Eeffects And Mechanism Of Insulin And Glucose On Liver Cell Proliferation In Goose

Foie gras is a kind of high grade nutritional food with high economic value.In the production of goose fatty liver,the goose was overfed with high energy carbohydrate feed.The fatty liver volume and weight increased by 5?10 times.Although the liver cells have fat degeneration after overfeeding,the liver cells still maintain their physiological functions.The current study shows that insulin and glucose play an important role in cell proliferation,so they may be associated with liver cell proliferation in the process of goose liver steatosis.The present study was aimed to investigate the effects of overfeeding on growth performance,serum insulin,glucose and TG concentrations,and the mRNA expression of related genes in cell proliferation;and the effects of insulin and glucose,PI3K-Akt-mTOR signal pathway,FoxO1 and Sirtl on liver cell proliferation;which was help for to elucidate mechanism of the goose fat liver formation induced-by carbohydrate,and the regulation mechanism of insulin and glucose on liver cells proliferation in goose.In addition,the goose fatty liver could be a good model for learning hepatic steatosis,the prevention and treatment of fatty liver and relating liver disease.Eighty healthy male geese of each breed of Tianfu meat goose and Gang goose,incubated at the same day,were raised until 13 weeks old.At the age of 13 weeks old,80 geese of each breed of Tianfu meat goose and Gang goose was divided into two groups,respectively.The treatment group contains 60 geese,the rest of 20 geese as control group.The treatment group was treated with the pre-overfeeding of 7 days and following the overfeeding of 21 days,and the control group was treated with normal feeding.At the age of 17 weeks old,all geese were slaughtered.Blood samples were collected at the time of slaughter.Serum was prepared by centrifugation at 3000 rpm for 10 mitutes and was stored at-20 ? for later analyze.After slaughter,parts of leg muscle,breast muscle,abdominal fat pad,subcutaneous adipose tissue and liver were collected and stored at-80? for later analyze.Studing the effects of overfeeding on body weight,leg muscle weight,breast muscle weight,abdominal fat pad weight,subcutaneous adipose tissue weight and liver weight,serum glucose,insulin and TG concentrations,and the mRNA expression of related genes in cell proliferation.At the same time,through the isolation and culture of Tianfu meat goose primary hepatocytes,the factors involved in the cell proliferation was measured using real time fluorescent quantitative PCR,Western blot,BrdU ELISA and relating techniques,and the regulation mechanism of overfeeding,insulin and glucose in cell proliferation of goose liver was discussed.The main results are as follows:1.The body weight,liver weight,leg muscle weight,abdominal fat weight,subcutaneous fat weight of Tianfu meat geese and Gang geese significantly increased after overfeeding.Meanwhile,the concentrations of glucose,insulin and TG in serum were increased significantly.The mRNA expression levels of PI3K and Aktl in Tianfu meat goose liver were significantly higher than those in leg muscle and breast muscle,however,the mRNA expression levels of Aktl and mTOR in Gang goose liver were significantly higher than those in leg muscle and breast muscle.The mRNA expression levels of rictor,raptor and S6K in Tianfu meat goose liver increased significantly,while the mRNA expression levels in Gang goose liver had no significant change.The expression levels of FoxO1 and Sirtl in Tianfu meat goose and Gang goose liver significantly decreased,while the mRNA expression level of CyclinDl/D2/D3 significantly increased.The protein content of CyclinDl in Tianfu meat goose and Gang goose liver,breast muscle,leg muscle increased significantly after overfeeding.2.Insulin stimulated the mRNA expression levels of the cell cycle initiation factor CyclinDl/D2/D3,and inhibited the mRNA expression levels of p21 and p27,which were involved in the proliferation of goose primary hepatocytes.In addition,insulin stimulated the mRNA expression levels and protein content of PI3K-Akt-mTOR signaling pathway.LY294002,Rapamycin and NVPBEZ235 are PI3K-Akt-mTOR pathway inhibitors that reduced CyclinDl/D2/D3 mRNA expression levels,increased p21 and p27 mRNA expression levels,however,insulin could reverse the inhibition of three kinds of inhibitors to some extent.5 mmol/L or 35 mmol/L glucose could stimulate the proliferation of primary liver cells and activate the PI3K-Akt-mTOR signaling pathway.The use of three kinds of PI3K-Akt-mTOR signal pathway inhibitors could significantly dismiss glucose-induced the up-regulation of cell proliferation in goose primary hepatocytes.The single treatment of glucose or insulin,and the co-treatment of glucose and insulin all significantly increased the mRNA expression levels of CyclinDl/D2/D3 and the rate of DNA synthesis.3.After miRNA-FoxO1 inhibited FoxO1,the mRNA expression levels of CyclinDl/D2/D3 and DNA synthesis rate in goose primary hepatocyte significantly increased,however,the mRNA expression levels of p21 and p27 significantly decreased.miRNA-FoxO1 evidently increased protein content of CyclinDl.Insulin,the single miRNA-FoxO1 treatment,or the co-treatment of insulin and miRNA-Fox01 all stimulated the proliferation of the goose primary hepatocytes.However,the up-regulation of on cell proliferation by miRNA-FoxO1 was decreased significantly by NVP-BEZ235.4.After treatment with vitamin PP,inhibitor as SIRT1,the DNA synthesis rate,the ratio of BrdU positive cells and the proliferation index of goose primary hepatocytes were all increased,the mRNA expression levels and protein content of CyclinDl/D2/D3 increased,while the mRNA expression level and protein content of FoxO1 decreased.After treatment with miRNA-FoxO1,the proliferation of goose primary hepatocytes was stimulated,the mRNA expression levels and protein content of CyclinDl/D2/D3 increased,however,the mRNA expression level and protein content of SIRT1 decreased.After the co-treatment with vitamin PP and miRNA-FoxO1,the proliferation of goose primary hepatocytes was also stimulated,the mRNA expression levels and protein content of CyclinDl/D2/D3 increased,while the mRNA expression levels and protein content of FoxO1 and Sirtl all decreased.After the co-treatment with vitamin PP and insulin,the proliferation of goose primary hepatocytes was stimulated,and the mRNA expression levels and protein content of CyclinDl/D2/D3 increased,while the mRNA expression level and protein content of p21 decreased.The inhibitors(LY294002,rapamycin and NVP-BEZ235)of PI3K-Akt-mTOR signal pathway all could dismiss the induction of cell proliferation by vitamin PP inhibitting Sirtl in goose hepatocytes.In summary,the formation of goose fatty liver was accompanied by the proliferation of hepatocytes.Overfeeding,insulin and glucose all can activate PI3K-Akt-mTOR signaling pathway,induce the mRNA expression and protein content of CyclinD1/D2/D3,inhibit the mRNA expression and protein content of p21 and p27,and promote goose liver cells proliferation.FoxOl and SIRT1 have inhibitory effect on cell proliferation in goose liver.The proliferation of goose hepatocytes may be necessary for hepatic steatosis,and for maintaining the normal physiological function of hepatocytes.