Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells

Abstract Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function....

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Autores principales:	A. F. Bekebrede, J. Keijer, W. J. J. Gerrits, V. C. J. de Boer
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/54d58d496fee41948179fda1ccf69fef
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spelling	oai:doaj.org-article:54d58d496fee41948179fda1ccf69fef2021-12-02T19:16:18ZMitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells10.1038/s41598-021-99460-02045-2322https://doaj.org/article/54d58d496fee41948179fda1ccf69fef2021-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-99460-0https://doaj.org/toc/2045-2322Abstract Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.A. F. BekebredeJ. KeijerW. J. J. GerritsV. C. J. de BoerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q A. F. Bekebrede J. Keijer W. J. J. Gerrits V. C. J. de Boer Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
description	Abstract Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.
format	article
author	A. F. Bekebrede J. Keijer W. J. J. Gerrits V. C. J. de Boer
author_facet	A. F. Bekebrede J. Keijer W. J. J. Gerrits V. C. J. de Boer
author_sort	A. F. Bekebrede
title	Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
title_short	Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
title_full	Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
title_fullStr	Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
title_full_unstemmed	Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
title_sort	mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/54d58d496fee41948179fda1ccf69fef
work_keys_str_mv	AT afbekebrede mitochondrialandglycolyticextracellularfluxanalysisoptimizationforisolatedpigintestinalepithelialcells AT jkeijer mitochondrialandglycolyticextracellularfluxanalysisoptimizationforisolatedpigintestinalepithelialcells AT wjjgerrits mitochondrialandglycolyticextracellularfluxanalysisoptimizationforisolatedpigintestinalepithelialcells AT vcjdeboer mitochondrialandglycolyticextracellularfluxanalysisoptimizationforisolatedpigintestinalepithelialcells
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Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells

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