Figure 3.

NADPH is the limiting reducing agent upon glucose deprivation. A, ROS accumulate upon glucose deprivation. ROS was measured by flow cytometry using CM-H₂DCFDA at the indicated time points. ROS accumulation steadily increased with time after glucose deprivation. B, glucose deprivation–sensitive T98 cells were deprived of glucose, and metabolites were extracted at the indicated time points. Accumulation of l-cystine and l-cysteine occurred within 10 min after glucose deprivation and steadily increased over the course of 2 h. C, NADPH is rapidly consumed following glucose deprivation. An LC-MS metabolomics method designed to detect NAD(P)H (35) was used at the same time points as in B. Depletion of NADPH preceded depletion of NADH and GSH, with NADPH dropping below the lower limit of detection within 60 min. D, Pearson correlation coefficient was calculated between each metabolite, and hierarchical clustering was performed on the Pearson correlation coefficients. Clustering revealed that l-cystine, l-cysteine, and l-proline behave differently from all other proteinogenic amino acids. Glycolysis metabolites and reducing agents clustered together, reflecting that they both deplete upon glucose deprivation. 18 of 20 proteinogenic amino acids also clustered together, reflecting that amino acid metabolism and glycolysis are uncoupled upon glucose deprivation. E, l-proline is steadily depleted after glucose deprivation in sensitive T98 cells. The de novo synthesis of l-proline requires NADPH to convert pyrroline-5-carboxylate to l-proline. Error bars are standard deviation of the mean.