Abstract 588: Corticotropin-releasing Hormone via Its Type2 Receptor Inhibits Insulin-stimulated NO Formation in HUVECs

Abstract

Background: Stress may provoke endothelial dysfunction thereby inducing atherosclerotic cardiovascular diseases (ASCVD). Corticotropin-releasing hormone (CRH) is recognized as a key regulator of hypothalamo-pituitary-adrenal axis under stress, however, its role in endothelial function and ASCVD is not known well. Insulin can produce NO formation in endothelial cells through eNOS activation. We studied effect of CRH and urocortin 2 (UCN2) on insulin-stimulated NO formation in HUVECs. Methods and Results: HUVECs were incubated in serum-free media for 8 h prior to the experiment. Treatment of HUVECs with insulin (100 nM) enhanced NO production detected by 5 μM 4,5-diaminofluorescein-diacetate. Fluorescent and phase contrast images were obtained using an fluorescence microscope (Em max : 515 nm), using an exposure time based on control conditions, and mean fluorescence intensity was calculated with ImageJ. Insulin-stimulated NO production was markedly inhibited by either CRH or UCN2 (100 nM each), which was reversed mostly by 100 nM astressin 2B (CRH receptor type 2 (CRHR2) inhibitor) and partly by 100 nM NBI35965 (CRHR1 inhibitor). CRH induced Rho-associated kinase (ROCK) and p-IRS-1(Ser). Insulin (2nM) stimulated p-eNOS (Ser1177), which was inhibited by CRH. Conclusions: Taken together, CRH/UCN2 via mainly its type 2 receptor signaling can inhibit insulin-stimulated eNOS/NO pathway. This CRH’s inhibitory effect on insulin signaling is mediated at least partly by activation of ROCK/ p-IRS-1 (Ser).