Podcasts about igfbps

Wachstum und Differenzierung sind zentrale Prozesse bei der Entstehung und Entwicklung des Lebens. Die Größe eines Zellverbandes wird vor allem durch die Balance zwischen Zellwachstum, Proliferation und die Apoptoserate definiert (Lupu et al. 2001). Unterschiedliche Größen von Organen können durch unterschiedliche Zellzahlen und/oder durch unterschiedliche Zellgrößen bedingt sein. So ist der Mensch beispielsweise größer als eine Maus, vor allem weil er mehr Zellen hat (Raff 1996). Im Vergleich dazu wurden bei verschiedenen Drosophila-Unterarten unterschiedliche Flügelgrößen aufgrund einer Änderung der Zellgröße entdeckt (Prout & Barker 1989). Allerdings ist bis heute nur unzureichend geklärt, wie das Wachstum durch Zellgröße und -zahl koordiniert ist und wie dadurch Struktur und Funktion von Organen bzw. Organismen entstehen. Ein zentrales regulatorisches System für die Kontrolle des Wachstums ist das GH/IGF (Wachstumshormon/Insulin-Like Growth Factor)-System. Ohne die endo- und parakrin wirksamen Komponenten GH und IGF-I ist in der Maus postnatal nahezu kein Wachstum möglich. Mäuse, die weder funktionelles GH noch IGF-I besitzen, erreichen nur 17 % der Größe und des Gewichts einer vergleichbaren adulten Maus. Damit stoßen sie an die kleinste gerade noch für ein Säugetier tolerierbare Größe (Lupu et al. 2001). Wichtige Vermittler der gewebespezifischen Wirkungen von IGF-I und -II sind die IGF-Bindungsproteine (IGFBPs). Bislang sind sechs unterschiedliche IGFBPs bekannt, die durch eine hohe Affinität für die IGFs definiert sind und damit deren biologische Wirkungen beeinflussen. Unter den IGFBPs nimmt IGFBP-2 eine besondere Stellung ein, weil es ein in vivo relevanter Inhibitor für Wachstum ist. Es konnte gezeigt werden, dass IGFBP-2 die Effekte von GH auf die Zellgröße modulieren kann. Während GH in Zona fasciculata Zellen der Nebenniere 11 Wochen alter Mäuse sowohl Zellzahl als auch Zellgröße stimulierte, wirkte sich die Koexpression von IGFBP-2 in einer Normalisierung der GH-induzierten Zellgröße, nicht jedoch der GH-induzierten Zellzahlerhöhung aus (Hoeflich et al. 2002). Dieses Modell eignet sich somit ideal, um die Regulation von Zellgröße und Zellzahl getrennt zu untersuchen. In der vorliegenden Arbeit wurden die molekularen Grundlagen dieser Veränderungen sowohl auf der Ebene des Transkriptoms, als auch der Proteine untersucht. Um Fragen zur Altersabhängigkeit der Wachstumsregulation zu bearbeiten, wurde die Untersuchung an zwei verschiedenen Altersgruppen durchgeführt.

Insulin-like growth factor I (IGF-I) and -II (IGF-II) are single chain peptides produced by many tissues, functioning in an endocrine, autocrine or paracrine fashion to regulate cellular proliferation, survival and differentiation. IGF actions are initiated upon binding to the insulin-like growth factor I receptor (IGF-IR) and are modulated through interactions with a family of six secreted IGF-binding proteins (IGFBP-1 to -6). IGF-I is necessary for normal growth and differentiation during both, embryonic and postnatal development. IGF-II is a stimulator of fetal growth but its functions in the postnatal period are still unclear. Notably, expression of IGF-II is shut down shortly after birth in rodents (but not in humans). Previous studies in phosphoenolpyruvate-carboxykinase (PEPCK)-IGF-II transgenic mice demonstrated that overexpression of IGF-II resulted in disproportionate growth of specific organs but a significant increase in body size was not observed. Homozygous IGF-I deficient mice were shown to be severely retarded in growth. The aim of