The Regulation Of STAT3 Phosphorylation In Hypothalamus And Its Effects On Leptin-Mediated Energy Metabolism

Metabolic diseases of animals are non communicable diseases,including nutritional diseases because of nutritional deficiency or excessive and metabolic diseases results from disorders of metabolic processes.Although it is clear that the deletion of STAT3 gene in hypothalamus leads to leptin resistance and massive obesity,which proves the importance of STAT3 in leptin signaling,we still do not know whether the phospho-STAT3 is declined or elevated in hypothalamus of wild-type obese animals with or without the stimulation of leptin.For this point of view,preliminary studies were performed in our laboratories.The results of repeated EMSA(electrophoretic mobility shift assay)showed that the obvious increase of both basal and leptin-stimulated phospho-STAT3 was cocomitant with the significant decrease of POMC expression in the hypothalamus of the diet-induced obese mice with leptin resistance.Furthermore,in an established in vitro system for the study of leptin signaling,the decline of phospho-STAT3 caused by the addition of ether a mutant of Foxo1 or S31-201,the STAT3 phosphorylation inhibitor,resulted in the elevation of POMC expression in response to leptin stimulation.Therefore we hypothesized here that the over-elevation of phospho-STAT3 in hypothalamus would be one of the reasons for the onset of leptin resistance;the reduction of hypothalamic phospho-STAT3 could promote leptin sensitivity.To prove this hypothesis,we used in vitro cell experiments,transgenic animals and ventricle injection of small molecule inhibitors to reduce the level of STAT3 phosphorylation in hypothalamus and the change of animal metabolic behavior.The main results are as follows:1.The reduction of STAT3 phosphorylation in 293 cells promotes the signal transduction of leptin.This study uses an effective in vitro cell test system,which can normally reflex the POMC pathway in the leptin signal transduction pathway in the body.The results showed that the activation of STAT3 is required for POMC transcription mediated by leptin,which is blocked whout STAT3 or the addition of cytokines SOCS3 and Fox O1.On the contrary,when the Fox O1 mutant Fox O1ΔDBD,lack of DNA binding domain,was added to the system,the phosphorylation of STAT3 was inhibited,but the signal transduction of leptin was enhanced.When the small molecule S3I-201,inhibitor STAT3 phosphorylation,was added to the system,the decrease of phosphorylation level of STAT3 was accompanied by the enhancement of leptin signal conduction.The above results indicate that reducing the phosphorylation level of STAT3 can improve the effect of leptin.2.The reduction of phosphorylation of STAT3 in the hypothalamus of transgenic mice results in the loss of weight in mice.The transgenic first mice of Fox O1 Delta DBD,obtained by commercial service,were bred and expanded.A stable genotype identification method was established,and the target gene highly transcribed was screened by fluorescence quantitative PCR in mouse strain Line7.The expression of target protein in the hypothalamus of mouse strain was identified by immunoprecipitation.The results showed that the expression of target protein Fox O1 Delta DBD in the hypothalamus of transgenic mice decreased the phosphorylation level of STAT3.The phenotypic analysis showed that the weight of the transgenic mice was significantly lower than that of the wild type mice in both chow diet and the high fat diet feeding.The transcription of UCP1 in brown adipose tissue of transgenic mice is higher than that of wild type mice.Metabolic cage and cold stimulation test show that the increase of self heat consumption of transgenic mice is one of the reasons of body weight loss in mice.Ventricle injection of S3I-201 reduced both the phosphorylation of STAT3 in the hypothalamus and the weight of the mice.The male rats of Kunming with similar age and weight were divided into experimental and control groups,and were fed alone in single cages.Mice were anesthetized with pentobarbital sodium fafter 48h’starvation,and ventriclely microinjected in the presence or absense of leptin.The experimental group was injected with STAT3 inhibitor(SI3-201),and the control group was injected with the same volume of DMSO.After 45 minutes,the hypothalamus was taken to do Western hybridization(3 in each group).The results showed that the activation of STAT3 phosphorylation by leptin was inhibited by SI3-201.After injection for 12,24 and 48 hours the body weight of experimental group,when the effect of surgery was excluded.,was significantly reduced at each time point compared with the control group(P < 0.05),demonstrating that intraventricular injection of S3I-201 can also have a significant effect on reducing the body weight of animals while inhibiting the phosphorylation of leptin-mediated STAT3.In conclusion,the inhibition of STAT3 phosphorylation mediated by leptin in the hypothalamus of mice was achieved through in vivo and in vitro experiments,and it was found that this inhibition promoted the signal transduction of leptin and the weight loss of mice.The results strongly support the new view that the high level of phosphorylation in the hypothalamus of DIO mice contribute to the compromise of leptin action.