The Characteristics Of Abnormal Bone Metabolism In Type 1 Diabetes Mellitus And The Intervention Effect Of Incretin Drugs And Its Mechanism

Part 1Differences in bone microarchitecture found via HR-pQCT in adult versus juvenile onset T1D diabetic Asian males and compared to non-diabetics-an observational cross-sectional pilot studyBackgrounds:Patients with type 1 diabetes(T1D)have an increased risk of fractures.In addition to decreased bone density,there are also abnormalities in bone microarchitecture.There are few reports about bone microarchitecture in T1D patients.So far,there have not been any reports about bone microarchitecture measured by high resolution peripheral quantitative CT(HR-pQCT)in Asian T1D patients.In addition,the age at T1D onset is an important factor affecting fracture risks,and the age at onset may also affect their bone microarchitecture.Objectives:We aimed to compare the differences in bone mineral density and microarchitecture measured by HR-pQCT between adult male T1D patients and control group to make up for the gap in HR-pQCT research in Asian T1D population;further to compare the differences in bone mineral density and microarchitecture between juvenile onset and adult onset T1D patients to explore the effect of disease onset age on bone density and bone microarchitecture.Methods:This cross-sectional study included 32 adult male T1D patients and 32 healthy subjects.T1D patients were further divided into juvenile onset group and adult onset group according to the age at disease onset before or after 18 years old.Demographic data,anthropometric parameters and biochemical indicators were collected.DXA was used to measure the areal bone mineral density of the lumbar spine and hip,and HR-pQCT was used to determine the volumetric bone density and bone microarchitecture of ultradistal non-dominant radius and tibia.Comparisons were made between overall T1D patients and overall controls,adult-onset T1 D patients and their corresponding controls,juvenile-onset T1D patients and their corresponding controls,and adult-onset T1D patients and juvenile-onset T1D patients.Results:1.General characteristics:of the 32 T1D patients,15 were juvenile-onset,with an average age of 32 years,an average age at disease onset of 10 years and a median disease duration of 11 years,and the other 17 were adult-onset,with an average age of 36 years,an average age at disease onset of 29 years and a median disease duration of 7 years.Six of the T1D patients had a history of fractures,all associated with trauma or falls.The average HbAlc was 7.0%,the average 25-hydroxyvitamin D was 20.7 ng/ml,and the average testosterone was 5.1 ng/ml.Compared to juvenile onset group,adult onset group had lower BMI and waist circumference(p=0.002 and p=0.003),shorter duration of diabetes(p<0.001),and lower total daily insulin dosage(p=0.003).Although there was no difference in β-CTX levels between the two groups,the serum ALP level in the adult onset group was lower than that of the juvenile onset group(p=0.037).There were no significant differences in age,HbAlc,blood pressure,urine ACR,serum calcium,phosphorus,25-hydroxyvitamin D and testosterone levels between the two groups.2.At radius,total T1D patients had significantly lower total vBMD,trabecular vBMD,trabecular BV/TV,trabecular thickness and cortical thickness(p<0.05),while higher trabecular seperation(p=0.024)than total controls.Adult-onset and juvenile-onset T1D patients both had lower total vBMD(p=0.009,p=0.006),trabecular vBMD(p=0.007,p<0.001),trabecular BV/TV(p=0.006,p=0.001)and Tb.Th(p=0.003,p<0.001)than controls.After adjustment for BMI,disease duration and insulin dosage,juvenile-onset patients tended to have lower trabecular vBMD(-1.6%,p=0.066),BV/TV(-4.3%,p=0.05),and Tb.Th(-8.3%,p=0.064)than adult-onset patients,while adult-onset patients tended to have higher Ct.Po(+22.6%,p=0.070)than juvenile-onset patients.3.At tibia,all T1D patients had significantly lower Tt.vBMD(p=0.006),Tb.Th(p=0.003)and Ct.Th(p=0.018)than all controls.Adult-onset patients had lower total vBMD(p=0.027),Ct vBMD(p=0.021),but higher Ct.Po(p=0.036)than controls,while juvenile-onset patients had lower Tb.Th(p<0.001)and 1/Tb.N.SD(p=0.023)than controls.After adjusting for covariates,adult-onset patients tended to have higher Ct.Po.Dm(+4.3%,p=0.061)than juvenile-onset patients.4.The lumbar spine and hip aBMD