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Biomedical subjects

J Zapf

Publications and source records attributed to J Zapf.

At least 73 records · Page 4Linked to original sources
PubMed1995

Cleavage analysis of insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants.

Cultured human fibroblasts and osteoblast-like cells secrete an insulin-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP-4) into two fragments of approximately 18 and 14 kDa. Edman degradation of the isolated proteins established the amino termini of the reaction products. Sequence analysis of the 14-kDa carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine at position 135 of the mature protein. Four variant IGFBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed. Wild-type and mutant IG-FBPs-4 bound IGF-I and IGF-II with equivalent affinities and, in the intact state, were equally effective inhibitors of IGF-I action. However, the IGFBP-4 mutants were relatively resistant to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected at this time. After a 24-h incubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa fragments. Pre-exposure of human fibroblasts in serum-free conditioned medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of DNA synthesis. When added with IGF-II, the protease-resistant mutant IG-FBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of IGF-I-stimulated DNA synthesis. These biological studies suggest that the IGFBP-4/IGFBP-4 protease system may play a role modulating local cellular response to IGF-I.

Amino Acid Sequence↗
PubMed1995

Immunohistochemical localization of insulin-like growth factor I and II in the endocrine pancreas of birds, reptiles, and amphibia.

Immunoreactive insulin-like growth factors I and II (IGF-I, IGF-II) were sought in the endocrine pancreas of representative birds, reptiles, and amphibia using antisera specific for mammalian IGF-I and IGF-II and the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM), and pancreatic polypeptide (PP) in double immunofluorescence. Both IGF-I and IGF-II immunoreactivities were present in the endocrine pancreas of all species. IGF-II immunoreactivity was exclusively found in INS-immunoreactive (-IR) cells, indicating evolutionary conservation of the islet IGF-II system. In contrast, IGF-I immunoreactivity was distributed differently among the species and never occurred in INS-IR cells. In the anuran Xenopus laevis, IGF-I immunoreactivity was present in islet cells showing coexistence of GLUC and PP immunoreactivities. In reptiles, the lizards (Lacerta viridis, Scincus officinalis) exhibited IGF-I immunoreactivity in PP-IR and SOM-IR cells and the snakes (Psamophis leniolatum, Coluber ravergieri) in SOM-IR and GLUC-IR cells. In birds, IGF-I immunoreactivity was located either in SOM-IR cells only (Gallus g. domesticus, Streptopelia roseogrisea) or in PP-IR and SOM-IR cells (Coturnix c. japonica). Thus, the distribution patterns of islet IGF-I immunoreactivities in birds, reptiles, and amphibia are equivalent to those in mammals and most bony fish. They differ, however, from those found in cartilaginous fish, cyclostomes, and protochordates, where a total or partial coexistence of IGF-I and INS immunoreactivities has been obtained. Therefore, the divergence of IGF-I and INS seems to have occurred early in vertebrate phylogeny. Furthermore, the existence of IGF-I immunoreactivity likely is common in the islets of all vertebrates. Finally, no phylogenetic trend to concentrate IGF-I immunoreactivity in a particular islet cell type is apparent.

Animals↗
PubMed1995

Proteolysis of IGFBPs by cathepsin D in vitro and in cathepsin D-deficient mice.

Affinity-purified lysosomal protease cathepsin D cleaved recombinant human IGFBP-1 to -5 in fragments of defined sizes, while IGFBP-6 was not degraded. To assess the role of cathepsin D for proteolytic processing of IGFBP in vivo, serum from cathepsin D-deficient mice and conditioned media from cathepsin D-deficient fibroblasts and organ explants were analyzed. No differences for the pattern and level of IGFBPs were detected. When conditioned media from fibroblasts were incubated at acid pH, proteolysis of IGFBP-1 and -4 was observed only in media derived from cathepsin D-expressing cells. Additional experiments showed that the proteolysis of IGFBP-4 is mediated by cathepsin D and not by a protease activated by cathepsin D. The IGFBP-4 degrading activities in media from organ explants from cathepsin D-deficient mice were found to be sensitive to inhibitors of aspartyl and cysteine proteases. The data indicate that different classes of acid pH-dependent proteases can contribute to the regulation of IGFBP-4 abundance.

Animals↗
PubMed1995

Expression, effects, and fate of IGFBP-5 are different in normal and malignant osteoblastic cells.

