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Analysis of Endothelial Fatty Acid Metabolism Using Tracer Metabolomics

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Book cover High-Throughput Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1978))

Abstract

Blood vessels are lined by a streamlined monolayer of quiescent endothelial cells (ECs). Although these cells can remain quiescent for years, different stimuli (ischemia, inflammation) and growth factors can activate them and drive a process of new vessel formation (angiogenesis). Emerging evidence reveals that cellular metabolism is a key determinant of the EC subtype specification. The use of stable isotope tracing and mass spectrometry analysis has been essential for the discovery that fatty acid metabolism contributes to EC proliferation and lymphatic EC differentiation. This chapter describes the methodology for setting up palmitate-based tracer metabolomics and the subsequent liquid chromatography-mass spectrometry (LC-MS)-based analysis. As such, tracer metabolomics can be used: (1) to identify the different metabolic pathways relying on carbons provided by fatty acid oxidation and (2) to quantify the relative contributions of palmitate-derived carbons. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailed procedures for the labeling conditions, sample preparation, and subsequent LC-MS analysis.

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Acknowledgments

J.K. is supported by the Research Foundation Flanders (FWO); S.M.F. is supported by Marie Curie CIG, grants from the Research Foundation Flanders (FWO-Vlaanderen) Belgium; P.C. is supported by the VIB TechWatch program, a Federal Government Belgium grant (IUAP7/03), long-term structural Methusalem funding by the Flemish Government, grants from the Research Foundation Flanders (FWO-Vlaanderen), Foundation against Cancer (2012-175 and 2016-078), and ERC Advanced Research grant (EU-ERC743074).

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Author notes

  1. Joanna Kalucka and Bart Ghesquière contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer Biology (CCB), VIB, Leuven, Belgium

    Joanna Kalucka & Peter Carmeliet

  2. Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium

    Joanna Kalucka & Peter Carmeliet

  3. Metabolomics Expertise Center, VIB Center for Cancer Biology (CCB), VIB, Leuven, Belgium

    Bart Ghesquière

  4. Department of Oncology, Metabolomics Expertise Center, KU Leuven, Leuven, Belgium

    Bart Ghesquière

  5. Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology (CCB), VIB, Leuven, Belgium

    Sarah-Maria Fendt

  6. Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium

    Sarah-Maria Fendt

Authors
  1. Joanna Kalucka
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  2. Bart Ghesquière
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  3. Sarah-Maria Fendt
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  4. Peter Carmeliet
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Corresponding author

Correspondence to Peter Carmeliet .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, USA

    Angelo D'Alessandro

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Kalucka, J., Ghesquière, B., Fendt, SM., Carmeliet, P. (2019). Analysis of Endothelial Fatty Acid Metabolism Using Tracer Metabolomics. In: D'Alessandro, A. (eds) High-Throughput Metabolomics. Methods in Molecular Biology, vol 1978. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9236-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9236-2_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9235-5

  • Online ISBN: 978-1-4939-9236-2

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