Influence of insulin, glucocorticoids and glucose on glycogen synthase activity in hepatocyte cultures.
A preparation method of monolayer cultures from rat hepatocytes with up to 95% plating efficiency is described. Cell number and DNA content per dish remained stable during 3 days in culture in hormone-free media containing 10 mM glucose. The cellular content of protein was decreased to 50% in the same time period. Glycogen content and specific activity of glycogen synthase present in the original hepatocyte suspension declined rapidly during 2 days in the absence of hormones. Addition of 20 nM insulin or 0.1 microM triamcinolone did not prevent this loss of cellular contents, however, in the simultaneous presence of both hormones the original levels of protein, glycogen and glycogen synthase were maintained for 2 days. In 2-day-old hepatocytes an increase of glycogen synthase activity could be evoked by insulin in the presence of 20 or even 5 mM glucose provided these cells had been precultivated in the presence of triamcinolone. Both the I-form and the activity determined at 6.7 mM glucose 6-phosphate rose after exposition of hepatocytes to insulin and 20 mM glucose, whereas a 'total activity' being unchanged by hormone treatment of the cells was only obtained if 50 mM glucose 6-phosphate were present during determination of enzyme activity. Addition of 10 microgram/ml cycloheximide during incubation of the cells reduced the effect of 20 mM glucose and abolished that of insulin on glycogen synthase and on the corresponding glycogen deposition. It is discussed that insulin and glucose activate glycogen synthase in two successive steps by regulating synthesis and activity of an interconverting enzyme.
D000818:Animals; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008112:Liver Glycogen / Q000378:metabolism; D008297:Male; D051381:Rats
D005938:Glucocorticoids; D007328:Insulin; D008112:Liver Glycogen; D003513:Cycloheximide; D006006:Glycogen Synthase; D005947:Glucose