Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids

The peroxisome proliferator-activated receptor-γ (PPARγ) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARγ include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARγ ligands are intermediates of lipid metabolism and oxidation that bind PPARγ with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO2-FA) are endogenous products of nitric oxide (•NO) and nitrite (NO2−)-mediated redox reactions that activate PPARγ at nanomolar concentrations. We report that NO2-FA act as partial agonists of PPARγ and covalently bind PPARγ at Cys-285 via Michael addition. NO2-FA show selective PPARγ modulator characteristics by inducing coregulator protein interactions, PPARγ-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO2-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

The rapidly expanding global burden of type II diabetes mellitus (DM) 3 and the concomitant increased risk for cardiovas-cular disease (1,2) have motivated better understanding of relevant cell signaling pathways and potential therapeutic strategies. One major characteristic of metabolic syndrome and DM is insulin resistance, leading to hyperglycemia and dyslipidemia. Following initial clinical use of TZDs as anti-hyperglycemic agents to treat DM in the late 1990s, the nuclear receptor PPAR␥ was discovered as their molecular target. This receptor is expressed primarily in adipose tissue, muscle, and macrophages, where it regulates glucose uptake, lipid metabolism/ storage, and cell proliferation/differentiation (3)(4)(5). Thus, PPAR␥ ligands and allied downstream signaling events play a pivotal role in both the development and treatment of DM (6,7). This is underscored by the observation that mutations in the C-terminal helix 12 of the ligand-binding domain (LBD) of PPAR␥ (e.g. P467L or V290M) are linked with severe insulin resistance and the onset of juvenile DM (8).
The oxidizing inflammatory milieu contributing to the pathogenesis of obesity, diabetes, and cardiovascular disease also promotes diverse biomolecule oxidation, nitrosation, and nitration reactions by O 2 and ⅐ NO-derived species. Although oxidized fatty acids typically propagate proinflammatory conditions, the recently detected class of NO 2 -FA act as anti-inflammatory mediators. Nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) have been detected in healthy human blood and murine cardiac tissue. The levels of free/unesterified OA-NO 2 are ϳ1-3 nM in human plasma (9,10), with OA-NO 2 produced at increased rates and present at higher concentrations during inflammatory and metabolic stress (11)(12)(13). The signaling actions of NO 2 -FA are primarily ascribed to the electrophilic olefinic carbon situated ␤ to the electron-withdrawing NO 2 substituent, facilitating kinetically rapid and reversible Michael addition with nucleophilic amino acids (i.e. Cys and His) (14). NO 2 -FA adduction of proteins and GSH occurs in model systems and clinically, with this reaction influencing apparent blood and tissue concentrations (15). The adduction of nucleophilic amino acids in multiple signaling mediators alters protein function and patterns of gene expression. This results in the inhibition of macrophage activation via S-nitroalkylation of the NFB p65 subunit (16), the inactivation the oxidant-generating enzyme xanthine oxidoreductase (17) S-nitroalkylation of Keap-1, activation of Nrf2-dependent phase 2 gene expression (18), and activation of heat shock factordependent gene expression (19). In addition to influencing the activities of these signaling mediators, OA-NO 2 and LNO 2 activate PPAR␥ (20). Although OA-NO 2 and LNO 2 transactivated PPAR␥ as potently as rosiglitazone (Rosi) in luciferase-based assay systems, NO 2 -FA-induced differentiation of pre-adipocytes to adipocytes and subsequent triglyceride accumulation were diminished when compared with Rosi (20,21). These results suggested that NO 2 -FA may act as partial rather than full agonists of PPAR␥ and motivated more detailed investigation.
The large, relatively nonselective ligand binding pocket of PPAR␥ binds eicosanoids and oxidized fatty acid derivatives at micromolar concentrations (22), conferring to this receptor an ability to sense and transduce signals generated by an oxidizing inflammatory milieu that also supports the nitration of fatty acids. Most proposed "natural" PPAR␥ ligands are intermediates of lipid metabolism and oxidation that bind with low affinity at concentrations orders of magnitude greater than physiological conditions. Among these natural ligands are saturated and unsaturated free fatty acids (palmitic acid, K d ϭ 156 M; and linoleic acid, K d ϭ 1 M), prostaglandins (15-deoxyprostaglandin-J 2 (15d-PGJ 2 ), K d Ϸ600 nM), leukotrienes, and other oxidized lipid derivatives (9-and 13 hydroxyoctadecadienoic acid, K d ϭ 10 -20 M; and epoxyeicosatrienoic acids, K d ϭ 1.1-1.8 M), and lysophosphatidic acid (22). Synthetic TZD ligands, such as Rosi (K d ϭ 40 -70 nM) (23,24), bind PPAR␥, increase insulin sensitivity (23), and alleviate symptoms associated with diabetes. Unfortunately, the full receptor activation of PPAR␥ by TZDs also results in undesirable side effects such as weight gain, edema, and an increase in adverse cardiovascular events (25,26). Consequently, there is significant motivation to identify PPAR␥ agonists with gene expression activation profiles that differ from those of TZDs.
