Effects of phytoceramide on PPAR transcriptional activity. (A) NIH/3T3 cells were transfected with 4 × PPRE firefly luciferase reporter plasmid along with expression plasmids for PPARs (PPARα, PPARβ or PPARγ) and RXR and an internal control TK Renilla luciferase vector. After 24 h cultivation, the cells were treated with the indicated concentration of phytoceramide or ceramide for 24 h. As positive control, 10 μM of WY-14643 (WY), L-165,041 (LD) or ciglitazone (CIG) were used. (B) NIH/3T3 cells were transfected as described above, expect for an empty luciferase reporter instead of 4 × PPRE firefly luciferase reporter. Twenty-four hours after transfection, phytoceramide was added to the cells for 24 h. The luciferase activity of the cell was measured, and firefly luciferase activity was normalized to Renilla luciferase activity. The activity of a vehicle control was expressed as 1 and the relative luciferase activities were presented as a fold induction to that of the vehicle control. All results are shown as means ± S.D. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the level of vehicle control (Dunnett's test).