Acquisition Of Complete MRNA And Genomic Sequences Of Goose IGFBP5 And Its Involvement In The Development Of Goose Fatty Liver

Unlike human non-alcoholic fatty liver disease(NAFLD),goose fatty liver is physiological without any overt pathological symptoms such as inflammation and fibrosis even under severe steatosis.As an organ playing an important role in insulin-mediated regulation of metabolism,especially glucose and lipid homeostasis,liver is a major site for synthesis of some endocrine factors such as insulin grow factor 1 and 2(IGF1 and IGF2)and their binding proteins(IGFBPs).IGFBP5 is one of the most evolutionarily conserved members of the IGFBP family and has a modest binding preference for IGF2 over IGF1.It can inhibit or potentiate IGF actions in a range of cells and has an important role in bone,ovary,mammary gland,kidney and liver physiology.Previous studies show that the level of IGFBP5 in serum is correlated with non-alcoholic fatty liver disease(NAFLD).Several lines of evidence indicate that IGFBP5 plays a role in the development of NAFLD.However,the expression and function of IGFBP5 in goose fatty liver remain unknown.The genomic and mRNA sequences of goose IGFBP5 gene previously published in the National Center for Biotechnology Information(NCBI)database were not complete.To acquire the full-length mRNA sequence of goose IGFBP5 gene,5’-RACE assay and the nested PCR were first employed in the present study.Primers were designed based on the newly obtained 5’-untranslated region(5’-UTR)sequence,using which the missing sequence within the first intron of goose IGFBP5 gene was amplified and determined.Based on the completed mRNA sequence,bioinformatics analysis indicated that goose IGFBP5 gene was 18481 bp long,containing three exons(187 bp,119 bp and 132 bp for exon 1,2 and 3,respectively)and three introns(17012 bp,intron 2:145 bp and 363 bp for intron 1,2 and 3,respectively).The putative protein of goose IGFBP5 gene consisted of 145 amino acid residues with two conserved domains including the IGFBP homolog domain and the thyroglobulin type-1 domain.Both newly assembled mRNA and genomic sequences were assigned GenBank accession numbers,i.e.,No.MW351707 and No.MW362807,respectively.These sequences laid a foundation for further study on the role of IGFBP5 in the formation of goose fatty liver.To determine the potential role of IGFBP5 in the formation of goose fatty liver,primary hepatocytes were isolated from Landes goose embryos and transfected with IGFBP5 overexpression vector vs.empty vector.Quantitative PCR analysis indicated that the mRNA abundance of IGFBP5 in goose primary hepatocytes transfected with IGFBP5 overexpression vector was significantly higher than that in the cells transfected with empty vector,which was followed by transcriptome analysis.A total of 777 differentially expressed genes(DEGs)were identified,including 80 downregulated and 697 upregulated in the hepatocytes transfected with IGFBP5 overexpression vector vs.empty vector.The quality of transcriptome analysis was validated by quantitative PCR analysis of some DEGs.The KEGG pathway analysis identified the DEGs were enriched in the following pathways:the focal adhesion pathway,the ECM-receptor interaction pathway,the regulation of the actin cytoskeleton pathway,the MAPK signaling pathway,and the GnRH signaling pathway.These pathways are known to be associated with the occurrence of inflammation and fibrosis in the liver.The results suggest that IGFBP5 may participate in the formation of goose fatty liver through these pathways,which needs to be verified by further investigation.To address the involvement of IGFBP5 in the development of goose fatty liver,quantitative PCR analysis and immunoblotting assay were performed on goose fatty liver vs.normal liver.Data showed that the mRNA abundance of the IGFBP5 gene in the fatty liver was significantly lower than that in the goose normal liver.It is consistent with the immunoblot analysis of IGFBP5 protein in goose fatty liver vs normal liver.This finding reconfirms our previously published result.Moreover,quantitative PCR analysis on the downstream genes of IGFBP5 showed that the expression of DBI and LOC106036132 genes was induced in goose fatty liver versus normal liver.Immunoblotting analysis on the p38 MAPK pathway,an inflammation related pathway,showed that the mRNA or protein expression levels of total and phosphorylated p38 MAPK were suppressed in goose fatty liver versus normal liver.This is in line with IGFBP5 functions revealed by transcriptome analysis of goose hepatocytes treated with IGFBP5 overexpression vector vs.empty vector.In addition,the expression of IGFBP5 in goose primary hepatocytes could be significantly inhibited by 100 nM estradiol and 10 nM insulin but not by 50 nM troglitazone and 50mmol glucose.These findings suggest that IGFBP5 is involved in the development of the goose fatty liver via a number of genes(e.g.,DBI,LOC106036132)and the p38 MAPK pathway.In conclusion,IGFBP5 suppression in goose fatty liver leads to the decreased expression of p38 MAPK protein,which may contribute to preventing goose fatty liver from the occurrence of pathological symptoms,including inflammation and fibrosis.