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Frontiers in Neuroenergetics ·

Volume 3 - 2011 | https://doi.org/10.3389/fnene.2011.00005

A Comprehensive Metabolic Profile of Cultured Astrocytes Using Isotopic Transient Metabolic Flux Analysis and 13 C-Labeled Glucose

  • Ana I. Amaral1,2
  • Ana P. Teixeira1,2
  • Bjørn I. Håkonsen3
  • Ursula Sonnewald3
  • Paula M. Alves1,2*corresponding author
  1. 11 Instituto de Tecnologia Química e Biológica – Universidade Nova de Lisboa Oeiras, Portugal
  2. 22 Instituto de Biologia Experimental e Tecnológica Oeiras, Portugal
  3. 33 Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology Trondheim, Norway
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Abstract

Metabolic models have been used to elucidate important aspects of brain metabolism in recent years. This work applies for the first time the concept of isotopic transient 13C metabolic flux analysis (MFA) to estimate intracellular fluxes in primary cultures of astrocytes. This methodology comprehensively explores the information provided by 13C labeling time-courses of intracellular metabolites after administration of a 13C-labeled substrate. Cells were incubated with medium containing [1-13C]glucose for 24 h and samples of cell supernatant and extracts collected at different time points were then analyzed by mass spectrometry and/or high performance liquid chromatography. Metabolic fluxes were estimated by fitting a carbon labeling network model to isotopomer profiles experimentally determined. Both the fast isotopic equilibrium of glycolytic metabolite pools and the slow labeling dynamics of TCA cycle intermediates are described well by the model. The large pools of glutamate and aspartate which are linked to the TCA cycle via reversible aminotransferase reactions are likely to be responsible for the observed delay in equilibration of TCA cycle intermediates. Furthermore, it was estimated that 11% of the glucose taken up by astrocytes was diverted to the pentose phosphate pathway. In addition, considerable fluxes through pyruvate carboxylase [PC; PC/pyruvate dehydrogenase (PDH) ratio = 0.5], malic enzyme (5% of the total pyruvate production), and catabolism of branched-chained amino acids (contributing with ∼40% to total acetyl-CoA produced) confirmed the significance of these pathways to astrocytic metabolism. Consistent with the need of maintaining cytosolic redox potential, the fluxes through the malate–aspartate shuttle and the PDH pathway were comparable. Finally, the estimated glutamate/α-ketoglutarate exchange rate (∼0.7 μmol mg prot−1 h−1) was similar to the TCA cycle flux. In conclusion, this work demonstrates the potential of isotopic transient MFA for a comprehensive analysis of energy metabolism.

Keywords

References

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Citation

Amaral, A. I., et al. (2011). A Comprehensive Metabolic Profile of Cultured Astrocytes Using Isotopic Transient Metabolic Flux Analysis and 13 C-Labeled Glucose. Frontiers in Neuroenergetics, Volume 3. https://doi.org/
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3 - 2011

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Copyright © 2011 Amaral, Teixeira, Håkonsen, Sonnewald and Alves.

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*Correspondence: Paula M. Alves

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