| Diabetes often leads to the development of osteoporosis and accelerates its progression.Type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM)affect bone mineral density(BMD)and increase the risk of fracture.And there is growing evidence that T2 DM and osteoporosis may share a common genetic predisposition and molecular basis of disease.Due to the correlation between the two disease,more and more attention has been paid to the influence of anti-hyperglycemia agents on bone metabolism.Glucagon-like peptide-1(GLP-1)receptor agonists(GLP-1RAs)are novel and promising drugs for T2 DM,which have clinical application value in bone-associated diseases.As a new class of hypoglycemic agent,GLP-1RA mimics the effects of incretins in the body.GLP-1 is synthesized and secreted by intestinal L cells stimulated by food intake,which enhances insulin release from islet beta cells and inhibits secretion of glucagon by alpha cells in a glucose concentration-dependent manner.In addition to the hypoglycemic effect,GLP-1 can also delay the gastric emptying and suppress the appetite by affecting vagal afferent fibers and the hypothalamus,eventually resulting in weight loss.These beneficial effects of GLP-1 on glucose homeostasis in vivo are mediated through the GLP-1 receptor.However,native GLP-1 is rapidly degraded in the circulation by dipeptidyl peptidase IV(DPP-IV).Liraglutide,a synthetic GLP-1RA,which has 97% homology to human GLP-1 structure,is resistant to inactivation of DPP-IV and has a longer half-life,making it a new-type anti-hyperglycemia drug for daily injections.In addition to its important value in the treatment of diabetes and obesity,the expression of the GLP-1 receptor in various tissues except the pancreatic β-cells and α-cells aroused great interest in exploring the cardiovascular,nerve,kidney protection,and other extra pancreatic functions.In vitro studies indicate that osteoblasts express functional receptors for GLP-1.Therefore,bone may also be a potential target organ for GLP-1RAs.Glucagon-like peptide-1(GLP-1)has been reported to increase bone density and improve bone quality.The relationship between GLP-1 and fracture is still under investigation.GLP-1 promotes bone formation and inhibits bone resorption,but the exact process and related molecular mechanisms remain unclear.Osteoblasts are mesenchymal cells that play a key role in maintaining bone homeostasis.They are responsible for the production of extracellular matrix proteins,regulating matrix mineralization,controlling bone remodeling and regulating osteoclast differentiation.Osteoblasts play an important role in the pathogenesis of many bone diseases,especially osteoporosis.For decades,the main treatment of osteoporosis is to inhibit osteoclast maturation,proliferation and activity;however,in recent years,abnormal differentiation or activity of osteoblasts has led to bone metabolism abnormalities and related diseases,which gains increased attention.The maintenance of osteoblasts function and number has become the focus of the current treatment of osteoporosis.In view of the close relationship between type 2 diabetes and osteoporosis,this study mainly focused on the effects of liraglutide on proliferation,differentiation and apoptosis of pre-osteoblastic MC3T3-E1 cells in vitro and explored the molecular mechanism of liraglutide on osteoblast formation ability,providing a new drug target and a new therapeutic strategy for diabetic patients with osteoporosis.Part one Liraglutide promotes proliferation,differentiation of MC3T3-E1 cells and GLP-1R is expressed in MC3T3-E1 cellsObjectives: To observe the effects of different concentrations of liraglutide on proliferation and differentiation of murine osteoblastic MC3T3-E1 cells at different time points and to confirm the expression of GLP-1R in MC3T3-E1 cells.Methods:1 Different concentrations of liraglutide(0,10,100,500,1000 nM)were applied to stimulate osteoblastic MC3T3-E1 cells cultured in vitro and MTT was utilized to assay the viability of cells at 24,48 and 72 h after cell fusion for evaluating the proliferative capacity of osteoblasts.2 The osteoblastic MC3T3-E1 cells were induced for differentiation with 50mg/L ascorbic acid and 10 mM sodium β-glycerophosphate in the presence of different concentrations of liraglutide(0,10,100,500,1000nM).The cells were collected on day 0,7,14,21,and 28,and the specific differentiation markers including