Effect Of Insulin Resistance On Lipid Metabolism Of Primary Hepatocytes From Laying Hens And Investigation On This Mechanism

Fatty liver hemorrhagic syndrome(FLHS)is one of main nutritional and metabolic diseases.Disordered insulin signal pathway is the key to many metabolic diseases,whereas its relationship with FLHS rarely has really been reported.Our previous research has found that laying hens with fatty liver hemorrhagic syndrome(FLHS)were not sensitive to insulin,and their insulin signal pathway also changed significantly.Currently,rare research has been found to investigate the relationship between disordered insulin signal pathway and FLHS pathogenesis.Therefore,the current research aims to further investigate the relationship between disordered insulin signal pathway and lipid metabolism of primary hepatocytes of laying hens in vivo.After primary hepatocytes of laying hens with peak egg production were successfully cultured and validated,serum free medium with high glucose concentrations were adopted to induce the insulin resistance model(IR),investigating the effect of IR on lipid metabolism of primary hepatocytes.Rapamycin,the mTOR inhibitor,was adopted to treat hepatocytes for 48 h in order to investigate its inhibiting effect on mTOR and lipid metabolism of hepatocytes.Under the condition of inhibiting mTOR,55mmol/L glucose was used to treat hepatocytes to research whether mTOR promotes lipid metabolism through feedback regulating IR.And results showed that:(1)Compared with the control group,the ability of hepatocytes to consume glucose in high glucose group(11,33,44 mmol/L)is significantly reduced and there is no effect on cell viability,showing a stable IR model was successfully induced.Specifically,55mmol/L glucose could significantly reduce hepatocyte ability to consume glucose at 12 h,24h and 36 h,therefore it was selected to induce IR model.(2)55mmol/L glucose successfully induced the IR model,compared to the control group,the m RNA expression of mTOR,S6K1 and 4EBP was significantly increased,the phosphorylated protein of S6K1 was also increased.Accordingly,mTOR feedback regulation was found in primary hepatocytes.Compared to control group,the hepatocyte TG and LDL-ch concentrations in the IR group was significantly higher,and HDL-ch was significantly lower than that in the control group(P<0.05);Glut-1 m RNA,Srebp-1c and FOXO-1 gene expression in IR group was significantly decreased,Glut-3 m RNA expression was significantly increased in the IR group,indicating IR could promote the lipid synthesis.(3)After treatment with different concentrations of mTOR inhibitor-rapamycin(20n M,50 n M,100 n M),the m RNA expression of mTOR,S6K1,4EBP were significantly inhibited,AKT and P-AKT protein expression increased,and the upstream target Ins R m RNA expression was significantly increased(P<0.05).Rapamycin significantly increased glucose consumption(P<0.05);concentrations of TG,LDL-ch and T-CHO in IR group were significantly lower than those of the control group.The m RNA expression level of FOXO-1 decreased significantly;the expression of Glut1 and Glut3 increased significantly.It suggests that there is a negative feedback of mTOR to regulate the conduction of insulin signal.(4)After 50 n M Rapamycin was used to inhibit mTOR in layer hepatocytes and then treated with 55 mmol/L glucose,it was found that the expression of mTOR,S6K1 and 4EBP in Rapa+ High Glu group primary hepatocytes was significantly lower than that of High Glu group,and the Ins R was significantly increased(P<0.05).Compared to High Glu group,the glucose consumption of hepatocytes significantly increased.Lipid synthesis in Rapa+High Glu treatment group cells was significantly lower than that in High Glu group,which was shown that TG,and T-CHO concentration was significantly reduced;FOXO-1 was significantly reduced and Glut1 and Glut 3 rose significantly.Western blot results showed that the total protein and phosphorylated protein of Akt was increased compared with High Glu group,suggesting that rapamycin abolished high glucose-induced hepatocyte IR by inhibiting mTOR.Conclusion: This experiment for the first time proved that IR is closely related to fat metabolism of laying hens.And the negative feedback regulation of mTOR on IR might be the cause for lipid accumulation in primary hepatocytes of laying hens.