The Research On The Role And Mechanism Of Key Enzymes For Selenoprotein Synthesis In High-fat Diet Induced Obesity

Background:The prevalence of obesity is increasing dramatically worldwide.At the same time,the complications of obesity including diabetes,hypertension,fatty liver disease,myocardial infarction and other risks,which seriously endanger people’s lives and health.Therefore,reducing the prevalence of obesity and obesity related complications is an urgent problem that we need to solve.Oxidative stress and inflammation are important pathophysiological processes involved in obesity.As a trace element,selenium is mainly involved in the synthesis of various selenoproteins and plays an important role in anti-inflammatory and anti-oxidation in the body.In addition,selenoprotein is related to the normal physiological function of adipocytes,and its abnormal expression may cause hypertension,insulin resistance,inflammatory response.In summary,selenoprotein may be closely related to obesity.Phosphoryl t RNA kinase（PSTK）and selenophosphate synthetase 2（SPS2）are key enzymes in selenoprotein synthesis,and their dysfunction can lead to decreased expression of selenoproteins.In view of the close relationship between selenoprotein and obesity,this research mainly studies the role and mechanism of key enzymes（PSTK and SPS2）for selenoprotein synthesis in high fat induced obesity model.Purpose:Firstly,we plan to verify the physiological role of PSTK and SPS2 through the gene knock out mice;secondly,whether PSTK or SPS2 plays an important role in high-fat diet induced obesity model,how it impacts the liver de novo lipogenesis pathway.Thirdly,the exact mechanism behind this effect.Methods:1.First,we used CRISPR-CAS9 technology to construct liver-specific Sps2 and Pstk knockout mice(Alb-cre,Sps2^fl/fl;Alb-cre,Pstk^fl/fl),we examined the selenoprotein expression and compared the pathological injury of liver in the two types gene knockout mice.2.Obesity model was developed by using high-fat diet（HFD,60%fat）or control diet（CTD,10%fat）for 8 weeks in Alb-cre,Sps2^fl/fl and Sps2^fl/flmice.We compared the differences in body weight,body fat distribution,energy metabolism,glucose metabolism,and the function and pathological injury of liver in each group mice after the high fat diet experiment.3.Alb-cre,Pstk^fl/fl and Pstk^fl/flmice were fed with a HFD or CTD for 8 weeks to construct an obesity model.The body weight,body fat distribution,and glucose metabolism of the mice in each group were compared.We also assessed the lipid deposits in the liver tissues of the mice.4.Using m RNA sequence technology to compare the differential genes in liver tissues of Alb-cre,Pstk^fl/fl+HFD and Pstk^fl/fl+HFD mice,searching the pathway that Pstk gene was significantly related.5.Using q RT-PCR and Western blot technology to verify the results from m RNA sequence of Alb-cre,Pstk^fl/fl+HFD and Pstk^fl/fl+HFD mice.6.We extracted mouse primary hepatocytes from Alb-cre,Pstk^fl/fl and Pstk^fl/flmice.In vitro,the primary hepatocytes were treated with palmitic acid and oleic acid（PA/OA）to induce the lipid accumulation.Experiments were divided into four groups:WT(wild type,Pstk^fl/fl),LKO(liver knock out,Alb-cre,Pstk^fl/fl),WT+PA/OA,LKO+PA/OA.We assessed lipid deposition and analyzed the key enzymes in liver de novo lipogenesis（DNL）pathway in the four groups.7.In vitro,we overexpressed PSTK gene in Hep G2 cell and treated these cells with PA/OA to induce the lipid accumulation.Experiments were divided into four groups:Hep G2+oe Con,Hep G2+oe PSTK,Hep G2+oe Con+PA/OA,Hep G2+oe PSTK+PA/OA.We assessed lipid deposition and analyzed the expression of key enzymes of liver DNL pathway in the four groups.8.To verify whether AMPK is involved in the lipid metabolism regulated by