マウス胎児線維芽細胞におけるオートファジー誘導の研究のための非標的メタボロミクスおよびリピドミクス

Autophagy is a complex self-degradative process that recycles cytoplasmic components through lysosomal degradation, enabling cells to maintain homeostasis during stress and nutrient deprivation. Despite major advances in understanding the basic mechanisms of autophagy, important gaps remain in translating them to human diseases. This study investigated the metabolic fingerprints and footprints of two mechanistically different autophagy inducers, Torin1 (mTOR-dependent) and Tat-Beclin1 (mTOR-independent), in mouse embryonic fibroblasts (MEF). Multi-platform untargeted metabolomics and lipidomics analyses were performed at 3 and 18 h exposure to elucidate both intracellular and extracellular metabolic changes using liquid chromatography-high-resolution mass spectrometry coupled to drift tube ion mobility, complemented by [¹³C]-glucose tracing. Torin1 exposure caused downregulation of TCA cycle intermediates, accumulation of purine degradation products, enhanced phospholipid catabolism, and triglycerides' enrichment. In contrast, Tat-Beclin1 preserved central carbon metabolism, promoted recovery of glutathione levels, and redirected diglycerides toward the biosynthesis of polyunsaturated phosphocholines (PC) and C18-containing phosphoethanolamines (PE). Despite these compound-specific responses, several common alterations were observed, including downregulation of ceramides, upregulation of ether-linked PEs, consistent enrichment of PC O-12:0_16:0, lyso-PE 22:6, PC 16:0_20:4, PC 16:0_22:5, and depletion of PE 32:1, PE 34:2, and PE 38:6, along with secretion of unsaturated fatty acids and uptake of sphingomyelin 35:1;O2 and cytosine from the extracellular compartment. Together, these results show that Torin1 and Tat-Beclin1 trigger distinct yet partly overlapping metabolic programs. The metabolic signatures identified here provide reference profiles for future mechanistic studies and highlight candidate biomarkers that may support early functional evaluation of autophagy modulators in disease-relevant settings.