Electrophilic Fatty Acids Research Articles

Role of electrophilic nitrated fatty acids during development and response to abiotic stress processes in plants.

Nitro-fatty acids are generated from the interaction of unsaturated fatty acids and nitric oxide (NO)-derived molecules. The endogenous occurrence and modulation throughout plant development of nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) suggest a key role for these molecules in initial development stages. In addition, NO2-Ln content increases significantly in stress situations and induces the expression of genes mainly related to abiotic stress, such as genes encoding members of the heat shock response family and antioxidant enzymes. The promoter regions of NO2-Ln-induced genes are also involved mainly in stress responses. These findings confirm that NO2-Ln is involved in plant defense processes against abiotic stress conditions via induction of the chaperone network and antioxidant systems. NO2-Ln signaling capacity lies mainly in its electrophilic nature and allows it to mediate a reversible post-translational modification called nitroalkylation, which is capable of modulating protein function. NO2-Ln is a NO donor that may be involved in NO signaling events and is able to generate S-nitrosoglutathione, the major reservoir of NO in cells and a key player in NO-mediated abiotic stress responses. This review describes the current state of the art regarding the essential role of nitro-fatty acids as signaling mediators in development and abiotic stress processes.

The Emerging Therapeutic Potential of Nitro Fatty Acids and Other Michael Acceptor-Containing Drugs for the Treatment of Inflammation and Cancer.

Nitro fatty acids (NFAs) are endogenously generated lipid mediators deriving from reactions of unsaturated electrophilic fatty acids with reactive nitrogen species. Furthermore, Mediterranean diets can be a source of NFA. These highly electrophilic fatty acids can undergo Michael addition reaction with cysteine residues, leading to post-translational modifications (PTM) of selected regulatory proteins. Such modifications are capable of changing target protein function during cell signaling or in biosynthetic pathways. NFA target proteins include the peroxisome proliferator-activated receptor γ (PPAR-γ), the pro-inflammatory and tumorigenic nuclear factor-κB (NF-κB) signaling pathway, the pro-inflammatory 5-lipoxygenases (5-LO) biosynthesis pathway as well as soluble epoxide hydrolase (sEH), which is essentially involved in the regulation of vascular tone. In several animal models of inflammation and cancer, the therapeutic efficacy of well-tolerated NFA has been demonstrated. This has already led to clinical phase II studies investigating possible therapeutic effects of NFA in subjects with pulmonary arterial hypertension. Albeit Michael acceptors feature a broad spectrum of bioactivity, they have for a rather long time been avoided as drug candidates owing to their presumed unselective reactivity and toxicity. However, targeted covalent modification of regulatory proteins by Michael acceptors became recognized as a promising approach to drug discovery with the recent FDA approvals of the cancer therapeutics, afatanib (2013), ibrutinib (2013), and osimertinib (2015). Furthermore, the Michael acceptor, neratinib, a dual inhibitor of the human epidermal growth factor receptor 2 and epidermal growth factor receptor, was recently approved by the FDA (2017) and by the EMA (2018) for the treatment of breast cancer. Finally, a number of further Michael acceptor drug candidates are currently under clinical investigation for pharmacotherapy of inflammation and cancer. In this review, we focus on the pharmacology of NFA and other Michael acceptor drugs, summarizing their potential as an emerging class of future antiphlogistics and adjuvant in tumor therapeutics.

Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids

Electrophilic nitro-fatty acids [NO2-FAs (fatty acid nitroalkenes)] showed beneficial signaling actions in preclinical studies and safety in phase 1 clinical trials. A detailed description of the pharmacokinetics (PK) of NO2-FAs is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly and become esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and distribution of the endogenously detectable and prototypical NO2-FA, 10-nitro-oleic acid (10-NO2-OA), in dogs after oral administration. Supported by HPLC-high-resolution-MS/MS analysis of synthetic and plasma-derived 10-NO2-OA-containing triacylglycerides (TAGs), we show that a key mechanism of NO2-FA distribution is an initial esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions confirmed time-dependent preferential incorporation of 10-NO2-OA into TAGs when compared with its principal metabolite, 10-nitro-stearic acid. Finally, new isomers of 10-NO2-OA were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we reveal that NO2-FAs display unique PK, with the principal mechanism of tissue distribution involving complex lipid esterification, which serves to shield the electrophilic character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution to target organs.

