The Relationship Between Metabolic Status And Bone Mineral Density In Men And The Effects Of Berberine On Bone Metabolism In Obese Male Rats

Background:Osteoporosis(OP)is a metabolic bone disorder caused by a variety of reasons,characterized by decreased bone mass,microarchitectural deterioration,resulting in decreased bone strength and prone to fragility fractures.People’s quality of life is affected by fractures associated with OP.The most serious fracture is hip fracture,which can lead to high disability rate and mortality rate.It is extremely expensive to treat osteoporosis every year,which brings heavy burden to the family and society.Therefore,it is a question for researchers to explore the risk factors of OP and effective intervention measures.The relationship between obesity and OP has attracted more and more attentions among many affecting factors of OP,but conclusions are controversial.Various studies have shown a protective role of obesity against osteoporosis.With the increasing of research,different results have emerged.Recent evidence suggests obesity may be a risk factor for low bone mass and related fractures.Obese individuals have a high risk of developing hypertension,diabetes mellitus,cardiovascular disease,dyslipidaemia and many other diseases.That is why obesity can reduce life expectancy.But not all obesity have the same risk for related diseases.Individuals in the same body mass index(BMI)category can have substantial heterogeneity of metabolic features.According to metabolic status and BMI,the population can be divided into metabolically healthy normal weight(MHNW)group,metabolically unhealthy but normal weight(MNNW)group,metabolically healthy obese(MHO)group and metabolically unhealthy obese(MUO)group.Studies have indicated that the different obesity subtypes based on metabolic status may be an indicator that can be used to predict the risk of some chronic diseases.Moreover,increasing evidences show that metabolic status are related to bone health.However,these data could not encompass the relationship between bone health and obesity subtypes taking metabolic status into accout.At present,most research on osteoporosis is related to women.With the increasing of OP in men year by year,we should pay more attention to the bone health of men.The purpose of this study is to investigate the relationship between obesity and forearm BMD in different metabolic status to provide theoretical basis for the precise prevention and treatment of OP.Objectives:To analyze the forearm bone mineral density(BMD)and its possible risk factors in a northern Chinese population and explore the relationship between obesity and bone health for providing theoretical basis for the precise prevention and treatment of OP in men.Methods:Data were from epidemiological investigation in Shungeng and Huaiyin communities of Jinan city in China,which were organized by Provincial Hospital affiliated to Shandong Universtity from 2009 to 2010.According to the inclusion and exclusion criteria,850 men were enrolled in the study.According to criteria of World Health Organization(WHO),individuals were divided into normal weight group,overweight group and obesity group.According to BMI,metabolic syndrome(MetS)and insulin resistance index(HOMA-IR),the population were divided into MHNW group,MNNW group,MHO group and MUO group.Analysis of covariance was performed to compare forearm BMD among the groups.The covariates included age,height,weight.Partial correlation analysis and multiple linear regression models were used to explore the association between forearm BMD and clinical parameters.Results:1.Baseline characteristics of subjectsIn the WHO group,no significant differences were found in age,height and smoking among the three groups(P>0.05).Weight,BMI,waist circumference(WC),systolic blood pressure,diastolic blood pressure,high-density lipoprotein(HDL-c),total cholesterol(TC),insulin(INS),low-density lipoprotein(LDL-c),fasting blood glucose(FBG),triglyceride(TG)and HOMA-IR in the overweight and obesity group were higher than that in the normal weight group(P<0.05 or 0.01).When taking metabolic status and BMI into accout for subtype analysis,Weight,BMI,WC,systolic blood pressure,diastolic blood pressure,HDL-c,TG,INS,FBG and HOMA-IR in MUNW group were higher than that in the MHNW group(P<0.05 or 0.01),but there was no difference in TC,LDL-c,age,height and smoking(P>0.05).Higher weight,BMI,WC,systolic blood pressure,diastolic blood pressure,TC,HDL-c,LDL-c,TG,INS,FBG