BACKGROUND: Previous work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine-a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction.
METHODS: mTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine.
RESULTS: mTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P < 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P < 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P < 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS.
CONCLUSIONS: Chloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle.
Trial registration NCT00891696. Registered 29 April 2009.
Digital Object Identifier (DOI)
This project is supported by a Grant from NIH R01 AR49877, P30 AG024832, T32 HD07539, NIDRR H133P110012, and in part by a NIH Clinical and Translational Science Award UL1TR001439 from the National Center for Advancing Translational Sciences.
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
Borack, Michael S.; Dickinson, Jared M.; Fry, Christopher S.; Reidy, Paul T.; Markofski, Melissa M.; Deer, Rachel R.; Jennings, Kristofer; Volpi, Elena; and Rasmussen, Blake B., "Effect of the Lysosomotropic Agent Chloroquine on mTORC1 Activation and Protein Synthesis in Human Skeletal Muscle" (2021). Athletic Training and Clinical Nutrition Faculty Publications. 5.