INTERACTIONS OF NUTRIENTS, INSULIN-LIKE GROWTH-FACTORS (IGFS) AND IGF-BINDING PROTEINS IN THE REGULATION OF DNA-SYNTHESIS BY ISOLATED FETAL-RAT ISLETS OF LANGERHANS

Insulin is a major regulatory hormone for optimal tissue growth and fu nction in utero. Its continued availability to the growing fetus depen ds on increasing islet cell mass. The purpose of the study was to exam ine the interactions between nutrient availability and insulin-like gr owth factor (IGF) release and action during DNA synthesis by isolated fetal rat islets of Langerhans. Specifically, we wished to determine ( a) whether the availability of glucose or total amino acids altered th e release of endogenous IGF-I or -II, (b) if both IGF-I and -II: were effective mitogens for pancreatic beta-cells, (c) whether islets relea sed IGF-binding proteins (IGFBPs) and their possible regulation by nut rient availability and (d) how IGFBPs might regulate the ability of IG Fs to alter islet DNA synthesis. Islets of Langerhans were isolated fr om fetal rat pancreata on day 22 of gestation by collagenase digestion . Islets enriched in beta-cells following a 5-day preincubation regime were maintained in various concentrations of glucose (1.4-16.7 mmol/l ) or amino acids (x 1- x 3 total concentrations), with or without exog enous IGF-I, -II, IGFBP-1 or IGFBP-2. The release of insulin and endog enous IGF-I and -II were each determined by radioimmunoassay, and IGFB P release characterized by Western ligand blot analysis. DNA synthesis was measured by the incorporation of [H-3]thymidine. Isolated islets demonstrated an increased release of insulin in response to increasing amounts of both glucose and amino acids, demonstrating functional via bility. Both classes of nutrients also increased the DNA synthetic rat e of islets. Islets released almost twice as much IGF-II (0.22 +/- 0.0 8 nmol/l, mean +/- S.E.M., n=4) as IGF-I (0.14 +/- 0.03 nmol/l) in cul tures containing 8.7 mmol glucose/l and x 1 amino acids. Lesser or gre ater concentrations of glucose did not alter the release of either IGF , but the release of IGF-II was significantly increased (0.53 +/- 0.08 nmol/l, P<0.01) in the presence of x 2 amino acids. Exogenous IGF-I w as fivefold more active in stimulating DNA synthesis by islets (half m aximal concentration (ED(50)) 1.6 +/- 0.4 nmol/l, n=3) than was IGF-II (ED(50) 8.1 +/- 0.6 nmol/l), regardless of glucose concentration. Iso lated islets released four species of IGFBP with molecular sizes of ap proximately 19, 25, 35 and 46 kDa respectively. The 35 kDa form was id entified by Western immunoblot as IGFBP-2. Increasing the glucose conc entration between l.4 mmol/l and 16.7 mmol/l caused a dose-related inc rease in the release of the 19, 25 and 35 kDa IGFBP species. Increasin g amino acid concentrations from x1 to x2 concentrations increased the relative amounts of all IGFBP species, but greater concentrations wer e inhibitory. Exogenous IGFBP-1 and BP-2 synergized with sub-effective concentrations of IGF-I or -II to increase DNA synthetic rate. The re sults show that isolated fetal rat islets release more IGF-II than IGF -I, but that IGF-I is a more potent stimulus to DNA synthesis. The abi lity of glucose to increase islet DNA synthesis was not accompanied by altered release of endogenous IGFs, but did result in increased relea se of IGFBPs. Increasing the concentration of total amino acids increa sed the release of both IGF-II and IGFBPs. Since exogenous IGFBPS were able to potentiate the mitogenic actions of IGFs, it is likely that n utrients, IGFs and IGFs and IGFBPs interact to promote islet cell hypl asia in late gestation.