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Endocrinology Vol. 148, No. 1 441-451
Copyright © 2007 by The Endocrine Society

Postnatally Elevated Levels of Insulin-Like Growth Factor (IGF)-II Fail to Rescue the Dwarfism of IGF-I-Deficient Mice except Kidney Weight

Corinna Moerth1, Marlon R. Schneider1, Ingrid Renner-Mueller, Andreas Blutke, Martin W. Elmlinger, Reinhold G. Erben, Cecilia Camacho-Hübner, Andreas Hoeflich and Eckhard Wolf

Institute of Molecular Animal Breeding and Biotechnology (C.M., M.R.S., I.R.-M., A.B., E.W.), Gene Center, Institute of Animal Physiology (R.G.E.), Veterinary Faculty, University of Munich, D-81377 Munich, Germany; Department of Pediatric Endocrinology (M.W.E.), Children’s Hospital, University of Tuebingen, D-72074 Tuebingen, Germany; Department of Endocrinology (C.C.-H.), William Harvey Research Institute, University of London, London E1 4NS, United Kingdom; and Research Unit Genetics and Biometry (A.H.), Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf, Germany

Address all correspondence and requests for reprints to: Professor Dr. Eckhard Wolf, Institute of Molecular Animal Breeding and Biotechnology, Gene Center, University of Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany. E-mail: ewolf{at}lmb.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study tested whether elevated levels of IGF-II in the postnatal period can rescue the dwarfism in IGF-I-deficient mice. Heterozygous Igf1 mutant mice [I+/– IIwt] were crossed with heterozygous Igf1 mutant, phosphoenolpyruvate carboxykinase promoter IGF-II transgenic mice [I+/– IItg], and [I+/+ IIwt], [I+/+ IItg], [I–/– IIwt], and [I–/– IItg] offspring were investigated. IGF-II levels were 11- and 6-fold higher in male and female [I–/– IItg] vs. [I–/– IIwt] animals. Western ligand blot analysis revealed markedly reduced activities of 30- and 32-kDa IGF binding proteins (IGFBPs) (most likely IGFBP-1 and IGFBP-2) and the 39- to 43-kDa IGFBP-3 double band in serum from IGF-I-deficient mice. These binding proteins were partially restored by overexpression of IGF-II. Analysis of weight data from the early postnatal period until d 60 showed that, in the absence of IGF-I, elevated levels of IGF-II have no effect on body weight gain. A detailed analysis of body proportions, bone parameters, and organ weights of 60-d-old mice also failed to show effects of IGF-II with one important exception: in Igf1 mutant and also Igf1 intact male mice, IGF-II overexpression significantly increased absolute (+32.4 and +28.6%; P < 0.01) and relative kidney weights (+29.0 and +22.4%; P < 0.001). These changes in kidney weight were associated with reduced phosphorylation of p38 MAPK. In summary, our genetic model shows that substantial amounts of IGF-II in the circulation do not rescue the postnatal growth deficit of IGF-I-deficient mice but increase absolute and relative kidney weights of normal and IGF-I-deficient male mice, suggesting a gender-specific role of IGF-II for kidney growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SIZE OF an animal depends on the number and volume of the cells it contains, with some contribution by extracellular matrix and fluids. In mammals, growth (increase in size) begins at preimplantation embryonic stages and lasts until a steady state is reached in the postnatal period. Appropriate growth is controlled by hormones and growth factors that bind to specific receptors, thereby activating intracellular signaling pathways. In mice, the IGF system has been unequivocally identified as a major determinant of both embryonic and postnatal growth (1). The IGF system consists of two ligands (IGF-I and IGF-II), two receptors (IGF1R and IGF2R), and several IGF binding proteins (IGFBPs) (reviewed in Ref. 2). The IGFs are small single-chain mitogenic peptides structurally similar to proinsulin and to each other. They are produced in several tissues and function in an autocrine/paracrine fashion (3) and, because they circulate in the plasma bound to specific IGFBPs, act also as classical hormones. Despite their structural homology, each growth factor has specific expression patterns and effects.

The main source of circulating IGF-I is the liver (4, 5), in which its expression is strongly regulated by GH. In other tissues, IGF-I expression is mostly GH independent (1, 6). The regulation of IGF-II expression is not as well understood, and species-specific differences appear to be important. Whereas adult humans display high levels of IGF-II, in mice and rats, expression of IGF-II is shut down in almost every tissue after birth, and very low levels of this growth factor are detectable in postnatal life (7).

