Metabolic Flux Analysis Tools to Investigate Brain Metabolism In Vitro

  • Ana I. AmaralEmail author
  • Paula M. Alves
  • Ana P. Teixeira
Part of the Neuromethods book series (NM, volume 90)


In recent decades, 13C nuclear magnetic resonance (NMR) spectroscopy and metabolic modeling tools allowed estimating the main cerebral metabolic fluxes in vitro and in vivo. These investigations contributed significantly to elucidate neuro-glial metabolic interactions, cerebral metabolic compartmentation, and the individual contribution of neurons and astrocytes to brain energetics. However, many issues in this field remain unclear and/or under debate.

Despite the valuable amount of data generated in cell culture studies involving 13C-labeled substrates and NMR spectroscopy or mass spectrometry, only a few studies have employed modeling approaches to fully explore the results obtained. Here, we present different Metabolic Flux Analysis (MFA) methodologies that, combined with information provided by isotopomers of key compounds derived from the metabolism of 13C-labeled precursors, allow for a more comprehensive investigation of cell metabolism in cultured brain cells. Overall, MFA is presented as a powerful tool to investigate particular aspects of cerebral metabolic compartmentation in the context of physiology and disease as it allows quantifying changes in the distribution of metabolic fluxes caused by pathological insults, drug treatments, or presence of different metabolic substrates.

Key words

Brain energy metabolism Metabolic flux analysis Primary cultures Astrocytes Neurons 13C glucose 13C NMR spectroscopy Gas chromatography–mass spectrometry 


