TitleAuthorsAbstractMethods SummaryReferencesMethodsAntibodiesAnimalsHuman fibroblastsCD74 knock-down by siRNAHeart-muscle incubationsHeart-muscle glucose transportCell-surface GLUT4 photolabellingMouse heart perfusionsIn vivo coronary occlusion/reperfusionImmunohistochemistryAMPK assayMIF assayRecombinant MIFImmunoblottingStatisticsMethods ReferencesFigure 1 Role of MIF in heart muscle AMPK signalling during hypoxia.Figure 2 Heart MIF expression and release triggered by ischaemia.Figure 3 Genetic MIF deletion impairs ischaemic heart AMPK activation and glucose uptake, and exacerbates post-ischaemic cardiac dysfunction and injury.Figure 4 Human MIF promoter genotype determines MIF secretion and AMPK activation during hypoxia.LETTERSMacrophage migration inhibitory factor stimulatesAMP-activated protein kinase in the ischaemic heartEdward J. Miller1*,JiLi1*{, Lin Leng2, Courtney McDonald2, Toshiya Atsumi5, Richard Bucala2,3*& Lawrence H. Young1,4*Understanding cellular response to environmental stress hasbroad implications for human disease. AMP-activated proteinkinase (AMPK) orchestrates the regulation of energy-generatingand -consuming pathways, and protects the heart against isch-aemic injury and apoptosis1. A role for circulating hormones suchas adiponectin2and leptin3in the activation of AMPK has receivedrecent attention. Whether local autocrine and paracrine factorswithin target organs such as the heart modulate AMPK isunknown. Here we show that macrophage migration inhibitoryfactor (MIF), an upstream regulator of inflammation4, is releasedin the ischaemic heart, where it stimulates AMPK activationthrough CD74, promotes glucose uptake and protects the heartduring ischaemia-reperfusion injury. Germline deletion of the Mifgene impairs ischaemic AMPK signalling in the mouse heart.Human fibroblasts with a low-activity MIF promoter polymorph-ism5have diminished MIF release and AMPK activation duringhypoxia. Thus, MIF modulates the activation of the cardioprotec-tive AMPK pathway during ischaemia, functionally linkinginflammation and metabolism in the heart. We anticipate thatgenetic variation in MIF expression may impact on the responseof the human heart to ischaemia by the AMPK pathway, and thatdiagnostic MIF genotyping might predict risk in patients withcoronary artery disease.Macrophage migration inhibitory factor (MIF) is a pleiotropiccytokine that controls the inflammatory ‘set point’ by regulatingthe release of other pro-inflammatory cytokines6. MIF is expressedin several cell types, including monocytes/macrophages7, vascularsmooth muscle8and cardiomyocytes9, and is released on stimulationfrom pre-formed storage pools. MIF is involved in the pathogenesisof inflammatory diseases, such as atherosclerosis8,10, rheumatoidarthritis5, sepsis4, asthma11and acute respiratory distress syndrome12.Human MIF gene expression is determined by promoter polymorph-isms, including a tetra-nucleotide CATT repeat at position –794 (ref.5). MIF signalling is known to activate ERK1/2 MAPK (ref. 13)through a receptor complex comprising CD74 (ref. 14) and CD44(ref. 15). In contrast, the chemokine receptors CXCR2 and CXCR4participate in MIF-mediated migratory function10.MIF also stimulates glycolysis during sepsis, increasing the syn-thesis of fructose 2,6-bisphosphate and cellular glucose uptake16. Thesignalling pathways by which MIF exerts its metabolic effects areunknown, but one candidate is the AMP-activated protein kinase(AMPK)—an important regulator of both glycolysis and glucoseuptake during cellular stress1. AMPK senses the cellular energy stateand affects diverse pathways to increase cellular ATP productionand limit energy consumption. AMPK activity is regulated by AMPbinding to its regulatory c-subunit17and by phosphorylation of thecatalytic a-subunit by upstream kinases, including LKB1 (ref. 18) andCaMKKb (ref. 19). In the heart, AMPK stimulates 6-phosphofructo-2-kinase activity and glycolysis20, induces glucose transporter-4(GLUT4, encoded by the SLC2A4 gene) translocation21, increasesischaemic glucose uptake1,22and limits myocardial injury andapoptosis1.AMPK phosphorylation is also modulated by the adipocyte-derived circulating hormones leptin3and adiponectin23, raising thepossibility that cytokines might also activate AMPK. We hypothe-sized that AMPK might be activated in an autocrine/paracrine fash-ion by MIF in the heart during ischaemia, linking the regulatorycontrol of inflammation and metabolism.Initial experiments examined whether MIF has a role in the stimu-lation of the AMPK pathway during hypoxia in rat heart muscles.Hypoxic activation of AMPK (Fig. 1a) was associated with a twofoldincrease in muscle MIF release (Fig. 1b), the latter consistent withprevious results in cardiomyocytes24. Pre-treatment with anti-MIFantibody reduced hypoxic AMPK activation by 67% (Fig. 1c). One ofthe important AMPK actions during hypoxia and ischaemia is toincrease glucose transport1,22. Hypoxic glucose transport was inhi-bited 38% by anti-MIF antibody (Fig. 1d), indicating that secretedextracellular MIF modulates downstream AMPK action.To investigate whether MIF modulates AMPK, we added MIF tonormoxic heart muscles. MIF caused time- and dose-dependentincreases in AMPK phosphorylation (Fig. 1e and f), and increasedheart muscle glucose uptake (Fig. 1g). Hypoxia and insulin-stimulated glucose uptake in the heart are mediated by translocationof the glucose transporter GLUT4 to the cell surface where it isphysiologically active21. We used a cell-membrane impermeantphotolabel compound and found significant translocation ofGLUT4 to the cell surface (Fig. 1h), elucidating the mechanismthrough which MIF increases glucose uptake.We next examined whether MIF modulates AMPK signalling inthe ischaemic heart. MIF is expressed by cardiomyocytes9,24, endothe-lial cells, monocytes and macrophages7. We used the isolated mouseheart perfused with crystalloid buffer, eliminating the potentialcontribution of MIF from circulating cells. MIF was highly expressedin cardiomyocytes, according to immunohistochemical data (Fig.2a). Ischaemia triggered cardiac MIF release into the coronaryvenous effluent and decreased heart MIF content after ischaemia-reperfusion (Fig. 2b).To determine whether MIF plays a part in ischaemic AMPK activa-tion, we used hearts from Mif2/2mice25and compared them to wild-type controls. Mif2/2mice demonstrated a normal baseline cardiacphenotype with respect to left ventricular size and