ACTIVATION AND GLUCAGON REGULATION OF MITOGEN-ACTIVATED PROTEIN-KINASES (MAPK) BY INSULIN AND EPIDERMAL GROWTH-FACTOR IN CULTURED RAT AND HUMAN HEPATOCYTES

Many hepatocellular activities may be proximally regulated by intracel lular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepa tocytes. Using Western immunoblots, rat and human hepatocytes were fou nd to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0.5-1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2.5 min exposure to EGF. Insulin treat ment produced phosphorylation of the insulin receptor beta subunit; sh c phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. I nsulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Go -treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocy tes; human hepatocyte MAPK was maximally stimulated by EGF alone. Gluc agon pretreatment blocked phosphorylation, gel mobility shift and kina se activity of MAPK induced by insulin but only partially blocked EGF- induced MAPK activation in human hepatocytes. Glucagon also reduced th e activation of MAPK by EGF in rat hepatocytes. Pre-treatments with fo rskolin or cyclic AMP analogues diminished in the insulin-, EGF-and in sulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicat e that although EGF and insulin may both signal through the MAPK/ras/r af/MAPK pathway, the response for MAPK differs between these ligands a nd between species. Further, in both rat and human, glucagon exerts it s effects through a cyclic AMP-dependent mechanism at a level in the i nsulin and EGF signal transduction pathways downstream of MAPK but pro mixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylat ion by glucagon in human hepatocytes provides further evidence for a r af-1-independent pathway for activation of MAPK. (C) 1998 John Wiley & Sons, Ltd.