Differential Potencies of Naturally Occurring Regioisomers of Nitrolinoleic Acid in PPARγ Activation

Previous studies demonstrated that the naturally occurring electrophile and PPARγ ligand, nitrolinoleic acid (NO₂-LA), exists as a mixture of four regioisomers [Alexander, R. L., et al. (2006) Biochemistry 45, 7889−7896]. We hypothesized that these alternative isomers have distinct bioactivities; therefore, to determine if the regioisomers are quantitatively or qualitatively different with respect to PPARγ activation, NO₂-LA was separated into three fractions which were identified by NMR (13-NO₂-LA, 12-NO₂-LA, and a mixture of 9- and 10-NO₂-LA) and characterized for PPARγ interactions. A competition radioligand binding assay showed that all three NO₂-LA fractions had similar binding affinities for PPARγ (IC₅₀ = 0.41−0.60 μM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC₅₀ = 0.25 μM). However, when PPARγ-dependent transcription activation was examined, there were significant differences observed among the NO₂-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO₂ derivative was the most potent with respect to maximum activation of PPARγ and an EC₅₀ of 0.045 μM (compare with the rosiglitazone EC₅₀ of 0.067 μM), while the 13-NO₂ and 9- and 10-NO₂ derivatives were considerably less effective with EC₅₀ values of 0.41−0.62 μM. We conclude that the regioisomers of NO₂-LA are not functionally equivalent. The 12-NO₂ derivative appears to be the most potent in PPARγ-dependent transcription activation, whereas the weaker PPARγ agonists, 13-NO₂ and 9- and 10-NO₂, may be relatively more important in signaling via other, PPARγ-independent pathways in which this family of nitrolipid electrophiles is implicated.