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

Abstract

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.

Fig. 1

4-OI alkylates cysteine 22 of GAPDH. a The orthogonal projection to latent structures discriminant analysis (OPLS-DA) score plots compared control (CON) and LPS-stimulated (LPS) RAW264.7 macrophages (24 h). Results are from seven independent experiments. b Metabolite levels in CON versus LPS-induced RAW264.7 macrophages. Blue and red dots represent metabolites significantly downregulated and upregulated after LPS stimulation, respectively. c Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by 4-OI (+242.15 Da) after 4-OI treatment for 4 h in GAPDH immunoprecipitated from RAW264.7 macrophages. d Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by itaconate (+130.02 Da) after LPS stimulation for 6 h in GAPDH immunoprecipitated from RAW264.7 macrophages. e Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by 4-OI (+242.15 Da) in recombinant mouse GAPDH treated with 4-OI (500 μM) for 4 h at 37 °C. Da daltons. f Schematic diagram of GAPDH thiol group was covalent modified by 4-OI

Fig. 2

4-OI inhibits GAPDH activity and glycolysis. a RAW264.7 macrophages were treated with 4-OI (62.5 μM) for 3 h and then subjected to LPS stimulation for 24 h. GAPDH enzyme activity assays of cell lysates were performed. b 4-OI dose- and time-dependent inhibition of GAPDH enzyme activity. Recombinant mouse GAPDH was incubated with the indicated drug concentrations. Aliquots were removed at various time points, followed by an enzyme activity assay. c–h RAW264.7 macrophages and BMDMs were treated with 4-OI for 3 h, followed by LPS stimulation for 24 h. c, d Lactate levels of RAW264.7 macrophages (c) and BMDMs (d) were determined by a lactate assay. e, f ECAR of RAW264.7 macrophages (e) and (f) BMDMs were measured by an ECAR assay as described in “Methods.” g, h OCR of RAW264.7 macrophages (g) and BMDMs (h) were assayed by an OCR assay as described in “Methods.” i RAW264.7 macrophages were treated with 125 μM 4-OI or vehicle and stimulated with 1 μg/mL LPS, in triplicate. Cells were then added with 12 mM U¹³C-glucose, and ¹³C-glucose labelling of glycolytic intermediates was measured by GC-MS. Colour key in heat map indicates the metabolite expression value: red represents the significant increases and blue represents the significant decreases. Heat map indicated blockade of glycolytic flux at GAPDH. All data shown are summarized from three independent experiments. Values represent the mean ± SEM at each time point. p Values were calculated using two-tailed Student’s t test or one-way ANOVA with Sidak’s correction for multiple comparisons. Source data are provided as a Source Data file

Fig. 3

4-OI alleviates inflammation by inhibiting GAPDH activity. a, b RAW264.7 macrophages (a) and BMDMs (b) were treated with vehicle or 4-OI at the indicated concentrations. After 3 h, RAW264.7 macrophages or BMDMs were stimulated with LPS (1 μg/mL or 100 ng/mL) for 24 h, and IL-1β in the supernatants was measured by ELISA. c, d LPS-induced iNOS protein expression and its relative quantification after 24 h in RAW264.7 macrophages (c) and BMDMs (d) pretreated with or without 4-OI for 3 h. Data were corrected based on the actin loading control. e Treating LPS-stimulated RAW264.7 macrophages with 5 mM 2-DG, a glycolysis inhibitor, replicated the effect of 4-OI on IL-1β production. f IL-1β secretion was measured by ELISA in BMDMs that were treated with LPS ± DMF for 24 h in either limiting (0.5 mM) or saturating (25 mM) concentrations of glucose. Data shown are representative of three independent experiments. p Values were calculated using one-way ANOVA with Sidak’s correction or two-way ANOVA with Turkey’s correction for multiple comparisons tests. All data show mean ± SEM. Source data are provided as a Source Data file

Fig. 4

Covalently modifying C22 of GAPDH mediates the anti-inflammatory effects of 4-OI. a LPS-stimulated RAW264.7 macrophages were treated with 3 μM heptelidic acid, a GAPDH inhibitor, which also decreased IL-1β release. b, c Treatment with heptelidic acid replicated the effects of 4-OI on iNOS expression in LPS-stimulated RAW264.7 macrophages (b) and BMDMs (c). d, e TNF-α mRNA expression and protein release were measured in 1 μg/mL LPS-treated (6 h) RAW264.7 cells after treatment with heptelidic acid or 4-OI. f, g LPS-induced NF-κB (24 h) expression in the nucleus (f) or cytosol (g) after treatment with vehicle or 4-OI as indicated. h Wild-type GAPDH (GAPDH-WT) or the Cys-22 mutant (GAPDH-C22A) was overexpressed in RAW264.7 macrophages for 24 h and then treated with LPS and 4-OI for 24 h, followed by measurement of IL-1β secretion by ELISA. Representative results are shown from three independent experiments. Data represent the mean ± SEM. p Values were determined by two-tailed Student’s t test or one-way ANOVA with Sidak’s correction for multiple comparisons test. Source data are provided as a Source Data file

Fig. 5

4-OI protects against LPS lethality. a Mice were injected with a single dose of 4-OI (50 mg/kg), followed 2 h later by an intraperitoneal injection of LPS (5 mg/kg), and the mice were then re-treated with 4-OI 24 and 48 h later (n = 20 mice/group). Log-rank (Mantel–Cox) test was used to compare the differences in survival rates between groups. b–d In parallel experiments, the serum levels of IL-1β (b) and IL-6 (c) at 2 h and lactate (d) at 24 h after intraperitoneal injection of LPS (5 mg/kg) were measured. Results are from three independent experiments. Data values are the mean ± SEM, p values were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons test. Source data are provided as a Source Data file. e Proposed model of the anti-inflammatory role of 4-OI, which may explain the physiologic negative feedback function of itaconate