Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double stranded RNA (dsRNA) activated protein kinase R (PKR) has been implicated in skeletal muscle dysfunction. However, pathological roles of PKR in ER stress in muscle are not fully understood. The current study aimed to investigate the effect of imoxin (IMX), a selective PKR inhibitor, on tunicamycin (TN)-induced promotion of ER stress and suppression of insulin signaling in C2C12 myotubes. Cells were pre-treated with 5 uM IMX for 1 hr, and exposed to 0.5 µg/ml TN for 23 hr. A subset of cells was stimulated with 100 nM insulin for the last 15 min. mRNA expression and protein levels involved in ER stress were measured by RT-PCR and Western blotting, respectively. TN significantly augmented PKR phosphorylation by 231%, which was prevented by IMX. In addition, IMX reduced mRNA and protein levels of ER stress-related markers including CCAAT-enhancer-binding protein homologous protein (CHOP, mRNA: 95% decrease; protein: 98% decrease), activating transcription factor 4 (ATF4, mRNA: 69% decrease; protein: 99% decrease), cleavage of ATF6, and spliced X-box binding protein 1 (XBP-1s, mRNA: 88% decrease; protein: 79% decrease) which were induced by TN. Furthermore, IMX ameliorated TN-induced suppression of phospho-insulin receptor beta (317% increase) and Akt phosphorylation (by 36% at Ser473 and 30% at Thr308) in myotubes, while augmenting insulin-stimulated AS160 phosphorylation and glucose uptake (by ~30%). These findings suggest that IMX may protect against TN-induced skeletal muscle ER stress and insulin resistance, which are potentially mediated by PKR.

中文翻译：

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Imoxin 抑制衣霉素诱导的内质网应激并恢复 C2C12 肌管中的胰岛素信号传导。

长期的内质网 (ER) 应激可介导炎症性肌病和胰岛素信号通路。双链 RNA (dsRNA) 激活蛋白激酶 R (PKR) 与骨骼肌功能障碍有关。然而，PKR 在肌肉内质网应激中的病理作用尚不完全清楚。目前的研究旨在研究伊莫辛 (IMX)（一种选择性 PKR 抑制剂）对衣霉素 (TN) 诱导的内质网应激促进和 C2C12 肌管胰岛素信号传导抑制的影响。细胞用 5 uM IMX 预处理 1 小时，并暴露于 0.5 µg/ml TN 23 小时。在最后 15 分钟内用 100 nM 胰岛素刺激细胞亚群。分别通过 RT-PCR 和蛋白质印迹法测量参与 ER 应激的 mRNA 表达和蛋白质水平。TN 显着增强 PKR 磷酸化 231%，这被 IMX 阻止了。此外，IMX 降低了 ER 应激相关标志物的 mRNA 和蛋白质水平，包括 CCAAT 增强子结合蛋白同源蛋白（CHOP，mRNA：减少 95%；蛋白质：减少 98%），激活转录因子 4（ATF4，mRNA：69 % 减少；蛋白质：减少 99%）、ATF6 的切割和剪接的 X-box 结合蛋白 1（XBP-1s，mRNA：减少 88%；蛋白质：减少 79%），这些都是由 TN 诱导的。此外，IMX 改善了肌管中 TN 诱导的磷酸化胰岛素受体 β（增加 317%）和 Akt 磷酸化（Ser473 处 36% 和 Thr308 处 30%）的抑制，同时增强了胰岛素刺激的 AS160 磷酸化和葡萄糖摄取（通过~ 30%）。这些发现表明 IMX 可以防止 TN 诱导的骨骼肌内质网应激和胰岛素抵抗，这可能由 PKR 介导。