| Background Bone used to be regarded as an inert organ primarily functioning in sclerotin metabolism of calcium and phosphorus.Recent studies whereas explicitly suggest that bone exhibits biological activity as well.By means of autocrine,paracrine or secretion into the blood,bone synthesizes or secretes a variety of active substances,such as bone regulatory protein,growth factors and inflammatory factors,participating in metabolism and functional regulation of specific organs and tissues.Therefore,bone also functions as an active endocrine organ and participates in various metabolic regulation of the body.Osteocalcin(OC)secreted by osteoblasts is involved in the regulation of energy consumption,glucose metabolism,insulin secretion and lipid metabolism,while patients with type 2 diabetes often suffer disorders of glycolipid metabolism,atherosclerosis(AS)and nonalcoholic fatty liver Disease(NAFLD).There is believed to be specific relation of osteocalcin with AS and NAFLD,while only a few studies has been conducted focusing on this issue.Furthermore,previous studies mostly focus on the correlation between serum OC and glucolipid metabolism among three types of glucose tolorence patients,in subjects with normal glucose tolerance,impaired fasting glucose regulation and type 2 diabetes mellitus(T2DM),without ruling out the interferences of hypoglycemic,hypolipidemic drugs and abnormal bone metabolism.If male T2DM patients with normal bone mineral density and without hypoglycemic and hypolipidemic therapy are selected as the research objects,studies excluding the interferences of hormone,hypoglycemic&hypolipidemic drugs and abnormal bone metabolism could be accomplished,making it possible to reveal the relation between OC and glucolipid metabolism in early phase of diabetes mellitus,to further clarify the function mechanism of OC,to provide new sights,guidance,and measures in treating diabetes mellitus and interrelated complications.Recent studies have shown that osteocalcin participates in the regulation of energy consumption,glucolipid metabolism and insulin secretion,and plays an important role in the complex pathways between bone and organs regulating energy metabolism.Liver,as a central metabolic organ,is also involved in many metabolic processes.Now one question may arise that whether their's a connection between OC and liver diseases caused by metabolic disorders such as NAFLD?At present,reports on the correlation between osteocalcin and diabetes mellitus complicated with NAFLD are rare and inconsistent.It is necessary to explore the correlation between OC and T2DM complicated with NAFLD,aiming to provide new principles and approaches for the prevention and treatment of diabetes mellitus and NAFLD.Besides,some studies reveal that glucose metabolism disorders,lipid metabolism disorders,NAFLD are among the risk factors of AS,and some other studies have suggested that OC and glucose metabolism,lipid metabolism as well as NAFLD are closely related,so what is the correlation between serum OC and AS?It is worthwhile to to evaluate the correlation between serum OC and CIMT in newly diagnosed males with T2DM,to determine the relation between serum OC and diabetic macroangiopathy,with the intention to provide evidence for early prediction,evaluation,diagnosis and treatment of diabetic macrovascular complications.In summary,newly diagnosed male T2DM patients,with normal bone mineral density,and without hypoglycemic/hypolipidemic therapy,are worthy of study in the matter of OC.By analyzing the correlation between OC and glycolipid metabolism,NAFLD and AS in this population,in-depth model and process of the interaction mechanism may be established,which would for certain contribute to lay a solid foundation for further studies of related diseases and clinical practices such as translational medicine intervention.Objective This study is initiated to investigate the relation between OC and glycolipid metabolism,the relation between OC and carotid atherosclerosis,and the relation between OC and NAFLD among newly diagnosed male T2DM patients.The results will provide guidelines for clinical intervention utilizing OC or OC analogues in the treatment of diabetic macrovascular disease and diabetes with NAFLD in the future.MethodsStudy1:(1)A total sample set of 105 male patients is collected.These patients are initially diagnosed as T2DM,with normal bone mineral density,and have not received hypoglycemic and hypolipidemic drug therapy.According to the level of HbA1c,these T2DM patients are divided into 3 groups:group 1A(HbA1c<7%),group 1B(7%<HbA1c<9%)and group 1C(HbA1c<9%).The median of serum OC is used as the dividing line,and all the samples are divided into 1D group and 1E group.