2 3 3 2h serine

The following isotopic tracers were purchased from the indicated sources: [2,3,3-²H]serine, [1-²H]glucose, [3-²H]glucose, [U-¹³C]glutamine and [U-¹³C]glucose (Cambridge Isotope Laboratories) and [2,2,3,3-²H]dimethyl succinate (Sigma-Aldrich). Isotope-labeled glucose and glutamine medium was prepared from phenol red–, glucose-, glutamine-, sodium pyruvate–, sodium bicarbonate–free DMEM powder (Cellgro) supplemented with 3.7 g/L sodium bicarbonate, 25 mM glucose and 4 mM glutamine. Isotope-labeled serine medium was prepared from scratch following the standard DMEM formula, by mixing together stock solutions containing vitamins, amino acids without serine, inorganic salts, and glucose, and thereafter supplemented with 42 mg/L [2,3,3-²H]serine. Isotope-labeled succinate medium was prepared from DMEM powder, 25 mM glucose, 4 mM glutamine supplemented with 2 mM [2,2,3,3-²H]dimethyl succinate. In HEK293T cells (but not the 3T3-L1 cells studied here) decreasing the glucose and glutamine levels in DMEM to 10 mM and 1 mM, respectively, increases fractional malate labeling from the tracer and thereby facilitates malic enzyme flux measurement. Isotopic medium was supplemented with 10% dialyzed FBS (Sigma-Aldrich) and, for differentiating cells only, 5 μg/ml insulin.