Normal osteoblasts from newborn rat calvaria and human osteosarcoma (Saos-2) cells express IGFBP-5 mRNA. IGF I increases IGFBP-5 mRNA levels in both cell types, whereas retinoic acid stimulates IGFBP-5 mRNA expression in calvaria but suppresses it in Saos-2 cells. IGFBP-5 mRNA expression is stimulated in normal bone cells by parathyroid hormone. 30 nM IGFBP-5 stimulates 3H-thymidine incorporation in calvaria (which produce IGF I contributing to basal proliferation in serum-free medium) but not in Saos-2 cells which produce little IGF I and IGF II. Among the 5 rhIGFBPs tested (IGFBP-2 to -6), only IGFBP-5 stimulates DNA synthesis in calvaria cells, and only IGFBP-6 in Saos-2 cells. RhIGFBP-5 displays a short half-life (approximately 30 min) in serum-free medium of calvaria cells and a long half-life (approximately 15 h) in the medium of Saos-2 cells. Fragments of 20 and 14 kDa accumulate in the media of both cell types. Intact (31 kDa) IGFBP-5 is associated and remains with the extracellular matrix of mature calvaria osteoblasts but not of Saos-2 cells. Among the IGFBPs produced IGFBP-5 is unique with regard to its marked affinity to matrix of normal bone cells, its short half-life when released, and its stimulatory effects on DNA synthesis.

Animals↗
PubMed1995

Intravenously injected insulin-like growth factor (IGF) I/IGF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats.

Insulin-like growth factor (IGF) circulates in blood in two large molecular mass forms of 150 and 40 kD. Under normal conditions, most of the IGF is bound to the 150-kD complex by which it is retained in the circulation and therefore unable to exert acute insulin-like actions. The aim of this study was to answer the question whether or not IGF in the 40-kD complex is bioavailable to insulin target tissues and thus can cause acute insulin-like effects in vivo. Intravenously injected 1:1 molar recombinant human (rh) IGF I/rhIGF binding protein (BP)-3 complex lowered blood glucose and stimulated glycogen synthesis in diaphragm of hypophysectomized, but not of normal rats. The serum half-lives of the two components of the complex were similar to each other, but considerably shorter in hypox than in normal rats. On neutral gel filtration of serum both components of the injected complex appeared predominantly in the 150-kD region in normal rats. In hypox rats which lack the 150-kD complex they were found in the 40-kD region and disappeared rapidly from the circulation. We conclude that in the absence of the 150-kD complex, IGF associated with the 40-kD complex can rapidly leave the vascular compartment, reach insulin or type 1 IGF receptors and exert acute insulin-like effects.

Animals↗
PubMed1995

Replacement of growth hormone (GH) in normally growing GH-deficient patients operated for craniopharyngioma.

Removal of a craniopharyngioma usually results in panhypopituitarism. Some children, however, grow normally or even excessively after extirpation of the tumor despite a proven lack of GH and have so far not been treated with hGH. We studied the effects of short (2-day) and long term (1-yr) administration of hGH on metabolism and growth in six patients receiving regular hormonal replacement therapy. During short term human (h) GH treatment, 15N retention was not significantly increased (mean +/- SEM, 115.4 +/- 9.6% of basal balance) and was not different from the control value. In contrast, 15N retention was 210.3 +/- 20.7% in children with GH deficiency from other causes. Long term administration of hGH (2 IU/m2.day, sc, for 12 months) did not influence growth velocity, but increased the calf circumference and decreased the body mass index and skinfold thickness in prepubertal patients. Insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP-3), and the 150-kilodalton IGFBP complex were decreased before and restored to normal during treatment. The reverse was observed for the 50-kilodalton IGFBP complex. Growth (velocity) in these patients did not correlate with any of the usual indicators of the growth status and remains unexplained. Although hGH did not affect growth, it had other beneficial effects and is recommended for these patients.

Adolescent↗
PubMed1994

Insulin-like growth factor I stimulates myofibril development and decreases smooth muscle alpha-actin of adult cardiomyocytes.

