Roles For Insulin And Insulin Receptor In The Mechanism Underlying The Process Of Skeletal Fluorosis

Objectives:The involvement of different form of osteocalcin in a bone-pancreas loop hasbeen demonstrated by previous studies and gives rise to concerns on the crosstalk andinteraction between bone and pancreas, which results in the role of insulin in themetabolism bone disease was noted again. Our previous study demonstrates the activebone formation and bone resorption in the pathogenesis of skeletal fluorosis as onekind of metabolism bone disease. The active commitment, differentiation and matureof osteoblast play a key part in the occurrence and development of skeletal fluorosis.The aim of thesis is to investigate a link between insulin level and characteristicchanges of osteopathology induced by excessive fluoride, and clarify the role ofinsulin in the osteopathology in vivo and stimulation of osteoblastic function in vitroinduced by fluoride.Methods and results:Rats were treated with fluoride by gavage two months to develop fluorosisanimal model. After one month’s period, the rats received Streptozotocin (STZ)through intraperitoneal injection for once to inhibit the secretion of insulin. The resultshowed that dental fluorosis was obvious of the fluoride-treated rats. And dentalfluorosis changed from the translucent streaks to chalky with the increase of fluoridedose. The teeth broken of fluoride-treated rats with STZ was more obvious thanfluoride-treated rats, and the group of high-dose fluoride showed teeth defect andfracture. The insulin, insulin sensitivity, blood glucose and HbA1C level wasanalyzed by immunohistochemistry, the results showed that fluorine couldstimulate the secretion of insulin, but the changed of blood glucose and HbA1C levelwere not obvious. The insulin level of fluoride-treated rats with STZ was increased inlow-dose fluoride group, which was not decreased in high-dose fluoride group. WhileSTZ treated rats could influence the hypoglycemic function of insulin. Insulin sensitivity experiment could reflect the insulin effectiveness of the body. We couldsee that low-dose of fluoride and co-administered with STZ could cause bloodglucose level backed to normal delayed from the trend of the change of bloodglucose, which probably was attributed to that a certain dose of fluoride could reducethe sensitivity of insulin and even caused insulin resistance. The insulin and theosteocalcin receptor GPRC6A expression in pancreas was analyzed byimmunohistochemistry. The results showed that fluoride-treated stimulated insulinsecretion, insulin secretion capacity of high fluoride group rats increased moresignificantly. STZ treated rats solely inhibited insulin secretion, and fluoride togetherwith STZ treated rats still stimulated the expression of insulin. The osteocalcin andbone density levels analysis showed that bone density decreased more obvious withthe increment of fluoride. The treated with STZ decreased bone mineral density andthe bone mineral density decreased more obvious when increased the fluoride. Therats of fluoride-treated solely induced the protein expression of GPRC6A, but thefluoride-treated rats with STZ decreased the receptor expression. Osteocalcinmoderately elevated under the condition of high fluoride, but the change ofosteocalcin was not significant between groups after combined with STZ treatment.Consideration of the relationship between the OCN and GPRC6A, fluoride couldstimulate GPRC6A expression lightly and improve the osteocalcin content in theblood. Observed from the front, the fluorine significantly stimulated insulinsecretion.At the same time, the STZ combined with fluoride treatment decreasedinsulin sensitivity and influenced the GPRC6A expressed in islet which leaded todecline. The quantitative analysis of osteogenesis and osteoclastogenesis relatedgenes expression in fluoride-treated rats with or without STZ through real-time PCR.The result showed that low-dose fluoride induced osteoblast differentiation earlyobviously, and the effect of high-dose fluoride was not obvious. STZ combined withfluoride treatment significantly inhibited osteogenesis early differentiation, but itwould be helpful for osteogenesis differentiation. Fluoride administrationsignificantly stimulated the function of osteoclasts and STZ reduced the function ofosteoclasts, and osteoclastogenesis function was not obvious. Osteoclastogenesisrelated genes expression showed that the fluoride administration stimulated osteoclastogenesis and bone resorption, significantly. STZ treatment did not inhibitedthe expression of osteoclastogenesis related genes, but combined with fluorideadministration significantly increased OPG expression and reduced the RANKL gene,showed the application both inhibited the activity of osteoclasts. The changed ofTGFβ、β-catenin and Wnt10genes illustrated that the TGFβ played an importantrole in the process of osteogenesis and osteoclastogenesis induced by fluoride, andplayed a role in the process of insulin sensitivity caused by STZ,which leadedosteogenesis and osteoclastogenesis induced by fluoride. While β-catenin and Wnt10mainly involved in the osteogenesis, osteoclastogenesis activity changed induced byfluoride. And STZ combined with fluoride treatment inhibited the rats bone β-cateninexpression on a certain extent, mainly embodies its expression caused by theinhibition of osteogenesis.Osteoblast differentiation experimental was conducted with mesenchymal