collagen-1(COL1),osteocalcin(OC),runt-related transcription factor-2)and osteoprotegerin(OPG)were detected by Realtime PCR.Alkaline phosphatase(ALP),an early indicator of osteogenic differentiation,was detected by colorimetric method for MC3T3-E1 cells at 0,7 and 14 days.Alizarin red staining was used to observe the extracellular matrix mineralized nodules after 21 days of cell mineralization induction and the relative absorbance was calculated.3 The mRNA and protein levels of GLP-1R in mouse MC3T3-E1 cells were detected by RT-PCR and Western blot analysis using mouse pancreas as a positive control.GLP-1R in mouse MC3T3-E1 cells was detected by confocal microscopy.The GLP-1R mRNA and protein levels in MC3T3-E1 cells were analyzed by RT-PCR and Western blot at 0,7,14,21,and 28 days after induction to determine GLP-1R expression trend.Results:1.Liraglutide increases the viability of osteoblast precursor MC3T3-E1 cells,reaching its peak at a median time(48 h)and concentration(100 nM).2.The mRNA level of osteoblast differentiation factor COL1,OC,RUNX2,OPG and ALP activity indicated that MC3T3-E1 cells undergo specific osteogenic differentiation and maturation.Liraglutide significantly enhanced the mRNA expression of COL1,OC,RUNX2 and OPG.COL1 and OPG increased most obviously on day 14,while OC and Runx2 achieved the maximum effect on day 21.Alizarin red staining were observed and semi-quantitative of calcium deposition by etylpyridinium chloride in osteoblasts was performed,which showed that liraglutide promoted the mineralization of MC3T3-E1 cells.3.GLP-1R is present in MC3T3-E1 cells.The result of RT-PCR showed that GLP-1R specific band appeared at 111 bp.Western blot analysis and confocal microscopy further confirmed this result.In addition,liraglutide increased GLP-1R mRNA and protein expression during MC3T3-E1 cell differentiation and reached its highest value at 21 days.Part two Liraglutide promotes proliferation and differentiation of MC3T3-E1 cells by activating PI3K/Akt,ERK1/2 and cAMP/PKA signaling pathway involving β-catenin via GLP-1RObjectives: To study the molecular mechanism of liraglutide-mediated regulation of proliferation and differentiation of osteoblastic MC3T3-E1 cells.Methods:1 To illustrate the activation of PI3K/Akt,ERK1/2 and cAMP/PKA signaling pathway,Western blot analysis was used to determine the phosphorylation of Akt,ERK1/2 and β-catenin at different time points(0-60min).The activation of cAMP was detected by ELISA.Western blot was used to detect the β-catenin in cytoplasm and nucleus of MC3T3-E1.The transcription level of TCF7L2 was analyzed by real-time fluorescence quantitative PCR.2 The signaling pathway rescue experiment was carried out by using PI3 K signal inhibitor LY294002,ERK inhibitor PD98059 and PKA inhibitor H89.PKA inhibitor H89 was used to confirm the effect of PKA activation on the phosphorylation of β-catenin protein.Meanwhile,three inhibitors were used to validate the effect of PI3K/Akt,ERK1/2 and cAMP/PKA signaling pathway on the level of β-catenin in cytoplasm and nucleus and the transcriptional level of TCF7L2,to clarify the activation of Wnt signaling pathway.3 Silencing GLP-1R and β-catenin by small interfering RNA(siRNA)transfection.The effect of silenced GLP-1R on the phosphorylation of Akt,ERK1/2 and β-catenin and the level of intracellular cAMP,the β-catenin protein level and the transcriptional level of TCF7L2 in cytoplasm and nucleus were examined.The effect of silencing β-catenin on cytoplasm and nucleus β-catenin protein and transcriptional level of TCF7L2 were examined.4 The effects of signaling inhibitors and small interfering RNAs on cell viability,essential molecular markers and mineralization of MC3T3-E1 cells during differentiation stages were examined.Results:1.Liraglutide up-regulated ERK1/2 and Akt phosphorylation,with the most obvious effect for ERK1/2 at 30 min and Akt at 45 min.The promotion of ERK and AKT phosphorylation by liraglutide was offseted by the specific inhibitors LY294002 and PD98059.ELISA showed that the optimal concentration of liraglutide for increasing cAMP was 100 nM and cAMP reached its maximum at 30 minutes.As liraglutide activated cAMP,phosphorylation of β-catenin at the C-terminal ser675 site reached its peak at the same time,and H89 attenuated phosphorylation of β-catenin at the C-terminal ser675 site,indicating that cAMP/PKA/β-catenin-ser675 signaling cascade