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  • br Materials and methods br Results br Conclusion br Conflic


    Materials and methods
    Conflict of interest
    Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFD0400302) and General Program of National Natural Science Foundation of China (31471602).
    Introduction A significant number of studies have evidenced the importance of nutrients in the etiology of chronic diseases related to different organs and human body systems (Michaelsen et al., 2012, Um et al., 2006, Xu et al., 2013, Yang and Huffman, 2013). Some studies indicate that the increased prevalence of these diseases is related to the excess of proteins obtained from dairy products or dietary supplementation with soluble milk proteins (whey protein), which are frequently present in modern diet. These foods and supplements are rich in Leucine (Leu), a branched-chain amino p2y receptor (BCAA) that acts on growth regulation, protein biosynthesis and cellular metabolism (Melnik, 2012a). Thus, Leu has been gaining importance for playing a key role in signaling metabolic pathways that are nutrient-sensitive, such as mTOR (mammalian target of rapamycin) (Tato et al., 2011, Torres-Leal et al., 2010). However, studies show that the excess of Leu may cause a superactivation of this pathway, leading to the senescence of β-pancreatic cells, excess of adipose tissue and disordered protein synthesis. These effects, observed in vitro and in vivo, mimic clinical metabolic alterations related to insulin signaling and to cellular growth, which in humans can lead to both type 2 diabetes mellitus and cancer (Nicastro et al., 2012, Rachdi et al., 2012, Tremblay et al., 2005). The Leu effects have been thoroughly studied in several types of cells. Leu has many functions, such as stimulating insulin release and regulating gene expression and protein synthesis. Studies in vitro and in vivo have shown that Leu is capable to stimulate p2y receptor proliferation and differentiation of liver, muscle, pancreatic and adipose cells (Melnik, 2012b). However, most studies have analyzed the effect of Leu under normal concentrations and supplemented culture medium, so that the effects of its excess are still unclear (Chong and Maiese, 2012, Nicastro et al., 2012, Rachdi et al., 2012, Tremblay et al., 2005). Therefore, it is relevant to analyze the effects of this nutrient on bone cells, considering that Leu is the main amino acid present in dairy products, which constitutes an important group of food related to bone health (Conigrave et al., 2008, Pereira, 2014). Beside this, surprisingly, there is limited evidence about the effects of Leu supplementation on bone tissue cells. In this way, studies in vitro using cell lineages, such as MC3T3-E1 cells, are useful to understand the underlying mechanisms of this process (Sudo et al., 1983). Thus, considering the role of Leu as an important nutrient for cell growth and due to its unclear effect on bone tissue, the present study aimed to analyze the effects of Leu supplementation on the proliferation of pre-osteoblasts MC3T3-E1 cells in vitro.
    Materials and methods
    Discussion In this study, we have showed that Leu supplementation reduces proliferation of MC3T3-E1 pre-osteoblast cells. The initial hypothesis of the study, that Leu would increase cell proliferation through the activation of mTOR (Chong and Maiese, 2012, Luo et al., 2005, Melnik, 2012a) was not confirmed. On the other hand, the antiproliferative effect observed was seen exclusively in cells treated with Leu, indicating that this amino acid is involved in some specific mechanism of cell growth inhibition. Aiming to identify the potential causes of the decreased cell proliferation, we have investigated toxicity levels of Leu supplementation on these cells, as well as apoptosis percentage and cell damage caused by oxidative stress. In order to assess cellular necrosis through cytotoxicity, we have evaluated cellular release of LDH into the supernatant. We have observed that the dose of 200μM (which represents >50% above normal levels) increased significantly the concentration of this enzyme in the supernatant, indicating that at doses of 50μM and 100μM, the antiproliferative effect was not associated to cytotoxicity. It is possible that the cytotoxic effect observed at 200μM is associated to a decrease of pH in the culture medium. Acidification of the culture medium, caused by excess of amino acids protons (hydrogen ions), can reduce osteoblast proliferation, differentiation and mineralization. This effect is related to an attempt of cells to buffer the acid pH, releasing minerals such as sodium, potassium, bicarbonate and calcium into the culture medium, which play an important role in these processes (Nicoll and Howard, 2014). On the other hand, a previous study has shown that when pre-osteoblasts differentiate, proliferation decreases. However, the differentiation of these cells in culture only occurs after a period of 10 to 14days (Barbara et al., 2004). It is important to highlight that experiments in the present study were performed in a period of 48h.