Hepatitis C virus E2 protein involve in insulin resistance through an impairment of Akt/PKB and GSK3β signaling in hepatocytes

Abstract

Background: Hepatitis C virus (HCV) infection may cause liver diseases of various severities ranging from primary acute infection to life-threatening diseases, such as cirrhosis or hepatocellular carcinoma with poor prognosis. According to clinical findings, HCV infection may also lead to some extra-hepatic symptoms, including type 2 diabetes mellitus (DM). Since insulin resistance is the major etiology for type 2 DM and numerous evidences showed that HCV infection associated with insulin resistance, the involvement of E2 in the pathogenesis of type 2 DM and underlying mechanisms were investigated in this study.

Methods: Reverse transcription and real-time PCR, Western blot assay, Immunoprecipitation, Glucose uptake assay and analysis of cellular glycogen content.

Results: Results showed that E2 influenced on protein levels of insulin receptor substrate-1 (IRS-1) and impaired insulin-induced Ser308 phosphorylation of Akt/PKB and Ser9 phosphorylation of GSK3β in Huh7 cells, leading to an inhibition of glucose uptake and glycogen synthesis, respectively, and eventually insulin resistance.

Conclusions: Therefore, HCV E2 protein indeed involved in the pathogenesis of type 2 DM by inducing insulin resistance.

Figure 1

IR and IRS-1 mRNA levels were unchanged with HCV E2 protein expression. Huh7 cells were transfected with FLAG-CMV2 or FLAG-E2 for 24 hours. After the confirmation of successful E2 expression (A), cells were subjected to semi-quantitative PCR (B) and RT-PCR (C &D) to show IR and IRS-1mRNA expression levels were not changed by E2 expression. The error bars represented standard deviation from triplicate experiments. NS means not significant.

Figure 2

IR and IRS-1 protein levels while HCV E2 protein expressed in Huh7. Huh7 cells were transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and then subjected to western blotting for IR and IRS-1 with β-actin acting as internal control (A). Relative photographic density was quantified and the results were shown as histograms mean relative IR (B) and IRS-1 (C) levels after being normalized by β-actin. While the protein level of IR was unchanged, that of IRS-1 protein expression was decreased by HCV E2 expression. The error bars represented standard deviation from triplicate experiments. * indicates p<0.05.

Figure 3

HCV E2 induce SOCS3 mRNA and protein levels in Huh7. Huh7 cells were transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and then subjected to semi-quantitative PCR for SOCS3 expression with GAPDH being an internal control (A). For western blotting, cell lysate samples were prepared from Huh7 cells transfected with FLAG-E2 for the indicated time and then subjected to western blotting for SOCS3 with β-actin and IL-6 treatment (100 ng/ml for 30 minutes) being an internal control and positive control, respectively. Results in (B) indicated that the expression levels of SOCS3 were increased by E2 expression.

Figure 4

HCV E2-caused down-regulation of IRS-1 may be through an ubiquitination. In the absence or presence of MG132 (10 and 20 nM), Huh7 cells were transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and then subjected to immunoblotting with the indicated antibodies (A). Results indicated that MG132, proteosomal proteolysis inhibitor, may restore the E2-related down-regulation of IRS1. Furthermore, cell lysates were subjected to immunoprecipitation with IRS-1 followed by immunoblotting with anti-ubiquitin antibody. Results in (B) indicated that HCV E2 caused an accumulation of ubiquitin-conjugated IRS-1.

Figure 5

Insulin-induced phosphorylation of Akt and GSK3β was inhibited by E2 protein. After being transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and a serum-free incubation 16 hours, cells were subjected to a insulin stimulation for an indicated time period (0, 15, 30 and 60 minutes) followed by western blotting with indicated antibodies to reveal a time-dependent increase of phosphorylation of Akt (A) and GSK3β (C). Furthermore, transfected or untransfected cells were treated insulin for 30 minutes and then subjected to western blotting to detect Ser473 and Thr308 phosphorylation of Akt (B) serine phosphorylation of GSK3β (D). Results suggested that an expression of E2 inhibited insulin-induced Thr308 phosphorylation of Akt and GSK3β phosphorylation.

Figure 6

Insulin-induced glucose uptake and glycogen synthesis were both decreased by an expression of E2 protein. After being transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and a serum-free incubation 16 hours, cells were subjected to insulin stimulation for 13 minutes and then glucose uptake analysis with 2-deoxy-d-[1,2-³ H] glucose. [³ H] glucose uptake levels were quantified with liquid scintillation counter and presented as a percentage to that of control (A). For glycogen synthesis analysis, the insulin stimulation was carried out for 9 hours before cells were subjected to glycogen assay kit (B). These results clearly indicated that E2 protein can inhibit insulin-induced cellular glucose uptake and glycogen synthesis. The error bars represented standard deviation from triplicate experiments. * indicates p<0.05 and ** indicates p<0.01.