and HOMA-IR were observed in MUO group than those in MHO group(P<0.05 or 0.01).No significant difflerences were found in age,height and smoking between MHO group and MUO group(P>0.05).2.Variation in forearm BMD among different groups(1)Variation in forearm BMD among different groups in WHO criteriaSubjects in overweight and obesity group have higher forearm BMD than normal weight participants in crude(P<0.01),no significant differences were found after adjusting age,height and weight(P>0.05).In middle-young-aged group,individuals in overweight and obesity group have higher forearm BMD than normal weight participants in crude(P<0.01),no significant differences were found after adjusting age,height and weight(P>0.05).In aged group,overweight group has higher forearm BMD than normal weight participants in crude(P<0.01),no significant differences between overweight group and normal weight group were found after adjusting age,height and weight(P>0.05).Obesity also has higher forearm higher forearm BMD than normal weight participants in crude,but there is no difference(P>0.05).(2)Variation in forearm BMD among different groups divided by metabolic status and BMIIn crude,MHO individuals had higher forearm BMD than subjects with MHNW、MUNW and MUO(all P<0.01),MUO individuals had higher forearm BMD than subjects with MHNW and MUNW(P<0.05 or 0.01).No significant difference was found between MHNW group and MUNW group.After adjusting age,height and weight,MHO group had higher forearm BMD than subjects with MUO,there were no significant differences among other groups(P>0.05).Middle-young-aged men with MHO had higher forearm BMD than subjects with MHNW and MUO in crude(P<0.01 or 0.05).After adjusting age,height and weight,MHO group had higher forearm BMD than subjects with MUO(P<0.01),no significant differences were found compared with MHNW group(P>0.05).Aged subjects with MHO had higher forearm BMD than subjects with MHNW and MUNW in crude(all P<0.01),same is true compared MUO group with MHNW and MUNW group(P<0.01 or 0.05).After adjusting age,height and weight,MHO group had higher forearm BMD than subjects with MHNW and MUNW(P<0.05).There were no significant differences compared MUO group with MHNW and MUNW group(P>0.05).3.Partial correlation analysis and multiple linear regressionAge-,height-,and weight-adjusted partial correlation analyses showed that WC,LDL-c,INS and HOMA-IR were negatively correlated with forearm BMD in middle-young-aged obese men.Multiple regression analyses presents HOMA-IR showed significant negative relationship with forearm BMD in middle-young-aged obese men.Conclusions:1.When obesity is defined only by BMI(WHO standard),weight is a good predictor for forearm BMD in middle-young-aged men.2.When taking metabolic status and BMI into accout for subgroup analysis of obesity,the bone health status of obesity with different metabolic status were different.①Obesity with normal metabolism in middle-young-aged men had higher forearm BMD than obesity with abnormal metabolism.②Obesity with normal metabolism in aged men had higher forearm BMD than subjects with normal weight.3.HOMA-IR is a negative predictor of forearm BMD in middle-young-aged obese men.Background:With the development of modern society,the relationship between obesity and osteoporosis(OP)has attracted more and more attentions.Meta analysis showed that the bone mineral density(BMD)of overweight and obese children was higher than that of normal weight children,but the long-term impact of childhood overweight and obesity on bone health at adulthood is not clear.Different study has shown that overweight and obese children have a higher risk of fracture than normal weight children.In part one of the study,after analysis of adult male epidemiological survey data,we found BMD in obesity is higher than that in normal weight,but no difference was found after adjusting for weight.Considering the bone health include not only BMD but also other indicators such as bone strength,the bone health status in obesity needs to be further confirmed.Over 75%of obese people have abnormal metabolism.In this study,obese male SD rats with abnormal metabolism were induced by high-fat diet,and the bone health status in juvenile and adulthood were observed.At present,the benefits for bone health have not been found in the use of drugs to lose weight and improve the metabolic status in obese patients.Berberine(BBR)is a main alkaloid purified from rhizoma coptidis.It has numerous positive effects,including