| Part1 The changes of serum ZAG levels in obese patinets with different metabolic statusObjectives:Obesity has become a common disease that threaten the human health worldwide.Compared with metabolically healthy obesity(MHO),patients with metabolically abnormal obesity(MAO)usually have more complications and a poorer prognosis.Zinc-alpha 2-glycoprotein(ZAG)is a novel adipocytokine.Our previous studies have shown that serum ZAG levels were significantly lower in obese patients,and were negatively associated with body weight,body mass index(BMI),waist circumference(WC),hip circumference,waist-to-hip ratio,etc.However,there is no relevant literature about serum ZAG levels in obese patients with different metabolic status.Therefore,this study aimed to compare serum ZAG levels in obese patients with different metabolic status,and further explore the efficacy of serum ZAG in distinguishing the different metabolically phenotypes of obesity.Methods:The study included 193 obese patients from the Obesity Clinic and 32 subjects from the Medical Center in the Peking Union Medical College Hospital.According to body weight and metabolism status,subjects were divided into 4 groups:metabolically healthy non-obese(MHNO,n=32),MHO group(n=40),MAO group(n=104),metabolically abnormal diabetic obese(MADO,n=49).The definition of obesity,metabolically abnormal,and type 2 diabetes mellitus were based on the Chinese Adult BMI Taxonomy,the revised Adult Treatment Panel III of the National Cholesterol Education Program(NCEP/ATPIII),the diagnostic criteria of the American Diabetes Association(ADA),respectively.Serum ZAG and adiponectin levels were measured by enzyme-linked immunosorbent assay(ELISA)kits.The t-test was adopted to compare the difference of measurement data of the two groups.The difference of measurement data between groups was determined by analysis of variance(ANOVA),while the comparison of count data was used by chi-square test.Pearson correlation analysis was used to determine the correlation between ZAG and adiponectin and the various parameters of each group.Linear regression analysis was used to analyze the independent influencing factors of ZAG and adiponectin.Multivariate logistic regression was used to analyze the risk of metabolic abnormalities.The receiver operating characteristic(ROC)curve analysis was used to determine the sensitivity and specificity of serm ZAG and adiponectin for diagnosing metabolic abnormalities.Results:1.Compared to the MHNO group,serum ZAG levels were significantly elevated in the MHO group(8.44±1.14?g/mL vs,8.02±0.98?g/mL,P<0.05).As the severity of metabolic abnormalities increased,serum ZAG levels were progressively decreased in MAO and MADO groups,which decreased by 5.81%and 10.55%of that in MHO group,respectively(P<0.05).No significant difference was found in serum adiponectin levels between MHNO and MHO groups(19.82±6.88?g/mL vs.19.39±6.18?g/mL,P>O.05).Compared with the MHO group,serum adiponectin levels in the MAO and MADO groups were significantly decreased,which decreased by 14.90%and 26.71%,respectively(P<0.05).2.The simple bivariate correlation analysis showed that in all participants,serum ZAG levels were positively correlated with total cholesterol(TC,r=0.145)and negatively correlated with fasting blood glucose(FBG,r=-0.206),2-hour postprandial blood glucose(r=-0.209),oral glucose tolerance test(OGTT)0 min PG(r=-0.206),OGTT 120 min PG(r=-0.209),homeostasis model assessment of insulin(HOMA-IR,r=-0.172)and homeostasis model assessment of adiponectin(HOMA-AD,r=-0.191)(P all<0.05).In all participants,serum adiponectin levels were positively correlated with age(r=0.179),gender(r=0.259),high density lipoprotein cholesterol(r=0.270),quantitative insulin sensitivity check index(QUICKI,r=0.375),and negatively correlated with BMI(r=-0.233),WC(r=-0.317),systemic blood pressure(r=-0.233),diastolic blood pressure(DBP,r=-0.221),alanine transaminase(r=-0.250),aspartate aminotransferase(r=-0.156),free fatty acids(r=-0.286),creatinine(r=-0.156),uric acid(UA,r=-0.314),FBG(r=-0.162),fasting insulin(r=-0.347),OGTT 0 min PG(r=-0.162),HOMA-IR(r=-0.351),adipose tissue insulin resistance(Adipo-IR,r=-0.333)and HOMA-AD(r=-0.558).By stepwise multiple logistic regression analysis in all subjects,total cholesterol(?