Effect of the lysosomotropic agent chloroquine on mTORC1 activation and protein synthesis in human skeletal muscle

Abstract

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.

Keywords: Amino acid sensing; Chloroquine; Muscle protein turnover; mTOR signaling.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Consort flow diagram for study recruitment

Fig. 2

Schematic of randomized experimental protocol. Participants ingested 10 g EAA following biopsy two. The small arrows represent blood draws whereas the large arrows represent biopsies. EAA, essential amino acids. FSR, fractional synthesis rate. FBR, fractional breakdown rate. n = 7 (CON), 7 (CHQ)

Fig. 3

Blood and Muscle Amino Acid Concentrations. Changes from rest in blood Leucine (A), muscle Leucine (B), blood Phenylalanine (C), and muscle Phenylalanine (D) at baseline and following ingestion of a 10 g EAA beverage. Data are mean ± SEM. N = 7 for both treatment groups. “a” different from resting values, P < 0.05. “b” difference between 1 h post and 2 h post, “c” difference between treatment groups, P < 0.05. CON, control. CHQ, chloroquine

Fig. 4

Muscle mTORC1 Signaling. Western blot analyses of mTORC1 (A) and mTOR pathway-related proteins: S6K1 (B), 4EBP1 (C) and rpS6 (D), at baseline and following a 10 g EAA beverage. Data are mean ± SEM. N = 7 for both treatment groups. *Different from pre, P < 0.05. #trend difference from pre, P = 0.07. CON, control. CHQ, chloroquine

Fig. 5

Fractional Synthetic Rate, Fractional Breakdown Rate, and Whole Body Proteolysis. A FSR (vastus lateralis) at baseline and for the two hour period post ingestion of the 10 g EAA beverage. Data are mean ± SEM. N = 7 for both treatment groups. *Different from rest, P < 0.05. #trend difference from pre, P = 0.06. FSR, fractional synthesis rate. CON, control. CHQ, chloroquine. EAA, essential amino acids. B FBR (vastus lateralis) for the 1 h period prior to ingestion of the 10 g EAA beverage. Data are mean ± SEM. N = 7 for both treatment groups. FBR, fractional breakdown rate. CON, control. CHQ, chloroquine. C Whole body proteolysis at baseline and for the two hour period post ingestion of the 10 g EAA beverage. Data are mean ± SEM. N = 7 for both treatment groups. *Different from pre, P < 0.05. CON, control. CHQ, chloroquine

Fig. 6

Myotube mTORC1 signaling and protein synthesis. Cells were starved for 8 h in HEPES-buffered saline. Control was starved for an additional 70 min in fresh HEPES. “Leucine” was starved for 60 min in fresh Hepes and 10 min in fresh Hepes with leucine. “Chloroquine + leucine” was starved in fresh HEPES with chloroquine for 60 min and 10 min in fresh HEPES with leucine. A Phosphorylation of mTORC1 at Ser 2448 in control, leucine (1 mM for 10 min), and chloroquine + leucine (2 mg/ml chloroquine for 60 min followed by 1 mM leucine for 10 min) conditions. Insert shows representative western blot for each condition. B Phosphorylation of S6K1 at Thr 389 (same conditions). Insert shows representative western blot for each condition. Data are mean ± SEM. N = 8 for both treatment groups. *Different from control, P < 0.05. &Different from chloroquine + leucine. Chq, chloroquine. C Western blot analyses of protein synthesis using the SUnSET technique. Nutrient Rich-Control and Chloroquine were serum starved for 16 h and nutrient starved for 1 h (2 mg/ml for Chloroquine group only) prior to 30 min puromycin (1 µM) exposure. Nutrient Starve groups were starved under the same conditions as described above for mTORC1 signaling prior to 30 min puromycin (1 µM) exposure. Data are mean ± SEM, N = 6. abcColumns with uncommon letters differ, P < 0.05