Effects of insulin on osteoblast and its post-receptor mechanism

AIM:To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS:The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P44/42MAPK were determined by Western blotting analysis.RESULTS:Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10-7 mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10-7 mol/L.When the insulin concentration beyond 10-7 mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10-6 mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group (P<0.05),but there was no statistically significant difference among insulin-stimulated groups (P>0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P44/42MAPK.The degree of tyrosine phosphorylation of P44/42MAPK was increased step by step along with the increasing doses of insulin from 0 to 10-7 mol/L (P<0.05,between groups).After insulin chronicity treatment,there was a marked reduction in the tyrosine phosphorylation of P44/42 MAPK (P<0.05,between groups).There was no significant change in protein level of P44/42MAPK.CONCLUSIONS:Insulin enhances the proliferation of osteoblasts as a growth factor at a suitable concentration,but this effect disappears at chronic high insulin stimulation.The MAPK may be involved in the proliferating effect of insulin on osteoblasts.Transient stimulation of insulin activates the P44/42MAPK,however chronic high insulin stimulation results in down-regulation of P44/42MAPK signal activity.