The possibility that NO 2 -FA act as partial PPAR␥ agonists led us to investigate the biochemical mechanisms and consequences of PPAR␥-NO 2 -FA binding, as well as physiological outcomes upon chronic NO 2 -FA treatment in vivo. We report herein the characterization of NO 2 -FA binding and activation of PPAR␥, the subsequent effects of NO 2 -FA on PPAR␥-coregulator interactions, and the ability of NO 2 -FA to restore insulin sensitivity in vivo. Specifically, NO 2 -FA covalently adduct PPAR␥, display coregulator interactions distinct from those of Rosi, and reduce insulin and glucose levels in Lep ob mice without inducing the weight gain typically induced by Rosi.

EXPERIMENTAL PROCEDURES
Materials-␤-Mercaptoethanol was from Sigma. Sequencing grade modified trypsin was from Promega (Madison, WI). 15-d-PG J 2 and rosiglitazone were from Cayman Chemicals (Ann Arbor, MI). A purified synthetic peptide containing the NO 2 -FA-reactive Cys-285 (IFQGCQFR) and identical to the predicted tryptic peptide upon PPAR␥ LBD digestion was prepared by the Peptide Synthesis Core Facility at the University of Pittsburgh.
NO 2 -FA Synthesis, Detection, and Handling-NO 2 -FA including OA-NO 2 , LNO 2 , and corresponding internal standards [ 13 C 18 ]OA-NO 2 and [ 13 C 18 ]LNO 2 were synthesized as described previously (21,27,28). NO 2 -FA were synthesized via nitroselenation. In particular, oleic acid (NuCheck Prep, Ͼ99%) (29) was converted to a nitrophenyl selenylated intermediate in the presence of mercuric salts and then oxidized with hydrogen peroxide (30% aqueous) to yield the nitroalkene product OA-NO 2 . The crude product was purified by multiple rounds of column chromatography on silica gel. The final product was analyzed for purity by 1 H NMR and HPLC-MS. OA-NO 2 produced by this method is an equimolar combination of 9-and 10-nitro-octadec-9-enoic acids. Specific OA-NO 2 regioisomers and allyl esters were synthesized and purified as described previously (27).
LC-MS Detection and Analysis of PPAR␥ Post-translational Modifications-First, 5 g of purified human recombinant PPAR␥ LBD (residues 206 -447, containing a His 6 tag) was incubated with ligands for 15 min in phosphate buffer, pH 7.4. PPAR␥ was then digested using mass spectrometry grade modified trypsin (Roche Applied Science) at a PPAR␥ to trypsin ratio of 50:1 overnight at 37°C. The resulting peptide digest was immediately analyzed by HPLC-MS/MS for post-translational modification. Analyses were performed using an Agilent 1200 Series HPLC system (Agilent) coupled to an LTQ mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray ionization source. HPLC was performed by injecting samples (3 l) in a C18 reverse phase column (Zorbax SB 0.5-mm inner diameter, 0.5-m particle size, and 150 mm, Agilent) at an 8 l/min flow rate using 97% solvent A (H 2 O, 0.1% formic acid) and 3% solvent B (acetonitrile, 0.1% formic acid). Peptides were eluted from the column using the following gradients: 7-min hold at 3% solvent B, and then the gradient was set to reach 65% solvent B in 50 min and then 100 in 1 min and hold at 100% solvent B for 5 min, and then re-equilibrated at initial conditions for 12 min. MS analysis was performed in the positive ion mode with the following source parameters that were optimized for the detection and identification of electrophilic fatty acid-modified cysteine-and histidine-containing peptides: source voltage 5 kV, capillary temperature 220°C, tube lens 70 V, capillary voltage 50 V, and collision energy of 35 V. The covered PPAR␥ sequence (Ͼ90%) included all peptides containing either Cys or His residues (nucleophilic targets of nitroalkenes) (15), allowing for a comprehensive analysis of putative target residues. All modifications were further confirmed using [ 13 C 18 ]OA-NO 2 -treated PPAR␥. The 13 C-labeled modified peptides had the same retention times, confirming the detected b and y ions by a predicted mass shift of 18 m/z. All modified peptide spectral profiles were compared with respective nonmodified peptides and fragmentations further confirmed. Quantification of peptides using different concentrations of ligands was done using Excalibur QuanBrowser (Thermo Fisher Scientific). For quantification, the LTQ mass spectrometer was set to continuously isolate and fragment the specific m/z values per period of the different peptides of interest (up to 15 ions per period). The most intense and characteristic b and y ions were selected and their areas integrated. The peptide FEFAVK derived from PPAR␥ trypsinization was chosen as an internal standard to account for processing, injection, and detection variability because it did not contain methionine, histidine, or cysteine and had good ionization and detection properties. The loss of native peptide was calculated by comparison with this standard peptide. In addition, the detection of the modified peptides was also analyzed.
