Su Xue Bessie, Fedeli Valeria, Liu Guizhen, Amma Meike, Boulasiki Paraskevi, Wang Jingyi, Bizzarri Mariano, Jessen Henning, Fiedler Dorothea, Riccio Antonella, Saiardi Adolfo
Laboratory for Molecular Cell Biology, University College London, London, England, United Kingdom of Great Britain and Northern Ireland, WC1E 6BT, GB.
Institute of Organic Chemistry, Albert-Ludwigs-University of Freiburg, Freiburg, Germany, 79104, DE.
Biochem J. 2025 May 13;482(11):675-90. doi: 10.1042/BCJ20253028.
Inositol plays key roles in many cellular processes. Several studies focussed on the quantitative analysis of phosphorylated forms of inositol, enabled by analytical tools developed to detect these highly charged molecules. Direct measurement of free inositol however has been challenging, because the molecule is uncharged and polar. As a result, the mechanisms maintaining the homeostasis of the inositol remains poorly understood. In this study, we overcome these challenges by developing a quantitative liquid chromatography - mass spectrometry (LC-MS) protocol that can resolve and quantify the three main sugar molecules present inside cells: glucose, fructose, and inositol, as well as distinguish the clinically relevant isomers of inositol: myo-, scyllo-, and chiro-inositol. The quantitative power of the new method was validated by accurately monitoring the changes of inositol levels under well-established conditions in Saccharomyces cerevisiae, where the endogenous synthesis of inositol is increased in the transcription repressor OPI1 knockout opi1D and decreased when wild type yeast is fed with exogenous inositol. The method also revealed a new layer of regulation that takes place when exogenous inositol is added to further boost endogenous inositol synthesis in opi1D in a positive feedback loop. Analyses of mammalian cell lines provided many new insights into inositol metabolism. First, different cell lines displayed distinct sugar profiles and inositol concentrations and responded differently to inositol starvation. Second, mammalian cells can synthesize and import scyllo- but not chiro-inositol. Importantly, our method lent direct evidence to the previous hypothesis that lithium treatment could significantly reduce inositol levels in primary cortical neurons, thus diminishing the pool of free inositol available to the phosphoinositide cycle.
肌醇在许多细胞过程中发挥着关键作用。多项研究聚焦于肌醇磷酸化形式的定量分析，这得益于为检测这些高电荷分子而开发的分析工具。然而，游离肌醇的直接测量一直具有挑战性，因为该分子不带电荷且具有极性。因此，维持肌醇稳态的机制仍知之甚少。在本研究中，我们通过开发一种定量液相色谱 - 质谱（LC-MS）方案克服了这些挑战，该方案能够分离和定量细胞内存在的三种主要糖类分子：葡萄糖、果糖和肌醇，还能区分临床上相关的肌醇异构体：肌醇、 scyllo-肌醇和手性肌醇。通过在酿酒酵母中已确立的条件下准确监测肌醇水平的变化，验证了新方法的定量能力。在酿酒酵母中，转录抑制因子OPI1基因敲除的opi1Δ菌株中肌醇的内源性合成增加，而野生型酵母在添加外源性肌醇时肌醇合成减少。该方法还揭示了一层新的调控机制，即在opi1Δ菌株中添加外源性肌醇以通过正反馈回路进一步促进内源性肌醇合成时发生的调控机制。对哺乳动物细胞系的分析为肌醇代谢提供了许多新见解。首先，不同的细胞系表现出不同的糖谱和肌醇浓度，并且对肌醇饥饿的反应不同。其次，哺乳动物细胞可以合成并导入scyllo-肌醇，但不能导入手性肌醇。重要的是，我们的方法为先前的假设提供了直接证据，即锂处理可显著降低原代皮层神经元中的肌醇水平，从而减少可用于磷酸肌醇循环的游离肌醇池。
