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

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.

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.

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.

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.