Zhang Jiping, Pollard Alice E, Pearson Eleanor F, Carling David, Viollet Benoit, Ellacott Kate L J, Beall Craig
Department of Clinical and Biomedical Sciences, Faculty of Health and Life Sciences, University of Exeter Medical School, Exeter, UK.
MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital, London, UK.
Diabet Med. 2025 Mar;42(3):e15456. doi: 10.1111/dme.15456. Epub 2024 Dec 24.
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.
急性低血糖会促进促炎细胞因子的产生，增加糖尿病患者发生心血管事件的风险。AMP激活的蛋白激酶（AMPK）受促炎细胞因子产生的调节并影响其产生。我们试图研究AMPK在低血糖诱导的促炎细胞因子巨噬细胞迁移抑制因子（MIF）变化中的机制作用，MIF在糖尿病患者中升高。
使用巨噬细胞系Raw264.7细胞、从野生型小鼠或AMPKγ1功能获得性小鼠获得的原代巨噬细胞骨髓来源巨噬细胞，以及AMPKα1/α2基因敲除小鼠胚胎成纤维细胞（MEF）。变构AMPK激活剂PF-06409577和BI-9774与抑制剂SBI-0206965联合使用。我们使用蛋白质印迹法检测蛋白质磷酸化/表达的变化，并使用免疫荧光法检测蛋白质定位。使用细胞外通量分析和基于荧光素酶的ATP测定评估代谢功能。通过酶联免疫吸附测定（ELISA）定量细胞因子释放。使用基于荧光的活性氧（ROS）测定检测氧化应激，并使用流式细胞术检查细胞活力。
暴露于低血糖的巨噬细胞显示AMPK有短暂且适度的激活，并且代谢向氧化磷酸化增加转变。此外，低血糖增加了氧化应激并增强了巨噬细胞MIF的释放。然而，PF-06409577和BI-9774对AMPK的药理学激活减弱了低血糖诱导的MIF释放，使用AMPKγ1功能获得性（D316A）小鼠进行基因激活也观察到类似趋势，其对低血糖诱导的MIF释放产生轻微影响。抑制NFκB信号传导适度减少了MIF释放，并且AMPK激活适度但显著减少了低血糖诱导的NFκB核转位。
综上所述，这些数据表明药理学上的AMPK激活可抑制能量应激引起的巨噬细胞释放MIF，这表明AMPK激活可能是减轻低血糖诱导的炎症的有用策略。
