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A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium

Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid...

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Autores principales: Krycer, James R., Lor, Mary, Fitzsimmons, Rebecca L., Hudson, James E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8819040/
https://www.ncbi.nlm.nih.gov/pubmed/34971704
http://dx.doi.org/10.1016/j.jbc.2021.101547
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author Krycer, James R.
Lor, Mary
Fitzsimmons, Rebecca L.
Hudson, James E.
author_facet Krycer, James R.
Lor, Mary
Fitzsimmons, Rebecca L.
Hudson, James E.
author_sort Krycer, James R.
collection PubMed
description Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.
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spelling pubmed-88190402022-02-11 A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium Krycer, James R. Lor, Mary Fitzsimmons, Rebecca L. Hudson, James E. J Biol Chem Methods and Resources Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research. American Society for Biochemistry and Molecular Biology 2021-12-29 /pmc/articles/PMC8819040/ /pubmed/34971704 http://dx.doi.org/10.1016/j.jbc.2021.101547 Text en Crown Copyright © 2021 Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Methods and Resources
Krycer, James R.
Lor, Mary
Fitzsimmons, Rebecca L.
Hudson, James E.
A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title_full A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title_fullStr A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title_full_unstemmed A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title_short A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
title_sort cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8819040/
https://www.ncbi.nlm.nih.gov/pubmed/34971704
http://dx.doi.org/10.1016/j.jbc.2021.101547
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