Biochemical Engineering Institute, Saarland University, Campus A1.5, D-66123 Saarbrücken, Germany.
J Biosci Bioeng. 2011 Dec;112(6):616-23. doi: 10.1016/j.jbiosc.2011.07.021. Epub 2011 Aug 23.
This study focused on analyzing active pathways and the metabolic flux distribution in human neuronal AGE1.HN cells that is a desirable basis for a rational design and optimization of producing cell lines and production processes for biopharmaceuticals. (13)C-labeling experiments and (13)C metabolic flux analysis were conducted using glucose, glutamine, alanine and lactate tracers in parallel experiments. Connections between cytosolic and mitochondrial metabolite pools were verified, e.g., flux from TCA cycle metabolite (13)C to glycolytic metabolites. It was also found that lactate and alanine are produced from the same pyruvate pool and that consumed alanine is mainly directly metabolized and secreted as lactate. Activity of the pentose phosphate pathway was low being around 2.3% of the glucose uptake flux. This might be compensated in AGE1.HN by high mitochondrial malic enzyme flux producing NADPH. Mitochondrial pyruvate transport was almost zero. Instead pyruvate carbons were channeled via oxaloacetate into the TCA cycle which was mainly fed via α-ketoglutarate and oxaloacetate during the investigated phase. The data indicate that further optimization of this cell line should focus on the improved substrate usage which can be accomplished by an improved connectivity between glycolytic and mitochondrial pyruvate pools or by better control of the substrate uptake.
本研究集中分析了人神经元 AGE1.HN 细胞中的活性途径和代谢通量分布，这为理性设计和优化生物制药的细胞系和生产工艺提供了基础。（13）C 标记实验和（13）C 代谢通量分析是在平行实验中使用葡萄糖、谷氨酰胺、丙氨酸和乳酸示踪剂进行的。细胞溶质和线粒体代谢物池之间的连接得到了验证，例如，三羧酸循环代谢物（13）C 到糖酵解代谢物的通量。还发现乳酸和丙氨酸是从同一个丙酮酸池产生的，消耗的丙氨酸主要是直接代谢并分泌为乳酸。磷酸戊糖途径的活性很低，约占葡萄糖摄取通量的 2.3%。在 AGE1.HN 中，通过高线粒体苹果酸酶通量产生 NADPH 来补偿。线粒体丙酮酸转运几乎为零。相反，丙酮酸碳通过草酰乙酸进入三羧酸循环，在研究阶段，三羧酸循环主要通过α-酮戊二酸和草酰乙酸进料。数据表明，进一步优化该细胞系应侧重于提高底物利用率，可以通过改善糖酵解和线粒体丙酮酸池之间的连通性或更好地控制底物摄取来实现。
