Inflammatory action of certain cytokines can influence cell

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 AH 6809 arrest has been already described in vitro (Chatterjee et al., 2015). On the other hand, MCP-1 is 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. Recently, autophagy has been investigated due to its important action on bone remodeling. Although autophagy is a mechanism of programmed cell death, in basal conditions, it may become an important process for maintaining the skeleton. On the other hand, situations of stress may increase cellular autophagy, due to the activation or inhibition of specific routes. Inhibition of mTOR, for instance, that occurs during deprivation of nutrients (glucose/amino acids) or by the action of Rapa, induces autophagy (Klionsky et al., 2016). Another mechanism involved in the activation of autophagy is the increased endoplasmic reticulum stress, which has been associated with pathological conditions of excessive nutrients, such as obesity. In vivo and in vitro studies indicate that excess energy and nutrients induces endoplasmic reticulum stress in adipose tissue cells, which is sufficient stimulus to promote autophagy (Nuñez et al., 2013, Zhou et al., 2010). In MC3T3-E1 pre-osteoblasts, the increase in autophagy, either by endoplasmic reticulum stress or by the action of drugs, present paradoxical effects that may negatively influence cell proliferation or even induce a protective effect against apoptosis (Wei et al., 2014, Yang et al., 2014). When cells were treated with excess of Leu, we were not able to induce autophagy, although it was possible to identify the antiproliferative effect, since a lower number of cells per field were visible. Images of cells stained with acridine orange obtained by fluorescence microscopy showed that it was possible to identify small red dots on the cells treated with Leu, although we could not find significant differences in the quantification of the percentage of autophagic cells, evaluated by flow cytometry. This result indicates that the decrease in cell proliferation caused by the supplementation with Leu in MC3T3-E1 cells is not caused by increased autophagy. The excess of nutrients has also been related to increased early senescence in several in vitro studies. Investigation of mechanisms that lead cells to senescence process has become a target for possible therapeutic actions, as this mechanism is involved in the proper development of healthy cells and in the control of tumor cell proliferation (Kuilman et al., 2010). Therefore, some studies investigating the action of amino acids, especially Leu, have been conducted in order to clarify these issues. An elegant study (Rachdi et al., 2012) evaluating the effect of Leu supplementation on the regulation of β-cell mass during pancreatic development, has shown that increased Leu consumption by pregnant rats resulted in a hyperglycemic and hypoinsulinemic fetuses with increased body weight, but without an adaptation to β-cell mass. In order to elucidate Leu effect on the regulation of β-cell mass, researchers used an in vitro bioassay that mimics major steps that occur during β-cell development from fetal pancreatic progenitor cells. The assay demonstrated that Leu increased the expression of hypoxia-inducible factor 1-α (HIF-1α), a repressor of the development of NGN3-positive pancreatic endocrine progenitor cells. Studies in tumor liver cells (HepG2) supplemented with Leu also identified an increase of cellular senescence (Nakano et al., 2013, Nishitani et al., 2013). The results presented in our study corroborate the previous findings mentioned above. Leu supplementation was able to promote senescence of pre-osteoblasts, as it was possible to identify in cell images labeled with DAPI and obtained by fluorescence microscopy. Senescent cells, although still viable and metabolically active, lose their proliferative capacity and differentiate from others for its increased nuclei.