Electrophilic nitro-oleic acid reverses obesity-induced hepatic steatosis

Non-alcoholic fatty liver disease (NAFLD) is linked to obesity and insulin resistance and is the most prevalent chronic liver disease. During the development of obesity and NAFLD, mitochondria adapt to the increased lipid load in hepatocytes by increasing the rate of fatty acid oxidation. In concert with this, reactive species (RS) generation is increased, damaging hepatocytes and inducing inflammation. Hepatic mitochondrial dysfunction is central to the pathogenesis of NAFLD via undefined mechanisms. There are no FDA approved treatments for NAFLD other than weight loss and management of glucose tolerance. Electrophilic nitro-oleic acid (NO2-OA) displays anti-inflammatory and antioxidant signaling actions, thus mitochondrial dysfunction, RS production and inflammatory responses to NO2-OA and the insulin sensitizer rosiglitazone were evaluated in a murine model of insulin resistance and NAFLD. Mice on HFD for 20 wk displayed increased adiposity, insulin resistance and hepatic lipid accumulation (steatosis) compared to mice on normal chow (NC). The HFD mice had mitochondrial dysfunction characterized by lower hepatic mitochondrial complex I, IV and V activity compared to mice on NC. Treatment with NO2-OA or rosiglitazone for the last 42 days (out of 20 wk) abrogated HFD-mediated decreases in hepatic mitochondrial complex I, IV and V activity. Notably, NO2-OA treatment normalized hepatic triglyceride levels and significantly reversed hepatic steatosis. Despite the improved glucose tolerance observed upon rosiglitazone treatment, liver weight and hepatic triglycerides were significantly increased over vehicle-treated HFD mice. These observations support that the pleiotropic signaling actions of electrophilic fatty acids limit the complex hepatic and systemic pathogenic responses instigated by obesity, without the adverse effects of thiazolidinedione drugs such as rosiglitazone.

Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells

Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2-mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.

Topical electrophilic nitro-fatty acids potentiate cutaneous inflammation.

Endogenous electrophilic fatty acids mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction and gene expression. Nitro-fatty acids and other electrophilic fatty acids may thus be useful for the prevention and treatment of immune-mediated diseases, including inflammatory skin disorders. In this regard, subcutaneous (SC) injections of nitro oleic acid (OA-NO2), an exemplary nitro-fatty acid, inhibit skin inflammation in a model of allergic contact dermatitis (ACD). Given the nitration of unsaturated fatty acids during metabolic and inflammatory processes and the growing use of fatty acids in topical formulations, we sought to further study the effect of nitro-fatty acids on cutaneous inflammation. To accomplish this, the effect of topically applied OA-NO2 on skin inflammation was evaluated using established murine models of contact hypersensitivity (CHS). In contrast to the effects of subcutaneously injected OA-NO2, topical OA-NO2 potentiated hapten-dependent inflammation inducing a sustained neutrophil-dependent inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and γδ T cells. Consistent with these results, HPLC-MS/MS analysis of skin from psoriasis patients displayed a 56% increase in nitro-conjugated linoleic acid (CLA-NO2) levels in lesional skin compared to non-lesional skin. These results suggest that nitro-fatty acids in the skin microenvironment are products of cutaneous inflammatory responses and, in high local concentrations, may exacerbate inflammatory skin diseases.

219 - Nitro-oleic Acid Protects Mice from Diet-Induced Hepatic Steatosis and Insulin Resistance without the Adverse Side Effects of Thiazolidinediones

