PDF(Pubmed)
Abstract：
Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5\'-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators.
摘要：
Torin1，一种靶向哺乳动物雷帕霉素靶蛋白（mTOR）的选择性激酶抑制剂，由于其强大的自噬诱导能力，仍然广泛用于自噬研究，不管它的非特定属性。认识到mTOR抑制对代谢的影响，我们的目标是开发一种可靠且彻底的非靶向代谢组学工作流程,以研究Torin1诱导的小鼠胚胎成纤维细胞(MEF)代谢变化.至关重要的是，我们的质量保证和质量控制（QA/QC）方案旨在通过降低假阳性的可能性来提高对报告结果的信心，包括验证实验，复制从样品制备到数据分析的所有实验步骤。本研究通过使用基于液相色谱-高分辨率质谱（LC-HRMS）的非靶向代谢组学平台，调查了Torin1暴露的代谢指纹。我们的工作流程在Torin1暴露后确定了67种改变的代谢物，结合单变量和多变量统计和验证实验的实施。特别是,细胞内神经酰胺，甘油二酯，磷脂酰胆碱,磷脂酰乙醇胺，谷胱甘肽,和5'-甲硫腺苷下调。溶血磷脂酰胆碱,溶血磷脂酰乙醇胺，甘油磷酸胆碱，甘油三酯，肌苷,和次黄嘌呤上调。进一步的生化途径分析提供了对报告的变化的更深入的见解。最终,我们的研究提供了一个有价值的工作流程，可以为将来研究其他化合物的影响而实施，包括更具体的自噬调节剂。
