| ObjectiveObserve hyperglucose and hyperinsulin environment impact on osteoblast function,by measuring protein expression of primary osteoblasts,glucose transporter 4(Glut4),osteocalcin(BGP),collagen Ⅰ(Coi Ⅰ),study the effect of glucose metabolism on the function of osteoblasts,so as to explore the mechanism of diabetic osteoporosis,seek new targets and breakthrough for the treatment of diabetic osteoporosis.MethodsIn vitro subculture primary mouse osteoblasts after the separation and extraction,then cells were induced to maturity,the cells were divided into four groups,normal control group(Group N,the glucose concentration in the culture medium for 5.5 mmol/L),the high insulin group(Group Y,insulin concentration in culture medium for 1 umol/L),the high glucose group(Group T,the glucose concentration in the culture medium for 25 mmol/L),high-glucose and high-insulin group(Group D,the glucose concentration for 25 mmol/L and insulin concentrations for 1 umol/L in the culture medium).Induced respectively for 6 h and 24 h,use Western Blot method to detect the expression of Glut4 in each groups,and Elisa method to detect content of BGP、Coi Ⅰ,observe the morphology of osteoblast though salkaline phosphatase staining,and the state of mineralized nodules in extracellular matrix though alizarin red staining.Results1、Compared with the normal control group,Glut4 expression increased in group Y,group T and group D induced by high glucose and high insulin for 6h(P< 0.05),Glut4 expression increased in group Y and decreased in group T for24 h induction(P < 0.05),and there was no difference between group D and group N after 24 h induction.2、Compared with the normal control group,BGP secretion increased in group T and group D in induction for 6h(P < 0.05),In induction for 24 h,BGP secretion increased in group Y and decreased in group T(P < 0.05),and there was no difference between group D and group N.3、Compared with the normal control group,Coi Ⅰ expression of group Y,group T,group D was no significant difference in different induced time.Conclusions1、Under hyperglucose and hyperinsulin induction,the specific functional protein BGP,secreted by osteoblasts,was consistent with the expression of Glut4,which was a key protein in glucose metabolism,Insulin promoted Glut4 expression prior to BGP secretion increasing.Hyperglucose suppressed Glut4 expression and BGP secretion.2、The Hyperglucose and hyperinsulin induction had no an impact on Coi Ⅰsecretion in primary osteoblasts.3、Insulin promoted Glut4 expression and BGP secretion,and improved the inhibitory effect of hyperglucose on the BGP secretion.Insulin promoted bone matrix synthesis and mineralization by improving the glucose metabolism in osteoblasts themselves.From the perspective of cellular energy metabolism,glucose utilization disorder in osteoblasts may partly explain the pathogenesis of diabetic osteoporosis,Glut4,a key protein in glucose metabolism of osteoblasts,was expected to be a new target for the treatment of diabetic osteoporosis. |
