Abstract
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.
Highlights
The development of vascular endothelial dysfunction, a key early step in atherogenesis, is associated with elevated circulating levels of interleukin (IL)-1β, tumour necrosis factor-α (TNFα) and IL-61
IL-1β-stimulated nuclear factor-κB (NFκB) and jun N-terminal kinase (JNK) activation occurs via a complex signalling mechanism, by which IL-1β binding to the IL-1 receptor stimulates formation of a signalosome including TGFβ-activated kinase-1 (TAK1) and inhibitor of NFκB (IκB) kinase (IKK) in a manner dependent on IL-1 receptor associated kinases (IRAKs)[4,5]
The extent of AMPK activation was further stimulated by increasing the canagliflozin concentration to 30 μmol/l, whereas stimulation of human umbilical vein endothelial cells (HUVECs) with concentrations of dapagliflozin and empagliflozin up to 30 μmol/l had no effect on AMPK activity (Fig. 1a)
Summary
The development of vascular endothelial dysfunction, a key early step in atherogenesis, is associated with elevated circulating levels of interleukin (IL)-1β, tumour necrosis factor-α (TNFα) and IL-61. Several recent studies have reported that administration of SGLT2 inhibitors improves pro-inflammatory IL-6, MCP-1 and ICAM-1 gene expression in blood vessels of rodent models of diabetes[13,15,16,17] These vascular effects of systemic SGLT2 inhibitor administration may be secondary to changes in glycaemia, blood pressure or actions on extra-cardiovascular tissues, yet may reflect a direct action on blood vessels. Empagliflozin has been reported to improve the reduced viability of human umbilical vein endothelial cells (HUVECs) cultured in high glucose concentrations[17], without altering basal HUVEC proliferation[14] It remains uncertain whether SGLT2 inhibitors have beneficial direct effects on blood vessels. Since we and others have identified anti-inflammatory actions of AMPK activation in vascular cells and systemic delivery of SGLT2 inhibitors has been reported to impair vascular inflammatory signalling and reduce atheroma in mice, we examined whether SGLT2 inhibitors influence AMPK activity to influence pro-inflammatory signalling pathways in cultured human endothelial cells
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