Study On The Mechanism Of Energy Metabolism In Diet Induced Obesity Rats By Leptin Regulate Autophagy

Objective1.To study the effects of leptin protein on the autophagy and the balance of energy metabolism in diet induced obesity rats.2.3T3-L1 preadipocyte were cultured in vitro and treated with leptin and autophagy tools drug.To observe the effect of leptin on the differentiation and phenotype of 3T3-L1 preadipocytes.To investigate the effect and mechanism of leptin on autophagy in 3T3-L1 preadipocytes.Method1.SD rats were fed with high fat diet to establish diet induced obesity(DIO)animal model.The weight gain and foot intake of rats was monitored during feeding.After 16 weeks,the rats in the high fat group(HFD)were further divided into diet induced obesity(DIO)group and obesity resistance group(DR)according to their weight.Rats fed normal diet served as control group.The levels of serum leptin were detected by EILSA.2.DIO,DR and CF groups were randomly divided into three groups,namely: leptin high concentration group(H),leptin low concentration group(L),and the control group(C).The body weight and food intake were monitored after 24 hours of intraventricular injection of leptin;The levels of serum leptin were detected by EILSA.The morphology of liver and adipose tissue was observed by HE staining;The expression of autophagy related factors LC3 B,Beclin-1 and P62 proteins in hypothalamus,liver and adipose tissue were detected by Western Blot.3.DIO,DR and CF groups were randomly divided in two groups,namely: leptin high concentration group(H)and the control group(C).The leptin was injected continuously for one week with lateral ventricle catheterization.Monitored body weight and food intake.The levels of serum leptin were detected by EILSA.The morphology of liver and adipose tissue was observed by oil red O staining and HE staining.The expression of autophagy related factors LC3 B,Beclin-1 and P62 proteins in hypothalamus,liver and adipose tissue were detected by Western Blot.4.3T3-L1 preadipocytes were cultured in vitro and induced to differentiate into mature adipocytes.The leptin was involved in the process of differentiation.The effect of leptin on the differentiation and phenotype of 3T3-L1 preadipocytes was observed by oil red O staining;mRNA expression was detected by RT-PCR,including adipocyte differentiation,triglyceride synthesis and decomposition,fatty acid beta oxidation,and brown fat Characteristic Genes;Leptin and autophagy tool drugs(Rapamyin and Bafilomycin A1)were intervened at different stages of cell differentiation,to detect the expression of autophagy related factor LC3 B protein level,to observe the changes of P62 in immunofluorescence microscopy;The levels of autophagy and mTOR signaling pathway related factors LC3 B,P62,Beclin-1,4E-BP1 and p-4E-BP1 were detected in 3T3-L1 preadipocytes before and after leptin intervention,to investigate whether leptin plays a role in adipocyte differentiation and autophagy via mTOR dependent signaling pathway.Result1.The weight and energy intake of DIO rats were higher than CF and DR rats.DIO rats have leptin resistance,the serum leptin levels in DIO rats were higher than CF and DR rats.2.After short-term intervention of leptin,In CF group,weight decreased,but energy intake increased more than before intervention;In DIO group,weight decreased,and energy intake in leptin high concentration group was lower than before intervention;In DR group,weight decreased,but there was no significant change in energy intake than before intervention.After intervention,the serum leptin levels decreased in CF、DIO and DR,but there was no significant change than before intervention.DIO and DR rats had hepatic steatosis,and the volume of fat cells in DIO rats increased significantly.After 24 hours of intervention with leptin,the morphology of hepatocytes and adipocytes was unchanged.Compared with the CF rats,the autophagy of DIO and DR rats was down regulated in liver tissues but it was up-regulated in adipose tissue and hypothalamus.After intervention with leptin,the autophagy of DIO and DR rats was up regulated in liver tissues but it was down regulated in adipose tissue and hypothalamus;The autophagy of CF rats up-regulated in liver,and down-regulation in adipose tissue and hypothalamus.3.After long-term intervention with leptin,In CF group,there was no significant change in body weight,but energy intake increased;In DIO and DR group,weight and energy intake has decreased;After the long-term prognosis of leptin,The serum leptin levels of CF、DIO and DR groups increased after the long-term intervention with Leptin,but the difference was not statistically significant;Long term intervention of leptin to improve steatosis in the liver tissue of DIO and DR rats;After intervention with leptin,the autophagy of DIO and DR rats was up regulated in liver tissues but it was down regulated in adipose tissue and hypothalamus;After intervention with leptin,the autophagy of CF rats up-regulated in liver,and down-regulation in adipose tissue and hypothalamus.4.The intervention of leptin had no effect on the morphology of 3T3-L1 cells,but the rate of adipogenesis decreased.After intervention with leptin,the expression of PPARγ and HSL mRNA was increased in the early stage of cell differentiation,and the expression of aP2,DGAT1 and DGAT2 mRNA was decreased at the late stage of cell differentiation,leptin increased expression of mRNA of UCP1 and PGC-1α.6.After intervention with leptin protein,the autophagy was up regulation in the early stage of 3T3-L1 preadipocyte differentiation,at the late stage of cell differentiation,autophagy was down regulated.The regulation of leptin on autophagy in 3T3-L1 preadipocytes is mediated by the mTOR signaling pathway.Conclusions1.After the central administration of leptin,In DIO、DR and CF group,the energy consumption increased;leptin regulated the autophagy of liver tissues,adipose tissue and hypothalamus.Maintain the balance of energy metabolism.2.The intervention of leptin had no effect on the morphology of 3T3-L1 cells,but the rate of adipogenesis decreased.After intervention with leptin protein,the autophagy was up regulation in the early stage of 3T3-L1 preadipocyte differentiation,at the late stage of cell differentiation,autophagy was down regulated.The regulation of leptin on autophagy in 3T3-L1 preadipocytes is mediated by the mTOR signaling pathway.