Supplementary Figure 8: GPD2 deficiency protects against induction of reverse electron transport (RET) through a mechanism independent of known negative regulators of oxidative metabolism in LPS-stimulated BMDMs.

(a) qPCR analysis of Irg1, Nos2, and Idh1 gene expression in BMDMs unstimulated or stimulated with LPS for 12h. (b) Steady-state metabolomic analysis of itaconate levels, shown as relative to unstimulated, in BMDMs stimulated with LPS for the indicated times (n=3). Data are from one experiment representative of three independent experiments (a) or from one experiment (b). Mean (a-b) +/- s.e.m. (b) shown. (c) Schematic depicting FET and RET during LPS activation and tolerance. During acute LPS exposure (LPS activation), electrons from oxidation of metabolic substrates (that is glucose) flow forward through the ETC (green dotted line), creating a proton motive force for ATP production and also returning electron acceptor molecules (that is NAD⁺) for continued oxidation of metabolic substrates. LPS-induced GPD2 activity initially boosts forward electron transport (FET; left) to support an increase in glucose oxidation for acetyl-CoA synthesis and induction of inflammatory genes by enhanced histone acetylation. However, sustained GPD2 activity may overwhelm the ubiquinone pool (Q), the common sink for electrons from Complex I, Complex II, and GPD2, leading to a thermodynamic environment that permits electron backflow (red dotted line). Such reverse electron transport (RET) decreases return of NAD⁺ molecules to the TCA cycle, impairing glucose oxidation for acetyl-CoA synthesis and histone acetylation at inflammatory genes. Therefore, we propose that GPD2-GPS activity acts as a rheostat for inflammatory gene induction in BMDMs, linking the duration of LPS exposure to the directionality of electron transport to control glucose oxidation and balance induction and suppression of inflammatory responses.