Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid.

The peroxisome proliferator-activated receptor-␥ (PPAR␥) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPAR␥ include lipids and antihyperglycemic drugs such as thiazolidinediones (TZDsThe rapidly expanding global burden of type II diabetes mellitus (DM) 3 and the concomitant increased risk for cardiovascular 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 (3-5). 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 (11-13). 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

Nitrated derivatives of fatty acids (NO 2 -FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO 2 -FA signal transduction lacks insight into how or if NO 2 -FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO 2 ) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO 2 or metabolites were detected under basal conditions, whereas administered 18:1-NO 2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO 2 -FA are also metabolized via ␤-oxidation, with high performance liquid chromatographytandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO 2 as metabolites. Additionally, a significant proportion of 18:1-NO 2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO 2 hydration or nitric oxide ( ⅐ NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO 2 detected by exchange to added ␤-mercaptoethanol. This, coupled with the observation of 18:1-NO 2 release from glutathione-18:1-NO 2 adducts, supports that reversible and exchangeable NO 2 -FAthiol adducts occur under biological conditions. After administration of [ 3 H]18:1-NO 2 , 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and ␤-oxidation reactions that terminate at the site of acyl-chain nitration.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, DeltaF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO 2 and LNO 2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M ؊1 s ؊1 and 355 M ؊1 s ؊1 , respectively, at pH 7.4 and 37°C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A 2 and 15-deoxy-⌬ 12,14 -prostaglandin J 2 . Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO 2 and LNO 2 , showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pK a of target thiols increased. Increasing concentrations of the detergent octyl-␤-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.