Analysis and Quantification of BME Adducts-The analysis and quantification of BME-adducted molecules were performed using a hybrid triple quadrupole-linear ion trap mass spectrometer (4000 Q Trap, Applied Biosystems/MDS Sciex) in the multiple reaction monitoring scan mode (13). BME adducts of OA-NO 2 and [ 13 C 18 ]OA-NO 2 were detected by monitoring for molecules that undergo a M Ϫ /[M-BME] Ϫ transition. The transitions used were 404.4/326.3 for BME-OA-NO 2 and

Mass spectrometric analysis of PPAR␥ electrophilic adduction
A, analysis of PPAR␥ tryptic digests. 5 g of control and electrophilic lipid-treated PPAR␥ was trypsinized overnight or for 4 h and then subjected to HPLC-MS/MS using an LTQ mass spectrometer as detailed under "Experimental Procedures." Sequence in green shows peptides that were routinely identified. Initial analysis was performed using Bioworks 3.1. All peptides were manually confirmed. All peptides containing histidine and cysteine nucleophilic residues were detected. B, liquid chromatography-MS/MS characterization of peptides containing nucleophilic residues. Table 1 summarizes the retention time of the different peptides, theoretical mass, observed mass to charge ratio, and charge of the different peptides. Peptides derived from PPAR␥ treated with OA-NO 2 under the conditions used throughout the study show a significant shift in retention time due to increased hydrophobicity that was used as an additional confirmatory tool. C, analysis of Michael addition reaction between electrophilic fatty acid derivatives and nucleophilic residues in PPAR␥. PPAR␥ (5 g) was treated as indicated under "Experimental Procedures" with the different lipids at PPAR␥/lipid ratios of 1.333, 0.133, and 0.022 for 15 min, trypsinized, and subjected to HPLC-MS/MS. The experiment was repeated three times at the different protein to lipid ratios, and the results summarized in this table. High represents modifications that were detected in all processed samples at all the different concentrations, with an extensive concomitant loss of native peptide. Medium represents peptides that were detected modified only at the higher concentrations of lipid used. Low represents peptides that were only detected modified in at least two separate experiments at only the highest concentrations of electrophile used with no significant loss of native peptide. Very low represents peptides that were found to be modified at the highest lipid concentrations of electrophile used with no loss of native peptide in at least one analysis. Integration of peak and calculation of peak areas was performed using Excalibur's QuanBrowser (Thermo Fisher).

Covalent PPAR␥ Binding by Nitro-fatty Acids
422.4/344.3 BME-[ 13 C 18 ]OA-NO 2 . Samples were separated by reverse-phase HPLC using a 20 ϫ 2-mm C18 Mercury MS column (3 m, Phenomenex). OA-NO 2 adducts of BME were separated and eluted from the column using a gradient solvent system consisting of solvent A (H 2 O containing 0.1% acetic acid) and solvent B (CH 3 CN containing 0.1% acetic acid) at 750 l/min under the following conditions: hold at 0% solvent B for 2 min, then 0 -90% solvent B (3 min; hold for 2 min), and 90 -0% B (0.1 min; hold for 2 min). The declustering potentials were Ϫ90 V and Ϫ50 V for GS-lipid and BME-lipid electrophilic adducts, respectively. The collision energies were set at Ϫ35 and Ϫ17 V for GS-lipid and BME-lipid electrophilic adducts, respectively. Zero grade air was used as source gas, and nitrogen was used in the collision chamber. Data were acquired and analyzed using Analyst 1.4.2 software (Applied Biosystems, Framingham, MA). Quantification was achieved by comparing peak area ratios between analytes and their corresponding internal standards, and analyte concentration was calculated using an internal standard curve. The isomeric distribution after covalent binding of OA-NO 2 to PPAR␥ and reaction with BME was analyzed and compared with pure 9-NO 2 and 10-NO 2 OA-NO 2 internal standards.
HEK 293T Cells-HEK 293T cells (ATCC, CRL-11268) were maintained in Dulbecco's modified Eagle's medium with high glucose supplemented with 10% fetal bovine serum (Hyclone) and grown at 37°C in 5% CO 2 . Cells were transiently transfected with 7 g of pcDNA3 empty, FLAG-tagged PPAR␥, or FLAG-tagged PPAR␥ C285A mutant expression vectors by calcium phosphate coprecipitation. The HEK 293T cells were then treated with different concentrations of OA-NO 2 for 2 h.
