Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-13C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained 13C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (13C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Ahn WS, Antoniewicz MR (2013) Parallel labeling experiments with [1,2-(13)C]glucose and [U-(13)C]glutamine provide new insights into CHO cell metabolism. Metabolic Engineering 15:34–47
Amaral AI, Teixeira AP, Håkonsen BI, Sonnewald U, Alves PM (2011) A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Frontiers in Neuroenergetics 3:1–5
Antoniewicz MR, Kelleher JK, Stephanopoulos G (2006) Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metabolic Engineering 8(4):324–337
Bak LK, Schousboe A, Waagepetersen HS (2006) The glutamate/GABA-glutamine cycle: Aspects of transport, neurotransmitter homeostasis and ammonia transfer. Journal of Neurochemistry 98(3):641–653
Bélanger M, Allaman I, Magistretti PJ (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metabolism 14(6):724–738
Bolanos JP, Peuchen S, Heales SJR, Land JM, Clark JB (1994) Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. Journal of Neurochemistry 63:910–916
Brown AM (2004) Brain glycogen re-awakened. Journal of Neurochemistry 89(3):537–552
Candelario KM, Shuttleworth CW, Cunningham LA (2013) Neural stem/progenitor cells display a low requirement for oxidative metabolism independent of hypoxia inducible factor-1 alpha expression. Journal of Neurochemistry 125(3):420–429
Carinhas N, Bernal V, Monteiro F, Carrondo MJT, Oliveira R, Alves PM (2010) Improving baculovirus production at high cell density through manipulation of energy metabolism. Metabolic Engineering 12(1):39–52
Carinhas N, Pais DAM, Koshkin A, Fernandes P, Coroadinha A, Carrondo MJT, Alves PM, Teixeira AP (2016) Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production. Scientific Reports 6:23529
Crown SB, Antoniewicz MR (2013) Publishing (13)C metabolic flux analysis studies: A review and future perspectives. Metabolic Engineering 20:42–48
Doetsch F, Caille I, Lim DA, Garcı JM, Alvarez-buylla A (1999) Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult Mammalian Brain. Cell 97:703–716
Folmes CDL, Nelson TJ, Martinez-Fernandez A, Arrell DK, Lindor JZ, Dzeja PP, Ikeda Y, Terzic CP, Terzic A (2011) Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metabolism 14(2):264–271
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. Biotechnology and Bioengineering 100(2):344–354
Johnson MA, Weick JP, Pearce RA, Zhang S-C (2007) Functional Neural Development from Human Embryonic Stem Cells: Accelerated Synaptic Activity Coculture. Journal of Neuroscience 27(12):3069–3077
Kleiderman SM, Sá JV, Teixeira AP, Brito C, Gutbier S, Evje LG, Hadera MG, Glaab E, Henry M, Sachinidis A, Alves PM, Sonnewald U, Leist M (2016) Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells. Glia 64(5):695–715
Knobloch M, Braun SMG, Zurkirchen L, von Schoultz C, Zamboni N, Araúzo-Bravo MJ, Kovacs WJ, Karalay Ö, Suter U, Machado RAC, Roccio M, Lutolf MP, Semenkovich CF, Jessberger S (2013) Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis. Nature 493(7431):226–230
Kriegstein A, Alvarez-Buylla A (2009) The glial nature of embryonic and adult neural stem cells. Annual Review of Neuroscience 32:149–184
McKenna M, Gruetter R, Sonnewald U, Waagepetersen H, Schousboe A (2012) Energy Metabolism of the Brain. In: Brady STS, Albers RW, Price DL (eds) Basic Neurochemistry: Principles of Molecular, Cellular, and Medical Neurobiology, 8th edn. Elsevier Academic Press, Oxford, UK, pp 200–299
Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, Jewell CM, Johnson ZR, Irvine DJ, Guarente L, Kelleher JK, Vander Heiden MG, Iliopoulos O, Stephanopoulos G (2012) Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481:380–384
Munger J, Bennett BD, Parikh A, Feng X, Rabitz HA, Shenk T, Rabinowitz JD (2008) Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol 26(10):1179–1186
Nedergaard M, Ransom B, Goldman SA (2003) New roles for astrocytes: Redefining the functional architecture of the brain. Trends in Neurosciences 26(10):523–530
Noh K, Wiechert W (2006) Experimental Design Principles for Isotopically Instationary C Labeling Experiments. Biotechnology and Bioengineering 94:234–251
