NUTRITION AND SOMATOMEDIN .26. MOLECULAR REGULATION OF IGF-I BY INSULIN IN CULTURED RAT HEPATOCYTES

Although decreased levels of circulating insulinlike growth factor I (IGF-I) and hepatic IGF-I mRNA in diabetic animal models support a role for insulin in IGF-I regulation, it has been difficult to study the effects of insulin in vitro. Few immortal cell lines exhibit hormone-responsive production of IGF-I, and studies of cultured hepatocytes have often been inconclusive because conventional methods may not reproduce the IGF-I transcripts recognized in vivo. We have modified the hepatocyte isolation and RNA extraction procedures to circumvent this problem, resulting in transcripts of 1, 2, and 7.5 kb. With increasing insulin from 10(-10) to 10(-6) M, dexamethasone from 10(-10) to 10(-7) M, and amino acids from 1 to 10 x rat arterial levels, IGF-I release rose by 226, 257, and 447%, respectively (P < 0.05), with correlated changes in IGF-I mRNA (r = 0.75, P < 0.005). In the presence of 5 x amino acids and 10(-7) M dexamethasone, insulin-stimulated IGF-I release was dose dependent, increasing 183% at 10(-6) M (P < 0.01). Across a broad range of amino acid concentrations (0.25-6.25 x), insulin provided consistent stimulation of both IGF-I mRNA content and release of IGF-I. Cells cultured for 2 days at 10(-10) M insulin and then 2 days at 10(-6) M insulin released 66% more IGF-I than cells cultured 4 days at 10(-10) M insulin (P < 0.01). Hepatocyte IGF-I release was correlated strongly with content of IGF-I mRNA, both sum of transcripts (r = 0.76, P < 0.001) and individual transcripts. Insulin appears to regulate IGF-I release at the mRNA level in hepatocyte primary culture. This system should be a useful tool for further studies of molecular mechanisms of IGF-I regulation.