Hypoglycaemic stimulation of macrophage cytokine release is suppressed by AMP-activated protein kinase activation.

Abstract

Aims: Acute hypoglycaemia promotes pro-inflammatory cytokine production, increasing the risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences the production of pro-inflammatory cytokines. We sought to examine the mechanistic role of AMPK in low glucose-induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in people with diabetes.

Methods: Macrophage cell line Raw264.7 cells, primary macrophage bone marrow-derived macrophages obtained from wild-type mice or AMPK γ1 gain-of-function mice, were used, as were AMPKα1/α2 knockout mouse embryonic fibroblasts (MEFs). Allosteric AMPK activators PF-06409577 and BI-9774 were used in conjunction with inhibitor SBI-0206965. We examined changes in protein phosphorylation/expression using western blotting and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was detected using a fluorescence-based reactive oxygen species (ROS) assay, and cell viability was examined using flow cytometry.

Results: Macrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Moreover, low glucose increased oxidative stress and augmented the release of macrophage MIF. However, pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPKγ1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFĸB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFĸB.

Conclusions: Taken together, these data indicate that pharmacological AMPK activation suppresses the release of MIF from macrophages caused by energy stress, suggesting that AMPK activation could be a useful strategy for mitigating hypoglycaemia-induced inflammation.