stemcells collected from femur and tibia of3weeks Wistar mice. The stabile and purifiedcell types of the third generation of BMSC were selected to be mineralized fluidincubation for28days and stained with alizarin red S. The cell fusion and multilayersuperposition in purple or pink mass was observed, which illustrated that the BMSCafter the third generation collected in this experiment was consisted of stem cells withosteogenesis potential, so as to provide the basis for the following osteogenesisinduction experiments. The fluoride or insulin treated cells could stimulateBMSC activity, and insulin combined with low and middle-dose fluorideenhanced cell activity, but insulin combined with high-dose fluoride aggravatedthe inhibition of cell activity, which reflected that the insulin played hyperplasia roleon stimulated osteogenesis with fluoride. The early differentiation of the cells wasobserved by Alkaline phosphatase (ALP) stained with Gomori modified Calcium-Cobalt (Ca-Co) method. Significantly the low and middle-dose fluoride stimulatedthe early differentiation of BMSC cells, but the high-dose fluoride inhibitedthe differentiation of the cells, so did the BMSC cells combined withinsulin treatment, and this synergistic effect mainly manifested the inhibitory effecton ALP at high-dose fluoride. MC3T3-E1cells were used as an in vitro osteoblast precursor of fluoride model.The experimental conditions of fluoride concentration were1,2,8mgF-/L and theinsulin concentration was50nmmol/L, which could induce the activity of osteoblastwere determined by the effects of different doses of fluoride and insulin on the cellproliferation activity. In order to observe the effect of insulin receptor on osteoblastfluorided induced the activity of osteogenesis and osteoclastogenesis, we used geneinterference (siRNA) effectively to reduce the insulin receptor expression inosteoblast. CCK-8method was used to observe the activity on osteoblast of thefluoride from low to high-doses combined with insulin. The results showed that theinfluence of fluoride on MC3T3-E1cell activity was concentrated in low and middle-dose fluoride treated group, and insulin stimulation not only itself could stimulate theactivity of osteoblast, but also could strengthen the effect of fluoride stimulated theactivity of osteoblast. The insulin receptor substrate in osteoblast and the relatedfactors Esp and FoxO1which osteocalcin regulated insulin secretion were analyzedby quantitative polymerase chain reaction. The results showed that after the insulinreceptor were effectively suppressed, the insulin receptor substrates were significantlyreduced, but expression of Esp gene was significantly stimulated. The expressionsof insulin receptor and insulin receptor substrate were induced in different degreesafter combining with fluoride, and Esp gene was reduced correspondingly. So wecould conclude that the fluoride could significantly stimulate the expression of insulinreceptor and play its regulatory role of osteoblastic cells by the coordination ofinsulin receptor substrate and Esp gene signal pathway. The further quantitativeanalysis was conducted to observe the changed of the expression of the regulationfactor of osteoblastic and osteoclastogenesis in osteoblast after the inhibition ofinsulin receptor expression. The insulin receptor mainly affected theosteogenic transcription factor-osterix and osteocalcin stimulated osteogenesis,and MAPK signaling pathway was involved in the process of osteogenic activityregulated by insulin. On the other hand, the changed of osteoclastdifferentiation factor explained that the insulin receptor regulated the activity ofosteoclast differentiation mainly by regulated the OPG gene expression. Conclusion:The aggravation of dental fluorosis was observed in rats treated by the higherdose of fluoride, Streptozocin (STZ) inhibited insuin secretion and aggravated thecharacter of fluoride toxicity. Fluoride stimulated GPRC6A expression, osteocalcinand insulin level in serum. The different concentrations of fluoride caused the varyingdegree of insulin sensitivity.Adminstration of STZ intefered with activity of insulinthrough reducing blood glucose and even induced insulin resistance. Rats treated withfluordie caused decrease of bone mineral density and higher dose of fluordieaggravated damage. Changes of markers of osteogenesis and osteoclastogenensissuggesed bone turnover occurred in rats induced by fluorid. The decrease of insulinactivity and sensitivity obviously influenced osteoblastic and osteoclasticdifferentiation and further aggravated bone injury. The TGFβ played key role onosteogenesis and osteoclastogenesis induced fluoride, and involved in the mechanismunderlying insulin regulated bone turnover induced by fluordie. Fluoride stimulatedosteogenesis and osteoclastogenesis by mediatedβ-catenin and Wnt10signalingpathway.The in vitro study verified the stimulation action of fluoride on bone marrowstromal cell and osteoblast precursors. Insulin stimulated cell viability of two kinds ofosteoblastic cells, but hardly indued their early differention. Insulin mainly modulatedosteogenesis and osteoclastogenesis induced by fluoride through mediating theinsulin receptor and Esp genes of insulin signaling pathway. Insulin primarilyinfluenced osterix and OCN to take part in the bone formation caused by fluoride. Onthe other hand, it mainly regulated expression of OPG and and take part in the boneresorption caused by fluoride.