| ChREBP is one of the important nuclear factors in regulating glycolysis and de novo lipogenesis, it has emerges as a central determinant of cellular carbohydrate and lipid metabolism through its regulate of key genes of the glycolysis and fatty acid synthase. In the present study, ChREBP sequence of goose was cloned by RACE and analyzed through bioinfomation, tissue relative mRNA distributions was analyzed. Overfeeding was carried out by providing Landes and Sichuan White goose. After two weeks of overfeeding, tissue relative mRNA, TG concentrations of liver and plasma parametric were detected. In cellular level, goose primary hepatocytes cultured with different concentration of glucose, insulin and T0901317 the relative mRNA level of ChREBP was measured. The results were as follows:(1) The length of goose ChREBP sequence is cloned by 3'RACE and 5'RACE. The relative molecular mass and isoelectric point of ChREBP protein were 35.62kDa and 5.36, respectively. BLAST analysis suggested that there was the most similiarity between the ChREBP mRNA sequences of goose and Gallus gallus, while the homology of amino acids was 92%.(2) The bioinformation analysis suggested that the complete cDNA sequence of goose ChREBP, which contained a 125 bp 5'UTR, a 945 bp open reading frame (ORF) encoding 314 amino acids, and a 192 bp 3'UTR. ChREBP amino acid sequence contained 17 phosphorylation sites, including 14 phosphorylation sites in Ser residue,2 phosphorylation sites in Thr residue,1 phosphorylation sites in tyr residue.There is one O-glycosylation site, no N-glycosylation site and conserved fuction motif.(3) The result of real time RT-PCR demonstrated that, the goose ChREBP mRNA was most abundant in liver, highly expressed in sebum, abdominal fat and intestine, hardly expressed in brain, testis and muscle tissue.(4) The mRNA expression of ChREBP gene was markedly decreased in the liver of Sichuan White goose and Landes goose after overfeeding two weeks, and the gene level was marked lower in Landes goose than that in Sichuan White goose (P<0.05).(5) The mRNA abundance of ChREBP in liver had negative correlations with the TG content in liver lipid, plasma insulin level and plasma glucose level in Landes goose. Especially had significant negative correlations with TG content in liver lipid and plasma insulin level (P<0.05). However, the mRNA abundance of ChREBP in liver of Sichuan White goose had only significant positive correlations with plasma glucose level.(6) The MTT assay was used for the quantification of cytoactive. After 24 h cultured with glucose, insulin and T0901317,30 mmol/L (P<0.05) glucose had an significantly inducing effect on the cytoactive, 5 mmol/L glucose and 50 nmol/L insulin had no effect on cell cytoactive,100,150 nmol/L insulin and 0.01,0.1,1,10μmol/L T0901317 had inducing effect on cell viability,200 nmol/L insulin had an inhibiting effect on the cytoactive.(7) 5 mmol/L glucose had no effect on intracellular TG concentration,30 mmol/L (P<0.05) glucose significantly increased TG accumulation. light concentration insulin had no effect on TG concentration, but significantly decreased with 200 nmol/L insulin. 0.01μmol/L T0901317 had no effect on TG concentration, with the concentration of T0901317 increasing, the TG concentration increased significantly (P<0.05).(8) Different concentration of glucose, insulin and T0901317 cultured with goose primary hepatocytes had evident effect on ChREBP expression. Compared with control group, the mRNA level of ChREBP in the experimental groups which were treated with 5 mmol/L and 30 mmol/L glucose was increased by 7.7 (P<0.01) and 8.3 (P<0.01) times, respectively; Treated with 50 nmol/L,100 nmol/L,150 nmol/L and 200 nmol/L insulin was increased by 2.41 (P<0.01),2.69 (P<0.01),3.10 (P<0.01) and 1.68 (P<0.05) times, respectively; Treated with 0.01,0.1,1 and 10μmol/L T0901317 was increased by 2.86 (P<0.01),4.19 (P<0.01),4.4 (P<0.01) and 7.02 (P<0.01) times, respectively. |
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