| Background and purposeThe main feature of type 1 diabetes（T1DM）is hyperglycemia caused by reduced insulin secretion.The effect of T1DM on bone metabolism is usually manifested by bone loss and decreased bone strength,leading to increased risk of fracture.Recent studies have shown that the inhibition of WNT/β-catenin signaling pathway in bone tissue is closely related to the pathogenesis of diabetic osteopenia.Indeed,the WNT/β-catenin signaling pathway is critical for the proliferation,differentiation and maturation of osteoblasts,and deeply involved in the regulation of skeletal development and bone remodeling.We previously found that the trabecular bone and cortical bone respond differently to WNT/β-catenin signaling,leading to differential expression levels in wild-type mice.Therefore,in the first part of the study,we systematically observed the differences between trabecular and cortical bone phenotypes in type 1 diabetic mice and tried to find the key regulatory factors.Furthermore,we investigated the different effect of osteoblasticβ-catenin activation on the trabecular and cortical bone of diabetic mice and the underlying mechanisms.The mainly negative effect of T1DM on bone metabolism is to inhibit the function and activity of osteoblasts,which leads to bone loss.The recombinant human parathyroid hormone PTH（1-34）is a representative bone-forming agent.The mechanism of PTH-induced osteoanabolic effect is partly mediated through activation of the WNT/β-catenin signaling pathway.In the second part of the study,we systematically evaluated whether or not the anabolic effects of intermittent PTH（1-34）treatment are maintained in a mouse model of T1DM.Then in the third part of the study,we usedβ-catenin transgenic mice to determine whether or not activation ofβ-catenin signaling in osteoblasts plus PTH treatment result in a combined anabolic effect on restoring bone structure and strength in the setting of T1DM.MethodsPart Ⅰ:For T1DM induction,mice were given intraperitoneal injections of STZ（50mg/kg）for 5 consecutive days.For the stabilization ofβ-catenin in committed osteoblasts,Col1-3.2kb-CreERTM;Catnblox（ex3）mice were initially generated and then intraperitoneally injected with tamoxifen（TM;10 mg/kg/day×4 consecutive days）to activate the function of the promoter.All mice were allotted to three groups:Control group with buffer injection;T1DM group with STZ injection;and T1-CA group with STZ injection plus osteoblasticβ-catenin activation.Six weeks after the first injection of STZ or buffer only,all mice were euthanized and dissected to obtain bone tissue.The distal femur,proximal tibia and the fifth lumbar vertebrae were considered as trabecular bone,while the middle part of long bones was used as cortical bone for analysis and comparison.The following experiments were performed:1.Analysis of bone morphology and bone mass by X-ray,Micro-CT scanning and H&E staining of paraffin sections;2.Biomechanical analysis of trabecular and cortical bones by the compression test and the three-point bending test,respectively;3.Bone histomorphometry analysis including measurement of cortical bone porosity,by fluorescence double labeling and undecalcificated bone sections;4.TRAP staining of tibial paraffin sections to investigate the morphology and quantity of osteoclasts;5.Von Kossa staining of femoral undecalcificated sections to observe bone mineralization;6.Immunohistochemistry of paraffin sections to observe the expression level of osteoblasts and osteoclast markers as well as key molecules of signaling pathways;7.Gene expression of osteoblasts and osteoclast markers and signaling pathway-related factors were detected by real-time quantitative PCR after RNA extraction of bone tissue;8.Mouse osteoblast cell line-MC3T3-E1 cells were cultured in vitro under high glucose environment,and total protein was extracted for Western-blotting assay to detect protein expression of signaling pathway-related factors;9.Primary osteoblasts of Catnblox（ex3）mice were cultured in a high glucose environment and infected with Cre adenovirus to activateβ-catenin.The protein level of related factor in cell culture medium were further detected,and the gene expressions were also detected by quantitative PCR after extracting cellular RNA.Part Ⅱ:Mice were first injected with STZ to induce T1DM.After 3 weeks to allow bone loss,the mice were assigned to receive human PTH（1–34）（75μg/kg/d）or vehicle（0.9%saline）daily by injection for 3 weeks.Accordingly,the mice were divided into four groups:a wild-type group（WT）,a wild-type PTH treatment group（WT+PTH）,a type 1 diabetes group（T1D）and a diabetic PTH treatment group（T1D+PTH）.All mice were euthanized by pentobarbital overdose 1 week after treatment,and the following experiments were performed:1.Analysis of bone morphology and bone mass of femur and fifth lumbar vertebra by Micro-CT scan;2.Bone histomorphometry analysis by fluorescence double labeling and undecalcificated bone sections;3.TRAP staining to investigate the morphology and quantity of osteoclasts;4.Cell proliferation was detected by BrdU immunohistochemical staining;5.Detection of cell apoptosis in bone tissue by TUNEL method;6.Immunohistochemistry of paraffin sections to observe the expression level of osteoblast markers andβ-catenin protein;7.Gene