Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter-2 (SNAT2) cDNA expression in myoblasts.

Timothy Pearson,Oskar Wendowski,Penny P Powell

doi:10.1002/jcsm.12707

Abstract

BackgroundSkeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system‐A sodium coupled neutral amino acid transporter (SNAT)‐2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters.MethodsIn this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis.ResultsIn control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P < 0.05) in myotubes than found in myoblasts. After transfection with a SNAT2‐eGFP cDNA plasmid, C2C12 myoblasts significantly increased perinuclear punctate SNAT2‐eGFP expression that persisted and was more cytoplasmic after differentiation into myotubes. Interestingly, transfected cells were significantly more responsive to the hormone 5α‐dihydrotestosterone (DHT, 4.5 nM, by 1.6×, n = 3 per group, P < 0.04). Starvation significantly enhanced the amino acid C14‐MeAIB transport (1.7×, n = 3 per group, P < 0.05) indicating increased function of SNAT2. Inhibiting SNAT2 with high concentrations of MeAIB (3.3 or 5 mM) significantly reduced C14‐Isoleucine transport by L‐type amino acid transporter (LAT2, 52.8% and 77%, respectively, n = 3 per group, P < 0.05). However, there was no increase in the LAT2 transport of C14‐isoleucine detectable in SNAT2‐eGFP transfected cells after DHT (4.5 nM) exposure. This indicated that small amino acid availability was not rate limiting to LAT2 function in myoblasts.ConclusionsOverall, these data show that transfection of SNAT2‐eGFP expression enhanced its function following starvation and treatment with physiological levels of DHT. Enhanced SNAT2 expression in muscle cells offers a viable epigenetic target in pathological conditions associated with altered amino acid transport.

Highlights

Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body.[1]
Studies of humans indicate that by the age of 70, there is a ~25–30% reduction in the cross-sectional area of skeletal muscle and a decline in muscle strength by ~30–40%.2. They have further shown a decrease in protein synthesis with ageing; the anabolic effect of amino acids is intact in elderly muscle[3] and argues an imbalance between protein synthesis and breakdown that implies that altered amino acid transport into skeletal muscle may be a contributing factor
SNAT2 expression was investigated in low passage myoblast (Figure 1B, top left) cells or in cells differentiated into myotubes for 5 days (Figure 1B, top right)

Summary

Introduction

Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body.[1] Studies of humans indicate that by the age of 70, there is a ~25–30% reduction in the cross-sectional area of skeletal muscle and a decline in muscle strength by ~30–40%.2. They have further shown a decrease in protein synthesis with ageing; the anabolic effect of amino acids is intact in elderly muscle[3] and argues an imbalance between protein synthesis and breakdown that implies that altered amino acid transport into skeletal muscle may be a contributing factor. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis

Methods

Results

Conclusion