A novel method for analysis of nuclear receptor function at natural promoters: Peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation

Peroxisome proliferator-activated receptor-gamma (PPAR gamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the me diation of the actions of antidiabetic drugs known as thiazolidinediones (T ZDs). PPAR gamma activates many target genes involved in lipid anabolism in cluding the adipocyte fatty acid binding protein (aP2). In this study, indu ction of aP2 gene expression by PPAR gamma agonists was examined in both cu ltured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quan titation, bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a tim e frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BR L-49653 on aP2 mRNA levels was detectable as early as 30 min after treatmen t (47% increase) and was maximal after 24 h of treatment (12-fold increase) . The effects of troglitazone on aP2 mRNA induction were similar to those o f BRL-49653 except that the maximal level of induction was consistently low er (e.g. 24 h treatment 4-fold increase). Dose-response relationships for b oth of the TZDs were also determined using the 24-h treatment time point. E C(50)s for both BRL-49653 and troglitazone were estimated to be 80 nM and 6 90 nM, respectively. A natural PPAR gamma ligand, 15-deoxy-Delta(12,14)-PGJ (2), was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 mu M. Since the PPAR gamma-retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodi mer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis- RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficac y (2-fold induction), the maximal effect was reached at 100 nM. No synergis m or additivity in aP2 mRNA induction was detected when 9-cis-RA was includ ed with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL- 49653 was also detected, indicating that the bDNA assay may be a simple met hod to monitor nuclear receptor target gene induction in vivo.