=0.223),2-hour postprandial blood glucose(?=-0.201)and HOMA-AD(?=-0.155)were independent variables to serum ZAG levels(P all<0.05).In addition,age(?=0.094),DBP(?=-0.115),UA(p=-0.127),HOMA-IR(?=1.119),HOMA-AD(?=-1.375),and QUICKI(?=0.242)were independent variables to serum adiponectin levels(P all<0.05).3.Next,all subjects were stratified into trisections according to ZAG tertiles(lowest:<7.582?g/mL;median:7.582-8.283?g/mL;highest:>8.283?g/mL),and multivariate logistic regression analysis was performed to explore metabolically abnormal risks according to tertiles of ZAG in all subjects.The results showed that compared with subjects in the highest tertile of ZAG,the odds ratio(OR)of metabolically abnormal were 2.406(95%CI 1.214-4.768)for the lowest tertile and 2.985(95%CI 1.474-6.046)for the median tertile.Even after controlling for the confounding effect of age,gender,BMI,blood pressure,blood glucose,blood lipid,uric acid,and other indicators,the increased probability of being metabolically abnormal remained significant in the lowest and median tertile of ZAG.These results indicate that the decreased serum ZAG levels were closely associated with the metabolically abnormal phenotype.Additionally,all subjects were stratified into trisections according to adiponectin tertiles(lowest:<12.620?g/mL;median:12.620-19.300?g/mL;highest:>19.300?g/mL),and multivariate logistic regression analysis was performed to explore metabolically abnormal risks according to tertiles of adiponectin in all subjects.The results showed that the odds ratio(OR)of metabolically abnormal in subjects with low adiponectin levels were 4.371-fold(95%CI 2.452-11.761,P=0.000)higher than those with high adiponectin levels.Even after controlling for the confounding effect of age,gender,BMI,blood pressure,blood glucose,blood lipid,uric acid,and other indicators,the increased probability of being metabolically abnormal in subjects with low adiponectin levels remained significant These results indicate that the decreased serum adiponectin levels were also closely associated with the metabolically abnormal phenotype.4.Serum ZAG could discriminate the metabolically abnormal phenotype with receiver operating characteristic(ROC)curve area of 0.622(95%CI,0.539-0.706,P<0.05).The sensitivity of the diagnosis was 50%and the specificity of the diagnosis was 73.2%.In addition,the area under the curve of the combination of serum ZAG and adiponectin was 0.703,the 95%confidence interval was 0.629-0.776,the diagnostic sensitivity was 62.7%,the specificity was 73.6%.The results showed that the diagnostic value of the combination of serum ZAG and adiponectin in metabolic abnormalities was better than ZAG alone(P<0.05).Conclusions:1.Serum ZAG levels were compensatory elevated in the MHO group,but were progressively decreased in MAO and MADO groups,suggesting that serum ZAG levels were closely related to the body s metabolic status.2.Patients with decreased serum ZAG levels have a significantly higher risk of metabolic abnormalities than subjects with high serum ZAG levels.3.Serum ZAG may be a potential biomarker for the diagnosis of metabolic abnormalities.Combined serum ZAG and adiponectin have greater diagnostic value for metabolic abnormalities.Part 2 The roles and mechanisms of ZAG overexpression/knockdown in the development of obesity/obese miceObjectives:ZAG is a novel adipokine that are closely related to the regulation of obesity and glycolipid metabolism.Our previous study found that ZAG overexpression significantly reduced body weight,fat content and increased insulin sensitivity in high-fat diet-induced obese mice,but whether ZAG early intervention could prevent obesity in mice fed a high-fat diet and the role of ZAG knockdown in obese mice are still unclear.Therefore,in this study,we constructed ZAG-adeno-associated virus(AAV)8 vector and ZAG-shRNA-AAV8 