Combining metabolic flux analysis tools and 13C NMR to estimate intracellular fluxes of cultured astrocytes.
In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase.
Teixeira
Ana P
AP
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Quimica e Biológica (IBET/ITQB), Apartado 12, P-2781-901 Oeiras, Portugal.
Santos
Sónia Sá
SS
Carinhas
Nuno
N
Oliveira
Rui
R
Alves
Paula M
PM
D000818:Animals; D001253:Astrocytes / Q000201:enzymology*; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D001923:Brain Chemistry / Q000502:physiology*; D002247:Carbon Isotopes; D002478:Cells, Cultured; D002952:Citric Acid Cycle / Q000502:physiology; D004734:Energy Metabolism / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D009448:Neurochemistry / Q000379:methods; D010085:Oxidative Phosphorylation; D011766:Pyruvate Carboxylase / Q000032:analysis / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
D002247:Carbon Isotopes; D011768:Pyruvate Dehydrogenase Complex; D005973:Glutamine; D018698:Glutamic Acid; D019289:Pyruvic Acid; D005974:Glutamate-Ammonia Ligase; D011766:Pyruvate Carboxylase; D005947:Glucose