Abstract
Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.
Highlights
Endothelial cells (ECs) are essential for modulation of vascular homeostasis and signal transduction [1], including the production and regulation of vascular tone, modulation of inflammatory responses and maintenance of an anti-atherogenic phenotype of vascular smooth muscle cells (VSMCs) [1]
Insulin acts as a vasodilator, stimulating calcium-independent NO synthesis in cultured human aortic ECs (HAECs) through Akt-mediated phosphorylation of endothelial nitric oxide synthase Ser1177 and Ser615 [6]
Stimulation of HUVECs with concentrations of insulin above 0.1 μM robustly stimulated phosphorylation of Akt at Ser473 and Thr308 (Supplementary Figure S1), similar to concentrations of insulin previously demonstrated to be required for insulin-stimulated NO synthesis in cultured endothelial cells [30]
Summary
Endothelial cells (ECs) are essential for modulation of vascular homeostasis and signal transduction [1], including the production and regulation of vascular tone, modulation of inflammatory responses and maintenance of an anti-atherogenic phenotype of vascular smooth muscle cells (VSMCs) [1]. Insulin acts as a vasodilator, stimulating calcium-independent NO synthesis in cultured human aortic ECs (HAECs) through Akt-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) Ser1177 and Ser615 [6]. Several AMPK activators have been shown to increase NO synthesis in HAECs in an AMPK-dependent manner [10] It is unclear whether all AMPK activators improve insulin sensitivity and vascular function, as previous studies have largely been conducted using a variety of compounds that activate AMPK by altering cellular nucleotide ratios, including rosiglitazone, resveratrol, metformin and canagliflozin or mimic AMP, such as AICAR (5-amino-4-imidazolecarboxamide ribonucleoside) [12,19,20,21,22]. The effects of A769662 on insulin signalling and insulin-stimulated Akt/eNOS axis activation in primary human ECs were determined
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
