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  • LY3023414 As previously described Leu is able

    2020-08-04

    As previously described, Leu is able to specifically activate the complex 1 of the mammalian target of rapamycin (mTORC1) (Tato et al., 2011). mTOR pathway is activated as a response to hormonal stimuli (insulin), growth factors (IGF-1) or nutrients (glucose and amino acids). On the other hand, this pathway is inhibited through the action of Rapa, an anticancer and antidiabetes agent, known for its potent antiproliferative effect on different types of cells and also upon MC3T3-E1 cells, in which no toxicity with dosages of 0.1 to 20nM was demonstrated. This effect is directly related to the inhibitory effect of the drug upon the mTORC1, which inactivates the PI3K/AKT pathway, blocking the phosphorylation of p70S6K and, consequently, of all cell growth cascade (Singha et al., 2008). Our results showed that the decreased pre-osteoblast cell proliferation induced by Leu supplementation occurred through a mTOR-independent pathway, once the amino LY3023414 could not block the insulin proliferative effect, as shown for Rapa. Results observed in studies with cell culture showed that treatment with Leu for over 20min induces a decrease in the activity of P70S6K, a serine/threonine kinase that indicates the activity of mTOR (Peyrollier et al., 2000). This indicates that in chronic conditions Leu supplementation may not exert the same effect on the activation of mTOR and, as a consequence, on cell proliferation. Likewise, another hypothesis could be related to an over-activation of mTOR (Melnik, 2012b). Leu was not capable to inhibit this pathway, as expected, but increasing evidence supports that this amino acid plays an important role in diseases related to mTOR, such as diabetes and cancer. Maximal mTOR stimulation leads to increased cell proliferation. However, persistently over-stimulated proliferation holds the risk of early senescence (Dor et al., 2004, Teta et al., 2007). Inflammatory markers that could be involved with cell proliferation pathways, as a potential mechanism for the reduction of pre-osteoblasts proliferation, were also investigated. TGF-β1 is increased when mTOR is inhibited by Rapa. Once increased, TGF-β1 is able to stop the cell cycle, impacting negatively on cell proliferation (Chatterjee et al., 2015). In our study, when mTOR was inhibited due to the action of Rapa, a three times increase in TGF-β1 levels in comparison with cells treated with Leu was observed. The inability of Leu to increase TGF-β1 levels strengthen the idea that MC3T3-E1 cell proliferation decrease is not associated to mTOR inhibition. Inflammatory action of certain cytokines can influence cell proliferative routes (Deshmane et al., 2009). When mTOR is inhibited, for instance by the action of Rapa, TGF-β1, which has an anti-inflammatory action, is increased. The relationship between high levels of TGF-β1 and cell cycle arrest has been already described in vitro (Chatterjee et al., 2015). On the other hand, MCP-1 is LY3023414 the monomeric polypeptide member of the CC chemokine superfamily expressed by MC3T3-E1 cells and involved in inflammation and bone remodeling (Takeshita et al., 1993). Data presented here showed no significant differences between groups, although Rapa has presented a tendency to decrease MCP-1.