title: Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt.
abstract: Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.
authors.affiliation: Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Christine F
authors.initials: CF
authors.lastname: Kohlhaas
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Morrow
authors.forename: Neelam
authors.initials: N
authors.lastname: Jhakra
authors.forename: Vrushali
authors.initials: V
authors.lastname: Patil
authors.forename: John M C
authors.initials: JM
authors.lastname: Connell
authors.forename: John R
authors.initials: JR
authors.lastname: Petrie
authors.forename: Ian P
authors.initials: IP
authors.lastname: Salt
mesh_terms: D001011:Aorta / Q000187:drug effects* / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*
chemical_list: D007328:Insulin; D009569:Nitric Oxide; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt