Blbas Safia, Watson Emma, Butler Heather, Brown Jeremy, Herbert Terence P, Stover Cordula M, Bevington Alan, Abbasian Nima
Department of Respiratory Sciences University of Leicester Leicester UK.
Department of Cardiovascular Sciences University of Leicester Leicester UK.
FASEB Bioadv. 2020 Oct 21;3(1):36-48. doi: 10.1096/fba.2020-00076. eCollection 2021 Jan.
Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na-dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6-G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6-G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor-mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co-inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP-tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome-dependent degradation of this functionally important transporter.
慢性代谢性酸中毒通过增强骨骼肌中的总蛋白水解作用在恶病质中发挥作用。糖皮质激素也会引发蛋白水解，并在酸中毒的作用中起到允许作用。A系统氨基酸转运体SNAT2/SLC38A2在包括肌肉在内的哺乳动物细胞中普遍表达，进行中性氨基酸的钠依赖性主动转运，并且受到低pH的强烈抑制。将大鼠骨骼肌细胞系L6-G8C5暴露于低pH下会迅速抑制SNAT2转运活性并提高总蛋白水解速率。对SNAT2进行药理抑制或沉默也会增强蛋白水解作用。本研究检验了以下假设：糖皮质激素地塞米松（DEX）与低pH一样，会抑制L6-G8C5肌管中的SNAT2活性，从而导致总蛋白水解。用500 nM DEX孵育4小时可使A系统氨基酸转运速率降至对照培养物中速率的一半。这种抑制作用依赖于糖皮质激素受体介导的基因转录，但SNAT2 mRNA水平不受DEX影响。相反，通过免疫印迹评估的SNAT2蛋白明显减少。DEX和低pH对A系统转运活性的共同抑制作用在刺激总蛋白水解方面具有累加性。与此机制一致，蛋白酶体抑制剂MG132显著减弱了DEX对SNAT2转运活性的抑制作用。在使用GFP标记的SNAT2融合蛋白在HEK293A细胞中进行重组表达的类似实验中，获得了原理验证。得出的结论是，DEX会急性消耗SNAT2转运蛋白，至少部分是通过该功能重要转运蛋白的蛋白酶体依赖性降解实现的。
