Effects Of Adipokine ZAG On The Browning Of White Adipose Tissue In Mice And Its Mechanism

Adipose tissue is one of the important metabolic organs in animals.It can be divided into white,beige and brown fat.White adipose tissue is mainly stored energy,while brown and beige adipose tissue is dominated by heat production.Some studies have reported that white fat can be transformed into the phenotype of beige adipocytes in cold or specific stimulant,thereby burning fat,increasing energy consumption and improving obesity.Other studies have found that adiponectin can be combined with T-cadherin to promote the browning of subcutaneous fat by increasing the proliferation of macrophages.And zinc alpha 2 glycoprotein(ZAG)is a new type of lipid mobilization factor was found in recent years.Reports think ZAG can promote lipolysis,increasing fatty acid oxidation,reduced weight and fat content.Recent studies have reported that ZAG can promote the browning of adipocytes,however,the study on the relationship between ZAG and browning of white fat is not clear.Therefore,this experiment aims to establish a model of obesity and cold stress mice,and explore the influence of ZAG on the browning of white adipose tissue and its molecular mechanism in both cases.1 Effects of ZAG plasmid injection on browning of white adipose tissue in high-fat induced obese miceIn this experiment,4 weeks year old C57BL/6J male mice were randomly divided into two groups.The control group was fed normal diet and high-fat group was fed high-fat diet.The obese model was established after 8 weeks,and the normal diet mice were divided into two groups:the injection of empty plasmid group(ND)and the injection of ZAG recombinant plasmid group(NDZ),The high-fat diet mice were also divided into two groups:the injection of empty plasmid group(HF)and the injection of ZAG recombinant plasmid(HFZ).Meanwhile,the mice were injected with plasmid once every three days for eight times.Recording the weight gain per week,and collected epididymal fat and blood samples.The results showed that the body weight in HF group was significantly increased compared with ND group(P<0.01).The ZAG protein levels in NDZ and HFZ groups were significantly increased compared with ND and HF group after injection plasmids(P<0.01),also,the weight of mice was significantly decreased(P<0.05).In high-fat-diet induced mice,ZAG recombinant plasmid significantly reduced the weight of epididymis fat in mice(P<0.05),but did not affect the normal diet mice.The level of serum NEFA was significantly decreased after injection of ZAG recombinant plasmid(P<0.05).The levels of serum CHOI,HDL-C and LDL-C in the HFZ group were significantly lower than HF group(P<0.05).The mRNA level of browning marker CD 137 in NDZ group was significantly higher than ND group(P<0.05),The mRNA levels of browning marker UCP1、CD137 and energy metabolism factor UCP3、C/EBPβ in HFZ group was significantly higher than HF group(P<0.05).In addition,the protein levels of browning and energy metabolism factor CD 137、C/EBPβ in NDZ and HFZ group were significantly higher than ND and HF group(P<0.05),there was no significant change in other lipid metabolism factors.The results showed that ZAG could reduce the fat deposition and improve the obesity by promoting the browning of epididymis fat and energy metabolism in high-fat-diet induced obese mice.2 Effects of ZAG gene deficiency on browning of white adipose tissue in cold stress miceIn this experiment,a cold stress model was used to induce the browning of white adipose tissue,and the effects of ZAG deletion were investigated on the browning of white fat and the molecular mechanism.Experiment using ZAG knockout C57BL/6J male mice of 8 weeks,and it will be randomly divided into two groups after a week of adaptation,a group in 22℃ environment,another group in 6℃ environment.There were also wild type mice as a control group,four groups of mice(WT 22℃,ZAG KO 22℃,WT 6℃,ZAG KO 6℃)were tested for a week and the weight of each group was recorded.At the end of the one week,anaesthesia was performed by intraperitoneal injection of pentobarbital sodium,and the blood was collected in the abdomen,and the subcutaneous fat,epididymis and brown fat were collected in mice,and the weight was recorded.The results showed that the protein level of ZAG was significantly increased after cold stress in wild-type(WT)mice(P<0.05).ZAG protein was not expressed in ZAG knockout mice.In addition,the ZAG mRNA levels of subcutaneous fat,brown fat and epididymis fat in wild-type mice were significantly increased after cold stress(P<0.05).In the case of cold stress,the weight of wild-type mice was significantly lower than ZAG knockout mice(P<0.01).And the weight of subcutaneous fat(inguinal fat and anterior-subcutaneous