Metformin promotes CAR phosphorylation by activating AMPK. (A) Human hepatocytes (HL#78 and #81) were treated with DMSO (0.1%), AICAR (100, 250 µM), PB (1 mM), CITCO (1 µM), or their combinations as indicated. CYP2B6 expression was assayed by real-time PCR and Western blot analysis. (B) Human hepatocytes (HL#74 and #83) were treated with 0.1% DMSO, PB (1 mM), CITCO (1 µM), MET (1 mM), Compound C (CC; 5, 10, 20 μM), or their combinations as outlined in Materials and Methods. Total RNA from each group was analyzed by real-time PCR for CYP2B6 expression. Homogenate proteins from each treated hepatocytes were used for Western blot analysis of CYP2B6, hCAR (Thr38), and β-actin. (C) Human hepatocytes (HL#81) were treated with MET (1 mM), CC (20 µM), or their combination for 0, 15 minutes, and 1, 4, and 24 hours. Homogenate proteins from treated hepatocytes were used for Western blot analysis of hCAR (Thr38), AMPK (Thr172), and β-actin. (D) Phosphorylation of AMPK (Thr172) was also measured in human hepatocytes (HL#78, #82, and #83) after the treatment of vehicle control (0.1% DMSO), PB (1 mM), or CITCO (1 µM) for 24 hours. Data represent the mean ± S.D. of three independent transfections (**P < 0.01).