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A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation.

ABSTRACT: The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARgamma subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2, 4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARgamma ligand that was a weak partial agonist of PPARgamma transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.

SUBMITTER: Oberfield JL

PROVIDER: S-EPMC26842 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation.

Oberfield J L JL Collins J L JL Holmes C P CP Goreham D M DM Cooper J P JP Cobb J E JE Lenhard J M JM Hull-Ryde E A EA Mohr C P CP Blanchard S G SG Parks D J DJ Moore L B LB Lehmann J M JM Plunket K K Miller A B AB Milburn M V MV Kliewer S A SA Willson T M TM

Proceedings of the National Academy of Sciences of the United States of America 19990501 11

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARgamma subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2, 4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix ...[more]

PMID: 10339548

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Project description:BackgroundHumans are exposed to a complex mixture of environmental chemicals that impact bone and metabolic health, and traditional exposure assessments struggle to capture these exposure scenarios. Peroxisome proliferator activated receptor-gamma (PPARγ) is an essential regulator of metabolic and bone homeostasis, and its inappropriate activation by environmental chemicals can set the stage for adverse health effects. Here, we present the development of the Serum PPARγ Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum.MethodsFirst, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPARγ and RXRα, the obligate DNA-binding partner of PPARγ, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPARγ ligand and/or LG100268, a synthetic RXR ligand, to characterize the dose response and determine the simplest and most efficacious format. Following optimization of the bioassay, we assessed the cumulative activation of PPARγ by ligands in serum from mice treated with a PPARγ ligand and commercial human serum samples.ResultsCos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPARγ. Co-transfection of an RXR expression vector with the PPARγ expression vector did not increase PPRE transcriptional activity induced by rosiglitazone. Treatment with an RXR ligand marginally increased PPRE transcriptional activity in the presence of transfected PPARγ, and co-treatment with an RXR ligand reduced rosiglitazone-induced PPRE transcriptional activity. Therefore, the final bioassay protocol consists of transfecting Cos-7 cells with a PPARγ expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10 μL of serum, and measuring luminescence and fluorescence after a 24 h incubation. Sera from mice dosed with rosiglitazone induced PPRE transcriptional activity in the SPAA in a dose-dependent and PPARγ-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPARγ-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands.ConclusionsThe SPAA can reliably measure total PPRE transcriptional activity in small volumes of serum. This system provides a sensitive, straightforward assay that can be reproduced in any cell culture laboratory.

Dataset Information

A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation.

Publications

A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation.

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