measured by DXA did not differ between juvenile-onset and adult-onset T1D patients.In addition,after adjusting BMI,no significant correlations were found between glycated hemoglobin,urine ACR,25-hydroxyvitamin D,insulin dose,testosterone levels and DXA or HR-pQCT related parameters.Conclusions:This was the first study reporting HR-pQCT derived bone microarchitecture in T1D patients of Asian male background.Our study found adult-onset and juvenile-onset Asian T1D patients both exhibited deficits in vBMD and microarchitecture compared with controls and demonstrated some differences in cortical and trabecular microarchitecture between them.Part 2Study on the mechanism of abnormal bone metabolism in type 1 diabetic mice and the intervention study of incretin-based therapiesBackgrounds:The mechanism of bone damage in type 1 diabetes(T1D)is complex and abnormal immune inflammation may play an important role in it,but evidence is scarce.Liraglutide and linagliptin can be used in combination with insulin in the treatment of T1D patients in specific situations,but there is very little research evidence on its effect on bone metabolism in T1D patients or animal models.Objectives:1.In the T1D mouse model,we aimed to observe the characteristics of abnormal bone metabolism,including changes in bone density,bone microarchitecture and bone turnover,and use transcriptome sequencing technology(RNA-seq)and bioinformatics analysis to explore the possible mechanisms involved,focusing on immunoinflammatory pathways.2.In the T1D mouse model,we used liraglutide and linagliptin as a single or combined with insulin as intervention therapies to explore the effect of liraglutide and linagliptin on bone density,bone microarchitecture and bone turnover in T1D mice.RNA-seq and bioinformatics analysis were used to explore their possible mechanisms.Methods:Female C57BL/6J and NOD mice,using streptozotocin to construct type 1 diabetes model,were divided into normal blood glucose control group,T1D model group,insulin treatment group,liraglutide treatment group,insulin+liraglutide treatment group,linagliptin treatment group,insulin+linagliptin treatment group.Random body weight and blood glucose were measured every week,and after 8 weeks of feeding,they were sacrificed and serum and bone tissue samples were taken.Serum specimens were tested for C-peptide,bone metabolism indexes(P1NP and CTX)and inflammation indexes;tibial bone was subjected to micro-CT to determine bone density and bone microarchitecture,including cortical volumetric bone mineral density(Ct.vBMD)and trabecular volumetric bone mineral density(Tb.vBMD),cortical bone thickness(Ct.Th),trabecular bone volume fraction(BV/TV),number of trabecular bone(Tb.N),trabecular bone thickness(Tb.Th),trabecular bone separation(Tb.Sp);femoral bone was examined by histopathological analysis to determine bone trabecular parameters and bone marrow adipocyte count;Selected tibia was examined for RNA-seq determination and subsequent bioinformatics analysis,and selected genes with significant differences were used for RT-qPCR verification.Results:1.The effect of liraglutide and linagliptin on body weight,glucose metabolism and serum C-peptide in T1D mice(1)The body weight of C57-T1D mice decreased significantly compared with the control group,and each hypoglycemic treatment group had no significant recovery compared with the untreated group,in which the insulin+liraglutide treatment group was even lower than the untreated group(p=0.013).The blood glucose of the T1D group was significantly higher than that of the control group(p<0.001),which maintained at about 20mmol/l.The blood glucose of the mice in each hypoglycemic treatment group had a downward trend compared with the untreated group,but none of them reached statistical difference(p all>0.05).In the C57-T1D group,the C-peptide decreased compared with the control group,but the difference was not statistically significant(p=0.206).In the liraglutide and ligagliptin treatment group,the C peptide decreased compared with the T1D group,while each group using insulin had an increased trend,in which the C-peptide elevation in the insulin+linagliptin treatment group reached a statistical difference(p=0.008).Overall,liraglutide could reduce body weight and lower blood sugar,and linagliptin had no significant effect on body weight or blood sugar.