title: 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.
authors.affiliation: Department of Pediatrics, University of Colorado Denver, Aurora, Colorado 80045, USA.
authors.forename: Ishtiaq
authors.initials: I
authors.lastname: Qadri
authors.forename: Mahua
authors.initials: M
authors.lastname: Choudhury
authors.forename: Shaikh Mizanoor
authors.initials: SM
authors.lastname: Rahman
authors.forename: Trina A
authors.initials: TA
authors.lastname: Knotts
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Janssen
authors.forename: Jerome
authors.initials: J
authors.lastname: Schaack
authors.forename: Mieko
authors.initials: M
authors.lastname: Iwahashi
authors.forename: Livia
authors.initials: L
authors.lastname: Puljak
authors.forename: Francis R
authors.initials: FR
authors.lastname: Simon
authors.forename: Gordan
authors.initials: G
authors.lastname: Kilic
authors.forename: J Gregory
authors.initials: JG
authors.lastname: Fitz
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Friedman
mesh_terms: D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000821:virology; D004790:Enzyme Induction; D005234:Fatty Liver / Q000201:enzymology / Q000821:virology*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D016679:Genome, Viral; D005943:Gluconeogenesis / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000502:physiology; D050356:Lipid Metabolism / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism; D015398:Signal Transduction; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D017362:Cyclic AMP Response Element-Binding Protein; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D005947:Glucose; D005952:Glucose-6-Phosphatase; D007328:Insulin; D008156:Luciferases; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D017361:Viral Nonstructural Proteins; C545012:CEBPB protein, human; C115163:CREB1 protein, human; C085126:FOXO1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase