Leucine activation of mTORC1 in skeletal muscle cells is inhibited by the lysosomotropic agent chloroquine (820.2)

Recent work in human embryonic kidney cells has uncovered many of the cellular mechanisms underlying amino acid activation of the mTORC1 signaling pathway. It is now known that mTORC1 translocation to the lysosomal surface is required for the amino acid activation of mTORC1 signaling. It is not known if the lysosome is required for amino acid activation of mTORC1 in skeletal muscle cells. To test this hypothesis, we exposed C2C12 muscle cells (that were plated and differentiated into myotubes for 7‐8 days) to both leucine and a potent lysosomal inhibitor (chloroquine). Cells were placed into three groups: control (1), leucine only (2) and leucine+chloroquine (3). The first two groups were taken out of media and starved in a Hepes buffer for 9hrs. Group 3 was starved for 8hrs followed by a 1hr starve in 2000ug/ml chloroquine in the same Hepes buffer. Groups 2 and 3 were given 1mM leucine for ten minutes before collection. Western blotting was used to determine phosphorylation of mTOR and its downstream targets. The leucine only group showed a significant increase in mTOR and p70s6k phosphorylation compared to control and leucine+chloroquine groups (p<0.05). It appears that disruption of lysosomal function by chloroquine blocks leucine activation of the mTORC1 signaling pathway in skeletal muscle cells. Future work will determine the role of the lysosome for amino acid activation of mTORC1 in vivo. Grant Funding Source : Supported by NIH AR049877