expression of apoptosis-related factors and PTH/β-catenin pathway-related factors in bone tissue were detected by real-time quantitative PCR after RNA extraction.Part Ⅲ:For the moderate activation of osteoblastβ-catenin signaling,the Col1-3.2 kb-CreERTM;Catnblox（ex3）mice were injected with a single dose of tamoxifen（TM,15 mg/kg）.Select the above-mentioned transgene mice and litter non-transgene mice to induce the type1 diabetes by STZ injection,and continued feeding for 3 weeks.The mice then received a single-dose injection of TM.3 days later,the transgene mice or the non-transgene mice were injected with PTH（1-34）or saline（75μg/kg/day）for 3 weeks.Accordingly,the mice were divided into four groups:Type 1 diabetes group（T1D*）,diabetic PTH treatment group（T1D*+PTH）,diabeticβ-catenin transient activation group（T1D*+TA）and diabetic combination treatment group（T1D*+TA+PTH）.All mice were euthanized 1 week after treatment,and the following experiments were performed:1.Analysis of bone morphology and bone mass by Micro-CT scanning and H&E staining of paraffin sections;2.Biomechanical analysis of femur by the three-point bending test;3.Bone histomorphometry analysis by fluorescence double labeling and undecalcificated bone sections;4.Von Kossa staining of femoral undecalcificated sections to observe bone mineralization;5.TRAP staining to investigate the morphology and quantity of osteoclasts;6.Immunohistochemistry for detection of BrdU cell proliferation and protein expression of osteoblast markers;7.Gene expressions of adipogenic related factors were detected by quantitative PCR after RNA extraction of bone marrow adipose tissue;8.Protein expressions of PTH and WNT/β-catenin signaling pathway-related factors were detected by Western-Blotting assay after total protein extraction of bone tissue;9.Isolation of mouse primary bone marrow stromal cells and osteogenic induction,then the cell RNA was extracted for quantitative PCR detection of gene expression of osteogenic differentiation factors,and the alizarin red staining was performed to detect osteogenic differentiation levels;10.Primary osteoblasts of mice were isolated and cultured,then the cell RNA was extracted for quantitative PCR detection of gene expression of PTH and WNT/β-catenin signaling pathway-related factors.ResultsPart Ⅰ Differential effects of type 1 diabetes mellitus and subsequent activation ofβ-catenin in osteoblasts on trabecular and cortical bone in a mouse model1.Successful establishment of a STZ-induced T1DM mouse model showed that the fasting blood glucose of mice was higher than 16.7 mmol/L.The diabetic mice exhibited typical diabetes symptoms of polyuria,polydipsia,and polyphagia,as well as significantly decreased body weight.2.Compared with Control mice,T1DM mice showed significant bone loss in both long bones and vertebral body.Moreover,the extent of bone loss was greater in trabecular bone than that in cortical bone.3.In T1DM mice,the morphometric indicators including N.Ob/T.Ar,Ob.S/BS and BFR,as well as the expression of osteoblast differentiation markers OSX and ALP,were all decreased compared with Control mice,and the extent of these reductions was greater in the trabecular bone,consistent with the trend of bone loss.However,the bone resorption indicators N.Oc/T.Ar and Oc.S/BS showed no significant changes in T1DM mice compared with Control mice,and also no significant difference between the trabecular bone and the cortical bone.4.The expression levels ofβ-catenin and its downstream factors LEF-1,TCF and Axin2also decreased in the bone tissue of T1DM mice,and the extent of decline was greater in trabecular bone.5.The expression of IGF-1R in bone tissue of T1DM mice was lower than that of Control mice,and the decrease in trabecular bone was significantly greater than that in cortical bone.Further in vitro experiments showed that the protein expressions of IGF1R,pAKT,GSK3βandβ-catenin were significantly inhibited in MC3T3-E1 cells cultured in high glucose environment.6.Compared with T1DM mice,T1-CA mice showed a significant increase in trabecular bone mass,as well as the morphometric indicators of bone formation and the expression levels of RunX2 and OSX in trabecular bone,and there was no change in the expression of osteocalcin.Meanwhile the morphometric indicators of bone resorption decreased in T1-CA mice,accompanied by higher expression of OPG and a decrease in the ratio of RANKL/OPG expression.In addition,the mineralization level and biomechanical parameters of trabecular bone in T1-CA mice were also significantly enhanced.7.The cortical bone mass slightly increased in T1-CA mice compared with T1DM mice,but to a lesser extent than that in trabecular bone.Trends of bone formation morphometric indicators and osteoblast markers in cortical bone were consistent with that in bone mass.8.The cortical bone of the T1-CA mice displayed an unexpected increase in bone resorption,manifested by the increase of N.Oc/T.Ar and Oc.S/BS with 125.6%and 86.9%,respectively.We also observed an increase in cortical bone porosity.Meanwhile,the mineralization level and biomechanical parameters of trabecular bone in T1-CA mice did not improve compared with T1D mice.9.Compared with T1DM mice,the expression of OPG in the cortical bone of T1-CA mice was increased while the ratio of