Effects Of β3-adrenergic Agonist On The Expressions Of Browning Genes And MicroRNAs In Adipose Tissue Of C57BL/6J Mice

BACKGROUNDWith development of economyand changes of lifestyle,incidenceof obesity increases rapidly, which has become a threat to human health and one of the most important public health problems worldwide. According to WHO,from1980to present, the global obesepopulation has doubled. In2008,there are more than1.4billion adults over20years who are overweight in the world, among them200million men and300million femaleare obese. It has been estimated that by2030, the global overweight population will exceed2.16billion and the obese population will reach to1.12billion.Obesity is a risk factor for diseases such as hypertension, coronary heart disease and diabetes. According to WHO, obesity has become the fifth leading cause of death in the world. The prevalence of obesity in adults is more than10%worldwide.In addition,44%of diabetes,23%of ischaemic heart disease,7%to41%different occurrence of tumor are closely related to obesity. Thus, obesity has become one of the most important health problems which need to be solvedurgently. Long-term of excessof energy intake leads toexcess white fat in the bodywhich cause obesity. Compared with restricted diet, people are more likely to increaseenergy metabolism to burn off excess fat. It’s a good way to exercise, but most people are difficult to ensure whether there is enough physical activity to achieve the goal of weight lossin the fast-paced modern society. In the treatment of obesity, most diet pills leave away from clinical application due to theirside effects. So we need to find a new approach for the treatment of obesityurgently. Brown adipose tissue (BAT), which has special physiological function,has become a new research targeton obesity treatment recently. Mammalian adipose tissue in the body can be divided into white adipose tissue(WAT) and brown adipose tissue(BAT). WATstores excess energy in the form of triglycerides, andalso secretes a variety of factors to regulate energy metabolism. While BAToxidatesfree fatty acids to produce heatso as to maintain the body temperature in cold environment. Brown fat cells contain many mitochondria, these mitochondrial membrane surface contains a lot of UCP1which has high tissue specificity. UCP1allows proton re-entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain.Itis very important in the process of adaptive thermogenesis under cold stimulation. Moreover, there are also lots of capillaries around brown fat, sympathetic nerve fibers can also go directly to brown adipose tissue membrane. If fully activated,50g BAT can consume the body20%of basal metabolic energy.We used to think that adultsdon’thave activated BAT, however, fiveindependent research groupsconfirmed metabolic active regionsin adult neck and supraclavicular regionsin2009throughPositron Emission Tomography and Computed Tomography (PET-CT),and adipose tissuesfrom neck were examined and the molecular markers were analysed which proved adults have metabolically active BAT. Cypess et al analyzed1972adult patients by usingPET-CT scan, found that5.4%of patients did have active BAT; They also found that the activity of brown adipose tissue revealed a negative correlation with age, body mass index, fasting blood glucose levels.Now BAT has been an important target to treat obesity.In recent years, researchers have devoted to turn WAT into BAT. Transcription factors are important factorsfor the transdifferentiation of brown fat. Study found that C/EBP3can induce3T3L1white fat cell line transdifferentiation to become browning cells in vitro; Whereas myoblast in PRDM16-C/EBP0transcription complex under the action of transdifferentiation become brown fat with physiological functions; In addition, PRDM16-C/EBPβ transcription complex can inducefibroblasts transdifferentiation to brown fat cell.In AP2-PRDM16transgenic mice,subcutaneous fat turnsinto brown, not in visceral fat.MicroRNAs are noncoding RNAs (ncRNAs), which can negatively regulate protein expression in the transcription level. Mammalian microRNAs regulatemore than60%protein-encoding genes, and each microRNA can regulate hundreds of different mRNAs. MicroRNAs in the process of fat cell transdifferentiation can also play a very important role. Subcutaneous fat and visceral fat appear obvious brownin ap2-miR-196a transgenic mice. Sun et al found overexpressmiR-193b/365in C2C12musclecell line, brown fat cells can be induced.Blocking miR-193b and/or miR-365in primary brown preadipocytesmarkedly impaired brown adipocyte adipogenesis. The latest research results show that miR-133regulates brown fat differentiation through PRDM16.Recent research also reveals the miR-155is key to BAT. UCP1expressionsare restrained in miR-155transgenic mice, howener, UCP1expressions are activated in miR-155knockout mice.For energy balance, increasing the heat productioncan be regarded as a kind of important means to fight obesity. With the existence