Cargando…

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

BACKGROUND: 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 ce...

Descripción completa

Detalles Bibliográficos
Autores principales: Quek, Lake-Ee, Liu, Menghan, Joshi, Sanket, Turner, Nigel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931697/
https://www.ncbi.nlm.nih.gov/pubmed/27379180
http://dx.doi.org/10.1186/s40170-016-0153-9
_version_ 1782440948242841600
author Quek, Lake-Ee
Liu, Menghan
Joshi, Sanket
Turner, Nigel
author_facet Quek, Lake-Ee
Liu, Menghan
Joshi, Sanket
Turner, Nigel
author_sort Quek, Lake-Ee
collection PubMed
description BACKGROUND: 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. RESULTS: Using [U-(13)C(6)]-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)C(6)]-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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40170-016-0153-9) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4931697
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-49316972016-07-05 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 Quek, Lake-Ee Liu, Menghan Joshi, Sanket Turner, Nigel Cancer Metab Research BACKGROUND: 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. RESULTS: Using [U-(13)C(6)]-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)C(6)]-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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40170-016-0153-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-07-04 /pmc/articles/PMC4931697/ /pubmed/27379180 http://dx.doi.org/10.1186/s40170-016-0153-9 Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Quek, Lake-Ee
Liu, Menghan
Joshi, Sanket
Turner, Nigel
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931697/
https://www.ncbi.nlm.nih.gov/pubmed/27379180
http://dx.doi.org/10.1186/s40170-016-0153-9
work_keys_str_mv AT queklakeee fastexchangefluxesaroundthepyruvatenodealeakycellmodeltoexplainthegainandlossofunlabelledandlabelledmetabolitesinatracerexperiment
AT liumenghan fastexchangefluxesaroundthepyruvatenodealeakycellmodeltoexplainthegainandlossofunlabelledandlabelledmetabolitesinatracerexperiment
AT joshisanket fastexchangefluxesaroundthepyruvatenodealeakycellmodeltoexplainthegainandlossofunlabelledandlabelledmetabolitesinatracerexperiment
AT turnernigel fastexchangefluxesaroundthepyruvatenodealeakycellmodeltoexplainthegainandlossofunlabelledandlabelledmetabolitesinatracerexperiment