Molecular mechanisms of ghrelin-mediated endothelial nitric oxide synthase activation

Abstract

Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.

Figure 1

Ghrelin stimulates eNOS phosphorylation and NO production in ECs. HUVECs were incubated with ghrelin (100 nm) at the indicated times (A–C) or with various doses for 15 min (D and E). The phosphorylation of eNOS at Ser-1179 (p-eNOS) and the level of eNOS expression in cell lysates were analyzed by Western blots. The medium was collected for chemiluminescence analysis of NO production. All quantitative data are the levels of phosphorylated eNOS normalized to those of total eNOS, and presented as mean ± sem (n = 4). *, P < 0.05 vs. control without ghrelin stimulation.

Figure 2

Akt is involved in ghrelin-induced eNOS activation. A–D, HUVECs were incubated with ghrelin (100 nm) at the indicated times (A and B, n = 4) or with various doses for 15 min (C and D, n = 4). E, HUVECs were pretreated with vehicle (dimethylsulfoxide), or 100 nm wortmannin for 30 min before exposure to 100 nm ghrelin for 15 min (n = 3). G, HUVECs were infected with 50 MOI (multiplicity of infection) Ad-LacZ or Ad-Akt-DN and then exposed to ghrelin (n = 3). The phosphorylation of Akt at Ser-473 (p-Akt) and eNOS at Ser-1177 (p-eNOS) and total levels of Akt and eNOS expression were analyzed. F and H, HUVECs were treated as in E and G except for a 30-min treatment of ghrelin (n = 3). NO production was detected by chemiluminescence analysis. *, P < 0.05 vs. control without ghrelin stimulation. #, P < 0.05 vs. the group treated with ghrelin.

Figure 3

Ghrelin activates AMPK that is also involved in eNOS activation. A–D, HUVECs were incubated with ghrelin (100 nm) at indicated times (A and B, n = 4) or with various doses for 15 min (C and D, n = 4). E, HUVECs were pretreated with vehicle [dimethylsulfoxide (DMSO)], or 10 μm compound C for 30 min before exposure to 100 nm ghrelin for 15 min (n = 3). G, HUVECs were pretreated with 100 nm control siRNA, or 100 nm AMPK siRNA for 48 h before exposure to 100 nm ghrelin for 15 min (n = 3). I, HUVECs were infected with 100 MOI (multiplicity of infection) Ad-LacZ or Ad-AMPK-DN and then exposed to ghrelin (n = 3). The phosphorylation of AMPK at Thr-172 (p-AMPK) and eNOS at Ser-1177 (p-eNOS) and total levels of AMPK and eNOS expression were analyzed. F, H, and J, HUVECs were treated the same as in E, G, and I, respectively, except for a 30-min treatment of ghrelin. NO production was detected by chemiluminescence analysis. *, P < 0.05 vs. control without ghrelin stimulation. #, P < 0.05 vs. the group treated with ghrelin.

Figure 4

GHSR mediates ghrelin activation of AMPK, Akt, and eNOS in a CaMKKβ-dependent manner. A, HUVECs were transfected with scrambled control siRNA (100 nm) or human GHSR siRNA (100 nm) for 48 h, and then exposed to ghrelin for 15 min (A) or 30 min (B). Cell lysates were analyzed for Western blots (n = 3). NO production in the medium was detected by chemiluminescence analysis (n = 3). C and D, HUVECs were pretreated with vehicle [dimethylsulfoxide (DMSO)], STO-609 at the indicated does for 30 min before exposure to 100 nm ghrelin for 15 min (C) or 30 min (D) (n = 3). E and F, HUVECs were pretreated with control siRNA or CaMKKβ siRNA and then exposed to 100 nm ghrelin for 15 min (E) or 30 min (F) (n = 3). The assays were performed the same as in A and B. *, P < 0.05 vs. control without ghrelin stimulation. #, P < 0.05 vs. the group treated with ghrelin. p-AMPK, Phosphorylation of AMPK at Thr-172; p-eNOS, phosphorylation of eNOS at Ser-1179.

Figure 5

GHSR activates eNOS, Akt, and AMPK in intact vessels ex vivo and in vivo. A and B, Fresh aortas were isolated from C57BL/6 mice, and then ex vivo perfused with 100 nm ghrelin or saline (control group) for 15 min. C and D, C57BL/6 mice were subjected to tail vein injection with ghrelin (25 ng/kg) or the same amount of saline (control group) for 30 min, and then aortas were isolated. Tissue extracts from aortas were analyzed by Western blotting with various antibodies as indicated. The data represent results of three independent sets of experiments from six animals. Representative immunoblots (A and C) and quantitative data of protein phosphorylation (B and D) were shown. *, p < 0.05 vs. control without ghrelin stimulation. p-AMPK, Phosphorylation of AMPK at Thr-172; p-eNOS, phosphorylation of eNOS at Ser-179.

Figure 6

Ghrelin activation of eNOS mediates its inhibition of monocyte adhesion on ECs. A, HUVECs were pretreated with vehicle [dimethylsulfoxide (DMSO)], or 200 μm L-NAME for 30 min before exposure to 100 nm ghrelin for 30 min, and then exposed to TNFα (5 ng/ml) or vehicle for 6 h. B, U937 monocyte adhesion on TNFα- or vehicle-stimulated HUVECs was analyzed. Images of U937 monocyte adhesion on HUVECs are shown. Data shown are pooled from four independent experiments. *, P < 0.05 vs. control (Con) without TNFα stimulation alone. #, P < 0.05 vs. the group treated with TNFα plus ghrelin.