Carlos Batthyany

Fatty Acid Transduction of Nitric Oxide Signaling: MULTIPLE NITRATED UNSATURATED FATTY ACID DERIVATIVES EXIST IN HUMAN BLOOD AND URINE AND SERVE AS ENDOGENOUS PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR LIGANDS*♦

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9- and 10-nitro-9-cis-octadecenoic acid; OA-NO2), structurally characterized and determined to be identical to OA-NO2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO2 were quantified in clinical samples using 13C isotope dilution. Plasma free and esterified OA-NO2 concentrations were 619 ± 52 and 302 ± 369 nm, respectively, and packed red blood cell free and esterified OA-NO2 was 59 ± 11 and 155 ± 65 nm. The OA-NO2 concentration of blood is ∼50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 μm. OA-NO2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPARγ, PPARα, or PPARδ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO2. PPARγ showed the greatest response, with significant activation at 100 nm, while PPARα and PPARδ were activated at ∼300 nm OA-NO2. OA-NO2 also induced PPAR γ-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazo-lidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.

Nitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-γ activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-κB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-κB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-α-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo

Nitric oxide (.NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon β to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC50 of ∼3 μmm for both nitroalkenes, an IC50 equivalent to the potent thiol oxidant peroxynitrite (ONOO-) and an IC50 30-fold less than H2O2, indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.

Fatty Acid Transduction of Nitric Oxide Signaling NITROLINOLEIC ACID IS A HYDROPHOBICALLY STABILIZED NITRIC OXIDE DONOR

The aqueous decay and concomitant release of nitric oxide (·NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ·NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and ·NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ·NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ·NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.

Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids SELECTIVE LIGAND ACTIVITY AND ANTI-DIABETIC SIGNALING ACTIONS

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 peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma 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 PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-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 NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

Detection and Quantification of Protein Adduction by Electrophilic Fatty Acids: Mitochondrial Generation of Fatty Acid Nitroalkene Derivatives

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

Conjugated Linoleic Acid is a Preferential Substrate for Fatty Acid Nitration

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 oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 105 greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO2-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate.

Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signalling mediators, based on their high-affinity PPARgamma (peroxisome-proliferator-activated receptor gamma) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO2 (cholesteryl-nitrolinoleate). CLNO2 levels were found to increase approximately 20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-gamma; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the (*)NO (nitric oxide) inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrow-derived cells) inflammatory responses were suppressed when activated in the presence of CLNO2 or LNO2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARgamma and *NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-kappaB (nuclear factor kappaB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids.

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO(2)) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO(2) via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO(2)-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO(2)-FAs stimulated the phosphorylation of eNOS at Ser(1179). These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO(2)-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.

Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 μm, limiting both purine oxidation and formation of superoxide \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{O}_{2}^{{\bar{{\cdot}}}})\) \end{document}. Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, β-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.