Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Exogenous Nitro-Oleic Acid Treatment Inhibits Primary Root Growth by Reducing the Mitosis in the Meristem in Arabidopsis thaliana.

Nitric oxide (NO) is a second messenger that regulates a broad range of physiological processes in plants. NO-derived molecules called reactive nitrogen species (RNS) can react with unsaturated fatty acids generating nitrated fatty acids (NO2-FA). NO2-FA work as signaling molecules in mammals where production and targets have been described under different stress conditions. Recently, NO2-FAs were detected in plants, however their role(s) on plant physiological processes is still poorly known. Although in this work NO2-OA has not been detected in any Arabidopsis seedling tissue, here we show that exogenous application of nitro-oleic acid (NO2-OA) inhibits Arabidopsis primary root growth; this inhibition is not likely due to nitric oxide (NO) production or impaired auxin or cytokinin root responses. Deep analyses showed that roots incubated with NO2-OA had a lower cell number in the division area. Although this NO2-FA did not affect the hormonal signaling mechanisms maintaining the stem cell niche, plants incubated with NO2-OA showed a reduction of cell division in the meristematic area. Therefore, this work shows that the exogenous application of NO2-OA inhibits mitotic processes subsequently reducing primary root growth.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages.

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO2-OA) on macrophage lipid metabolism. Pure synthetic NO2-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.

Profile of Arachidonic Acid-Derived Inflammatory Markers and Its Modulation by Nitro-Oleic Acid in an Inherited Model of Amyotrophic Lateral Sclerosis.

The lack of current treatments for amyotrophic lateral sclerosis (ALS) highlights the need of a comprehensive understanding of the biological mechanisms of the disease. A consistent neuropathological feature of ALS is the extensive inflammation around motor neurons and axonal degeneration, evidenced by accumulation of reactive astrocytes and activated microglia. Final products of inflammatory processes may be detected as a screening tool to identify treatment response. Herein, we focus on (a) detection of arachidonic acid (AA) metabolization products by lipoxygenase (LOX) and prostaglandin endoperoxide H synthase in SOD1G93A mice and (b) evaluate its response to the electrophilic nitro-oleic acid (NO2-OA). Regarding LOX-derived products, a significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) levels was detected in SOD1G93A mice both in plasma and brain whereas no changes were observed in age-matched non-Tg mice at the onset of motor symptoms (90 days-old). In addition, 15-hydroxyeicosatetraenoic acid (15-HETE) levels were greater in SOD1G93A brains compared to non-Tg. Prostaglandin levels were also increased at day 90 in plasma from SOD1G93A compared to non-Tg being similar in both types of animals at later stages of the disease. Administration of NO2-OA 16 mg/kg, subcutaneously (s/c) three times a week to SOD1G93A female mice, lowered the observed increase in brain 12-HETE levels compared to the non-nitrated fatty acid condition, and modified many others inflammatory markers. In addition, NO2-OA significantly improved grip strength and rotarod performance compared to vehicle or OA treated animals. These beneficial effects were associated with increased hemeoxygenase 1 (HO-1) expression in the spinal cord of treated mice co-localized with reactive astrocytes. Furthermore, significant levels of NO2-OA were detected in brain and spinal cord from NO2-OA -treated mice indicating that nitro-fatty acids (NFA) cross brain-blood barrier and reach the central nervous system to induce neuroprotective actions. In summary, we demonstrate that LOX-derived oxidation products correlate with disease progression. Overall, we are proposing that key inflammatory mediators of AA-derived pathways may be useful as novel footprints of ALS onset and progression as well as NO2-OA as a promising therapeutic compound.