Second to glucose, glutamine, the most abundant amino acid in the human blood3, can serve as a ready source of carbon to support energy generation and biomass accumulation. Glutamine plays a pleiotropic role in cellular functions4. coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism. Introduction Proliferating cancer cells comprehensively rewire their metabolism to Cetirizine sustain growth and survival in the harsh conditions, such as hypoxia and nutrition deficiency1. Upon the resurgence of research interest into cancer metabolism, aberrant glucose utilization has been centrally studied recently. As a famous hallmark of cancers, aerobic glycolysis, termed the Warburg effect, is characterized by the increased metabolic flux of glucose to secretory lactate2. This process leads to the lack of carbon source from glucose to make building bricks, especially lipids, for cell proliferation. Therefore, the alternative carbon source is required for cell growth. Second to glucose, glutamine, the most abundant amino acid in the human blood3, can serve as a ready source of carbon to support energy generation and biomass accumulation. Glutamine plays a pleiotropic role in cellular functions4. Directly, glutamine can be incorporated to protein, and regulate protein translation and trafficking5. Through catabolic transformations, glutamine provides carbon and nitrogen for the biosynthesis of non-essential amino acids5 and nucleotides6,7. In addition, glutamine can also forward fuel the citric acid Cetirizine cycle (CAC)8,9. Under hypoxia, the glutamine consumption in proliferating cells is elevated, and it preferentially provides carbon for fatty acid biosynthesis through reductive carboxylation10, by which glutamine-derived -ketoglutarate is reduced to citric acid by isocitrate dehydrogenases with NADPH oxidizing to NADP+. One glutamine contains five carbon atoms and two nitrogen atoms in the forms of amine and amide groups. When cells begin to addict to glutamine carbon, which usually happens on proliferating cancer cells under hypoxia4, how do they deal with the potentially overflowed nitrogen? It has long been supposed that glutamine offers -ketoglutarate for cells by deamination through glutaminase (GLS)11 and glutamate dehydrogenase (GLUD)9. Concomitantly with these processes, the increasing amount of ammonia is produced and could be toxic to cells12,13. Although a recent report showed that breast cancer cells could slightly recycle ammonia to generate amino acids through GLUD14, GLUD-mediated conversion of ammonia and -ketoglutarate to glutamate does not efficiently occur in most of cancer cells4,15. To avoid over-accumulating ammonia, the best way for proliferating cancer cells is to reduce its generation. Therefore, how glutamine nitrogen is coordinatively metabolized to avoid releasing ammonia deserves to be further determined. Different elements in a metabolite usually have different metabolic fates, thus their coordinative metabolism is critical to maintain the metabolic homeostasis in cells. Once the changed microenvironment perturbs the homeostasis, re-building a new coordinative metabolism is required. Here we show that hypoxia alters glutamine metabolism and drives a new metabolic homeostasis of its carbon and nitrogen. Results Cetirizine Requirement of glutamine-nitrogen for cell survival Glutamine Cetirizine is required for cell survival16C19, and its loss induced cell death (Supplementary FGFR2 Fig.?1a). Supplementation with nucleosides, but not -ketoglutarate and non-essential amino acids including glutamate, significantly suppressed cell death in MCF-7, HeLa, and A549 cells induced by glutamine loss (Supplementary Fig.?1aC1c), supporting the well-established notion that glutamine is necessary for nucleotide biosynthesis6. In fact, glutamine can be potentially synthesized from glutamate by glutamine synthetase (GS) (Supplementary Fig.?2a). However, glutamine deprivation led to a dramatic loss of cellular glutamine (about 5% of the control) but showed no or less effect on other nonessential amino acids and the intermediates in the CAC in MCF-7 and HeLa cells (Supplementary Fig.?2b, c). Notably, the culture medium did not contain nonessential amino acids including glutamate. It suggests that cells could synthesize glutamate from -ketoglutarate (Supplementary Fig.?2a). We then used the labeled carbon source, 13C6-glucose, to culture MCF-7 and HeLa cells, and the 13C tracing analysis showed that -ketoglutarate and glutamate were substantially labeled by 13C even in the presence of glutamine but the glucose-derived fraction significantly increased in the absence of glutamine (Supplementary Fig.?2d). Nonetheless, glutamine was not labeled at all in the presence of glutamine but slightly labeled, when compared to -ketoglutarate and glutamate, in the absence of glutamine (Supplementary Fig.?2d). These results suggest that glutamine cannot be efficiently synthesized in cells even upon its scarcity, and it could be attributed to the low level of GS. We then over-expressed GS in MCF-7 cells (Supplementary Fig.?1d), and found.