BackgroundTwo-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.ResultsBy applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly 13C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in 13C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from 13C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.ConclusionsTaken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.

中文翻译：

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2-羟基戊二酸在结肠直肠癌细胞中不易代谢的证据

背景双羟基戊二酸 (2HG) 作为 L 和 D 对映异构体以低浓度存在于健康哺乳动物细胞中。L 和 D 对映异构体都涉及通过仅部分表征的机制调节细胞生理学。在多种人类癌症中，D 对映异构体由于异柠檬酸脱氢酶 (IDH) 的功能获得性突变而积聚，并被假设通过尚未完全了解的机制驱动恶性肿瘤。虽然很多注意力都集中在确定 2HG 合成的路线上，但尚未详细研究 2HG 的代谢命运。然而，2HG 的代谢可能具有影响细胞功能和癌症发病机制的重要机制后果，例如调节氧化还原电位或产生具有独特作用模式的未知产物。结果通过应用我们基于同位素的代谢组学平台，我们公正而全面地筛选了 IDH1 突变的 HCT116 结直肠癌细胞中的 L- 和 D-2HG 产物。在均匀 13C 标记的 2HG 中孵育 HCT116 细胞 24 小时后，我们使用液相色谱/质谱法跟踪小分子中的标记碳。引人注目的是，我们没有从我们筛选的数千种代谢组学特征中识别出任何 2HG 代谢产物。与这些结果一致，我们没有检测到在添加 L、D 或 2HG 的外消旋混合物后，在 13C 标记的葡萄糖中培养的野生型或 IDH1 突变细胞的三羧酸循环代谢物的标记模式有任何显着变化。一项更灵敏、更有针对性的分析揭示了痕量水平的同位素富集 (< 1%) 在一些来自 13C 标记的 2HG 的中心碳代谢物中。然而，我们发现细胞在 48 小时内不会以高于我们检测限的水平从培养基中消耗 2HG。结论 综上所述，我们得出结论，2HG 碳在 HCT116 细胞系中不容易转化。这些数据表明 2HG 诱导的表型改变不是其代谢产物的结果。