The signaling of both IGFs is mediated by the IGF1R, a heterotetrameric transmembrane glycoprotein with extracellular ligand-binding and intracellular tyrosine kinase domains (8). The IGF1R binds IGF-I and IGF-II with a high affinity (~10–10 M), and insulin with about 100-fold lower affinity (9). Binding of IGFs to the IGF1R is modulated by the IGFBPs. In contrast to the IGF1R, the structurally unrelated IGF2R is a single-chain polypeptide consisting of a large extracellular domain and a small cytoplasmic tail lacking kinase activity (10). In mammals, this receptor is bifunctional: on the one hand, it serves as the cation-independent mannose 6-phosphate receptor and is involved in the trafficking of lysosomal enzymes carrying a mannose 6-phosphate recognition tag; on the other hand, it regulates the turnover of IGF-II by receptor-mediated endocytosis.

To gain insight into the functions of the IGF system during embryogenesis and postnatal development, several transgenic and knockout mouse models for the members of the IGF family have been established over the last decades (reviewed in Ref. 11). Homozygous IGF-I-deficient mice have approximately 60% of normal weight at birth but reach approximately 30% of normal weight at an age of 2 months, indicating that IGF-I not only is necessary for normal embryonic growth but also has a continuous function throughout postnatal development (12, 13, 14). The infertility (15), delay of skeletal development (13), and altered bone structure (16) of these mutants have been particularly well characterized. The description of intrauterine and postnatal growth failure in patients bearing a deletion or mutation of the IGF1 gene indicates a similar function of IGF-I in humans (17, 18, 19). Homozygous Igf2 mutant mice are also born with approximately 60% of normal weight but do not display a further size reduction, suggesting that this growth factor is essential for normal embryonic but not for postnatal growth (20).

Overexpression of IGF-I after birth results in a consistent increase in body weight (21), which is also present in the absence of GH (22). Excess of IGF-II in the postnatal period, however, induced by infusion of the growth factor in rats (23), transplantation of IGF-II-secreting tumors into nude rodents (24), or overexpression in transgenic mice (reviewed in Ref. 25), has not resulted in a considerable increase in body weight or size. Although overexpression of IGF-II in transgenic mice did not cause a significant increase in body size, disproportionate growth of specific organs was observed. This information, together with the phenotype of IGF-II-deficient mice (in which postnatal growth is not further impaired; see above) and the fact that IGF-II expression is down-regulated after birth in mice, led to the concept that the major function of IGF-II is to stimulate embryonic growth or, in other words, to function as a fetal analog of IGF-I (7), at least in rodents. However, at present the role of postnatal IGF-II signaling through the IGF1R is unclear, especially in species with high levels of IGF-II after birth, such as humans.

To shed additional light on what has been called the IGF-II enigma (26), we investigated the effect of postnatally increased IGF-II levels in the absence of IGF-I by a genetic approach. For this purpose, we designed a two-step mating scheme of Igf1 mutant mice (14) with phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II transgenic mice (27) to generate mice lacking IGF-I and overexpressing IGF-II at the same time. To test whether IGF-II is able to rescue phenotypic consequences of IGF-I deficiency in mice, regulatory effects on serum IGFBPs as well as effects on body and organ growth and bone parameters were systematically analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal breeding
IGF-I-deficient mice (kindly provided by Dr. Lyn Powell-Braxton, Genentech Inc., South San Francisco, CA, via Dr. Pieter Doevedans, University of Maastricht, Maastricht, The Netherlands) were genotyped as previously described (14). Because the survival rate of homozygous Igf1 knockout mice was very low on the original genetic background, we mated the heterozygous animals with NMRI outbred mice, resulting in 50% NMRI background. PEPCK-IGF-II transgenic mice were maintained on NMRI background and genotyped as previously described (27).

Due to the infertility of IGF-I-deficient mice, two mating steps were necessary. First, heterozygous Igf1 mutant mice [I+/– IIwt] were mated with hemizygous PEPCK-IGF-II transgenic mice [I+/+ IItg]. In a second step, [I+/– IIwt] and [I+/– IItg] mice were mated to obtain the following six genotypes: [I+/+ IIwt], [I+/+ IItg], [I+/– IIwt], [I+/– IItg], [I–/– IIwt], and [I–/– IItg]. All resulting groups had 75% NMRI genetic background. The animals were maintained under specified pathogen-free conditions in a closed barrier system and had free access to a standard rodent diet (V1534; Ssniff, Soest, Germany) and tap water. All animal experiments were approved by the author’s institutional committee on animal care and carried out in accordance with the German Animal Protection Law with permission from the responsible veterinary authority.

