Fast exchange fluxes around the pyruvate node: a leaky cell model to explain the gain and loss of unlabelled and labelled metabolites in a tracer experiment

Glucose and glutamine are the two dominant metabolic substrates in cancer cells. In (13)C tracer experiments, however, it is necessary to account for all significant input substrates, as some natural (unlabelled) substrate in the medium, often derived from serum, can be metabolised by cells despite not showing signs of net consumption.

The current work established the possibility that a cell's range of significant input substrates may be broader than anticipated. Metabolite exchange can affect intracellular enrichments. In particular, we showed that pyruvate was more strongly connected to lactate than to upstream glycolytic intermediates and that a fast lactate exchange may alter the outcome of flux analyses. Nevertheless, the leaky cell model may be an opportunity in disguise-the ability to continuously monitor metabolism using only the enrichments of extracellular metabolites.

Using [U-(13)C6]-glucose tracers and measuring extracellular metabolite enrichments by GC-MS, we found that pancreatic cells HPDE and PANC-1 secrete lactate, pyruvate, TCA cycle metabolites and non-essential amino acids synthesised from glucose. Focusing our investigations on pyruvate exchange in HEK293 cells, we observed that the four metabolites pools, intracellular and extracellular lactate and pyruvate, had similar (13)C enrichment trajectories. This indicated that these metabolites can mix rapidly. Using a hybrid (13)C-MFA, we followed to show that the lactate exchange flux had increased when extracellular lactate concentration was increased by 10-fold. By allowing rapid exchange fluxes around the pyruvate node, (13)C-MFA revealed that PANC-1 cells cultured in [U-(13)C6]-glucose doubled the conversion of unlabelled substrates to pyruvate when treated with TNF-α. Conclusions: The current work established the possibility that a cell's range of significant input substrates may be broader than anticipated. Metabolite exchange can affect intracellular enrichments. In particular, we showed that pyruvate was more strongly connected to lactate than to upstream glycolytic intermediates and that a fast lactate exchange may alter the outcome of flux analyses. Nevertheless, the leaky cell model may be an opportunity in disguise-the ability to continuously monitor metabolism using only the enrichments of extracellular metabolites.