Analysis of the stimulative effect of arginine on translation initiation of protein synthesis in skeletal muscle
Authors: Suzuki Daisuke , Takami Yuki , Toyoshima Yuka , Yoshizawa Fumiaki 
Published: May 2024

Introduction: Amino acids are not only important precursors for the synthesis of proteins, but also participate in the regulation of major metabolic pathways. Specific amino acids stimulate protein synthesis. Previous studies have shown that arginine (Arg) stimulates protein synthesis at the level of translation initiation through the activation of the mechanistic target of rapamycin complex 1 (mTORC1) in cell culture system; however, little is known about the effect of Arg on protein synthesis in vivo. Here, we evaluated whether Arg stimulates protein synthesis at the level of translation initiation and compared the effects of Arg on translation initiation with those of leucine (Leu), which is well-studied the detail of its stimulative mechanism, in mouse skeletal muscle. Methods: Eighteen hour-fasted mice were intraperitoneally injected with saline or 4.6 mmol/ kg/ body weight of Arg or Leu and subsequently sacrificed and excised gastrocnemius muscles at 1 h after injection. In addition, we used mouse-derived C2C12 myotubes treated with Arg to examine the mechanism by which Arg stimulates translation initiation in mouse skeletal muscle. The cells were preincubated with several kinds of inhibitors for appropriate time and then treated with Arg or Leu. Using homogenate of skeletal muscle and cell lysate, the phosphorylation states of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1), key regulatory factors involved in the translation initiation were measured by immunoblotting as indicators of translation initiation activity. Results: Intraperitoneal injection of either Arg or Leu significantly increased S6K1 phosphorylation compared to the saline-injection, but the increase in 4E-BP1 phosphorylation was observed in Leu-injected mice not in Arg-injected mice. These results suggest that Arg can stimulate translation initiation in skeletal muscle of fasted mice. In C2C12 myotubes, as with Leu, Arg increased both 4E-BP1 and S6K phosphorylation, and these increases were abolished by the treatment of rapamycin, a mTORC1 inhibitor. These data indicate that Arg directly activates mTORC1 pathway as well as Leu. In addition, the treatment of a phosphoinositide 3-kinase (PI3K) inhibitor and a AKT inhibitor completely cancelled Arg-dependent 4E-BP1 and S6K1 phosphorylation; however, the inhibition of Leu-induced 4E-BP1 and S6K1 phosphorylation by these inhibitors were in partial. Furthermore, the inhibitor of the G protein‐coupled receptor family C, group 6, subtype A (GPRC6a), which has been shown to associate with regulation of PI3K/AKT pathway and mTORC1 activation, completely suppressed the increases of 4E-BP1 and S6K1 phosphorylation by Arg. Conclusion:Our data indicate that Arg has the ability to activate translation initiation in mouse skeletal muscle. In addition, the results of our experiments using skeletal muscle cells support the idea that Arg stimulates translation initiation via GPRC6a/PI3K/AKT/mTORC1 pathway in mouse skeletal muscle. Unlike the effects of Arg, it is likely that besides the PI3K/AKT pathway, Leu activates another signaling pathways upstream of mTORC1 to stimulate translation initiation in skeletal muscle. This research was funded by JSPS KAKENHI, grant number 18K05501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.