Immunoprecipitation-Cell lysates were prepared by washing with phosphate-buffered saline, and lysis was performed in TGH buffer (150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 10% glycerol) supplemented with protease inhibitors. Lysates were incubated with FLAGagarose M2 beads (Sigma) at 4°C overnight. Immunoprecipitation fractions were obtained by centrifugation at 14,000 ϫ g for 1 min, washed with Tris-buffered saline three times, and then eluted with 3ϫ FLAG peptides (Sigma) in Trisbuffered saline, pH 7.4, at 4°C for 1 h. Supernatants, containing specific FLAG-tagged proteins, were assayed for NO 2 -FA content via BME protein-to-BME trans-nitroalkylation reaction (13). Proteins were additionally resolved and quantified using the gel electrophoresis and silver staining (Invitrogen).
Immunoblotting-Total cell extracts or lysates were resolved on 10 or 11% SDS-PAGE and transferred onto nitrocellulose or polyvinylidene difluoride membranes (Bio-Rad) in Tris/glycine buffer (Bio-Rad). The membranes were probed with either monoclonal anti-FLAG M2 or polyclonal aP2 antibodies (Cell Signaling) and normalized using a polyclonal ␤-actin antibody (Sigma).
In Vitro Competitive Binding Studies-The GST-tagged PPAR␥ ligand-binding domain (PPAR␥ LBD), terbium-labeled anti-GST antibody, and Fluormone TM Pan-PPAR Green tracer were added to ligand in a black 384-well assay plate (Corning Glass catalog no. 677) for final assay concentrations of 5 nM PPAR␥, 5 nM antibody, and 5 nM tracer. After a 1-h incubation at 25°C, the terbium emission at 495 nm and the FRET signal at 520 nm were measured following excitation at 340 nm using a Tecan Ultra 384 microplate reader (Tecan Group Ltd., Maennedorf, Switzerland).
to ligand in a black 384-well assay plate (Corning Glass catalog no. 3677) for final assay concentrations of 5 nM PPAR␥, 5 nM antibody, and 125 nM peptide. For fold-change experiments, CBP-1, PGC-1␣, TRAP/DRIP-2, NCoR ID2, and SMRT ID2 peptides (Invitrogen) were used at final assay concentrations of 500 nM. After a 2-h incubation at 25°C, the terbium emission at 495 nm and the FRET signal at 520 nm were measured following excitation at 340 nm using a TECAN Ultra 384 microplate reader (Tecan Group Ltd., Maennedorf, Switzerland).
PPAR␥ Activation Reporter Assay-PPAR␥-UAS-bla 293T division-arrested cells (Invitrogen) were used for the PPAR␥ reporter cell-based assay contained a PPAR␥ ligand-binding domain/GAL4 DNA-binding domain chimera stably integrated into a parental cell line previously engineered with a ␤-lactamase reporter gene under control of an upstream-activating sequence-response element. PPAR␥-UAS-bla 293T division arrested cells were thawed and immediately plated in black-walled clear bottom and poly-D-lysine-coated 384-well plates at a density of 30,000 cells per well in assay medium (phenol red-free Dulbecco's modified Eagle's medium with 0.1% charcoal-stripped fetal bovine serum (Invitrogen)). Cells were then treated with serial dilutions of various ligands as indicated and incubated in a 37°C and 5% CO 2 incubator for 18 h. Following incubation, LiveBLAzer TM -FRET B/G loading solu-tion (Invitrogen) was added to the cells, and they were incubated at room temperature for 2 h. Fluorescence intensity at 460 and 530 nm emission following excitation at 406 nm was measured using a TECAN Safire2 (Tecan Group Ltd., Maennedorf, Switzerland) with optimal gain settings determined by the instrument. After subtraction of fluorescence background from cell-free wells, the ratio of fluorescence intensity at 460 versus 530 nm (designated as 460:530 nm) was calculated. Data are expressed as mean Ϯ S.D. and evaluated by a one-way ANOVA, post hoc Bonferroni test.
Pre-adipocyte 3T3-L1 Cell Studies-3T3-L1 cells were treated with OA, Rosi, OA-NO 2 , or media alone and harvested after 3, 6, or 9 days. RNA was isolated using TRIzol (Invitrogen) and aP2 gene product amplified with Taqman probe Fabp4 (Applied Biosystems). 3T3-L1 cell differentiation was induced 2 days after confluence by incubating cells with Dulbecco's modified Eagle's medium and 10% fetal bovine serum (Hyclone) supplemented with 1 M dexamethasone, 0.5 mM 1-methyl-3isobutylxanthine, and 10 g/ml insulin (DMI) Ϯ agonist (3 M Rosi, 3 M OA, 3 M OA-NO 2 , 3 M linoleic acid, or 3 M LNO 2 ) for 3 days, after which cells were maintained with 10 g/ml insulin Ϯ agonist for an additional 2 days. At day 5, cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum Ϯ agonist for an additional 2 days. RNA was isolated from differentiated treated cells using TRIzol, and complementary cDNA was generated. Quantitative real time PCR for GLUT4 and actin (GLUT4 forward, ATG AGA AAC GGA AGT TGG AGA GA; GLUT4 reverse, GTG GGT GCG GCT GCC; and actin forward, AGA GGG AAA TCG TGC GTG AC; actin reverse, CAA TAG TGA TGA CCT GGC CGT) were performed using the SYBR Green method. Expression of GLUT4 was compared with the housekeeping gene actin using the ⌬⌬Ct method. For Oil Red O staining, cells were stained on day 7. Oil Red O images were collected by scanner and processed in Corel Photopaint by selecting circular areas representing 95% of the surface area of the plate to avoid edge effects (no adjustments to color, brightness/contrast, intensity, etc. were performed).
