Abstract

Abstract Objectives Bone marrow mesenchymal stem cells are the precursor cells for adipocytes and osteoblasts. The accumulation of adipocytes in bone marrow at the expense of osteoblastogenesis is a major factor contributing to age-related bone loss. Grapefruit flavonoid naringenin (NAR) was shown to promote osteogenesis but the mechanism behind these effects is not clear. In this study, we propose to demonstrate that the osteogenic effects of NAR are mediated through the activation of adenosine monophosphate-activated kinase (AMPK) pathway. Methods MC3T3-E1 pre-osteoblast cells were cultured using alpha MEM medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Osteogenic differentiation was induced by using differentiation medium purchased from Stem Cell Technologies. Western blotting, ELISA and alizarin red assays were conducted to test our hypothesis. Results NAR increased calcium deposition in MC3T3 cells compared to DMSO control after 21-day differentiation period. Additionally, in the presence of AMPK inhibitor Ara-A, NAR-induced calcium deposition was suppressed compared to NAR alone. Furthermore, while NAR increased phosphorylation of AMPK, the combination of NAR and Ara-A significantly decreased AMPK phosphorylation. Finally, Ara-A suppressed NAR-induced expression of osteogenic markers including Runx2, osteoprotegerin and osteocalcin suggesting that NAR-induced osteogenesis is partly mediated through AMPK pathway. All data were expressed as the mean ± SEM, minimum n = 3. Comparisons were made by using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. Conclusions The suppression of osteogenic markers and calcium deposition in the presence of NAR plus Ara-A, an AMPK inhibitor, compared to NAR alone provide evidence that the observed osteogenic effects of NAR are in part mediated through the activation of AMPK pathway. These data combined with our previous reports of anti-adipogenic effects of NAR suggest that NAR may have potential therapeutic applications for osteoporosis by not only promoting osteogenesis but also inhibiting adipogenesis in the bone marrow. Funding Sources Division of Research, PCOM and NIH-1R03 AI128254–01A1.

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