| Objective:Osteoporosis has become a high-risk disease in China as a kind of metabolic bone diseases.At present,the prevalence of osteoporosis in females over 50years old is 20.7%and that in males is 14.4%,and the prevalence is still on the rise[1].The incidence of fracture is also increasing year by year,which results in a significant reduction in the life quality of patients[2,3].Therefore,more and more attention has been paid to the pathogenesis of bone metabolic diseases and the methods of intervention and treatment.It is well known that bone tissue is constantly undergoing the process of bone remodeling.In this process,old bone is absorbed and new bone is formed.Bone resorption induced by osteoclasts and bone formation induced by osteoblasts are the key links to maintain the integrity of bone structure and appropriate bone strength in the bone reconstruction cycle[4,5].Osteoblasts are the main functional cells of bone formation.They can synthesize specific bone matrix proteins such as type I collagen,osteocalcin and bone phosphate protein.In the process of osteogenesis,once the osteoblasts are embedded by bone matrix in the surrounding,they become to osteoblasts.Recent study has shown that the energy of osteoblast metabolism mainly comes from glucose metabolism[5,6].Glucose uptake is mainly dependent on glucose transporters.There are three kinds of glucose transporters expressed in osteoblasts:GLUT1(glucose transporter 1),GLUT3(glucose transporter 3)and GLUT4(glucose transporter 4)[6-8].The expression level of GLUT1 in osteoblasts was two orders of magnitude higher than that of other transporters.The glucose uptake rate of GLUT1-/-osteoblasts was about 75%lower than that of the control group,which suggested that GLUT1 is the main transporter of glucose uptake in osteoblasts.Animal experiment has shown that glucose uptake mediated by GLUT1 plays an important role in the differentiation of osteoblasts during growth process.The rats whose osteoblasts tured GLUT1-/-exhibited osteogenesis disorder before birth and osteoporosis and glucose intolerance after birth[6].It needs to be further studied for whether glucose metabolism of osteoblases and bone formation can be improved by adjusting the expression level of activity of GLUT1.Osteocalcin(OC)synthesized by mature osteoblasts is one of the most abundant noncollagen proteins in bone[18].In osteoblasts,after protein translation at the endoplasmic reticulum,osteocalcin undergoes carboxylation at the 17,21 and 24glutamic acid(Glu)residues by?-glutamyl carboxylase.Actually,there is a small amount of osteocalcin containing one or more empty Glu residue(s),which is called undercarboxylated osteocalcin(ucOC).Under acidic conditions,the Gla residues of carboxylated osteocalcin can be decarboxylated and osteocalcin converts to decarboxylated osteocalcin(dcOC)[18].Undercarboxylated osteocalcin and decarboxylated osteocalcin can be called uncarboxylated osteocalcin(Glu OC),which is related to energy metabolism closely[19].Up to now,studies have confirmed that G protein-coupled receptor family C group 6 subtype A(GPRC6A)was the receptor of carboxylated osteocalcin and Glu OC,which participated in the physiological response and glucose metabolism induced by Glu OC[12,13,20].In Vitro,incubation of Glu OC promoted skeletal muscle cells and adipocytes glucose uptake[14,15].Since Glu OC could increase glucose uptake in multiple cells,it is not clear whether dcOC,as a kind of Glu OC,can improve bone formation by increasing glucose uptake by osteoblast-like cells.Previous studies have shown that activation of PI3K/Akt(phosphoinositide3-kinase/protein kinase B)and HIF-1?(hypoxia-inducible factor-1?)signaling pathway was associated with GLUT1-mediated glucose uptake[16,17].Whether dcOC can increase glucose uptake by osteoblast-like cells via PI3K/Akt pathway and HIF-1?is unclear and needs further experimental verification.In our study,decarboxylated osteocalcin was used to intervene MG63 cells(human osteoblast-like sarcoma cells)for a short time and a long time in vitro to observe the changes of glucose uptake,cell proliferation and signal factors that may be involved of MG63 cells.Then decarboxylated osteocalcin was injected into mice to observe its effects on glucose metabolism,bone formation markers,bone mineral density and bone microstructure in mice.We intended to explore the effect of decarboxylated osteocalcin on glucose metabolism of osteoblasts and the possible molecular mechanism,and to provide new intervention targets and research strategies for metabolic osteopathy such as osteoporosis.The paper is divided into three parts,and its main contents are as follows:1.To explore the effect of dcOC on glucose uptake in MG63 cells through GLUT1.2.To explore the role of PI3K/Akt signaling pathway and HIF-1?in regulation of glucose uptake in MG63 cells by dcOC.3.To explore the effect of dcOC on bone formation and microstructure in mice.Methods:1.MG63 cells,HEK-293 cells and Jurkat cells were cultured in vitro.DcOC medium with concentration of 0,0.3,3,10 and 30 ng/ml was prepared on the day before the experiment.Different concentrations of dcOC were used to intervene MG63 cells for1 hour or 72 hours.2.We investigated the expression of GPRC6A in MG63 cells using western blot analysis(HEK-293 cells were used as negative control and Jurkat cells as positive control).3.We investigated the fluorescence intensity of 2-NBDG uptaken by MG63 cells after dcOC intervention using flow cytometry analysis.The optimal concentration of dcOC for intervening glucose uptake in MG63 cells for 1 hour or 72 hours was determined by this method.4.We investigated the expression of GPRC6A,Akt,p-Akt(phospho-protein kinase B),HIF-1?,GLUT1 and Runx2(runt-related transcription factor 2)in MG63 cells after dcOC intervention for 1 hour or 72 