4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects

Activated macrophages switch from oxidative phosphorylation to aerobic glycolysis, similar to the Warburg effect, presenting a potential therapeutic target in inflammatory disease. The endogenous metabolite itaconate has been reported to regulate macrophage function, but its precise mechanism is not clear. Here, we show that 4-octyl itaconate (4-OI, a cell-permeable itaconate derivative) directly alkylates cysteine residue 22 on the glycolytic enzyme GAPDH and decreases its enzyme activity. Glycolytic flux analysis by U13C glucose tracing provides evidence that 4-OI blocks glycolytic flux at GAPDH. 4-OI thereby downregulates aerobic glycolysis in activated macrophages, which is required for its anti-inflammatory effects. The anti-inflammatory effects of 4-OI are replicated by heptelidic acid, 2-DG and reversed by increasing wild-type (but not C22A mutant) GAPDH expression. 4-OI protects against lipopolysaccharide-induced lethality in vivo and inhibits cytokine release. These findings show that 4-OI has anti-inflammatory effects by targeting GAPDH to decrease aerobic glycolysis in macrophages.

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Metabolomic analysis were analysed using R package XCMS (version 3.2) and SIMCA14.1 software package (V14.1, Sartorius Stedim Data Analytics AB, Umea, Sweden). Image Lab software was used for Western blot. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analysed by Wave (Agilent Technologies, Inc. Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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All studies must disclose on these points even when the disclosure is negative. We have used at least three biological replicates for all experiments. The design was based on prior assay experience and similar experiments reported in the literature.
No data were excluded from the analyses.
All experiments were highly reproducible.
Samples were processed in random order.
All experimenters were blinded to group allocation.
All antibodies had validation statements and results on the manufacturer's websites.
Murine-derived macrophage RAW 264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
RAW264.7 cells are very distinct in morphology and have been tested for expression of known markers.
All cells tested negative for mycoplasma contamination.
No commonly misidentified cell lines were used.