fat),epididymis fat,muscle and the ratio of subcutaneous fat to body weight in WT mice were significantly lower than ZAG knockout mice(P<0.05).In the case of cold stress,the expression of subcutaneous fat browning marker CD 137 and Tbxl in wild-type mice was significantly higher than ZAG knockout mice(P<0.05),while no significant changes were observed under normal temperature conditions.In cold stress condition,subcutaneous lipolysis factors HSL and ATGL,energy metabolism factor PGC1α,UCP3 and C/EBPP levels in WT mice was significantly higher than ZAG knockout mice(P<0.05).The mitochondrion-encoded gene in subcutaneous fat of wild-type mice was significantly higher than ZAG knockout mice(P<0.05).In cold stress condition,the subcutaneous fat protein levels of β3-AR,PPAR-y and P-P38 in the WT mice was significantly higher than ZAG knockout(P<0.05).Under the cold stress condition,the mRNA levels of browning marker,lipolysis and energy metabolism factor of epididymal fat and energy metabolism factor of brown fat in wild-type mice was significantly higher than ZAG knockout mice(P<0.05).The above results show that cold stress increases the expression of ZAG in the body.ZAG deficiency inhibits energy consumption and lipid mobilization in adipose tissue,and inhibits the browning of white fat.This effect is achieved through theβ3-AR/P-P38/PPAR-y signaling pathway.3 Effects of ZAG plasmid injection on browning of white adipose tissue of ZAG knockout mice in cold stressIn this experiment,ZAG plasmid was injected into ZAG knockout mice and explores the effect of ZAG on browning of white adipose tissue of ZAG knockout mice in cold stress.Experiment using ZAG knockout C57BL/6J male mice of 8 weeks,and it will be randomly divided into two groups,a group of injection of empty plasmid(Control 6℃),another group of injection of ZAG recombinant plasmid(ZAG 6℃).Two groups of mice were placed in 6℃ environment in captivity for a week.The subcutaneous fat,epididymis and brown fat were collected and weighed after a week.The results showed that the expression of ZAG in subcutaneous fat increased significantly after injection of ZAG plasmid.The weight of subcutaneous fat(including inguinal fat and anterior-subcutaneous fat)and epididymal fat after injection of ZAG plasmid was significantly lower than control group(P<0.05).In the case of cold stress,the protein levels of the subcutaneous fat browning marker UCP1,Tbx1 and energy metabolism factor PGC1,UCP3,and C/EBPβ in the ZAG plasmid group were significantly higher than in the control group(P<0.05).The mitochondrial structure of subcutaneous adipose tissue of was better in the ZAG group,and the internal structure of mitochondrial in the control group was not complete.In cold stress condition,epididymal fat browning marker UCP1,Prdm16,CD137 and lipolysis factor ATGL,energy metabolism factor UCP3,C/EBPβ,CPT1A mRNA levels in the ZAG group was significantly higher than in the control group(P<0.05).The mRNA levels of energy metabolism factor UCP3,C/EBP and CPT1B in ZAG group were significantly higher than in the control group in brown fat(P<0.05).The above results showed that the exogenous addition of ZAG increased the fat consumption and decreased the fat deposition in ZAG knockout mice of cold stress.And ZAG promotes the browning of white fat,while improving mitochondrial function,increasing energy consumption and promoting heat production.4 Effects of transfection ZAG plasmid on browning of 3T3-L1 adipocytesIn the previous study,we found that ZAG promotes the browning of white fat in obese and cold stress mice.In this part of the study,we selected 3T3-L1 adipocytes for the experiment,and studied the effect of ZAG on the browning of adipocytes in vitro.The results showed that the expression of ZAG in adipocytes increased significantly after injection of ZAG plasmid(P<0.05).Overexpression of ZAG significantly increased the protein level of browning marker UCP1,Tbxl and energy metabolism factor PGCla and CPT1A(P<0.05).The mitochondrial membrane potential was negatively correlated with the uncoupling protein,and ZAG significantly reduced the mitochondrial membrane potential in adipocytes(P<0.05).The increase of mitochondria content was a key characteristic of the Browning of white fat,and the mitochondrial content of adipocytes in the ZAG group was significantly higher than in the control group(P<0.05).In addition,ZAG significantly increased the expression of β3-AR and P-P38 protein(P<0.05).The above results show that ZAG overexpression can induce the 3T3-L1 adipocytes transformation into the beige fat cells,accompanied by increased mitochondrial content and the increase of the energy metabolism.The possible mechanisms of this regulation are dependent on the β3-AR/P-P38 signaling pathways.