Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients.HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function.The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress,but the detailed vasoprotective mechanism remains elusive.The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction.Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo.Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy.Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo.Exendin-4 decreased the expression of ER stress markers (e.g.,ATF4,spliced XBP1,and phosphorylated elF2α) in human umbilical vein endothelial cells (HUVECs),and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor).Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries.Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α),an important ER chaperone in endothelial cells,and this effect was mediated by AMPK activation.Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production,thus ameliorating HHcy-induced endothelial dysfunction.The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries.AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.