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. 2005 Feb 15;102(7):2340-5.
doi: 10.1073/pnas.0408384102. Epub 2005 Feb 8.

Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor gamma ligand

Francisco J Schopfer  1 , Yiming Lin, Paul R S Baker, Taixing Cui, Minerva Garcia-Barrio, Jifeng Zhang, Kai Chen, Yuqing E Chen, Bruce A Freeman
Affiliations

Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor gamma ligand

Francisco J Schopfer et al. Proc Natl Acad Sci U S A. .

Abstract

Nitroalkene derivatives of linoleic acid (nitrolinoleic acid, LNO2) are formed via nitric oxide-dependent oxidative inflammatory reactions and are found at concentrations of approximately 500 nM in the blood of healthy individuals. We report that LNO2 is a potent endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARgamma; Ki approximately 133 nM) that acts within physiological concentration ranges. This nuclear hormone receptor (PPARgamma) regulates glucose homeostasis, lipid metabolism, and inflammation. PPARgamma ligand activity is specific for LNO2)and not mediated by LNO2 decay products, NO donors, linoleic acid (LA), or oxidized LA. LNO2 is a significantly more robust PPARgamma ligand than other reported endogenous PPARgamma ligands, including lysophosphatidic acid (16:0 and 18:1), 15-deoxy-Delta12,14-PGJ2, conjugated LA and azelaoyl-phosphocholine. LNO2 activation of PPARgamma via CV-1 cell luciferase reporter gene expression analysis revealed a ligand activity that rivals or exceeds synthetic PPARgamma agonists such as rosiglitazone and ciglitazone, is coactivated by 9 cis-retinoic acid and is inhibited by the PPARgamma antagonist GW9662. LNO2 induces PPARgamma-dependent macrophage CD-36 expression, adipocyte differentiation, and glucose uptake also at a potency rivaling thiazolidinediones. These observations reveal that NO-mediated cell signaling reactions can be transduced by fatty acid nitration products and PPAR-dependent gene expression.

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Figures

Fig. 1.
Fig. 1.
LNO2 is a potent PPAR ligand. (A) CV-1 cells, transiently cotransfected with different nuclear receptor ligand-binding domains fused to the Gal4 DNA-binding domain and the luciferase reporter gene under the control of four Gal4 DNA-binding elements, were incubated with vehicle (methanol) or LNO2 (3 μM, 2h, n = 4). (Inset) Dose response of LNO2-dependent PPARγ ligand-binding domain activation (n = 4). (B) Dose response of LNO2-dependent PPARγ, α, and δ activation (n = 4). The luciferase reporter gene was under the control of three PPRE. (C) Response of CV-1 cells transfected with PPARγ and a luciferase reporter under the control of PPRE after exposure to LNO2 and other reported PPARγ ligands [1 and 3 μM each of ciglitazone, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPA 16:0), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPA 18:1), 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (AzPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (AzPC ester), Δ9,11-conjugated LA (CLA1), and Δ10,12-conjugated LA (CLA2), with n = 3–5]. For 15-deoxy-Δ12,14-PGJ2, the concentration was 3 and 5 μM. “Vector” indicates empty vector. (Inset) By using the same reporter construct, the dose response of PPARγ activation by LNO2, rosiglitazone, 15-deoxy-Δ12,14-PGJ2, and LA was measured (n = 3). All values are expressed as mean ± SD. *, significantly different (P < 0.05) from vehicle control, using Student's t test. All experiments were repeated at least three times.
Fig. 2.
Fig. 2.
Characterization of the PPARγ ligand activity of LNO2. (A) By using CV-1 cells cotransfected with PPARγ and PPRE-controlled luciferase expression plasmids, the activation of PPARγ by LNO2 was evaluated in the presence of PPARγ-specific antagonist GW9662 added 1 h before LNO2 addition or upon coaddition of the RXR coactivating ligand 9-cis-retinoic acid (n = 3). PPARγ activation by LNO2 was inhibited in a dose-dependent manner by GW9662 and was enhanced in the presence of the coactivator 9-cis-retinoic acid. (B) The action of LNO2 as a PPARγ ligand was compared with LNO2 decay products. Effective LNO2 concentrations after selected decay periods were measured by liquid chromatography-MS with electrospray ionization by using [13C]LNO2 as internal standard (9). PPARγ activation was assessed by means of PPRE reporter analysis in CV-1 cells (n = 3). (C) Potential PPARγ ligand activity of LNO2 decay products was measured by means of PPRE reporter analysis (n = 4). (D) Competition of LNO2, linoleate, and unlabeled rosiglitazone for PPARγ-bound [3H]rosiglitazone. For A–C, all values are expressed as mean ± SD. *, significantly different (P < 0.05) from vehicle control; #, significantly different from LNO2 alone, using Student's t test. All experiments were repeated at least three times.
Fig. 3.
Fig. 3.
LNO2 induces CD36 expression in macrophages and adipogenesis of 3T3-L1 preadipocytes. (A) Mouse RAW264.7 macrophages at ≈90% confluence were cultured in DMEM with 1% FBS for 16 h and then treated with various stimuli for 16 h as indicated. The PPARγ-specific antagonist GW9662 was added 1 h before the treatment. The cell lysate was immunoblotted with anti-CD36 and anti-β-actin Abs. (B and C) After reaching confluence (2 d), 3T3-L1 preadipocytes were cultured for 14 d and stained by using Oil red O (18) (B) or treated with various stimuli as indicated, and the cell lysate was immunoblotted with anti-PPARγ, anti-aP2, and anti-β-actin Abs (C). (D) LNO2 increases 2-deoxy-d-[3H]glucose uptake in 3T3-L1 adipocytes. (Left) The dose-dependent effects of LNO2 on 2-deoxy-d-[3H]glucose uptake in 3T3-L1 adipocytes. (Right) PPARγ-specific antagonist GW9662 was added 1 h before the treatment. 2-deoxy-d-[3H]glucose uptake assay was performed as described in Materials and Methods. All experiments were repeated at least three times. Values are expressed as mean ± SD (n = 6). Statistical analysis was done by using Student's t test (*, P < 0.05 vs. vehicle control; #, P < 0.05 vs. GW9662 untreated groups). Veh, vehicle; Rosi, rosiglitazone; 15-d-PGJ2, 15-deoxy-PGJ2.
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