Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, UK.
J Cachexia Sarcopenia Muscle. 2021 Jun;12(3):811-822. doi: 10.1002/jcsm.12707. Epub 2021 May 13.
Skeletal 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.
In 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.
In 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 C -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 C -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 C -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.
Overall, 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.
骨骼肌的质量和功能部分依赖于氨基酸的供应，改变氨基酸的转运是导致虚弱的一个重要原因，虚弱会随着年龄的增长导致独立性下降，并减缓创伤的恢复。由 SLC38A2 基因家族编码的系统 A 钠偶联中性氨基酸转运体（SNAT）-2 产生一个 506 个氨基酸的 56 kDa 蛋白，是骨骼肌中氨基酸的重要转运体。衰老与 SNAT2 转运体的表达减少有关。
在这项研究中，我们使用 C2C12 细胞系，使用成肌细胞和分化为肌管的细胞。我们研究了 SNAT2 DNA 的表达是否会增强细胞内氨基酸水平，并增加其用于蛋白质合成的可用性。
在对照成肌细胞和肌管中，我们发现 SNAT2 的表达明显低于肌管中的成肌细胞（每组减少 6.5 倍，n = 4，P < 0.05）。用 SNAT2-eGFP cDNA 质粒转染后，C2C12 成肌细胞的核周点状 SNAT2-eGFP 表达明显增加，在分化为肌管后仍然存在，并且更多地分布在细胞质中。有趣的是，转染细胞对激素 5α-二氢睾酮（DHT，4.5 nM，增加 1.6 倍，n = 3 个/组，P < 0.04）的反应明显更敏感。饥饿显著增强了 C-MeAIB 的氨基酸转运（增加 1.7 倍，n = 3 个/组，P < 0.05），表明 SNAT2 的功能增强。用高浓度 MeAIB（3.3 或 5 mM）抑制 SNAT2 显著减少 L 型氨基酸转运体（LAT2）对 C-异亮氨酸的转运（分别减少 52.8%和 77%，n = 3 个/组，P < 0.05）。然而，在 DHT（4.5 nM）暴露后，在 SNAT2-eGFP 转染细胞中没有检测到 C-异亮氨酸的 LAT2 转运增加。这表明在成肌细胞中，小氨基酸的可用性不是 LAT2 功能的限速因素。
总的来说，这些数据表明，SNAT2-eGFP 表达的转染增强了饥饿和生理水平的 DHT 处理后其功能。增强肌肉细胞中的 SNAT2 表达为与氨基酸转运改变相关的病理条件下提供了一个可行的表观遗传靶标。
