Study On The Role Of IGFBP2 In The Development Of Goose Fatty Liver And Related Mechanism

The capacity of goose liver to deposit fat is strong.Goose liver can form severe steatosis in a short period of time.In mammals,severe hepatic steatosis is usually accompanied by irreversible pathological symptoms,such as hepatitis,fibrosis and injury.However,goose fatty liver(or foie gras)can return to normal under certain condition without any obvious damages,suggesting that goose liver may have a unique mechanism underlying its development.Mammalian fatty liver studies indicate that insulin resistance is an important mechanism underlying fatty liver formation,and inflammation,endoplasmic reticulum stress,ceramide signaling pathway can promote the occurrence of insulin resistance.However,our group revealed that insulin resistance plays an important role in the formation of goose fatty liver,but inflammation,endoplasmic reticulum stress and ceramide signaling pathway are not activated in goose fatty liver.Therefore,uncovering new mechanism underlying insulin resistance in goose fatty liver will help address the mechanism underlying the formation of goose fatty liver.IGFBP2 is one of the insulin-like growth factor(IGFBPs)family genes.IGFBP2 is primarily secreted by the liver and it is involved in energy and fat metabolisms in the cell.Therefore,addressing the role of IGFBP2 in goose fatty liver and the related mechanism will promote the understanding of the mechanism underlying the formation of goose fatty liver.In this study,quantitative PCR(qRT-PCR),cell culture,gene interference(overexpression or knockdown)were carried out to elucidate the role of IGFBP2 in the formation of goose fatty liver and the related mechanism by determing the expression of IGFBP2 in the development of goose fatty liver,screening the factors that affect the expression of IGFBP2,revealing the function of IGFBP2,and the potential mechanism.The main results are as follows:1.Study on the expression of IGFBP2 in the development of goose fatty liver.In this study,real-time PCR was used to determine the expression of IGFBP2 in the liver,muscle and fat of both the overfed and normally-fed geese.The data showed that the expression of IGFBP2 in these tissues was significantly inhibited by overfeeding,suggesting that it plays an important role in the development of goose fatty liver.2.The effect of fatty liver-related factors on the expression of IGFBP2 in goose primary hepatocytes.In this study,goose primary hepatocytes were treated with different dosages of glucose,insulin,fatty acids and cholesterol,and the expression of IGFBP2 was subsequently determined by qPCR.The results showed that glucose,insulin and linoleic acid reduced the expression of IGFBP2 significantly compared to control cells,while the expression of IGFBP2 was up-regulated by cholesterol,but palmitate and oleate had no effect on the expression of IGFBP2.3.Screening for transcription factor of IGFBP2.First,upstream sequence of IGFBP2 was amplified by PCR with primers that were designed based on turkey genomic sequence.The PCR products were subsequently sequenced and verified.Using the upstream sequence,multiple transcription factor(s)including Oct-1 were predicted with online 'Gene Regulation' software.To verify Oct-1 as a transcription factor of IGFBP2,the agonist of Oct-1,retinoic acid,was used to treat goose primary hepatocytes.Data indicated the expression of IGFBP2 was up-regulated by retinoic acid treatment,suggesting Oct-1 is a potential transcription factor of IGFBP2.4.The effect of IGFBP2 on fat deposition in goose primary hepatocytes.Data showed that overexpression of IGFBP2 gene in goose primary hepatocytes after 48 hours of transfection significantly inhibited fat deposition in the IGFBP2-overexpressing cells compared to the control cells that were transfected with empty vector.In contrast,siRNA-medicated knocking-down of IGFBP2 gene significantly increased fat deposition in goose primary hepatocytes compared to the cells transfected with control siRNA.Together,these findings indicated that IGFBP2 gene could regulate lipid metabolism,which was in line with the observation that IGFBP2 gene was inhibited in different tissues during the development of goose fatty liver.5.The regulation of IGFBP2 on the expression of key genes in PI3K-Akt pathway.In the IGFBP2-overexpressing primary hepatocytes,the expression of IGF-1,PI3K and AKTIP was significantly inhibited and the RTK gene was induced.However,knocking-down IGFBP2 gene did not resulted in the opposite result.The expression of all the genes was not significantly changed by IGFBP2 knocking-down,though the expression of IGF-1 was induced by IGFBP2 interference.This is probably due to the redundant function of other IGFBP family genes.Overexpression of IGFBP2 gene indicated that IGFBP2 gene could regulate PI3K-Akt signaling through transcriptional regulation.In summary,these findings suggest that inhibition of IGFBP2 expression may promote fat deposition in the cell by regulating the PI3K-AKT signaling pathway and high glucose,insulin and linoleic acid can lead to the inhibition of IGFBP2,thus the IGFBP2 gene plays an important role in the development of goose fatty liver.