Reciprocal interference between insulin and interferon-alpha signaling in hepatic cells: a vicious circle of clinical significance?

UNLABELLED: Insulin resistance (IR) is common in chronic hepatitis C (CHC) and associates with reduced virological response to pegylated-interferon (PEG-IFN)/ribavirin therapy, but the underlying mechanisms are still unclear. We have previously shown that, in CHC patients, insulin plasma levels are inversely related to antiviral effect induced by PEG-IFN. Therefore, we investigated the in vitro effect of insulin on interferon alpha (IFN-α) intracellular signaling as well as that of IFN-α on insulin signaling. HepG2 cells, preincubated with or without insulin, were stimulated with IFN-α2b and messenger RNA (mRNA) and protein expression of IFN-stimulated genes (ISGs) were measured at different timepoints. The role of intracellular suppressors of cytokine signaling 3 (SOCS3) was evaluated with the small interfering RNA (siRNA) strategy. To assess the effect of IFN-α on insulin signaling, HepG2 were preincubated with or without IFN before addition of insulin and cells were then analyzed for IRS-1 and for Akt/PKB Ser473 phosphorylation. Insulin (100 and 1000 nM) significantly reduced in a dose-dependent fashion IFN-induced gene expression of PKR (P=0.017 and P=0.0017, respectively), MxA (P=0.0103 and P=0.00186), and 2'-5' oligoadenylatesynthetase 1 (OAS-1) (P=0.002 and P=0.006). Insulin also reduced IFN-α-induced PKR protein expression. Although insulin was confirmed to increase SOCS3 expression, siRNA SOCS3 did not restore ISG expression after insulin treatment. IFN-α was found to reduce, in a dose-dependent fashion, IRS-1 gene expression as well as Akt/PKB Ser473 phosphorylation induced by insulin. CONCLUSION: These results provide evidence of reciprocal interference between insulin and IFN-α signaling in liver cells. These findings may contribute to understand the role of insulin in CHC: IR might be favored by endogenous cytokines including IFN-α, and the resulting hyperinsulinemia then reduces the antiviral response to exogenous IFN in a vicious circle of clinical significance.

Comparable performance of TMA and Real-Time PCR in detecting minimal residual hepatitis C viraemia at the end of antiviral therapy.

BACKGROUND: Antiviral therapy for chronic hepatitis C may cause transient on-treatment response followed by post-treatment relapse. OBJECTIVES: We have compared the prognostic value for post-treatment relapse of minimal hepatitis C residual viraemia detected at end-of-therapy by transcription mediated assay (TMA) and by Abbott RealTime PCR HCV assay. STUDY DESIGN: Minimal residual viraemia was investigated in 202 HCV patients who had completed a full course of Pegylated Interferon (PEG-IFN) plus ribavirin and were HCV-RNA negative by conventional PCR in two separate serum samples obtained during the last week of therapy and the results were then correlated with post-treatment outcome. RESULTS: Minimal residual viraemia was detected in 22/202 (11%, 95% CI: 7-16%) and in 28/202 (13.8%, 95% CI 10-19%) patients by TMA and by Abbott RealTime HCV assay, respectively, with 92% concordant results. Post-treatment relapse was seen in 81.8% (95% CI: 60-93%) of TMA positive and in 82.1% (95% CI: 64-92%) of Abbott RealTime HCV assay positive cases compared to 16.6% (95% CI: 12-23%) of TMA negative and 17.2% (95% CI: 12-23%) of Abbott RealTime HCV assay negative patients. CONCLUSIONS: These results indicate that TMA and the Abbott RealTime HCV assay have comparable sensitivity and specificity in identifying minimal residual viraemia at the end of antiviral therapy, with excellent predictive value for post-treatment relapse.

Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes.

BACKGROUND/AIMS: The hepatitis C virus NS5A protein is phosphorylated by several cellular kinases, including casein kinase 2 (CK2). Little is known about CK2 phosphorylation of NS5A from different HCV genotypes and clinical isolates. METHODS: NS5A from patients with HCV-1a (24 cases), HCV-1b (9) or HCV-3 (16) was analyzed by direct sequencing and CK2 phosphorylation sites were defined using a well-validated prediction rule. In vitro phosphorylation assays were performed using recombinant CK2 and synthetic peptides or full-length NS5A. In vivo phosphorylation by endogenous CK2 of NS5A expressed in hepatoma cells was also investigated. RESULTS: The mean number of CK2 sites within full-length NS5A, was significantly higher in HCV-3 compared to HCV-1a (P<0.01) and HCV-1b (P<0.01). The number of CK2 sites was more homogeneous in HCV-3 variants compared to HCV-1a and HCV-1b variants (P<0.05). The number of predicted CK2 sites correlated with the degree of in vitro and in vivo phosphorylation of NS5A by CK2. CONCLUSIONS: CK2-dependent phosphorylation of NS5A is heterogeneous among different HCV genotypes and clinical isolates. This might have an influence on virus biology and pathogenicity.

