An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

Abstract

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1-3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) alpha-subunit (Ser(52))]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser(240/244)) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h (P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2 alpha phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA (P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.

Fig. 1.

Leucine kinetics indicating arterial concentration (A), delivery (B), and transport (C) into the muscle and intracellular concentrations (D) in the basal state (Basal) and 1, 2, and 3 h following essential amino acid (EAA) ingestion. Each time point represents hourly averages as described in materials and methods. *Significantly different from Basal (n = 7, P < 0.05).

Fig. 2.

L-type amino acid transporter [LAT1/SLC7A5 mRNA (A) and protein (C) and CD98/SLC3A2 mRNA (B) and protein (D)] expression in young, healthy subjects in the basal state and 1, 2, and 3 h following EAA ingestion. *Significantly different from Basal (n = 7, P < 0.05).

Fig. 3.

A-type and amino acid transporter [SNAT2/SLC38A2 mRNA (A) and protein (C), PAT1/SLC36A1 mRNA (B), and PAT2/SLC36A2 mRNA (D)] expression in young, healthy subjects in the basal state and 1, 2, and 3 h following EAA ingestion. *Significantly different from Basal (n = 7, P < 0.05).