Effects Of Ceramide On High Glucose-induced Deregulation Of PKB/eNOS Signaling Pathway In Human Umbilical Vascular Endothelial Cells

ObjectiveThis study aims to identify the following points:1. Observe the changes of ceramide in the vascular endothelial cells cultured in high D-glucose.2. To investigate the effects of ceramide accumulation induced by high D-glucose on NO production and IRS-1/PI3K/PKB (Akt)/eNOS signaling pathway, thus to study whether the accumulation of ceramide is responsible for the high D-glucose-induced endothelial dysfunction through targeting insulin-induced PKB/eNOS signaling pathway.MethodsMaterialsHUVECs exposed to5mmol/L D-glucose and30mM D-glucose medium for4,8,12,16,24hours to observe the levels of ceramide and NO accumulation, respectively; we selected some HUVECs that were incubated with D-glucose at various concentrations (5,10,20,30mM) for16h to observe the changes of ceramide and NO levels,respectively; HUVECs is stimulated with insulin at50nM for16hours to observe the changes of NO levels; followed by culture supernatant harvest and/or cell lysate preparation for assays described below. We used de novo ceramide synthesis blocker50μM myriocin and2μM DES, to attenuate the antagonistic effect of HG toward the Akt/eNOS pathway. Moreover, the inhibition of the ceramide degradation pathways blocker150μM NOE and50μM PDMP inducing ceramide build up augmented the inhibitory effect of HG on PI3K/PKB/eNOS signaling pathways.MethodsThe total proteins of HUVECs were extracted by Cytoplasmic Protein Extraction Kit. The ceramide level was determined by liquid chromatography-mass spectrometry. The NO level was determined by diazonium reduction method. The protein expressions of Akt and eNOS were determined by Western blot.Results1. High D-glucose concomitantly induced the accumulation of ceramide in dose-dependent and time-dependent manners in HUVECs (p <0.05), reaching a maximum value at16h; High L-glucose had no significant effect on the accumulation of ceramide in HUVECs (p>0.05).2. High D-glucose also decreased NO generation in dose-dependent and time-dependent manners in HUVECs (p<0.05)3. Exposing HUVECs to50nM insulin for4,8,12,16h, recceptively, NO increased significantly and reached the maximum value at4hours, and decreased to lowest value at24hours.(p<0.05)4. High D-glucose stimulated ceramide accumulation and blocks insulin signaling by preventing the activation of PKB and eNOS,(p<0.05)5. Myriocin, the blocker of de novo ceramide synthesis, completely inhibited ceramide accumulation, and also attenuated the antagonistic effect of high D-glucose on PKB and eNOS phosphorylation. At the same time, NO production was significantly improved. Similarly, another inhibitor DES also prevented the high D-glucose effect on ceramede.(p <0.05).6. NOE or PDMP which block the degradation pathway of ceramide increased ceramide accumulation, NOE decreased the phosphorylation of protein expression of PKB and eNOS and decreased NO generation (p <0.05).Conclusions:High glucose induces the accumulation of ceramide, which appears to mediate HG’s inhibitory effects on the Akt/eNOS pathway in endothelial cells, leading to a significant decrease in NO generation; Myriocin or DES reduces ceramide accumulation induced by high D-glucose, improving the activity of Akt/eNOS, and increase NO production; NOE and PDMP induce ceramide accumulation, augment the inhibitory effect of high glucose on PI3K/PKB/e NOS signaling pathways and decrease NO production. Therefore, ceramide is involved in dysfunction of vascular endothelial cells through the Akt/eNOS signaling pathway.