PSTK,we detected the changes of AMPK and p AMPK under the following conditions:Alb-cre,Pstk^fl/fl+HFD and Pstk^fl/fl+HFD mice liver,in vitro PA/OA stimulation-induced hepatocyte lipid accumulation model.9.In order to study the effect of AMPK on PSTK-regulated fatty acid metabolism,we treated Hep G2 cell overexpressing PSTK gene with AMPK activator metformin（Met）in PA/OA condition.The experiments were divided into four groups:Hep G2+oe Con+PA/OA,Hep G2+oe Con+PA/OA+Met,Hep G2+oe PSTK+PA/OA,Hep G2+oe PSTK+PA/OA+Met.We assessed the lipid deposition and the m RNA levels of the key enzymes in the liver DNL pathway in the four groups.Results:1.⑴We successfully constructed Alb-cre,Sps2^fl/fl,Alb-cre,Pstk^fl/fl mice.⑵Compared to control Sps2^fl/fl or Pstk^fl/fl mice,Alb-cre,Sps2^fl/fl or Alb-cre,Pstk^fl/fl mice showed the decreased expression of liver selenoproteins GPX1,CPX2,GPX3,GPX4,TXNRD2,but not TXNRD1.There were no obvious changes in plasma SEPP1 and liver pathological damage in both mice.2.After 8 weeks of high-fat diet,all Alb-cre,Sps2^fl/fl+HFD and Sps2^fl/fl+HFD mice developed an obese phenotype.There was no difference in body weight,food intake,fat and muscle mass,and energy metabolism between Alb-cre,Sps2^fl/fl+HFD and Sps2^fl/fl+HFD obese mice;there was also no diversity in the glucose and insulin tolerance test,pathological injury of liver between the two groups of mice.3.In the obesity mouse model,compared with Pstk^fl/fl+HFD mice,Alb-cre,Pstk^fl/fl+HFD mice had significantly reduced body weight and fat mass,increased food intake;but no difference in glucose metabolism.Further,Alb-cre,Pstk^fl/fl+HFD mice had reduced liver lipid deposition、liver total cholesterol（TC）,Triglyceride（TG）and plasma TC levels.4.According to the results of m RNA sequence,the liver Pstk gene knockout resulted in significant enrichment of fatty acid metabolism pathway.Then,the related differential genes of this pathway were further clustered and analyzed,the important transcriptional regulator gene Srebp1（also called Srebf1）and key enzyme genes Scd1、Fasn of the liver DNL pathway were significantly reduced in Alb-cre,Pstk^fl/fl+HFD mice.5.Comparing with Pstk^fl/fl+HFD obesity models,the transcriptional regulator SREBP1c and key enzymes FAS、p ACC1、SCD1 of liver DNL pathway were decreased in Alb-cre,Pstk^fl/fl+HFD mice;but these genes were not significant different between Sps2^fl/fl+HFD and Alb-cre,Sps2^fl/fl+HFD mice.6.Comparing with WT+PA/OA group,lipid deposits were obvious alleviated,the TC and TG levels were reduced,and SREBP1c、FAS、p ACC1、SCD1 of liver DNL were reduced in LKO+PA/OA group.7.In the Hep G2+oe PSTK+PA/OA group,the lipid deposition and the levels of TC and TG of hepatocytes was significantly increased,the expression of SREBP1c、FAS、SCD1 was higher than Hep G2+oe Con+PA/OA group.8.Compared with Pstk^fl/fl+HFD mice,the increased level of p AMPK was found in liver of Alb-cre,Pstk^fl/fl+HFD mice.LKO+PA/OA also had increased p AMPK level than WT+PA/OA.Furthermore,Hep G2+oe PSTK+PA/OA showed decreased p AMPK level than Hep G2+oe Con+PA/OA group.9.In vitro PA/OA induced lipid accumulation model,the hepatocytes treated with Met could significantly reduce SREBP1c,FASN,SCD1,ACC1 m RNA levels and the lipid deposition and TC and TG levels caused by PSTK overexpression.Conclusion:1.The knockout of Pstk or Sps2 gene in mice liver can reduce the expression of selenoproteins but without obvious liver injury.2.Knockout of liver Sps2 gene has no obvious effect on high fat induced mice obesity;however,the knockout of liver Pstk gene can protect from high fat induced mice obesity and non-alcoholic fatty liver injury.3.The effect of hepatic Pstk gene knockout in reducing hepatic lipid deposition mainly by regulating the AMPK-SREBP1c-ACC1/FAS/SCD1 pathway.