Glutamine has pleiotropic role of positively regulating respiration and maintaining redox balance selectively in high‐invasive ovarian cancer (OVCA) cells Oxygen consumption rate (OCR) was measured in high‐invasive (SKOV3 and SKOV3ip), low‐invasive (IGROV1 and OVCAR3) and moderately invasive (OVCA420) cells in media containing Gln, aminooxyacetate (AOA) and α‐ketoglutarate (α‐KG). Glutamine has pleiotropic role of positively regulating respiration and maintaining redox balance selectively in high‐invasive ovarian cancer (OVCA) cells Oxygen consumption rate (OCR) was measured in high‐invasive (SKOV3 and SKOV3ip), low‐invasive (IGROV1 and OVCAR3) and moderately invasive (OVCA420) cells in media containing Gln, aminooxyacetate (AOA) and α‐ketoglutarate (α‐KG). OCR was normalized with value before injections. Analysis of proliferation of OVCA cell lines with range of concentrations of 6‐diazo‐5‐oxo‐L‐norleucine (L‐DON), a Gln analog which inhibits Gln's conversion to glutamate. Data were normalized with complete media conditions without L‐DON. Analysis of proliferation of OVCA cell lines with range of concentrations of epigallocatechin gallate (EGCG, an inhibitor of glutamate dehydrogenase, which converts glutamate into α‐KG) used to inhibit glutamate entering the tricarboxylic acid (TCA) cycle. α‐KG was used to rescue reduction of cell proliferation by EGCG. Data were normalized with complete media conditions without EGCG. Analysis of proliferation of OVCA cell lines when glutaminolysis was inhibited with range of concentrations of AOA. Data were normalized with complete media conditions without AOA. Lactate and citrate generation from glucose through direct glycolysis or through both oxidative pentose phosphate pathway (PPP) and glycolysis. Black circles represent labeled carbons, empty circles represent unlabeled carbons, pink circles represent unlabeled carbons in oxidative PPP. Lactate and citrate generation from glutaminolysis. Blue circles represent labeled carbons from malic enzymes, and green circles are labeled carbons from canonical TCA cycle. Glucose deprivation effect on lactate secretion rate. Comparison of M3 pyruvate and M3 lactate derived from either glucose (1:1 mixture of U‐13C6 glucose and 1‐13C1 glucose) or Gln (U‐13C5 Gln) in high‐ and low‐invasive OVCA cells. Since all conditions have complete media, their total pyruvate and lactate content should be the same. Comparison of M2 citrate labeling from glucose and M6 citrate labeling from Gln in OVCAR3 and SKOV3 cells. Total pyruvate and lactate content in all conditions should be equal as explained for (H). Oxidative pentose phosphate pathway fluxes estimated using the mathematical relation (percentage of unlabeled M0 lactate—percentage of labeled M1 lactate) to represent relative oxidative PPP fluxes in OVCAR3 and SKOV3 cells cultured with 1:1 mixture of 13C6 glucose and 1‐13C‐labeled glucose. Role of Gln in maintaining mitochondrial membrane potential (MMP) levels in low‐ and high‐invasive cells. Gln maintains ATP content selectively in high‐invasive OVCA cells. Gln reduces reactive oxygen species induced by H2O2 in high‐invasive cells, but not in low‐invasive cells. Gln maintains NADPH level in high‐invasive OVCA cells. Gln increases ratio of NADPH/total NADPH ratio selectively in high‐invasive OVCA cells. Glucose is unable to provide enough reducing equivalents in high‐invasive OVCA cells. Gln/glucose deprivation's effect on total glutathione and reduced glutathione level in cancer cells. Data information: Data in (A–N) are expressed as mean ± SEM, n ≥ 9, **P < 0.01, ***P < 0.001. Data in (O, P) (n ≥ 6) are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. Lifeng Yang et al. Mol Syst Biol 2014;10:728 © as stated in the article, figure or figure legend