Effect Of Glucagon-like Peptide1on Endothelial Nitric Oxide Production With High Free Fatty Acids And Its Underlying Mechanism

Objective:To investigate the effect of GLP-1on reducing nitric oxide (NO) production of human umbilical vein endothelia cells (HUVECs) induced by PA and explore the underlying mechanism.Methods:To find out the proper concentration of PA that could significantly decrease NO production in HUVECs. HUVECs were cultured with different concentrations of palmitic acid (PA) which were0,100μM,200μM,400μM and600μM respectively. Nitric Oxide assay kit was used to detect NO concentration of cell culture media, Western Blot was used to detect the expression level of p-eNOS(Ser1177).HUVECs were cultured with different concentration of GLP-1(Ong/ml、1ng/ml、5ng/ml、10ng/ml), with the aim of exploring the effective concentration of GLP-1, We use the same method as above to detect NO concentration of culture medium and expression level of p-eNOS(Ser1177). This research focused on the upregulation effect of GLP-1on NO production damaged by PA, we add GLP-1to HUVECs which have been pre-incubated with PA,with or without PI3K inhibitor, to explore its possible mechanism. In this research we establish four groups as follows: A blank control group containing1%BSA in the culture medium, B FFAs group with400μM PA, C FFAs+GLP-1group culture with400μM PA+1ng/ml GLP-1, D FFAs+GLP-1+LY294002culture with400μM PA+1ng/ml GLP-1+20μM LY294002. Sample was extracted from each group and the concentration of NO was measured meanwhile Western Blot was used to detect the expression level of p-Akt (Ser473) and p-eNOS (Ser1177).Result:After incubation of HUVECs with different concentration of PA, the amount of NO and p-eNOS(Ser1177) decreased while concentration of PA increased, they were even lower than those of control group when the concentration of PA surpass200μM (P<0.05or P <0.01). Under different GLP-1concentration used (0ng/ml,lng/ml,5ng/ml,10ng/ml),both NO and p-eNOS(Ser1177) increased in the concentration of1ng/ml (P<0.05or P<0.01). Observations from our4HUVECs cultures(A Blank Control Group, B PA Group, C PA+GLP-1Group,D PA+GLP-1+LY294002Group) found that the amounts of NO, p-eNOS(Ser1177) and p-Akt (Ser473) all increased in Group C when compared to B(P<0.05), but the amounts of NO and p-Akt (Ser473) were lower than the Blank Control Group (P<0.05). Another finding showed the amounts of NO and p-Akt (Ser473) in Group D being lower than C(P<0.05), but higher than B (P<0.05).Conclusions:First, high free fatty acids inhibit eNOS activity, reduces vascular endothelial NO production. Second, GLP-1can upregulate eNOS activity and NO production. Third, GLP-1can partially reverse endothelial damage caused by high FFAs, by upregulating PI3K-Akt-eNOS pathway, though there may be other signal pathways.