PT - JOURNAL ARTICLE AU - Martin Schubert AU - Stefanie Becher AU - Maria Wallert AU - Marten B. Maeß AU - Masoumeh Abhari AU - Knut Rennert AU - Alexander S. Mosig AU - Silke Große AU - Regine Heller AU - Michael Grün AU - Stefan Lorkowski TI - The Peroxisome Proliferator–Activated Receptor (PPAR)-<em>γ</em> Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPAR<em>δ</em> and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages AID - 10.1124/mol.119.117887 DP - 2020 Mar 01 TA - Molecular Pharmacology PG - 212--225 VI - 97 IP - 3 4099 - http://molpharm.aspetjournals.org/content/97/3/212.short 4100 - http://molpharm.aspetjournals.org/content/97/3/212.full SO - Mol Pharmacol2020 Mar 01; 97 AB - Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ.SIGNIFICANCE STATEMENT Peroxisome proliferator–activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.