Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis.
HEK-293 is the most extensively used human cell line for the production of viral vectors and is gaining increasing attention for the production of recombinant proteins by transient transfection. To further improve the metabolic characterization of this cell line, we have performed cultures using ¹³C-labeled substrates and measured the resulting mass isotopomer distributions in lactate by LC/MS. Simultaneous metabolite and isotopomer balancing allowed improvement and validation of the metabolic model and quantification of key intracellular pathways. We have determined the amounts of glucose carbon channeled through the PPP, incorporated into the TCA cycle for energy production and lipids biosynthesis, as well as the cytosolic and mitochondrial malic enzyme fluxes. Our analysis also revealed that glutamine did not significantly contribute to lactate formation. An improved and quantitative understanding of the central carbon metabolism is greatly needed to pursue the rational development of engineering approaches at both the cellular and process levels.
Henry
Olivier
O
Chemical Engineering Department, École Polytechnique de Montréal, C.P. 6079, Succ. Centre-ville, Montréal, QC H3C3A7, Canada. olivier.henry@polymtl.ca
Jolicoeur
Mario
M
Kamen
Amine
A
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D002853:Chromatography, Liquid / Q000379:methods; D002952:Citric Acid Cycle / Q000502:physiology*; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism*; D008055:Lipids / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008293:Malates / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism; D010427:Pentose Phosphate Pathway / Q000502:physiology
D000596:Amino Acids; D002247:Carbon Isotopes; D008055:Lipids; D008293:Malates; D005973:Glutamine; D062907:Oxaloacetic Acid; D019344:Lactic Acid; D000105:Acetyl Coenzyme A; D002244:Carbon; C030298:malic acid; D005947:Glucose