Proliferation of adult rat hepatocytes in primary culture induced by insulin is potentiated by cAMP-elevating agents.
We investigated whether or not insulin and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early and late phases of primary culture. Adult rat hepatocytes synthesized a significant amount of DNA when cultured in the presence of 10(-7) M insulin for 3 h. Under these conditions, the number of nuclei increased within 4 h. Hepatocyte DNA synthesis and proliferation were not essentially affected by the initial plating densities. Other cAMP-elevating agents, such as glucagon, forskolin and dibutyryl cAMP, as well as beta-adrenoceptor agonists (i.e., metaproterenol and isoproterenol) alone had no effect on either hepatocyte DNA synthesis or proliferation in primary culture. In contrast, these agents potentiated both processes at concentrations as low as 10(-7) M when cultured in combination with 10(-7) M insulin. The stimulatory effects of beta-adrenoceptor agonists and other cAMP-elevating agents were significantly blocked by the cAMP-dependent protein kinase inhibitor, H-89 (N-[2-(p-(bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10(-7) M). The mitogenic effect of insulin upon hepatocytes was almost completely suppressed by genistein (5 x 10(-6) M), wortmannin (10(-7) M) and by rapamycin (10 ng/ml). These results show that insulin rapidly induced the proliferation of adult rat hepatocytes in primary culture. The mitogenic effects of insulin were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. The effects of beta-adrenoceptor agonists and cAMP-elevating agents may be mediated through cAMP-dependent protein kinase. In addition, the activation of receptor tyrosine kinase, phosphoinositide 3-kinase and p70 ribosomal protein S6 kinase may be involved in the insulin signal transduction pathway.
Kimura
M
M
Biochemical Pharmacology Group, Faculty of Pharmaceutical Sciences, Josai University, Keyakidai Sakado, Saitama, Japan.
Ogihara
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D009921:Metaproterenol / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology; D015398:Signal Transduction
D000319:Adrenergic beta-Antagonists; D007328:Insulin; D018341:Receptors, Adrenergic, alpha-2; D009921:Metaproterenol; D004815:Epidermal Growth Factor; D003994:Bucladesine; D004247:DNA; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine