Objective—We investigated whether NADPH oxidase–dependent production of superoxide contributes to activation of NF-B in endothelial cells by the saturated free fatty acid palmitate.

Methods and Results—After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 µmol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a >2-fold increase in superoxide levels, an effect associated with activation of NF-B signaling as measured by phospho-IB, NF-B activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of 3 different interventions—pretreatment with superoxide dismutase (SOD), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA—attenuated palmitate-mediated NF-B signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitate-mediated superoxide production was not affected by overexpression of a phosphorylation mutant IB (NF-B super repressor) that blocks cellular inflammation downstream of IKK/NF-B. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4^-/- mice, compared to low-fat fed controls.

Conclusions—These results suggest that NADPH oxidase–dependent superoxide production links palmitate-stimulated TLR4 activation to NF-B signaling in endothelial cells.