Abstract
BackgroundAmino acid availability stimulates protein synthesis via the mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. In response to an increase in cellular amino acid availability, translocation of cytosolic mTORC1 to the lysosomal surface is required to stimulate mTORC1 kinase activity. However, research elucidating the amino acid responsive mechanisms have thus far only been conducted in in vitro models.Our primary objective was to determine whether an increase in amino acid availability within human skeletal muscle in vivo would alter the expression of genes associated with amino acid sensing, transport and mTORC1 regulation. Our secondary objective was to determine whether an acute perturbation in lysosomal function would disrupt the normal pattern of muscle amino acid responsive gene expression.MethodsWe recruited 13 young adults into one of two groups: The first group ingested 10 g of essential amino acids (EAA). The second group ingested 10 g of EAA in the presence of chloroquine (CQ), a lysosomotropic agent. The subjects from each group had biopsies of the vastus lateralis taken before and after EAA ingestion. We determined the relative mRNA expression of 51 potential amino acid responsive genes using RT-qPCR.ResultsThere was a differential mRNA expression for 22 genes, with 15 mRNAs significantly changing (P < 0.05) in response to EAA ingestion (e.g., REDD1: +209 ± 35%; SLC38A9: +31 ± 9%; SLC38A10: +57 ± 15%). In the CQ group, EAA ingestion resulted in a differential expression as compared to EAA alone (i.e., 11 out of the 22 genes were different (P < 0.05) between the two groups.)ConclusionsExpression of several amino acid sensing, transport, and mTORC1 regulatory genes in human skeletal muscle are responsive to an increase in amino acid availability. Furthermore, potential acute disruption of lysosomal function by ingestion of chloroquine interferes with the normal pattern of gene expression following feeding. Our in vivo data in humans provide preliminary support for the in vitro work linking amino acid sensing pathways to mTORC1 translocation to the lysosome.Trial RegistrationNCT00891696. Registered 29 April 2009.
Highlights
Amino acid availability stimulates protein synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway
We found that a total of 22 genes related to amino acid sensing, amino acid transport and mTORC1 regulation in human skeletal muscle changed significantly as a result of the ingestion of grams of essential amino acids in the EAA group, with another genes showing a trend (See Additional file 1: Table S2)
Both hypotheses are supported by the data: 1) an increase in amino acid availability in human skeletal muscle increases the expression of genes associated with amino acid sensing, as well as other genes we term as amino acid responsive and 2) administration of chloroquine to alter lysosomal function results in a differential pattern of gene expression
Summary
Amino acid availability stimulates protein synthesis via the mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. Our primary objective was to determine whether an increase in amino acid availability within human skeletal muscle in vivo would alter the expression of genes associated with amino acid sensing, transport and mTORC1 regulation. Several other proteins are involved in controlling mTORC1 translocation to the lysosome and subsequent activation. These other proteins include an amino acid transporter located within the lysosome (SLC38A9), several GTPases (e.g., RagA/B, RagC/D), and scaffolding complexes (e.g., Ragulator) [8,9,10, 15, 16]
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