Boyle, James Graham
The effect of metformin on vascular function and AMP-activated protein kinase activation in Type 2 diabetes.
MD thesis, University of Glasgow.
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Obesity, metabolic syndrome and Type 2 diabetes are clinical states associated with insulin resistance and an increased risk of vascular disease. The prevalence of Type 2 diabetes is reaching epidemic proportions and there is a growing need for new therapeutic targets. There is a significant body of research that demonstrates an association between obesity, insulin resistance, endothelial dysfunction and accelerated atherosclerosis, but the molecular mechanisms underlying this link are incompletely understood. Biguanides and thiazolidinediones are widely used in the treatment of Type 2 diabetes to improve glycaemia but the precise site, mode and extent of their actions remain uncertain. For example, in the United Kingdom Prospective Diabetes Study (UKPDS), metformin treatment reduced the incidence of myocardial infarction by 39% in comparison with conventional treatment which is more than would have been predicted by the difference in achieved HbA1c (0.6%). In addition, when the metformin group was compared with a group treated with sulphonylurea or insulin, to control for glycaemia, there was still a significant reduction in the incidence of stroke and any other diabetes-related end-point. The enzyme AMPK is a novel therapeutic target in Type 2 diabetes and the development of specific tissue specific AMPK activators is an attractive prospect for the future. We therefore investigated the role of AMPK in the action of biguanides and thiazolidinediones. Accordingly, the aims of this project were to first determine if metformin improves vascular endothelial function and large artery stiffness in patients with Type 2 diabetes. In parallel, the thesis examines if metformin exerts its beneficial effects in patients with Type 2 diabetes in association with altered AMPK activity in human adipose. Finally, the thesis determines if insulin sensitizers such as metformin and the thiazolidinediones, acting directly on vascular endothelial cells, increase NO production by increasing AMPK activity thus accounting for beneficial effects on endothelial function and, in metformin’s case, cardiovascular outcome.
Twenty men with Type 2 diabetes were randomised to metformin (500 milligrams three times daily for ten weeks) and gliclazide (80 milligrams twice daily for ten weeks) in a double-blinded, glycaemia controlled, cross-over design. There was a six week run in and washout period. At the end of each ten week phase of therapy routine and non routine blood sampling (including plasma ADMA), PWV and an adipose biopsy were performed to determine resistance artery function and adipose AMPK activity. The effects of metformin, its closely related analogue phenformin and rosiglitazone were characterised in cultured human aortic endothelial cells (HAECs). AMPK activity was assessed using a peptide kinase assay and the quantification of phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC) in HAECs incubated in rosiglitazone, phenformin or metformin. Nitric oxide (NO) release was evaluated with a Sievers NO meter.
The mean age and BMI of subjects in the clinical study was 56.5 years and 31kg/m2. Mean HbA1c after metformin therapy was 8.3% compared with 7.8% following gliclazide therapy. There was no significant difference between resistance artery function and PWV. ADMA was marginally lower following metformin therapy (p=0.019). AMPK activity was 2 fold higher following metformin therapy when compared with gliclazide therapy (p=0.002). Stimulation of human aortic endothelial cells with phenformin and 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 Ser1177. Infection of endothelial cells with a virus encoding a dominant negative AMPK mutant attenuated both phenformin and rosiglitazone-stimulated Ser1177 phosphorylation and NO production. Furthermore, the stimulation of AMPK and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated-receptor-inhibitor, GW9662. Incubation of HAECs with metformin, however, had no effect on AMPK activity or NO synthesis.
Taken together, the data in this thesis provides further insight into the molecular mechanisms underlying the interactions between insulin sensitizing drugs, the enzyme AMPK and both the human vascular endothelium and adipose. Further work leading to an increased understanding of the molecular mechanisms by which anti-diabetic drugs activate AMPK in both the vascular endothelium and adipose tissue may identify novel therapeutic targets in patients with Type 2 diabetes.
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