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 insulin-signalling mechanism. This study investigates the mechanisms by which EGF affects the basal rate and the insulin stimulation of glycogen synthesis. The basal rate of glycogen synthesis is higher at low than at high cell density. EGF inhibits the basal rate of glycogen synthesis at low cell density but not in confluent cultures and abolishes the 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 difference in rates of glycogen synthesis due to cell density, neither does it induce responsiveness to EGF at high cell density, establishing that responsiveness to EGF is a function of cell density and not of the basal rate and that inhibition of the insulin stimulation also cannot be accounted for by the higher rate of glycogen synthesis. Cytochalasin D and phalloidin, which alter cell morphology through interactions with the microfilament cytoskeleton, mimic the cell-density-dependent inhibition of glycogen synthesis by EGF. The inhibition of glycogen synthesis by EGF and cytochalasin D is additive and cytochalasin D potentiates the inhibition of glycogen synthesis by EGF, suggesting involvement of a cytoskeletal mechanism. Exogenous phospholipase C inhibits glycogen synthesis at both low and high cell density and the inhibition at low cell density is not additive with that caused by either EGF or cytochalasin D, suggesting that these agonists inhibit glycogen synthesis through changes in Ca2+ and/or diacylglycerol. The inhibition of glycogen synthesis by EGF in the absence of insulin stimulation is blocked by neomycin, which inhibits Ca2+ release from intracellular stores but not by antagonists of protein kinase C. It was also inhibited by pertussis toxin (50%), suggesting that it may involve GTP-binding-protein-mediated release of Ca2+ from intracellular stores. The inhibition of the stimulation of glycogen synthesis by insulin was not affected by neomycin and was only marginally inhibited by pertussis toxin or guanosine 5'-O-[3-thio]triphosphate (GTP[S]). We infer from these findings that the inhibition by EGF of the basal rate of glycogen synthesis and of the insulin stimulation are mediated by different mechanisms. The latter is pertussis toxin insensitive and independent of cell density, whereas the former is expressed only at low cell density, it is potentiated by cytochalasin D and inhibited by pertussis toxin.
Peak
M
M
Department of Medicine, Medical School, University of Newcastle upon Tyne, England.
Agius
L
L
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D006003:Glycogen / Q000096:biosynthesis*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D008099:Liver / Q000378:metabolism*; D008297:Male; D037342:Pertussis Toxin; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D007211:Indoles; D007773:Lactates; D010566:Virulence Factors, Bordetella; D015638:Cytochalasin D; D019344:Lactic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004815:Epidermal Growth Factor; D006003:Glycogen; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C064758:Ro 31-8220