Qualitative and quantitative RT-PCR analysis
Tissues were homogenized in TRIzol reagent (Invitrogen, Karlsruhe, Germany), and total RNA was extracted according to the manufacturer’s instructions. For reverse transcription, 400 ng of DNase-digested total RNA were denatured for 5 min at 65 C and incubated with reverse transcriptase and buffer (SuperScript II; Invitrogen) according to the manufacturer’s instructions.

Conventional RT-PCR to detect transgene-specific IGF-II sequences used the primers hIGF-II no. 5 (sense): 5'-ATG GGA ATC CCA ATG GGG AAG-3' and IGF-II no. 10 (antisense): 5'-CGG GGT CTT GGG TGG GTA GAG-3'. To confirm the correct reverse transcription, a fragment of the housekeeping gene ß-actin was amplified as described previously (28).

Quantification of transgene-specific mRNA abundance was performed with TaqMan assays using an ABI PRISM 7700 sequence detector (Applied Biosystems, Weiterstadt, Germany). The TaqMan gene expression assay Hs01005963_m1 (Applied Biosystems) containing both primers and FAM-labeled probe was used. Gene expression was normalized for ß-actin expression quantified by use of ß-actin-specific primers (accession no. NM 007393.1: 1010F, TGA CAG GAT GCA GAA GGA GA, 1084R, GTA ACG AGG AGG ACT CGC) and probe 106 (CTC TGG CT) from the Universal Probe Library (Roche, Mannheim, Germany; intron-spanning assay, amplicon length: 75 bp). Each assay included triplicates of: cDNA for the gene of interest, a no-template control, and four dilutions of cDNA pooled from all groups to calculate the corresponding amplification efficiency as described before (29).

Measurement of IGFs and IGFBPs in serum
Serum was obtained by centrifugation of blood samples and stored at –20 C until further analysis. Serum levels of IGF-I and -II were quantified by RIAs (Mediagnost, Reutlingen, Germany) as previously described (30) by following the accepted principles for IGF measurements (31). The IGF-I assay (sensitivity 0.02 ng/ml) uses an excess of IGF-II to eliminate the interference by IGFBPs. For the IGF-I RIA, the interassay coefficient of variation (CV) was 7.4% at 50% B/B0, the intraassay CV was less than 6.5%. The RIA for IGF-II (sensitivity 0.1 ng/ml) has a cross-reactivity of less than 0.05% with IGF-I and vice versa. The interassay variation of the IGF-II assay is 7.9% and the intraassay CV did not exceed 5.4%.

Western ligand blot analysis of IGFBPs present in serum was done according to a standard protocol (32), with modifications as described before (27). [125I]IGF-II-binding proteins were visualized and quantified on PhosphorImager-Storm (Molecular Dynamics, Inc., Krefeld, Germany).

Analysis of body and organ growth
Body weight of selected litters was recorded daily, starting from d 3 or 4 after birth until d 45. From this date on, mice were weighed twice a week until d 60 when mice were killed by bleeding from the retroorbital sinus under ether anesthesia. Body weight data were corrected to a weighing age of n x 3 by linear interpolation. At necropsy, nose-rump length was measured as the distance between nose and base of the tail. Organs were dissected, blotted dry on tissue paper, and weighed to the nearest milligram. For bilateral organs the paired weight was recorded and the mean organ weight was calculated. After removal of internal organs and skin, the carcass (without head and tail) was weighed to the nearest 0.1 g.

Bone histology and bone mineral density (BMD) measurements
The right femur was fixed in 4% paraformaldehyde for 24 h at 4 C and washed overnight in PBS (pH 7.40) containing 10% sucrose. Subsequently bones were dehydrated and embedded undecalcified in methylmethacrylate as previously described (33). Three-micrometer-thick sections were prepared using a HM 360 microtome (Microm, Walldorf, Germany) and stained with von Kossa/McNeal (34).

BMD of the left femur was measured by peripheral quantitative computed tomography (pQCT) using a XCT Research M+ pQCT machine (Stratec Medizintechnik, Pforzheim, Germany). One slice (0.2 mm thick) in the middiaphysis of the femur and three slices in the distal femoral metaphysis located 1.5, 2.0, and 2.5 mm proximal to the articular surface of the knee joint were measured. BMD values of the distal femoral metaphysis were calculated as the mean over three slices. A voxel size of 0.070 mm and a threshold of 600 mg/cm3 were used for calculation of cortical BMD. Values for bone mineral content (BMC) are given as milligram per 1-mm-thick bone slice.