those on blood glucose,blood lipids,antidiarrheal and antibacterial,as well as anti-inflammatory,antitumor et al.In addition,data showed that BBR could improve bone health in some OP animal models.However,the effects of BBR on bone metabolism in obesity with abnormal metabolism has not been reported at home and abroad.This study aims to evaluate the effects of BBR on bone health in obese rats with abnormal metabolism.Objectives:1.To observe the bone metabolism of obese male Sparague Dawley(SD)rats with abnormal metabolism in juvenile and adulthood,including serum calcium(Ca),phosphorus(P),bone formation markers,bone resorption markers,inflammatory indicators,BMD,bone microstructure,bone biomechanical properties et al.2.To observe the effects of BBR on bone metabolism in obese male SD rats with abnormal metabolism.Methods:After acclimatized for 1 week,50 male SD rats(4-week-old)were randomly assigned to one of two diets:normal control diet(ND group,n=20)or high fat diet(HFD group,n=30).Body weight were tested weekly.HFD-induce obese models were successfully established,when body weight and fat percentage of rats in HFD group were significantly higher compared with that in ND group.Then these rats were randomly divided into HFD diet+BBR group(HB group,n=10)and HFD group(HFD diet,n=10),which were respectively intraperitoneally administrated with BBR at a dose of 3mg/kg or equivalent volume of dimethyl sulfoxide(DMSO)once a day for another 10 weeks.Meanwhile,Rats ND group were intraperitoneally administrated with the same volume of DMSO like those in HFD group.Dynamically observe bone health of obese male SD rats in juvenile and adulthood and observe the effects of BBR on bone health in obese male SD rats.Body composition,intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)was measured when rats were at 17 week olds.The blood glucose levels of IPGTT and IPITT in obese male SD rats of juvenile were significantly higher than that of ND group,which suggests obese rats with abnormal metabolism.After being starved for 12 h,the rats(n=10)were anesthetized and sacrificed.The serum was centrifuged at room temperature to test Ca,P,bone formation markers type 1 N-terminal propeptide(P1NP),bone resorption markers c-telopeptide of type 1 collagen(CTX-1),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α).Three-point bending test was performed on the left femur.Micro computed tomography(microCT)examination was conducted on the right femur to observe changes in bone microstructure.After 10 weeks of BBR intervention,the rats at 27 week olds were anesthetized and sacrificed.The serum was collected to test Ca,P,P1NP,CTX-1,IL-1β and TNF-α.Three-point bending test was conducted on the left femur to oberserve bone strength.Bone microarchitecture was investigated by microCT examination on the right femur.Left tibias were stained with HE to observe the number of trabecular and bone marrow adipocytes.Results:1.Body composition,IPGTT and IPITT of obese male SD rats in juvenile(1)Body weight and body composition of obese male SD rats in juvenileRats in HFD group had higher body weight and total fat percentage at the end of 12 week in comparison with ND diet(P<0.01).Body weight in HFD group increased 21.2%compared with that in ND group at the end of 12 weeks.HFD-induce obese male SD rat models were successfully established.(2)IPGTT and IPITT in obese male SD rats in juvenileThe blood glucose levels of IPGTT and IPITT in obese male SD rats of juvenile were significantly higher than that of ND group(P<0.01).The results in IPITT showed insulin sensitivity was significantly declined in obese male SD rats of juvenile compared with ND group.Results show that abnormal glucose tolerlance and low insulin sensitivity in obese rats.2.BMD,bone microstructure and biomechanical properties in obese male SD rats in juvenileWhole body BMD,left femur BMD and spinal BMD in obese rats were significantly higher compared with ND group(all P<0.05).No significant differences were found between them after adjusting weight(all P>0.05).The trabecular Volume bone mineral density(Tb.vBMD),trabecular bone volume/total volume(Tb.BV/TV),trabecular number(Tb.N)were decreased(all P<0.05)and the trabecular separation(Tb.Sp)was increased(P<0.05)in obese rats compared with the ND group.Meanwhile,the cortical volume bone mineral density(Ct.vBMD),cortical bone volume/total volume(Ct.BV/TV)and cortical bone thickness(Ct.Th)had no significant differences between two groups(all P>0.05).No significant differences were found in the maximum load,maximum fracture load,ultimate tensile strength,stiffness,energy absorption,and elastic modulus between two groups in juvenile(all P>0.05).3.BBR improved glucose and lipid metabolism properties in adult obese male SD rats(1)BBR improved glucose metabolism in adult obese male SD ratsAfter 10 weeks of BBR intervention,the blood glucose levels of IPGTT and IPITT in adult obese male SD rats were significantly higher than that of ND group(all P<0.05).However,SD rats in HB group displayed lower levels of blood glucose throughout the IPGTT and IPITT compare with the obese rats(all P<0.05).