The Effect And Related Mechanism Of Orphan Nuclear Receptor NR4A1 On Adipocyte Differentiation And Lipid Accumulation

Obesity is a chronic metabolic disease.Nowadays,the rate of obesity is increasing with the development of modern life.When the calory of a human body intaking exceeds that of comsuming,the extra-calory will be stored in adipose tissue.When the body lipid accumulation is further more than that the normal physiological requirement,obesity will be developed.Lipid accumulation in the body mainly means the enlargement of adipose tissue,which is composed of adipocytes.The mature adipocytes are differentiated from preadipocytes(adipose tissue-derived stromal cells),thereafter,lipids are continuously accumulated in the cytoplasm of adipocyte;the volume of adipocytes increases with the lipid accumulation,the lipid droplet takes the major volume of the cytoplasm in the adipocyte,the number of the organelles in the cytoplasm is also reduced accordingly.The process of adipogenesis in vitro includes adipocyte differentiation and lipid accumulation,and a variety of factors are involved in the regulation of this process,such as PPARy and C/EBPs.The orphan nuclear receptor NR4A1 can also be called Nur77,TR3 or NGFI-B.It can exist in various tissues.It can receive a variety of stimuli or signals,and can be involved in the regulation of cell functions in a variety of ways.It has important regulatory effects,including cell proliferation,apoptosis,immunity and so on.As a transcription factor,it has also been reported to be involved in glucose and lipid metabolism.The main object of our study is to research the regulatory role and the related mechanism of NR4A1 in adipogenesis.In our study,NR4A1 enhances the transcription of GATA binding protein 2(GATA2),which in turn inhibits the transcriptional expression of PPARy.Meanwhile,NR4A1 suppresses the expression of the sterol regulatory element combining with the transcription factor 1(SREBP1)and its downstream gene,fatty acid synthase(FAS),via increasing the expression of p53.Finally,it can achieve the purpose of inhibiting adipogenesis and controlling weight.The study of NR4A1 in adipogenesis will provide a new target and clues for the prevention and treatment of obesity.Part ?:The effect of NR4A1 on obesity in vivoAim:To identify the genotype and the expression of NR4A1 in mice,and to study the effect of NR4A1 on weight and obesity in mice.Methods:1.A small piece of tail was obtained from mice,and then the genomic DNA of the NR4A1 KO mice and the control(wild type,WT)mice was extracted by routine method,and PCR was performed with the primers suggested by Jackson Laboratory to identify the genotype of mice.2.RNA in the epididymis adipose tissue of mice was extracted,and the mRNA expression of NR4A1 was detected by qPCR.We certified that the mRNA expression of NR4A1 in the adipose tissue from KO mice was lost.3.Protein was extracted from mouse epididymal adipose tissue,and the protein expression of NR4A1 was detected by Western blot.We certified that the protein expression of NR4A1 in the adipose tissue from KO mice was lost.4.We fed the NR4A1 KO mice and WT mice with high fat feed(containing 60%fat)for 14 weeks,measured the weight of mice every week,and compared the different weight changes between KO and WT mice.The volume of adipose tissue was detected and the body fat rate of the mice was calculated.5.The epididymis adipose tissue of mice was extracted,and the surface area size of adipocytes was compared via using frozen sections and HE staining techniques.6.The blood glucose changes of the two mice were detected by intraperitoneal injection of glucose(GTT)or insulin(ITT),and the differences of glucose or insulin tolerance between the two mice were analyzed.7.The mRNA expressions of related lipolysis and adipogenesis genes in the epididymis adipose tissue were detected by qPCR.8.The protein expressions of related lipolysis and adipogenesis genes in the epididymal adipose tissue were detected by Western blot.Results:1.The result of genotype PCR verification showed that wild type mice had an amplified band of 180bp DNA fragment,while the KO mice had a band of 350bp DNA fragment,the heterozygous mice had two bands both of 180bp and 350bp in DNA agrose gel electrophorisis.2.Taking wild type mice as a control,NR4A1 KO mice showed defective mRNA expression of NR4A1 in the epididymal adipose tissue.3.Taking wild type mice as a control,NR4A1 KO mice showed defective protein expression of NR4A1 in the epididymal adipose tissue.4.During the 14 weeks of high-fat feeding,NR4A1 OK mice gained weight much faster.After 14 weeks,the adipose tissue of the NR4A1 KO mice was larger and had higher body fat rates than that of the wild type mice.5.The analysis of HE staining indicated that the adipose tissue of NR4A1 KO mice had larger cell surface area.6.The result of GTT showed that compared with wild type mice,NR4A1 KO mice reflected faster and more elevated blood glucose after glucose inj ection,and it was more difficult to restore normal blood glucose levels.The result of ITT showed no difference in blood glucose changes between the two mice after insulin injection.7.After detecting the mRNA level in epididymal adipose tissue,we found out that there was no significant difference between wild type mice and NR4A1 KO mice in the expression of related lipolysis genes HSL,ATGL,LPL and related adipogenesis genes C/EBR? or adiponectin,while the mRNA expression of two adipogenesis genes PPARy and FAS increased significantly.8.We found that the protein expression of related fat synthesis gene FABP4 had no difference in the epididymal adipose tissue,while