Transforming growth factor beta (TGF-beta), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-beta. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-beta was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [125I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CCl4-exposed rat livers. We found specific binding of [125I]IGF-II only on CCl4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 +/- 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 = 165 pmol/L. With a mink lung epithelial cell (Mv1Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-beta function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [ 125

In order to obtain more information about a possible role of somatomedin (SM) in vivo, increase in length and weight, costal cartilage activity (35SO4 = and 3H-methyl-thymidine incorporation), organ weight and serum glucose levels were studied in Snell dwarfmice, during treatment with human growth hormone (hGH), l-thyroxine (T4), insulin and a semipurified SM-preparation (SM-P1). SM-P1, hGH and T4 caused a significant increase in length and weight, whereas insulin was not effective in a dosage similar to the insulinlikeactivity of the SM-P1 preparation measured by radioreceptorassay. The effect obtained with the SM-preparation cannot be due to contamination of GH, prolactin, T4, insulin or testosterone. Costal cartilage activity was measured three days after initiation of treatment by incubation immediately after sacrifice in plasma free medium. Both sulphate and thymidine incorporation were increased by the administration of SM-Pl and hGH, compared to buffer treated controls. After treatment for four weeks no differences between treated and untreated animals in cartilage activity could be observed anymore. Both SM-P1 and hGH induced weight gain in most organs. An outspoken effect was seen on lymphoid organs, the submandibular salivary glands and muscle, a sizable effect was present on the kidneys and a small effect, not reaching significance for the SM-Pl treated group on brain weight. An impression of divergence was seen with liver weight, which increased on hGH while there was a small and not significant rise on SM-P1. A definite discrepancy was found in skinfold thickness, which was essentially unchanged on hGH and increased on SM-P1. Testes and epididymal fat, though probably increasing their weights, were in insufficient number to do statistics on. Serum T4 is low in untreated dwarfmice. It is increased to a subnormal level after hGH administration whereas no effect is observed with SM-P1. The dose dependent effect of insulin, measured by radioimmunoassay, on the induction of hypoglycaemia is markedly similar to the effect of nonradioimmunoactive insulin-like activity of SM-P1 as measured by radioreceptorassay. The hypoglycaemia induced with insulin and the SM-preparation is reduced by adding 10 % glucose to the diet. In conclusion: SM-P1 shows growth stimulating and insulin-like actions in vivo. The pattern of effects obtained with hGH, T4 and insulin differs from that of SM-P1.

Specific binding to and proliferative actions of insulin-like growth factors-I and -II (IGF-I and -II) on fetal mouse osteoblasts were tested. Membranes of mouse osteoblasts were shown by binding competition studies to possess specific binding sites for IGF-I and IGF-II. When IGF-I was used as a tracer, half-maximal displacement was obtained with 1.11 micrograms IGF-I/l and with 14 micrograms IGF-II/l. Displacement of 125I-labelled IGF-I was accomplished with 2.33 micrograms IGF-II/l and with 55 micrograms IGF-I/l. Affinity cross-linking showed bands of 130 kDa 125I-labelled IGF-I and 260 kDa 125I-labelled IGF-II under reducing conditions, further indicating the presence of classical type-I and -II receptor sites on mouse osteoblasts. Mitogenic effects of IGFs were weak; a combination with epidermal growth factor or fibroblast growth factor showed strong synergistic action however. The possibility of autocrine/paracrine actions of IGFs is discussed.

Background: Detection of incompletely processed precursor forms of insulin-like growth factor-II (“big” IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68–88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma. Methods: The ELISA used a solid-phase antibody to E(68–88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C18 extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration. Results: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean ± SD, 22.6 ± 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 ± 0.8 nmol/L) were low. Antibodies directed against either E(68–88) or E(13–134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry. Conclusions: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.