The Role Of ChREBP And ACC1 Gene In Glyco-and Lipometabolism In Hepatocyte

Liver tissue is a major organ of glycometabolism (glycolysis and hepatic glycogen synthesis) and lipometabolism (lipid synthesis). The metabolic system plays an important action during the maintain energy balance. Lipid synthesis are regulated by some pivotal enzymes which activated by allosterism and contravalence modification. Most of the enzymes linked to glycometabolism and lipometabolism are regulated by the diet. And glucose is one of the significant nutrients. Glucose could act on the transcription of the genes.Carbohydrate response element-binding protein (ChREBP) and Acetyl-CoA carboxylase (ACCs) are both important factors of glycometabolism and lipometabolism in liver. ChREBP is a member of the bHLH/ZIP transcription factor family. It is expressed in the liver significantly. The high carbohydrate diets could up regulate the expression of ChREBP, as the high lipogene diets would suppress its expression. It could bind to the ChoRE region of many lipogenic enzymes to actived their gene transcription. Meanwhile, its activity would be regulated by insulin, glucose and antidiabetiecs, that why we need to study the mechanism of ChREBP.ACC has two isoforms:ACC1 and ACC2. ACC1 is mainly expressed in the cytoplasm of liver and adipose tissue, and proposed to be involved in fatty acid synthesis, whereas ACC2 is predominantly expressed in skeletal muscle and heart, which is believed to be responsible for fatty acidβ-oxidation. ACC is one of the factors-induced by carbohydrate response element-binding protein (ChREBP). It is regulated by diet, hormone and other physiological factors else.In this study, by choosing pig and mouse ChREBP and ACC1 and using real-time PCR, transfection and western blotting, we studied the gene characteristics, transcriptional regulation and function of ChREBP, and transcriptional regulationg of ACC1 and got the following results:1. By the comparative analysis, according mouse and human ChREBP sequences, pig ChREBP gene was cloned. By semi-quantitative RT-PCR, the tissue expression profile of ChREBP in eleven tissue samples was determined. By radiation hybrid (RH) mapping analysis, pig ChREBP was located and compared with the result of human ChREBP.2. With different time and different dose treatment of glucose and insulin on HepG2 hepatoma cell line, by real-time PCR, the mRNA expression of ChREBP, sterol regulatory element binding protein-1c (SREBP-1c), ACC1 and fatty acid synthetase (FAS). The results indicated that glucose could increased the expression of ChREBP, SREBP-1c, ACC1 and FAS separately.3. By RT-PCR, CDS of ChREBP and SREBP were cloned. Subsequently, their expression vectors were constructed, respectively. By transfection or co-transfection, their effects on transcription of ACC1 and FAS were investigated. The results indicated that there is no cooperation but competition between ChREBP and SREBP-1c.4. By using arachidonic acid (AA), ACC1 was obviously activated while as the expression of ChREBP and SREBP-1c was reduced. The expression of liver X receptor (LXR) and peroxisome proliferators-activated receptorα(PPARα) which bind to the promoter of ACC1 were reduced while as cAMP-response element binding protein 1 (CREB1) was activated. It suggests that the stimulation of AA was not via the glycometabolism approaches which have been studied.5. By real-time PCR, the expression of the symbol genes in cells linked to glucose absoption and fatty acid synthetic, such as glucose transport 2 (GLUT2), glucose-6-phosphatase (G6Pase) and FAS was checked after dealing with AA. Measured the wastage of glucose in culture media and the creation of triacylglycerol (TG) in cells. The results domanstrated that the inducement of AA on ACC1 can promote fatty acid synthesis but has no relation to glycometabolism.6. Constructing the expression vector of CREB1, by transfecting and co-transfecting, its effect on transcription of ChREBP, SREBP-1c and ACC1 were investigated. Subsequently, the promoter vector of ACC1 was constructed. By co-transfecting with the expression vector of CREB1, the activity of firefly luciferase was checked. The datas showed that CREB1 could bind to the PⅡpromoter of ACC1. It is match the result of AA stimulation. It confirmed the effect of AA on ACC1 expression via activation of CREB1.