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Measuring Glycolytic and Mitochondrial Fluxes in Endothelial Cells Using Radioactive Tracers

  • Koen Veys
  • Abdiel Alvarado-Diaz
  • Katrien De Bock
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1862)

Abstract

Endothelial cells (ECs) form the inner lining of the vascular network. Although they can remain quiescent for years, ECs exhibit high plasticity in both physiological and pathological conditions, when they need to rapidly form new blood vessels in a process called angiogenesis. EC metabolism recently emerged as an important driver of this angiogenic switch. The use of radioactive tracer substrates to assess metabolic flux rates in ECs has been essential for the discovery that fatty acid, glucose, and glutamine metabolism critically contribute to vessel sprouting. In the future, these assays will be useful as a tool for the characterization of pathological conditions in which deregulation of EC metabolism underlies and/or precedes the disease, but also for the identification of anti-angiogenic metabolic targets. This chapter describes in detail the radioactive tracer substrate assays that have been used for the determination of EC metabolic flux in vitro.

Key words

Endothelium Metabolic flux rate Radioactive tracer substrates Glycolysis Fatty acid oxidation Glucose oxidation Glutamine oxidation 

References

  1. 1.
    Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473:298–307. https://doi.org/10.1038/nature10144 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8:464–478. https://doi.org/10.1038/nrm2183 CrossRefPubMedGoogle Scholar
  3. 3.
    De Bock K, Georgiadou M, Schoors S et al (2013) Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154:651–663. https://doi.org/10.1016/j.cell.2013.06.037 CrossRefPubMedGoogle Scholar
  4. 4.
    Wilhelm K, Happel K, Eelen G et al (2016) FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature 529:216–220. https://doi.org/10.1038/nature16498 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Doddaballapur A, Michalik KM, Manavski Y et al (2015) Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3. Arterioscler Thromb Vasc Biol 35:137–145. https://doi.org/10.1161/ATVBAHA.114.304277 CrossRefPubMedGoogle Scholar
  6. 6.
    Cantelmo AR, Conradi L-C, Brajic A et al (2016) Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy. Cancer Cell:1–48. https://doi.org/10.1016/j.ccell.2016.10.006 CrossRefGoogle Scholar
  7. 7.
    Schoors S, De Bock K, Cantelmo AR et al (2014) Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Cell Metab 19:37–48. https://doi.org/10.1016/j.cmet.2013.11.008 CrossRefPubMedGoogle Scholar
  8. 8.
    De Bock K, Georgiadou M, Carmeliet P (2013) Role of endothelial cell metabolism in vessel sprouting. Cell Metab 18:634–647. https://doi.org/10.1016/j.cmet.2013.08.001 CrossRefPubMedGoogle Scholar
  9. 9.
    Yu P, Wilhelm K, Dubrac A et al (2017) FGF-dependent metabolic control of vascular development. Nat Publ Group 545:224–228. https://doi.org/10.1038/nature22322 CrossRefGoogle Scholar
  10. 10.
    Schoors S, Bruning U, Missiaen R et al (2015) Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature 520:192–197. https://doi.org/10.1038/nature14362 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wong BW, Wang X, Zecchin A et al (2017) The role of fatty acid β-oxidation in lymphangiogenesis. Nature 542:49–54. https://doi.org/10.1038/nature21028 CrossRefPubMedGoogle Scholar
  12. 12.
    Kim B, Li J, Jang C, Arany Z (2017) Glutamine fuels proliferation but not migration of endothelial cells. EMBO J 36:2321–2333. https://doi.org/10.15252/embj.201796436 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Huang H, Vandekeere S, Kalucka J et al (2017) Role of glutamine and interlinked asparagine metabolism in vessel formation. EMBO J 36:2334–2352. https://doi.org/10.15252/embj.201695518 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Leopold JA, Walker J, Scribner AW et al (2003) Glucose-6-phosphate dehydrogenase modulates vascular endothelial growth factor-mediated angiogenesis. J Biol Chem 278:32100–32106. https://doi.org/10.1074/jbc.M301293200 CrossRefPubMedGoogle Scholar
  15. 15.
    Leopold JA, Dam A, Maron BA et al (2007) Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity. Nat Med 13:189–197. https://doi.org/10.1038/nm1545 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rohlenova K, Veys K, Miranda-Santos I, et al (2017) Endothelial cell metabolism in health and disease. Trends Cell Biol. doi: https://doi.org/10.1016/j.tcb.2017.10.010 CrossRefGoogle Scholar
  17. 17.
    Eelen G, de Zeeuw P, Treps L et al (2018) Endothelial cell metabolism. Physiol Rev 98:3–58. https://doi.org/10.1152/physrev.00001.2017 CrossRefPubMedGoogle Scholar
  18. 18.
    Uebelhoer M, Iruela-Arispe ML (2016) Cross-talk between signaling and metabolism in the vasculature. Vasc Pharmacol 83:4–9. https://doi.org/10.1016/j.vph.2016.06.002 CrossRefGoogle Scholar
  19. 19.
    Sawada N, Arany Z (2017) Metabolic regulation of angiogenesis in diabetes and aging. Physiology. doi: https://doi.org/10.1152/physiol.00039.2016;wgroup:string:Physio
  20. 20.
    Kler RS, Sherratt HSA, Turnbull DM (1992) The measurement of mitochondrial β-oxidation by release of 3H2O from [9,10-3H]hexadecanoate: application to skeletal muscle and the use of inhibitors as models of metabolic disease. Biochem Med Metab Biol 47:145–156. https://doi.org/10.1016/0885-4505(92)90018-t CrossRefPubMedGoogle Scholar
  21. 21.
    Drynan L, Quant PA, Zammit VA (1996) Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over β-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states. Biochem J 317:791–795. https://doi.org/10.1042/bj3170791 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Clem B, Telang S, Clem A et al (2007) Small molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth. Mol Cancer Ther 6:A85–A85CrossRefGoogle Scholar
  23. 23.
    Lee Y-J, Kang I-J, Bünger R, Kang Y-H (2003) Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells. Microvasc Res 66:91–101CrossRefGoogle Scholar
  24. 24.
    Pandolfi PP, Sonati F, Rivi R et al (1995) Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J 14:5209–5215CrossRefGoogle Scholar
  25. 25.
    Leighton B, Curi R, Hussein A, Newsholme EA (1987) Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovine pulmonary endothelial cells. FEBS Lett 225:93–96CrossRefGoogle Scholar
  26. 26.
    Sanchez EL, Carroll PA, Thalhofer AB, Lagunoff M (2015) Latent KSHV infected endothelial cells are glutamine addicted and require Glutaminolysis for survival. PLoS Pathog 11:e1005052. https://doi.org/10.1371/journal.ppat.1005052 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Durante W, Liu X-M, Yates B et al (2017) Glutaminase 1 promotes the proliferation of endothelial cells via the induction of cyclin a. FASEB J 31:1065.4–1065.4. https://doi.org/10.1096/fj.1530-6860 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Koen Veys
    • 1
    • 2
  • Abdiel Alvarado-Diaz
    • 3
  • Katrien De Bock
    • 3
  1. 1.Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer BiologyVIBLeuvenBelgium
  2. 2.Laboratory of Angiogenesis and Vascular Metabolism, Department of OncologyKU LeuvenLeuvenBelgium
  3. 3.Laboratory of Exercise and Health, Department of Health Sciences and TechnologyETH ZürichZürichSwitzerland

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