vector,through subcutaneous white adipose tissue injection in order to explore(1)the roles and mechanisms of ZAG overexpression/knockdown in the development of obesity in mice;(2)the roles and mechanisms of ZAG overexpression/knockdown in obese mice.At the cellular level,we explored(1)the effect of stable transfection of ZAG,ie,ZAG endogenous overexpression on the expression of white adipose tissue browning-related genes during differentiation of 3T3L1 cells;(2)the effect of exogenous mouse/human ZAG pure treatment on the expression of white adipose tissue browning-related genes in mature 3T3L1 cells.Methods:Part 2 included animal experiments and cell experiments.Animal experiments are mainly divided into the following four parts:1.The construction of ZAG-AAV8 and ZAG-shRNA-AAV8 virus vectors.Firstly,the pcDNA3.1(-)-mZAG plasmid and the plasmid containing the ZAG-shRNA sequence were verified in 3T3L1/Hepal-6 cells.Then the verified overexpression/knockdown plasmids were separately sent to Shandong Weizhen and Shanghai Heyuan companies,in order to construct ZAG-AAV8 and ZAG-shRNA-AAV8 viral vectors.2.The effect of ZAG-AAV8 intervention(ZAG overexpression)on body weight of high fat fed mice.Firstly,twelve 8-week male C57BL6/J mice were randomly divided into control group,low-dose tail vein group,median-dose tail vein group,high-dose tail vein group,low-dose subcutaneous fat group,high-dose subcutaneous fat group,in order to explore the applicable injection site and dose of ZAG-AAV8 virus vector.Then,forty 8-week-old male C57BL6/J mice were randomly divided into normal diet control group(Group 1,n=10),high-fat diet control group(Group 2,n=10),high-fat diet ZAG overexpression group(Group 3,n=10),high fat diet positive drug group(Group 4.n=10).The bilateral inguinal subcutaneous adipose tissue of the group 1,the group 2 and the left inguinal subcutaneous adipose tissue of the group 3 were injected with 100?L(concentration of 1*1012vg)control virus.The right inguinal subcutaneous adipose tissue of the group 3 was injected with 100?L(concentration of 1*10 2vg)ZAG-AAV8 virus.The group 4 was given empagliflozin(10mg/kg/d)by intragastric administration.From the day of the experimental intervention,the group 1 was given 10%normal feed;the other 3 groups were given 45%high fat diet.Four weeks later,the intraperitoneal glucose tolerance test(IPGTT)and the insulin tolerance test(IPITT)were performed intraperitoneally.The mice were sacrificed 7 weeks later,and the body weight,body fat and blood biochemical parameters of the mice were measured.The morphology of the subcutaneous adipocytes was observed by HE staining.The expression levels of ZAG and uncoupling protein 1(UCP1)in subcutaneous adipose tissue were observed by immunohistochemical staining.The expression levels of ZAG and genes related to glucose and lipid metabolism in epidydimal adipose tissue and liver were measured by RT-qPCR.The differentially expressed genes in subcutaneous adipose tissue of groups 1-3 were searched by RNA sequencing,the signal pathways and biological functions were analyzed by bioinformatics software.3.The effect of ZAG-shRNA-AAV8 intervention(ZAG knockdown)on body weight of high fat fed mice.Twenty-one 8-week-old male C57BL6/J mice were randomly divided into control group(concentration of 1*10 vg,n=5),very low dose group(concentration of 5*109vg,n=4),low dose group(concentration of 5*1010vg,n=4),middle dose group(concentration of 2.5*10 vg,n=5)and high dose group(concentration of 1*1012vg,n=3).All 1ice were injected in the bilateral inguinal subcutaneous adipose tissue.From the day of the experimental intervention,all mice were fed with 45%high fat diet.After 6 weeks,IPGTT and IPITT were performed.After 7 weeks,the mice were sacrificed,and the body weight,body fat and blood biochemical parameters of the mice were measured.The expression levels of ZAG and UCP1 in subcutaneous adipose tissue were observed by immunohistochemical staining.The expression levels of ZAG and genes related to glucose and lipid metabolism in subcutaneous adipose