The PROactive study recently demonstrated that pioglitazone, a thiazolidinedione class drug, reduces macrovascular morbidity and mortality in patients with type 2 diabetes. This class of drug has been reported to exert PPAR gamma receptor dependent as well as receptor independent effects, possibly via AMP kinase activation (AMPK) but the precise mode of action resulting in improved cardiovascular outcome remains uncertain.
We studied the effects of rosiglitazone in cultured human aortic endothelial cells (HAEC). Cell lysates were prepared after pre-incubation of cells with rosglitazone in the presence or absence of the PPAR gamma receptor antagonist GW9662 or a dominant-negative AMPK construct. Nitric oxide (NO) release was measured with a Sievers NO meter. AMPK was immunoprecipitated from lysates and assayed using the AMARA substrate peptide.
Rosiglitazone caused acute stimulation NO production. This reached a maximum 2.1 fold increase at 60 minutes with 0.2 mM (P<0.05) and was dose dependant. AMPK was activated by 0.2 mM rosiglitazone within 10 minutes, reaching a maximum 10.6 fold stimulation at 60 minutes (P<0.05). Pre-incubation with GW9662 had no effect on rosiglitazone related NO production or AMPK activity (P<0.05). When HAECs were infected with a dominant-negative AMPK, stimulation of NO by 0.2mM of Rosiglitazone for 1 hour was significantly reduced (P<0.05).
These data support the hypothesis that the beneficial effects on cardiovascular outcome of thiazolidinediones may be partly explained by a PPAR gamma independent increase in endothelium derived NO. This may be explained by direct and rapid effects on mitochondrial respiration which reduce ATP levels so activating the enzyme AMPK. Further investigation of the way in which thiazolidinediones affect intracellular signalling in vascular endothelial cells may provide an understanding of the relative importance of receptor independent versus receptor dependent actions and facilitate more selective treatments for the management of diabetes and vascular disease.
01 - 05 Apr 2006
European Society of Endocrinology