  1. 1.
    Lee K, Berthiaume F, Stephanopoulos GN, Yarmush ML (1999) Metabolic flux analysis: a powerful tool for monitoring tissue function. Tissue Eng 5(4):347–368PubMedCrossRefGoogle Scholar
  2. 2.
    Varma A, Palsson BO (1994) Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl Environ Microbiol 60(10):3724–3731PubMedPubMedCentralGoogle Scholar
  3. 3.
    Bernal V, Carinhas N, Yokomizo AY, Carrondo MJ, Alves PM (2009) Cell density effect in the baculovirus-insect cells system: a quantitative analysis of energetic metabolism. Biotechnol Bioeng 104(1):162–180PubMedCrossRefGoogle Scholar
  4. 4.
    Niklas J, Schneider K, Heinzle E (2010) Metabolic flux analysis in eukaryotes. Curr Opin Biotechnol 21(1):63–69PubMedCrossRefGoogle Scholar
  5. 5.
    Quek LE, Dietmair S, Kromer JO, Nielsen LK (2010) Metabolic flux analysis in mammalian cell culture. Metab Eng 12(2):161–171PubMedCrossRefGoogle Scholar
  6. 6.
    Vallino JJ, Stephanopoulos G (1993) Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol Bioeng 41(6):633–646PubMedCrossRefGoogle Scholar
  7. 7.
    Bonarius HP, Hatzimanikatis V, Meesters KP, de Gooijer CD, Schmid G, Tramper J (1996) Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng 50(3):299–318PubMedCrossRefGoogle Scholar
  8. 8.
    Carinhas N, Bernal V, Monteiro F, Carrondo MJ, Oliveira R, Alves PM (2010) Improving baculovirus production at high cell density through manipulation of energy metabolism. Metab Eng 12(1):39–52PubMedCrossRefGoogle Scholar
  9. 9.
    Forbes NS, Meadows AL, Clark DS, Blanch HW (2006) Estradiol stimulates the biosynthetic pathways of breast cancer cells: detection by metabolic flux analysis. Metab Eng 8(6):639–652PubMedCrossRefGoogle Scholar
  10. 10.
    Nadeau I, Sabatie J, Koehl M, Perrier M, Kamen A (2000) Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis. Metab Eng 2(4):277–292PubMedCrossRefGoogle Scholar
  11. 11.
    Amaral AI, Teixeira AP, Hakonsen BI, Sonnewald U, Alves PM (2011) A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Front Neuroenergetics 3:5PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Teixeira AP, Santos SS, Carinhas N, Oliveira R, Alves PM (2008) Combining metabolic flux analysis tools and 13C NMR to estimate intracellular fluxes of cultured astrocytes. Neurochem Int 52(3):478–486PubMedCrossRefGoogle Scholar
  13. 13.
    Amaral AI, Teixeira AP, Martens S, Bernal V, Sousa MF, Alves PM (2010) Metabolic alterations induced by ischemia in primary cultures of astrocytes: merging 13C NMR spectroscopy and metabolic flux analysis. J Neurochem 113(3):735–748PubMedCrossRefGoogle Scholar
  14. 14.
    Amaral AI, Teixeira AP, Sonnewald U, Alves PM (2011) Estimation of intracellular fluxes in cerebellar neurons after hypoglycemia: importance of the pyruvate recycling pathway and glutamine oxidation. J Neurosci Res 89(5):700–710PubMedCrossRefGoogle Scholar
  15. 15.
    Keibler MA, Fendt SM, Stephanopoulos G (2012) Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism. Biotechnol Prog 28:1409–1418PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3(3):195–206PubMedCrossRefGoogle Scholar
  17. 17.
    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) (13)C-based metabolic flux analysis. Nat Protoc 4(6):878–892PubMedCrossRefGoogle Scholar
  18. 18.
    Noh K, Wiechert W (2011) The benefits of being transient: isotope-based metabolic flux analysis at the short time scale. Appl Microbiol Biotechnol 91(5):1247–1265PubMedCrossRefGoogle Scholar
  19. 19.
    Noh K, Wahl A, Wiechert W (2006) Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions. Metab Eng 8(6):554–577PubMedCrossRefGoogle Scholar
  20. 20.
    Hofmann U, Maier K, Niebel A, Vacun G, Reuss M, Mauch K (2008) Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng 100(2):344–354PubMedCrossRefGoogle Scholar
  21. 21.
    Noh K, Gronke K, Luo B, Takors R, Oldiges M, Wiechert W (2007) Metabolic flux analysis at ultra short time scale: isotopically non-stationary 13C labeling experiments. J Biotechnol 129(2):249–267PubMedCrossRefGoogle Scholar
  22. 22.
    Wiechert W, Noh K (2005) From stationary to instationary metabolic flux analysis. Adv Biochem Eng Biotechnol 92:145–172PubMedGoogle Scholar
  23. 23.
    Zamboni N (2011) 13C metabolic flux analysis in complex systems. Curr Opin Biotechnol 22(1):103–108PubMedCrossRefGoogle Scholar
  24. 24.
    Noack S, Noh K, Moch M, Oldiges M, Wiechert W (2010) Stationary versus non-stationary (13)C-MFA: A comparison using a consistent dataset. J Biotechnol 154:179–190PubMedCrossRefGoogle Scholar
  25. 25.
    Schaub J, Mauch K, Reuss M (2008) Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data. Biotechnol Bioeng 99(5):1170–1185PubMedCrossRefGoogle Scholar
  26. 26.
    Lemons JM, Feng XJ, Bennett BD, Legesse-Miller A, Johnson EL, Raitman I, Pollina EA, Rabitz HA, Rabinowitz JD, Coller HA (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8(10):e1000514PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Maier K, Hofmann U, Reuss M, Mauch K (2008) Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part II. Flux estimation. Biotechnol Bioeng 100(2):355–370PubMedCrossRefGoogle Scholar
  28. 28.
    Maier K, Hofmann U, Bauer A, Niebel A, Vacun G, Reuss M, Mauch K (2009) Quantification of statin effects on hepatic cholesterol synthesis by transient (13)C-flux analysis. Metab Eng 11(4–5):292–309PubMedCrossRefGoogle Scholar
  29. 29.
    Olstad E, Olsen GM, Qu H, Sonnewald U (2007) Pyruvate recycling in cultured neurons from cerebellum. J Neurosci Res 85(15):3318–3325PubMedCrossRefGoogle Scholar
  30. 30.
    Drejer J, Larsson OM, Kvamme E, Svenneby G, Hertz L, Schousboe A (1985) Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellum in vivo. Neurochem Res 10(1):49–62PubMedCrossRefGoogle Scholar
  31. 31.
    Drejer J, Schousboe A (1989) Selection of a pure cerebellar granule cell culture by kainate treatment. Neurochem Res 14(8):751–754PubMedCrossRefGoogle Scholar
  32. 32.
    Richter-Landsberg C, Besser A (1994) Effects of organotins on rat brain astrocytes in culture. J Neurochem 63(6):2202–2209PubMedCrossRefGoogle Scholar
  33. 33.
    Klamt S, Saez-Rodriguez J, Gilles ED (2007) Structural and functional analysis of cellular networks with Cell NetAnalyzer. BMC Syst Biol 1:2PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Wang NS, Stephanopoulos G (1983) Application of macroscopic balances to the identification of gross measurement errors. Biotechnol Bioeng 25(9):2177–2208PubMedCrossRefGoogle Scholar
  35. 35.
    Klamt S, Schuster S (2002) Calculating as many fluxes as possible in underdetermined metabolic networks. Mol Biol Rep 29(1–2):243–248PubMedCrossRefGoogle Scholar
  36. 36.
    Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41(5):1484–1487PubMedCrossRefGoogle Scholar
  37. 37.
    Shank RP, Leo GC, Zielke HR (1993) Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of d-[1-13C]glucose metabolism. J Neurochem 61(1):315–323PubMedCrossRefGoogle Scholar
  38. 38.
    Mawhinney T, Robinett R, Atalay A, Madson M (1986) Analysis of amino acids as their tert-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry. J Chromatogr 358:231–242PubMedCrossRefGoogle Scholar
  39. 39.
    Biemann K (1962) Mass spectrometry in organic chemistry applications. McGraw, New York, NY, pp 223–227Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Ana I. Amaral
    • 1
    Email author
  • Paula M. Alves
    • 2
    • 3
  • Ana P. Teixeira
    • 2
    • 3
  1. 1.The Anne McLaren Laboratory for Regenerative Medicine and Department of Clinical Neurosciences, Wellcome Trust—Medical Research Council Cambridge Stem Cell InstituteUniversity of CambridgeCambridgeUK
  2. 2.IBET, Instituto de Biologia Experimental e TecnológicaOeirasPortugal
  3. 3.Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal

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