Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media
Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media
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DOI：
10.1021/bp0702673
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发表时间：
2008-03-01
影响因子：
2.9
通讯作者：
Reichl, Udo
中科院分区：
文献类型：
--
作者：
Sidorenko, Yury;Wahl, Aljoscha;Reichl, Udo
在哺乳动物细胞培养物中，已知由于谷氨酰胺代谢而释放到培养基中的氨和由于葡萄糖不完全氧化而分泌的乳酸盐都基本上抑制细胞的生长。对于某些细胞系，例如杂交瘤细胞，排泄的氨也对产物形成有影响。虽然谷氨酰胺通常被认为是哺乳动物细胞的主要能量来源，但最近发现，各种贴壁细胞系（MDCK、CHO-K1和BHK 21）也可以在无谷氨酰胺的培养基中生长，只要谷氨酰胺被丙酮酸盐取代。在这样的培养基中，氨和乳酸盐释放的水平显著降低。在这项研究中，代谢流分析（MFA）应用于Madin达比犬肾（MDCK）细胞培养在含谷氨酰胺和无谷氨酰胺的培养基。MFA的结果允许进一步研究在贴壁细胞的不同生长阶段期间，用丙酮酸取代谷氨酰胺对MDCK细胞代谢的影响，例如，早期指数期和晚期接触抑制期。丙酮酸似乎直接进入TCA循环，而消耗的大部分葡萄糖以乳酸盐的形式排出。虽然确切的机制目前尚不清楚，但这导致在无谷氨酰胺培养基中细胞代谢所需的葡萄糖摄取减少。此外，消耗的ATP无效循环似乎显着减少时，取代谷氨酰胺丙酮酸。这些发现意味着无谷氨酰胺培养基有利于细胞更有效地利用营养物质。然而，在所考虑的两种培养中，许多代谢通量是相似的，例如，大部分氨基酸的摄取和降解速率或通量通过TCA循环的分支，将β-酮戊二酸转化为苹果酸，其负责线粒体ATP合成。此外，在两种培养物中，细胞生长的比速率大致相同。因此，从含谷氨酰胺到无谷氨酰胺的丙酮酸盐培养基的转换提供了一系列益处，而没有细胞代谢的显著变化。
In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO-K1, and BHK21) can grow as well in glutamine-free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine-containing and glutamine-free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact-inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine-free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine-free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting (x-ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine-containing to glutamine-free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.