Adult rat cardiomyocytes in long-term culture express type 1 insulin-like growth factor (IGF) receptors. In contrast to insulin receptors, type 1 IGF receptors are up-regulated during culturing. IGF-I added to the cells at plating increased granular density and pseudopodia number per cell after 7 days. After 16 days, IGF-I-treated cells showed, as compared with controls, a dramatic increase of the number of newly built sarcomeres and were packed with myofibrils. At the same time, IGF-I suppressed the accumulation of smooth muscle alpha-actin (sm-alpha-actin) in a dose-dependent manner. Under the conditions of this in vitro system, growth hormone had no effect on cell morphology or sm-alpha-actin. sm-alpha-Actin, a nonsarcomeric isoform of actin expressed in early fetal cardiac development, reappears both during long-term culture of adult rat cardiomyocytes and during heart hypertrophy. This study shows that type 1 IGF receptors are up-regulated in adult rat cardiomyocytes in long-term culture and that IGF-I enhances myofibril development and concomitantly down-regulates sm-alpha-actin. This protein forms stress-fiber-like structures and may temporarily serve as a scaffold for the formation of new sarcomeres until myofibrils have developed throughout the cell and the scaffold is no longer needed. Our findings thus allow us to propose another hypothesis for the mechanism leading to overload heart hypertrophy.

Actins↗
PubMed1994

Role of muscle insulin-like growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4.

The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.

Animals↗
PubMed1994

Tumour-induced hypoglycaemia due to 'big' IGF-II.

A 57-year-old woman with a slowly growing intraabdominal leiomyosarcoma developed life-threatening hypoglycaemia. Plasma C-peptide levels were low (0.08 nmol L-1 and < 0.05 nmol L-1, reference interval 0.18-0.63 nmol L-1). Total IGF-II was normal (760 ng mL-1) whilst 'big' IGF-II was markedly elevated (440 ng mL-1). After surgical tumour reduction, 'big' IGF-II levels in plasma normalized (117 ng mL-1) and the patient experienced no new episodes of hypoglycaemia. Life-threatening hypoglycaemia due to tumour production of IGF-II can be ameliorated by reduction of the tumour burden and should be kept in mind even in incurable patients.

Female↗
PubMed1994

Growth hormone and parathyroid hormone stimulate IGFBP-3 in rat osteoblasts.

Osteoblast-like cells prepared from calvaria of newborn rats produce insulin-like growth factor (IGF) I and several insulin-like growth factor binding proteins (IGFBPs) in vitro. Among the IGFBPs found in conditioned cell culture medium, IGFBP-3 is the most abundant. Intact IGFBP-3, as assessed by 125I-labeled IGF-II ligand blot analysis, is more abundant in culture media of cells exposed to growth hormone (GH) or to parathyroid hormone (PTH), both at 5 x 10(-9) mol/l, for 24 h. At the same time, concentrations of IGF-I are increased in media of cells exposed to PTH but not to GH, compared with hormone-free control cultures. IGFBP-3 mRNA is increased in osteoblasts exposed to PTH or to GH but not in response to 5 x 10(-9) mol/l IGF-I. PTH exerts a rapid (within 2 h) stimulatory effect on IGF-I and IGFBP-3 production, both at the message and peptide levels, whereas GH increases only IGFBP-3, both at the message and peptide levels (after 24 h). We conclude that IGF-I does not mediate increased IGFBP-3 production by rat osteoblasts in response to GH and PTH.

Animals↗
PubMed1994

Role of insulin-like growth factor II and IGF binding proteins in extrapancreatic tumor hypoglycemia.

Serum from patients with extrapancreatic tumor hypoglycemia (EPTH) contains elevated levels of big (pro) IGF II which disappears after successful removal of the tumor. Nevertheless, total IGF II serum levels are mostly found in the normal range both before and after operation. Why then do these patients become hypoglycemic? Oversecretion of big IGF II leads to suppression of growth hormone (GH). As a consequence, formation of a GH-dependent 150-kD IGF binding protein (BP) complex is impaired which normally carries 70-80% of total serum IGF II and largely restricts its bioavailability. Impaired formation of the 150-kD complex leads to a shift of IGF II to a 50-kD IGFBP complex, resulting in a 30-fold shorter serum half-life of IGF II, increased turnover and enhanced bioavailability. Insulin target organs are thus exposed to an enormous insulin-like potential which is continuously provided by oversecreted big IGF II and causes increased glucose consumption by skeletal muscle, inhibition of hepatic glucose production, inhibition of lipid mobilisation from adipose tissue, and pronounced hypoglycemia.

Biological Availability↗
PubMed1994

Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells.

Animals↗
PubMed1994

Comparison of the effects of growth hormone and insulin-like growth factor I on substrate oxidation and on insulin sensitivity in growth hormone-deficient humans.