of brown fat in adults, the researchers hope to treat and prevent obesity and its related diseases by adjusting the heat production of brown fat. Looking to the future, there are two ways to increase the amount of brown fat and activity:one is through the small molecule drugs or growth factors to stimulate growth, differentiation and activation of BAT in the body. The second is to induce mature brown fat cells through stem cells in vitro which can be transplanted to obese populations.Obesity has become a serious threatto people’s health in modern society.And as we mentioned earlier, obesity has a close relation withBAT. MicroRNAs can inhibit the process of translation or make mRNA degradation and widely involved in the process of development, cell proliferation and apoptosis. Recently, researchers found thatMicroRNAs play an important role in the process of the browning ofWAT. However,whethermore MicroRNAs involvedin the process needs further study. UCP1gene is key to the brown fat adaptive thermogenesis, but report about microRNAs targeted UCP1is little. So we focuse on the microRNAs targeted UCP1,which mayindicate the process of browningwhite fatclearlyand lay a good foundation for the prevention and treatment of obesity.Research is divided into the following two parts:Chapter1Effects ofp3-adrenergic agonist on theexpressions of browning genes in adipose tissue of C57BL/6J miceObjective:Stuty effects of β3-adrenergic agonists (CL316243),the expressions ofbrowning genesin epididymal fat,subcutaneous fat,brown adipose tissue.Methods:1.14male C57BL/6J mice(6weeks) were randomly divided into2groupsaveragely, Daily intraperitoneal inject β3-adrenergic agonists (CL316243) or saline to mice according to their body weight for10days. Sacrifice the mice with cervical vertebra dislocation method24hours after the last injection.Collect epididymal fat, subcutaneous fat,brown adipose tissue which immediately were put into-80℃for storage.2. Detect UCP1, CIDEA, DIO2and CPT1B and PGC-la,C/EBPβ,PRDM16, PPAR γ2genes mRNA expressionsin epididymal fatthroughquantitative real-timePCR method.3. DetectUCP1,CIDEA,PGC-1α,C/EBPβ,PRDM16,PPARγ2genesmRNA expressions insubcutaneous fatthrough quantitative real-time PCR method.4. Detect UCP1, CIDEA, PGC-la, C/EBPβ, PRDM16, PPARγ2genes mRNA expressions in BATthrough quantitative real-time PCR method.5. HE staining was used to observe morphological changes of epididymal fat in mice with between two groups,.6. Statisticalanalysis:Alldataareexpressedasmeans±SD.Statisticalanalysiswereperfor medusingtheSPSS13.0softwarePackage.Statisticalsignificanceofdifferences between two groups wasevaluated byt test.P<0.05wasconsideredassignificant.Results:1. Compared with the control group, adipogenesis related gene PPARy2mRNA expression was significantly reduced in EP and SUBwith CL316243treated (P<0.05).PPARγ2mRNA expression werenoStatisticaldifferences in BAT.2. Compared with the control group,BAT specific genes UCP1, CIDEA, DIO2and fatty acid oxidation related gene CPT1B mRNA expression were significantly raised in epididymal fat with CL316243treated(P<0.05)..The expressions ofother BAT differentiation genesPGC-1α, C/EBPβ, PRDM16mRNA have no Statistical differences (P>0.05).2. Compared with the control group, BAT specific genes UCP1, CIDEA and BAT differentiation genes PGC-1α, C/EBPβ, PRDM16mRNA expression had no Statistical differences with CL316243treatedin subcutaneous fat (P>0.05).3. Compared with the control group, BAT specific genes UCP1, CIDEA and BAT differentiation genes PGC-1α, C/EBPβ, PRDM16mRNA expression had no Statistical differenceswith CL316243treated in brown adipose tissue(P>0.05).4. In control group,adipocyte present a unilocular fat droplet, nucleus werelocated peripheralin HE staining, In CL316243group,adipocytescontained smaller cells with richcytoplasmic staining and multilocular lipid droplets.Conclusion:1. Adipogenesiscouldbe restrainedin subcutaneous fat and epididymal fat with β3-adrenergic agonist CL316243treated;2. Epididymal fatcould be made browning and fatty acid oxidation levelareelevated with β3-adrenergic agonist CL316243treated.So the level of energy metabolism were raised in mice. Chapter2:Effects of β3-adrenergic agonist on the expressions of browning microRNAs in adipose tissue of C57BL/6J miceObjective:Study the expressions of microRNAsthat target UCP1in epididymal fat with β3-adrenergic agonist CL316243treated.Methods:1. According to miranda database, search microRNAswhich target UCP1and screen out possible microRNAs.2. Detect the expressions of miR-9, miR-338-3p and nonrelated miR-let7g mRNA expressionsthrough quantitative real-time PCR.Results:Compared with control group, the expressions of miR-9and miR-338-3pwere significantly restrainedin epididymal fatwithCL316243treated(P<0.05), and miR-let7g expression was no Statistical differences (P>0.05).Conclusion:MiR-9and miR-338-3p may be involved in the process of the browning of white adipose tissuethrough regulating UCP1gene expression.