Molecular Mechanism Of Trib1 Regulating Brown Fat Energy Metabolism And The Intervention Effect Of Berberine

BackgroundDue to changes in lifestyle and diet and the role of genetic factors,the prevalence of obesity is increasing year by year.Due to the long-term imbalance of energy intake and consumption by the body,excess energy is stored in white fat cells in the form of triglycerides,which manifests as obesity.At present,obesity is mainly treated by surgery or drugs to reduce the body’s energy intake.But this has many side effects associated with it,and proven methods are still lacking.Therefore,it has been proposed that obesity can be treated by increasing energy expenditure.Brown adipose tissue(BAT)is a beneficial fat located in the scapula of the human body,and contains a large number of mitochondria,in which high levels of uncoupling proteins consume triglycerides and glucose to generate heat,so regulating brown fat function is of great significance for the treatment of obesity.Significance.Tribbles homolog 1(Trib1)belongs to the Tribbles pseudokinase family and plays akey role in tumorigenesis and macrophage polarization.Genome-wide analysis showed that Trib1 expression was highly correlated with blood lipid levels,but the relationship between Trib1 and adipose tissue metabolism was unclear.Therefore,this project used CRISPR/Cas9 technology to establish Trib1 knockout mice to explore the effect of Trib1 on energy metabolism in brown adipose tissue.Berberine is an isoquinoline alkaloid isolated from the traditional Chinese medicine Coptis chinensis Franch.Modern pharmacological studies have found that berberine can reduce the body weight and blood lipids of obese mice,and improve the inflammatory response and insulin resistance caused by hyperlipidemia.,promoting the browning of white fat and increasing energy expenditure,is a potential drug for the treatment of obesity.Some studies have found that berberine can reduce the serum triglyceride level in mice by increasing the protein expression of LDLR and TRIB1 in the liver of mice,but the effect of berberine on the energy metabolism of TRIB1 and BAT in obesity models is still unclear.AimThis project explored the regulatory effect of Trib1 on energy metabolism in brown adipose tissue from both in vitro and in vivo,and clarifies the molecular mechanism of berberine to improve brown fat metabolism in the treatment of obesity,so as to provide a scientific basis for the treatment of metabolic diseases.Methods1.Perform RNA-Sequencing on myocardial tissue of db/db mice aged 1,6 and 12 months to detect common differential genes in the development of dyslipidemia and conduct target screening.RT-PCR was used to determine the expression levels of Tribl in various tissues and organs of C57BL/6J mice and in different differentiation stages of 3T3-L1 adipocytes.C57BL/6J mice were intraperitoneally injected with β3adrenoceptor agonist(1 mg/kg),and the expression levels of Tribl and thermogenesisrelated proteins and genes were detected by HE staining,Western blot and RT-PCR.2.Use CRISPR/Cas9 technology to establish Trib1 knockout mice for breeding and identify their genotypes.Body fat meter,tissue HE staining,ELISA and animal metabolism analysis system were used to detect the effects of Trib1 knockout on body weight,blood lipids and heat production in mice.Trib1 knockout mice were intraperitoneally injected with β3-adrenoceptor agonist(1 mg/kg).Effects of brown adipose tissue metabolic levels.3.The TRIB1-GST fusion protein was constructed by prokaryotic plasmids for protein binding in vitro(Pull down assay),and RNA-Sequencing was performed on the brown adipose tissue of Trib1 knockout mice and wild-type mice to detect the key molecules that Trib1 regulates brown fat function.Western blot,RT-PCR,mitochondrial complex enzyme activity detection kit and ATP level detection kit were used to explore the effect of Trib1 knockout on brown fat mitochondrial respiratory chain.Trib1 was further overexpressed on 3T3-L1 adipocytes by eukaryotic plasmids,and the targets and mechanisms of Trib1 regulating brown fat function were verified by RNA-Sequencing,cell oil red staining,Mitotracker mitochondrial staining and