the expressions of two adipogenesis genes(PPARy and FAS)in NR4A1 KO mice were increased.Conclusions:Taking wild type mice as a control,NR4A1 knockout mice gains weight much faster under the condition of high-fat feeding,and the obesity signs in KO mice such as fat volume,body fat rate and adipocyte surface area are more obvious.NR4A1 regulates adipocyte differentiation and lipid accumulation by affecting the expression of PPARy and FAS rather than lipolysis.Part ?:The mechanism of NR4A1 on adipocyte differentiation and lipid accumulationAim:To explore the effect of NR4A1 on adipocyte differentiation and lipid accumulation,and to figure out the related mechanism of it.Methods:1.Preadipocytes 3T3-L1 were cultured and induced with chemical cocktail to differentiate into mature adipocytes,Western blot was applied to detect the dynamic expression change of protein such as NR4A1,PPARy and FAS during the process of differentiation.2.Preadipocytes 3T3-L1 were infected with lentivirus overexpressing NR4A1 and control lentivirus.Thereafter,the cell lines stably overexpression of NR4A1(OV cells)and the control cell lines(NC cells)were obtained through puromycin drug selection and clonal screening,and the overexpression efficiency of NR4A1 was detected by Western blot or qPCR.3.The preadipocytes(NC and OV cells)were induced to differentiate into mature adipocytes.The differentiation process was detected on day 0(DO),day 4(D4)and day 8(D8)with oil red O staining,to identify the degree of adipocyte differentiation and compare the differences between NC and OV cells4.We harvested the cells for the protein extraction during the process of differentiation on DO,D2,D4,D6 and D8,we examined the protein expression of PPARy and FAS between NC and OV cells.5.Through the analysis of dual luciferase reporter assay,we detected the effect of NR4A1 on the transcripition of promoters such as PPARy,FAS and GATA2.6.ChIP-qPCR experiments were used to identify whether the potential binding sites on the promoters of PPARy,FAS,and the two potential binding sites on the GATA2 promoter can be physically associated with NR4A1.7.Western blot was used to measure the difference of GATA2 protein expression in epididymal adipose tissue between WT and KO mice,and qPCR was used to measure the mRNA expression difference of GATA2 in epididymal adipose tissue between WT and KO mice.8.The stable cell lines knocking down GATA2(KD cells)were obtained by infection with lentivirus encoding GATA2-siRNA.And the stable cell lines knocking down GATA2 and overexpressing NR4A1(KD-OV)were obtained via virus infection overxepressing NR4A1 after KD cell were knocked down the expression of GATA2.Normal control cells(NC cells),KD cells and KD-OV cells were used to induce adipocyte differentiation,and the protein or RNA were extracted on the fourth day of adipogenesis induction.Western blot and qPCR were applied to detect the expression levels of PPARy in NC,KD and KD-OV cells.9.The mRNA expression level of GATA2 during adipocyte differentiation was detected by qPCR.10.The protein expression of SREBP1c in NC and OV cells during adipocyte differentiation was analyzed.Western blot and qPCR were used to measure the protein and mRNA expression levels of p53 in epididymal adipose tissue between wild type mice and NR4A1 knockout mice.Results:1.The protein expression level of NR4A1 had the similar trend with PPAR? and FAS during the adipocyte differentiation.2.The stable cell lines were obtained,and the mRNA and protein expressions of NR4A1 were significantly higher in OV cells compared with NC cells.3.The analysis of Oil red O staining showed that NC and OV cells were induced successfully into mature adipocytes,and the ratio of mature adipocytes in OV cells was much smaller than that of NC cells.4.In the process of adipogenesis,the protein expression of PPARy and FAS in OV cells were much reduced compared with NC cells.5.The analysis of dual luciferase reporter assay showed that the transcripition of PPARy was inhibited by NR4A1,while the transcription of FAS was not impacted by NR4A1.More importantly,we found that the transcriptional activation of GATA2 was enhanced by NR4A1.6.The results of ChIP-qPCR showed that NR4A1 had no direct binding with PPARy or FAS.However,it had physical association with the potential binding site(from-1125bp to-1120bp)on GATA2 promoter,while it had no physical association with another potential binding site(from-936bp to-93 1bp).7.The protein and mRNA expressions of GATA2 in the epididymal adipose tissue of NR4A1 KO mice were reduced compared to WT mice.8.Taking NC cells as the control,the protein and mRNA expressions of PPARy were increased in KD cells.And the protein and mRNA expressions of PPARy were not increased in KD-OV cells compared with NC cells.9.During the process of adipogenesis in different preadipocytes(NC and OV cells),the expression of GATA2 were reduced,but the expression of GATA2 in OV cells was always more than that in NC cells.10.During adipocyte differentiation,the expression of SREBP1c,which is the upstream gene of FAS,was decreased in OV cells.And the protein and mRNA expressions of p53 in the epididymal adipose tissue of NR4A1 KO mice were decreased compared with WT mice.Conclusions:The transcription factor NR4A1 activates the transcriptional activity of GATA2 by directly binding to its promoter,and GATA2 inhibits the transcriptional expression of PPARy as a transcription factor.In addition,NR4A1 activates the expression of p53 indirectly,and p53 inhibits the expression of SREBPlc and FAS.In summary,NR4A1 inhibits adipocyte differentiation and lipid accumulation through these two pathways.