Adipocyte-specific Nrf2 deletion negates nitro-oleic acid benefits on glucose tolerance in diet-induced obesity.

Obesity is commonly linked with white adipose tissue (WAT) dysfunction, setting off inflammation and oxidative stress, both key contributors to the cardiometabolic complications associated with obesity. To improve metabolic and cardiovascular health, countering these inflammatory and oxidative signaling processes is crucial. Offering potential in this context, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by nitro-fatty acids (NO2-FA) promote diverse anti-inflammatory signaling and counteract oxidative stress. Additionally, we previously highlighted that nitro-oleic acid (NO2-OA) preferentially accumulates in WAT and provides protection against already established high fat diet (HFD)-mediated impaired glucose tolerance. The precise mechanism accounting for these protective effects remained largely unexplored until now. Herein, we reveal that protective effects of improved glucose tolerance by NO2-OA is absent when Nrf2 is specifically ablated in adipocytes (ANKO mice). NO2-OA treatment did not alter body weight between ANKO and littermate controls (Nrf2fl/fl) mice on both the HFD and low-fat diet (LFD). As expected, at day 76 (before NO2-OA treatment) and notably at day 125 (daily treatment of 15 mg/kg NO2-OA for 48 days), both HFD-fed Nrf2fl/fl and ANKO mice exhibited increased fat mass and reduced lean mass compared to LFD controls. However, throughout the NO2-OA treatment, no distinction was observed between Nrf2fl/fl and ANKO in the HFD-fed mice as well as in the Nrf2fl/fl mice fed a LFD. Glucose tolerance tests revealed impaired glucose tolerance in HFD-fed Nrf2fl/fl and ANKO compared to LFD-fed Nrf2fl/fl mice. Notably, NO2-OA treatment improved glucose tolerance in HFD-fed Nrf2fl/fl but did not yield the same improvement in ANKO mice at days 15, 30, and 55 of treatment. Unraveling the pathways linked to NO2-OA's protective effects in obesity-mediated impairment in glucose tolerance is pivotal within the realm of precision medicine, crucially propelling future applications and refining novel drug-based strategies.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes.

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

Circulating plasma factors in Parkinson's disease enhance nitric oxide release of normal human neutrophils.

Nitric oxide (*NO)-mediated toxicity has been involved in neurodegenerative diseases, including Parkinson's disease (PD). We have recently reported an increase of about 50% in *NO production rate in PMA-activated polymorphonuclear leukocytes (PMN) from either newly diagnosed or chronically treated PD patients. As humoral factors in sera from PD patients could inhibit cell dopaminergic activity, the aim of this study was to determine whether a plasma circulating factor from PD patients could modify *NO metabolism in PMN from healthy control subjects. To this purpose, we determined simultaneously the maximal production rate of *NO and hydrogen peroxide (H2O2) of PMA-activated PMN isolated from healthy control subjects in the presence of aliquots of plasma of PD patients. The results showed that, after 30 min incubation, plasma from newly diagnosed (n=4) or from L-Dopa chronically treated (n=7) PD patients enhanced *NO release in neutrophils isolated from healthy controls by about 50% and 47% respectively, with respect to non-parkinsonian control plasma (n = 10); in the same condition, H2O2 production did not differ among the groups. These data suggest that an overproduction of *NO related to plasma circulating factors, already detected at initial stages of the disease, participates in the pathophysiology of Parkinson's disease.

Nitro-oleic acid targets transient receptor potential (TRP) channels in capsaicin sensitive afferent nerves of rat urinary bladder.

Nitro-oleic acid (9- and 10-nitro-octadeca-9-enoic acid, OA-NO(2)) is an electrophilic fatty acid nitroalkene derivative that modulates gene transcription and protein function via post-translational protein modification. Nitro-fatty acids are generated from unsaturated fatty acids by oxidative inflammatory reactions and acidic conditions in the presence of nitric oxide or nitrite. Nitroalkenes react with nucleophiles such as cysteine and histidine in a variety of susceptible proteins including transient receptor potential (TRP) channels in sensory neurons of the dorsal root and nodose ganglia. The present study revealed that OA-NO(2) activates TRP channels on afferent nerve terminals in the urinary bladder and thereby increases bladder activity. The TRPV1 agonist capsaicin (CAPS, 1 µM) and the TRPA1 agonist allyl isothiocyanate (AITC, 30 µM), elicited excitatory effects in bladder strips, increasing basal tone and amplitude of phasic bladder contractions (PBC). OA-NO(2) mimicked these effects in a concentration-dependent manner (1 µM-33 µM). The TRPA1 antagonist HC3-030031 (HC3, 30 µM) and the TRPV1 antagonist diaryl piperazine analog (DPA, 1 µM), reduced the effect of OA-NO(2) on phasic contraction amplitude and baseline tone. However, the non-selective TRP channel blocker, ruthenium red (30 µM) was a more effective inhibitor, reducing the effects of OA-NO(2) on basal tone by 75% and the effects on phasic amplitude by 85%. In bladder strips from CAPS-treated rats, the effect of OA-NO(2) on phasic contraction amplitude was reduced by 65% and the effect on basal tone was reduced by 60%. Pretreatment of bladder strips with a combination of neurokinin receptor antagonists (NK1 selective antagonist, CP 96345; NK2 selective antagonist, MEN 10,376; NK3 selective antagonist, SB 234,375, 1 µM each) reduced the effect of OA-NO(2) on basal tone, but not phasic contraction amplitude. These results indicate that nitroalkene fatty acid derivatives can activate TRP channels on CAPS-sensitive afferent nerve terminals, leading to increased bladder contractile activity. Nitrated fatty acids produced endogenously by the combination of fatty acids and oxides of nitrogen released from the urothelium and/or afferent nerves may play a role in modulating bladder activity.

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.