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Assessment of total, ligand-induced peroxisome proliferator activated receptor ? ligand activity in serum.

ABSTRACT: BACKGROUND:Humans 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 (PPAR?) 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 PPAR? Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum. METHODS:First, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPAR? and RXR?, the obligate DNA-binding partner of PPAR?, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPAR? 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 PPAR? by ligands in serum from mice treated with a PPAR? ligand and commercial human serum samples. RESULTS:Cos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPAR?. Co-transfection of an RXR expression vector with the PPAR? 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 PPAR?, 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 PPAR? expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10??L 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 PPAR?-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPAR?-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands. CONCLUSIONS:The 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.

SUBMITTER: Edwards L

PROVIDER: S-EPMC6506953 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Assessment of total, ligand-induced peroxisome proliferator activated receptor γ ligand activity in serum.

Edwards Lariah L Watt James J Webster Thomas F TF Schlezinger Jennifer J JJ

Environmental health : a global access science source 20190509 1

<h4>Background</h4>Humans 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 ( ...[more]

PMID: 31072366

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Project description:Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ, and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2-8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms.

Dataset Information

Assessment of total, ligand-induced peroxisome proliferator activated receptor ? ligand activity in serum.

Publications

Assessment of total, ligand-induced peroxisome proliferator activated receptor γ ligand activity in serum.

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