The Effects Of FoxO1 Regulating Lipid Metabolism In Goose Primary Hepatocytes

Objective: The liver plays an important role in maintaining the balance of the body glucolipid metabolism. In recent years, the Forkhead box protein 01 (FoxO1) play an improtant role in lipid metabolism, which became the research target gene of insulin resistance, obesity and type 2 diabetes. In this experiment,using goose primary hepatocytes as experimental material, by transfection FoxO1 interference plasmid, adding insulin and NVP - BEZ235 in hepatocytes, the related factors of fatty acid synthesis, oxidation and VLDL - TG secretion were detected to reflect the lipid metabolism change with the RT-PCR, western blotting, Elisa and oil red O staining method,which could be used to analyze the role of insulin and FoxO1 in regulating goose hepatocytes lipid metabolism, explore it is whether the regulation of FoxO1 on liver lipid metabolism is mediated by PI3K-Akt signaling pathway, and further clarify the role of FoxO1 transcription factors in the process of goose liver lipid metabolism. It could provide theoretical basis for exploring the molecular mechanism and gene regulation mechanism of insulin, PI3K Akt signaling pathway and FoxO1 in liver lipid metabolism.Results:1.After 24 hours of transfection pcDNA6.2 - GW/EGFP - miR FoxOl plasmid, total FoxO1 protein significantly decreased, and fluorescent quanfitation PCR detection showed FoxO1 interference efficiency reaced 64.3%, which showed FoxO1 was inhibited in goose liver cells.2.The insulin treatment increased FoxO1 phosphorylation activity in goose hepatocytes, and the NVP - BEZ235 treatment increased FoxO1 gene mRNA expression and protein expression (P< 0.05), which suggested that insulin can inhibit FoxO1 activity and NVP-BEZ235 can increase its activity.3.FoxO1 interference can significantly reduce the Aktl mRNA expression level, and increase the mTORcl mRNA expression levels (P< 0.05); Western blotting tests showed that FoxO1 interference can reduce AKT phosphorylation levels and increase mTORcl protein expression, which indicated that FoxO1 can regulate Aktl and mTORcl gene expression.4.FoxO1 interference significantly reduced the mRNA expression and the kinase expression of FAS, ACCa, CPT1, ACOX1, MTP and ApoB gene (P< 0.05), increased DGAT1 mRNA expression, however, the gene mRNA expression of SREBP1, DGAT2, PPARa and PPARy showed no significant difference (P> 0.05). After FoxO1 interference, Western blot test showed that the protein content of ACCaand MTP was significantly lower than the control group, but CPT1 had no significant change. Oil red O staining showed the goose hepatocytes FoxO1 interference multiply increased lipid droplets and lipid deposition, meanwhile, increased intracellular and extracellular TG content, decreased extracellular VLDL content.5.After insulin treatment, CPT1, ACOX1, MTP, ApoB, PPARa and PPARy gene mRNA expression decreased in goose hepatocytes, and the ACCa, FAS, SREBP1, DGAT1 and DGAT1 gene expression increased. The result of Western blotting and Elisa showed the protein level of CPT1 and MTP was similar with the mRNA expression level. In addition, oil red O staining found that fat droplets increased. In addition, after insulin treatment, transfection FoxOl interference plasmid together in goose hepatocytes, CPT1, ACOX1, MTP, ApoB, PPARa and PPARy gene mRNA expression was significantly lower than the control group, and more lower than insulin treatment significantly (P< 0.05). However, the expression level of SREBP1 and DGAT1 increased, the ACCa and FAS gene mRNA expression levelshowed no significant difference with control group. Meawhile, intracellular and extracellular TG content were higher than the control group, and extracellular VLDL content reduced; At the same time, the result of oil red O staining showed cell lipid drops granules and lipid deposition increased.6.After NVP - BEZ235 inhibiting PI3K-Akt-mTORcl signaling pathways, the mRNA expression of ACCa, FAS, SREBP1, DGAT1 and DGAT1 decreased, and the mRNA expression of CPT1, ACOX1, MTP, ApoB, PPARaand PPARy gene increased (P< 0.05). The result of Oil red O staining showed that intracellular lipid droplets particles decreased significantly, while intracellular and extracellular TG content decreased, extracellular VLDL content increased. However, after NVP-BEZ235 inhibiting PI3K-Akt-mTORcl signaling pathway, FoxO1 interference plasmid transfection together did not significantly change the mRNA expression of ACCa, FAS, SREBP1, DGAT1, DGAT1, CPT1, ACOX1, MTP, ApoB, PPARa and PPARy gene, and intracellular lipid droplets content tended to normal.