cGPDH is a direct PPARα/γ target gene. Mouse cGPDH reporter constructs containing 2240, 560, or 280 bp of immediate upstream promoter region were transfected into NIH-3T3 cells together with a PPARα (A) or PPARγ (B) expression vector. Normalized activity of the full-length cGPDH reporter in the absence of PPAR and ligands was set at 1. (C) Binding of the PPAR/RXR heterodimer to putative response elements, as determined by electrophoretic mobility shift assay. The double-stranded response elements cGPDH-PPRE1 (lanes 1–8). Fold-excess of specific (SC) or nonspecific (NSC) cold probe is indicated. (D) Expression of cGPDH during 3T3-L1 adipogenesis as determined by Q-PCR. Expression at day 8 was set at 100%. ChIP of PPRE within mouse cGPDH promoter using anti-mPPARγ or anti-mPPARα antibodies. Gene sequences spanning the putative PPREs (+1020 to +782) and a random control sequence (+2519 to +2124) were analyzed by PCR in the immunoprecipitated chromatin of 3T3-L1 preadipocytes and adipocytes (E), fed and fasted wild-type and PPARα-null mice (F), and wild-type and PPARα-null mice treated or not with Wy14643 (G). Preimmune serum was used as a control. (H) Transcriptional activity of site-directed mutants (mut) of the cGPDH promoter. Mouse cGPDH reporter constructs containing double nucleotide changes in PPRE1, PPRE2, or both, were transfected into HepG2 cells together with a PPARγ expression vector. Normalized activity of the reporter in the absence of PPAR and ligand was set at 1. Error bars in A, B, and H represent SEM. Cntl, random control sequence; PI, preimmune serum; prom, promoter; Veh, vehicle; Wy, Wy14643; for, forward primer; rev, reverse primer.