PPARα governs glycerol metabolism
J. Clin. Invest. David Patsouris, et al. 114:94 doi:10.1172/JCI20468 [
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Figure 4cGPDH 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.