(2)Personal and medical treatment history of each patient is collected.Clinical data including height,body weight,body mass index(BMI),fasting overnight venous blood,serum biochemical indicators,serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and liver function are collected from each patient.Liver function indicating parameters,such as Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),?-Glutamyl Transpeptadase(GGT)are collected with standard enzymatic method.Fasting blood glucose(FBG)level is measured with hexokinase method;fasting insulin(FINS)is determined by electrochemiluminescence;glycosylated hemoglobin(Hemoglobin A1,HbA1c)is determined by phase chromatography;fasting serum OC levels is determined by electrochemiluminescence.(3)We use HOMA-IR(Homeostasis Model Assessment of Insulin Resistance index)to evaluate insulin resistence activity,and HOMA-?(Homeostasis Model Assessment of?cell)to evaluate function of insulin?cells.Formulas are given:HOMA-IR=FBG×FINS/22.5;HOMA-?=20×FINS/(FBG-3.5)(%).(4)Data collecting and logging is carried out using electronic spreadsheet code Excel,statistical analysis is carried out using SPSS(Statistical Package for the Social Sciences)V23.0.The variation tendency of serum OC among each group and its correlation with other metabolic indices are analyzed to clarify the relation of serum OC with glucose metabolism and lipid metabolism.Study2:(1)A total of 90 newly diagnosed male patients are selected,with T2DM,with normal bone mineral density,without hypoglycemic and hypolipidemic drug therapy.SBP,DBP data is collected.Serum Albumin(ALB)is measured by automatic biochemical analyzer.C-Reactive Protein(CRP)is measured by scattering nephelometry method.Homocysteine(HCY)is measured by cyclic enzymatic method.Blood uric acid(BUA)is measured by oxidase assay method.Blood routine test is performed by Sysmex XE2100 automatic blood analyzer.The rest of clinical data is collected and processed the same way as in Study1.(2)According to the guidelines for diagnosis and treatment of non-alcoholic fatty liver disease(Chinese Liver Disease Association,revised edition 2010),patients can be diagnosed as NAFLD in presence of these symptoms:1)No history of drinking or alcohol consumption does not exceed140g/week for men.2)Excluding viral hepatitis,drug-induced liver disease,total parenteral nutrition,hepatolenticular degeneration,and other factors which can lead to specific diseases of fatty liver.3)Histological changes of liver biopsy conformed to the pathological diagnostic criteria of fatty liver disease.Since liver biopsy histological diagnosis is usually difficult to obtain,clinical diagnosis of NAFLD in medical practice is based on:1)Imaging of liver are consistent with diagnostic criteria for diffuse fatty liver and there are not other causes available to explain it;2)Serum transaminase and/or glutamyl transpeptidase(GGT)levels in patients with metabolic syndrome and its related components are continuously increasing for more than half a year.Right after weight loss and insulin resistance are improved,the abnormal zymogram and imaging findings are improved or restored to normal,and the diagnosis of NAFLD can be clearly made.(3)Patients are divided into 2 groups:group 2A(non-fatty liver group,T2DM without NAFLD)and group 2B(fatty liver group,T2DM with NAFLD).Set NAFLD Fibrosis Score(NAFLDFS)equals 0.676 as the cut-off point,group 2B is divided into 2 subgroups:group 2C(low risk of liver fibrosis group,NAFLDFS<0.676)and group 2D(liver fibrosis group,NAFLDFS<0.676).(4)Statistical analysis is performed utilizing SPSS V23.0.Serum OC levels and other metabolic indices are analyzed among each group and compared between groups.The relation between serum OC and NAFLD in newly diagnosed males with diabetes is further explored.Study3:(1)A total of 61 newly diagnosed male patients are enrolled,with T2DM,with normal bone mineral density,without hypoglycemic and hypolipidemic drug therapy.Clinical data is collected as in Study1 and Study2.(2)Device is GE LOGIQ9,the peripheral blood vessel probe frequency is set to 7.5MHz.During the examination,the distal wall of the bilateral common carotid artery and the proximal segment of the internal carotid artery are separated from the vessel wall at 1.0-1.5cm.The vertical distance between the anterior and posterior intima of the artery and the endocardial interface are measured.The intima-media thickness of the anterior and posterior wall of the common carotid artery,as well as carotid artery intima-media thickness(CIMT)i.e.the vertical distance between the anterior and posterior intima of the artery including the endocardial interface,are measured respectively.CIMT<1.0mm is regarded as normal;1.0mm?CIMT<1.5mm is regarded as intima thickening;CIMT?1.5 mm,with irregular wall swelling and thickening,and with local structural changes of convex lumen,are regarded as atherosclerotic plaque formation.According to CIMT magnitude,newly diagnosed male diabetes mellitus patients are divided into normal carotid intima-media group(group 3A)and abnormal carotid intima-media group(group 3B),the latter including 2 sug-groups,i.e.carotid intima-media thickening group(group 3C)and carotid atherosclerotic plaque group(group 3D).