Hepatitis C Virus Core Protein Upregulates Serine Phosphorylation of Insulin Receptor Substrate-1 and Impairs the Downstream Akt/Protein Kinase B Signaling Pathway for Insulin Resistance

Generation of cell lines and cell culture-grown HCV.HepG2 and Huh-7 cells stably transfected with HCV core or HCV_1-2962 plasmid DNA (HCV-FL) were generated as previously described (8, 46). Immortalized human hepatocytes (IHH) were generated by stable transfection of an HCV core (genotype 1a) genomic region into primary hepatocytes (9, 44). HCV genotype 1a (clone H77) was grown in IHH as recently described (30). Virus growth was measured from cell culture supernatant filtered through a 0.45-μm-pore-size cellulose acetate membrane (Nalgene, Rochester, NY) by using a fluorescent focus-forming assay. The HCV titer was calculated to be ∼10⁵ focus-forming units/ml. HCV genotype 2a (clone JFH1) was grown in Huh-7 cells as previously described (30).

Reagents.Human recombinant TNF-α (Calbiochem), recombinant human IL-6 (BD Pharmingen), human recombinant insulin (Roche), phospho-IRS-1 (Ser³¹²) antibody, and IRS-1 antibody (Upstate Biotechnology, Lake Placid, NY), phospho-specific antibodies to Akt (Ser⁴⁷³, Thr³⁰⁸), antibody to tAkt and anti-phosphotyrosine antibody (Santa Cruz Biotechnology), β-actin antibody (Santa Cruz Biotechnology), and 2-deoxy-d-[³H] glucose (Amersham) were purchased from the indicated suppliers.

Western blot analysis.Cells were treated with or without TNF-α (25 ng/ml) and IL-6 (25 ng/ml) for 30 min after 16 h of serum starvation in Dulbecco modified Eagle medium. Cells were washed with phosphate-buffered saline (PBS) and lysed in TNTG buffer (30 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100). After freeze-thawing, the cell debris was removed by centrifugation. Samples were heated at 95°C for 5 min in sample reducing buffer, and the proteins were resolved by sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis. Resolved proteins were transferred onto nitrocellulose membrane (Bio-Rad) and blocked with 3% nonfat dry milk. The membrane was incubated with a primary antibody, followed by a secondary antibody coupled to horseradish peroxidase for detection of protein bands by using chemiluminescence (Amersham). Cellular β-actin was detected by using a specific antibody (Santa Cruz Biotechnology) for a relative comparison of the protein load in each lane. The results are representative of at least three independent experiments.

Glucose uptake by hepatocytes.Cells were seeded in a 12-well plate for glucose uptake assay as described earlier (15, 25). After serum starvation overnight in 0.1% bovine serum albumin-Dulbecco modified Eagle medium, the cells were treated with TNF-α (25 ng/ml) for 30 min and incubated for 2 h with glucose-free medium. The cells were subsequently incubated in 1 ml of PBS containing 100 nM insulin for 30 min at 37°C. After being washed with PBS, cells were incubated in 1 ml of PBS containing 1 μCi of 2-deoxy-d-[³H]glucose/ml for 5 min. The reaction was stopped by adding 0.1 mM cytochalasin B on ice. The cells were washed with ice-cold PBS and solubilized in 0.4 ml of NaOH (0.05 M). Radiolabeled glucose uptake by cells was measured in 4 ml of scintillation fluid by using a Beckman LS6500 scintillation counter. Glucose uptake is presented from the mean of multiple experiments.

