A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose.
A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose.
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DOI：
10.3389/fnene.2011.00005
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Alves PM
中科院分区：
文献类型：
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作者：
Amaral AI;Teixeira AP;Håkonsen BI;Sonnewald U;Alves PM
近年来，代谢模型已被用于阐明脑代谢的重要方面。这项工作适用于第一次同位素瞬态13 C代谢通量分析（MFA）的概念，估计在原代培养的星形胶质细胞的细胞内通量。该方法全面探索了13 C标记的底物给药后细胞内代谢物的13 C标记时间过程所提供的信息。将细胞与含有[1- 13 C]葡萄糖的培养基一起孵育24小时，然后通过质谱法和/或高效液相色谱法分析在不同时间点收集的细胞上清液和提取物样品。通过拟合碳标记网络模型来估计代谢通量，以实验确定的同位素分布。该模型能很好地描述糖酵解代谢物库的快速同位素平衡和TCA循环中间体的缓慢标记动力学。通过可逆氨基转移酶反应与TCA循环相关的大量谷氨酸和天冬氨酸可能是观察到的TCA循环中间体平衡延迟的原因。此外，据估计，星形胶质细胞摄取的葡萄糖的11%被转移到戊糖磷酸途径。此外，相当大的流量通过丙酮酸羧化酶[PC; PC/丙酮酸脱氢酶（PDH）的比例= 0.5]，苹果酸酶（总丙酮酸产量的5%），和catalysts的支链氨基酸（占总乙酰辅酶A的40%）证实了这些途径的重要性星形胶质细胞代谢。与维持细胞溶质氧化还原电位的需要一致，通过苹果酸-天冬氨酸穿梭和PDH途径的通量是相当的。最后，估计的谷氨酸/α-酮戊二酸交换速率（约0.7 μmol mg prot−1 h−1）与TCA循环通量相似。总之，这项工作表明了同位素瞬态MFA的能量代谢的综合分析的潜力。
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.