OBJECTIVE: To investigate how ceramide mediates the effects of high-glucose-induced inhibition of the Akt/endothelial nitric oxide synthase (eNOS) signalling pathway in human vascular endothelial cells (HUVECs). MATERIALS AND METHODS: NO levels were determined by ELISA. Endogenous ceramide levels were determined using a liquid chromatography-mass spectrometry assay. Akt and eNOS protein expressions were determined by Western blotting. RESULTS: High-glucose levels induce ceramide accumulation in a dose- and time-dependent manner (p<0.05). We also show that exposure of HUVECs to high-glucose conditions inhibits the insulin-mediated activation of Akt/eNOS signalling and the subsequent NO generation in a dose-dependent manner (p<0.05). Preventing de novo ceramide synthesis attenuated the antagonistic effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05); conversely, inducing ceramide build-up augmented the inhibitory effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05). CONCLUSION: Ceramide is both necessary and sufficient for mediating the inhibition of the Akt/eNOS signalling pathway by high-glucose levels in endothelial cells.
OBJECTIVE: To investigate how ceramide mediates the effects of high-glucose-induced inhibition of the Akt/endothelial nitric oxide synthase (eNOS) signalling pathway in human vascular endothelial cells (HUVECs). MATERIALS AND METHODS: NO levels were determined by ELISA. Endogenous ceramide levels were determined using a liquid chromatography-mass spectrometry assay. Akt and eNOS protein expressions were determined by Western blotting. RESULTS: High-glucose levels induce ceramide accumulation in a dose- and time-dependent manner (p<0.05). We also show that exposure of HUVECs to high-glucose conditions inhibits the insulin-mediated activation of Akt/eNOS signalling and the subsequent NO generation in a dose-dependent manner (p<0.05). Preventing de novo ceramide synthesis attenuated the antagonistic effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05); conversely, inducing ceramide build-up augmented the inhibitory effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05). CONCLUSION:Ceramide is both necessary and sufficient for mediating the inhibition of the Akt/eNOS signalling pathway by high-glucose levels in endothelial cells.