(2)The body weight of the NOD-T1D mouse group was significantly lower than that of the control group(p<0.001),and the body weight of each hypoglycemic treatment group was higher than that of the untreated group.The weight of the insulin treatment group was statistically higher than that of the untreated group(p=0.027).The blood glucose in the T1D group was significantly higher than that in the control group(p<0.001),and gradually increased to maintain at about 30mmol/l.The blood glucose in each treatment group was slightly lo wer than that in the untreated group,and none of them was controlled to normal,only insulin+liraglutide treatment group dropped to about 20 mmol/l,reaching statistical difference(p=0.007).In the NOD-T1D group,the C-peptide decreased compared with the control group,but the difference was not statistically significant(p=0.314).Except for the insulin treatment group,C-peptide decreased in the other treatment groups,but there was no statistical difference(All p>0.05).In general,liraglutide could reduce body weight,liraglutide combined with insulin treatment could slightly reduce blood glucose,and linagliptin had no significant effect on body weight or blood sugar.2.The bone density and microarchitecture characteristics of T1D mice and the therapeutic effect of liraglutide and linagliptin(1)Parameters measured by micro-CT in C57-T1D mice:Tb.vBMD and Ct.vBMD in T1D group were significantly lower than those in control group(all p<0.001).After treatment,only Tb.vBMD increasement in liraglutide treatment group reached statistical difference(p=0.007),Ct.vBMD rebounded in each group of hypoglycemic treatment to reach statistical difference(p<0.05).Compared with the control group,the cortical and trabecular microarchitecture of T1D mouse were abnormal,showing that BV/TV,Tb.N,Ct.Th decreased significantly,Tb.Sp increased significantly(all p>0.05),Liraglutide monotherapy or combined with insulin therapy tended to improve trabecular microarchitecture,but none of them reached statistical significance,and each hypoglycemic treatment group could significantly increase Ct.Th(p<0.05).The bone trabecular parameters and the number of bone marrow adipocytes determined by bone histopathological morphology did not reach statistical differences among the groups.In short,the tibia bone density of C57-T1D mice decreased compared with the control group,and manifested cortical and trabecular bone deficts.Liraglutide alone or combined with insulin could improve its reduced bone density and abnormal bone microarchitecture.Insulin,linagliptin alone or combined insulin had no improvement on trabecular bone density or microarchitecture,but could only improve its cortical bone density and cortical thickness.(2)Parameters measured by micro-CT in NOD-T1D mice:Tb.vBMD in the T1D group was significantly lower than that in the control group(p=0.043),and Ct.BMD did not reach statistical difference(all p<0.05).The increasement in Tb.vBMD in the insulin-treated group,the liraglutide-treated group,and the insulin+liraglutide treated group all reached statistically significant differences(p=0.007).The microarchitecture parameters of trabecular bone and cortical bone tended to be abnormal in the T1D group,but they did not reach statistical differences.The bone trabecular parameters determined by bone histopathological morphology tended to be abnormal in the T1D group,but did not reach statistical differences.Overall,the trabecular bone density of NOD-T1D mice decreased significantly,and the trabecular bone microarchitecture tended to be abnormal,but there were few abnormal parameters.It might be considered that the duration of T1D was only 6 weeks,and no significant morphology had yet appeared.Insulin,liraglutide monotherapy or combination insulin treatment could improve its reduced trabecular bone density,and linagliptin or combined insulin treatment had no effect on bone density and microarchitecture.3.Serum bone turnover indexes and inflammation indexes in T1D mice and the therapeutic effects of liraglutide and linagliptin(1)The PINP of C57-T1D mice was slightly higher than that of the control group,and did not reach statistical difference(p=0.138).Only the reduction of P1NP in the linagliptin group reached statistical difference(p=0.016).There was no statistical difference in CTX between groups(p=0.405).There was no statistical difference in TGFβ among the groups(p=0.353).