this study was to test whether elevated levels of circulating IGF-II can rescue the dwarfism in IGF-I deficient mice and thereby function as a stimulator of postnatal growth in the absence of IGF-I. For this purpose, we crossed heterozygous IGF-I deficient mice [I+/- IIwt] with heterozygous IGF-I deficient mice carrying PEPCK-IGF-II transgenes [I+/- IItg]. The resulting offspring comprised six different groups: homozygous IGF-I knockout and PEPCK-IGF-II wildtype mice [I-/- IIwt], homozygous IGF-I knockout and PEPCK-IGF-II transgenic mice [I-/- IItg], animals lacking one IGF-I allele and wildtype for the PEPCK-IGF-II transgene [I+/- IIwt], lacking one IGF-I allele and harbouring the PEPCK-IGF-II transgene [I+/- IItg], wildtype for the IGF-I mutation and carrying the PEPCK-IGF-II transgene [I+/+ IItg], and completely wildtype [I+/+ IIwt]. The genotype of all mice was determined by PCR. Body weight of mice was recorded daily until the age of 8 weeks. The nose-rump length (NRL) and the weights of individual organs and of the carcass were recorded and the femurs and lumbar vertebras prepared for further investigations. At an age of 8 weeks, mean serum concentrations of IGF-I were beyond detection level in [I-/- IIwt] and [I-/- IItg] mice, intermediate in [I+/- IIwt] and [I+/- IItg] animals and highest in [I+/+ IIwt] and [I+/+ IItg] mice. IGF-II levels were significantly increased in animals harbouring the PEPCK-IGF-II transgene ([I-/- IItg], [I+/- IItg], and [I+/+ IItg]) when compared to their wildtype counterparts ([I-/- IIwt], [I+/- IIwt], and [I+/+ IIwt]). This reflected the genotype, demonstrating the appropriateness of our experimental model. Analysis of body weight data from day 3-4 after birth until day 60 revealed that in the absence of IGF-I, elevated levels of IGF-II have no effect on body weight gain. The same was found for the nose-rump length and the carcass. The weight of specific organs, however, was altered. Compared to the wildtype counterparts ([I-/- IIwt]), the relative kidney weight in [I-/- IItg] mice was significantly increased. IGF-I is known to play an important role in bone growth and in cancellous bone homeostasis. Investigations of geometric and structural bone parameters showed that in the presence or absence of IGF-I, an increase in the circulating levels of IGF-II was without effect on the skeleton and could not substitute for the skeletal functions of IGF-I in IGF-I-ablated mice. Homozygous IGF-I deficient mice are known to have elevated levels of growth hormone (GH). To demonstrate that the lack of effect on growth in our [I-/- IItg] animals was not due to a loss of these elevated GH-levels, a GH-Western immunoblot was performed, revealing that, despite elevated levels of IGF-II, increased levels of GH were still present in [I-/- IItg] animals. Evaluation of the serum levels of IGFBPs by Western ligand blot analysis demonstrated that IGFBP-1 and IGFBP-4 levels were similar in all groups, whereas the levels of IGFBP-2 and IGFBP-3 were strongly reduced in [I-/- IIwt] animals. In the presence of IGF-II ([I-/- IItg]), they were partially restored but the amounts were still smaller than in the IGF-I wildtype animals ([I+/+ IIwt] and [I+/+ IItg]). In summary, these results show that under our experimental conditions, IGF-II is not able to rescue the postnatal growth deficit of IGF-I knockout mice and apparently does not exert a negative feedback on the secretion of growth hormone. However, it could be demonstrated, that the IGFs have differentiated effects on the regulation of the expression/stability of individual IGFBPs.

