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Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells

Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the...

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Autores principales: John, Scott, Calmettes, Guillaume, Xu, Shili, Ribalet, Bernard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10621844/
https://www.ncbi.nlm.nih.gov/pubmed/37917627
http://dx.doi.org/10.1371/journal.pone.0286660
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author John, Scott
Calmettes, Guillaume
Xu, Shili
Ribalet, Bernard
author_facet John, Scott
Calmettes, Guillaume
Xu, Shili
Ribalet, Bernard
author_sort John, Scott
collection PubMed
description Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the known cardio-protective role of HK in cardiac muscle, we have investigated the regulation of lactate and pyruvate metabolism by hexokinases (HKs), utilizing wild-type HEK293 cells and HEK293 cells in which the endogenous HKI and/or HKII have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. This decrease was rapidly reversed upon inhibition of the malate aspartate shuttle by aminooxyacetate, or inhibition of mitochondrial oxidative respiration by NaCN. These results suggest that in the absence of HKs, pyruvate-dependent activation of the TCA cycle together with the malate aspartate shuttle facilitates lactate transformation into pyruvate and its utilization by mitochondria. With replacement by overexpression of HKI or HKII the cellular response to pyruvate and NaCN was modified. With either hexokinase present, both the decrease in lactate due to the addition of pyruvate and the increase following addition of NaCN were either transient or suppressed altogether. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN), abolished the effects of HK replacement. These results suggest that blocking of the malate aspartate shuttle by HK may involve activation of the pentose phosphate pathway and increased NADPH production.
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spelling pubmed-106218442023-11-03 Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells John, Scott Calmettes, Guillaume Xu, Shili Ribalet, Bernard PLoS One Research Article Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the known cardio-protective role of HK in cardiac muscle, we have investigated the regulation of lactate and pyruvate metabolism by hexokinases (HKs), utilizing wild-type HEK293 cells and HEK293 cells in which the endogenous HKI and/or HKII have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. This decrease was rapidly reversed upon inhibition of the malate aspartate shuttle by aminooxyacetate, or inhibition of mitochondrial oxidative respiration by NaCN. These results suggest that in the absence of HKs, pyruvate-dependent activation of the TCA cycle together with the malate aspartate shuttle facilitates lactate transformation into pyruvate and its utilization by mitochondria. With replacement by overexpression of HKI or HKII the cellular response to pyruvate and NaCN was modified. With either hexokinase present, both the decrease in lactate due to the addition of pyruvate and the increase following addition of NaCN were either transient or suppressed altogether. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN), abolished the effects of HK replacement. These results suggest that blocking of the malate aspartate shuttle by HK may involve activation of the pentose phosphate pathway and increased NADPH production. Public Library of Science 2023-11-02 /pmc/articles/PMC10621844/ /pubmed/37917627 http://dx.doi.org/10.1371/journal.pone.0286660 Text en © 2023 John et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
John, Scott
Calmettes, Guillaume
Xu, Shili
Ribalet, Bernard
Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title_full Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title_fullStr Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title_full_unstemmed Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title_short Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
title_sort real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10621844/
https://www.ncbi.nlm.nih.gov/pubmed/37917627
http://dx.doi.org/10.1371/journal.pone.0286660
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