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 function in utero. Its continued availability to the growing fetus depends on increasing islet cell mass. The purpose of the study was to examine the interactions between nutrient availability and insulin-like growth 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 the release of endogenous IGF-I or -II, (b) if both IGF-I and -II: were effective mitogens for pancreatic beta-cells, (c) whether islets released IGF-binding proteins (IGFBPs) and their possible regulation by nutrient availability and (d) how IGFBPs might regulate the ability of IGFs to alter islet DNA synthesis. Islets of Langerhans were isolated from 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 exogenous IGF-I, -II, IGFBP-1 or IGFBP-2. The release of insulin and endogenous IGF-I and -II were each determined by radioimmunoassay, and IGFBP 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 viability. Both classes of nutrients also increased the DNA synthetic rate of islets. Islets released almost twice as much IGF-II (0.22 +/- 0.08 nmol/l, mean +/- S.E.M., n=4) as IGF-I (0.14 +/- 0.03 nmol/l) in cultures containing 8.7 mmol glucose/l and x 1 amino acids. Lesser or greater 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 was fivefold more active in stimulating DNA synthesis by islets (half maximal 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. Isolated islets released four species of IGFBP with molecular sizes of approximately 19, 25, 35 and 46 kDa respectively. The 35 kDa form was identified by Western immunoblot as IGFBP-2. Increasing the glucose concentration between l.4 mmol/l and 16.7 mmol/l caused a dose-related increase in the release of the 19, 25 and 35 kDa IGFBP species. Increasing amino acid concentrations from x1 to x2 concentrations increased the relative amounts of all IGFBP species, but greater concentrations were inhibitory. Exogenous IGFBP-1 and BP-2 synergized with sub-effective concentrations of IGF-I or -II to increase DNA synthetic rate. The results 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 ability of glucose to increase islet DNA synthesis was not accompanied by altered release of endogenous IGFs, but did result in increased release of IGFBPs. Increasing the concentration of total amino acids increased the release of both IGF-II and IGFBPs. Since exogenous IGFBPS were able to potentiate the mitogenic actions of IGFs, it is likely that nutrients, IGFs and IGFs and IGFBPs interact to promote islet cell hyplasia in late gestation.