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Insulin-like growth factor I (IGF-I), a potent mitogenic peptide, is present in considerable quantities in most mammalian milks, but its importance for the neonate is unknown. To test the hypothesis that milk-borne IGF-I is an important factor in the regulation of neonatal growth, as well as that of the gastrointestinal tract, rat pups were fed a rat milk substitute (RMS) devoid of growth factors via gastrostomy. These animals were compared with those given RMS supplemented with recombinant human IGF-I added at a concentration of 500 ng/ml. Animals given RMS + IGF-I gained mere weight than controls, although skeletal growth as represented by elongation of the tail was no different. Animals fed RMS + IGF-I had increased brain and liver wet weights as well as increased liver and small intestine protein contents. Serum IGF-I concentrations in the IGF-I-supplemented group were more than twofold above RMS controls and were similar to dam-fed rat pups. Semiquantification of serum IGF-binding proteins (IGFBP) in these animals documented that in IGF-I-supplemented pups the amount of 38- to 40-kDa molecular mass IGFBP species was also greater than in RMS controls. The rate of migration of enterocytes from crypts in duodenum and proximal jejunum was greater in IGF-I-supplemented animals than in rats fed RMS alone. These studies suggest that milk-borne IGF-I is important in modulation of somatic and gastrointestinal tract growth in the neonatal rat.

The organ distribution of intravenously injected 125I-IGF-I at a dose of 2-4 x 10(6) cpm or 5-10 ng/animal was studied in 10- to 12-day-old Sprague-Dawley rats at 5 and 30 min after injection. Results of the study suggest that, although the main portion of intravenous IGF-I remains in the circulation, significant amounts are also found in the carcass, liver and kidney. Blood radioactivity fell by 50% 30 min after injection, but concentrations in the carcass, liver, kidney and skin either remained stable or increased. Gel chromatography demonstrated that significant portions of radioactivity recovered from serum, liver and kidney coeluted in a position identical to the injected IGF-I. In addition, the extracted peptide bound competitively to a membrane IGF-I receptor preparation. Studies performed on liver and kidney from these animals 5 min after injection showed that on a per gram wet weight basis, these organs contained equivalent amounts of 125I-IGF-I. However, although by 30 min, 65% of the intact labelled IGF-I has been removed from the liver, the amount remaining in kidney tissue was equal to that noted 5 min after injection. Bile was collected over a 2-hour period and contained approximately 2% of the injected radioactivity and a significant portion (30%) of this radioactivity coeluted with native IGF-I. This material also bound competitively in a radioreceptor assay, suggesting 'intactness' of this peptide. From this study, we conclude that (a) IGF-I, when administered intravenously, remains for at least 30 min in a receptor-active form in blood and several organs; (b) IGF-I derived from the circulation is cleared from the liver more quickly than from the kidney of suckling rats, and (c) that IGF-I is transferred from blood to bile.

Milk-borne insulin-like growth factors I and II (IGF-I and -II) may be of importance in the differentiation of the gastrointestinal tract of the suckling. To test this hypothesis, 10- to 11-d-old suckling rats were given via an orogastric tube 125I-IGF-I (n = 6) or 125I-IGF-II (n = 6) in rat milk and killed 30 min later. The results of this study demonstrated that approximately 40% of the radioactivity administered was detected in the gastrointestinal tract for both 125I-IGF-I and 125I-IGF-II experiments. Gel chromatography of acid extracts of homogenates of gastrointestinal tissues and luminal contents demonstrated that a significant fraction of recovered radioactivity eluted in a position identical to "native" IGF. These findings were confirmed by subjecting similarly treated samples to high performance liquid chromatography. In addition, radioactive material recovered from M(r) 7,500 fractions bound specifically to crude membrane IGF-I and -II receptor preparations, further suggesting the preservation of biologic activity of the recovered peptides. Although skin homogenates contained large peptide fragments of 125I-IGF-I, no "intact" IGF was found in the blood or other tissues. These findings suggest that milk-borne IGFs are stable in the neonatal gastrointestinal tract and remain biologically active for as long as 30 min postingestion.

