Coordinative Metabolism Of Glutamine Carbon And Nitrogen In Proliferating Cancer Cells Under Hypoxia

Objectives Under hypoxia,most of glucose is converted to secretory lactate,which leads to cancer cells to find alternative carbon sources to support energy generation and biomass accumulation.Glutamine,the most abundant amino acid in human blood,has been shown to provide carbon for fatty acid biosynthesis through reductive carboxylation.Different elements in a metabolite usually have different metabolic fates,one glutamine contains five carbon atoms and two nitrogen atoms in the form of amino and amide groups.When cells begin to addict to glutamine carbon,which usually happens on proliferating cancer cells under hypoxia,how do they deal with the potentially overflowed nitrogen? We set out to explore the molecular mechanisms of glutamine carbon and nitrogen metabolism in cancer cells.Methods1.Add carbon source,amino acid and nucleotide metabolites to study whether they can suppressed cell death induced by glutamine loss.Use metabolic flux analysis technology to study whether other metabolites can be converted to glutamine.2.Using metabolic mass spectrometer,metabolic flux analysis technology and automatic biochemical analyzer to detect the derivation of glutamine carbon and glutamine-related nitrogen compounds in cancer cells under normoxia and hypoxia.3.Metabolomics techniques were used to screen for differences in nitrogen-containing metabolites in cancer cells under normoxia and hypoxia.4.Metabolic flux technique is used to detect the metabolic fate of glutamine-derived aspartate under normoxia and hypoxia.5.Under normoxia and hypoxia,metabolic flux technique was used to detect the amino acid and nucleotide production derived from glutamine.MCF7,He La,A549,HCC-LM3,SCG7901,4T1 were cultured under different conditions to detect metabolite changes in the culture medium.A carbon source is added to detect changes in metabolites in cancer cells under hypoxia.6.Western blot was used to detect the protein expression changes of glutamine metabolism in MCF7 and He La at different time points under hypoxia.The changes of NADH/NAD+ under normoxia,hypoxia and Antimycin A conditions were examined.The metabolism of glutamine under Antimycin A was examined.The pyruvate analogue AKB was supplemented to detect glutamine metabolism under different conditions.7.Metabolic mass spectrometry is used to detect metabolites in serum of normal people,breast cancer,lung cancer,gastric cancer and liver cancer patients.Hela cells were inoculated into nude mice to detect changes in metabolites in serum and tumor tissues of tumor-bearing mice and normal mice.The enzymes on the metabolic pathway were knocked down to construct stable cell lines to detect cell proliferation under normoxia and hypoxia.The cell lines were inoculated into nude mice,and the effect of knockdown-related enzyme on cancer proliferation was verified in vivo.Results1.Glutamine nitrogen is required for cell proliferation.2.Hypoxia increases glutamine uptake and decreases ammonia production.3.We screen the nitrogen metabolome including 226 nitrogen-contained metabolites.Five of the six compounds significantly increased under hypoxia,including dihydroorotate,orotate,IMP,guanosine,and inosine,were involved in the nucleotide biosynthesis pathway.4.Boosting of glutamine-derived aspartate into pyrimidine precursors under hypoxia.5.Enrichment of glutamine-nitrogen in excretory dihydroorotate under hypoxia.We observed a significantly increased amount of dihydroorotate,but not orotate,in hypoxic medium of various cell lines.6.Hypoxia-induced electron accumulation promotes biosynthesis and excretion of dihydroorotate.7.The orotate in the serum of cancer patients was significantly increased.It was confirmed in mice that elevated dihyroorotate,not orotate was secreted into serum and converted into orotate.Increased dihydroorotate biosynthesis is required for in vivo tumor growth.Conclusions We found that glutamine nitrogen is necessary to nucleotide biosynthesis,but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia.Dihyroorotate,not orotate,is then secreted out of cells.Furthermore,we found that the specific metabolic pathway occurs in vivo and is required for tumor growth.The identified metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis,with the rest carbon and nitrogen being safely removed.Therefore,our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia,and provide a comprehensive understanding on glutamine metabolism.