In Vivo Studies-All animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Approval 0905750). Osmotic mini-pumps (ALZET, DURECT Corp., Cupertino, CA) containing either vehicle, OA (8 mg/kg/day), Rosi (6 mg/kg/day), or OA-NO 2 (8 mg/kg/day) were subcutaneously implanted in C57BL/6J and Lep ob (ob/ob) male mice (for 28 days), 8 -10 weeks of age (The Jackson Laboratories, Bar Harbor, ME), using isoflurane anesthesia. Food intake and mouse weight were monitored three times weekly. Blood glucose levels were assessed using a True Track glucometer (Home Diagnostics Inc., Ft. Lauderdale, FL). Insulin levels were measured via immunoassay (Alpco Diagnostics, Salem, NH). Tolerance tests, including oral glucose (2 mg/g body weight) on fasted mice (12-15 h), and intraperitoneal insulin (1.5 units/kg body weight) on nonfasted mice, were performed between 19 and 21 days of treatment, and blood samples were taken at various time points (0 -120 min). Data are expressed as mean Ϯ S.D., where asterisk is p Ͻ 0.01 (oneway ANOVA, post hoc Neuman-Keuls).
Statistical Analysis-All data were evaluated with a one-way ANOVA followed by the post hoc test indicated above. Data are expressed as means Ϯ S.D.

Characterization of Covalent Modification of PPAR␥ by
OA-NO 2 -The PPAR␥ LBD was incubated in the absence and presence of different concentrations of OA-NO 2 at mole ratios ranging from 1:1 to 1:100 (OA-NO 2 /PPAR␥) (Table 1A). At OA-NO 2 /PPAR␥ mol ratios of 1:1, modifications of Cys-285, His-266, His-323, His-425 and His-449 were detected, although most histidines were modified at very low yields (Table 1, B and C). When the OA-NO 2 /PPAR␥ ratio was lowered to 0.01, Cys-285 was the only residue undergoing covalent reaction with OA-NO 2 . Overall, this indicates that histidines are minor targets for electrophilic fatty acids, and thus effects induced by peripheral alkylation reactions and conformational changes were considered negligible. The modification and fragmentation pattern of the Cys-285-containing peptide was further confirmed by comparison of the OA-NO 2 -adducted PPAR␥ LBD tryptic peptide with a synthetic peptide adducted with either OA-NO 2 or [ 13 C 18 ]OA-NO 2 (Fig. 1A). The reactivity of Cys-285 toward electrophilic PPAR␥ agonists was then evaluated. We selected OA-NO 2 and LNO 2 as exemplary NO 2 -FA, 15d-PGJ 2 as a representative ␣,␤-unsaturated keto moiety, and OA-NO 2 having an allyl-adducted carboxylic acid as a negative control. The reactivity of 15d-PGJ 2 toward Cys-285 was much less than that of OA-NO 2 . Moreover, the OA-NO 2 reactivity toward PPAR␥-Cys-285 was similar to the one obtained for LNO 2 (Fig. 1B and Table 1C). These data are concordant with the lower K i values displayed toward PPAR␥ by 15d-PGJ 2 compared with LNO 2 and OA-NO 2 , as well as with the higher transactivation potency of NO 2 -FA compared with 15d-PGJ 2 . Moreover, PPAR␥ reactivity was (a) completely blocked by the allyl derivatization of OA-NO 2 ( Fig. 1B and Table 1C), and (b) 10-nitro-octadec-9-enoic acid (C10-OA-NO 2 ) was more reactive toward the Cys-285 than 9-nitro-octadec-10-enoic acid (C9-OA-NO 2 ) (Fig. 2). Control studies did not support the possibility of postincubation adduction reactions or trans-nitroalkylation reactions during trypsinization for MS analysis. Short term trypsinization was also performed, and the results did not differ from longer incubations (data not shown). Intracellular Modification of PPAR␥ by OA-NO 2 -The cellular environment contains a high concentration of nucleophilic targets, including GSH and protein Cys and His residues. Although the reaction of NO 2 -FA with nucleophiles is reversible, the cellular concentration of nucleophiles may act more as a sink rather than a reservoir of NO 2 -FA. In addition, NO 2 -FA are rapidly metabolized by mitochondria via ␤-oxidation (30). Thus, the uptake, transport, and binding of NO 2 -FA to nuclear PPAR␥ can be limited by multiple competing