To determine the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the development of the pancreas, and specifically of the islets of Langerhans, we have examined the cellular distribution and developmental changes in the expression of IGFs and IGFBPs in the pancreas of the fetal and neonatal rat between 19.5 days of gestation and postnatal day 28. This represents a period of substantial growth and restructuring of the beta cell component in islets of this species. IGF-I, IGF-II, and IGFBPs-1 to -6 mRNAs were localized by in situ hybridization, and peptides by immunohistochemistry, in histological sections. IGF-II mRNA was highly expressed in islet cells and some ductal epithelial cells in late fetal and early neonatal life, but was barely detectable by postnatal day 28. IGF-II peptide showed a similar distribution. IGF-I mRNA was barely detected in the fetus or neonate and was localized predominantly in the ductal and acinar tissues after postnatal day 7. IGF-I immunoreactivity was associated with some islet cells in the fetus and neonate, suggesting an endocrine rather than a paracrine source. We performed co-localization studies to assess whether the distribution of IGFs within the pancreas might be due to a sequestration by locally produced IGFBPs. The presence of mRNAs for both IGFBPs-1 and -2 was minimal in the pancreas prior to postnatal day 7, although subsequently IGFBP-1 mRNA was seen in islet cells, while IGFBP-2 mRNA was localized in both islets and acinar tissues. In contrast, both IGFBPs-1 and -2 immunoreactivities were identified in islets from late fetal life, suggesting a circulatory source for these IGFBPs during early pancreatic development. IGFBPs-3 to -5 mRNAs and immunoreactivities were identified within islet cells throughout fetal and neonatal life, with IGFBPs-3 and -5 being mainly associated with the alpha cell-rich islet mantle. The results show a compartmentalization of IGFs within pancreatic tissue, reflecting both paracrine and endocrine sources. The localization and action of IGFs in pancreas likely involves sequestration and distribution by endogenous as well as circulating IGFBPs.

ABSTRACT

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 β-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 β-cells following a 5-day preincubation regime were maintained in various concentrations of glucose (1·4–16·7 mmol/l) or amino acids (×1–×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 [³H]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/1 and × 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 × 2 amino acids. Exogenous IGF-I was fivefold more active in stimulating DNA synthesis by islets (half maximal concentration (ED₅₀) 1·6 ± 0·4 nmol/l, n = 3) than was IGF-II (ED₅₀ 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 1·4 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 × 1 to × 2 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 IGFBPs interact to promote islet cell hyperplasia in late gestation.

Journal of Endocrinology (1993) 138, 401–412