The Effect And Regulatory Mechanism Of Osteocalcin On Insulin Secretion Disorder Caused By Lipid Toxicity In Beta-TC-6 Cells

Objectives:To study the role of overexpressing insulin receptor in osteoblast on its differentiation and osteocalcin expression,and to further explore whether osteocalcin secreted by osteoblasts can heal insulin secretion disorder caused by lipotoxicity in beta-TC-6 cell and explore its possible mechanism.Methods:1.Experimental design: The focus of this study is on the cross-talks between osteocalcin and insulin,and the cells we selected are mouse osteoblast precursor cells MC3T3-E1 and mouse insulinoma cells BetaTC-6.The experiment is divided into two parts.In the first part,we overexpress the Ins R gene in MC3T3-E1 cells,and explores its effect on the differentiation disorder caused by hyperglycosemia and high fat.In the second part,we study whether osteocalcin secreted by osteoblasts can promote the release of insulin in Beta-TC-6 cells,and heal β-cell glucose-stimulated insulin secretion(GSIS)disorder caused by lipotoxicity.Then we try to explore its potencial mechanism.2.MC3T3-E1 cells were transfected with lentiviral plasmids to overexpress the insulin receptor.Then we wanted to explore its effect on the differentiation of osteoblasts and the expression of osteocalcin.At last we collected the differentiated conditioned medium to intervene Beta-TC-6 cells.We wanted to detect the changing of osteocalcin receptor(Gprc6a),GSIS-related glucose transporter(Glut2)and insulin of Beta-TC-6 cells by Western blot,q PCR and explore whether it can improve insulin secretion function.3.Beta-TC-6 cells were intervened with exogenous osteocalcin and high fat.Then we detected the changing of Gprc6 a,Glut2 and PI3K/AKT/Fox O1/Pdx1 signaling pathways by western blot,GSIS test to detect the secretion of insulin,q PCR was used to detect the changes of Gprc6 a,Glut2,and insulin m RNA.Flow cytometry was used to detect the apoptosis of β cells.PI3 K inhibitors were further used to observe whether the above effects of osteocalcin could be blocked.4.Use the lentiviral plasmid transfection to knock down the Gprc6 a gene of islet Beta-TC-6 cells,then use Fox O1 inhibitors and Pdx1 activators to intervene the key molecules of the PI3K/AKT/Fox O1/Pdx1 pathway respectively,and explore the mechanisms of osteocalcin on insulin secretion.Results:1.The expression of osteocalcin and Runx2 protein reached a peak(P<0.05)after 14 days of induced differentiation,and the deposits of mineralized nodules is numerous,which proved that the optimal induction time for this experiment was 14 days.2.Compared with the control group,the high-sugar and high-fat intervention can significantly reduce Runx2 and osteocalcin levels(P<0.05),and reduce mineralized nodules,which proves that glycolipid toxicity can inhibit the differentiation of the osteoblast.3.Overexpressing Ins R gene of osteoblasts can increase the expression of Runx2 and osteocalcin,promote the differentiation of osteoblasts,and heal the differentiation disorder caused by high sugar and high fat(P<0.05).4.The conditioned medium of overexpressed Ins R gene of osteoblast can increase the expression of Glut2,Gprc6 a and the level of insulin m RNA in Beta-TC-6 cells,increaing the level of Glut2 and insulin caused by lipotoxicity.While these effects can be inhibited by osteocalcin antibody(P<0.05).5.Exogenous incomplete carboxylation of osteocalcin can significantly increase the expression of Gprc6 a in Beta-TC-6 cells,activate the PI3K/AKT/Fox O1/Pdx1 signaling pathway,increase p-AKT/t-AKT,PFox O1/t-Fox O1 ratio,and up-regulate the expression of Pdx1,insulin,and Glut2;GSIS results show that osteocalcin stimulates β-cell insulin secretion under high glucose environment,and flow cytometry results show that osteocalcin can improve Beta-TC-6 cell apoptosis caused by lipotoxicity.After adding PI3 K inhibitor,both the ratio of p-AKT/t-AKT and p-Foxo1/t-FOXO1 decreased,and the expression of Pdx1 and Glut2 decreased(P<0.05),which indicated that blocking PI3 K can inhibit the protective effect of osteocalcin on lipotoxic insulin secretion disorders6.Knocking down Gprc6 a gene in Beta-TC-6 cells can block the expression of Glut2 and insulin stimulated by osteocalcin.Adding Pdx1 agonists and Fox O1 inhibitors respectively can partially offset the down-regulation of Glut2 and insulin secretion disorder caused by knocking down Gprc6a(P<0.05),which indicates that Gprc6 a is an upstream molecular with Fox O1 and Pdx1.Conclusions:1.Insulin signal pathway of osteoblast can improve its differentiation disorder caused by glycolipid toxicity and increase the expression of osteocalcin.2.Osteocalcin can promote the synthesis of insulin by activating theβ-cell Gprc6a/PI3K/AKT/Fox O1/Pdx1,and promote the release of insulin by up-regulating the expression of Glut2.3.Mutual cross-talk between osteocalcin and insulin maybe form an endocrine ring.