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A Roadmap for Interpreting 13C Metabolite Labeling Patterns from Cells

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A Roadmap for Interpreting 13C Metabolite Labeling Patterns from Cells Available online at www.sciencedirect.com ScienceDirect 13 A roadmap for interpreting C metabolite labeling patterns from cells 1,2 3,* 4,* Joerg M Buescher , Maciek R Antoniewicz , Laszlo G Boros , 5,* 6,* 7,* Shawn C Burgess , Henri Brunengraber , Clary B Clish , 8,* 9,* 10,* Ralph J DeBerardinis , Olivier Feron , Christian Frezza , 1,2,* 11,* 12,* Bart Ghesquiere , Eyal Gottlieb , Karsten Hiller , 13,* 14,* 15,* Russell G Jones , Jurre J Kamphorst , Richard G Kibbey , 16,* 17,* 18,* Alec C Kimmelman , Jason W Locasale , Sophia Y Lunt , 11,* 19,* 20,* Oliver DK Maddocks , Craig Malloy , Christian M Metallo , 21,22,* 23,24,* Emmanuelle J Meuillet , Joshua Munger , 25,* 26,* 27,28,* Katharina No¨ h , Joshua D Rabinowitz , Markus Ralser , 29,* 30,* 31,* Uwe Sauer , Gregory Stephanopoulos , Julie St-Pierre , 32,* 33,* Daniel A Tennant , Christoph Wittmann , 34,35,* 11,* Matthew G Vander Heiden , Alexei Vazquez , 11,* 36,37,* 29,* Karen Vousden , Jamey D Young , Nicola Zamboni and 1,2 Sarah-Maria Fendt 13 Measuring intracellular metabolism has increasingly led to Goodman Cancer Research Centre, Department of Physiology, McGill 13 University, Montreal, QC, Canada important insights in biomedical research. C tracer analysis, 14 13 Institute of Cancer Sciences, University of Glasgow, Glasgow, UK although less information-rich than quantitative C flux 15 Internal Medicine, Cellular and Molecular Physiology, Yale University analysis that requires computational data integration, has been School of Medicine, New Haven, CT, USA 16 established as a time-efficient method to unravel relative Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA pathway activities, qualitative changes in pathway 17 Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA contributions, and nutrient contributions. Here, we review 18 13 Department of Biochemistry and Molecular Biology, Michigan State selected key issues in interpreting C metabolite labeling University, East Lansing, MI, USA 19 patterns, with the goal of drawing accurate conclusions from Advanced Imaging Research Center-Division of Metabolic steady state and dynamic stable isotopic tracer experiments. Mechanisms of Disease and Department of Radiology, The University of Texas Southwestern Medical Center, Dallas, TX, USA 20 Addresses Department of Bioengineering, University of California, San Diego, La 1 Vesalius Research Center, VIB, Leuven, Belgium Jolla, CA, USA 2 21 Department of Oncology, KU Leuven, Leuven, Belgium L’Institut des Technologies Avance´ es en Sciences du Vivant (ITAV), 3 Department of Chemical and Biomolecular Engineering, University of Toulouse Cedex 1, France 22 Delaware, Newark, DE, USA The University of Arizona Cancer Center, and Department of 4 Department of Pediatrics, UCLA School of Medicine, Los Angeles Nutritional Sciences, The University of Arizona, Tucson, AZ, USA 23 Biomedical Research Institute at the Harbor-UCLA Medical Center and Department of Biochemistry, University of Rochester Medical Center, Sidmap, LLC, Los Angeles, CA, USA Rochester, NY, USA 5 24 Advanced Imaging Research Center-Division of Metabolic Department of Biophysics, University of Rochester Medical Center, Mechanisms of Disease and Department of Pharmacology, The Rochester, NY, USA 25 University of Texas Southwestern Medical Center, Dallas, TX, USA Institute of Bio- and Geosciences, IBG-1: Biotechnology, 6 Department of Nutrition, Case Western Reserve University School of Forschungszentrum Ju¨ lich GmbH, Ju¨ lich, Germany 26 Medicine, Cleveland, OH, USA Department of Chemistry and Lewis–Sigler Institute for Integrative 7 Broad Institute of Harvard and MIT, Cambridge, MA, USA Genomics, Princeton University, Princeton, NJ, USA 8 27 Children’s Medical Center Research Institute, UT Southwestern Cambridge Systems Biology Centre and Department of Biochemistry, Medical Center, Dallas, TX, USA University of Cambridge, Cambridge, UK 9 28 Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Division of Physiology and Metabolism, MRC National Institute for Expe´ rimentale et Clinique (IREC), Universite´ catholique de Louvain, Medical Research, London, UK 29 Brussels, Belgium Institute of Molecular Systems Biology, ETH Zurich, Zurich, 10 MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Switzerland 30 Centre, Cambridge Biomedical Campus, Cambridge, UK Department of Chemical Engineering, Massachusetts Institute of 11 Cancer Research UK, Beatson Institute, Glasgow, UK Technology, Cambridge, MA, USA 12 31 Luxembourg Centre for Systems Biomedicine, University of Goodman Cancer Research Centre, and Department of