Essential amino acid ingestion alters expression of genes associated with amino acid sensing, transport, and mTORC1 regulation in human skeletal muscle

Abstract

Background: Amino 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.

Methods: We 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.

Results: There 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.).

Conclusions: Expression 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 registration: NCT00891696. Registered 29 April 2009.

Keywords: Chloroquine; Lysosome; Muscle; Protein synthesis; mTORC1.

Figures

Fig. 1

Amino Acid Transporters. SLC38A10 and SLC38A9 are both sodium-coupled neutral amino acid transporters, with SLC38A9 implicated in activating RAGULATOR and hence mTORC1 and SLC38A10 having an unknown function. SLC7A1 is a cationic transporter (e.g., arginine) and SLC7A8 codes for the LAT2 light subunit. x-axis: EAA = essential amino acid only group, CQ = essential amino acid plus chloroquine group, mean = mean of group ± standard error; y-axis units is fold change in gene expression from the basal to fed state using the 2-∆∆Ct method; bar indicates independent t-test comparison between EAA and CQ, symbols over means indicate paired t-test comparison of fold change with normalized baseline; * = p ≤ 0.05, ** = p ≤ 0.01, # = p < 0.10, NSD or no label = p > 0.10; the female subjects (n = 3 in CQ) are delineated by darker data points

Fig. 2

GATOR Complex. a GATOR1 is a negative regulator of mTORC1, acting as a GAP towards Rag A/B. b GATOR2 is a positive regulator of mTORC1, acting as a negative regulator of GATOR1. x-axis: EAA = essential amino acid only group, CQ = essential amino acid plus chloroquine group, mean = mean of group ± standard error; y-axis units is fold change in gene expression from the basal to fed state using the 2-∆∆Ct method; bar indicates independent t-test comparison between EAA and CQ, symbols over means indicate paired t-test comparison of fold change with normalized baseline; * = p ≤ 0.05, ** = p ≤ 0.01, # = p < 0.10, NSD or no label = p > 0.10; the female subjects (n = 3 in CQ) are delineated by darker data points

Fig. 3

a TSC Complex. The TSC complex has 3 subunits, TSC1, TSC2 and TBC1D7. TSC1 increased in EAA, but TSC2 and TBC1D7 decreased in CQ. The TSC complex is a negative regulator of mTORC1, acting as a negative regulator of Rheb. b REDD1 and REDD2. REDD1 and REDD2 have the same function, negatively regulating mTORC1 through the TSC complex. The mRNAs coding for REDD1 (DDIT4) and for REDD2 (DDIT4L) changed in an opposite manner. x-axis: EAA = essential amino acid only group, CQ = essential amino acid plus chloroquine group, mean = mean of group ± standard error; y-axis units is fold change in gene expression from the basal to fed state using the 2-∆∆Ct method; bar indicates independent t-test comparison of EAA and CQ, symbols over means indicate paired t-test comparison of fold change with normalized baseline; * = p ≤ 0.05, ** = p ≤ 0.01, # = p < 0.10, NSD or no label = p > 0.10; the female subjects (n = 3 in CQ) are delineated by darker data points

Fig. 4

Simplified Schematic of mTORC1 Regulation. In the post-prandial state an increase in amino acid and insulin availability results in Akt phosphorylation by PDK1 at serine 308, and Redd1/2 and the TSC complex are inhibited. Under these conditions, Rheb is active at the lysosomal membrane and can, in turn, activate mTORC1 kinase activity. With sufficient amino acids in the cell, the Rag heterodimer is recruited to the lysosome where it interacts with Rheb to increase mTORC1 kinase activity. This results in increased protein synthesis and inhibition of autophagy via phosphorylation of S6K1 and 4E-BP1, and ULK1 and TFEB, respectively. In the current study, EAA ingestion in the group that did not receive chloroquine, we found that the following proteins/protein complexes depicted in the schematic had increased mRNA expression (p < 0.05 or p < 0.10): SNAT9, CAT1, LAT2, PAT1, SESTRIN2, REDD1, TSC-TSC2 complex, Ragulator, GATOR1 and GATOR2, PI3K, mTORC1, and TFEB. For a complete listing of the genes and how the conditions of the study changed mRNA expression, refer to Additional file 1: Tables S1 and S2 in the supplement. AA = amino acids, LEU = leucine, ARG = arginine, GLN = glutamine, GAP = GTPase activating protein, GEF = guanine exchange factor, GTP = guanine triphosphate, GDP = guanine diphosphate. H + =hydrogen ion (proton), O2 = oxygen. Text within shapes = protein or subunit names. Dotted lines and arrows indicate repressed functions under the stated conditions