Insulin-like growth factors (IGF-I and IGF-II) are expressed in many cell types and tissues and act in endocrine, autocrine or paracrine manner to regulate cellular proliferation, survival and differentiation. IGF actions are initiated upon binding to the type I IGF receptor (IGF-IR) and are modulated through interactions with a family of six secreted IGF-binding proteins (IGFBP-1 to -6). Although the six conserved IGFBPs are structurally related, each of them has specific characteristics and may have specific functions. Most knowledge about the IGFBPs has been gained from the numerous in vitro studies, their specific roles in vivo are largely unknown. Transgenic mice overexpressing a particular IGFBP allow us to investigate the specific functions of the corresponding IGFBP in vivo. To this end, IGFBP-4- and IGFBP-6-overexpressing models were established and analyzed in the present study. First, an expression vector containing the murine H-2Kb promoter and a human beta-globin splicing cassette was used to construct the transgenes, to obtain ubiquitous expression of the mouse Igfbp4 and Igfbp6 cDNA. Two lines of H-2Kb-mcIGFBP-4 and ten lines of H-2Kb-mcIGFBP-6 transgenic mice were generated. The transgene was ubiquitously expressed at RNA level in both transgenic models, however, at protein level, transgene expression was only detected in the spleen, thymus, lung and kidney of both H-2Kb-mcIGFBP-4 transgenic lines, but in no organ of H-2Kb-mcIGFBP-6 transgenic mice. Phenotypic analyses of the H-2Kb-mcIGFBP-4 transgenic model revealed that overexpression of IGFBP-4 had no significant effect on the postnatal body and organ growth, except that the weight and volume of thymus in 8- and 12-week-old transgenic mice were significantly reduced (p < 0.05) compared to the controls. Histomorphometric analysis demonstrated that the volume of the thymic cortex was significantly decreased in transgenic mice (p < 0.05), whereas that of the thymic medulla was not changed. The fractions of various cell types in the bone marrow, thymus, spleen, lymph node and peripheral blood were determined by flow cytometry. No significant difference was found between transgenic and control groups, suggesting that IGFBP-4 excess in the lymphoid organs did not affect the development of the lymphatic cells. The proliferative capacity of the splenocytes of transgenic animals was significantly reduced after Con A and LPS stimulation (p < 0.05), but not altered after the stimulation by anti-CD3 and anti-IgM/IL2. This is probably due to transgenic IGFBP-4 expression restricted in the non-lymphatic cells. However, detailed expression of the transgene warrants further investigation. In order to realize IGFBP-6-overexpressing mice, a second construct was designed, namely CMV-mgIGFBP-6, in which the mouse Igfbp6 genomic sequence was cloned under the control of the cytomegalovirus (CMV) promoter. Four independent lines of transgenic mice were generated. Transgene expression was high in the exocrine pancreas and relatively low in the lung and liver. The activities of serum IGFBPs were not different between transgenic mice and controls. In transgenic mice, high levels of active IGFBP-6 were detected in the luminal content of the duodenum, but neither in the luminal contents of other segments of the gastrointestinal tract (GIT), nor in tissue extracts of all GIT segments. Glucose homeostasis was not altered by IGFBP-6 expression. Postnatal body and organ growth was not affected in transgenic mice, except for the absolute and relative weight and length of duodenum which were significantly reduced in 4-month-old transgenic mice as compared to controls (p < 0.05). This reduction was mainly due to a significantly smaller volume and surface area of the tunica mucosa as determined by histomorphometric analsis. Our analysis of the first IGFBP-6 transgenic mouse model provides direct evidence for inhibition of intestinal growth by luminal IGFBP-6 excess. This finding is important in the context of neonatal intestinal growth of mammals, considering the fact that milk contains large amount of IGFBPs which may at least in part arrive intact in the intestine.

Die Vorhersage der Fertilität und die Aufklärung von Zusammenhängen zwischen der Befruchtungsfähigkeit und Parametern im Organismus ist in der Reproduktionsmedizin ein zentrales Anliegen, um eine effizientere Selektion hochfertiler Tiere zu erreichen. Das Seminalplasma wurde als geeigneter Parameter zur Beurteilung der Fertilität identifiziert, da die positive Beeinflussung der Spermienmotilität und die Vorverlegung der Ovulation durch Seminalplasma nachgewiesen wurde. Diese Arbeit hatte das Ziel anhand verschiedener Pietrainebergruppen und Hybridebergruppen einerseits Komponenten des IGF-Systems im Eberseminalplasma zu identifizieren und ihren Einfluss auf die Fertilität zu überprüfen, andererseits mittels der Proteomics-Technik das komplexe Proteingemisch im Eberseminalplasma zu analysieren. Es wurde das Vorhandensein von IGF-I/II im Eberseminalplasma nachgewiesen, ein Zusammenhang mit der Fertilität bestand nicht. Die Untersuchung der IGFBP-Aktivität ergab beim Vergleich der verschiedenen Pietrainebergruppen signifikante Unterschiede in der relativen Intensität der IGFBP-Banden, die Hybrideber bestätigten diese Ergebnisse nicht. Um eine möglichst einfache, nicht radioaktive Testung dieser IGFBP-Aktivität zu ermöglichen, war es nötig, zu ermitteln, um welche IGFBPs es sich bei diesen Banden handelte. In einer Seminalplasmaprobe wurde IGFBP-5 mittels MALDI-MS identifiziert. Ein Western-Blot mit gegen IGFBP-5 generierten Antikörpern wäre ein effektives Verfahren zur Ermittlung der IGFBP-5-Aktivität im Eberseminalplasma. Die Nutzung der Proteomics-Technik zur Darstellung des Proteinmusters im Eberseminalplasma erwies sich als problematisch, da eine vertikale Streifung und die starke Intensität einzelner Spots die Identifizierung erschwerte. Es wurden mittels MALDI-TOF-TOF PSP-I und b-Mikroseminoprotein identifiziert.Um möglicherweise von PSP-I maskierte Proteine geringerer Intensität darstellen zu können, war es nötig, den PSP-I Gehalt vor der 2D-Gelelektrophorese zu verringern. Antikörper, die gegen zwei Hauptisoformen des PSP-I generiert wurden, zeigten hohe Titer gegen PSP-I und waren auch in der Lage natives PSP-I zu erkennen. Damit ist die affinitätschromatographische Abreicherung von PSP-I möglich und dadurch die Verbesserung der Trennleistung, so dass die 2D-Gelelektrophorese zur Identifizierung positiv wirksamer Proteinkomponenten im Seminalplasmas effizient genutzt werden kann.

