Nitro‐Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes

Abstract

Nitro-fatty acids possess anti-atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro-oleic acid (OLA-NO2) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti-oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA-NO2 (0-1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7-dichlorofluorescein diacetate, decreased dose-dependently, but the anti-oxidative effects of OLA-NO2 were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA-NO2 treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA-NO2 vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA-NO2 vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA-NO2 treated cells demonstrated down-regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone-sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA-NO2 vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein-mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA-NO2 modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti-oxidants and triglyceride metabolizing enzymes.

New Insights into the Anti‐Inflammatory and Antioxidant Properties of Nitrated Phospholipids

“…Incubation of macrophages with either physiological concentrations of OLA‐NO 2 or with equivalent levels of OLA decreased dose dependently the cellular ROS generation levels. Additionally, compared with native OLA, OLA‐NO 2 was found to have modest but significant beneficial effects on macrophage oxidative status (Rosenblat et al, ). However, for the ABTS •+ and DPPH • assays, no antioxidant activity was observed for nonmodified POPC.…”

“…Indeed, OLA was already shown to be able to protect PC12 rat cells against nanoparticle-induced oxidative stress, enhancing cell survival (Khatchadourian & Maysinger, 2009). Also, another recent study, aiming to compare the effects of OLA-NO 2 with those of OLA on cellular oxidative status of J774A.1 macrophages, evaluated their effects on intracellular ROS generation (Rosenblat, Rom, Volkova, & Aviram, 2016). Incubation of macrophages with either physiological concentrations of OLA-NO 2 or with equivalent levels of OLA decreased dose dependently the cellular ROS generation levels.…”

Leucine supplementation attenuates macrophage foam‐cell formation: Studies in humans, mice, and cultured macrophages

“…VLDL and LDL were isolated from plasma of healthy humans by discontinuous density gradient ultracentrifugation as previously described (47,49). VLDL fraction was separated at d= 1.006 g/mL, and LDL fraction at d= 1.063.…”

“…[ 3 H]-labels counts were normalized to cellular protein levels, and expressed as cpm/mg cell protein.2.5.4. Triglycerides biosynthesis rateMacrophages were washed with PBS (1x) and the rate of triglycerides biosynthesis was assessed after a 4 h-incubation period at 37°C with [ 3 H]-oleic acid (3.3 µCi/mL) in serum-free DMEM supplemented with 0.2% BSA(34,47,48).At the end of the incubation time, cells were washed with PBS (x2), and lipids were extracted. After evaporation of the hexane phase, lipids were dissolved in CHCl 3 and separated by TLC on silica gel plates, and developed in hexane:ether:acetic acid (130:30:1.5, v:v:v).…”

Inflammatory signaling and metabolic regulation by nitro-fatty acids

“…Still, administration of NO 2 -FA in pre-clinical models of metabolic disorders suggests an active role in lipid metabolism, with observed improved lipid profiles and liver steatosis, with decreased plasma triglycerides [62; 63] and reduced expression of lipoprotein-associated phospholipase A2 (Lp-PLA2), a biomarker of cardiovascular risk [64]. In macrophages, NO 2 -FA prevents triglyceride accumulation via down-regulation of diacylglycerol acyltransferase 1 (DGAT1), and induced expression of triglyceride hydrolysis enzymes, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) [65]. Novel evidence demonstrates an active incorporation of free NO 2 -FA into monoacyl-, diacyl- and triglycerides [66], suggesting that the aforementioned lipases might be putative targets of NO 2 -FA nitroalkylation.…”