(2)The P1NP of NOD-T1D mice was lower than that of the control group,but the difference was not statistically significant(p=0.162).The difference between each treatment group and the untreated group was not significant.There was no significant difference in CTX between the groups(p=0.252).There was no significant difference in TGFβ among the groups(p=0.998).4.RNA-seq and bioinformatics analysis results C57-T1D mice tibiaFrom the C57-T1D mice,we randomly selected three specimens from each group of control group,T1D model group,and liraglutide treatment group as RNA-seq analysis objects.A total of 1031 differential expression genes were found between control group and T1D group,with 936 gene up-regulated and 95 genes down-regulated.Compared with T1D group,a total of 1280 differential genes were found in liraglutide treatment group,of which 443 genes were up-regulated and 837 genes were down-regulated.KEGG pathway analysis showed that these differential genes were mainly enriched in immune inflammation-related signaling pathways.Among them,bone metabolism-related pathways included TNFα signaling pathway,osteoclast differentiation signaling pathway,NFκB signaling pathway,Th 17 cell differentiation pathway,and IL-17 signaling pathway,and PPARγ signaling pathway,etc.5.RT-qPCR verification of differential genes screened by RNA-Seq in C57-T1D mice(1)The expressions of osteogenesis-related genes(Runx2 mRNA,OC mRNA)and osteoclast-related genes(c-fos mRNA,TRAP mRNA)in bone tissue of C57-T1D mice both increased significantly(all p<0.05),and RANKL/OPG mRNA ratio also increased significantly(p=0.033),indicating that the osteoclast process tended to be more active;the expression of PPARy mRNA,TNFa mRNA and IL-17 mRNA in bone tissue of C57-T1D mice all increased significantly(all p<0.05),TGF-β mRNA was not significantly different from control group(p>0.05).(2)Runx2 mRNA,OC mRNA and TRAP mRNA in the liraglutide treatment group were significantly lower than those in the untreated group(all p<0.05),and OC mRNA and TRAP mRNA in the insulin+liraglutide group were significantly lower than those in the untreated group(both p<0.05),the RANKL/OPG mRNA ratio in the liraglutide group and insulin+ liraglutide group was not significantly different from that in the untreated group.In the liraglutide group,PPARγ mRNA and TNFa mRNA were significantly lower than those in the untreated group(both p<0.05),and IL-17 mRNA and TNFa mRNA in the insulin+ liraglutide group were significantly lower than those in the untreated group(p<0.05).(3)The OC mRNA of the linagliptin-treated group was further increased than that of the untreated group,reaching significant difference(p=0.044),and the ratio of RANKL/OPG mRNA in the insulin+linagliptin group was significantly lower than that of the control group(p<0.001).IL-17 mRNA was significantly higher in the linagliptin-treated group than in the untreated group(p=0.007).These results showed that the expression of both osteogenetic and osteoclast-related genes in T1D mice increased,but the increase in the ratio of RANKL/OPG indicated that the osteoclast process tended to be more active;meanwhile,the expression of PPARy and inflammatory factors IL-17 and TNFa all increased.Liraglutide monotherapy or combined insulin therapy inhibited the expression of osteogenetic and osteoclast-related genes,had little effect on the ratio of RANKL/OPG,and inhibited the expression of PPARy and inflammatory factors.RANKL/OPG ratio was significantly reduced in linagliptin+insulin treatment group,indicating osteoclast activity was inhibited.Conclusions:1.There was a decrease in bone density and abnormal bone microarchitecture in T1D mice,as well as excessive osteoclast activation and increased expression of IL-17 and TNFα,suggesting that IL-1 7/TNF α might participate in compromised T1D bone metabolism by activating osteoclast activity,which has laid the foundation for the subsequent in-depth study of the mechanism.2.Liraglutide monotherapy or combined insulin therapy could improve the decreased bone density and abnormal bone microarchitecture in T1D mice while linagliptin had no such effect,and he bone protective effect of liraglutide may be related to its inhibition of inflammatory factors and thus the activity of osteoclasts,which provided new experimental evidence for the clinical selection of hypoglycemic drugs.