Nutrition is an essential factor that affects bone health. Osteoporosis condition may benefit from nutritional changes. Betaine (BET) is a component of several foods and is an indispensable osmolyte and a source of methyl groups. BET also has an antioxidant activity. Moreover, BET stimulates via Insulin like Growth Factor I (IGF-I) in hepatocytes and skeletal muscle. In particular, our group showed that BET enhances neo myotubes formation and differentiation while promoting IGF-I pathway in C2C12 murine myoblasts. The processes of myogenesis and osteogenesis are characterized by sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. In order to verify this assume the effects of BET were tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible action of BET on hObs differentiation were evaluated by Real-time PCR, Western blot and Immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. Real-time PCR results indicated that BET stimulated significantly the expression of principal bone transcription factors (RUNX2, osterix) and specific proteins (bone sialoprotein and osteopontin). Western blot and Immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. In details, BET induced CaMKII signaling activation and a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels. A significant rise in IGF-I mRNA at 3 h and 6 h and a significant increase of IGF-I protein at 6 h and 24 h after BET stimulus was detected. Furthermore, BET was able to improve significantly both SOD2 gene expression and protein content. In conclusion, our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are promoted by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. These data, within our previous results obtained in muscle cell, suggest that BET could be a promising nutraceutical therapeutic agent in the approach to counteract the senile frailty and related mortality.
Betaine promotes cell differentiation of human osteoblasts in primary culture / P. Senesi, A. Montesano, F. Vacante, I. Villa, A. Ferraretto, A. Spinello, M. Bottani, S. Bolamperti, A. Rubinacci, L. Luzi, I. Terruzzi. ((Intervento presentato al 39. convegno Società Italiana di Endocrinologia tenutosi a Roma nel 2017.
Betaine promotes cell differentiation of human osteoblasts in primary culture
P. Senesi;A. Montesano;A. Ferraretto;M. Bottani;L. Luzi;I. Terruzzi
2017
Abstract
Nutrition is an essential factor that affects bone health. Osteoporosis condition may benefit from nutritional changes. Betaine (BET) is a component of several foods and is an indispensable osmolyte and a source of methyl groups. BET also has an antioxidant activity. Moreover, BET stimulates via Insulin like Growth Factor I (IGF-I) in hepatocytes and skeletal muscle. In particular, our group showed that BET enhances neo myotubes formation and differentiation while promoting IGF-I pathway in C2C12 murine myoblasts. The processes of myogenesis and osteogenesis are characterized by sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. In order to verify this assume the effects of BET were tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible action of BET on hObs differentiation were evaluated by Real-time PCR, Western blot and Immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. Real-time PCR results indicated that BET stimulated significantly the expression of principal bone transcription factors (RUNX2, osterix) and specific proteins (bone sialoprotein and osteopontin). Western blot and Immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. In details, BET induced CaMKII signaling activation and a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels. A significant rise in IGF-I mRNA at 3 h and 6 h and a significant increase of IGF-I protein at 6 h and 24 h after BET stimulus was detected. Furthermore, BET was able to improve significantly both SOD2 gene expression and protein content. In conclusion, our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are promoted by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. These data, within our previous results obtained in muscle cell, suggest that BET could be a promising nutraceutical therapeutic agent in the approach to counteract the senile frailty and related mortality.
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