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Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism
Journal article   Open access   Peer reviewed

Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism

Sophie Trefely, Joyce Liu, Katharina Huber, Mary T. Doan, Helen Jiang, Jay Singh, Eliana von Krusenstiern, Anna Bostwick, Peining Xu, Juliane G. Bogner-Strauss, …
Molecular metabolism (Germany), v 30, pp 61-71
01 Dec 2019
DOI: https://doi.org/10.1016/j.molmet.2019.09.004
PMID: 31767181
Featured in Collection :   UN Sustainable Development Goals @ Drexel
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https://doi.org/10.1016/j.molmet.2019.09.004View
Published, Version of Record (VoR)CC BY-NC-ND V4.0,  Open

Abstract

Endocrinology & Metabolism Life Sciences & Biomedicine Science & Technology
Objective: The dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of C-13-labeled substrates targeting central metabolic pathways. Methods: We designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS). Results: We show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondria! and cytosolic metabolic analyses. Conclusions: Our data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment. (C) 2019 The Author(s). Published by Elsevier GmbH.

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Web of Science research areas
Endocrinology & Metabolism
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