ARS | Publication request: Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß)

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 5, 2012
Publication Date: September 6, 2012
Citation: Qadri, I., Choudhury, M., Rahman, S.M., Knotts, T.A., Janssen, R.C., Schaack, J., Iwahashi, M., Puljak, L., Simon, F.R., Kilic, G., Fitz, J.G., Friedman, J.E. 2012. Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß). Journal of Biological Chemistry. 10.1074.

Interpretive Summary: Hepatitis C virus (HCV) infection is one of the leading causes of liver disease. HCV typically results in a prolonged, clinical course that may progress over decades toward liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma. HCV infection is strongly associated with nonalcoholic fatty liver disease (NAFLD) and the development of diabetes. Mounting evidence suggests that HCV-associated insulin resistance may be an underlying cause of hepatic steatosis and progression to diabetes. Initially, the HCV core was implicated in the pathogenesis of steatosis and insulin resistance in patients with HCV infection; however, recent studies suggest that nonstructural component-5A (NS5A) represents a candidate protein that could contribute to the pathogenesis of lipid formation and by extension insulin resistance. Whereas a role for NS5A in HCV-induced steatosis has been suggested, its role in the regulation of genes involved in hepatic glucose metabolism, particularly the rate-limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), remains unclear. The contribution of C/EBPß to activation of the gluconeogenic and lipogenic program in HCV cells has thus far not been addressed experimentally. In the present study, we provide novel information that HCV-replicating cells, in the absence of inflammatory cytokines, dramatically activate PEPCK gene expression and genes coding for lipid uptake and the de-novo pathway for lipogenesis. This is accompanied by inhibition of insulin signaling and increased lipid accumulation; all important characteristics underlying the progression to NAFLD. Our results reveal that both NS5A and C/EBPß knockdown separately suppress several key genes important for gluconeogenesis and de-novo lipogenesis indicating that C/EBPß, in addition to NS5A, may control genes critical for the progression to diabetes in HCV-infected cells.

Technical 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 upregulated in Huh.8 cells which stably express an HCV subgenome replicon. HCV increased activation of CREB, C/EBPß, FOXO1, and PGC-1a and involved activation of the CRE in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPß-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1a 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, while 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 IRS- 1(Ser312), and decreased Akt(Ser473) activation in response to insulin. C/EBPß-RNAi normalized lipogenic genes SREBP-1c, PPARgamma, and LXRa, 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/EBPß, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPß and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.