FUNCTIONAL ANTAGONISM BETWEEN CCAAT ENHANCER BINDING PROTEIN-ALPHA AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA ON THE LEPTIN PROMOTER/

The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass, Recent work has suggested that the antidiabetic ag ents, the thiazolidinediones (TZ), which are also high affinity ligand s of peroxisome proliferator-activated receptor-gamma (PPAR gamma), in hibit leptin expression in rodents, To examine the effects of this cla ss of drug on the leptin gene in adipocytes we performed Northern anal ysis on primary rat adipocytes cultured in the presence or absence of TZ, TZ reduced leptin mRNA levels by 75%, To determine whether this ef fect was mediated at the transcriptional level, we isolated 6510 base pairs of 5'-flanking sequence of the leptin promoter and studied repor ter constructs in primary rat adipocytes and CV-1 cells, Sequence anal ysis demonstrated the presence of a consensus direct repeat with a 1-b ase-pair gap site between -3951 and -3939 as well as a consensus CCAAT /enhancer binding protein (C/EBP) site between -55 and -47, Our functi onal analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promot er constructs ranging from -6510 to +9 to -65 to +9, In CV-1 cells, wh ich contain endogenous PPAR gamma, TZ treatment alone had little effec t on these constructs, However, TZ treatment did inhibit C/EBP alpha-m ediated transactivation of the leptin promoter, This down-regulation o f leptin reporter constructs mapped to a -65 to +9 promoter fragment w hich binds C/EBP alpha in gel mobility shift assays but does not bind PPAR gamma 2 alone or as a heterodimer with 9-cis-retinoic acid recept or, Conversely, the promoter (-5400 to +24 base pairs) of the aP2 gene , another adipocyte-specific gene, was induced 7.3-fold by TZ, Co-tran sfection with C/EBP alpha minimally stimulated the aP2 promoter from b asal levels but notably blocked activation by TZ, These data indicate that PPAR gamma and C/EBP alpha can functionally antagonize each other on at least two separate promoters and that this mechanism may explai n the down-regulation of leptin expression by thiazolidinediones.