Skip to main content
SpringerLink
Log in

Metabolic Flux Analysis Tools to Investigate Brain Metabolism In Vitro

  • Protocol
  • First Online:
Brain Energy Metabolism

Part of the book series: Neuromethods ((NM,volume 90))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 139.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lee K, Berthiaume F, Stephanopoulos GN, Yarmush ML (1999) Metabolic flux analysis: a powerful tool for monitoring tissue function. Tissue Eng 5(4):347–368

    Article  PubMed  CAS  Google Scholar 

  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):3724–3731

    PubMed  CAS  PubMed Central  Google Scholar 

  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):162–180

    Article  PubMed  CAS  Google Scholar 

  4. Niklas J, Schneider K, Heinzle E (2010) Metabolic flux analysis in eukaryotes. Curr Opin Biotechnol 21(1):63–69

    Article  PubMed  CAS  Google Scholar 

  5. Quek LE, Dietmair S, Kromer JO, Nielsen LK (2010) Metabolic flux analysis in mammalian cell culture. Metab Eng 12(2):161–171

    Article  PubMed  CAS  Google Scholar 

  6. Vallino JJ, Stephanopoulos G (1993) Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol Bioeng 41(6):633–646

    Article  PubMed  CAS  Google Scholar 

  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):299–318

    Article  PubMed  CAS  Google Scholar 

  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):39–52

    Article  PubMed  CAS  Google Scholar 

  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):639–652

    Article  PubMed  CAS  Google Scholar 

  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):277–292

    Article  PubMed  CAS  Google Scholar 

  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 3:5

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  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):478–486

    Article  PubMed  CAS  Google Scholar 

  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):735–748

    Article  PubMed  CAS  Google Scholar 

  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):700–710

    Article  PubMed  CAS  Google Scholar 

  15. Keibler MA, Fendt SM, Stephanopoulos G (2012) Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism. Biotechnol Prog 28:1409–1418

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3(3):195–206

    Article  PubMed  CAS  Google Scholar 

  17. Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) (13)C-based metabolic flux analysis. Nat Protoc 4(6):878–892

    Article  PubMed  CAS  Google Scholar 

  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):1247–1265

    Article  PubMed  Google Scholar 

  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):554–577

    Article  PubMed  Google Scholar 

  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):344–354

    Article  PubMed  CAS  Google Scholar 

  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):249–267

    Article  PubMed  Google Scholar 

  22. Wiechert W, Noh K (2005) From stationary to instationary metabolic flux analysis. Adv Biochem Eng Biotechnol 92:145–172

    PubMed  CAS  Google Scholar 

  23. Zamboni N (2011) 13C metabolic flux analysis in complex systems. Curr Opin Biotechnol 22(1):103–108

    Article  PubMed  CAS  Google Scholar 

  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 154:179–190

    Article  PubMed  Google Scholar 

  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):1170–1185

    Article  PubMed  CAS  Google Scholar 

  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):e1000514

    Article  PubMed  PubMed Central  Google Scholar 

  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):355–370

    Article  PubMed  CAS  Google Scholar 

  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):292–309

    Article  PubMed  CAS  Google Scholar 

  29. Olstad E, Olsen GM, Qu H, Sonnewald U (2007) Pyruvate recycling in cultured neurons from cerebellum. J Neurosci Res 85(15):3318–3325

    Article  PubMed  CAS  Google Scholar 

  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):49–62

    Article  PubMed  CAS  Google Scholar 

  31. Drejer J, Schousboe A (1989) Selection of a pure cerebellar granule cell culture by kainate treatment. Neurochem Res 14(8):751–754

    Article  PubMed  CAS  Google Scholar 

  32. Richter-Landsberg C, Besser A (1994) Effects of organotins on rat brain astrocytes in culture. J Neurochem 63(6):2202–2209

    Article  PubMed  CAS  Google Scholar 

  33. Klamt S, Saez-Rodriguez J, Gilles ED (2007) Structural and functional analysis of cellular networks with Cell NetAnalyzer. BMC Syst Biol 1:2

    Article  PubMed  PubMed Central  Google Scholar 

  34. Wang NS, Stephanopoulos G (1983) Application of macroscopic balances to the identification of gross measurement errors. Biotechnol Bioeng 25(9):2177–2208

    Article  PubMed  CAS  Google Scholar 

  35. Klamt S, Schuster S (2002) Calculating as many fluxes as possible in underdetermined metabolic networks. Mol Biol Rep 29(1–2):243–248

    Article  PubMed  CAS  Google Scholar 

  36. Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41(5):1484–1487

    Article  PubMed  CAS  Google Scholar 

  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):315–323

    Article  PubMed  CAS  Google Scholar 

  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 358:231–242

    Article  PubMed  CAS  Google Scholar 

  39. Biemann K (1962) Mass spectrometry in organic chemistry applications. McGraw, New York, NY, pp 223–227

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Anne McLaren Laboratory for Regenerative Medicine and Department of Clinical Neurosciences, Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, West Forvie Building—Forvie Site, Robinson Way, CB2 0SZ, Cambridge, UK

    Ana I. Amaral

  2. IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901, Oeiras, Portugal

    Paula M. Alves & Ana P. Teixeira

  3. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157, Oeiras, Portugal

    Paula M. Alves & Ana P. Teixeira

Authors
  1. Ana I. Amaral
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paula M. Alves
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana P. Teixeira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ana I. Amaral .

Editor information

Editors and Affiliations

  1. Carl-Ludwig-Institute for Physiology, University of Leipzig, Leipzig, Germany

    Johannes Hirrlinger

  2. Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Helle S. Waagepetersen

Appendix

Appendix

Table 10 Detailed biochemical reactions included in the metabolic network of cerebellar granule neurons
Full size table
Table 11 Detailed biochemical reactions included in the metabolic network of astrocytes
Full size table
Table 12 Stoichiometric matrix (metabolic reactions) of the isotopic transient MFA model describing astrocytic metabolisma (Chap. 4)
Full size table
Table 13 Mass isotopomer balances of metabolites represented in the network describing astrocytic metabolism
Full size table

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Amaral, A.I., Alves, P.M., Teixeira, A.P. (2014). Metabolic Flux Analysis Tools to Investigate Brain Metabolism In Vitro. In: Hirrlinger, J., Waagepetersen, H. (eds) Brain Energy Metabolism. Neuromethods, vol 90. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1059-5_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1059-5_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1058-8

  • Online ISBN: 978-1-4939-1059-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation