INHIBITION OF GLYCOGEN-SYNTHESIS BY EPIDERMAL GROWTH-FACTOR IN HEPATOCYTES - THE ROLE OF CELL DENSITY AND PERTUSSIS-TOXIN-SENSITIVE GTP-BINDING PROTEINS

Epidermal growth factor (EGF) counteracts the stimulation of glycogen synthesis by insulin in hepatocytes, but it is not known whether this is due to inhibition of glycogen synthesis or to inhibition of the ins ulin-signalling mechanism. This study investigates the mechanisms by w hich EGF affects the basal rate and the insulin stimulation of glycoge n synthesis. The basal rate of glycogen synthesis is higher at low tha n at high cell density. EGF inhibits the basal rate of glycogen synthe sis at low cell density but not in confluent cultures and abolishes th e difference due to density. However, EGF inhibits the stimulation of glycogen synthesis by insulin irrespective of cell density. Increasing glycogen synthesis by increasing the [glucose] does not abolish the d ifference in rates of glycogen synthesis due to cell density, neither does it induce responsiveness to EGF at high cell density, establishin g that responsiveness to EGF is a function of cell density and not of the basal rate and that inhibition of the insulin stimulation also can not be accounted for by the higher rate of glycogen synthesis. Cytocha lasin D and phalloidin, which alter cell morphology through interactio ns with the microfilament cytoskeleton, mimic the cell-density-depende nt inhibition of glycogen synthesis by EGF The inhibition of glycogen synthesis by EGF and cytochalasin D is additive and cytochalasin D pot entiates the inhibition of glycogen synthesis by EGF suggesting involv ement of a cytoskeletal mechanism. Exogenous phospholipase C inhibits glycogen synthesis at both low and high cell density and the inhibitio n at low cell density is not additive with that caused by either EGF o r cytochalasin D, suggesting that these agonists inhibit glycogen synt hesis through changes in Ca2+ and/or diacylglycerol. The inhibition of glycogen synthesis by EGF in the absence of insulin stimulation is bl ocked by neomycin, which inhibits Ca2+ release from intracellular stor es but not by antagonists of protein kinase C. It was also inhibited b y pertussis toxin (50%), suggesting that it may involve GTP-binding-pr otein-mediated release of Ca2+ from intracellular stores. The inhibiti on of the stimulation of glycogen synthesis by insulin was not affecte d by neomycin and was only marginally inhibited by pertussis toxin or guanosine 5'-O-[3-thio]triphosphate (GTP[S]). We infer from these find ings that the inhibition by EGF of the basal rate of glycogen synthesi s and of the insulin stimulation are mediated by different mechanisms. The latter is pertussis toxin insensitive and independent of cell den sity, whereas the former is expressed only at low cell density, it is potentiated by cytochalasin D and inhibited by pertussis toxin.