Effect of Glucagon and Insulin on the Growth of Neonatal Rat Hepatocytes in Primary Tissue Culture

Abstract

Commercial (bovine-porcine) glucagon added in a single dose between 10-12 and 10-7 M to neonatal rat hepatocytes in primary cultures with subsequent incubation for 20-24 h, stimulated their entry into the DNA synthesis phase as revealed by [3H]thymidine-labeling and radioautography; about 14 h of incubation was required before an effect was observed. Commercial (bovine) insulin at doses between 10-11 and 10-7 M apparently stimulated the entry of hepatocytes into S phase. However, insulin''s effect, which needed 20 h for induction, was due to the release of a wave of synchronized hepatocytes from an earlier produced block near the G1/S boundary of their growth-division cycle. Equimolar mixtures of glucagon with insulin from 10-15-10-7 M increased the fraction of hepatocytes synthesizing DNA 1st at 4-8 h, and then at 20-24 h. Effective doses of glucagon, insulin and glucagon plus insulin also increased the entry of hepatocytes into mitosis, as found after a 4 h incubation with colchinine (0.1 mM). Withholding inactivated fetal bovine serum from the growth medium for 24 h did not change the mitotic activity either of the untreated or of the glucagon- and glucagon plus insulin-stimulated hepatocytes, but it increased the proliferogenic effect of bovine insulin. Highly purified crystalline (porcine) glucagon, insulin and glucagon plus insulin also stimulated the growth of hepatocytes in the presence or absence of serum. Equimolar (10-14 M) mixtures of glucagon with (Bu)2c[dibutyryl cyclic]GMP and of insulin with (Bu)2cAMP increased the hepatocytic replication as efficiently as did glucagon plus insulin at the same dose. Glucagon and insulin are synergistic, intracycle regulators of the growth of neonatal rat hepatocytes. Cyclic nucleotides may mediate at least partly the hepatotrophic effects of the pancreatic hormones.