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bioRxiv · 10.1101/2023.10.16.562445

Pharmacological AMP-activated protein kinase activation suppresses low glucose-dependent macrophage migration inhibitory factor release from macrophages

Zhang, J.·Pollard, A. E.·Carling, D.·Benoit, V.·Ellacott, K. L.·Beall, C.
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Abstract

Aims/hypothesisAcute hypoglycemia promotes pro-inflammatory cytokine production, increasing risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences production of pro-inflammatory cytokines. We tested the mechanistic role of AMPK in low glucose induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in patients with diabetes. MethodsMacrophage cell line Raw264.7 cells, primary macrophage bone marrow derived macrophages obtained from wild type mice or AMPK {gamma}1 gain-of-function mice were utilized, as were AMPK1/2 knockout mouse embryonic fibroblasts (MEF). Allosteric AMPK activators PF-06409577 and BI-9774 were used, in conjunction with inhibitor SBI-0206965 were also used. 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 ELISA. Oxidative stress was detected using a fluorescence-based ROS assay, and cell viability was examined using flow cytometry. ResultsMacrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Low glucose induced oxidative stress and increased release of macrophage migration inhibitory factor (MIF). 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 AMPK{gamma}1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFB. AMPK activation did not alter low glucose-induced oxidative stress in macrophages but application of AMPK inhibitor SBI-0206965 enhanced oxidative stress in macrophages and in AMPK knockout MEFs, suggesting an AMPK-independent mechanism Conclusions/interpretationTaken together, these data indicate that pharmacological AMPK activation suppresses release of MIF from macrophages. This is mediated by reduced activation of NFB signalling in response to low glucose-induced oxidative stress and suggests that pharmacological AMPK activation could be a useful strategy for mitigating hypoglycemia-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/562445v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d3364borg.highwire.dtl.DTLVardef@1919395org.highwire.dtl.DTLVardef@704923org.highwire.dtl.DTLVardef@1d1cc59_HPS_FORMAT_FIGEXP M_FIG Tweet Low glucose induces pro-inflammatory MIF release from macrophages, and pharmacological AMPK activation suppresses the release of MIF. AMPK/ NFB signalling pathway is involved, which may be a new strategy to attenuate the pro-inflammatory response in hypoglycemia. C_FIG

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Zhang, J., Pollard, A. E., Carling, D., Benoit, V., Ellacott, K. L., Beall, C.. 2023-10-19. Pharmacological AMP-activated protein kinase activation suppresses low glucose-dependent macrophage migration inhibitory factor release from macrophages. https://doi.org/10.1101/2023.10.16.562445

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