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Bielohuby M.,Ludwig Maximilians University of Munich | Zarkesh-Esfahani S.H.,University of Isfahan | Zarkesh-Esfahani S.H.,Isfahan University of Medical Sciences | Zarkesh-Esfahani S.H.,University of Sheffield | And 10 more authors.
DMM Disease Models and Mechanisms | Year: 2014

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7-3.3% coefficient of variation) and linearity (90.4-105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists. © 2014. Published by The Company of Biologists Ltd

Reyer A.,Leibniz Institute for Farm Animal Biology | Schindler N.,Leibniz Institute for Farm Animal Biology | Ohde D.,Leibniz Institute for Farm Animal Biology | Walz C.,Leibniz Institute for Farm Animal Biology | And 6 more authors.
American Journal of Physiology - Endocrinology and Metabolism | Year: 2015

Recent studies suggest that insulin-like growth factor-binding protein-2 (IGFBP-2) affects both growth and metabolism. Whereas negative growth effects are primarily due to negative interference with IGF-I, the mechanisms for metabolic interference of IGFBP-2 are less clear. As we demonstrate, overexpression of IGFBP-2 in transgenic mice is correlated with a decelerated clearance of blood glucose after oral administration. IGFBP-2 carries an integrin-binding domain (RGD motif), which has been shown to also mediate IGF-independent effects. We thus asked if higher serum levels of IGFBP-2 without an intact RGD motif would also partially block blood glucose clearance after oral glucose application. In fact, transgenic mice overexpressing mutated IGFBP-2 with higher levels of IGFBP-2 carrying an RGE motif instead of an RGD were not characterized by decelerated glucose clearance. Impaired glucose tolerance was correlated with lower levels of GLUT4 present in plasma membranes isolated from muscle tissues after glucose challenge. At the same time, activation of TBC1D1 was depressed in mice overexpressing wild-type but not mutated IGFBP-2. Although we do not have reason to assume altered activation of IGF-I receptor or PDK1/Akt activation in both models, we have identified increased levels of integrin-linked kinase and focal adhesion kinase dependent on the presence of the RGD motif. From our results we conclude that impaired glucose clearance in female IGFBP-2 transgenic mice is dependent on the presence of the RGD motif and that translocation of GLUT4 in the muscle may be regulated by IGFBP-2 via RGDdependent mechanisms. © 2015 the American Physiological Society.

Hoeflich A.,Leibniz Institute for Farm Animal Biology | Wirthgen E.,Ligandis GbR | David R.,Reference and Translation Center for Cardiac Stem Cell Therapy | Classen C.F.,University of Rostock | And 2 more authors.
Frontiers in Endocrinology | Year: 2014

IGFBP-2 (1) has been described as a brain tumor oncogene (2) and is widely expressed in cancers from different origins (3-8). IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9) and also has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10). Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2. © 2014 Hoeflich, Wirthgen, David, Classen, Spitschak and Brenmoehl.

Hoeflich A.,Leibniz Institute for Farm Animal Biology | Reyer A.,Leibniz Institute for Farm Animal Biology | Ohde D.,Leibniz Institute for Farm Animal Biology | Schindler N.,Leibniz Institute for Farm Animal Biology | And 12 more authors.
Aging Cell | Year: 2016

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E- mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously. © 2016 The Anatomical Society and John Wiley & Sons Ltd.

Wirthgen E.,Ligandis GbR | Wirthgen E.,Leibniz Institute for Farm Animal Biology | Hoflich C.,Ligandis GbR | Spitschak M.,Leibniz Institute for Farm Animal Biology | And 6 more authors.
Growth Hormone and IGF Research | Year: 2016

The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P < 0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P < 0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P < 0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P < 0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P < 0.001). The total IGFBP amount of Toggenburg goats was reduced (P < 0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. © 2015 The Authors.

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