Palmitate evokes ceramide‐dependent reactive oxygen species (ROS) generation from sources other than NADPH oxidase in bovine aortic endothelial cells (BAECs)

Abstract

In preliminary studies we showed that basal and insulin‐mediated endothelial nitric oxide (NO) synthase phosphorylation (p‐eNOS) and NO production are impaired when BAECs are treated with the saturated fatty acid palmitate (P, 500 uM) vs. vehicle (V) for 3 hours (h). Co‐incubation of BAECs with the ceramide synthesis inhibitor myriocin (M, 10 μM) negated these effects. ROS can lower NO bioavailability. We determined the contribution from ceramide to P‐induced ROS production, and investigated sources of ROS. DCFDA fluorescence was used to assess ROS after BAECs were incubated for 3h with V ± P, M, 10 μM KCN (a mitochondrial ROS inhibitor), or 1 μM NVN (an NADPH oxidase inhibitor). Relative to V, P‐evoked increases (2.8±0.1‐fold, p<0.05, n=48) in ROS production were blunted (15±5%; p<0.05) but were not completely abolished by M, suggesting the existence of ceramide‐dependent and –independent pathways. KCN, NVN, and KCN+NVN lowered (p<0.05) P‐evoked ROS production by 37±4%, 18±7%, and 39±12% (n=8–32), respectively, suggesting that in addition to mitochondrial and NADPH oxidase – dependent sources of ROS, additional sources of palmitate‐induced ROS exist. P‐induced ROS production decreased further when BAECs were co‐incubated with M+NVN (46±4%, p<0.05, n=16) vs. NVN alone, but not when M was co‐incubated with KCN (n=12) or KCN+NVN (n=16). Moreover, P‐evoked increases (p<0.05, n=12) in NADPH oxidase activity were similar in the absence (1.6±0.2‐fold) and presence (2.1±0.2‐fold) of M. Thus, P‐evoked and ceramide‐mediated ROS production in BAECs is not derived from NADPH oxidase but may arise from mitochondria. NIHR15HL091493, ADA7‐08‐RA‐164