Increased phosphoenolpyruvate carboxykinase gene expression and steatosis during hepatitis C virus subgenome replication: role of nonstructural component 5A and CCAAT/enhancer-binding protein β

Abstract

Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPβ and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.

FIGURE 1.

Pictorial view of HCV subgenomic replicon expressed in Huh.8 cells. Huh.8 cells contain the stable integration of HCV nonstructural components NS2, NS3, NS4A, NS4B, NS5A, and NS5B, whereas Ava.1 contains NS2, NS3, NS4A, NS4B, NS5B, and mutated NS5A with a 47-amino acid deletion.

FIGURE 2.

HCV protein expression increases PEPCK gene transcription in a CRE-dependent manner. A, luciferase activity of PEPCK-LUC was measured in Huh7, Huh.8, and Ava.1 cells as outlined under “Experimental Procedures.” The luciferase activity was corrected for transfection efficiency. The values are presented as the means ± S.E. *, p < 0.05 (n = 3). B and C, PEPCK RNA levels were measured by qPCR in uninfected control cells and cells infected with either Ad-GFP or Ad-NS5A in both Huh7 cells and isolated primary rat hepatocytes, respectively. The values are presented as the means ± S.E. *, p < 0.05 (n = 3). D, equal amounts of nuclear extracts were collected from HepG2 cells transfected with either Ad-GFP or Ad-NS5A, and subjected to Western blot analysis with NS5A and TATA binding protein (TBP). Representative blots are presented. E, PEPCK mRNA levels were measured by qPCR in cells infected with either Ad-GFP or Ad-NS5A, and the data were normalized to ubiquitin C. *, p < 0.001 (n = 4). F, glucose production was measured in 2-h medium samples of HepG2 cells infected with Ad-GFP or Ad-NS5A (n = 4). The values are presented as the means ± S.E. p = 0.054. G, overexpression of NS5A increased CREB phosphorylation in HepG2 cell line. Equal amounts of nuclear extracts were collected from HepG2 cells infected with Ad-GFP and Ad-NS5A and subjected to Western blot analysis. Representative blots are presented.

FIGURE 3.

PEPCK promoter analysis and gene expression in Huh.8 cells. A, PEPCK transcriptional activity was measured in Huh.8 cells by DNA transfections of promoter-less control construct (pGL3-LUC), PEPCK-LUC, CRE mutant PEPCK-LUC (CRE mutant), and GRE mutant PEPCK-LUC (−430-LUC mutant). The values are presented as the means ± S.E. *, p < 0.05 (n = 3). B, comparison of gluconeogenic gene expression in Huh7 and Huh.8 cells. Transcription factor and coactivator gene expression were analyzed by qPCR in Huh7 and Huh.8 cells, and the data were normalized to reference gene RPL13A (n = 3–4). The values are presented as the means ± S.E. *, p < 0.05.

FIGURE 4.

Insulin action on PEPCK, G-6-Pase, and pFOXO1 expression in NS5A-overexpressed HepG2 cells. HepG2 cells were infected with either Ad-GFP or Ad-NS5A and treated with or without 100 nm insulin for 6 h. A, PEPCK and G-6-Pase RNA levels were measured by qPCR. The values are presented as the means ± S.E. *, p < 0.05 versus no insulin; #, p < 0.005 versus Ad-GFP; **, p < 0.02 versus Ad-GFP + insulin (n = 3). B, equal amounts of nuclear/cytoplasmic (Cyto) extracts were collected from the similar treatment and subjected to Western blot analysis with pFOXO1 (Ser-256), total FOXO1, and TATA binding protein (TBP) or tubulin as a loading control. The representative blot is a composite of a larger blot in which all the samples were run simultaneously together, and the composite was made by splicing complete lanes together for presentation purposes. Quantification by densitometry was expressed as ratio of nuclear pFOXO1 (Ser-256) to total FOXO1 and nuclear to cytoplasmic total FOXO1. The values are presented as the means ± S.E. (n = 3). #, p < 0.05 versus Ad-NS5A control; *, p < 0.05 versus Ad-GFP control.

FIGURE 5.

