ИСТИНА

INTRODUCTION: IGF1 plays an important role in the regulation of connective tissue, bone and muscle homeostasis in adults. Experiments in rodents demonstrated that leucine (Leu) intake increases the systemic IGF1 level (Teodoro et al., 2012; Pedrosa et al., 2013; Pedroso et al., 2014). Studies in HeLa, HEK293T and COS7 cells revealed an obligate two-step mechanism of mTORC1 regulation: pre-activation (priming) by arginine (Arg) and then activation by Leu (Dyachok et al., 2016). The aim of our research was to investigate effect of Leu on expression of IGF1 and IGF-1-dependent genes in human myoblasts. We have assumed that Leu may regulate the gene expression in human myotubes, and this effect may be improved by pre-activation with Arg. METHODS: After starvation (1 h), myotubes were incubated with Arg (0,4 mM, 30 min), or Leu (0,8 mM, 30 min), or Arg and then Leu. The 4E-BP1Thr37/46 and S6K1Thr389 phosphorylation (targets of mTORC1) was evaluated by Western blot. In the second experiment, after starvation (1 h) myotubes were incubated with Leu (0,8 mM, 24 h), or Arg (0,4 mM, 24 h) and then Leu (24 h). Expression of IGF1, COL1A1, COL3A1, COL5A1, SREBF1, HMGCR, LOX genes was measured by qPCR. RESULTS: Arg did not affect the S6K1Thr389 or 4E-BP1Thr37/46 phosphorylation, whereas Leu increased proteins phosphorylation (10-fold and 3,5-fold, respectively), but the most significant effect was related with consecutive Arg-Leu stimulation. Leu had little effect on the IGF1 and IGF1-dependent genes expression. The consecutive Arg-Leu stimulation increased expression of IGF-1Ea, MGF, COL1A1, COL5A1 and SREBF1 (3-fold, 5,8-fold, 3,6-fold, 1,4-fold, and 1,4-fold, respectively). CONCLUSION: Two-step mTORC1 activation by Arg and Leu in human myoblasts was shown. The same effect for leucine-stimulated gene expression was found: the consecutive Arg-Leu stimulation leads to the most significant activation of genes expression. It means that expression of IGF1 and IGF1-dependent genes is regulated by amino acids via a two-step mechanism. The study was supported by the Science research program of RAS presidium «Fundamental research for biomedical technologies» for 2018-2020 yy.