Fig. 1

Carbon sources for D-2-HG production and ¹³C tracing studies. a Schematic model of metabolic pathways involved in the production of D-2-HG from extracellular glucose, glutamine, and glutamate; the ¹³C labeling patterns following incubation in culture medium containing ¹³C-labeled glucose (light circles) and glutamine or glutamate (dark circles). b Example of ¹³C NMR spectra of extracts of HCT116-IDH1^wt/R132H and HCT116 cells cultured in medium with [1-¹³C]-glutamine (Gln*, blue) or [1-¹³C]-glutamate (Glu*, red). [1-¹³C]-D-2-HG (D-2-HG*) was detected at 181.8 ppm and could not be observed in parental HCT116 cells. c Quantified NMR results showing levels of Glu*, Gln*, and D-2-HG* in HCT116-IDH1^wt/R132H cells, after incubation in medium with Gln* (blue) or Glu* (red). d LC-MS analysis of D-2-HG pool fractional enrichments in HCT116-IDH1^wt/R132H cells after incubation with ¹³C-labeled substrates. Bar I displays the total amount of D-2-HG and the fractions that were derived from Gln* (blue) and Glc* (gray). Results were obtained from two separate measurements: in the first measurement cells were incubated in DMEM with Glc and Gln*; in the second measurement DMEM was supplemented with Glc* and Gln*. The difference in labeled fractions from these two measurements was assigned as the Glc* fraction. In a parallel experiment Gln and Gln* were substituted by Glu and Glu* respectively. Bar II shows the total amount of D-2-HG and the fractions that were derived from Glu* (red) and Glc* (gray). A third experiment was performed with DMEM containing Glu*, Gln, and Glc. Bar III displays the total amount of D-2-HG and the fraction that was derived from Glu* (red). Provided that both Glu and Gln are available from the culture medium, production of Glu-derived D-2-HG (red) was substantial. Gln* and Glc* fractions were not measured in experiment III. Supplemented metabolite concentrations were always the same: 5.5 mM glucose and 4 mM glutamine and/or 4 mM glutamate