Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis

Abstract

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.

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

“…As a proof of concept, RESPYR was expressed in either rat pancreatic b cells (INS-1E) in which ATP is predominantly produced through OXPHOS (Spacek et al, 2008) or HEK293 cells in which glycolysis provides the main source of energy (Dietmair et al, 2012;Henry et al, 2011). In both cell types, addition of pyruvate induced a similar rapid increase in BRET signal ( Figure 3B), showing that RESPYR was expressed and functional.…”

Influence of HEK293 metabolism on the production of viral vectors and vaccine

“…The ratio of glucose and lactate: Y Lact/Glc indicates proportion of carbon channeled to lactate rather than entering mitochondria for further ATP synthesize. For HEK293 cells, this ratio varies between 1.0 and 2.35 [29,[44][45][46][47]. Lactate can also be produced from amino acids, through reverse TCA in mitochondria, which explains how ratio over 2 can be obtained in some cases.…”

“…Similarly to other cell lines, glutamine is the most consumed AA in HEK293 cell culture, followed by serine, asparagine/aspartate, leucine, valine and arginine [46,51,65]. AA consumption contributes to the generation of toxic by-products, especially lactic acid and ammonia.…”

“…Different MFA studies of HEK293 cells show that over 50% of the carbon entering glycolysis goes to lactate production [46,50]. A batch culture of HEK293 cells supplemented with C 13 labeled glucose and glutamine was used by Henry et al [46] to investigate carbon repartition in different pathways of HEK293 cell central metabolism.…”

“…A batch culture of HEK293 cells supplemented with C 13 labeled glucose and glutamine was used by Henry et al [46] to investigate carbon repartition in different pathways of HEK293 cell central metabolism. They found that glucose was utilized mostly for glycolysis, with smaller (15%) use in PPP for NADPH and ribose production.…”