Palm T, Bolognin S, Meiser J, Nickels S, Träger C, Meilenbrock R-L, Brockhaus J, Schreitmüller M, Missler M, Schwamborn JC (2015) Rapid and robust generation of long-term self-renewing human neural stem cells with the ability to generate mature astroglia. Scientific Reports 5:16321
Pellerin L, Bouzier-Sore A-K, Aubert A, Serres S, Merle M, Costalat R, Magistretti PJ (2007) Activity-Dependent Regulation of Energy Metabolism by Astrocytes: An Update. Glia 55:1251–1262
Pellerin L, Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proceedings of the National Academy of Sciences of the United States of America 91(22):10625–10629
Phatnani H, Maniatis T (2015) Astrocytes in Neurodegenerative Disease. Cold Spring Harbor Perspectives in Biology 7(6):a020628
Rouach N, Koulakoff A, Abudara V, Willecke K, Giaume C (2008) Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission. Science 322:1551–1555
Sá, J. V., Duarte, T. M., Carrondo, M. J. T., Alves, P. M., and Teixeira, A. P. (2015). Metabolic Flux Analysis: A Powerful Tool in Animal Cell Culture. In M. Al-Rubeai (Ed.), Animal Cell Culture (Vol. 521–539, p. 785). Springer International Publishing
Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Molecular Systems Biology. doi:10.1038/msb4100109
Seri B, Garcı JM, Mcewen BS, Alvarez-buylla A (2001) Astrocytes Give Rise to New Neurons in the Adult Mammalian Hippocampus. The Journal of Neuroscience 21(18):7153–7160
Simard M, Nedergaard M (2004) The neurobiology of glia in the context of water and ion homeostasis. Neuroscience 129(4):877–896
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., and Barres, B. a. (2001). Control of synapse number by glia. Science (New York, N.Y.), 291, 657–661
Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg Effect : Cell Proliferation. Science 324:1029–1034
Walls, A. B., Bak, L. K., Sonnewald, U., Schousboe, A., and Waagepetersen, H. S. (2014). Metabolic Mapping of Astrocytes and Neurons in Culture Using Stable Isotopes and Gas Chromatography-Mass Spectrometry (GC-MS). In HirrlingerJohannes & H. S. Waagepetersen (Eds.), Brain Energy Metabolism (pp. 73–105)
Westergaard, N., Banke, T. U. E., Wahl, P., Sonnewaldt, U., and Schousboe, A. (1995). Citrate modulates the regulation by Zn2 + of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release, 92(April), 3367–3370
Westergaard N, Sonnewald U, Unsgård G, Peng L, Hertz L, Schousboe A (1994) Uptake, release, and metabolism of citrate in neurons and astrocytes in primary cultures. Journal of Neurochemistry 62(5):1727–1733
Wiechert W (2001) 13C metabolic flux analysis. Metabolic Engineering 3(3):195–206
Yeo H, Lyssiotis CA, Zhang Y, Ying H, Asara JM, Cantley LC, Paik J-H (2013) FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells. The EMBO Journal 32(19):2589–2602
Young, J. D. (2014). INCA: A computational platform for isotopically nonstationary metabolic flux analysis. Bioinformatics (Oxford, England), 30, 11–13
Young JD, Walther JL, Antoniewicz MR, Yoo H, Stephanopoulos G (2008) An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis. Biotechnology and Bioengineering 99(3):686–699
Zhang J, Nuebel E, Daley GQ, Koehler CM, Teitell MA (2012) Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal. Cell Stem Cell 11(5):589–595
Zwingmann C, Leibfritz D (2003) Regulation of glial metabolism studied by 13C-NMR. NMR in Biomedicine 16(6–7):370–399
Support from iNOVA4Health - UID/Multi/04462/2013, a program funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Educação e Ciência and co-funded by FEDER under the PT2020 Partnership Agreement, is acknowledged. This research has also received support from FCT through the project MITP-TB/ECE/0013/2013, from the German research foundation (RTG1331, KoRS-CB) and the German ministry for science (BMBF-DynaMeTox). JV Sá is a recipient of a Ph.D. fellowship from FCT (PD/BD/52474/2014). The expert technical assistance of Lars Evje with GC-MS is gratefully acknowledged. We are also thankful to Nuno Carinhas for his help on fluxome analysis.
S.I. : In honor of Dr. Mary McKenna.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Sá, J.V., Kleiderman, S., Brito, C. et al. Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis. Neurochem Res 42, 244–253 (2017). https://doi.org/10.1007/s11064-016-1907-z
- Neural stem cells
- Astrocytic differentiation
- Metabolic flux analysis
- Carbon labelling cultures