hour using western blot analysis.5.We used LY294002 which was the PI3K inhibitor to inhibit the activity of PI3K.DcOC was used to intervene MG63 cells for 1 hour.Then flow cytometry analysis was used to investigate the fluorescence intensity of 2-NBDG uptaken by MG63 cells.Western blot was used to investigate the expression levels of Akt,p-Akt and GLUT1.6.MG63 cells were intervened by dcOC for 72 hours while HIF-1?was silenced by siRNA.Then flow cytometry analysis was used to investigate the fluorescence intensity of 2-NBDG uptaken by MG63 cells.Western blot was used to investigate the expression levels of HIF-1?and GLUT1.7.The effect of dcOC on the proliferation of MG63 cells was detected by Cell counting kit-8(CCK-8)assay.8.The effect of dcOC on ALP activity in MG63 cells was detected by Alkaline phosphatase test kit.9.Kunming mice were divided into three groups and given intraperitoneal injection of dcOC at different doses(0ng/g/d,3ng/g/d,30ng/g/d)for 8 weeks.The mice were weighed before execution.10.The levels of serum calcium,serum phosphorus and fasting plasma glucose in mice were measured by automatic biochemical analyzer.11.The levels of osteoprotegerin(OPG)and procollagen 1 N-terminal propeptide(P1NP)in serum of mice were determined by enzyme-linked immunosorbent assay(ELISA).12.Immunohistochemical assay was used to verify the expression and observe variation tendency of GPRC6A receptor and GLUT1 protein in osteoblasts of mice.13.We used microCT to scan the mice femora to measure cortex bone mineral density(BMD),trabeculae BMD,bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Th.n).Results:1.The result of western blotting assay confirmed that MG63 cells expressed GPRC6A receptor.HEK-293 cells didn't express GPRC6A as the negative control,while Jurkat cells did express GPRC6A as positive control.2.Compared with control group,glucose uptake of MG63 cells was significantly increased following the treatment of dcOC for both 1h and 72h.When data were analyzed for dcOC dose-response effects,the optimum concentration of dcOC to increase glucose uptake in MG63 cells for 1 hour stimulation was 3ng/ml.The optimum concentration of dcOC to increase glucose uptake in MG63 cells for 72hours stimulation was 10ng/ml.3.MG63 cells were treated with dcOC at different concentration for 1 hour.DcOC triggered Akt phosphorylation in a dose-dependent manner.And the most significant concentration of dcOC was 3ng/ml(P<0.01).On the other hand,there was no increase in the expression of the GPRC6A,HIF-1?,GLUT1 and Runx2.4.MG63 cells were treated with dcOC at different concentrations for 72 hours.DcOC increased the protein level of HIF-1?,GLUT1 and Runx2,triggered Akt phosphorylation in the dose-dependent manner.And the most effective concentration of dcOC was 10ng/ml(P<0.01).On the other hand,no increase in the expression levels of the GPRC6A.5.The effect of dcOC on the proliferation of MG63 cells was confirmed by CCK-8assay.Compared with the control group,the proliferation ability of the cells at 24h,48h and 72h after dcOC intervention was significantly increased,which was dose-dependent.At the concentration of 10ng/ml,the effect of dcOC on cell proliferation was the most obvious(P<0.01).6.The result of ALP activity test showed that compared with the control group,the activity of ALP increased significantly after the dcOC intervention of 3ng/ml and10ng/ml for 72 hours(P<0.01).7.LY294002 was used to inhibit the activity of PI3K,and then 3ng/ml dcOC solution was used to intervene MG63 cells for 1 hour.Compared with dcOC group,the effects on increasing Akt phosphorylation and 2-NBDG uptake in LY294002+dcOC group were inhibited(P<0.01).But there was no effect on GLUT1 protein expression in the dcOC group and LY294002+dcOC group(P>0.05).8.The HIF-1?expression of MG63 cells was inhibited by siRNA before MG63 cells were treated with optimal concentration of dcOC(10ng/ml)for 72 hours.Compared with dcOC group,the effects on increasing 2-NBDG uptake and GLUT1 protein expression were disappeared in the HIF-1?siRNA+dcOC group(P<0.01).9.There were no significant differences in body weight,fasting plasma glucose,fasting plasma insulin of mice among the groups after intraperitoneal injection of dcOC for 8 weeks(P>0.05).10.There were no significant differences in serum calcium and phosphorus levels of mice among the groups after intraperitoneal injection of dcOC for 8 weeks(P>0.05).Compared with the control group,serum P1NP level was higher in the HOC group(P<0.05).There was no significant difference in serum OPG level among the three groups(P>0.05).11.The results of immunohistochemistry assay showed definitly that GPRC6A receptor and glucose transporter 1 were expressed in osteoblasts of mice.Neither3ng/g/d nor 30ng/g/d dcOC treatment could change the expression level of GPRC6A in osteoblasts of mice(P>0.05).30ng/g/d dcOC treatment could increased the expression level of GLUT1(P<0.05).12.DcOC had no effect on bone mineral density,trabeculae bone mineral density,BV/TV,trabecular thickness and trabecular number of mice(P>0.05).Conclusions:1.In vitro,short-term dcOC intervention(1 hour)stimulated glucose uptake in MG63cells by activating the PI3K/Akt signaling pathway without insulin intervention,but didn't increase the expression level of GLUT1 protein.Long-term intervention(72hours)can increase the expression of GLUT1 protein in MG63 cells by increasing the expression of HIF-1?in the absence of insulin and normal glucose.In turn,glucose uptake of MG63 cells was promoted.2.DcOC could increase the proliferation activity,ALP activity and cell differentiation of MG63 cells.3.In vivo,intraperitoneal injection of dcOC(30ng/g/d)for 8 weeks increased the expression level of GLUT1 and might increase osteoblast activity of mice. |
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