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Abstract
Background
Oxidative stress and inflammation are important factors in the pathogenesis of diabetes and contribute to the development of diabetic complications. To understand the mechanisms that cause vascular complications in diabetes, we examined the effects of high glucose and/or free fatty acids on the production of superoxide from neutrophils and their role in endothelial cell damage.
Methods
Human neutrophils were incubated in the media containing 5.5 mM D-glucose, 30 mM D-glucose, 3 nM oleic acid, or 30 µM oleic acid for 1 hour to evaluate superoxide production through NAD(P)H oxidase activation. Human aortic endothelial cells were co-cultured with neutrophils exposed to high glucose and oleic acid. We then measured neutrophil adhesion to endothelial cells, neutrophil activation and superoxide production, neutrophil-mediated endothelial cell cytotoxicity and subunits of neutrophil NAD(P)H oxidase.
Results
After 1 hour of incubation with various concentrations of glucose and oleic acid, neutrophil adherence to high glucose and oleic acid-treated endothelial cells was significantly increased compared with adhesion to low glucose and oleic acid-treated endothelial cells. Incubation of neutrophils with glucose and free fatty acids increased superoxide production in a dose-dependent manner. High glucose and oleic acid treatment significantly increased expression of the membrane components of NAD(P)H oxidase of neutrophil (gp91phox). Endothelial cells co-cultured with neutrophils exposed to high glucose and oleic acid showed increased cytolysis, which could be prevented by an antioxidant, N-acetylcysteine.
Figures and Tables
Fig. 1
Effect of high glucose and/or oleic acid on neutrophil adhesion to endothelial cells. *P < 0.01; †P < 0.01; ‡P < 0.001 vs. cells treated with normal glucose or low oleic acid for 1 h. §P < 0.001 vs. cells treated with high glucose and high oleic acid. NAC, N-acetylcystein.
Fig. 2
Effect of high glucose and/or oleic acid on neutrophil activation. *P < 0.01; †P < 0.01; ‡P < 0.001 vs. cells treated with normal glucose or low oleic acid for 1 h.
Fig. 3
Effect of high glucose and/or oleic acid on neutrophil superoxide production. *P < 0.01; †P < 0.01; ‡P < 0.001 vs. cells treated with normal glucose or low oleic acid for 1 h. §P < 0.001 vs. cells treated with high glucose and high oleic acid. NAC, N-acetylcystein.
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