Faculty of Kinesiology and Physical Education, University of Toronto, Ontario, Canada.
Faculty of Medicine, University of Toronto, Ontario, Canada.
Am J Physiol Cell Physiol. 2022 Jan 1;322(1):C94-C110. doi: 10.1152/ajpcell.00357.2021. Epub 2021 Dec 1.
Following anabolic stimuli (mechanical loading and/or amino acid provision), the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or before translocation (i.e., in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25 g/kg protein and 0.75 g/kg carbohydrate) alone [ = 7; 23 ± 5 yr; 76.8 ± 3.6 kg; and 13.6 ± 3.8% body fat (BF), FED] or following a whole body resistance exercise bout ( = 7; 22 ± 2 yr; 78.1 ± 3.6 kg; and 12.2 ± 4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300 min following anabolic stimuli. RPS6 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated ( = 0.76, < 0.001). Peripheral staining intensity of p-RPS6 increased above PRE in both FED and EXFED at 120 min (∼54% and ∼138%, respectively, < 0.05) but was greater in EXFED at both poststimuli time points ( < 0.05). The peripheral-to-central ratio of p-RPS6 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120 min irrespective of stimulus ( = 0.006) before returning to PRE at 300 min. These data confirm that RPS6 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.
在合成代谢刺激（机械负荷和/或氨基酸供应）后，雷帕霉素复合物 1（mTORC1）作为蛋白质合成的主要调节因子，向细胞外周易位。然而，尚不清楚 mTORC1 介导的磷酸化事件是否发生在这些外周区域，或者在易位之前（即在中央区域）发生。因此，我们旨在确定在合成代谢刺激后，人骨骼肌中 mTORC1 介导的磷酸化事件 RPS6 的细胞位置。14 名年轻、健康的男性要么单独摄入蛋白质-碳水化合物饮料（0.25 克/千克蛋白质和 0.75 克/千克碳水化合物）[=7；23±5 岁；76.8±3.6 千克；和 13.6±3.8%体脂（BF），FED]，要么在全身抗阻运动后摄入[=7；22±2 岁；78.1±3.6 千克；和 12.2±4.9%BF，EXFED]。在休息（PRE）和合成代谢刺激后 120 和 300 分钟时，分别从股外侧肌活检。通过免疫荧光染色或免疫印迹测量 RPS6 磷酸化，结果呈正相关（=0.76， < 0.001）。在 FED 和 EXFED 中，p-RPS6 的外周染色强度在 120 分钟时均高于 PRE（分别增加约 54%和 138%， < 0.05），但在两个刺激后时间点均高于 EXFED（ < 0.05）。即使校正了总 RPS6 分布，p-RPS6 染色的外周-中央比值也表现出相似的模式，表明 RPS6 磷酸化在纤维的外周发生得更为广泛。此外，在 120 分钟时，无论刺激如何，paxillin 阳性区域（粘着斑复合物的标志物）内的 p-RPS6 强度均升高（=0.006），然后在 300 分钟时恢复到 PRE。这些数据证实，在合成代谢刺激后，mTOR 易位到人类肌纤维区域，RPS6 发生磷酸化，并确定粘着斑复合物为体内 mTORC1 调节的潜在部位。