tissue,epididymal adipose tissue and liver were measured by RT-qPCR.The differentially expressed genes in subcutaneous adipose tissue of group 1 and group 4 were searched by RNA sequencing,the signal pathways and biological functions were analyzed by bioinformatics software.4.The effect of ZAG-AAV8 intervention(ZAG overexpression)/ZAG-shRNA-AAV8 intervention(ZAG knockdown)on body weight of high-fat diet-fed obese mice.538-week-old male C57BL6/J mice were randomly divided into normal diet group(n=10)and high-fat diet group(n=43).After 8 weeks,the high-fat diet-fed obese mice model was successfully established,and then all mice were divided into normal diet over-expression control group(Group1,n=6);high-fat diet over-expression control group(Group 2,n=11);high-fat diet ZAG overexpression group(Group 3,n=10);normal diet knockdown control group(Group 4,n=6);high-fat diet knockdown control group(Group 5,n=10);high-fat diet ZAG knockdown group(Group 6,n=12).The bilateral inguinal subcutaneous fat of group 1 and group 2 were injected with 100?L over-expression control virus(concentration of 5*1011vg).The bilateral inguinal subcutaneous fat of group 4 and group 5 were injected with 100?L knockdown control virus(concentration of 2.5*1011vg).The bilateral inguinal subcutaneous fat of group 3 was injected with 100?l ZAG-AAV8 virus(concentration of 5*1011vg).The bilateral inguinal subcutaneous fat of group 6 was injected with 100?l ZAG-shRNA-AAV8 virus(concentration of 2.5*1011vg).After 7 weeks,IPGTT and IPITT were performed,and the mice were sacrificed 8 weeks later,the body weight,body fat and blood biochemical parameters of the mice were measured.Cell experiments are mainly divided into the following two parts:1.3T3L1 cell stably transfected with pcDNA3.1-mZAG was established,and the effect of endogenous overexpression of ZAG on white adipose tissue browning-related genes expression during cell differentiation was explored.2.After 3T3L1 cells were differentiated to mature cells,different concentrations of human/mouse ZAG pure products were administed for 24 hours to observe the effect of exogenous ZAG treatment on the expression of white adipose tissue browning-related genes in differentiated mature 3T3L1 cells.Results:The results of the animal experiment were as follows:1.The construction of ZAG-AAV8 and ZAG-shRNA-AAV8 virus vectors.Compared with the control group that transfected with pcDNA3.1(-)plasmid,after transfection with pcDNA3.1(-)-mZAG,the expression of ZAG gene in 3T3L1 cells was significantly increased,which increased to 18.75-fold of the control group(P<0.05).Then the verified pcDNA3.1(-)-mZAG plasmid was sent to Shandong Weizhen company in order to construct the ZAG-AAV8 vector.The concentration of the control virus provided by the company was 5.31×1013vg/mL,and for ZAG-AAV8 virus the concentration was 5.21 × 1013vg/mL.In addition,the sequence of shRNA3 desighned by Shanghai Heyuan company was GCCAACAAGAAGCTAGCAT.Compared with the control group,after transfection with shRNA3,the mRNA expression of ZAG gene in 3T3L1 and Hepal-6 cells were significantly decreased,which decreased by 34.40%and 59.05%of the control group(P all<0.05).After transfection with shRNA3,the protein levels of ZAG in 3T3L1 and Hepal-6 cells were also significantly decreased,which decreased by 9.78%and 43.81%of the control group(P all<0.05).The ZAG-shRNA3 sequence was further provided to Shanghai Heyuan company for the construction of the ZAG-shRNA-AAV8 vector.The concentration of the control virus provided by the company was 1.73 × 1013vg/mL,and for ZAG-shRN A-AAV8 virus the concentration was 1.44 × 1013vg/mL.2.The effect of ZAG-AAV8 intervention(ZAG overexpression)on body weight of high fat fed mice.ZAG overexpression can reduce the body weight and the weight of subcutaneous,perirenal and brown adipose tissue in high fat fed mice.The results of HE staining of subcutaneous adipose tissue showed that the subcutaneous fat cell volume of the high-fat diet control group(Group 2)was significantly increased compared with the normal diet control group(Group 