Untargeted metabolomic profiling reveals mTORC1-dependent regulation of amino acid utilization in lymphatic...

The lymphatic vascular system plays essential roles in tissue fluid drainage, dietary fat absorption and transport, and immune cell trafficking. To support these physiological functions, the lymphatic vasculature forms an extensive and highly organized network throughout the body. We have recently discovered that the mechanistic target of rapamycin complex 1 (mTORC1), with RAPTOR as an indispensable component, directs glycolysis and glutaminolysis in lymphatic endothelial cells (LECs) to promote lymphatic vessel formation. However, the role of mTORC1 in regulating LEC metabolism remains incompletely understood. Here, by conducting untargeted metabolomic profiling of control and RAPTOR-deficient LECs, we uncover a global impact of mTORC1 inhibition on amino acid utilization. Specifically, RAPTOR deficiency impairs the conversion of glutamine to glutamic acid, resulting in decreased levels of glutamic acid and aspartic acid, as well as reduced abundance of N-acetyl-glutamic acid and N-acetyl-aspartic acid-two metabolites unexpectedly detected in LECs. Integrated metabolomic and transcriptomic analyses further reveal that impaired glutaminolysis in RAPTOR-depleted LECs is accompanied by an increase in intracellular asparagine, arginine, and metabolites associated with arginine catabolism, potentially driven by upregulation of their respective transporters. In addition, RAPTOR depletion results in abnormal accumulation of branched-chain amino acids (BCAAs) and other essential amino acids primarily involved in protein synthesis. Mechanistically, our data suggest that defective BCAA catabolism and impaired translational control contribute to these metabolic alterations. Collectively, these findings reveal an important role of mTORC1 signaling in coordinating amino acid utilization and suggest that this regulation is critical for lymphatic vessel formation.