Recombinant insulin-like growth factor-II inhibits the growth-stimulating effect of growth hormone on the liver of Snell dwarf mice.

The actions and interactions of recombinant insulin-like growth factor-I and -II (IGF-I and IGF-II), alone or in combination with human GH on body growth and the growth of several organs were studied in the Snell dwarf mouse. IGF-I and -II stimulate to a similar extent sulfate incorporation into cartilage, and both IGFs increase body length and weight. IGF-II as well as IGF-I have clear effects on the size of the submandibular salivary glands, kidneys, and spleen. IGF-II, however, did not influence the weight of the lung, in contrast with IGF-I. GH treatment alone resulted in growth of the liver, whereas both IGFs were inactive. Surprisingly, IGF-II and, to a lesser extent, IGF-I inhibited GH-induced growth of the liver. Glycogen storage in the liver was decreased by treatment with IGF-II alone or in combination with GH, as shown by histological examination. It was not affected by GH, IGF-I, or GH plus IGF-I. Also, the size of the centrilobular hepatocytes was decreased by treatment with IGF-II and IGF-II plus GH; GH alone had a hypertrophic effect, whereas IGF-I or GH plus IGF-I had none. In contrast to GH, IGFs did not increase polyploidy. Treatment with IGF-II increased the level of IGFBP-3, as did IGF-I or GH treatment, as shown by Western ligand blotting. The IGFs appeared to have a greater effect on the induction of 38.5-kilodalton IGFBP-3 than GH, suggesting a different role in the regulation of glycosylation. In conclusion, IGF-I and IGF-II as well as GH have a stimulatory effect on general body growth and are effective in the stimulation of serum IGFBP-3, sulfate incorporation into cartilage, as well as the growth of specific organs in Snell dwarf mice. Both IGFs, alone or in combination with GH, show distinct effects on the growth of the liver with respect to several histological parameters, which require further exploration.

Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I-stimulated body growth but stimulates growth of the kidney in snell dwarf mice.

The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline-treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes.

Modulation of insulin-like growth factor (IGF) action by IGF-binding proteins in normal, benign, and malignant smooth muscle tissues.

The insulin-like growth factor (IGF) system is involved in the growth of uterine leiomyomas (L), as these tumors have higher IGF-II messenger ribonucleic acid levels, type I IGF receptor levels, and IGF-I peptide concentrations than myometrium (M). Furthermore, cultured L smooth muscle cells (SMC) respond with greater efficiency to IGF-I than M SMC. Here we investigate a possible modulating role of the binding proteins for the IGFs (IGFBPs) on the actions of IGFs. IGFBP-3 is the most predominant IGFBP in conditioned medium from SMC, with levels ranging from 13-288 ng/mL. Incubation of SMC cultures with IGF-I and the IGF-I analogs long-R3IGF-I and des(1-3)-IGF-I, which have decreased affinity for IGFBPs, revealed a facilitating effect of IGFBPs on the growth-stimulating activity of a high concentration of IGF-I in cell lines with high IGFBP-3 levels. Both a decreased level of IGFBP-3 and a low concentration of the growth factors added were a disadvantage for the facilitating effect. In M and L tissue sections, IGFBP-3 was found exclusively bound to the constituting cells, not in the extracellular matrix. This suggests that a negative modulating role of IGFBP-3 due to sequestration of IGF-I, as occurs in culture medium, is less relevant in vivo. In leiomyosarcoma sections, IGFBP-3 levels are decreased, indicating a decreasing, role for this binding protein in malignant smooth muscle tissues.

Insulin-like growth factors-I and -II and their binding proteins during postnatal development of dwarf Snell mice before and during growth hormone and thyroxine therapy.

The ontogeny of serum insulin-like growth factors (IGFs)-I and -II and their binding proteins (IGFBPs) was studied in normal and dwarf Snell mice. IGF-I concentrations in serum of normal mice increased between 4 and 8 weeks of age; dwarf mice had very low serum IGF-I levels. In both normals and dwarfs, serum IGF-II levels were highest soon after birth and dropped steadily thereafter. Western ligand blots of serum IGFBPs with 125I-IGF-II as tracer revealed the expected bands of 41.5, 38.5, 30-32 and 24 kDa. In normal mice the IGFBP-3 doublet was already detectable at 2 weeks of age, and its intensity increased with age. In dwarf mice the IGFBP-3 doublet was hardly detectable. The changes of IGFs and their IGFBPs were studied in sera of dwarf mice after treatment with growth hormone (GH) and/or thyroxine (T4) for 4 weeks. In spite of a comparable growth response obtained using these hormones, serum IGF-I was increased only by GH treatment; a small but significant decrease of serum IGF-II was obtained following GH or T4 treatment. An increase of the IGFBP-3 doublet was only obtained with GH; T4 and GH + T4 had no effect. The rise of IGFBP-3 after GH treatment was accompanied by the formation of the IGFBP 150 kDa complex, as measured by neutral gel chromatography. The size distribution of 125I-IGF-II was restored to normal, while with 125I-IGF-I only a small peak at 150 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein kinase C phosphosite(s) in B-50 (GAP-43) are confined to 15K phosphofragments produced by Staphylococcus aureus V8 protease.

