Activated mTORC1 by eccentric contractions contributes to Induce LAT1 mRNA expression in mouse skeletal muscle
Authors: Takada Hiroaki , Tando Yuki , Nunomiya Aki , Osana Shion , Tsunekawa Haruka , Murakami Taro , Nagatomi Ryoichi 
Published: November 2023

INTRODUCTION

Essential amino acids, especially leucine, induce protein synthesis through activation of the mammalian target of rapamycin complex‐1 (mTORC1) and ATP synthesis in skeletal muscle. The resistance exercise elicits activated mTORC1 and highly demand of leucine on increase of protein composition, ATP consumption by acute muscle contraction. When increased the demand for leucine by muscle contraction, leucine entered intracellular amino acid pool from extracellular amino acid pool. Plasma membrane transport system L, L‐type amino acid transporter 1 (LAT1) is the pathway responsible for importation of large neutral amino acids, such as leucine. LAT1 is a heterodimeric protein complex composed of glycosylated heavy subunit (CD98). LAT1 operates as an obligatory 1:1 heteroexchanger facilitating uptake of leucine in exchange for certain cytoplasmic amino acids accumulated via secondary active transporters (SNAT2) such as glutamine (Murakami et al., 2013, Hundal et al., 2009). Resistance exercise induction protein synthesis through activated mTORC1 and ATP synthesis, but the associate of amino acid transporters is unknown. We hypothesized that amino acid transporters were induced in skeletal muscle highly demand of leucine for increased protein synthesis through activation mTORC1 and ATP synthesis after the eccentric contractions (EC) like resistance exercise. In the present study, we determined whether EC would induce the expression of LAT1, CD98, SNAT2 in mouse skeletal muscle. Furthermore, we determined whether expression of these amino acid transporters would be suppressed by EC on fasted condition and rapamycin‐treated condition for induced inactivation mTORC1.

METHODS

All experiments were performed on male C57BL/6J mice (10 week of age). These mice were divided into free fed groups (n=28) and overnight fasted groups (n=28) (Method: 1) or treated rapamycin groups (n=28) and vehicle groups (n=28) before 3h of EC (Method: 2). The EC by electrical stimulation (ES) was delivered to sciatic nerve of left that was stimulated by the wire electrode with 4V current at a frequency of 100 Hz using a constant current stimulator on anesthetization by isoflurane. The entire protocol lasted 22min and consisted of 10 sets of six stimuli (Gordon et al., 2014). Unilateral eccentric EC of the gastrocnemius muscle of all mice were conducted. After 0, 1, 3, 6, 12, 24 h of the EC protocol, mice were anesthetized with isoflurane, and the gastrocnemius muscle was quickly extracted. Activated mTORC1 levels by EC was measured by phosphorylated 4E‐BP1 and phosphorylated p70 S6K1 (Thr389). To determine the expression levels of amino acid transporters was measured by LAT1 mRNA expression and CD98 mRNA, SNAT2 mRNA expression.

RESULTS

mTORC1 pathway in gastrocnemius muscle was activated by EC under fed condition. However, mTORC1 pathway was not activated by EC under fasted condition and rapamycin‐treated condition. LAT1 mRNA expression was unchanged by EC under fasted condition. LAT1 mRNA expression was increased by EC under fed condition to 179% (P < 0.05) and fasted condition to 197% (P < 0.05) of noncontracting control values. Importantly, the inhibitation of mTORC1 activation induced by rapamycin treatment suppressed the upregulation of LAT1 mRNA by EC of noncontracting control expression values.

CONCLUSIONS

The induction of LAT1 mRNA expression by EC was regulated by mTORC1. However, fasted condition may regulate it through other pathways.