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Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1β-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1β-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1β-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1β-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1β signalling.

Peroxynitrite is an endothelium-independent vasodilator that induces relaxation via membrane hyperpolarization. The activation of IP3 receptors triggers the opening of potassium channels and hyperpolarization. Previously we found that relaxation to peroxynitrite was maintained during the development of atherosclerosis due to changes in the expression of calcium-regulatory proteins. In this study we investigated: (1) the mechanism of peroxynitrite-induced relaxation in the mouse aorta, (2) the effect of atherosclerosis on relaxation to peroxynitrite and other vasodilators, and (3) the effect of atherosclerosis on the expression and function of the IP3 receptor. Aortic function was studied using wire myography, and atherosclerosis was induced by fat-feeding ApoE -/- mice. The expression of IP3 receptors was studied using Western blotting and immunohistochemistry. Relaxation to peroxynitrite was attenuated by the IP3 antagonists 2-APB and xestospongin C and also the K v channel blocker 4-aminopyridine (4-AP). Atherosclerosis attenuated vasodilation to cromakalim and the AMPK activator A769662 but not peroxynitrite. Relaxation was attenuated to a greater extent by 2-APB in atherosclerotic aortae despite the reduced expression of IP3 receptors. 4-AP was less effective in ApoE -/- mice fat-fed for 4 months. Peroxynitrite relaxation involves an IP3-induced calcium release and K V channel activation. This mechanism becomes less important as atherosclerosis develops, and relaxation to peroxynitrite may be maintained by increased calcium extrusion.

This thesis, entitled: ‘Role of AMP-protein kinase (AMPK) in regulation of perivascular adipose tissue (PVAT) function’, has been submitted by author Tarek Ali Mohamed Almabrouk for a degree of Doctor of Philosophy (PhD) in the College of Medical, Veterinary and Life Sciences at the University of Glasgow, October 2016. Apart from the cerebral circulation, all vasculature is surrounded by layers of adipose tissue known as perivascular adipose tissue (PVAT). In health, PVAT can function as an endocrine organ to produce a wide range of adipocytokines which can attenuate vascular contraction. The exact mechanism of this anti-contractile effect is still ill-defined, although much evidence suggests that PVAT-released adipocytokines may activate K+ channels on VSMCs or eNOS on endothelial layer possibly via AMP-activated protein kinase (AMPK). However, obesity results in oxidative stress and inflammation of the PVAT leading to abnormal adipocytokine release and PVAT dysfunction. AMPK is a serine/threonine kinase with many potential physiological functions, including regulation of energy heamostasis. AMPK is expressed in the three layers of the blood vessel: smooth muscle (VSM), the endothelium and PVAT and it is known that activation of AMPK leads to vascular dilatation via both endothelium- and non-endothelium-dependent mechanisms. Although it is known that AMPK can modulate VSM and endothelial function, it is unknown whether AMPK can influence the anti-contractile activity of PVAT. Therefore, this project aimed to investigate the mechanism of the anticontractile effect of PVAT by determining the functions of AMPK within adipocytes, as well as to assess the importance of vascular AMPK to the PVAT anti-contractile function. Experiments were conducted using wild type (WT) and global AMPKα1 knockout (KO) mice aortae. The phenotypic features of the PVAT were assessed by both histological, immunohistochemical and immunofluorescent methods. Secretory function of the PVAT was tested using an immunoblotting array and ELISA, whereas the anti-contractile effect of PVAT was studied using wire myography. Immunoblotting methods were used to test AMPK activity in the PVAT and VSMCs. Aortic rings from WT and KO mice were denuded of endothelium and mounted on a wire myograph in the presence and absence of PVAT. The responses to an AMPK activator (AICAR) and the AMPK-independent vasodilator cromakalim were subsequently assessed. Relaxation responses to AICAR or cromakalim in the Sv129 (wild type) mouse were significantly enhanced in the presence of endogenous attached or unattached PVAT, an effect that was absent in vessels from KO mice. Furthermore, enhanced relaxation was observed in vessels from KO mice incubated with PVAT from Sv129 mice, whereas PVAT from KO mice had no effect on relaxation of vessels from Sv129 mice. Furthermore, conditioned medium (CM) transfer experiments demonstrated the presence of an anticontractile factor released from PVAT that was absent in KO mice. Adiponectin secretion was reduced in PVAT from KO mice and PVAT-enhanced relaxation was attenuated in the presence of adiponectin blocking peptide. Adipokine array and ELISA demonstrated that adiponectin release is significantly reduced in the KO conditioned media in comparison with wild type CM. Globular adiponectin restores the relaxation response in both wild type aortae without PVAT and in KO aortae with and without PVAT. High fat diet (HFD) fed mice showed a reduction in the relaxation response to cromakalim in wild type vessels with intact PVAT in comparison with animals fed a normal chow diet (ND). HFD animals had increased inflammatory infiltrates in the PVAT which were associated with reduced AMPK activity and adiponectin release in comparison with ND fed WT mice. In KO mice, AMPK activity was also reduced and increased inflammatory infiltration was observed in both ND and HFD mice. In conclusion, the current project demonstrates that AMPKα1 has a critical role in maintaining PVAT’s anti-contractile effect; likely mediated through altered adiponectin secretion or sensitivity, and through protection of PVAT against inflammation. Marked reduction in AMPK activity in WT PVAT, accompanied with the reduction in the release of adiponectin in HFD and KO animal may explain the impaired vascular function observed in obesity.