The Effects Of Adenovims-mediated SREBP-1c Gene Overexpression On Fat Deposit In Calf Hepatocytes Cultured In Vitro

Fatty liver in cows is a nutritional and metabolic disease, which occurs primarilyin the lactation period. The development of fatty liver is closely related to sterolregulatory element binding protein-1c (SREBP-1c) in insulin signaling pathway.SREBP-1c is an important nuclear transcription factor contributing to lipid synthesiswithin the hepatocytes. Despite of its affect on expression of lipid transporter genes,most of its target genes of downstream are key rate-limiting enzyme in the lipidsynthesis. The ability of cows to export fat from liver is low in comparison to that ofmonogastric animals, which thus contributes to its morbidity of fatty liver increasingremarkablely. Possibly, these results arise from lipid synthesis mediated by SREBP-1c.Therefore, identifying effects of SREBP-1c on key genes in lipid metabolism mayprovide a basis to reveal its role in the development of fatty liver disease and toprevent the development of fatty liver disease. Calf hepatocytes cultured in vitro wastransfected by newly constructed recombinant adenovirus of SREBP-1c gene. Theconstructed recombinant adenovirus was assessed by Enzyme digestion and theexpression of genes in fatty acid synthesis, oxidation and transport was determined byqRT-PCR, Western blotting and ELISA.SREBP-1c gene was first transfered to a shuttle vector and inserted intoadenovirus plasmid. After digestion assessment, the recombinant adenoviruslinearized by Pac I transfected HEK293cells. Viral titer was determined with TCID50method after amplification. The results showed that the number of strips was in linewith expectations and the virus titer reached1.2×1012PFU/mL. Results demonstratedthat the recombinant adenovirus plasmid carrying SREBP1c gene was constructedsuccessfully. Hepatocytes cultured in vitro was infected by recombinant adenovirus with MOI(100). The results showed the expression of SREBP-1c gene and protein wasupregulated (P＜0.01). The levels of ACC-1gene and protein were highlyexpressed(P＜0.01). The expression of ACSL1gene raised and its activity increasedsignificantly (P＜0.01). Hower, the expression of genes and enzyme activity involvedin fatty acid oxidation declined to some degree. The expression of CPT-1and ECHgene (P＜0.01) also decreased except for that of CPT-2, LCAD, HADH and KT gene(P＜0.05). The activity of CPT-1, CPT-2, LCAD, HADH, ECH and KT decreasedsignificantly (P＜0.05). The genes involved in assembly and secretion of VLDLvaried inconsistently. The expression of MTTP (P＜0.05), ApoE (P＜0.05) andLDLR (P＜0.01) gene was upregulated, while the expression of ApoB (P＜0.01)gene was downregulated. The levels of ApoE protein increased significantly (P＜0.05). Interestingly, there was no significant change in the levels of MTTP protein (P＞0.05). The concentrations of TG and VLDL varied conversely. The concentrationsof TG increased, while the secretion of VLDL decreased. These results indicated thatthe SREBP-1c gene was highly expressed in hepatocytes cultured in vitro, which canpromote fatty acid synthesis and inhibit fatty acid oxidation and the secretion ofVLDL. SREBP-1c is a key regulatory transcription factor involved in lipid synthesis,and it is an important target gene for the therapy of fatty liver in cattle.