Seahorse energy metabolism instrument.4.After 8 weeks of oral administration of berberine(100 mg/kg)to ob/ob mice,the effect of berberine on body weight and blood lipids of obese mice was detected by insulin tolerance test,body fat meter,tissue oil red staining and ELISA.and inflammatory effects.The effects of berberine on brown fat thermogenesis in obese mice were investigated by animal metabolic analysis system,temperature measurement in cold environment,RT-PCR and Western blot.Trib1 was further knocked down by siRNA,and the effect of Trib1 knockdown on the regulation of energy metabolism by berberine was detected by RT-PCR,Western blot,Bodipy staining and Seahorse energy metabolism analyzer.Results1.In vivo experiments showed that in the myocardial tissue of db/db mice,the expression of Trib1 was significantly down-regulated with the progress of lipid metabolism disorders in mice.The gene expression level of Trib1 was significantly higher in brown fat of C57BL/6J mice than in other tissues.In a model in which cold stimulation and β3-adrenergic agonists enhanced brown fat function,the expression level of Trib1 was significantly increased.In vitro experiments showed that with the increase of lipid droplet accumulation in 3T3-L1 adipocytes,the expression of Trib1 gradually decreased,indicating that Trib1 is directly involved in adipose tissue metabolism.2.Trib1 knockout mice were used to determine their phenotype.The results showed that Trib1 knockout mice significantly increased blood lipid levels,increased lipid droplets in brown adipocytes,and significantly decreased the expression of the key protein UCP1,which was accompanied by leptin and insulin.resistance,suggesting that Trib1 has a regulatory effect on brown fat metabolism.Trib1 knockout mice were further treated with β3-adrenoceptor agonists,and the results showed that Trib1 knockout impairs the lipid metabolism and thermogenesis of brown fat,swells the brown fat mitochondria in mice,and destroys the mitochondrial membrane structure.3.At the animal level,through RNA-Sequencing and Pull Down experiments,it was found that Tribl knockout disrupted mitochondrial dynamic homeostasis by affecting the activity of the electronic respiratory chain,resulting in structural damage and lipid metabolism dysfunction.In this study,Trib1 was overexpressed in 3T3-L1 preadipocytes to validate it.The results of in vitro experiments showed that the overexpression of Trib1 increased the basal metabolic level of cells,and the effect of Trib1 was inhibited after adding the inhibitor.4.Berberine treatment of ob/ob spontaneously obese mice for 8 weeks can reduce mouse body weight and blood lipid level,and increase the expression of Tribl and mitochondrial respiratory chain complexes Ⅰ,Ⅱ and Ⅲ in brown fat of ob/ob mice increased mitochondrial copy number and expression levels of mitochondrial fatty acid oxidation-related genes,and enhanced mitochondrial function in brown fat in obese mice.The ability of berberine to reduce lipid droplets and increase cellular respiratory metabolism was inhibited after adding siRNA to interfere with the expression of Tribl in 3T3-L1 adipocytes.These findings suggest that berberine inhibits obesity by increasing the expression of Tribl and promoting brown adipocyte metabolism.ConclusionsTribl knockout disrupts brown fat thermogenesis and induces hyperlipidemia and obesity in mice.It was further demonstrated that Trib1 knockdown reduced mitochondrial respiratory chain complex activity,resulting in an imbalance of mitochondrial fusion and fission,which subsequently led to mitochondrial structural damage and lipid metabolism dysfunction.Overexpression of Tribl in vitro can reduce lipid droplet accumulation,increase mitochondrial number,and enhance the level of respiratory metabolism in adipocytes.The ability of berberine to reduce lipid droplets and increase cellular respiratory metabolism was inhibited after adding siRNA to interfere with the expression of Trib1 in adipocytes.It is shown that Trib1 regulates the function of mitochondria and brown adipocytes in mice,and is expected to be a new target for the treatment of metabolic diseases such as hyperlipidemia,fatty liver and obesity.