Although the stimulating effect of insulin-like growth factor I (IGF-I) on adrenal steroidogenesis has been well established, the role of IGF-II in the adult adrenal gland remains unknown. We, therefore, investigated the effect of recombinant human IGF-II on cortisol and cAMP synthesis from adult bovine adrenocortical cells. IGF-II, time and dose dependently, stimulated basal cortisol secretion maximally 3-fold. In combination with ACTH, IGF-II (13 nM) synergistically increased cortisol secretion from 1-fold (10(-8) M ACTH) to 28-fold of untreated control levels. In contrast, IGF-I at equimolar concentrations did not show an effect on basal cortisol secretion, and in combination with ACTH elicited a significant weaker stimulatory effect than IGF-II (22-fold increase). The synergistic effect of IGF-II on ACTH-promoted cortisol secretion was paralleled by accumulation of cAMP in the culture medium. Although both IGF receptors are present in adult bovine adrenocortical cells, the effect of IGF-II seems to be mediated through interaction with the IGF-I receptor, as [Arg54,55]IGF-II, which only binds to the IGF-I receptor, was equipotent to native IGF-II, whereas [Leu27]IGF-II, which preferentially binds to the type II IGF receptor, did not show any effect. By Western ligand blotting, four different molecular forms of IGF-binding proteins (IGFBPs) were identified in conditioned medium of bovine adrenocortical cells with apparent molecular masses of 39-44, 34, 29, and 24 kilodaltons. ACTH treatment increased the abundance of all binding proteins, on the average, 2.3-fold, except for the 29-kDa band, which was predominantly induced 6.8-fold. Additionally, [des1-3]IGF-I, a truncated IGF variant that exhibits only minimal binding to IGFBPs, was significant more potent than IGF-I and elicited the same maximum stimulatory effect on cortisol secretion as IGF-II and [des1-6]IGF-II. In conclusion, these results demonstrate that 1) IGF-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I; 2) this effect is mediated through interaction of IGF-II with the IGF-I receptor; 3) bovine adrenocortical cells synthesize various IGFBPs that are induced differentially by ACTH; and 4) IGFBPs apparently play a modulatory role in IGF-induced stimulation of adrenal steroidogenesis. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the interactions among locally produced IGFs, IGFBPs, and the IGF-I receptor can be evaluated.

The putative effects of diabetes and metabolic control on circulating levels of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) remain controversial. In the present study, serum levels of IGF-I and IGF-II and IGFBP-1, -2, and -3 were measured in 58 patients (age, 0.8-17 yr) with treated (51 subjects) or untreated (7 subjects) insulin-dependent diabetes mellitus (IDDM) and were compared with the levels in normal subjects. In the untreated patients IGF-I and IGF-II were decreased as compared with the healthy controls. In the treated diabetics IGF-I and IGF-II were reduced; IGFBP-2 (only in prepubertal subjects) and IGFBP-3 were increased. Furthermore, age-adjusted values of IGF-I, IGF-II, and IGFBP-3 were lower in prepubertal than in pubertal patients. Regression analysis revealed a negative correlation between hemoglobin (Hb)A1c and standard deviation scores (SDS) of IGF-I and a positive association between HbA1c and IGFBP-1 SDS or IGFBP-2 SDS. In the treated patients HbA1c was positively related to IGFBP-1 SDS and IGFBP-2 SDS when applying simple regression analysis and to IGFBP-2 SDS when using a multiple regression model. Strong correlations were observed between height SDS and IGF-I SDS, IGF-II SDS, and IGFBP-3 SDS in prepubertal subjects who had had IDDM for at least 2 yr, but not in adolescents. Such correlations have also been found in healthy children and adolescents. In conclusion; 1) IDDM is associated with alterations of the IGF-IGFBP system, which are partially accounted for by differences in metabolic control and pubertal status; 2) the lower plasma concentrations of serum IGF-I may play a role in the pathogenesis of growth impairment of poorly controlled prepubertal, but not pubertal, children and adolescents with IDDM; and 3) in addition, a potential role of the altered IGF-IGFBP system for the development of diabetic late complications is hypothesized.