RANKL/OPG expression was decreased,which is similar to the trend in trabecular bone.10.The expression level of WNT16 in the cortical bone of T1-CA mice decreased significantly compared with T1D mice,but the expression of WNT5a showed no difference.The protein expression level of WNT16 in cortical bone was significantly higher than that in trabecular bone in each group of mice.In vitro experiments confirmed that activation ofβ-catenin in osteoblasts in high glucose environment inhibited the expression of WNT16,but had no significant effect on WNT5a.Part Ⅱ PTH-induced increase in bone mass and bone formation was attenuated in T1D mice compared with non-diabetic controls,and the reason could be that PTH treatment failed to activateβ-catenin signaling in T1D mice.1.Intermittent PTH（1-34）treatment for 3 weeks can increase bone mass in T1D mice,and the increase of BV/TV（distal femur）,BA/TA（femoral shaft）and L5.BV/TV（vertebral body）in T1+PTH mice were+16%.+8%and+21%.However,the increase of the above three parameters in WT+PTH mice compared with WT mice were+67%,+31%and 75%,respectively.2.The bone histomorphometry results showed that the increases of trabecular and cortical BFR/BS in TID+PTH mice compared with T1D mice were significantly less than that in WT+PTH mice compared with WT mice But the bone resorption parameters N.Oc/T.Ar and Oc.S/BS increased in both TID+PTH mice and WT+PTH mice,and the extent of increments showed no difference.3.The results of BrdU immunohistochemistry showed that the number of positive cells in TID+PTH mice did not increase compared with T1D mice,while it increased greatly in WT+PTH mice corresponding to WT mice.TUNEL analysis revealed that the number of apoptotic cells were decreased after PTH treatment in both T1D and WT mice.In addition,mRNA expression of the pro-apoptotic gene,Bax,and the anti-apoptotic gene,Bcl-2,was inhibited and enhanced by PTH in the bone tissue derived from T1D or WT mice,respectively.4.Compared with T1D mice,the mRNA expression of PTH1R and LRP6 in bone tissue was significantly increased in TID+PTH mice,but the expression ofβ-catenin and its downstream target genes TCF,LEF-1 and CyclingD1 showed no obvious changes.Part Ⅲ Moderate pre-activation of osteoblasticβ-catenin can significantly enhance the osteoanabolic effect of PTH（1-34）on type 1 diabetic mice,result in greatly improved bone mass and bone strength.1.Pre-treatment with moderate activation of osteoblasticβ-catenin followed by PTH treatment for 3 weeks in T1D mice led to obvious osteoanabolic effect.The Micro-CT bone mass parameters of the vertebral body and femur in T1D*+TA+PTH mice were significantly higher than that in T1D*+PTH mice or T1D*+TA mice.2.Three-point bending test and Von Kossa staining showed that biomechanical parameters and bone mineralization levels of T1D*+TA+PTH mice were significantly improved compared with T1D*+PTH mice or T1D*+TA mice,respectively.3.The results of bone metabolism related factors showed that the osteoblast proliferation and early differentiation of pre-osteoblasts in bone marrow of T1D*+TA+PTH mice were significantly promoted,and the level of bone marrow adipose tissue was also greatly improved,compared with T1D*+TA mice or T1D*+PTH mice.4.The results of Western-blotting showed that the expression levels of PTH1R/LRP6/β-catenin pathway related and downstream target factors were all significantly increased in the bone tissue of T1D*+TA+PTH mice,while the expression of SOST,a WNT/β-catenin pathway inhibitor,was significantly inhibited.5.In vitro experiments also confirmed that the osteogenic differentiation of primary bone marrow mesenchymal cells（BMSCs）in T1D*+TA+PTH mice and the mRNA expression level of the above signaling pathways factors in its primary osteoblasts were higher than other groups.Conclusions1.The trabecular bone loss is greater than cortical bone loss in the type 1 diabetic mice,accompanied by greater impairment of bone formation in trabecular bone than that in cortical bone.These differences might be related to a greater inhibitory signal of the WNT/β-catenin system in trabecular bone than that in cortical bone.2.Constitutive activation ofβ-catenin in osteoblasts can significantly improve trabecular bone loss and enhance trabecular mineralization and biomechanical strength in type 1 diabetic mice.However,it could increase the endocortical osteoclasts activity by inhibiting the expression of WNT16 in the cortical bone,and lead to increased cortical porosity,which is a potential risk for the bone strength.3.Intermittent PTH（1-34）treatment can partially rescue the bone loss in type 1 diabetic mice,but the osteoanabolic effects were blunted compared with non-diabetic mice.This could be attributed to the direct inhibition ofβ-catenin signaling by high-glucose conditions,which dampen PTH-induced activation of the LRP6/β-catenin pathway.4.Pre-treatment with moderate activation of osteoblasticβ-catenin followed by PTH（1-34）treatment can significantly enhance the osteoanabolic effect of PTH on type 1 diabetic mice,result in greatly improved bone mass and bone strength.Thus,there is a clear advantage of developingβ-catenin as a target to improve efficacy of PTH therapy in the treatment of T1DM-related osteopenia... |
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