FIGURE 2.
Inhibition of PEPCK-C reporter by AICAR in HepG2 cells in the presence and the absence of forskolin. AICAR or the GSK3β inhibitor SB-415286 has the potential to down-regulate CRE-dependent transcription in the presence and in the absence of 10 μm forskolin. HepG2 cells were transfected with the PEPCK-C promoter fused to the luciferase reporter plasmid (pGL4.10-PEPCK-C: 0.5 μg) with an internal reporter, pRL-TK (0.03 μg). HepG2 cells were serum-starved overnight before incubation with AICAR (2 mm) or insulin (10 nm) with or without 10 μm forskolin. After a 6-h incubation, cells were harvested for the reporter assay. A, the PEPCK-C-dependent reporter activities that had been normalized with internal reporter activities were expressed as -fold activities of the empty reporter (pGL4.10). B, HepG2 cells were transfected with the CRE-reporter plasmid (pTAL-CRE: 0.25 μg) with the internal reporter pRL-TK (0.03 μg). HepG2 cells were serum-starved overnight before incubation with AICAR (2 mm), insulin (10 nm), the selective AMPK inhibitor Compound C (20 μm), GSK3β inhibitor, or SB-415286 (30 μm) with or without 10 μm forskolin. After a 6-h incubation, cells were harvested for the reporter assay. C, AMPK (WT, DN) was overexpressed by adenoviral (Ad) gene transfer, and expressions were detected by anti-AMPK antibody (upper blot in the right panel). AMPK activity was monitored by antiphospho-Ser79-ACC (lower blot in the right panel). The cells were transfected with the CRE-reporter plasmid (pTAL-CRE, 0.25 μg) with the internal reporter pRL-TK (0.03 μg), and the reporter assay was performed.