The Research Of Leptin Function Influenced By Fox O1?DBD

Leptin is an important discovery in the field of energy metabolism,and Fox O1 plays a negative feedback role in leptin signaling pathway.To improve leptin sensitivity,the mutant Fox O1?DBD of Fox O1 was constructed in our laboratory.However,it is unclear that the mechanism of energy metabolism mediated by leptin.In order to study the effect of Fox O1?DBD on leptin,the following aspects were studied:1.A recombinant plasmid p EF1?-myc-Fox O1?DBD was constructed and transfected into 293 Rb cells and detect the expression of mutant Fox O1?DBD by western blotting.The recombinant plasmid p EF1?-myc-Fox O1?DBD was successfully constructed and the expression of mutant Fox O1?DBD was detected in 293 Rb cells.2.A transgenic mouse model containing the mutant Fox O1?DBD is prepared by using a DNA prokaryotic microinjection method,and a mouse tail genomic DNA is taken as a template to identify the genotype by PCR.Ten first-born mice were obtained,10 breeding lines were established,and the genotype identification method of high G-C content PCR was successfully applied to the first-born mice to F5 generation mice.3.The expression of Fox O1?DBD at transcription level and translation level was detected by fluorescence quantitative PCR and immunoprecipitation,and the expression of Fox O1?DBD gene was successfully detected.4.The phenotype of Fox O1?DBD transgenic mice was analyzed.The results showed that the energy consumption of the transgenic mice was higher than that of wild-type mice under normal diet and high fat diet.Compared with wild-type mice,transgenic mice can regulate blood glucose concentration more quickly under the condition of common diet and have stronger glucose tolerance and better sensitivity to insulin.The changes of UCP1 expression level and rectal temperature in transgenic male mice under normal and cold stimulation showed that UCP1 was more adaptable to low temperature environment.The metabolic cage experiment showed that the body weight and food intake had little change,the oxygen consumption,carbon dioxide production and calorific value of transgenic male rats were lower than those of wild type male rats,and there were significant differences in several sites at night.The respiratory exchange rate,dynamic behavior and total behavior activity of transgenic male mice were consistent,and showed an increasing trend at night,but the peak of wild-type male mice was higher than that of transgenic male mice,and the decline of wild-type male mice was rapid,so that transgenic male mice were higher than wild-type male mice in daytime,and there were significant differences.From the transcription level of autophagy related genes,tf EB gene expression level is consistent with UCP1,suggesting autophagy in transgenic mice.5.Three recombinant plasmid p GL3-CD36 promoters were constructed and transfected into Hep G2 cells,and the promoter activity of p GL3-CD36 was detected and the results showed that p GL3-CD36 promoter was not active.6.The recombinant plasmid PCMV5-myc-PPAR? was constructed and transfected into Hep G2 cells.The expression of PPAR? by western blotting.And the promoter activity of p GL3-SRB?were detected.The expression of PPAR? was detected in Hep G2 cells.The promoter activity of p GL3-SRB? was decreased under the stimulus of leptin.To sum up,Fox O1?DBD has an effect on leptin,Fox O1?DBD can improve the energy metabolism,insulin sensitivity,glucose tolerance of animals,which lays a foundation for the study of Fox O1?DBD function and mechanism and provides a theoretical basis for the treatment of diabetes,obesity and other energy metabolism diseases.