Study The Mechanism Of NR4A1 Up-regulating The Density Of Mitochondria In Adipocytes

Obesity is a chronic metabolic disease affected by many factors,which can induce a series of complications,thereby affecting people’s health.Differentiated adipocytosis and excessive accumulation of lipids in adipocytes can lead to obesity.Therefore,obesity intervention can be based on inhibiting the differentiation of pre-adipocytes and increasing the lipid consumption in adipocytes or reducing lipid accumulation.To prevent obesity,the most important thing is to find the right molecular target.We found that NR4A1-deficient mice were more likely to be obese than wild-type mice,and exhibited abnormalities in body weight,body fat ratio,and metabolic regulation.Our previous work has demonstrated that the lone nuclear receptor NR4A1 molecule can inhibit the expression of sterol regulatory element binding transcription factor 1(SREBP1)and its downstream gene fatty acid synthase(FAS),thereby inhibiting lipid synthesis and accumulation.Mitochondria are organelles related to energy consumption,and their functional activity and quantity can affect the lipid accumulation of fat cells,NR4A1 has been reported to promote the expression of uncoupled protein 3(UCP3),and the NR4A family can regulate uncoupled protein 1(UCP1)molecules,promoting the transfer of stored energy into heat to increase energy consumption.In this study,we will continue to explore the mechanism of NR4A1 affecting obesity via up-regulating the density of mitochondria in adipocytes.In this study,adipose tissue,primary adipose stem cells from mice and 3T3L1 adipocytes were selected as research materials,NR4A1 knockout mice(NR4A1-KO mice)were generated with gene editing strategy,and cell lines overexpressing NR4A1 were constructed by lentivirus infection and drug selection.We found that the content of the marker molecule Tomm20 of mitochondria from the adipose tissue of NR4A1KO mice was lower than that from wild-type mice,and primary adipose stem cells were also analyzed.Primary adipose stem cells were isolated from the adipose tissues from WT or NR4A1-KO mice and induced for differentiation in vitro,and after their maturity,they were stained by Mitogreen mitochondrial probe dye,and it was found that the mitochondrial content in the adipocytes from NR4A1-KO mice was significantly lower than that from wild-type mice.These indicate a lack of NR4A1 or may cause a decrease in the number of mitochondria in fat cells.To explore the mechanism by which NR4A1 affects mitochondrial number,we examined the mitochondrial transcription factor TFAM in fat cells or tissues,as well as the mitochondrial DNA polymerase POLG and the PGC1-αassociated with mitochondrial biogenesis.Our data showed that the protein content of TFAM and POLG in the adipose tissue from wild-type mice was higher than that from KO mice,but no significant difference in PGC1-α expression was detected.In the adipocytes overexpressing NR4A1 from 3T3L1 preadipocytes,both mRNA and protein levels of POLG increased,and we also found that overexpression NR4A1 did enhance the transactivation of the POLG promoter by dual luciferase reporter assay,and we later on measured the copy amount of mitochondrial DNA,and found that in the 3T3L1 adipocytes overexpressing NR4A1 had much more mitochondrial DNA(mtDNA)compared with the control group,which indicated that NR4A1 increased the expression of POLG,which in turn increased the copy number of mtDNA and ultimately increased the number of mitochondria in adipocytes.Bioinfomatics analysis showed that there were 34 genes related to mitochondria were regulated by NR4A1,POLG was one of these genes.The elevated levels of TFAM protein caused by NR4A1 should also play an important role in modulating mitochondrial numbers in adipocytes.To summarize our study,the solitary nuclear receptor NR4A1 regulates mitochondrial copy capacity and mitochondrial germination by regulating TFAM and POLG molecules.This plays an important role in maintaining the copy volume and number of mitochondria,and also in the treatment of obesity and mitochondria-related diseases.The mechanism by which NR4A1 affects TFAM expression is currently unclear and needs to be further explored.