Glutamine–derived proline biosynthesis and lipogenesis compensate each other under hypoxia. ΔY/GMR refers to the overall ΔY value for the equivalent of each glutamate-associated metabolic reaction. (A) A model to show the glutamate-initiated palmitate biosynthesis. (B) A model to show glutamate–derived secretory proline. Cells use exogenous lipids/palmitates, and glutamate is used to synthesize proline that is secreted out of cells. Amino acids are absorbed or assumed to synthesize from the alanine amino group. (C) Proliferation rate of P5CS-expressing (HeLa/Cas9 and 4T1/Cas9) and P5CS-null (HeLa/KO#1, HeLa/KO#2, 4T1/KO#1, and 4T1/KO#2) cells. Gray boxes show amino acids; black arrows show the active pathways. (D and E) Mass isotopomer analysis of proline and acetyl-CoA in HeLa/wt, HeLa/P5CS-KO, and HeLa/KO-mP5CS cells cultured with [U-¹³C]-glutamine in the proline-contained medium under hypoxia or normoxia. (F) Proliferation rate of HeLa cells treated with or without ACC1 inhibitor PF-05175157 (PF; 5 µM or 10 µM). (G) Mass isotopomer analysis of proline in HeLa cells cultured with [U-¹³C]-glutamine in the proline-contained medium under hypoxia or normoxia. Cells were treated with or without 10 µM of PF-05175157. (H) Proliferation rate of HeLa/wt, HeLa/P5CS-KO, and HeLa/KO-mP5CS cells in the proline-contained medium treated with or without 10 µM of PF-05175157 under normoxia and hypoxia. (I) Labeled carbons in cholesterol in HeLa cells cultured with [U-¹³C]-glucose for 48 h under the normal condition in the presence or absence of lovastatin (1 µM). The relative pool size is also shown. (J) Labeled carbons in palmitate in HeLa cells cultured with [U-¹³C]-glucose for 48 h under the normal condition in the presence or absence of lovastatin (1 µM). The relative pool size is also shown. (K) Labeled carbons in palmitate in HeLa cells cultured with [U-¹³C]-glutamine for 48 h under hypoxia in the presence or absence of lovastatin (1 µM). The relative pool size is also shown. (L and M) Proliferation rate of HeLa/wt, HeLa/P5CS-KO, and HeLa/KO-mP5CS cells in the proline-contained medium treated with or without 1 µM of lovastatin and/or 10 µM of PF under normoxia and hypoxia. Data in C, F, H, L, and (M were from triplicate experiments, and all experimental data were verified in at least two independent experiments. Data in D, E, G, and I–K were from three independent cultures. Error bars represent mean ± SD. **, P < 0.01; Student’s t test. AcCoA/Ac-CoA, acetyl-CoA; Contrl, control; Hypo, hypoxia; Lact, lactate; Lova, lovastatin; Norm, normoxia; OAA, oxaloacetate; Palm, palmitate.