The neuron-specific phosphoprotein B-50 (= GAP-43/F1/pp46/P57) is an endogenous substrate of protein kinase C (PKC) in rat brain. To examine the location of the PKC phosphosites, phosphorylated B-50 was digested by Staphylococcus aureus V8 protease (SAP). The products migrated in SDS-polyacrylamide gel electrophoresis as two phosphoprotein bands of apparent molecular weight of 15 and 28 kDa (indicated as 15 and 28 K). This study reports further characterization of the 15 and 28 K phosphobands. ACTH(1-24), a characteristic inhibitor, inhibited equally effective the [(32)P]phosphate-incorporation into the 15 and 28 K phosphobands formed by SAP from B-50 endogenously phosphorylated in synaptosomal plasma membrane (SPM). Tests using immunoprecipitation or immunoblotting showed that all polyclonal rabbit B-50 antisera recognized the 28 K phosphoband, but only a minor population B-50 antibodies of a recently developed antiserum 8613 reacted with the 15 K phosphoband. The time course of the SAP digestion indicated that B-50 is degraded first to the 28 K band and then to the 15 K band. [(32)P]phosphate incorporated in B-50 was totally recovered in these phosphobands. Isoelectric focusing resolved the SAP products into one 28 K phosphopeptide of isoelectric point (IEP) 4.8 and the 15 K phosphofragment in at least 4 phosphopeptides, with IEP of 6.1, 6.6, 6.9 and 7.0, respectively. SAP digests of extensively phosphorylated B-50 analysed by isoelectric focusing in narrow pH gradients, showed microheterogeneity in the undigested B-50, the 28 and 15 K phosphofragments. The time course of SAP digestion of B-50 in endogenously phosphorylated SPM and in phosphorylated nerve growth cone membranes, demonstrated that in both types of membranes, 28 and 15 K phosphofragments are consecutively formed, with IEP identical to the phosphofragments derived from isolated B-50. Our findings suggest that the PKC phosphosite(s) in the B-50 protein are restricted to neutral 15 K peptides of the B-50 molecule.

Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II.

Previously, transgenic mice were constructed overexpressing human insulin-like growth factor II (IGF-II) under control of the H2kb promoter. The IGF-II transgene was highly expressed in thymus and spleen, and these organs showed an increase in weight. In the current study we have analyzed the sites of IGF-II mRNA expression, the distribution of IGF-II, IGF-I, and both IGF receptors, and histomorphometrical changes in thymus and spleen. With in situ mRNA hybridization, expression of the IGF-II transgene is found with high intensity in the thymic medulla and in the white pulp/marginal zone of the spleen, whereas there were scattered positive cells in the thymic cortex and in the splenic red pulp. Hybridization was restricted to non-lymphocytic cells. Immunohistochemistry revealed intense IGF-II peptide staining with the same distribution as IGF-II mRNA. There was additional intense IGF-II staining of all elements in the splenic red pulp (including trabeculae) and diffuse, low level staining in the thymic cortex. These findings were not observed in control mice. In the thymic medulla, most IGF-II producing cells co-labelled with keratin, whereas a minor population also stained for the monocyte/ macrophage marker MOMA-2. In the spleen, co-labelling of IGF-II producing cells was found with MOMA-1 (marginal zone), or with the dendritic cell marker NLDC-145 (red pulp). IGF-I and both IGF receptors were found in these organs in nearly all cell types, with a similar pattern in transgenic mice and in control animals. Histomorphometric analysis revealed a marked increase of thymus cortex size and an increased trabecular size in the spleen. This suggests that IGF-II overproduction induces local effects (auto/paracrine) in the thymic cortex, but not in the thymic medulla. Trabecular growth in the spleen most likely is a distant effect (paracrine or endocrine) of IGF-II overproduction.

Differential patterns of insulin-like growth factor-I and -II mRNA expression in medulloblastoma.

Insulin-like growth factors (IGFs) play an important role in tumour growth and development. We hypothesized that this is also the case for medulloblastomas, which are highly malignant cerebellar brain tumours usually occurring in children. In these tumours the expression patterns of IGF-I and -II mRNA were studied. Tumour specimens obtained from 12 children and two adults at diagnosis were hybridized in situ with digoxigenin-labelled cRNA probes for hIGF-I and hIGF-II mRNAs. In all cases, tumour cells showed abundant expression of IGF-I mRNA. Nine of the 14 tumours showed variable but significant IGF-II expression. In these tumours, the hybridization signal almost exclusively colocalized with a subpopulation of Ki-M1P positive cells that were identified as ramified microglia (RM) cells. In the five tumours without IGF-II expression, microglia/brain macrophages with a more rounded amoeboid-like morphology predominated. RM cells in normal cerebellar tissues, residing abundantly in areas of the white and, to a less extent, in the grey matter, were IGF-II mRNA-negative. These RM cells showed a thinner and more extensively branched appearance and were more evenly distributed than those encountered in medulloblastoma. Probably, during the transformation from the resting ramified towards the amoeboid morphology (or vice versa) IGF-II mRNA expression is only temporarily induced. The physiological meaning of the induction of IGF-II mRNA expression by these cells in medulloblastoma remains unclear but any IGF-II peptide synthesized could exert unfavourable mitogenic and antiapoptotic effects on adjacent tumour cells. However, in this relatively small number of cases we could not find any indications for a relationship between clinical characteristics of the various cases and the extent of IGF-II mRNA expression.