participates in this process.2.GLP-1R was silenced by siRNA and liraglutide-induced activation of cAMP/PKA /β-catenin-ser675,PI3K/Akt and ERK1/2 significantly decreased,indicating that liraglutide regulates downstream signaling pathways through GLP-1R.Liraglutide increased the content of β-catenin in the cytoplasm and enhanced nuclear translocation of β-catenin and up-regulated TCF7L2 expression.PKA inhibitor H89 and siGLP-1R down-regulated the nuclear localization of β-catenin and TCF7L2 expression.In addition,LY294002 and PD98059 partially inhibited the accumulation of β-catenin in the cytoplasm and nucleus and TCF7L2 transcription,indicating that PI3K/AkT and ERK1/2 may interact with β-catenin to activate Wnt signaling.3.Treatment with the inhibitor PD98059 caused the most obvious attenuation effect;LY294002 showed the least but still significant decrease,and H89 exhibited a level of inhibition somewhere in between them.The mRNA expression of osteoblastic differentiation markers after inhibitor treatment was much lower than that in the blanks.PD98059 and LY294002 preferentially inhibited the expression of Runx2,leaving H89 with the lowest level of OC expression,but they all restrained matrix calcification based on a mineralization staining assay Silencing of GLP-1R and β-catenin by small interfering RNAs counteracts the promotion of liraglutide on cell viability,alkaline phosphatase activity,osteogenic differentiation markers and mineralization in osteoblasts.Part three liraglutide inhibits serum deprivation-induced apoptosis of MC3T3-E1 cells through cAMP/PKA/β-catenin and PI3K/AKT/GSK3β signaling pathwayObjectives: To study the effect of liraglutide on apoptosis of osteoblasts and its underlying mechanism.Methods:1 The expression of GLP-1R in mouse osteoblast MC3T3-E1 was detected by RT-PCR,Western blot and laser scanning confocal microscope.2 MC3T3-E1 cells were stimulated with different concentrations of liraglutide(0,10,100,1000 nM)respectively,and then stained with Hoechst 33258.The apoptosis of MC3T3-E1 cells was detected by flow cytometry and ELISA.Western blot was used to detect the protein expression of Bcl-2,Bax and caspase-3.3 Liraglutide stimulated MC3T3-E1 cells,Western blot was used to detect phosphorylation of Akt,β-catenin and p-GSK3β expression,Western blot was used to detect the cytoplasmic and nuclear β-catenin protein levels in MC3T3-E1 cells.The transcription level of TCF7L2 was analyzed by real-time fluorescence quantitative PCR.PI3 K inhibitor LY294002 and PKA inhibitor H89 combined with small interfering RNA(siRNA)to silence the expression of GLP-1R and β-catenin to study the molecular mechanism of liraglutide inhibiting MC3T3-E1 cell apoptosis.Results:1.Liraglutide inhibits serum-induced apoptosis of MC3T3-E1 cells,promotes Bcl-2 protein expression,and inhibits Bax and caspase-3 protein expression not in a dose-dependent manner.2.100 nM liraglutide induced Akt phosphorylation and increased intracellular cAMP levels.Meanwhile,Akt phosphorylation was blocked by inhibitor LY294002.Liraglutide significantly enhanced the phosphorylation of cytoplasmic β-catenin at ser-675 and decreased the phosphorylation of phospho-glycogen synthase kinase 3β associated with Akt phosphorylation,accompanied by increased β-catenin in cytoplasm and nucleus and up-regulated TCF7L2 mRNA expression.These effects were abolished by PKA inhibitor H89 and PI3 K inhibitor LY294002,respectively.In addition,blockade of GLP-1R by siRNA inhibits intracellular cAMP activity and Akt and beta-catenin phosphorylation.These results indicate that liraglutide inhibits MC3T3-E1 cell apoptosis through GLP-1R-mediated cAMP/PKA and PI3K/Akt signaling pathways.3.Specific signaling inhibitors LY294002 and H89,small interfering RNA silencing GLP-1R and β-catenin can eliminate the effect of liraglutide on serum-induced apoptosis in MC3T3-E1 cells.Conclusions:1.Liraglutide promotes proliferation and differentiation of pre-osteogenic MC3T3-E1 cells and GLP-1R is expressed in MC3T3-E1 cells.2.Liraglutide promotes proliferation and differentiation of MC3T3-E1 cells by activating the the PI3K/Akt,ERK1/2 and cAMP/PKA signaling pathways cross-talking with β-catenin to target Wnt signaling pathway,which is mediated by GLP-1R.3.Liraglutide inhibits serum deprivation-induced apoptosis of MC3T3-E1 cells by cAMP/PKA/β-catenin and PI3K/AKT/GSK3β signaling pathway. |
PDF Full Text Request