Non-alcoholic fatty liver disease (NAFLD) is linked to obesity and insulin resistance and is the most prevalent chronic liver disease. During the development of obesity and NAFLD, mitochondria adapt to the increased lipid load in hepatocytes by increasing the rate of fatty acid oxidation. In concert with this, reactive species (RS) generation is increased, damaging hepatocytes and inducing inflammation. Hepatic mitochondrial dysfunction is central to the pathogenesis of NAFLD via undefined mechanisms. There are no FDA approved treatments for NAFLD other than weight loss and management of glucose tolerance. Electrophilic nitro-oleic acid (NO2-OA) displays anti-inflammatory and antioxidant signaling actions, thus mitochondrial dysfunction, RS production and inflammatory responses to NO2-OA and the insulin sensitizer rosiglitazone were evaluated in a murine model of insulin resistance and NAFLD. Mice on HFD (60% kcal from fat) for 20 weeks displayed increased adiposity, insulin resistance and hepatic lipid accumulation (steatosis) compared to mice on normal chow (NC). The HFD mice had mitochondrial dysfunction characterized by lower hepatic mitochondrial complex I, IV and V activity compared to mice on NC. Treatment with NO2-OA or rosiglitazone for the last 6.5 weeks (out of 20) abrogated the HFD-mediated decrease in hepatic mitochondrial complex I, IV and V activity. Both NO2-OA and rosiglitazone restored glucose tolerance and decreased pro-inflammatory cytokine levels and markers of oxidative stress. Notably, NO2-OA normalized hepatic triglyceride levels and significantly reversed hepatic steatosis. Despite the improved glucose tolerance observed with rosiglitazone treatment, liver weight and hepatic triglycerides were significantly increased over vehicle-treated HFD mice. These observations support that the pleiotropic signaling actions of electrophilic fatty acids limit the complex pathogenic events instigated by obesity without the adverse effects of thiazolidinedione drugs such as rosiglitazone.

Electrophilic nitro-fatty acids suppress allergic contact dermatitis in mice.

Reactions between nitric oxide (NO), nitrite (NO2-), and unsaturated fatty acids give rise to electrophilic nitro-fatty acids (NO2 -FAs), such as nitro oleic acid (OA-NO2 ) and nitro linoleic acid (LNO2 ). Endogenous electrophilic fatty acids (EFAs) mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. Hence, there is considerable interest in employing NO2 -FAs and other EFAs for the prevention and treatment of inflammatory disorders. Thus, we sought to determine whether OA-NO2 , an exemplary nitro-fatty acid, has the capacity to inhibit cutaneous inflammation. We evaluated the effect of OA-NO2 on allergic contact dermatitis (ACD) using an established model of contact hypersensitivity in C57Bl/6 mice utilizing 2,4-dinitrofluorobenzene as the hapten. We found that subcutaneous (SC) OA-NO2 injections administered 18 h prior to sensitization and elicitation suppresses ACD in both preventative and therapeutic models. In vivo SC OA-NO2 significantly inhibits pathways that lead to inflammatory cell infiltration and the production of inflammatory cytokines in the skin. Moreover, OA-NO2 is capable of enhancing regulatory T-cell activity. Thus, OA-NO2 treatment results in anti-inflammatory effects capable of inhibiting ACD by inducing immunosuppressive responses. Overall, these results support the development of OA-NO2 as a promising therapeutic for ACD and provides new insights into the role of electrophilic fatty acids in the control of cutaneous immune responses potentially relevant to a broad range of allergic and inflammatory skin diseases.

131 - Nitro-Fatty Acid Pharmacokinetics in the Adipose Tissue Compartment

Metabolic and redox reactions of nitric oxide and nitrite induce the nitration of biomolecules such as proteins, nucleic acids and unsaturated fatty acids. Nitrated unsaturated fatty acids (NO2-FAs) promote adaptive and anti-inflammatory cell signaling responses as a result of an electrophilic character that supports post-translational protein modifications. By reversibly adducting nucleophilic amino acids such as cysteine, NO2-FAs induce salutary enzymatic and transcriptional regulatory protein responses. NO2-FAs are found endogenously in a broad range of living organisms, become elevated in concentration during metabolic and inflammatory stress and at present, synthetic NO2-FA homologs are being tested in humans as a drug strategy for treating kidney disease. A unique pharmacokinetic profile is expected for electrophilic fatty acids because of an ability to undergo both Michael addition with biological nucleophiles and esterification into complex lipids. We now report new methods for the qualitative and quantitative analysis of complex lipids containing esterified NO2-FAs. This new capability supports the more complete definition of the distribution and metabolism of electrophilic fatty acid derivatives in tissue lipid compartments. Herein we report via quantitative whole-body autoradiography analysis of rats gavaged with radiolabeled 10-nitro-[14C]oleic acid (10-NO2-[14C]OA). This NO2-FA preferentially accumulates in adipose tissue over two weeks. To define the metabolism and incorporation of NO2-FA and their metabolites in adipose tissue lipids, adipocyte cultures were supplemented with 10-NO2-OA, nitro-stearic acid (NO2-SA), nitro-conjugated-linoleic acid (NO2-CLA) and nitro-linolenic acid (NO2-LnA). Then, quantitative high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was performed on adipocyte neutral and polar lipid fractions, both before and after acidic hydrolysis of esterified fatty acids. Electrophilic nitroalkene derivatives preferentially incorporated in the monoacyl- and diacyl-glyceride than in the triacylglyceride lipid pools of adipocytes, while the non-electrophilic NO2-SA showed an opposite distribution. This differential distribution profile was confirmed in vivo in the adipose tissue of NO2-OA-treated mice. This pattern of electrophilic nitroalkene deposition lends new insight into the unique pharmacokinetics and pharmacologic actions displayed by this chemically-reactive class of endogenous signaling mediators and synthetic drug candidates.

