Figure 1.

Human RPE utilizes nitrogen from proline to generate amino acids and exports them into the media.A, human RPE cells were grown for 20 weeks and switched into DMEM with or without 1 mM ¹⁵N proline. The spent media were collected at 0, 24, and 48 h, and cells were collected at 48 h for metabolite analysis with GC–MS. B, a schematic of proline nitrogen metabolism into amino acids (colored in blue) through proline dehydrogenase (PRODH), pyrroline-5-carboxylic acid (P5C) dehydrogenase (P5CDH), ornithine transaminase (OAT), glutamate decarboxylase (GAD), glutamine synthetase (GS), and aminotransferase (AT). These amino acids in proline metabolism were selected for GC–MS analysis. C and D, ¹⁵N fraction of metabolites derived from ¹⁵N proline in RPE cells and the total abundance of all isotopologs relative to control RPE cells without ¹⁵N proline. ∗p < 0.05, ∗∗∗p < 0.001, N = 3. E and F, ¹⁵N fraction of metabolites derived from ¹⁵N proline in RPE spent media at different time points and the total abundance of all isotopologs relative to control RPE culture media without ¹⁵N proline at 24 h or 48 h. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N = 3. Error bars represent SD. DMEM, Dulbecco's modified Eagle's medium; RPE, retinal pigment epithelium.