1).After ZAG-AAV8 injection,the subcutaneous fat adipocytes volume in group 3 was significantly reduced not only in the right subcutaneous white adipose tissue,but also in the left subcutaneous white adipose tissue.In addition,ZAG overexpression can improve glucose tolerance and increase insulin sensitivity in high-fat fed mice.The results of IPGTT showed that after ZAG overexpression,glucose levels at 60 minutes and 90 minutes and the area under the curve of 60-90 minutes of high fat fed mice were significantly lower than that of high fat control group(P all<0.05).IPITT results showed that after ZAG overexpression,glucose level at 30 minutes and the area under the curve of 0-30 minutes of high fat fed mice were significantly lower than the high-fat control group(P all<0.05).Serum biochemical tests found that after ZAG overexpression,serum free fatty acid levels were significantly increased,but uric acid levels were significantly reduced in high-fat fed mice(P all<0.05).The results of immunohistochemistry staining showed that after ZAG-AAV8 injection,compared with the high-fat diet control group(Group 2),the expression levels of ZAG and UCP1 in group 3 were significantly increased not only in the right subcutaneous white adipose tissue,but also in the left subcutaneous white adipose tissue.After ZAG overexpression,the expression of ZAG in epidydimal adipose tissue of high-fat fed mice was significantly increase,which reached to 69.53-fold of the high-fat diet control group(P<0.05);the expression of UCP1 had a tendency to increase;the expression of FAS was reduced,which reduced to 60.96%of the high-fat diet control group(P<0.05).RNA-seq results found that after ZAG intervention,a total of 6588 differentially expressed genes were found in the subcutaneous adipose tissue of mice.A total of 35 signaling pathways were found,including regulation of lipolysis in adipocytes,PI3K-Akt,thermogenesis signaling pathways and so on.3.The effect of ZAG-shRNA-AAV8 intervention(ZAG knockdown)on body weight of high fat fed mice.After ZAG knockdown,the weight of the body weight,epididymal,perirenal and brown adipose tissue of the high fat fed mice have a trend to increase.In addition,ZAG knockdown can aggravate glucose intolerance and insulin resistance in high-fat-fed mice.The IPGTT and IPITT results showed that after ZAG knockdown,the glucose levels at 0 minutes,30 minutes,60 minutes,90 minutes and 120 minutes of the high fat fed mice were tend to be higher than that of high fat control group.The results of immunohistochemistry staining showed that after ZAG knockdown,the expression levels of ZAG and UCP1 in subcutaneous white adipose tissue were significantly decreased when compared with the high-fat diet control group.After ZAG knockdown,the expression of ZAG in subcutaneous adipose tissue and epididymal adipose tissue had a tendency to decrease,which decreased to 40.15%and 83.18%of the high-fat diet control group.RNA-seq results found that after ZAG knockdown,a total of 160 differentially expressed genes were found in the subcutaneous adipose tissue of mice.3 signaling pathways were found.4.The effect of ZAG-AAV8 intervention(ZAG overexpression)/ZAG-shRNA-AAV8 intervention(ZAG knockdown)on body weight of high-fat diet-fed obese mice.In the high-fat diet induced obese mice,ZAG overexpression reduced the body weight of obese mice,while the weight of obese mice tended to increase after ZAG knockdown.The weight of subcutaneous,epididymal and perirenal adipose tissue of obese mice tend to decrease after ZAG overexpression,but tend to increase after ZAG knockdown.In addition,ZAG overexpression can improve glucose tolerance and increase insulin sensitivity in obese mice.IPGTT results showed that after ZAG overexpression,the glucose levels at 30 minutes and the area under the curve of 0-30 minutes and 30-60 minutes of obese mice were significantly lower than the high-fat control group(P all<0.05).IPITT results showed that after ZAG overexpression,the glucose levels at 30 minutes and 60 minutes and the area under the curve of 30-60 