Insulin-like growth factor-II (IGF-II) is an important regulator of embryonic growth and differentiation, but its function in postnatal life is unclear. To address this point, we generated transgenic mice harboring fusion genes in which a human IGF-II complementary DNA is placed under the transcriptional control of the rat phosphoenolpyruvate carboxykinase promoter. Transgene-specific messenger RNA was detected in liver, kidney, and several parts of the gut. Serum IGF-II levels in transgenic mice were 2-3 times higher than those in controls and increased after starvation. Circulating IGF-I correlated negatively and IGF-binding protein-2 (IGFBP-2) positively with IGF-II levels, suggesting that IGF-I is displaced from IGFBPs by IGF-II and that IGFII is a major regulator of IGFBP-2. Serum levels of IGFBP-3 and IGFBP-4 tended to be higher in phosphoenolpyruvate carboxykinase- IGF-II transgenic mice than in controls, as evaluated by ligand blot analysis. Starvation reduced serum IGF-I, but increased IGFBP-2 in transgenic mice more markedly than in controls. Fasting insulin levels were significantly reduced in transgenic mice, whereas glucose levels were not influenced by elevated IGF-II. The body growth of 4- and 12- week-old mice was not significantly influenced by elevated IGF-II, but transgenic mice displayed increased kidney and testis weight at the age of 4 weeks, and increased adrenal weight at the age of 12 weeks. Our results demonstrate that elevated IGF-II in postnatal life has multiple endocrine consequences and subtle time-specific effects on organ growth.

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor has been detected in many cells and tissues. In the rat, there is a dramatic developmental regulation of IGF-II/M6P receptor expression, the receptor being high in fetal and neonatal tissues and declining thereafter. We have systematically studied the expression of the human IGF-II/M6P receptor protein in tissues from 10 human fetuses and infants (age 23 weeks gestation to 24 months postnatal). We have asked 1) whether there is differential expression among different organs, and 2) whether or not the human IGF-II/M6P receptor is developmentally regulated from 23 weeks gestation to 24 months postnatal. Protein was extracted from human tissues using a buffer containing 2% sodium dodecyl sulfate and 2% Triton X-100. Aliquots of the protein extracts were analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting using an anti-IGF- II/M6P receptor antiserum (no. 66416) and 125I-protein A or an immunoperoxidase stain. IGF-II/M6P receptor immunoreactivity was detected in all tissues studied with the highest amount of receptor being expressed in heart, thymus, and kidney and the lowest receptor content being measured in brain and muscle. The receptor content in ovary, testis, lung, and spleen was intermediate. The apparent molecular weight of the IGF-II/M6P receptor (220,000 kilos without reduction of disulfide bonds) varied among the different tissues: in brain the receptor was of lower molecular weight than in other organs. Immunoquantitation experiments employing 125I-protein A and protein extracts from human kidney at different ages revealed a small, albeit not significant, difference of the receptor content between fetal and postnatal tissues: as in other species, larger amounts of receptor seemed to be present in fetal than in postnatal organs. In addition, no significant difference of the receptor content between human fetal liver and early postnatal liver was measured employing 125I-protein A- immunoquantitation in three fetal and five postnatal liver tissue samples. The distribution of IGF-binding protein (IGEBP) species, another abundant and major class of IGF binding principles, was also measured in human fetal and early postnatal lung, liver, kidney, muscle, and brain using Western ligand blotting with 125I-IGF-II: as with IGF-II/M6P receptor immunoreactivity there was differential expression of the different classes of IGFBPs in the various organs.