Utilizing systems biology to reveal cellular responses to peroxisome proliferator-activated receptor γ ligand exposure

Peroxisome proliferator-activated receptor gamma (PPAR gamma) 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 PPAR gamma plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPAR gamma-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 PPAR gamma ligand exposure on genome-wide PPAR gamma 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 PPAR gamma agonist) and GW 9662 (a reference PPAR gamma antagonist) on human hepatocarcinoma (HepG2) cell viability, PPAR gamma 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 PPAR gamma-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 PPAR gamma 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 PPAR gamma-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 PPAR gamma ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPAR gamma-dependent and PPAR gamma-independent mechanisms.