Programmed cellular death or apoptosis
Programmed cellular death or apoptosis is a process genetically controlled that plays an important role in cellular homeostasis, being an important defense mechanism to remove cells that have been infected, damaged or mutated (Smith and Smith, 2012, Wlodkowic et al., 2011). Nevertheless, apoptosis seems not to be related to cell proliferation decrease after treatment with Leu, considering that there was no statistically significant difference in the percentage of apoptotic cells between groups. Another hypothesis that could possibly explain cell proliferation decrease would be related to an oxidative stress increase. During oxidative stress, cellular production of free radicals and reactive oxygen species exceeds the physiologic capacity of the antioxidant defense system in removing these compounds (Bloomer and Goldfarb, 2004). A greater quantity of amino acids in the culture medium can increase expression of L-type amino Benzoylhypaconitine transporter-1 (LAT-1), responsible for the entry of Leu into the cells. Increased expression of these transporters enhances the influx of this amino acid into cells and, consequently, increases glutamate efflux out of the cell (Kim et al., 2006, Poncet and Taylor, 2013, Taylor, 2014). A lower concentration of intracellular glutamate reduces the production of glutathione, which affects negatively antioxidant cell protection. These events increase oxidative stress and reduce proliferative activity of MC3T3-E1 cells (Arakaki et al., 2013, Uno et al., 2007, Yang et al., 2014). Nonetheless, such hypothesis was not confirmed, considering that Leu was not capable of increasing generation of intracellular ROS and TBARS production, unlike demonstrated for cells treated with H2O2. 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 acid 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.