(2)BBR improved hepatic ester level and decreased the number of bone marrow adipocytes in adult obese male SD ratsHepatic total cholesteryl,free cholesteryl and triglyceride ester in adult obese male SD rats were significantly higher than that of ND group(all P<0.01).However,SD rats in HB group displayed lower levels of hepatic triglyceride ester compared with the obese rats(P<0.01),hepatic total cholesteryl and free cholesteryl hepatic total cholesteryl,free Cholesteryl and triglyceride ester decreased,but there was no significant difference(P>0.05).The results of HE staining in the left tibia samples showed the number of bone marrow adipocytes in obese rats was increased compared with the ND group(p<0.01),while it was dramatically decreased in the HB group compared with obese rats(p<0.01).4.Effects of BBR on Ca,P,bone metabolic index(PlNP and CTX-1),inflammatory marker(IL-1β,TNF-α)NO significant difference were found in Ca,P among three groups(all P>0.05).The bone formation markers P1NP were not changed between obese rats and the ND group in juvenile and adult.Compared with obese rats,P1NP was increased in the HB group(P<0.05).Compared with the ND group,CTX-1 was increased in adult obese rats(P<0.05).Though CTX-1 was decreased in the HB group than that in adult obese rats,there was no significant(P>0.05).Compared with the ND group,IL-1β and TNF-α were increased in obese rats in juvenile and adult(P<0.05 or 0.01).IL-1β and TNF-α were decreased in the HB group than that in adult obese rats(all P<0.01).5.BBR ameliorated bone biomechanical injury in adult obese male SD ratsCompared with the ND group,the maximum load,maximum fracture load,stiffness,energy absorption,ultimate tensile strength were decreased(P<0.01 or 0.05)in adult obese rats.The maximum load,maximum fracture load,stiffness,energy absorption were significantly enhanced(P<0.01 or P<0.05)in the HB group compared with obese rats.Ultimate tensile strength and elastic modulus were improved,but there was no significant difference between the HB group and obese rats(all P>0.05).6.BBR improved bone microstructure damage in adult obese male SD ratsCompared with the ND group,the trabecular volume bone mineral density(Tb.vBMD),trabecular bone volume/total volume(Tb.BV/TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)were significantly decreased(all P<0.01),and the trabecular separation(Tb.Sp)and structure model index(SMI)were increased(all P<0.01)in obese SD rats.Tb.vBMD,Tb.BV/TV,Tb.N,Tb.Sp,SMI were improved,but no significant differences were found(P>0.05)in the HB group compared to obese rats.The cortical volume bone mineral density(Ct.vBMD),cortical bone volume/total volume(Ct.BV/TV)and cortical bone thickness(Ct.Th)had no significant differences among three groups(all P>0.05).The results of HE staining in the left tibia samples showed that the trabecular number was decreased in obese rats compared with the ND group(P<0.01).Conclusions:1.The body weight and total fat percentage of male SD rats fed with HFD in 12 week were significantly increased.The inflammatory indexes of obese rats were significantly higher than those of control group.Abnormal glucose tolerlance and low insulin sensitivity exsit in obese male SD rats.2.Trabecular bone injury has occurred in juvenile obese male SD rats,but the bone strength had no significant change.In adult obese rats,the bone resorption marker increased significantly,and the trabecular bone injury further aggravated,and bone strength parameters were declined.3.BBR improved glucose metabolism and insulin resistance in obese male SD rats,improve inflammatory makers,reduced triglyceride ester in liver and the number of bone marrow adipocytes.4.BBR increased bone formation marker P1NP,reduced bone resorption marker CTX-1,and improved bone microstructure and bone strength parameters,especially in bone strength parameters.