cytosolic and nuclear reactions. To reveal whether NO 2 -FA reach intracellular compartments and covalently modify and activate the receptor, HEK 293T cells were transfected with either empty vector or FLAG-PPAR␥ and treated with 0 -5 M OA-NO 2 for 2 h. PPAR␥ was immunoprecipitated from cell lysates, and the extent of thiol adduction was determined by HPLC-MS/MS upon exchange of the adducted electrophile to BME (13,30). Expression levels of cellular proteins in general and PPAR␥ specifically were similar between different OA-NO 2 treatments and for cells transfected with a C285A mutant of PPAR␥ (Fig. 3, A-C, and Fig. 2A). Cell treatment with increasing concentrations of OA-NO 2 resulted in increased amounts of PPAR␥adducted OA-NO 2 that could be captured by an exchange reaction to BME (Fig. 3B). This was reflected by the appearance of PPAR␥-derived BME-OA-NO 2 adducts (Fig. 3D). Cells trans-fected with C285A PPAR␥ lacking the electrophilic Cys-285 displayed nonspecific OA-NO 2 adduction that was similar to empty vector-transfected cells (Fig. 3C). Thus, in both in vitro and in cell models, PPAR␥ is a relevant target for low concentrations of OA-NO 2 .
Characterization of OA-NO 2 Binding to PPAR␥-TR-FRET analysis permitted more detailed characterization of ligand/receptor interactions. Using TR-FRET, the impact of covalent PPAR␥ adduction by NO 2 -FA was analyzed, and competitive PPAR␥ LBD ligand displacement was quantified. GST-purified PPAR␥ LBD (0.5 nM) was incubated for 1 h with fluorescent ligand and the ligand of interest. Critical insight came from the use of different DTT concentrations in this analysis, as DTT is required in most ligand binding assays to maintain Cys-285 thiol reduction and receptor activity. Lowering DTT concen-  Table indicates ligand binding curves for OA-NO 2 , 9-OA-NO 2 , 9-SA-NO 2 , 12-SA-NO 2 , Rosi, GW9662 (a ligand that binds PPAR␥ covalently), and OA with concentrations ranging from 0.17 to 10,000 nM and containing 125 M DTT. Curve-fitting equations and EC 50 s were determined by XLfit4.

TABLE 3 Comparison of rosiglitazone and OA-NO 2 modulation of coregulator interactions with PPAR␥
Fold-changes in TR-FRET ratios (ligand/no ligand) were determined for the ligands Rosi (2 M), and OA-NO 2 (2 M) and for indicated coregulator peptides 62.5 nM.
tration decreased the assay window for Rosi (Fig. 4A) and revealed that DTT competitively reacts with added NO 2 -FA, resulting in a right shift of EC 50 with increasing DTT concentrations (Fig. 4B). Thus, measuring EC 50 values for ligands at varying DTT concentrations allowed extrapolation of an EC 50 value for OA-NO 2 of Ͻ1 nM in the absence of DTT (Fig. 4C). For comparing various ligands, 125 M DTT was uniformly used, with an appreciation that actual EC 50 values of PPAR␥ for electrophilic fatty acids can still be Ͼ10-fold less than observed (OA-NO 2 and LNO 2 , 13 and 36 nM, respectively, and Rosi, 31 nM, see Fig. 4D). The EC 50 values of oleic acid and linoleic acid were 1900 and 870 nM, respectively.
The adduction of OA-NO 2 with the LBD was also studied in the context of the competitive binding of ligands. The covalent nature of NO 2 -FA binding should result in a leftshift of the EC 50 value over time, as more and more of the receptor becomes adducted. GST-purified PPAR␥ LBD (0.5 nM) was incubated with fluorescent ligand and the ligand of interest, and then terbium emission and FRET signals were measured at various times during reactions (3, 10, 20, and 60 min). This revealed a decreased EC 50 value over time for OA-NO 2 , indicating that the dissociation rate of OA-NO 2 from PPAR␥ is much lower than for Rosi or the nonelectro-  philic homolog of OA-NO 2 , nitro-stearic acid ( Table 2). This significant time-dependent reduction in NO 2 -FA EC 50 , compared with ligands that lack electrophilic reactivities (Rosi, oleic acid, and nitro-stearic acid), further affirmed the covalent interaction of OA-NO 2 with PPAR␥.