(3)Statistical analysis is performed using SPSS V23.0 to investigate difference between the gropus regarding OC level and the relationship between serum OC and CIMT in each group.ResultsStudy1:(1)Comparison of indices between the groups in strict accordance with HbA1c level:There are significant differences in HOMA-IR and HOMA-?among group 1A,1B and 1C(P<0.05).From group A to group B and then to group C,HOMA-IR shows a gradual upward trend and HOMA-?a gradual downward trend.Serum OC decreases from group A to group B and then to group C(P<0.05).TG and FBG increases gradually from group A to group B and then to group C.TG and FBG values in group1B and group 1C are significantly higher than those in group 1A(P<0.05),but there is no significant difference between groups 1B and 1C(P>0.05).FINS increases gradually from group A to group B and then to group C.FINS in group 1C is higher than that in group 1A and group 1B(P<0.05),whereas there is no significant difference between group 1A and group 1B(P>0.05).(2)Comparison of various indices in accordance with OC levels:There are no significant differences in Age,TC,FINS,LDL-C and HDL-C between group 1D and group 1E.Compared with group 1E,BMI,HbA1,FBG,HOMA-IR and TG increases significantly(P<0.05),while HOMA-?decreases(P<0.05).(3)Pearson correlation analysis between serum OC and other indicators:Pearson correlation analysis shows that there is no correlation between serum OC and Age,FINS,TC,HDL-C and LDL-C(P>0.05).Serum OC is negatively correlated with BMI,FBG,HOMA-IR,TG and HbA1c(P<0.05),and positively correlated with HOMA-?(P<0.05).(4)Multiple linear regression analysis of serum OC and various metabolic indicators.In order to evaluate which variables are independent influencing factors of serum OC,multiple linear regression analysis was performed with serum OC as the dependent variable.The results show that FBG(?=-1.512,P=0.042),HbA1c(?=-0.032,P<0.001)and HOMA-IR(?=-1.077,P=0.042)are independent negative factors for serum OC.Study2:(1)Comparing the clinical data and biochemical indicators between group 2B(T2DM with NAFLD group)and group 2A(T2DM without NAFLD group),the indices including Age,BMI,HbA1c,FINS,HOMA-IR,TG,ALT,AST and CRP of group 2B are significantly higher than those in group 2A(P<0.05).(2)In comparison of group 2A,group 2C(low-risk group of liver fibrosis)and group 2D(liver fibrosis group),results shows that FINS,CRP in group 2D are significantly higher than those in group 2A and group 2C(P<0.05);HbA1c,FINS,HOMA-IR,TG,ALT and CRP of group 2C are significantly higher than those of group 2A(P<0.05).The level of serum OC decreases gradually from group 2A to group 2C and then to group 2D,and there is significant difference between these three groups(P<0.05).(3)Linear correlation analysis between OC and various indicators:OC is negatively correlated with age,BMI,fatty liver(normal group,low-risk group of liver fibrosis,liver fibrosis group),HbA1c,HOMA-IR,TG,ALT,AST and GGT(P<0.05);and is positively correlated with HOMA-?.(4)Correlation analysis between NAFLDFS and clinical/biochemical indicators.Pearson correlation analysis of NAFLDFS and various indicators in all patients with fatty liver shows that,excluding the variables constituting NAFLDFS formula such as age,BMI,AST and ALT,NAFLDFS,NAFLD is positively correlated with HbA1c,FINS,HOMA-IR,TG,CRP and other variables(P<0.05),and negatively correlated with variables such as OC and GGT(P<0.05).As for Logistic regression,we define liver fibrosis as grouping variable and its assignment,id est,liver fibrosis group=1;liver fiber low-risk group=0.Liver fibrosis as dependent variables,HbA1c,FINS,HOMA-IR,TG,GGT,CRP and OC as independent variables,the results show that HOMA-IR is an independent risk factor for liver fibrosis in T2DM patients with NAFLD;OR(Odds Ratio)is 1.134;95%CI is(1.046,1.229).OC is a protective factor for liver fibrosis in T2DM with NAFLD,with an OR of 0.451,with 95%CI(0.207,0.985).