20 - PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states

Abstract

Introduction Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotin-switch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression. Results PKM2 induction, -glutathionylation, and phosphorylation were all enhanced in metabolically stressed BMDMs but prevented in Grx1-overexpressing BMDMs. The dimer/tetramer ratio was increased in response to metabolic stress as was HIF1a-dependent expression of GLUT1 and IL-1b. Compared to monocytes isolated from HFD-fed mice, monocyte from Grx1Mactg mice showed reduced priming, reduced expression of PKM2, GLUT1 and IL-1b and diminished PKM2 phosphorylation. Conclusion Metabolic stress-induced PKM2 S-glutathionylation promotes PKM2 tetramer dissociation, phosphorylation and subsequent nuclear translocation, which, via HIF1a-dependent expression of glycolytic enzymes and inflammatory cytokines, reprograms monocyte-derived macrophages into a hyper-inflammatory phenotype. This mechanism may contribute to chronic inflammatory diseases associated with metabolic disorders.