OA-NO 2 Is a Selective PPAR␥
Modulator-TR-FRET-based analysis of Ͼ20 coregulator peptides indicated that PPAR␥ binding by OA-NO 2 induces few of the changes in coregulator peptide interactions typically induced by Rosi, supporting a role for NO 2 -FA as partial agonists of PPAR␥ (Table  3). Significant coregulator peptide binding changes were observed for peptides derived from the following coregulators (Fig. 5A): cAMPresponse element-binding protein-binding protein, motif 1 (CBP-1), thyroid hormone receptor-activated protein 220, also known as VDR-interacting protein, motif 2 (TRAP220/DRIP-2), PPAR␥ coactivator 1␣ (PGC-1␣), nuclear corepressor protein interacting domain 2 (NCoR ID2), and the silencing mediator of retinoid and thyroid hormone receptors, interacting domain 2 (SMRT ID2) (Fig. 5A). In contrast to Rosi, which both strongly recruits the coactivator peptides of CBP-1, TRAP220/DRIP-2, and PGC-1␣ and strongly displaces the corepressors NCoR ID2 and SMRT ID2, OA-NO 2 induced a weaker recruitment and displacement of coregulator peptides, indicating a partial PPAR␥ agonist activity (Fig. 5A). This partial agonist behavior of OA-NO 2 is also exemplified by the lower extent of SMRT ID2 displacement that is induced by OA-NO 2 compared with Rosi (Fig. 6). The modified PPAR␥ interaction with coregulators is further confirmed by the partial agonism induced by NO 2 -FA in a cell-based reporter assay using the chimeric Gal4 DNAbinding domain fused to the LBD of PPAR␥ with a ␤-lactamase reporter (Fig. 5B). The EC 50 value of NO 2 -FA for receptor activation is considerably lower than for Rosi, with potency in the low nanomolar range. The partial agonism of OA-NO 2 observed in this test system is also supported by analysis of PPAR␥ ligand-induced preadipocyte differentiation to adipocytes. OA-NO 2 induced Glut4 expression (Fig. 5C), and at the same time induced less adiponectin expression (data not shown) and triglyceride accumulation than that induced by Rosi (Fig. 5D), again supporting partial agonist activity of NO 2 -FA.

OA-NO 2 Normalizes Hyperglycemia in Diabetic
Mice-Diabetic ob/ob mice are obese and severely insulin-resistant. Moreover, TZDs such as Rosi exert PPAR␥-mediated anti-hyperglycemic effects in ob/ob mice but display adverse side effects such as accelerated weight gain (31). The selective modulator actions of OA-NO 2 were tested in vivo for anti-hyperglycemic activity and possible side effects. Administration of 8 mg/kg OA-NO 2 via an osmotic mini-pump over a period of 4 weeks gave a mean plasma level of 32.2 Ϯ 4.5 nM OA-NO 2 in obese ob/ob mice. OA-NO 2 significantly normalized blood glucose levels within 4 days of pump implantation, comparable with those induced by Rosi (Fig. 7A). Insulin levels, a hallmark of type II DM, were also significantly reduced by Rosi and OA-NO 2 but not by vehicle or native oleic acid administration (Fig. 7B). There were no changes in blood glucose or insulin levels in all treatment groups of nondiabetic wild type C57BL/6J mice (Fig. 8). Insulin resistance in vehicle and oleate-treated ob/ob mice was confirmed by intraperitoneal insulin and oral glucose tolerance tests. In contrast, intraperitoneal insulin administration in OA-NO 2 -treated mice revealed a significant and rapid decrease in glucose levels compared with vehicle and oleate treatments (Fig. 7C, right and left panels). Notably, Fig. 7D shows improved insulin sensitivity when glucose is administered orally in OA-NO 2 -treated mice (compared with vehicle and oleate treated mice). Insulin and glucose tolerance tests showed no changes in treated groups in non-insulin-resistant wild type C57BL/6J mice (Fig. 8). An adverse side effect of Rosi, weight gain, reflects both edema and adipogenesis. OA-NO 2treated mice did not gain weight at rates greater than vehicle and native fatty acid controls, as opposed to Rosi-treated mice (Fig.  7F), with all groups having similar caloric intakes (data not shown). In 3T3-L1 adipocytes, Rosi but not OA-NO 2 stimulated RNA transcription and translation of the PPAR␥-regulated gene, aP2 (Fig.  7E). This fatty acid trafficking protein mediates early and late stages of adipogenesis, affecting adipocyte number and triglyceride content. This difference in aP2 expression is consistent with the observation that Rosi induced greater extents of adipocyte lipid accumulation than OA-NO 2 (Fig. 5D).