Analysis of signal transduction in the kidney
Kidneys from six mice per group were homogenized in extraction buffer as described previously (30), and protein content was quantified using the bicinchoninic acid method. Twenty micrograms of protein were separated on 12% SDS-PAGE gels and transferred to polyvinyl difluoride membranes (Millipore, Eschborn, Germany). To verify equal loading and proper transfer, membranes were stained using Coomassie blue according to standard procedures. Membranes were blocked (5% dry milk and 1% Tween 20 in Tris-buffered saline) and incubated with primary antibodies overnight at 4 C. We studied activation of p38 MAPK, p42/44 MAPK, and phosphoinositide-dependent kinase (PDK)-1 (dilution 1:1000; Cell Signaling, Danvers, MA; no. 9211, 4376, and 3061, respectively; New England Biolabs, Frankfurt, Germany). To demonstrate specific activation of p38 MAPK and equal loading of the gels, all blots were stripped [2% SDS, 100 mM 2-mercaptoethanol in 62.5 mM Tris (pH 6.7) for 1 h at 70 C] and incubated with antibodies specific for total p38 MAPK (dilution 1:1000; Cell Signaling; no. 9212) and mouse actin monoclonal antibody (MAB1501, 1:10000; Chemicon, Hampshire, UK). After three washings in Tris-buffered saline containing 1% Tween 20, membranes were incubated with horseradish peroxidase-coupled goat antirabbit IgG (1:2000, Cell Signaling no. 7074; New England Biolabs) or horse radish-conjugated goat antimouse secondary IgG (1:7000; Dianova, Hamburg, Germany) for 1 h at room temperature. Finally, bound antibodies were detected using an enhanced chemiluminescence detection kit (ECL Advance Western blotting detection kit; GE Healthcare, Freiburg, Germany) and a Kodak Image Station 440CF (Eastman Kodak Co., Rochester, NY). Band intensities were quantified using the ImageQuant software package (GE Healthcare). All signal activities were normalized for the Coomassie blue stain. Separate blots were prepared for Igf1 intact and Igf1 knockout mice (n = 6 for each genotype) to evaluate effects of IGF-II expression and gender on the activity of signaling molecules. A direct comparison of IGF-I- and IGF-II-induced effects on p38 activity was made by using blots prepared from kidney protein extracts of male mice (n = 5 for [I+/+ IIwt]; n = 6 for all other genotypes).

Statistical analysis
Observed vs. expected frequencies of the individual genotypes in offspring from [I+/– IIwt] x [I+/– IItg] crosses were statistically evaluated by {chi}2 analysis. IGF measurements, body weight, and organ weight data and BMD values were analyzed by ANOVA using the general linear models procedure (SAS Institute, Inc., Cary, NC), taking the effect of the IGF-I status (I+/+, I–/–), IGF-II status (IIwt, IItg), sex (male, female), and interactions between these factors into account. Western ligand blots were performed for male mice only; therefore, sex was omitted from the model. Signaling data were analyzed separately for Igf1 intact and for Igf1 knockout mice to estimate effects of IGF-II status and sex, taking the effect of the individual blot into account. In addition, effects of IGF-I status and IGF-II status were estimated using the data of blots prepared from kidney protein extracts of male mice from all four genotypes. Signaling data are shown as least squares means (LSMs) and SE of least squares means. LSMs were compared using Student t tests. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genetic crosses
For our experiments, we used two genetically modified mouse models carrying either a loss-of-function mutation of the Igf1 locus (14) or an IGF-II expression vector (27). By a two-step mating scheme, we obtained six different genotypes of which the following four were used for further investigations: [I+/+ IIwt], [I+/+ IItg], [I–/– IIwt], and [I–/– IItg]. PCR analysis revealed a genotype distribution of Igf1 wild-type and heterozygous mice in accordance to the expected Mendelian ratios (data not shown). The expected proportion of [I–/– IIwt] and [I–/– IItg] animals was 12.5% each. However, we observed only 5.3% [I–/– IIwt] and 6.8% [I–/– IItg] mice, indicating a mortality rate (before weaning) of 57.6 and 46.6%, respectively. The proportions of surviving [I–/– IIwt] and [I–/– IItg] mice were significantly smaller than expected ({chi}2 = 13.0; P < 0.001, and {chi}2 = 7.6; P < 0.01); however, there was no significant difference in survival rate between these two groups ({chi}2 = 0.8; P = 0.37).

