Myristic Acid Stimulates Endothelial Nitric-oxide Synthase in a CD36- and an AMP Kinase-dependent Manner

Abstract

Dietary free fatty acids have been reported to have various effects on the endothelium including the generation of nitric oxide. The goal of the current study was to determine the mechanism whereby free fatty acid causes an increase in nitric oxide synthesis. The specific hypothesis tested was that free fatty acid association with CD36, a class B scavenger receptor, induces the activation of endothelial nitric-oxide synthase (eNOS). A human microvascular endothelial cell line and a transfected Chinese hamster ovary cell system were used to determine which free fatty acids stimulate eNOS. Surprisingly, only myristic acid, and to a lesser extent palmitic acid, stimulated eNOS. The stimulation of eNOS was dose-and time-dependent. Competition experiments with other free fatty acids and with a CD36-blocking antibody demonstrated that the effects of myristic acid on eNOS required association with CD36. Further mechanistic studies demonstrated that the effects of myristic acid on eNOS function were not dependent on PI 3-kinase, Akt kinase, or calcium. Pharmacological studies and dominant negative constructs were used to demonstrate that myristic acid/CD36 stimulation of eNOS activity was dependent on the activation of AMP kinase. These data demonstrate an unexpected link among myristic acid, CD36, AMP kinase, and eNOS activity.Dietary fatty acids are involved in the development of numerous cardiovascular diseases; however, the reported molecular mechanisms and the physiological end points are often controversial. The controversy is in part because dietary fatty acids affect multiple systems (e.g. liver, lipoproteins, endothelium, macrophages) and often have synergistic and/or antagonistic influences on the development of disease and because different fatty acids have different effects (1-4). In the present study we focused on the effects of free fatty acids on endothelial nitric-oxide synthase (eNOS) 1 activity. Few studies have examined the link between free fatty acids and eNOS activity. However, numerous studies have demonstrated that acylation of eNOS with myristic acid and palmitic acid can alter the subcellular localization of eNOS with subsequent profound effects on eNOS activity (5-8). A recent study by Esenabhalu et al. (9) showed that overloading endothelial cells with oleic acid caused an attenuation of calcium signaling, which resulted in a decrease in nitric oxide production. However, Lynn et al. (10) demonstrated that lower concentrations of oleic acid did not affect nitric oxide production. Interestingly, Lynn et al. also demonstrated that eicosapentaenoic acid did increase insulinstimulated nitric oxide production (10). These findings suggest that both the concentration and type of fatty acid can influence the activity state of eNOS and the subsequent production of nitric oxide. The mechanism(s) of how extracellular free fatty acid influences eNOS activity is not understood. One possibility is through interaction with CD36, a class B scavenger receptor that is expressed on the surface of numerous ce...

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

“…studies in which we examined the effects of individual fatty acids, which were delivered using albumin as the carrier, on eNOS activity in endothelial cells (38). In contrast with the present study, myristic acid complexed with albumin specifically and potently stimulated the activity of eNOS (38), whereas other fatty acids had little or no effect. The exact reasons for these discrepancies are presently unknown but almost certainly lie with how myristic acid is presented to the cell.…”

“…As a critical control, HDL enriched with palmitic acid did not affect NO generation, which further indicates that the inhibition is due to the presence of myristic acid and not the fatty acid enrichment procedure. Importantly, myristic acid-albumin stimulated NO production as we previously reported (38). In contrast with myristic acid-HDL, myristic acid-albumin and palmitic acidalbumin complexes did not affect acetylcholine stimulation.…”

“…We previously showed that myristic acid-albumin complexes stimulate eNOS activity; therefore, it seemed unlikely that myristic acid associated with HDL would inhibit eNOS (38). To test whether HDL-associated myristic acid affected eNOS activation, HDL from nondiabetic women was isolated, enriched with myristic acid or palmitic acid, and reisolated by size exclusion chromatography.…”

“…HME cells were then incubated with 0.75 Ci/ml of [ 3 H]arginine for 15 min to label the intracellular arginine pool. Without removal of the radiolabel, the cells were incubated with 1 g/ml of ionomycin, 10 g/ml of nondiabetic HDL, 10 g/ml of nondiabetic HDL enriched with myristic acid or palmitic acid, 10 M of myristic acidalbumin, or 10 M of palmitic acid-albumin [a ratio of 0.5 fatty acid to albumin as we previously described (38)] for 15 min at 37°C. The same treatments were also tested in the presence of 2 M acetylcholine to determine whether they affected acetylcholine-induced NO production.…”

Developmental programming of lipid metabolism and aortic vascular function in C57BL/6 mice: a novel study suggesting an involvement of LDL-receptor

“…Palmitic acid, an SFA, was increased in the aortic vessels of female S/S, S/C, and C/S offspring that showed reduced aortic contractile responses. Palmitic acid was recently shown to induce endothelial NO synthesis in conjunction with CD36, a class B scavenger receptor (63). Thus, it is likely that palmitic acidmediated increase in basal NO levels was responsible for the reduction of contractile responses observed in our study.…”