Biochemistry, Luxembourg, Esch-Belval, Luxembourg McGill University, Montreal, Quebec, Canada www.sciencedirect.com Current Opinion in Biotechnology 2015, 34:189–201 190 Systems biology 32 School of Cancer Sciences, College of Medical and Dental Sciences, conclusions on metabolic rates (fluxes), or the direction University of Birmingham, Edgbaston, Birmingham, UK of the flux changes, since an increase in metabolite 33 Institute of Systems Biotechnology, Saarland University, Saarbru¨ cken, concentration can both be indicative of increased activity Germany 34 of metabolite producing enzymes, but also decreased Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Broad Institute of Harvard and MIT, Cambridge, activity of metabolite consuming enzymes. MA, USA 35 Department of Medical Oncology, Dana-Farber Cancer Institute, In combination with growth rates (which provide global Boston, MA, USA 36 information on metabolic fluxes to biomass production), Department of Chemical and Biomolecular Engineering, Vanderbilt metabolite uptake/secretion rates provide a macroscopic University, Nashville, TN, USA 37 picture of overall metabolism. For instance, measuring Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA the rate of glucose depletion from the media reports the rate of glucose used by cells in a culture system. However, Corresponding author: Fendt, Sarah-Maria (sarah-maria.fendt@vib- these data alone are insufficient to reveal intracellular kuleuven.be) fluxes throughout the different metabolic pathways. * Authors are listed in alphabetic order. To examine intracellular fluxes (metabolite amount con- 13 Current Opinion in Biotechnology 2015, 34:189–201 verted/cell/time), heavy isotope (most frequently C) This review comes from a themed issue on Systems biology labeled nutrients (tracers) are commonly utilized [22– 13 29]. In formal C flux analysis, labeling patterns in Edited by Sarah Maria Fendt and Costas D Maranas intracellular metabolites resulting from metabolizing a 13 C labeled nutrient, cellular uptake and secretion rates, and prior knowledge of the biochemical reaction network http://dx.doi.org/10.1016/j.copbio.2015.02.003 are combined to computationally estimate metabolic 0958-1669/# 2015 Elsevier Ltd. All rights reserved. fluxes [11,30 ,31–33,34 ]. In practice, resolving meta- bolic fluxes from measured data can be time and data- intensive. In many cases, however, direct interpretation 13 13 of C labeling patterns (without formal C flux analysis) is sufficient to provide information on relative pathway activities, qualitative changes in pathway contributions Introduction via alternative metabolic routes, and nutrient contribu- Investigating cellular metabolism has a long-standing tion to the production of different metabolites. We refer 13 13 history in various research areas such as biochemistry, to this direct interpretation of C labeling patterns as C biotechnology and cellular physiology. A widely applica- tracer analysis. Here, we discuss selected important 13 ble toolbox to quantitatively measure intracellular me- aspects to consider when performing C tracer analysis tabolism has been developed in the context of to ensure correct data interpretation and to increase the biochemical engineering [1]. In light of the emerging insight obtained by stable isotopic tracer experiments. realization that altered cellular metabolism contributes to many diseases including cancer, metabolic syndromes, Metabolic steady state versus isotopic steady and neurodegenerative disorders, these approaches are state being increasingly applied to address biomedical research Metabolic steady state requires that both, intracellular questions [2–8,9 ]. metabolite levels and intracellular metabolic fluxes of a cell or a cell population are constant (Figure 1a) [35]. Cellular metabolism can be characterized by many pa- Controlled culture systems that ensure metabolic steady rameters including nutrient uptake and metabolite secre- state are continuous cultures (known as chemostats), where tion rates, intracellular metabolite levels, intracellular cell number and nutrient concentrations are maintained metabolic rates (fluxes), nutrient contributions to metab- constant throughout the experiment [36]. More commonly, olite and macromolecule synthesis, and pathway activities experiments are performed at pseudo-steady state, where [2,3,9 ,10–12]. changes in metabolite concentrations and fluxes are mini- mal on the timescale over which the measurement is being Metabolomics, which provides absolute or relative intra- made. In adherent mammalian cell culture, perfusion cellular or extracellular metabolite levels, is a broad and bioreactors and nutrostats [37,38], where nutrient concen- sensitive method to detect differences in metabolic states trations
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