Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

Abstract

Aim: To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs), and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.

Methods: HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR.

Results: Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177. Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin, which abolished GLP-1(9-36) formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.

Conclusion: GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

Figure 1

GLP-1 promotes endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated for 30 min in the absence or presence of GLP-1 (5–5000 pmol/L). Nitric oxide production was assayed using the fluorescent probe DAF-FM in the presence or absence of L-NAME (1 mmol/L). The experiment was repeated 3 times, and the values are means±SD. ^bP<0.05, ^cP<0.01 compared with control.

Figure 2

Effects of GLP-1 on eNOS Ser-1177 phosphorylation, mRNA and protein expression in HUVECs. (A) Cells were incubated in the presence of GLP-1 (5000 pmol/L) for 0 (control), 5, 10, and 30 min to investigate the time course of the effect on eNOS phosphorylation. (B) Cells were incubated in the absence (control) or presence of GLP-1 (5–5000 pmol/L) for 5 min to investigate the dose-dependence of the effect. eNOS phosphorylated at ser-1177 (P-eNOS 1177) was examined by Western blot analysis. Representative experiments are both shown in the upper parts. Band intensities, after normalization to β-actin, are expressed as ratios of control. Mean±SD. n=4. ^bP<0.05, ^cP<0.01 compared with control. (C and D) Before being harvested, cells underwent 0- (as control), 6-, 12-, 24-, and 48-h exposure to GLP-1 (5000 pmol/L). (C) eNOS mRNA levels, determined by real-time RT-PCR and normalized to β-actin mRNA, were expressed as mean±SD. (D) Total eNOS protein levels, determined by Western blot analysis and normalized to β-actin protein, are expressed as mean±SD. Representative experiment is shown in the upper part. n=4. ^bP<0.05 compared with control.

Figure 3

GLP-1R-dependent pathway and GLP-1(9–36)-related pathway are involved in effects of GLP-1 on eNOS levels in HUVECs. (A) GLP-1R and DPP-4 proteins were detected in HUVECs by Western blot analysis. Cells were incubated in the presence or absence (control) of GLP-1, GLP-1(9–36) or exenatide (GLP-1R agonist) (all at 5000 pmol/L) for the following times (B, C, D, E). Cells were incubated with GLP-1 in the presence of exendin (9–39) (G+E) or sitaglipin (G+S) or both (G+E+S) for the following times (F, G, H). (B, F) After 30-min incubation, eNOS activity was determined by NO content in cells. (C, G) After 5-min incubation, phosphorylation of eNOS at ser-1177 was examined by Western blot analysis. (D, H) After 48-h incubation, total eNOS protein level was measured by Western blot analysis. The upper parts of C, D, G, and H show representative experiments. Data are mean±SD after normalization to β-actin level. (E) After 48-h incubation, eNOS mRNA level was quantified by real-time RT-PCR. Data are mean±SD after normalization to β-actin level. ^bP<0.05, ^cP<0.01 compared to control .