Insulin signaling in Huh7 and Huh.8 cells. A, Huh7 and Huh.8 cells were serum-starved and treated with insulin (100 nm) or left untreated. Equivalent amounts of extracted proteins were subjected to Western blot analysis for Tyr-1146-phosphorylated IR-β relative to total IR-β, Ser-473-phosphorylated Akt relative to total Akt, and basal Ser-312-phosphorylated IRS-1 relative to total IRS-1. Representative blots are presented (for IR-β and AKT, these are composites of a larger blot where samples were run simultaneously). Insulin-stimulated fold induction relative to Huh7 (set to 1) is shown in bar graphs for pIR-β (Tyr-1146) and pAkt (Ser-473), normalized to total IR-β and Akt, respectively; and basal fold change relative to Huh7 is shown for pIRS-1 (Ser-312) normalized to total IRS-1. The values are the means ± S.E. *, p < 0.05 (n = 3). B, inhibition of the cellular response to insulin in HCV-replicating cells was measured by fluorescence assay. Mobilization of insulin-responsive vesicles was assessed by real time imaging using FM1-43 fluorescence. The rate of FM1-43 fluorescence provides a direct measurement of the cellular exocytotic activity. The dark bars represent the rates of constitutive exocytosis, and the light bars represent the rates of exocytosis after stimulation with insulin. The values are the means ± S.E. *, p < 0.05 (n = 3). Representative Huh7, Huh.8, and Ava.1 cells are shown with and without insulin treatment.

FIGURE 6.

Involvement of CREB and C/EBPβ in HCV-mediated activation of PEPCK transcription. A, equal amounts of protein extracts (whole cell or nuclear) were collected from Huh7, Huh.8, and Ava.1 cell lines and subjected to Western blot analysis with CREB or pCREB (Ser-133). Representative blots are shown. B, Huh7 and Huh.8 cells were infected with recombinant adenovirus encoding dominant-negative CREB (Ad-ACREB) or Ad-GFP control and transfected with PEPCK-LUC. Luciferase activities of PEPCK-LUC were then measured and compared in both cell lines. The values are presented as the means ± S.E. *, p < 0.05 (n = 3). C, equivalent amounts of nuclear protein extract were collected from Huh7 and Huh.8 cell lines and subjected to Western blot analysis with C/EBPβ. Representative blots are presented. D, Huh.8 cells were infected with nontargeted or C/EBPβ shRNA adenoviruses, and expression of C/EBPβ was analyzed by Western blot. The values are presented as the means ± S.E. *, p < 0.05 (n = 2). E, comparison of transcription factor gene expression under C/EBPβ knockdown conditions in Huh7 and Huh.8 cells. RNA expression was analyzed by qPCR in Huh7 and Huh.8 cells, and the data was normalized to RPL13A expression. The values are presented as the means ± S.E. *, p < 0.05 Adcntl versus AdC/EBPβ (n = 3–5).

FIGURE 7.

Intracellular lipids in Huh.8 cells. Huh7, Huh.8, and Ava.1 cells were stained with Nile Red, and representative cells are shown. Quantitative measurement of lipids in Huh7, Huh.8, and Ava.1 cells are shown as relative fluorescence. The values are the means ± S.E. *, p < 0.05 versus Huh7 and Ava.1 cells (n = 3).

FIGURE 8.

Comparison of gene expression and TG accumulation in Huh7 and Huh.8 cells and the role of C/EBPβ knockdown. A, lipogenic gene expression was analyzed by qPCR in Huh7 and Huh.8 cells, and the data were normalized to RPL13A expression. The values are presented as the means ± S.E. *, p < 0.05 (n = 3–5). B, identical lipogenic gene expression was measured in Huh7 and Huh.8 cells after infection with nontargeting or C/EBPβ shRNA adenoviruses. The values are presented as the means ± S.E. *, p < 0.05 versus Ad-shcntl (n = 3–5). C, Huh7 and Huh.8 cells were infected with nontargeting or AdC/EBPβ shRNA adenoviruses, and TG accumulation was measured as detailed under “Experimental Procedures.” The values are presented as the means ± S.E. *, p < 0.05 versus Huh7 control (n = 2). D, fatty acid oxidation and mitochondrial transcription factor gene expression was analyzed by qPCR in Huh7 and Huh.8 cells as in A and B, and the data were normalized to RPL13A expression. The values are presented as the means ± S.E. *, p < 0.05 versus Huh7 cntl; #, p < 0.05 versus Ad-shcntl (n = 3–5).