14-3-3β Mediated Effect Of Hepatic Glucagon Receptor On Gluconeogenesis Regulation

Objective:Glucagon combines with hepatic glucagon receptor and plays an important role on the regulation of glucose and lipid metabolism.The study aslo finds 14-3-3βprotein(YWHAB)combined with glucagon receptor.However,at present the expression of these two proteins after hepatic steatosis and the regulation of glucose metabolism in the liver of the bounding protein,are unclear.This study was designed to investigate the expression levels of GCGR、YWHAB protein after hepatic steatosis in tissue and cellular levels and YWHAB mediates effect of hepatic glucagon receptor on regulation of gluconeogenesis.Methods:We use quantitative PCR and Western blot to detect gcgr and ywhab mRNA and protein expression in HepG2 cell lines,mouse tissues,HepG2 cells after steatosis and ob/ob mice liver.The localization of GCGR and YWHAB in the liver of ob/ob mice and wild littermates was achieved by immunohistochemistry.Co-immunoprecipitation assay were used to confirm the interaction between YWHAB and GCGR in HepG2 cells.We established cell models of overexpression and silencing YWHAB,when they treated with or without glucagon,key enzymes like G6 Pase and PEPCK were examined by QPCR and Western Blot.Results:1.Glucagon receptor and YWHAB was expressed in the liver and HepG2 cells;The results of immunohistochemistry showed: glucagon receptor expressed in the cell membrane and YWHAB mostly expressed in the cytoplasm and nucleus.2.Compared to the control,the GCGR and YWHAB expression levels in ob/ob mice liver and HepG2 cells after oleic acid inducing steatosis were also significantly decreased.3.When the YWHAB overexpressed,glucagon significantly reduced the mRNA and protein expression levels of key enzymes in glucagon-induced gluconeogenesis pathway like the G6 Pase and PEPCK.After the transfection of YWHAB shRNA,glucagon significantly increased the mRNA and protein expression levels of G6 Pase and PEPCK.Conclusion:Glucagon receptor and YWHAB was expressed in the liver and HepG2 cells.After the occurrence of hepatic steatosis,both the expression of glucagon receptor and YWHAB was reduced.Glucagon receptor could combine with YWHAB,they inhibited hepatic gluconeogenesis under glucagon stimulation.