| Energy-metabolic disorders(such as obesity and type 2 diabetes)are serious threats to human health and are receiving increasing attention worldwide.The study found that excess Foxo1 in the hypothalamus can inhibit the signaling pathway of leptin and induce leptin resistance in animals,leading to energy metabolism disorders,but the mechanism of its action remains unclear.In the laboratory experiments,it was found that a mutant Foxo1(Foxo1?DBD)lacking the DNA binding domain can promote the signaling of leptin,suggesting that this mutant may promote the overall energy metabolism of the animal.To prevent the occurrence of energy metabolism disorders.To this end,a transgenic animal of Foxo1?DBD was prepared.In this paper,the transgenic animal was used as the main experimental object,and the regulation of Foxo1?DBD on the overall energy metabolism of animals was studied.Combined with the experiments of cell biology,the possible mechanism of action was discussed.1.Expansion of mice.Ten Foxo1?DBD transgenic primary mice were prepared in the laboratory and propagated into lines.On this basis,this experiment carried out the expansion of Line7 of transgenic mice.Hybrid and wild genotype mice were mated and heterozygous and wild type were identified from progeny mice.A total of 6 generations of F4-F9 generation mice were bred and identified.The genotype was identified by PCR.The results showed that the method was accurate and reliable,and the transgene was stably inherited.2.Determination of body weight and feed intake.Three 16-week-old Foxo1?DBD transgenic male mice and 16 wild-type mice(F7)were selected,and the body weight and feed intake of the mice were measured every three days for 72 days.The experimental results showed that under standard Chow diet conditions,there was no significant difference in body weight between F7 generation Foxo1?DBD transgenic mice and wild type mice.The body weight of F7 generation Foxo1?DBD transgenic mice under high fat diet was not significantly different from that of wild type mice.At the age of 65 weeks in the F4 generation,body weight and feed intake were measured again for 66 days.The weight of Foxo1?DBD transgenic mice was significantly smaller than that of wild-type mice(P <0.05),and the feed intake during this period was the same as that of wild-type mice.There were no significant differences(P> 0.05).3.Metabolic cage experiment.Metabolic cage experiments were performed on F4 generation 73-week-old Foxo1?DBD transgenic mice and 8 wild-type male mice.The results showed that the oxygen consumption(P < 0.001),CO2 production(P < 0.01)and calorific value(P < 0.01)of Foxo1?DBD transgenic mice were significantly higher than those of wild-type mice.4.Transcriptome sequencing and Real Time PCR validation.The differentially expressed genes in Foxo1?DBD transgenic mice were screened and verified by QPCR.Four 8-week-old male mice(three Foxo1?DBD transgenic mice and three wild-type mice)were selected from F7 generation,brown adipose tissue(BAT)was isolated,and differentially expressed genes in BAT were screened by transcriptome sequencing.By analysis,206 differentially expressed genes were found in Foxo1?DBD transgenic mice and wild-type mouse BAT(P < 0.05,| Log2 Fold Change | > 1),and 8-week-old male mice(Foxo1?DBD transgenic mice and wild type)were selected.Eight of the mice were subjected to QPCR verification of the transcription of 9 genes such as uncoupling protein 1(UCP1)and iodothyronine deiodinase 2(Dio2),and the results were consistent with the transcriptome sequencing results.5.UCP1 promoter activity assay.In order to verify the promoter effect of Foxo1?DBD transgene on UCP1 gene expression in animals,the UCP1 promoter plasmid p GL3-UCP1 was constructed,and p GL3-UCP1,p CMV-myc-Foxo1?DBD or p CMV-myc-Foxo1 was co-transfected into 3T3 L1 cell line,and Foxo1 was found to be significant.The activity of the UCP1 promoter was inhibited(P < 0.05),and the proportion of Foxo1?DBD exceeding 75% eliminated this inhibition.6.Foxo1 binds to the UCP1 promoter.To understand the mechanism of elevated UCP1 expression in transgenic mouse BAT,chromatin immunoprecipitation(Ch IP)was used to detect the binding of Foxo1 to UCP1 promoter in mouse BAT.The results showed that the binding of Foxo1 to the UCP1 promoter was detected in wild-type mice,but almost no such binding was detected in Foxo1?DBD transgenic mice,indicating that Foxo1?DBD competitively inhibited the binding of Foxo1 to the UCP1 promoter.Thereby,the inhibitory effect of Foxo1 on the UCP1 promoter was released.7.Determination of plasma T3 and T4 levels.Since the expression of the Dio2 gene in the BAT of Foxo1?DBD transgenic mice was significantly increased,it was speculated that the expression product promoted the process of T3 deiodination to produce T3,so 16 male mice of 8 weeks old(Foxo1?DBD transgenic mice and wild type mice)8)plasma total triiodothyronine(TT3),total tetraiodothyronine(TT4),free triiodothyronine(FT3)and free tetraiodothyronine(FT3)The assay showed that the levels of TT3 and FT3 in Foxo1?DBD transgenic mice were significantly higher than those in wild-type mice(P < 0.05),which was in line with expectations.8.The lateral ventricle was injected with the S3I-201 experiment.Foxo1?DBD is associated with a decrease in STAT3 phosphorylation during up-regulation of energy metabolism.S3I-201 is a STAT3 phosphorylation inhibitor.We can simulate the role of Foxo1?DBD by intraventricular injection of S3I-201.Therefore,close 8-week-old male KM mice were divided into four groups,and leptin and STAT3 phosphorylation inhibitor S3I-201(n = 10)were injected into the lateral ventricle,respectively.The results showed that mice injected with S3I-201 showed There was no significant decrease in feed intake and a significant decrease in body weight(P < 0.05).Conclusion: Foxo1?DBD can affect the energy metabolism of animals and increase the metabolic level of mice.The initial mechanism is that Foxo1?DBD eliminates the negative regulation of Foxo1 on UCP1 promoter to up-regulate the expression of UCP1 gene in BAT,and the up-regulation of Dio2 expression leads to the increase of T3.UCP1 expression.At the same time,the simulation of the regulation of STAT3 phosphorylation by Foxo1?DBD in the leptin signaling pathway was used to investigate the changes in the energy metabolism level of mice under this influence.The experiment found that Foxo1?DBD affects the energy metabolism of animals and conducts preliminary research on its mechanism,providing a theoretical basis for the treatment of energy metabolism diseases such as obesity and diabetes. |
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