Impact of NS5A sequences of Hepatitis C virus genotype 1a on early viral kinetics during treatment with peginterferon- alpha 2a plus ribavirin.

UNLABELLED: BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. METHODS: Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. RESULTS: At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. CONCLUSIONS: Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.

BACKGROUND & AIMS: Hepatic steatosis is frequent in chronic hepatitis C. Several mechanisms might be implicated, including metabolic cofactors and direct viral effects on intracellular lipid pathways. In a transgenic mouse model, hepatitis C virus (HCV) was shown to inhibit microsomal triglyceride transfer protein (MTP) activity, which is essential for hepatic lipoprotein assembly and secretion. No data are available on liver MTP activity in HCV-infected patients. We therefore investigated liver MTP gene expression and its lipid transfer activity in untreated cases infected with the major HCV genotypes showing variable degrees of hepatic steatosis. METHODS: MTP messenger RNA (mRNA) levels were measured by real-time polymerase chain reaction, and MTP activity was assessed by fluorescent assay in liver biopsy specimens of 58 HCV-positive patients. A set of metabolic and serum lipid markers was also measured at the time of liver biopsies. RESULTS: MTP mRNA levels showed a statistically significant (P = .001) inverse correlation with the degree of steatosis, independently of the HCV genotype. MTP mRNA levels also had an inverse correlation with serum insulin (P = .0002), homeostasis model assessment-insulin resistance (HOMA-IR) (P = .005), and body mass index (P = .02) in patients with HCV-1 and HCV-2 and with serum HCV-RNA (P = .02) in HCV-3 patients. Liver MTP-specific activity was significantly reduced in HCV-3 patients compared with those with other HCV genotypes (P = .004) and correlated with reduced serum cholesterol, apo B, and low-density lipoproteins. CONCLUSIONS: MTP may play a central role in HCV-related steatosis, being modulated by different genotype-specific mechanisms, mainly hyperinsulinemia in non-HCV-3 patients, and more profound and direct virus-related effects in HCV-3-infected individuals.

Hepatitis C minimal residual viremia (MRV) detected by TMA at the end of Peg-IFN plus ribavirin therapy predicts post-treatment relapse.

BACKGROUND/AIMS: Around 15-25% of chronic hepatitis C patients treated with Peg-IFN plus ribavirin become HCV-RNA negative by PCR during therapy but relapse after its withdrawal. We investigated whether minimal residual viremia (MRV) might be detected in these cases by Transcription-Mediated Amplification (TMA). METHODS: Two hundred and ninety-two consecutive patients (143 HCV-1, 82 HCV-2, 56 HCV-3 and 11 HCV-4) were prospectively treated with a standard schedule of Peg-IFNalpha 2b plus ribavirin combination and end-of-therapy response was assessed by conventional PCR using 2 protocol serum samples obtained 6-8h before the last two scheduled weekly injections of Peg-IFN. PCR negative samples were re-tested by TMA and the results were then correlated with the virological outcome after therapy withdrawal. RESULTS: Among 208 patients who were repeatedly HCV-RNA negative by PCR at the end-of-therapy, 26 (12.5%) were found HCV-RNA positive by TMA. Twenty-two of them, (96%) were PCR-relapsers after therapy withdrawal, compared to only 14% of the 182 TMA negative patients (P<0.0001). This virological profile was more frequent in HCV-1 and HCV-3 infected patients and correlated with a slower virological response during therapy. CONCLUSIONS: At the end of Peg-IFN plus ribavirin therapy, TMA is superior to PCR in identifying patients with sustained HCV-RNA clearance.

PKR gene expression and response to pegylated interferon plus ribavirin therapy in chronic hepatitis C.

Pegylated interferon (PEG-IFN) alpha combined with ribavirin is the current standard treatment for hepatitis C, but around 50% of patients do not respond for reasons that are not fully understood. To explore the regulation of IFN-inducible protein kinase (PKR), we have measured PKR mRNA levels in peripheral blood mononuclear cells (PBMCs) and in liver biopsies from patients with chronic hepatitis C. PBMCs were also analysed after in vitro incubation with IFN and during antiviral therapy. Non-responders to PEG-IFN plus ribavirin had pre-treatment PKR mRNA levels in PBMCs (0.1+/-0.0074) and in liver (0.102+/-0.051) that were significantly higher than those of responders (PBMCs: 0.023+/-0.014, P=0.0005; liver: 0.034+/-0.020; P=0.0002). On the other hand, PKR mRNA levels in PBMCs were similar in non-responders and in responders after in vitro exposure to IFN (0.434+/-0.301 vs 0.403+/-0.222; P=NS) and during therapy (0.31+/-0.10 vs 0.30+/-0.12; P=NS). These results indicate that in hepatitis C, non-responsiveness to IFN-alpha is associated with pre-treatment up-regulation of the PKR gene, evidence that the infecting hepatitis C virus is able to stimulate endogenous IFN production, being resistant to its antiviral effect. On the other hand, the PKR gene response to exogenous IFN was similar in responders and non-responders, at least in PBMCs, suggesting that variations in its activation are not major determinants of the outcome of antiviral treatment.