Rosiglitazone Stimulates Nitric Oxide Synthesis in Human Aortic Endothelial Cells via AMP-activated Protein Kinase*

James G Boyle,Pamela J Logan,Marie-Ann Ewart,James A Reihill,Stuart A Ritchie,John M.C Connell,Stephen J Cleland,Ian P Salt

doi:10.1074/jbc.m710048200

Abstract

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-gamma inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.

Highlights

Type 2 diabetes is associated with a greatly increased risk of atheromatous vascular disease, and vascular endothelial dysfunction has been demonstrated in type 2 diabetic patients (4 – 6)
We examined the ability of rosiglitazone to modulate NO production and AMPK activity in human aortic endothelial cells (HAECs)
To determine whether AMPK activation was required for rosiglitazone-stimulated NO synthesis, HAECs were infected with control (Ad.Null) adenoviruses or adenoviruses expressing dominant negative AMPK (Ad.␣1DN) prior to incubation with rosiglitazone (200 ␮M) for 1 or 24 h, and NO production was assessed

Summary

Introduction

Type 2 diabetes is associated with a greatly increased risk of atheromatous vascular disease, and vascular endothelial dysfunction has been demonstrated in type 2 diabetic patients (4 – 6). Preincubation of HAECs with the PPAR␥ inhibitor GW9662 (5 ␮M) was without effect on both basal or rosiglitazone-stimulated AMPK activity and NO synthesis (Fig. 1B). Preincubation of cells with the CaMKK inhibitor STO-609 had no significant effect on rosiglitazone-stimulated AMPK activity, AMPK Thr-172 phosphorylation, or phosphorylation of the AMPK substrate acetyl CoA carboxylase (ACC), yet completely inhibited vascular endothelial growth factor-stimulated AMPK activity (Fig. 3).

Results

Conclusion