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

Abstract

Previous studies demonstrated that the naturally occurring electrophile and PPARgamma ligand, nitrolinoleic acid (NO(2)-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 PPARgamma activation, NO(2)-LA was separated into three fractions which were identified by NMR (13-NO(2)-LA, 12-NO(2)-LA, and a mixture of 9- and 10-NO(2)-LA) and characterized for PPARgamma interactions. A competition radioligand binding assay showed that all three NO(2)-LA fractions had similar binding affinities for PPARgamma (IC(50) = 0.41-0.60 microM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC(50) = 0.25 microM). However, when PPARgamma-dependent transcription activation was examined, there were significant differences observed among the NO(2)-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO(2) derivative was the most potent with respect to maximum activation of PPARgamma and an EC(50) of 0.045 microM (compare with the rosiglitazone EC(50) of 0.067 microM), while the 13-NO(2) and 9- and 10-NO(2) derivatives were considerably less effective with EC(50) values of 0.41-0.62 microM. We conclude that the regioisomers of NO(2)-LA are not functionally equivalent. The 12-NO(2) derivative appears to be the most potent in PPARgamma-dependent transcription activation, whereas the weaker PPARgamma agonists, 13-NO(2) and 9- and 10-NO(2), may be relatively more important in signaling via other, PPARgamma-independent pathways in which this family of nitrolipid electrophiles is implicated.

Gas-Phase Fragmentation Analysis of Nitro-Fatty Acids

“…NO 2 -LA positional isomers have been previously described and confirmed by NMR [4, 32]. The CID fragmentation of 9-NO 2 -LA resulted in the formation of three main product ions containing structural information with m/z values of 168.1, 210.1, and 224.1 (Figure 2A).…”

Electrophilic nitro-fatty acids: anti-inflammatory mediators in the vascular compartment

“…NO 2 -FAs and keto-fatty acid derivatives have high binding affinities for all three PPAR isotypes, with PPARγ the most robustly-activated receptor (followed by α and then δ) [31, 32•]. Reporter cell transactivation studies revealed that different regioisomers of NO 2 -FA behave as partial agonists [33•]. Receptor-ligand-binding analysis indicates that NO 2 -FA covalently react with the ligand binding domain Cys285 residue of PPARγ in both biochemical reaction systems and cells.…”

Biomimetic Nitration of Conjugated Linoleic Acid: Formation and Characterization of Naturally Occurring Conjugated Nitrodienes

“…Both methods are nonregiospecific, producing a mixture of positional isomers of NO 2 -LA with nitration at the 9-, 10-, 12-, and 13-positions ( 4–7 ). Studies that isolate and compare individual isomers, 21 synthesize regiospecific isomers 22 ( 5 and analogs 23 ), and characterize the degradation manifolds of particular isomers 24 ( 4 ) have been reported. These initial reports focused on understanding the nitration of LA because of its natural abundance and the presence of a reactive bisallylic methylene that favors hydrogen abstraction reactions.…”