HCV core protein expression upregulates Ser³¹² phosphorylation status of IRS-1.IRS-1 represents one of the four IRS isoforms that varies in molecular mass between 60 and 180 kDa (60). Serine sites subjected to phosphorylation play a key role in regulating IRS-1 function. Several serine residues were identified, and among them Ser³¹² has been extensively studied as a molecular indicator of insulin resistance (4, 25, 34). TNF-α, an inflammatory cytokine, plays an important role in the development of insulin resistance in individuals infected with HCV (51). Therefore, to understand the mechanism of insulin resistance, we first examined the Ser phosphorylation status of IRS-1 by Western blot analysis after TNF-α treatment of hepatocytes. Huh-7 cells exhibited an increase in the Ser³¹² phosphorylation level of IRS-1 upon treatment with TNF-α at 25 ng/ml as a positive control (Fig. 1A). Circulating IL-6 levels are known to increase insulin-resistant states (22, 37, 53). IL-6 inhibits insulin signaling in hepatocytes (49, 50). Therefore, we also examined whether IL-6 exerts an effect that is similar to TNF-α-mediated Ser phosphorylation of IRS-1. For this, we incubated hepatocytes expressing HCV protein with or without IL-6 (25 ng/ml), and the phosphorylation status of IRS-1 was determined by Western blot analysis. The results suggested that IRS-1 Ser³¹² phosphorylation is increased in IL-6-treated Huh-7 cells compared to the basal level (Fig. 1A). Interestingly, HCV core or HCV polyprotein expression from an FL clone also led to an increase in Ser³¹² phosphorylation of IRS-1 in Huh-7 cells (Fig. 1B and C) compared to Huh-7 cells alone (Fig, 1A). However, treatment with TNF-α or IL-6 did not significantly enhance the phosphorylation status of IRS-1 at Ser³¹² site in Huh-7 cells stably transfected with HCV core or full-length genomic region. Since Huh-7 cells support HCV genotype 2a (clone JFH1), the role of cell culture-grown HCV on IRS-1 Ser³¹² phosphorylation status was separately examined. The IRS-1 Ser³¹² phosphorylation level was compared between mock- and virus-infected cells. Western blot analysis suggested a higher level of Ser³¹² phosphorylated IRS-1 in virus-infected Huh-7 cells than in control Huh-7 cells (Fig. 1D). All of the experiments described above were repeated multiple times and suggested reproducible results. Similar results were observed with HepG2 cells (data not shown), suggesting that the observations are not specific to the Huh-7 cell line. Together, these results provided evidence that HCV core protein expression upregulates Ser³¹² phosphorylation in human hepatocytes, which is not augmented after TNF-α or IL-6 treatment.

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FIG. 1.

HCV core protein enhances Ser³¹² phosphorylation status of IRS-1 in hepatocytes. The results are shown from Western blot analysis with phospho-specific and total IRS-1 specific antibodies from Huh-7 cells (A), Huh-7 cells stably transfected with HCV core genomic region (B), or full-length genome of HCV (C). Cellular actin was used as an internal control to verify the level of protein load in each lane. The molecular masses of the specific protein bands were verified from the positions of prestained molecular mass markers (Cambrex). (D) Huh-7 cells were also infected with cell culture-grown HCV genotype 2a (clone JFH1) at a multiplicity of infection of 1, and the IRS-1 Ser³¹² phosphorylation level was compared between mock- and virus-infected cells.

Inhibition of Ser³¹² phosphorylation in IRS-1 by kinase inhibitors.JNK plays an important role by phosphorylating IRS-1 at the Ser³¹² residue (34). Although the molecular mechanisms of insulin resistance are multiple, evidence suggests that attenuation of insulin signaling by JNK may be a central part of the pathobiology of insulin resistance. We and others have shown previously that HCV core protein activates JNK and MAPK (21, 24, 27, 52, 58). Since a direct involvement of JNK in insulin signaling has been established (3, 4, 28), we determined the role of JNK inhibitor SP600125 (treated with 20 μM for 30 min) upon IRS-1 Ser³¹² phosphorylation status in the presence of HCV core protein. Our results suggested a significant inhibition of the IRS-1 Ser³¹² phosphorylation in Huh-7 core cells in the presence of SP600125 (Fig. 2A). On the other hand, the activation of PI-3K is thought to be critical in allowing insulin to stimulate both the uptake of glucose and the translocation of a specialized glucose transporter GLUT4 to the plasma membrane. We similarly determined whether HCV core protein-induced Ser³¹² phosphorylation of IRS-1 could be blocked by the PI-3K inhibitor LY294002. For this, HCV core expressing Huh-7 cells were serum starved for 16 h and incubated with LY294002 (20 μM for 30 h). Cell lysates were subjected to Western blot analysis for comparison of Ser³¹² phosphorylation status of IRS-1 between LY294002-treated cells and an untreated control. We observed an inhibition of IRS-1 Ser³¹² in Huh-7 core expressing cells in the presence of LY294002 (Fig. 2B). Together, our results demonstrated that both JNK and PI3-K inhibitors reduce HCV core-mediated upregulation of phospho-IRS-1.

