Duan Likun, Cooper Daniel E, Scheidemantle Grace, Locasale Jason W, Kirsch David G, Liu Xiaojing
Department of Molecular and Structural Biochemistry, NC State University, Raleigh, NC, 27695, USA.
Department of Radiation Oncology, Duke University School of Medicine, Durham, NC, 27708, USA.
Cancer Metab. 2022 Jul 7;10(1):11. doi: 10.1186/s40170-022-00287-8.
C tracer analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. The distinct C labeling patterns (e.g., M + 1 species in vivo but not in vitro) of metabolites from [U-C]-glucose or [U-C]-glutamine tracing in vivo and in vitro have been previously reported by multiple groups. However, the reason for the difference in the M + 1 species between in vivo and in vitro experiments remains poorly understood.
We have performed [U-C]-glucose and [U-C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). C enrichment of metabolites in cultured cells and tissues was determined by LC coupled with high-resolution mass spectrometry (LC-HRMS). All p-values are obtained from the Student's t-test two-tailed using GraphPad Prism 8 unless otherwise noted.
We observed distinct enrichment patterns of tricarboxylic acid cycle intermediates in vivo and in vitro. As expected, citrate M + 2 or M + 4 was the dominant mass isotopologue in vitro. However, citrate M + 1 was unexpectedly the dominant isotopologue in mice receiving [U-C]-glucose or [U-C]-glutamine infusion, but not in cultured cells. Our results are consistent with a model where the difference in M + 1 species is due to the different sources of CO in vivo and in vitro, which was largely overlooked in the past. In addition, a time course study shows the generation of high abundance citrate M + 1 in plasma of mice as early as few minutes after [U-C]-glucose infusion.
Altogether, our results show that recycling of endogenous CO is substantial in vivo. The production and recycling of CO from the decarboxylation of [U-C]-glucose or [U-C]-glutamine is negligible in vitro partially due to dilution by the exogenous HCO/CO source, but in vivo incorporation of endogenous CO into M + 1 metabolites is substantial and should be considered. These findings provide a new paradigm to understand carbon atom transformations in vivo and should be taken into account when developing mathematical models to better reflect carbon flux.
碳示踪分析越来越多地用于监测体内和完整细胞中的细胞代谢，但数据解读仍然是揭示代谢活动复杂性的关键因素。多个研究小组此前已报道了体内和体外[U-¹³C]-葡萄糖或[U-¹³C]-谷氨酰胺示踪代谢物的不同碳标记模式（例如，体内存在M + 1物种而体外不存在）。然而，体内和体外实验中M + 1物种差异的原因仍知之甚少。
我们在荷肉瘤小鼠（体内）和癌细胞系（体外）中进行了[U-¹³C]-葡萄糖和[U-¹³C]-谷氨酰胺示踪。通过液相色谱与高分辨率质谱联用（LC-HRMS）测定培养细胞和组织中代谢物的¹³C富集情况。除非另有说明，所有p值均使用GraphPad Prism 8通过双侧学生t检验获得。
我们观察到体内和体外三羧酸循环中间体的不同富集模式。正如预期的那样，柠檬酸M + 2或M + 4是体外的主要质量同位素异构体。然而，柠檬酸M + 1出乎意料地是接受[U-¹³C]-葡萄糖或[U-¹³C]-谷氨酰胺输注的小鼠中的主要同位素异构体，但在培养细胞中并非如此。我们的结果与一个模型一致，即M + 1物种的差异是由于体内和体外CO的来源不同，而这在过去很大程度上被忽视了。此外，一项时间进程研究表明，在[U-¹³C]-葡萄糖输注后几分钟内，小鼠血浆中就会产生高丰度的柠檬酸M + 1。
总之，我们的结果表明内源性CO在体内的循环相当可观。[U-¹³C]-葡萄糖或[U-¹³C]-谷氨酰胺脱羧产生的CO及其循环在体外可忽略不计，部分原因是被外源性HCO₃⁻/CO₂源稀释，但内源性CO在体内掺入M + 1代谢物中是相当可观的，应予以考虑。这些发现为理解体内碳原子转化提供了一个新的范例，在开发数学模型以更好地反映碳通量时应予以考虑。