minutes of obese mice were significantly lower than the high-fat control group(P all<0.05).ZAG knockdown can aggravate glucose intolerance and insulin resistance in obese mice.IPGTT results showed that after ZAG knockdown,the glucose levels at 60 minutes and the area under the curve of 30-60 minutes of obese mice were significantly higher than the high-fat control group(P all<0.05).IPITT results showed that after ZAG knockdown,the glucose levels at 0 minutes and 120 minutes and the area under the curve of 0-30 minutes of obese mice were significantly higher than the high fat control group(P all<0.05).Serum biochemical tests found that after ZAG overexpression,serum fasting blood glucose and uric acid levels tended to be reduced in obese mice.After ZAG konckdown,serum alanine aminotransferase was significantly increased but free fatty acid was significantly decreased in obese mice(P all<0.05).The results of the cell experiment were as follows:1.After transfection with pcDNA3.1(-)-mZAG,the mRNA expression of UCP1,fibroblast growth factor 21 and white adipose tissue browning-related genes,such as peroxisome proliferator-activated receptor gamma coactivator 1 alpha,cell death-inducing DFFA-like effector a and peroxisome proliferator-activated receptor gamma 2 in 3T3L1 cells were significantly increased,which increased to 2.41-fold,2.40-fold,1.18-fold,1.25-fold and 2.83-fold of the control cells.2.After 3T3L1 cells were induced to differentiate into mature cells,the mature cells were treated with different concentrations of mouse ZAG pure product(0?g/mL,2?g/mL,5?g/mL,lO?g/mL,15?g/mL,20?g/mL)for 24 hours.RT-qPCR and western blot results showed that 5?g/mL mouse ZAG pure product could significantly increase white adipose tissue browning related genes such as UCP1,CIDEA and FGF21 expression levels in differented mature 3T3L1 cells(P all<0.05).In addition,after treated with 10?g/mL human ZAG pure product for 24h,the expression of UCP1 in the differentiated mature 3T3L1 cells have a tendency to increase,but did not reach statistical difference.Conclusions:1.Subcutaneous adipose tissue injections of adeno-associated virus vectors are more effective than tail vein injections.After ZAG-AAV8 intervention(ZAG overexpression),the expression of ZAG in the subcutaneous adipose tissue of mice was significantly increased.After ZAG-shRNA-AAV8 intervention(ZAG knockdown),the expression of ZAG in the subcutaneous adipose tissue of mice was significantly decreased.2.During the process of obesity,ZAG overexpression reduced the body weight and the subcutaneous,perirenal,brown fat content of mice fed with high fat,improved glucose tolerance and increased insulin sensitivity in high-fat fed mice;while ZAG knockdown had a tendency to increase the body weight and fat content of high fat fed mice.The above effects of ZAG may be achieved by activating signal pathways involved in regulation of lipolysis in adipocytes,PI3K-Akt and thermogenesis.In addition,after ZAG overexpression,the expression of UCP1 in subcutaneous adipose tissue of high-fat fed mice had a tendency to increase.At the cellular level,the expression levels of white adipose tissue browning-related genes in 3T3L1 preadipocytes stably transfected with pcDNA3.1(-)-mZAG plasmid were also significantly increased,which suggests that white adipose tissue browning may be another important mechanism of ZAG,and this effect may be a direct effect.3.After the establishment of the obesity model,ZAG overexpression can reduce the body weight and the fat content,improve the glucose tolerance and increase the insulin sensitivity of obese mice.ZAG knockdown can aggravate the impaired glucose tolerance and insulin resistance of obese mice.At the cellular level,exogenous ZAG treatment can promote the expression of white adipose tissue browning related genes in the differentiated mature 3T3L1 cells.The results of cell experiments showed that white adipose tissue browning is one of the important mechanisms of ZAG,and this effect is a direct effect. |
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