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FIG. 2.

Inhibition of Ser³¹² phosphorylation in IRS-1 by kinase inhibitors. (A) The phosphorylation status of Ser³¹² of IRS-1 in untreated and JNK inhibitor SP600125-treated Huh-7 cells stably expressing HCV core protein was analyzed by Western blotting. (B) A similar analysis was performed with PI-3K inhibitor LY294002 to study the phosphorylation status of Ser³¹² in IRS-1. Cellular actin was used as an internal control to verify the level of protein load in each lane. A difference in electrophoretic mobilities of Ser³¹² of IRS-1 could be due to processing of the phosphorylated protein.

Infection of hepatocytes by cell culture grown HCV modulates Akt phosphorylation.Akt signaling regulates cell proliferation and survival, cell growth, glucose metabolism, cell motility, and angiogenesis (56). Akt is a downstream target of PI-3K, and proper regulation of the PI-3K-Akt pathway is critical for the prevention of insulin resistance. In the present study, we investigated Ser⁴⁷³ and Thr³⁰⁸ phosphorylation status of Akt in hepatocytes infected with cell culture-grown HCV genotype 2a (clone JFH1) or genotype 1a (clone H77). For this, Huh-7 cells were infected with HCV JFH1 and IHH were infected with HCV H77, since the respective human hepatocyte cell line supports virus growth. The results suggested that Ser⁴⁷³ phosphorylation of Akt is increased in HCV JFH1-infected Huh-7 hepatocytes, whereas the Thr³⁰⁸ phosphorylation level was not significantly altered after infection with HCV (Fig. 3). Similar results were obtained from three independent experiments. Interestingly, infection with cell culture-grown HCV H77 displayed a much greater effect upon Akt phosphorylation compared to mock-infected IHH. This difference could be due to the two distinct hepatic cell lines used in our experiments or to the inherent differences of the two infecting HCV genotypes. Nevertheless, a clear elevation of the phosphorylation status at Ser⁴⁷³ was noted in each case. We also have observed that phosphorylated Akt antibodies recognized other polypeptides. At this time, we do not know whether HCV induces other isoforms of pAkt, and this remains to be elucidated. Activation of Akt is a mediator of glucose transport by insulin stimulation. Full activation of Akt by insulin appears to require phosphorylation of Thr³⁰⁸ and Ser⁴⁷³ by PDK1 and TORc2, respectively. Our results indicated that HCV infection does not significantly modulate the Thr³⁰⁸ phosphorylation status of Akt in human hepatocytes, as it does with pSer⁴⁷³.

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FIG. 3.

Ser⁴⁷³/Thr³⁰⁸ phosphorylation status of Akt in hepatocytes infected with cell culture grown HCV. (A) Mock- or HCV JFH1-infected Huh-7 cells were analyzed for the phosphorylation status of Ser⁴⁷³ and Thr³⁰⁸ by Western blotting. (B) A similar analysis was performed with mock- or HCV H77-infected IHH. The total Akt (tAkt) level is shown at the bottom of each lane. Antibodies to phosphorylated Akt recognized other polypeptides and may represent isoforms of pAkt.

HCV core protein expression inhibits insulin-induced phosphorylation of Akt at Thr³⁰⁸.Insulin is known to activate Ser⁴⁷³ and Thr³⁰⁸ phosphorylation of Akt. The results from the studies described above demonstrated that infection of Huh-7 or IHH by cell culture-grown HCV genotype 1a or 2a alters the phosphorylation status of Akt, leading to activation in the absence of external mediators. Since we could not infect HepG2 cells with HCV clone JFH1 or H77, we investigated whether insulin-induced Ser⁴⁷³/Thr³⁰⁸ phosphorylation of Akt is modulated upon HCV core protein expression alone in HepG2 cells. Western blot analysis was performed with antibodies to phosphorylated Ser⁴⁷³ or Thr³⁰⁸ of Akt. Ser⁴⁷³ phosphorylation of Akt was increased after insulin treatment of HepG2 control cells and in HepG2 cells expressing HCV core protein (Fig. 4A). Treatment of insulin and TNF-α together did not significantly alter Ser⁴⁷³ phosphorylation status. On the other hand, Thr³⁰⁸ phosphorylation of Akt was induced by insulin in HepG2 control cells and was inhibited by TNF-α (Fig. 4B). Interestingly, insulin-induced Thr³⁰⁸ phosphorylation was not altered in HepG2 cells expressing HCV core protein. We have observed similar results from three independent experiments. Inhibition of Thr³⁰⁸, but not Ser⁴⁷³ phosphorylation of Akt, by 7-hydroxystaurosporine (a nonspecific inhibitor of PKC) has been suggested as a basis for decreased insulin-stimulated glucose transport (32, 59). We have also shown that phosphorylation of IRS-1 Ser³¹² is related to Akt phosphorylation status. Therefore, the present observations suggested that HCV core protein-induced insulin resistance may occur via the downregulation of Thr³⁰⁸ phosphorylation of Akt, which may serve as a signature molecule in HCV core-mediated insulin resistance.