Whereas insulin-like growth factor I (IGF-I) has been found in various body fluids from different species, the presence or absence of IGF and associated binding proteins (IGFBPs) in bile has not been clearly defined. Bile concentration of IGF-I was measured in this study and found to be highest in the neonate and lowest in adult rats [133 +/- 15.9, 79.4 +/- 10.5, 45.3 +/- 12.7 ng/ml (mean +/- SE) in 12-day-old, 33-day-old, and adult rats, respectively]. When bile delivery rates of IGF-I (i.e., the product of IGF-I concentration in bile and the biliary flow rate) were calculated, IGF-I delivery was highest in weanling rats (469 pg.h-1.g body wt-1). When expressed as amount of IGF-I in bile delivered per day, however, delivery rates rose from 0.2 micrograms/day in the suckling and remained constant at 1.6-1.7 micrograms/day in both weanling and adult animals. Bile samples exposed to a placental membrane IGF receptor preparation showed significant dose-dependent inhibition of binding of native IGF-I. Because no IGF binding proteins were identified by Western ligand blot or by Sephadex gel chromatography, the results suggest the presence of biologically significant quantities of bioactive IGF-I in bile. We speculate that IGF-I in bile may play an important role in the growth of the gastrointestinal tract, both in the suckling as well as later in life.

Insulin like growth factor (IGF) 1 and 2 which are present and actively synthesised in the wound fluid stimulate several cell types involved in the process of wound healing. To investigate the role of IGF-1 and 2 and in addition, the association between IGF and their carrier proteins, IGF binding proteins (IGFBP), we have used a newly established model for human wound healing in fresh biopsy material. Histological examination shows that IGF-1 stimulates efficient reepithelialisation of the wounds both alone and in the presence of recombinant IGFBP-1. In contrast, IGF-2 stimulates healing only when used in combination with IGFBP-1. These findings suggest that the two IGFs and their carrier proteins may function during different phases of wound healing and that both IGF-1 and 2 act as potent inducers of wound healing; this may have direct clinical implications.

The effect of biosynthetic human insulin-like growth factor I (IGF-I) and IGF-II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG-CSF), granulocyte-macrophage CSF (rhGM-CSF), or interleukin-3 (rhIL-3), was investigated. IGF-I and IGF-II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF-I induced an increase of 62 +/- 16%, 84 +/- 13%, and 107 +/- 18% in granulocyte colony numbers in the presence of G-CSF, GM-CSF, or IL-3, respectively. The values for IGF-II were 66 +/- 13%, 96 +/- 12%, and 91 +/- 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G-CSF and GM-CSF-stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL-3 in such cultures. The growth-promoting effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against the IGF-I (Type I) membrane receptor. IGF-I and IGF-II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor.

The present report describes the mitogenic effect of recombinant IGF-2 on cultured human keratinocytes and fibroblasts compared to that of IGF-1. Furthermore, the modulating effect of a recently expressed recombinant form of placental-derived IGF-binding protein 1 (IGFBP-1) on IGF-induced proliferation was examined. A dose-dependent increase, up to 100%, in cell proliferation was seen in cultured human keratinocytes with IGF-2 and -1 and the proliferative response was comparable to the effect of epidermal growth factor (EGF). In human fibroblasts, IGF-1 stimulated DNA synthesis up to 300% for IGF-1 and up to 200% for IGF-2. The mitogenic effect of IGF-1 was enhanced by IGFBP-1 in both cell types. In contrast, the IGF-2-induced mitogenic effect was unperturbed. These findings indicate that the interaction between IGFs and their binding proteins may induce different responses depending upon the ligand and the target cell.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

The erythroid-potentiating effects of biosynthetic human insulin-like growth factor-I (IGF-I) and IGF-II were evaluated and compared in serum-free cultures of human marrow erythroid progenitors. IGF-I and IGF-II enhanced the in vitro growth of relatively mature (CFU-E) and primitive (BFU-E) erythroid progenitors at similar dose-dependent magnitudes. Significant elevations in erythroid colony counts were detected at 0.2-0.6 ng/mL of both peptides, with a maximal increase in CFU-E and BFU-E numbers detected at 2-6 ng/mL. Similar enhancement of erythroid progenitor cell growth by both peptides was also detected in cultures of marrow cells that had been depleted of accessory cells or in cultures of highly enriched hemopoietic progenitors. IGF-I and IGF-II enhanced erythroid progenitor cell growth at both limiting and saturating concentrations of recombinant human erythropoietin or granulocyte-macrophage colony-stimulating factor, but did not alter the sensitivity of CFU-E and BFU-E to their respective hemopoietic regulators. The erythroid-potentiating effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against IGF-I membrane receptors. IGF-I and IGF-II thus appear to exert their effects on human marrow erythroid progenitors via a direct mechanism involving the type I IGF receptor.

