Effect Of Liraglutide On Marker Gene Expression In Brown Adipose Tissue Of High Fat Fed Rats

Obesity is a clinical condition caused by variety of factors,with increase in the volume and the number of fat cells in the body.It is an important risk factor for many diseases,such as type 2 diabetes,metabolic syndrome,cardiovascular diseases,nonalcoholic fatty liver disease,etc.However,not all the adipose tissue is "baleful" in the body.According to the structure and function of the fat cells,adipose tissue in the body is divided into two types: white adipose tissue and brown adipose tissue.White adipose tissue,know as "adipose tissue",is widely distributed in the subcutaneous and internal organs,mainly composed of white fat cells,its function is storing excess energy in the form of triglycerides in the body;Brown adipose tissue is mainly distributed in nape,armpit and scapular region,and it is composed of brown fat cells,which are rich in mitochondria.The physiological functions of brown adipose tissue is shivering thermogenesis,it can resolve and consume excess fat by uncoupling action.In addition to the classic brown fat cells and white fat cells,people recently found the beige fat cells,which were induced in white adipose tissue depots in response to "browning process",beige fat cells appear in white fat depots and have similarcharacteristics with brown adipocytes.Liraglutide is an analogue of glucagon-like peptide(GLP-1),it can stimulate insulin secretion,and reduceglucagon secretion in glucose concentration dependent patternsat the same time.Therefore,liraglutide is mainly used for the treatment of type 2 diabetes in clinical.However,recent studies have found that GLP-1 can promote differentiation of brown fat precursor cells to brown fat cells,and by injecting GLP-1 agonists in ventricle,it can increase the activity of brown adipose tissue,and promote thermogenesis of brown adipose tissue.This study discusses the influence of liraglutide on the function of brown adipose tissue,reveals the possible mechanisms of liraglutide reducing weight,by injecting liraglutide to high fat fed rats,and detecting expression of marker gene in brown adipose tissue.This study can provide new ideas for the prevention and treatment of obesity and related metabolic diseases in future.Objective: To research effect of liraglutide on marker gene expression in brown adipose tissue of high fat fed rats,and to discuss the relationship between this effect and the concentration of liraglutide.Methods:Male healthy SD rats(weighted 240g-260g)were used in the study.After a week of acclimation feeding,the rats were divided into 5groups: normal control group(Con),high-fat control group(HF),low dose of liraglutide group(Lira-L)(0.4mg/kg/d),middle dose of liraglutide group(Lira-M)(0.6mg/kg/d),and high dose of liraglutide group(Lira-H)(0.8mg/kg/d),n=8.Con group were fed with standard chow diet with the energy contents as follows: 65.5% calories from carbohydrate,10.3% calories from fat,and 24.2% calories from protein with total energy of 348kcal/100g;HF,Lira-L,Lira-M,and Lira-H group were fed with a high-fat-diet with the energy contents as follows: 20.1% calories from carbohydrate,59.8% calories from fat and 20.1% calories from protein with total energy of 501kcal/100 g.The energy were equal among each group every day.The 6mg(1ml)liraglutide was dissolved in 11 ml normal saline,the concentration of this mixture is 0.5mg/ml.Lira-L group,Lira-M group,and Lira-H group were subcutaneously injected with 0.4mg/kg/d,0.6mg/kg/d,0.8mg/kg/d liraglutide respectively,Con and HF group were subcutaneously injected with equal normal saline.At the end of 12 th week,all the rats were weighed,after weigh the rats were sacrificed,brown adipose tissue in scapular region were collected for weigh and detection.HE staining was used to observe morphology of brown adipose tissue;RT-PCR were used to analysis the mRNA expression of uncoupling protein 1(UCP1),peroxisome proliferatorsactivated receptorsγ(PPARγ),peroxisome proliferator-activated receptor-γcoactivator 1α(PGC-1α),cell death-inducing DFFA-like effector A(CIDEA),CCAAT enhancer binding proteinβ(CEBPβ),PR domain containing 16(PRDM16);Western Blot was performed to evaluat the protein expression levels of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβ,PRDM16.Results:1.Rats weight and brown fat weight at the end of 12 th week:(1)body weight: There was no significant difference between two groups in body weight at the beginning of the experiment.After 12-week intervention,the body weight in Lira-L group,Lira-M group were lower than HF group and Con group,the body weight in Lira-H group were lower than HF group,the difference was statistically significant(P<0.05),but there was no statistical difference in Lira-L group,Lira-M group and Lira-H group(P>0.05).(2)brown fat weight: There was no significant difference of brown fat weight between groups(P>0.05).(3)brown fat weight/body weight: the ratio of brown fat weight/body weight in Lira-L group,Lira-M group,Lira-H group was more than HF group(P<0.05),but there was no difference in ratio between Lira-L group,Lira-M group and Lira-H group(P>0.05).2.Serum biochemical assays:Compared with Con group,serum glucose and triglyceride levels were increased in HF group(P<0.05).Compared with HF group,serum glucose and triglyceride levels were decreased in Lira-L group,Lira-M group and Lira-H group(P<0.05).3.HE staining results:Compared with HF group,HE staining showed physalides in brown fat cells are relatively smaller,and cytoplasm is more abundant in Lira-L group,Lira-M group and Lira-H group;compared with Con group,there was no obvious difference in morphology.4.The expression of marker gene mRNA in brown adipose tissue at the end of 12 th week:Compared with Con group,the mRNA expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 was down-regulated in HF group(P<0.05);Compared with HF group,liraglutide intervene resulted in the mRNA expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 up-regulation(P<0.05);the mRNA expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 among Lira-L group,Lira-M group and Lira-H group was no statistical difference(P>0.05).5.The expression of marker gene protein in brown adipose tissue at the end of 12 th week:Compared with Con group,the protein expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 was down-regulated in HF group(P<0.05);Compared with HF group,liraglutide intervene resulted in the protein expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 up-regulation(P<0.05);the protein expression of UCP1,PPARγ,CIDEA,PGC-1α,CEBPβand PRDM16 among Lira-L group,Lira-M group and Lira-H group was no statistical difference(P>0.05).Conclusion:1.Liraglutide intervene can obviously reduce the body weight of high fat fed rats,and increase the relative weight of brown adipose tissue.2.HE staining results showed that liraglutide can increase the amount of brown fat cells and reduce the lipid content in brown fat cells of high fat fed rats.3.Liraglutide intervene can increase thermogenesis of brown adipose tissue by increasing brown fat cells differentiation in high fat fed rats.4.There is no obvious correlation between the concentration of liraglutide and the effect of liraglutide to relative content and function of brown adipose tissue of high fat fed rats.