scrutiny though the relative importance of some of these
interrelated effects to the clinical picture remains to be fully determined.
Chronic inflammation as a non-specific consequence of hepatic inflammation
due to HCV infection leads to IR associated with metabolic liver disease
through increased levels of IL-1, TNF-α, IL-6 and leptin, and reduced levels of
adiponectin. This will complement obesity effects which involve simulation of
inflammatory mediator IkB kinase β and phosphorylation of IRS-1[26].
Multiple pathways for direct effects of the virus on IR have been identified.
These include impairment of insulin signaling through stimulation of
proteosomal inactivation of IRS-1 and IRS-2, viral induction of excess TNF
giving enhanced production of suppressor cytokine signal proteins (SOCS) in
a genotype specific manner[42] which act on the Akt pathway (also downregulated
by HCV-stimulated PP2A activity) giving impairment of GLUT-4
glucose transporter activation[43]; and action of HCV core protein as an
inhibitor of PPAR α (peroxisome proliferator-activated receptors)[44]. In
addition, HCV NS5a protein induces mitochondrial ROS (reactive oxygen

species) production of which activate release of a cascade of inflammatory
mediators through activation of nuclear factor-κB (NF-κB) and HCV NS3, and
induce endoplasmic reticulum stress signals. Progressive fibrosis induces IR
due to impairment in insulin clearance with resulting insulin levels[17,31].

Conclusions
Insulin resistance may be induced by HCV infection irrespective of the
severity of liver disease; insulin resistance starts at an initial phase of HCV
infection and facilitates the progression of fibrosis and development of HCC.
In chronic hepatitis C infection, HCV viral replication per se may be dominant
in the overall process of hepatocarcinogenesis associated with HCV infection.
This study complements studies of HCV infection in other populations and
indicates that in the Egyptian population suffering from a high burden of
hepatitis C genotype-4 virus the strikingly high rates of hepatocarcinogenisis
result from a combination of this direct viral effect and the influence of an
array of metabolic factors resulting from virus-induced insulin resistance.
Our results lay a foundation for a follow up cohort study with larger sample
size and including diabetic and non-diabetic CHC patients with and without
fibrosis that would have value to illustrate the role of IR as a separate
metabolic factor for HCC development and enable investigation of the role of
other individual metabolic signals such as adiponectin, and leptin which may
have particular involvement in hepatocarcinogenesis in patients infected with
HCV-4. A better understanding of IR involvement in HCV-4 mediated HCC
may point to potential for benefit of therapeutic interventions aimed at
control of IR, and IR-associated signals, in these patients.

Competing interests
The authors declare that they have no competing interests
Authors’ contributions
AAM performed the research; SAL developed the plan of the study and wrote
the paper; JC analyzed the molecular data and evaluated and edited the draft
manuscript; AGMH contributed analytical tools and reagents and IS collected
samples, gathered clinical data and contributed to the drafting data tables.
All authors have read and approved the final manuscript
Acknowledgements
Dr. Zeinab Aly el Din, Tropical Medicine Department, Faculty of Medicine,
Ain Shams University is thanked for her great help in collection of specimens
and data from the patients enrolled in the study. The authors gratefully
acknowledge financial support from Ain Shams University, Cairo, Egypt.

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