Protection of nitro-fatty acid against kidney diseases

Nitrated derivatives of unsaturated fatty acids are endogenously formed under oxidative and nitrative stress condition and are defined as electrophilic fatty acids containing a nitro group to a carbon-carbon double bond. Among the most studied nitro derivatives of unsaturated fatty acids are nitro-oleic acid (OA-NO2) and nitro-linoleic acid (LNO2). These products exhibit novel protective actions in a variety of rodent disease models. Diverse signaling events are responsible for effects of nitrated fatty acid, including activating peroxisome proliferator-activated receptor-dependent gene expression, suppressing NF-κB-induced inflammation, inhibiting oxidative stress, and increasing both endothelial nitric oxide synthase- and Nrf2-dependent gene regulation. Nitrated fatty acids have been emerging not only as a unique class of signaling molecules produced endogenously and but also as multipotent modulators of cell signaling pathways in cardiovascular and renal diseases. In this review, we discuss biochemical properties of nitrated fatty acid and its signaling pathways in the modulation of cellular events. A major focus is to review recent knowledge of nitrated fatty acid on the treatment of kidney diseases and its therapeutic potential for inflammation and metabolic disorders, with special emphasis on acute kidney injury and diabetic kidney disease.

Electrophilic Nitroalkenes Cause Degradation of NFκB RelA Protein in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and metastatic cancer. Current therapy for TNBC is limited, and it is an unmet need for novel therapeutic agents with improved efficacy. Electrophilic fatty acids are endogenously‐generated signaling mediators that modulate cell differentiation and proliferation via post‐translational modification of nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFkB, PPARgamma, and Keap1/Nrf2. Preliminary studies have revealed that an exemplary electrophilic fatty acid nitroalkene, nitro‐oleic acid (OA‐NO2), displays potential therapeutic value in TNBC cells, by preferentially reducing the growth and viability of two human TNBC cell lines, but not normal breast cells. Essential to proliferation and survival, NFkB signaling is highly activated in ER‐negative breast cancer cells and primary tumors. Notably, NFkB RelA protein level was diminished in TNBC cells upon OA‐NO2 treatment. Therefore, it is hypothesized that electrophilic fatty acids reduce TNBC cell proliferation and survival through down‐regulation of NFkB expression and signaling. We have demonstrated that OA‐NO2 down‐regulates transcript levels of NFkB –regulated genes, cyclin D1 and the pro‐survival genes, bcl‐xL and survivin, in TNBC cells. Moreover, OA‐NO2 promotes RelA protein polyubiquitination via nitroalkylation of RelA in TNBC cells. Overall, this study will serve as a prelude to the clinical study of electrophilic nitrated lipids as therapeutic agents that may display a high selectivity for killing TNBC tumors.

15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways

Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic “inactivation” of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators.

Topical electrophilic nitro-fatty acids potentiate cutaneous immune responses. (MUC7P.775)

Abstract Endogenous electrophilic fatty acids mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. In this regard, there is considerable interest in employing nitro-fatty acids and other electrophilic fatty acids for the prevention and treatment of inflammatory disorders. We evaluated nitro-oleic acid (OA-NO2) in a preclinical murine model of allergic contact dermatitis (ACD) as a potential topical therapy for the treatment of ACD. In marked contrast to previous findings, topical OA-NO2 potentiated hapten-dependent inflammation inducing a sustained psoriasis-like inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and γδ T cells. Interestingly, analysis by HPLC-MS-MS of skin from psoriasis patients revealed a significant increase in nitro-fatty acids in lesional skin compared to non-lesional skin. Overall, when applied topically to the cutaneous microenvironment nitro-fatty acids can potentiate inflammatory responses. Thus, it is paramount that a better understanding be obtained of how electrophilic fatty acid derivatives regulate cutaneous immunity due to the widespread use of fatty acids in topical creams and the abundant formation of electrophilic nitro-fatty acids during inflammation.