Insulin-like growth factor 1 (IGF-1) regulates growth of the brain. In order to characterize the variant IGF-1 present in human fetal brain we have determined the cDNA sequence for human fetal brain IGF-1. Using PCR to amplify cDNA obtained from isolated human fetal brain mRNA, two cDNA sequences encoding precursor proteins which correspond to IGF-1a and IGF-1b were obtained. This is the first characterisation of IGF-1 and its IGF-1a and IGF-1b precursors in the nervous system.

Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125I-insulin was 0.9 +/- 0.2% of total 125I-insulin added, and the IC50-value was estimated to 8.9 pmol/l. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125I-insulin-like growth factor-I after 4 h incubation at 10 degrees C was five times higher than the specific binding of insulin (4.4 +/- 0.4% of total 125I-insulin-like growth factor-I added), and the IC50-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3H-thymidine incorporation into DNA: insulin-like growth factor-I greater than B10 Asp greater than insulin-like growth factor-II greater than insulin greater than or equal to B28 Asp greater than B9 Asp/B27 Glu greater than proinsulin. The maximal effect of insulin-like growth factor-I on 3H-thymidine incorporation was 71 +/- 16% higher than the maximal effect of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) genes in the human brain and in glioma was studied by slot blot and Northern analysis following hybridization of IGF-I and IGF-II cDNA probes to isolated mRNA. In the fetal brain there were major IGF-I transcripts of 7.5 and 4.4 kilobases (kb) and one major IGF-II transcript of 6.0 kb. IGF-II mRNA was less abundant in the adult brain, with IGF-II mRNA being scarcely detectable. All glioma tissue examined displayed a marked enhancement of both IGF-I and IGF-II gene expression when compared to normal brain. IGF-I transcripts of 4.4 and 2.2-1.8 kb were identified. Similar to fetal brain a major IGF-II transcript of 6.0 kb was found in the glioblastoma examined.

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.

A truncated form of insulin-like growth factor 1 (IGF-1), which lacked the aminoterminal tripeptide Gly-Pro-Glu has been isolated from human fetal and adult brain. This truncated IGF-1 displayed more potent cross-reactivity and biological action on brain cells than IGF-1 isolated from human serum. We now present data on a recombinant DNA-derived truncated IGF-1 lacking the aminoterminal tripeptide. Recombinant truncated IGF-1 was 1.4-5-times more potent than recombinant and natural IGF-1 in displacing [125 I]IGF-1 from human fetal and adult brain and placenta membranes. These differences were slightly enhanced when truncated IGF-1 was used as radioligand. The relative potencies compared to insulin-like growth factor 2 (IGF-2) in displacing [125I]IGF-2 from rat liver membranes were recombinant truncated IGF-1, 0.3% and recombinant IGF-1, 0.2%. Recombinant truncated IGF-1 displayed 100-fold reduced affinity for the low molecular weight binding protein (IGF-BP) isolated from human amniotic fluid when compared to recombinant IGF-1. Likewise, the IGF-BP was 100-fold less potent in inhibiting the receptor binding of recombinant truncated IGF-1 than that of recombinant IGF-1. Recombinant truncated IGF-1 was 4-times more potent than recombinant and natural IGF-1 in stimulating DNA synthesis in fetal rat brain cells. This biological activity of recombinant truncated IGF-1 was not affected by the IGF-BP at concentrations which abolished the biological activity of recombinant IGF-1. The hypothesis that IGF-BP bound intact IGF-1 represents the endocrine form of IGF-1, whereas truncated IGF-1 represents the paracrine or autocrine form of IGF-1, is proposed.

Somatomedins are anabolic hormones that may stimulate growth during the perinatal period. To test this hypothesis, neonatal rats were injected with a biosynthetic somatomedin, insulin-like growth factor 1 (IGF-1) twice daily for the first 2 wk of life. Two biosynthetic IGF-1 preparations of different potency were tested as well as a preparation of human growth hormone in five litters of rats. When compared to saline-injected rats, IGF-1 injected rats had increased body weight and tail length as well as specific increases in weights of liver, brain, heart, and testes. In addition, significant increases in bone marrow erythropoietic cell precursors were apparent after IGF-1 injection. IGF-1-treated neonatal rats also exhibited precocious eye opening as a sign of epithelial cell differentiation. Five additional litters of rats received similar injections but were exposed to postnatal nutritional deprivation via artificially increasing litter size. Although IGF-1 caused stimulation of bone marrow erythropoiesis and precocious eye opening, no effects of IGF-1 on somatic or organ growth could be documented. This represents the first demonstration in vivo of the anabolic effects of IGF-1 in rapidly growing neonatal rats but suggests that nutritional sufficiency may also be necessary for the full expression of somatomedin effects.