BackgroundGlucose and glutamine are the two dominant metabolic substrates in cancer cells. In 13C tracer experiments, however, it is necessary to account for all significant input substrates, as some natural (unlabelled) substrate in the medium, often derived from serum, can be metabolised by cells despite not showing signs of net consumption.ResultsUsing [U-13C6]-glucose tracers and measuring extracellular metabolite enrichments by GC-MS, we found that pancreatic cells HPDE and PANC-1 secrete lactate, pyruvate, TCA cycle metabolites and non-essential amino acids synthesised from glucose. Focusing our investigations on pyruvate exchange in HEK293 cells, we observed that the four metabolites pools, intracellular and extracellular lactate and pyruvate, had similar 13C enrichment trajectories. This indicated that these metabolites can mix rapidly. Using a hybrid 13C-MFA, we followed to show that the lactate exchange flux had increased when extracellular lactate concentration was increased by 10-fold. By allowing rapid exchange fluxes around the pyruvate node, 13C-MFA revealed that PANC-1 cells cultured in [U-13C6]-glucose doubled the conversion of unlabelled substrates to pyruvate when treated with TNF-α.ConclusionsThe current work established the possibility that a cell’s range of significant input substrates may be broader than anticipated. Metabolite exchange can affect intracellular enrichments. In particular, we showed that pyruvate was more strongly connected to lactate than to upstream glycolytic intermediates and that a fast lactate exchange may alter the outcome of flux analyses. Nevertheless, the leaky cell model may be an opportunity in disguise—the ability to continuously monitor metabolism using only the enrichments of extracellular metabolites.

中文翻译：

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丙酮酸节点周围的快速交换通量：用于解释示踪剂实验中未标记和标记代谢物的增益和损失的渗漏细胞模型

背景葡萄糖和谷氨酰胺是癌细胞中的两种主要代谢底物。然而，在 13C 示踪剂实验中，有必要考虑所有重要的输入底物，因为培养基中的一些天然（未标记）底物（通常来自血清）可以被细胞代谢，尽管没有显示出净消耗的迹象。结果使用 [U -13C6]-葡萄糖示踪剂并通过 GC-MS 测量细胞外代谢物富集，我们发现胰腺细胞 HPDE 和 PANC-1 分泌乳酸、丙酮酸、TCA 循环代谢物和由葡萄糖合成的非必需氨基酸。专注于我们对 HEK293 细胞中丙酮酸交换的研究，我们观察到四种代谢物库，细胞内和细胞外乳酸和丙酮酸，具有相似的 13C 富集轨迹。这表明这些代谢物可以快速混合。使用混合 13C-MFA，我们发现当细胞外乳酸浓度增加 10 倍时，乳酸交换通量增加。通过允许丙酮酸节点周围的快速交换通量，13C-MFA 表明，当用 TNF-α 处理时，在 [U-13C6]-葡萄糖中培养的 PANC-1 细胞使未标记的底物转化为丙酮酸的转化率加倍。细胞的重要输入底物范围可能比预期的更广。代谢物交换可以影响细胞内富集。特别是，我们发现丙酮酸与乳酸的联系比与上游糖酵解中间体的联系更紧密，并且快速的乳酸交换可能会改变通量分析的结果。尽管如此，