Michael S Borack1,2,3, Jared M Dickinson2,4,5, Christopher S Fry2,4,6, Paul T Reidy1,2,7, Melissa M Markofski4,8, Rachel R Deer1,4, Kristofer Jennings9,10, Elena Volpi11,4, Blake B Rasmussen12,13. 1. Division of Rehabilitation Sciences, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. 2. Department of Nutrition and Metabolism, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. 3. Center for the Study of Aging and Human Development, Duke University, Durham, NC, USA. 4. Sealy Center On Aging, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. 5. Department of Health Sciences, Central Washington University, Ellensburg, WA, USA. 6. Department of Athletic Training and Clinical Nutrition, University of Kentucky, Lexington, KY, USA. 7. Department of Kinesiology, Nutrition and Health, Miami of Ohio University, Oxford, OH, USA. 8. Department of Health and Human Performance, University of Houston, Houston, TX, USA. 9. Department of Preventive Medicine and Population Health, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. 10. Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, TX, USA. 11. Department of Internal Medicine/Geriatrics, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. 12. Department of Nutrition and Metabolism, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. blrasmus@utmb.edu. 13. Sealy Center On Aging, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-1124, USA. blrasmus@utmb.edu.
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 withchloroquine-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.
RCT Entities:
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.
Entities:
Keywords:
Amino acid sensing; Chloroquine; Muscle protein turnover; mTOR signaling
Authors: Tracy G Anthony; Brent J McDaniel; Peter Knoll; Piyawan Bunpo; Greg L Paul; Margaret A McNurlan Journal: J Nutr Date: 2007-02 Impact factor: 4.798