Supplementary Figure 3: LPS activation increases GPD2 and GPS activity in BMDMs.

(a) Depiction of the spatial and biochemical position of GPD2 at the nexus between glycolysis and electron transport. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) generates NADH from oxidation of glucose in the cytosol, supplying electrons for reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) by cytosolic glycerol 3-phosphate dehydrogenase (GPD1). Electrons from G3P are passed directly to the electron transport chain (ETC) through the FAD cofactor of mitochondrial glycerol 3-phosphate dehydrogenase (GPD2), reducing ubiquinone (Q) to ubiquinol (QH₂) thus contributing to the proton motive force for oxidative phosphorylation. As the glycerol phosphate shuttle (GAPDH-GPD1-GPD2) directly links glycolysis to the ETC and resupplies NAD⁺ to the former, increased GPD2 activity serves as a metabolic node by which the ETC may regulate the rate of glucose utilization. LPS activation increases GPS flux, permitting the acquisition of a high metabolic state characterized by increased glucose oxidation and GPD2-driven mitochondrial respiration. (b) Immunoblot analysis of GPD2 protein in WT and Gpd2^−/− BMDMs unstimulated or stimulated with LPS for the times indicated. (c) Seahorse extracellular flux analysis of OCR in WT BMDMs stimulated with LPS for the indicated times, permeabilized, and treated with 10 mM glycerol 3-phosphate, 2 μM rotenone, and 1 mM ADP. Mean values shown. (d) FACS analysis of F4/80 and CD11b expression on BMDMs from WT and Gpd2^−/− mice. Numbers indicate percent of cells in gate. Data are from one experiment representative of three independent experiments.