The Regulatory Effects Of SREVPs On Lipid Mechanism In Bovine Preadipocytes And Fibroblasts

Sterol regulatory element binding proteins (SREBPs) belongs to membrane-boundprotein. It is the main activator of some key enzymes in the synthesis process of fatty acidand cholesterol. It also plays an important role in maintaining the stability of cytoplasm.Presently, the mechanism of SREBPs regulating preadipocytes differentiation and thetransdifferentiation of fibroblast into adipocytes is not clear. In order to detect the function ofSREBPs in the process of cell differentiation, we mainly focused on SREBP1and SREBP2ofSREBPs family in our present study. Bovine primary preadipocytes and primary fibroblastwas used as the media. Adenovirus mediated gene overexpression and inhibition was used asthe main biotechnology. Then the function of these two genes in the process of preadipocytesdifferentiation and fibroblast transdifferentiation was explored. Meanwhile, we detected theinteraction of these two genes on cellular level; these two genes’ effects on the expressionpattern of the genes having important effects on lipid metabolism were also examined. Themain results of this research were as follows:(1) The complete CDS regions of bovine SREBP1and SREBP2were successfullyobtained. The nucleotide length of these two genes were3441bp and3423bp, coding1146and1140amino acid residues respectively.(2) Through screening, one targeted shRNA was obtained with the interference efficiencyof87.4%, named shRNA-1053. Then, it was recombined with adenovirus-backbone-vectorpAd/PL-DEST in vitro and recombinant vector pAd-1053was obtained. The shutter vectorcarrying bovine SREBP1and SREBP2complete CDS regions was successfully constructed,and further packaged in E.coli BJ5183cell, and then we got the recombinant adenovirusexpression vector pAd-SREBP1and pAd-SREBP2. pAd-SREBP1and pAd-SREBP2wereamplified in HEK293A cell line. The viral titer was determined by fluorescence labellingmethod of GFP. Results demonstrated that the virus titer of Ad-SREBP1、Ad-SREBP2andAd-CMV-NC was1.5×109、7×108and1.3×109GFU/mL, respectively. The virus titer ofAd-1053and Ad-U6-NC was7×108and9×108GFU/mL.(3) SREBPs could promote the accumulation of lipid droplet in adipocytes. Results ofoil O staining demonstrated that over-expression of both SREBP1and SREBP2or just over expressing one of them in preadipocytes or fibroblast can improve the accumulation of lipiddroplet, significantly promote the differentiation of preadipocytes and successfully induce thetransdifferentiation of fibroblast into adipocytes-like cells. After the inhibition of SREBP1,the content of lipid droplet in preadipocytes decreased notably; but the content increasedslightly in fibroblast. Adenovirus–mediated interference of SREBP1combined with the overexpression of SREBP2can remarkably increase the content of lipid droplet in fibroblast.(4) Real-time PCR results indicated that there was mutual promotion relationship withinSREBPs family. In preadipocytes and fibroblast, over-expression and inhibition of SREBP1can promote and inhibit the expression of SREBP2respectively; over-expression of SREBP2can also enhanced expression level of SREBP1.(5) SREBPs can regulate some genes associated with lipid metabolism. Our resultsindicated that over-expression of SREBP1or SREBP2upregulated the mRNA expressionlevel of stearoyl-CoA desaturase (SCD), fatty acid synthase (FAS), acetyl-CoA carboxylasealpha (ACC-α), lipoprotein lipase (LPL), fatty acid binding protein-3(FABP3) and leptin1;while, down-regulation of SREBP1reduced the expression level of these genes. SREBP1justshowed effects on fatty acid transport protein-1(FATP1) at the early stage of differentiation.However, SREBP2showed no significant effects on the expression pattern of FATP1andFABP4.(6) Over-expression of SREBP1and SREBP2had no remarkable effects on PPARγ andC/EBPα gene at cellular level in bovine. However, down-regulation of SREBP1cansignificantly reduce the mRNA expression level of PPARγ and C/EBPα at the initial stage ofadipocyte differentiation.In conclusion, adenovirus mediated overexpression and inhibition biotechnology wasused to detect the function of SREBP1and SREBP2regulating preadipocytes differentiationand fibroblast transdifferentiation; we subsequently explored the interaction of these twogenes at transcriptional level and their effects on the functional genes related to lipidmetabolism. Our present research not only provided theory evidence for exploring theaccurate process of preadipocytes differentiation and fibroblast transdifferentiation, but alsolaid foundation for studying the mechanism of bovine fat formation and deposition.