This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Burris, T. P. Articles by Demarest, K. T. Search for Related Content PubMed PubMed Citation Articles by Burris, T. P. Articles by Demarest, K. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB TRANS-RETINOIC ACID

A Novel Method for Analysis of Nuclear Receptor Function at Natural Promoters: Peroxisome Proliferator-Activated Receptor Agonist Actions on aP2 Gene Expression Detected Using Branched DNA Messenger RNA Quantitation

Endocrine Therapeutics Department of Drug Discovery The R.W. Johnson Pharmaceutical Research Institute Raritan, New Jersey 08869

Peroxisome proliferator-activated receptor- (PPAR), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPAR activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPAR agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time 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 BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (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 of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC₅₀s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPAR ligand, 15-deoxy-^12,14-PGJ₂, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 µM. Since the PPAR:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer 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 efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included 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 method to monitor nuclear receptor target gene induction in vivo.

This article has been cited by other articles:

				T. P. Burris, C. Montrose, K. A. Houck, H. E. Osborne, W. P. Bocchinfuso, B. C. Yaden, C. C. Cheng, R. W. Zink, R. J. Barr, C. D. Hepler, et al. The Hypolipidemic Natural Product Guggulsterone Is a Promiscuous Steroid Receptor Ligand Mol. Pharmacol., March 1, 2005; 67(3): 948 - 954. [Abstract] [Full Text] [PDF]

				Y. D. Tchoukalova, M. G. Sarr, and M. D. Jensen Measuring committed preadipocytes in human adipose tissue from severely obese patients by using adipocyte fatty acid binding protein Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1132 - R1140. [Abstract] [Full Text] [PDF]

				Z. He, T. Jiang, Z. Wang, M. Levi, and J. Li Modulation of carbohydrate response element-binding protein gene expression in 3T3-L1 adipocytes and rat adipose tissue Am J Physiol Endocrinol Metab, September 1, 2004; 287(3): E424 - E430. [Abstract] [Full Text] [PDF]

				G. Cao, Y. Liang, C. L. Broderick, B. A. Oldham, T. P. Beyer, R. J. Schmidt, Y. Zhang, K. R. Stayrook, C. Suen, K. A. Otto, et al. Antidiabetic Action of a Liver X Receptor Agonist Mediated By Inhibition of Hepatic Gluconeogenesis J. Biol. Chem., January 3, 2003; 278(2): 1131 - 1136. [Abstract] [Full Text] [PDF]

				A. N. Player, L.-P. Shen, D. Kenny, V. P. Antao, and J. A. Kolberg Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization J. Histochem. Cytochem., May 1, 2001; 49(5): 603 - 612. [Abstract] [Full Text]

				M. Gurnell, J. M. Wentworth, M. Agostini, M. Adams, T. N. Collingwood, C. Provenzano, P. O. Browne, O. Rajanayagam, T. P. Burris, J. W. Schwabe, et al. A Dominant-negative Peroxisome Proliferator-activated Receptor gamma (PPARgamma ) Mutant Is a Constitutive Repressor and Inhibits PPARgamma -mediated Adipogenesis J. Biol. Chem., February 25, 2000; 275(8): 5754 - 5759. [Abstract] [Full Text] [PDF]