| Background:As one of the most serious and challenging health problems in the 21st century, diabetes mellitus affects millions of individuals worldwide. Recently, increasing attention has been paid to the relationship between diabetes and osteoporotic fractures[1]. Many clinical studies have reported that osteoporosis is one of the chronic complications associated with diabetes mellitus[2-4]. A higher risk of fractures was found in both type 1 and type 2 diabetes compared to the non diabetic population[5,6].Moreover, both diabetes and osteoporotic fractures affect a large proportion of older adults and pose a considerable burden on healthcare resources. Therefore, it is more important and urgent to reduce the risk of fractures in diabetic subjects than in non-diabetic counterparts.As an anti-hyperglycemic agent, metformin(Met) is commonly used in the treatment of type 2 diabetes by improving insulin resistance. Metformin could decrease the fracture rate in patients[7] through a direct osteogenic effect on osteoblasts in culture[9]. Recent findings showed that metformin could prevent bone loss in ovariectomized (OVX) rats[10] and inhibit receptor activator of nuclear factorκB ligand (RANKL)-induced osteoclast differentiation in Raw264.7 macrophage cells[11].However, mechanisms underlying the differentiation and function of osteoblast and osteoclast by metformin remain unclear.The osteoclast is a specialized macrophage polykaryon, and its differentiation is principally regulated by macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factorκB ligand (RANKL) and osteoprotegerin (OPG), the latter two cytokines are secreted predominantly by osteoblasts[12,13]. The RANKL, upon binding to receptor activator of nuclear factorκB (RANK) on the cell surface of osteoclasts or its precursors, functions to induce differentiation, activation and survival of osteoclasts, whereas OPG, a decoy receptor for RANKL, functions to inhibit osteoclastogenesis[14,15].Objectives:Considering the important role of OPG/RANKL in osteoclastogenesis regulation and prevention effects of bone loss in OVX rats by metformin, we hypothesized that metformin could regulate osteoclast differentiation by affecting the expression of OPG and RANKL in osteoblasts, as well as to provide futher experimental evidence for exploring the mechanism of metformin prevented bone loss.Methods:We observe the expression of OPG/RANKL mRNA and protein activity in osteoblasts and MC3T3-E1 by RT-PCR, ELISA and Western blotting, and kinds of inhibitors are used to discuss its probable signal transduction pathways. A Raw264.7 cells and osteoblasts co-culture system is used to further verify the above conclusions. Meanwhile, we observe the expression of OPG/RANKL protein activity in OVX rats given metformin.To study the effects of metformin on the cancellous and cortical bone in OVX rats by means of DEXA, histomorphometry and TRAP staining in osteoclasts.Results:1,Effects of metformin on OPG/RANKL mRNA and protein expression in osteoblasts. A,Dose-and time-dependent stimulation of OPG mRNA expression in osteoblasts by metformin.RT-PCR assay:metformin stimulates OPG mRNA expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1 dose-and time-dependently. After 48 h of culture in mouse calvarial osteoblasts, the OPG mRNA expression at 200-800μmol/L metformin was greater than that of controls (P<0.05). At 200μmol/L metformin, the OPG mRNA expression increased greatly (1.31±0.023 of control, P <0.05). At 400 or 800μmol/L metformin, the OPG mRNA expression increased more obviously (1.626±0.172 and 1.749±0.050 of control, respectively, P<0.01). The stimulatory effects of metformin on OPG mRNA expression are similar with that of estradiol (E2) in both cells examined. After 24-72 h in culture with 400μmol/L metformin, the OPG mRNA expression increased several-fold above that seen in controls (P<0.05). After being in culture for 72 h, the OPG mRNA expression increased slightly compared with control (2.03±0.279 of control, P=0.000).B,AMP-activated protein kinase pathway is involved in metformin-induced OPG protein expression in osteoblasts.ELISA detection:metformin induced OPG production in media in a dose-dependent manner in osteoblasts. Treatment with 50-800μmol/L metformin for 48 h caused a significant increase in OPG production compared with controls (P<0.001). At 50μmol/L metformin, the OPG production increased slightly compared with controls (P<0.05). At 200-800μmol/L metformin in culture, the OPG production increased more significantly (P<0.001), which reached a plateau at 800μmol/L. To determine the signal transduction pathways involved in metformin-induced OPG synthesis, various kinds of inhibitors were used. We found that inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, but not MEK1 inhibitor PD98059,the p38 MAP kinase inhibitor SB203580 and the NF-κB inhibitor Bay-117028, suppressed endogenous and metformin-induced OPG secretion in osteoblasts.C,Metformin reduces the RANKL mRNA and protein expression in osteoblasts. It was revealed that mRNA levels of RANKL in osteoblasts are decreased by metformin treatment in a dose-dependent manner. Western blot analysis using RANKL antibody further confirmed that protein levels of RANKL in osteoblasts are reduced by metformin treatment.D,Metformin prevents osteoclast differentiation in vitro. Raw264.7 cells were cultured in the supernatants of calvarial osteoblasts treated with metformin. TRAP-positive multi-nucleated cells (MNCs) containing more than three nuclei were counted as osteoclasts under microscope. We found that metformin reduced the number of TRAP-positive MNCs in a dose-dependent manner.To confirmed the differentiation of osteoclasts in this culture system, we further examined TRAP and cathepsin K mRNA expression in Raw264.7 cells by RT-PCR. It was found that the supernatants of osteoblast increased the TRAP and cathepsin K mRNA expression in a time-dependent manner in Raw264.7 cells (p< 0.05). These findings suggest that metformin may inhibit osteoclast differentiation by regulating cytokines production in osteoblasts.2,Effects of metformin on OPG/RANKL related changes in protein activity in OVX ratsA,Metformin stimulates OPG and suppresses RANKL expression in vivo. The results showed that metformin could increase the serum levels of OPG and decrease RANKL expression in bone marrow cells in metformin-treated OVX ratsB,Metformin prevents bone loss and suppresses osteoclastogenesis in OVX rats Bone mineral density measurement:In the left femur BMD was found significant differences in all groups (P＜0.001). OVX was significantly lower than Sham group (P＜0.001); OVX+Met were significantly higher than OVX group (P<0.001). Von Kossa and TRAP staining:The results of Von Kossa demonstrated the attenuation effect of metformin on bone loss induced by OVX. The normalized cell number of active TRAP-positive osteoclasts in bone resorption pits of proximal tibiae bone was 8.15 folds higher in OVX rats than in Sham rats. But this increase in osteoclastogenesis was markedly reduced following oral treatment of metformin.Conclusions:These results of studies in vivo and in vitro suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, and thereby inhibits osteoclast differentiation and prevents bone loss in OVX rats. |
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