Glutamine supplementation stimulates RANKL-induced osteoclast generation and diminishes apoptosis in RAW 267.4 macrophages

F SCOTTO, V SANTOS, C SOUSA, M CASTRO, E GROTZ, C PARRADO,; ABRAHAM MF, AC PARRADO, N SACCODOSSI, A RUGNA, E REY, MT GENTILE, A CANELLADA; T GENTILE, ; S MENEZES FREIRE, A NEY FREIRE; AM CANELLADA

Congreso; XXXIX Congress of the Brazilian Society of Immunology. Immuno Buzios 2014; 2014

INTRODUCTION: Osteoclast (OC) is a giant multinucleate cell that resorbs calcified matrix during bone formation and remodeling. Molecular mechanisms underlying metabolic adaptation to the energetic demands of osteoclastic bone resorption remain poorly understood. Murine macrophage RAW 264.7 cells have been widely used as an in vitro model of OC differentiation. Cytokines produced by activated immune cells, as RANKL, TNF-a, IL-17, induce OC differentiation. Glutamine (Gln) is a nonessential amino acid with antioxidant capacity that maintains normal immunological function under stress or pathological conditions. It was demonstrated that the RANKL-mediated osteoclastogenesis transiently generate reactive oxygen species that can trigger apoptosis. OBJECTIVE: To evaluate the effect of Gln on the OC differentiation of RAW cells in vitro. METHODS: RAW cells were cultured during 5 days with RANKL (0 or 50 nM) and Gln (0-2-4-7 mM), or in the presence or absence of supernatants of Balb/c mouse spleen cells that had been previously cultured during 24 h with Gln (2-4-7 mM) and concanavalin A (ConA, 0 or 5ug/mL). OCs were measured by Tartrate Resistant Acid Phosphatase (TRAP) expression and matrix metaloprotease activity (MMP), determined by cytochemistry and gel zymography respectively. Apoptosis was evaluated in RAW cells by analysis of DNA integrity in agarose gels, and by tunel assay. RESULTS: We found that in the absence of Gln in the culture media, OCs generation did not occur in response to RANKL. In the presence of Gln (2mM), RANKL increased 2 fold the osteoclast differentiation compared to control unstimulated cells (p minor 0.05). Raising Gln concentration to 4mM increased 23 % the RANKL-induced OC differentiation compared to Gln 2mM. Supernatants of spleen cells cultured with ConA and 7 mM Gln induced 2 fold OC differentiation of RAW cells compared to control supernatants (spleen cells cultured with ConA and 2mM Gln). In order to investigate whether the effect of Gln could be related to a inhibition of cell death, apoptosis was induced in RAW cells by serum starvation. We found that Gln 4 and 7 mM diminished 487% (p minor 0,001) the apoptosis of RAW cells. CONCLUSION: Gln supplementation improved the generation of OCs in response to RANKL, and diminished apoptosis of RAW cells. Whether the effect of Gln on OCs differentiation is related to a sequential decrease in ROS generation and apotosis of RAW cells induced by RANKL, deserves further investigation.