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FIG. 4.

Ser⁴⁷³/Thr³⁰⁸ phosphorylation status of Akt in insulin treated hepatocytes. Parental HepG2 and HepG2 cells stably transfected with HCV core gene were treated with insulin alone or together with TNF-α. Untreated control or treated cell lysates were subjected to Western blot analysis for phosphorylation status of Ser⁴⁷³ (A) and Thr³⁰⁸ (B) and total Akt (tAkt).

HCV core protein expression decreases glucose uptake in human hepatocytes.A prominent mechanism linking steatosis and fibrogenesis is insulin resistance, although the molecular basis of insulin resistance remains unknown at present (6). TNF-α induces insulin resistance in experimental animals and cultured cells, and high levels of TNF-α have been observed in chronic hepatitis C patients (33). Akt is an enzyme that can be activated by insulin via a PI-3K-dependent mechanism, leading to the promotion of glucose uptake (63). Therefore, impairment of Akt activation leads to decreased glucose uptake. To understand the role of HCV core protein alone or in the context of other viral proteins in insulin resistance, the metabolic activity of insulin (100 nM) was evaluated with regard to radiolabeled glucose uptake by Huh-7 cells stably transfected with HCV genomic regions or infected with cell culture-grown virus. Approximately 1 multiplicity of infection of cell culture-grown HCV was used to infect most of the Huh-7 cells in culture. Glucose uptake was significantly reduced (∼5-fold) in Huh-7 cells stably transfected with HCV core, FL, or infected with HCV genotype 2a (clone JFH1) compared to its basal level in the control cell line (Fig. 5A). Interestingly, the exogenous addition of insulin or TNF-α did not significantly alter glucose uptake in Huh-7 cells expressing HCV protein(s). On the other hand, glucose uptake was increased in the presence of insulin in control Huh-7 cells, and cotreatment with insulin and TNF-α reduced glucose uptake to a level approaching that of untreated controls. Hepatocytes expressing HCV core protein were treated with the JNK inhibitor (SP600125) for 30 min prior to the measurement of 2-deoxy-d-[³H]glucose uptakes in the presence of insulin. The inhibition of glucose uptake by insulin-stimulated hepatocytes was significantly relieved by using 10 or 20 μM concentrations of the JNK inhibitor (Fig. 5B). However, PI-3K inhibitor (LY294002) did not impair glucose uptake, as was expected due to its mechanism of action through the downstream Akt signaling pathway. Therefore, our results suggested that insulin-induced glucose uptake is impaired in HCV core protein expressing human hepatocytes involving the JNK pathway.

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FIG. 5.

Insulin-stimulated glucose uptake by hepatocytes expressing HCV proteins. (A) Glucose uptake was determined by measuring the incorporation of 2-deoxy-d-[³H]glucose in HCV protein expressing or HCV JFH1-infected Huh-7 cells with or without treatment with insulin (100 nM) alone or together with TNF-α (25 ng/ml). The results from four different experiments are shown as bar diagrams with the standard errors indicated. (B) Hepatocytes expressing HCV core protein were treated with JNK inhibitor SP600125 (10 μM) or PI-3K inhibitor LY294002 (25 μM) for 30 min prior to measurement of 2-deoxy-d-[³H]glucose uptake in the presence of insulin. The results from four independent experiments are presented as bar diagrams with the indicated standard errors.