Fatty acids, inflammation, and asthma

Fatty acids and consequently diet play an essential role in the formation of inflammatory mediators involved in the pathogenesis of asthma. Because intake variations of omega-6 (n-6) and omega-3 (n-3) fatty acids ultimately determine cell membrane incorporation, changes in diet have the potential to modify downstream production of inflammatory mediators derived from these compounds. It has long been hypothesized that decreasing the n-6/n-3 ratio could reduce the production of more proinflammatory mediators while increasing the formation of downstream metabolites that can serve to limit or resolve inflammation. In turn, these changes would result in improved asthma outcomes or would lower the risk for asthma incidence. This review will focus on the role of fatty acid inflammatory and resolving mediators and will summarize the clinical and epidemiologic data on how diet and obesity alter fatty acid profiles that can contribute to asthma.

Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity

Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Redox-Dependent Anti-Inflammatory Signaling Actions of Unsaturated Fatty Acids

Unsaturated fatty acids are metabolized to reactive products that can act as pro- or anti-inflammatory signaling mediators. Electrophilic fatty acid species, including nitro- and oxo-containing fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,β-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases.

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.

Inhibition of Obesity-Induced Pulmonary Arterial Hypertension by Electrophilic Fatty Acids

Inhibition of Obesity-Induced Pulmonary Arterial Hypertension by Electrophilic Fatty Acids

Formation and signaling actions of electrophilic fatty acid derivatives

The modification of cell proteins by chemical reactions stemming from metabolic and environmental stimuli significantly expands the functional proteome. These post-translational protein modifications (PTPM) allow cells to dynamically regulate metabolism, growth, differentiation and immune responses. Notably, PTPMs continue to emerge as a critical component of NO signaling. In addition to its initially-appreciated role in activating guanylate cyclase via heme–iron coordination, NO reacts with superoxide, lipid radicals and can be further oxidized by metalloproteins to yield reactive secondary species. These products include peroxynitrite and nitrogen dioxide that expand the breadth of reactions that transduce redox-dependent signaling. One class of reactive species byproducts, electrophilic unsaturated fatty acids, induce PTPM by reacting with protein thiols and other nucleophilic amino acids. Notably, multiple transcriptional regulatory mechanisms have protein constituents with functionally-significant electrophile-reactive amino acids. This highly conserved property provides cells with a capability to undergo stress-related adaptive signaling reactions. We present new data regarding the mechanisms of formation of electrophilic nitro- and keto-fatty acid derivatives and how to detect these reactive species and the PTPM they induce. The molecular targets, acute signaling responses, transcriptional regulation, cell signaling responses and physiological actions that are induced by low concentrations of electrophilic fatty acids are anti-inflammatory in nature. In aggregate, this presentation will advance the concept that NO and lipid electrophile-mediated PTPM reactions link cell function with inflammatory and metabolic status.

Electrophilic Fatty Acids Regulate Matrix Metalloproteinase Activity and Expression

Nitro-fatty acids (NO(2)-FA) are electrophilic signaling mediators formed by reactions of nitric oxide and nitrite. NO(2)-FA exert anti-inflammatory signaling actions through post-translational protein modifications. We report that nitro-oleic acid (OA-NO(2)) stimulates proMMP-7 and proMMP-9 proteolytic activity via adduction of the conserved cysteine switch domain thiolate. Biotin-labeled OA-NO(2) showed this adduction occurs preferentially with latent forms of MMP, confirming a role for thiol alkylation by OA-NO(2) in MMP activation. In addition to regulating pro-MMP activation, MMP expression was modulated by OA-NO(2) via activation of peroxisome proliferator-activated receptor-γ. MMP-9 transcription was decreased in phorbol 12-myristate 13-acetate-stimulated THP-1 macrophages to an extent similar to that induced by the peroxisome proliferator-activated receptor-γ agonist Rosiglitazone. This was affirmed using a murine model of atherosclerosis, ApoE(-/-) mice, where in vivo OA-NO(2) administration suppressed MMP expression in atherosclerotic lesions. These findings reveal that electrophilic fatty acid derivatives can serve as effectors during inflammation, first by activating pro-MMP proteolytic activity via alkylation of the cysteine switch domain, and then by transcriptionally inhibiting MMP expression, thereby limiting the further progression of inflammatory processes.