Insulin-like growth factor-I (IGF-I) is considered to be the mediator of the growth-promoting effects of growth hormone (GH). The metabolic effects of these two hormones, however, are different. Whereas GH treatment leads to elevated insulin and glucose levels, reduced insulin sensitivity, and impaired glucose tolerance, IGF-I treatment leads to reduced insulin and GH levels and enhanced insulin sensitivity. IGF-I may, therefore, not only be the mediator of the growth-promoting effects of GH but also a modulator of the effects of GH on insulin action and glucose metabolism. To study the influence of GH and IGF-I on substrate metabolism and insulin sensitivity (assessed by euglycemic, hyperinsulinemic clamping combined with indirect calorimetry and glucose tracer infusion), we have treated eight GH-deficient adults with GH (2 IU/m2 daily subcutaneously [s.c.]), IGF-I (10 micrograms/kg.h s.c.), or both hormones together for 7 d, respectively, and compared the effects of these treatment regimens with a control phase. Our findings suggest that (a) both GH and IGF-I promote lipolysis and lipid oxidation, albeit by different mechanisms; (b) treatment with either hormone is followed by enhanced energy expenditure and reduced protein oxidation; and (c) IGF-I reverses the insulin resistance induced by GH.

Adult↗
PubMed1994

Proteolysis of insulin-like growth factor binding protein-5 by pregnancy serum and amniotic fluid.

Incubation of iodinated recombinant human insulin-like growth factor binding protein (rhIGFBP)-5 with pregnancy serum or amniotic fluid resulted in the formation of 22- and 15 kDa fragments. Non-pregnancy serum did not contain IGFBP-5 proteolytic activity. Size fractionation revealed the proteolytic activity both in serum and amniotic fluid in a > 100 kDa fraction which co-eluted in gel filtration with proteins of approx. 200 kDa. The IGFBP-5 protease activity was inhibited by EDTA, phenanthroline and PMSF. The formation of proteolytic fragments was also observed using 125I labeled rhIGFBP-3 and -4 but not with rhIGFBP-1 or -6 as substrate. The data demonstrate that pregnancy serum and amniotic fluid contain a very similar cation-dependent serine protease which degrades IGFBP-3, -4 and -5.

Amniotic Fluid↗
PubMed1994

Expression and regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein messenger ribonucleic acid levels in tissues of hypophysectomized rats infused with IGF-I and growth hormone.

The expression and regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-2 (IGFBP-2), -3, -4, and -5 messages were studied in liver, kidney, spleen, thymus, heart, brain, skeletal muscle, testes, and epididymal (white) adipose tissue (WAT) from hypophysectomized rats infused with saline, recombinant human (rh) IGF-I, or rhGH and compared with tissue messenger RNA (mRNA) levels in age-matched normal rats. The IGF-I message was present in all of these tissues. It was most abundant in liver and WAT, but was barely detectable in kidney, brain, and thymus. GH dependence was most pronounced in liver, skeletal muscle, and WAT and less so in heart, testes, kidney, spleen, and thymus. The IGF-I message in brain was not influenced by hypophysectomy. IGF-I infusion induced a small increase in its own mRNA in skeletal muscle and WAT, whereas it decreased its own message in liver. IGFBPs were expressed in a tissue-specific manner; IGFBP-2 mRNA was most abundant in testes and hypophysectomized liver, IGFBP-3 mRNA was most abundant in spleen, kidney, WAT, and liver, IGFBP-4 mRNA was most abundant in liver, and IGFBP-5 mRNA was most abundant in kidney, WAT, and skeletal muscle. After hypophysectomy, significant decreases in IGFBP expression were observed in liver (except IGFBP-2), skeletal muscle, brain, WAT (except IGFBP-4), and testes (except IGFBP-2), in contrast to heart, kidney, spleen, and thymus. GH infusion did not affect IGFBP-2 mRNA levels in liver (in contrast to IGF-I infusion) or brain. Like GH, IGF-I normalized IGFBP-3 mRNA levels in liver, but, in contrast to GH, had no effect on IGFBP-5 mRNA in WAT. It was considerably less effective than GH in raising IGFBP-5 mRNA levels in skeletal muscle. Thus, GH infusion can exert different effects on IGF-I and IGFBP expression than infused rhIGF-I. Differences may be due to direct actions of GH at the tissue level, including auto/paracrine effects of locally produced IGF-I.

Animals↗