The Role Of Ceramide In The Palmitate-induced Deregulation Of PKB/eNOS Signaling Pathway In Human Umbilical Vein Endothelial Cells

ObjectiveTo investigated how free fatty acids palmitate,palmitoleate,oleate,and linoleate affect intracellular ceramide contents,thus to evaluate the mechanism of ceramide accumulation in endothelial cells.Methods①HUVECs were incubated with 600μM free fatty acids(palmitate,palmitoleate, oleate,linoleate)for 16 hours.Ceramide contents was detected by liquid chromatography/ion sprayionization tandem mass sepectrometry(LCfMS/MS).②HUVECs were incubated with palmitate in a series of concentrations(200μM,400μM,600μM)for various time(4,8,16,24,32 hours).Intracellular ceramide was detected by liquid chromatography/ion spray ionization tandem mass sepectrometry.③HUVECs were incubated with 600μM palmitate and 10μM myriocin or 10μM FB1 for 16 hours.Ceramide concentration was detected by liquid chromatography/ion spray ionization tandem mass sepectrometry.Results①The intracellular ceramide increased in a concentration -dependent manner after treating with high palmitate,and it got to the peak after 16h.Intracellular ceramide incubated with palmitoleate,oleate or linoleate had no changes.②Pretreating HUVECs with myriocin,a fungal toxin that inhibits serine palmitoyltransferase,completely prevented the palmitate-induced increase in ceramide levels.Fumonisin B1,a fungal toxin that inhibits ceramide synthase,also abolished the palmitate effect on ceramide.ConclusionPalmitate induced ceramide accumulation and this was mediated by the actions of ceramide synthesis.Palmitoleate,oleate,linoleatel had no effect on intracellular ceramide accumulation. ObjectiveTo investigated the effects of ceramide accumulation induced by palmitate on NO production and IRS-1/PI3K/PKB/eNOS signaling pathway,thus to study whether accumulation of ceramide is responsible for the palmitate-induced endothelial dysfunction through targeting insulin-induced PKB/eNOS signaling pathway.Methods①HUVECs were incubated with 5OhM insulin or 50nM insulin +palmitate in a series ofconcentrations(200μM,400μM,600μM)for various time(4,8,16,24,32 hours).Concentration of NO were measured using Griess Reaction in cell culture supernatants.②HUVECs were incubated with 50nM insulin or 50nM insulin +palmitate in a series of concentrations(200μM,400μM,600μM)for 16 hours.Expression of IRS-1,PI3K,PKB,eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.③HUVECs were incubated with 600μM palmitate and 10μM myfiocin or 10μM FB1 for 16 hours.Expression of PKB and eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.Concentration of NO were measured using Griess Reaction in cell culture supernatant.④HUVECs were incubated with 600μM palmitate and 50μM PDMP or 150μM NOE for 16 hours.Expression of PKB and eNOS,phosphorylation of PKB and eNOS were measured by Western Blot. Results①Concentration of NO decreased in a concentration -dependent manner after treating with high palmitate.②Palmitate stimulates ceramide accumulation and blocks insulin signaling by preventing the activation of PKB and eNOS,whereas it has no effect on "upstream" signaling events(i.e.insulin stimulation of IRS-1 and PI3-kinase).③Myriocin completely inhibited ceramide accumulation,and also abolished the inhibitory effect of palmitate on PKB and eNOS phosphorylation.At the same time, NO production was significantly improved.Similarly,another inhibitor Fumonisin B1 also prevented the palmitate effect on ceramide.④Treating HUVECs with either PDMP or NOE increased cellular ceramide to levels comparable with those achieved with palmitate alone,and both drugs markedly inhibited insulin-stimulated PKB and eNOS phosphorylations(but did not affected the total protein expression of PKB and eNOS.ConclusionPalmitic acid induces accumulation of ceramide,which appears to mediate palmitic acid's inhibitory effects on the PKB/eNOS pathway,leading to a significant decrease in NO generation.Thus,ceramide is a necessary and sufficient intermediate mediating the inhibition of the PKB /eNOS signaling pathway by palmitate in endothelial cells.The ceramide-mediated FFA actions on the insulin signaling may potentially contribute to the development of endothelial dysfunction and atherosclerosis in diabetes. Chapter three Effects of Rosiglitazone on Inhibition of the PKB/eNOS Signaling Pathway and Ceramide Accumulation by Palmitate in Human Vascular Endothelial CellsObjectiveTo investigated the effects of rosiglitazone on the ceramide accumulation,NO production and PKB/eNOS signaling pathway in HUVECs cultured with palmitate.Methods①HUVECs were incubated with rosiglitazone in a series of concentrations(5μM,10μM,30μM)for 16 time hours;HUVECs were incubated with 10μM rosiglitazone for for various time(4,8,16,24,32 hours).Concentration of NO were measured using Griess Reaction in cell culture supernatants.②HUVECs were incubated with rosiglitazone in a series of concentrations(5μM,10μM,30μM)for 16 hours,expression of PKB,eNOS, phosphorylation of PKB and eNOS were measured by Western Blot.③Treated HUVECs with 10μM rosiglitazone before were incubated with palmitate for 16 hours,Concentration of NO were measured using Griess Reaction in cell culture supernatants and expression of PKB,eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.④HUVECs were incubated with rosiglitazone in a series of concentrations(5μM,10μM,30μM)before cultured with 600μM palmitate for 16 hours;Intracellular ceramide was detected by liquid chromatography/ion spray ionization tandem mass sepectrometry(LC/MS/MS). Results①Rosiglitazone increased endothelial NO production in a dose dependent manner in cultured HUVECs.②Rosiglitazone increased expression PKB and eNOS phosphoraylation in concentration -dependent with no alteration in expression of PKB and eNOS in cultured HUVECs.③Treatment with 10μM Rosiglitazone increase phosphoraylation of PKB and eNOS and upregulated NO production in cultured HUVECs with palmitate.10μM Rosiglitazone inhibited ceramide accumulation,and also abolished the inhibitory effect of palmitate on Akt/PKB and eNOS phosphorylation.ConclusionTreatment with Rosiglitazone increased phosphoraylation of PKB and eNOS through a inhibition-ceramide-mediated mechanism in cultured HUVECs with high palmitate.