DISCUSSION
The crystallographic analysis of LNO 2 bound to the PPAR␥ pocket revealed close proximity between the electrophilic carbon ␤ to the nitro group and Cys-285 (32). This spatial arrangement motivated a more detailed examination of the interactions of NO 2 -FA with PPAR␥. It has been previously reported that 15d-PGJ 2 reacts covalently with Cys-285 in vitro, inducing a particular nuclear receptor conformational change (33). More recently, the covalent modification of PPAR␥ Cys-285 was shown to lead to unique receptor conformational changes that resulted in differential regulation of downstream gene transcription (34). Of relevance, the interaction and alkylation of nucleophilic residues distal to the binding pocket may induce additional conformational changes that result in unique coregulator associations and interactions. Thus, as a first approach to defining the mechanism of PPAR␥ activation by nitroalkene fatty acid derivatives, the nucleophilic amino acids of PPAR␥ that react with nitrated fatty acids were identified. The results from this approach reinforced the concept that the environment of Cys-285 renders it very electrophile-reactive, probably by reducing the pK a of the thiol and favoring the thiolate anion. This also suggests that additional stabilization of a covalent ligand-receptor complex can occur and has important implications for the pharmacokinetics of PPAR␥-dependent cell signaling when the receptor is covalently adducted by ligand.
PPAR␥ activation by full agonists such as Rosi induces the expression of proteins that regulate cell differentiation, lipid trafficking, glucose metabolism, and inflammation, thus increasing insulin responsiveness and decreasing blood glucose. However, full PPAR␥ agonists also stimulate adipocyte differentiation in vitro and induce weight gain in vivo, motivating a search for selective modulators that activate a subset of PPAR␥-regulated genes with reduced side effects (7,35). Here, we reveal that electrophilic NO 2 -FA derivatives, products of the oxidizing and nitrating conditions promoted by inflammation, bind to and react avidly with the redox-sensitive LBD Cys-285 of PPAR␥ (Fig. 3). This NO 2 -FA adduction may also serve to protect the oxidation-sensitive Cys-285 of the LBD from inflammatory-derived reactive species. These data reinforce the potent and unique nature of PPAR␥ binding by NO 2 -FA. TZDs bind and activate PPAR␥ reversibly through both hydrophobic and hydrogen bonding interactions (36). In contrast, electrophilic NO 2 -FA bind and activate PPAR␥ by not only hydrophobic and hydrogen bonding interactions but also by covalent reaction with the nucleophilic LBD Cys-285, a scenario where saturation binding kinetics no longer apply.
The specific ligand, cell type, and cell signaling conditions all combine to define the available coregulatory protein pool that interacts with PPAR␥. Hundreds of coregulator proteins have been identified that are either protein-modifying enzymes or scaffolds for transcriptional machinery that promote (coactivators) or prevent (corepressors) transcription. Ligand-dependent coregulator protein recruitment dictates the specificity of transcriptional responses that in turn lead to changes in metabolic homeostasis. Notably, coregulators are also pharmacological targets being pursued as modulators of metabolic adaptation. Rosi recruits and displaces a specific pattern of coregulators upon PPAR␥ binding, inducing a biological response characteristic of full PPAR␥ agonists. In contrast, binding of partial agonists induces a modified coregulator-PPAR␥ interaction pattern that differentially transactivates target genes (37). Although OA-NO 2 and Rosi both bind PPAR␥ with high affinity, distinctively different coregulator protein interactions result. This crucial aspect of ligand-specific receptor conformation and subsequent patterns of transcriptional complex assembly, receptor-DNA binding, and gene expression lends a second level of specificity to PPAR␥ signaling and defines how a particular ligand influences downstream events such as adipocyte differentiation, lipid metabolism, renal fluid regulation, and cellular bioenergetics (38 -42). PPAR␥ agonism by electrophilic NO 2 -FA manifests unique binding kinetics and induces conformational changes in PPAR␥ that result in coregulator interactions unique to these inflammatory by-products. These properties suggest that NO 2 -FA might induce physiological responses that differ from Rosi (22,32,36,37). These findings also support that the pro-adipogenic actions of Rosi, i.e. fat accumulation, can be linked in part to characteristic recruitment of the coactivators TRAP220/ DRIP-2 and CBP-1. Related to this, the weak corepressor displacement (NCoR ID2 and SMRT ID2) by OA-NO 2 may result in inhibition of adipogenesis (39).
In summary, this is the first demonstration of NO 2 -FA adduct formation with PPAR␥ and in vivo evidence for NO 2 -FA as anti-hyperglycemic agents that favorably modulate adipogenesis and circumvent the accelerated weight gain associated with TZD administration. The presence of endogenous NO 2 -FA in healthy human plasma at nanomolar levels and the increased formation of fatty acid nitration products during metabolic and inflammatory stress (11,13) underscore the potential ability of NO 2 -FA to modulate PPAR␥ signaling. Plasma levels of ϳ30 nM in vivo were sufficient to result in a pharmacological effect. This selective PPAR␥ ligand property of NO 2 -FA is also borne out by cell and receptor-coregulatory protein interaction studies. Inasmuch as cardiovascular disease incidence is increased in the diabetic population and TZD-based PPAR␥ agonists increase risk for adverse cardiovascular events, the unique PPAR␥ interactions of electrophilic fatty acids encourage further model systems and clinical investigation.