The Mechanism Of Insulin And Glucagon Regulating Lipid Metabolism By The AMPK Signaling Pathway In Calf Hepatocytes

Insulin (INS) and glucagon (GLN) are the two major pancreatic hormones in the energymetabolism and play a crucial role in energy homeostasis of the whole body. Lipid-relatedmetabolic disorder of liver is the cause of many diseases which often occur in the periparturientperiod, for example, ketosis and fatty liver. The metabolic profiles of ketotic cows include lowconcentration of serum insulin, high concentration of serum glucagon and low ratio of INS/GLN.But high concentration of INS and glucose are found in some cases of Ⅱ type ketosis and fattyliver, which indicate that insulin and glucagon have a correlation with the lipid metabolismdisorders. However, the regulating mechanism of insulin and glucagon on lipid metabolismremains unclear in cows. Therefore, the objective of this study was to investigate the mechanismof insulin and glucagon in the regulation of lipid metabolism by adenosine5’-monophosphate(AMP)-activated protein kinase (AMPK) signal pathway in calf hepatocytes cultured in vitro andunderstand the effect on lipid metabolic disorder of cows by insulin and glucagon.Hepatocytes from a newborn calf were incubated with100nmol/L insulin and glucagon for0,0.5,1,3,6,12h, respectively. The p-AMPKα/AMPKα and the AMPKα activity had a nadir at1hafter INS-treatment. The protein level of p-AMPKα/AMPKα and the AMPK activity were bothhigher after1h GLN-treatment compared with0h, and the AMPKα activity was higher at1h.These results indicate that the effect of INS and GLN on AMPKα was largest after1hINS-treatment or GLN-treatment, respectively.Hepatocytes were co-cultured in vitro with different concentrations of INS in the presence orabsence of5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR), an AMPKαactivator). The results showed that INS increased the phosphorylation of insulin receptor (INSR)and decreased phosphorylation of AMPKα. Moreover, INS increase the expression andtranscriptional activity of the carbohydrate responsive element-binding protein (ChREBP) andsterol regulatory element-binding protein-1c (SREBP-1c), resulted in the upregulation of lipogenic genes. Furthermore, INS decreased the expression and transcriptional activity of peroxisomeproliferators-activated receptor-α (PPARα), resulted in the downregulation of lipid oxidation genesand fatty acid transpotant genes. In addition, TG content was significantly increased and VLDLwas lower in insulin-treated groups compared with control group. Collectively, these resultsindicated that insulin suppresses the AMPKα signaling pathway through INSR to increaselipogenic and decrease lipid oxidation.Hepatocytes were co-cultured in vitro with different concentrations of GLN in the presenceor absence of BML-275(an AMPKα inhibitor). The results showed that GLN increased theexpression of GLNR and phosphorylation of AMPKα. Moreover, GLN decreased the expressionand transcriptional activity of the ChREBP and SREBP, resulted in the downregulation oflipogenic genes. Furthermore, GLN increased the expression and transcriptional activity ofPPARα, resulted in the upregulation of lipid oxidation genes and fatty acid transpotant genes. Inaddition, the TG content was significantly decreased and VLDL was higher in GLN-treated groupscompared with control group. Collectively, these results indicated that GLN activates the AMPKαsignaling pathway through GLNR to mediate the lipid metabolism.In conclusion, INS and GLN regulate the AMPKα signaling pathway through INSR andGLNR, respectively, to mediate the lipid oxidation, lipid synthesis and fatty acid transport in cowshepatocytes.