(5)Logistic regression analysis of influencing factors of T2DM combined with NAFLD:taking fatty liver(no fatty liver=0,with fatty liver=1)as the dependent variable and osteocalcin as the independent variable(samples are classified into 4groups by quartile division method according OC level).For the four groups,the first group had the lowest level of osteocalcin and the fourth group had the highest level of osteocalcin.Take parameters such as age,TC,TG,LDL-C,AST,ALT,GGT,HbA1c and BUA as covariates.The factor two-category logistic regression model is analyzed,and the independent variables and covariates are entered into the equation using Enter method.After adjusting age,TC,TG,LDL-C,AST,ALT,GGT,HbA1c and BUA in turn,we find that osteocalcin is still an independent negative factor for fatty liver.The risk of fatty liver in the first group is 8.049 times higher than that in the fourth group,indicating that the lower the osteocalcin,the higher the risk of developing fatty liver,and the relationship is independent of age,blood lipid,BUA,liver function,blood uric acid,blood glucose and other factors.Study3:(1)Comparison of clinical data and biochemical characteristics between normal CIMT group(group 3A)and abnormal CIMT group(group 3B):Compared with normal CIMT group,age,HbA1c,SBP,TG and CRP significantly increases in abnormal CIMT group(P<0.05),and OC significantly decreases(P<0.05).(2)Comparison of clinical data and biochemical characteristics between group 3A,3C and3D.There are significant differences in OC,HAB1C,TG and CRP among the three groups(P<0.05).HbA1c and TG increases from group 3A to group 3C and then to group 3D(P<0.05).SBP and TG in group 3C and 3D are significantly lower than that in group 3A(P<0.05),but there is no significant difference between group 3C and group 3D(P>0.05).From group 3A to group 3C and then to group 3D,the level of serum OC exhibits a decreasing trend(P<0.05).There are significant differences in serum OC level between the three groups(P<0.05).(3)Comparison of smoker percentage.The percentage of smokers is 29.41%in group 3A,43.48%in group 3C,and 66.67%in group 3D.There is no significant difference among the three groups(?~2=5.473,P=0.065).The percentage of smokers is 29.41%in group 3A,and 54.54%in3B.There is no significant difference between the two groups(?~2=3.106,P=0.078).(4)Comparison of incident fatty liver.The percentage of fatty liver is 35.29%in group3A,69.57%in group 3C and 76.19%in group 3D.There are significant differences among the three groups(?~2=7.520,P=0.023).The percentage of fatty liver in 3A group was 35.29%,and 72.73%in group 3B.There is significant difference between the two groups(?~2=7.315,P=0.007).(5)Correlation analysis between serum OC and other indicators.Serum OC is negatively correlated with FBG,HOMA-IR,TG,HbA1c and CRP(P<0.05),and is positively correlated with HOMA-?(P<0.05).(6)Spearman correlation analysis between CIMT and various metabolic indicators.Correlation analysis of CIMT(assignment:normal=0;thickening=1;plaque=2)and relevant indicators show that CIMT is positively correlated with age,SBP,HbA1c,HOMA-IR,TG CRP and BUA;is negatively correlated with serum OC.Choosing CIMT as the dependent variable,multiple linear regression analysis is performed with age,HbA1c,SBP,TG,CRP,OC,and T2DM combined with NAFLD(assignment definition:without=0;with=1),and the results show that:1)HbA1c(?=0.860,P=0.017)is an independent positive(risky)influencing factor of CIMT,OR is 1.446,95%CI is(1.102,4.654);2)T2DM combined with NAFLD(?=0.387,P=0.041)is an independent positive(risky)influencing factor of CIMT,OR is 1.180,95%CI is(1.026,5.357);3)Serum OC(?=-1.005,P=0.034)is an independent negative(protective)influencing factor,OR is-0.823,95%CI is(0.322,0.967).Conclusions Taking the newly diagnosed male T2DM population as the research object,the following conclusions are drawn.1)With the increase of glucose metabolism disorders,insulin disorder worsens,insulin resistance worsens,islet?-cell function declines,serum OC level decreases.With the increase of osteocalcin level,BMI decreases,glucolipid metabolism disorder is improved,insulin resistance is improved,and islet?cell function is improved.These observations suggest that OC may be involved in the regulation of glycolipid metabolism and in the improvement of insulin resistance.2)T2DM complicated with NAFLD patients have earlier onset age,higher body mass index,more severe insulin resistance,and more severe disorders of glucolipid metabolism than patients with T2DM alone.Osteocalcin is probably a protective factor for T2DM complicated with NAFLD case and T2DM combined with NAFLD fibrosis case.Insulin resistance is probably a risk factor for T2DM with NAFLD fibrosis case.3)T2DM complicated with CIMT abnormalities patients are more severe in glucolipid metabolism disorders and chronic inflammation than those with T2DM alone,and higher prevalence of complicated NAFLD.HbA1c and T2DM complicated with NAFLD are two independent risky factors for CIMT abnormalities;OC is an independent protective factor for CIMT abnormalities.OC may improve atherosclerosis by improving glycolipid metabolism,insulin resistance,and NAFLD. |
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