PEPCK-IGF-II transgene expression
Previous studies of adult PEPCK-IGF-II transgenic mice revealed expression of this transgene in liver, kidney, and several parts of the gut (27). To determine the onset of PEPCK-IGF-II expression at the major expression sites, we performed conventional RT-PCR analysis of total RNA from liver and kidneys of fetuses at d 17.5 and 19.5 as well as pups at d 4 postnatal. Transgene expression of PEPCK-IGF-II in the liver could be detected from embryonic stage 19.5 onward, and on postnatal d 4, the obtained signal was visibly stronger (Fig. 1AGo). In contrast to the liver, PEPCK-IGF-II transgene expression could not be detected in the kidney with this method in any of these early stages (data not shown). However, by using the more sensitive TaqMan technology, transgene-specific mRNA expression was detectable in kidney samples from PEPCK-IGF-II transgenic mice at all stages investigated (Fig. 1BGo).


Figure 1
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  		FIG. 1. IGF-II transgene expression. A, Conventional RT-PCR showing transgene expression in the liver of wild-type (wt) and PEPCK-IGF-II transgenic animals (tg) at the indicated ages. B, Quantitative RT-PCR analysis of transgene expression in the kidney of the same animals at the indicated ages (two animals per group were investigated). Igf2 gene expression was analyzed by use of TaqMan assays as described in Materials and Methods. Data are expressed as percent of ß-actin mRNA expression.

 
Serum IGF-I, IGF-II, and IGFBPs
IGF-I was undetectable in serum from 8-wk-old [I–/– IIwt] and [I–/– IItg] mice (Table 1Go). Interestingly, serum IGF-I levels were significantly (P < 0.001) higher in [I+/+ IIwt], compared with [I+/+ IItg] mice. Circulating IGF-II levels were 11- and 6-fold higher in male and female [I–/– IItg] vs. [I–/– IIwt] animals (P < 0.001). The increase of IGF-II levels by the presence of the PEPCK-IGF-II transgene was greater (P < 0.05) on an IGF-I-deficient background than in mice with intact Igf1 genes. Furthermore, serum IGF-II levels were significantly (P < 0.01) higher in male than female PEPCK-IGF-II transgenic mice, in both Igf1 mutant and intact animals. Western ligand blot analysis was used to determine the binding activities of serum IGFBPs in [I+/+ IIwt], [I+/+ IItg], [I–/– IIwt], and [I–/– IItg] mice (Fig. 2AGo). This analysis was performed for male mice, which exhibited higher PEPCK-IGF-II transgene expression. The activities of 30- and 32-kDa IGFBPs (most likely IGFBP-1 and IGFBP-2, respectively) and the characteristic 39- to 43-kDa IGFBP-3 double band were markedly reduced in serum of IGF-I-deficient mice as compared with Igf1 intact mice. These binding proteins were partially restored by expression of the PEPCK-IGF-II transgene but were still reduced in abundance, compared with Igf1 wild-type animals (Fig. 2BGo).


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  		TABLE 1. Serum IGF levels in 8-wk-old mice

 

Figure 2
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  		FIG. 2. Serum IGFBP levels. A, A representative Western ligand blot of serum samples from animals of the indicated genotypes using [125I]IGF-II as a tracer. B, PhosphorImager data for bound [125I]IGF-II were quantified and statistically evaluated as described in Materials and Methods. The figure shows means and SDs. Within IGFBP, means with different superscripts are significantly different (P < 0.05). Four male mice per group were investigated. wt, Wild-type; tg, transgenic.

 
Body weight gain
We closely monitored the body weight gain by weighing the animals from the first few days after birth up to d 60, an age at which final body size is attained to a large extent in mice. No difference at all could be observed between the [I–/– IItg] and the [I–/– IIwt] groups in both genders at any time point, demonstrating that, in the absence of IGF-I, elevated levels of circulating IGF-II have no effect on body weight gain (Fig. 3Go, A and B). In the groups with intact Igf1 alleles, the body weight was significantly (P < 0.05) higher in males carrying the PEPCK-IGF-II transgene from d 9 to 51. The difference decreased from about 20% on d 9 to less than 10% on d 51. At the end of the weighing period, body weight of PEPCK-IGF-II transgenic Igf1 intact males was not significantly different from that of control males. In females, the presence of the PEPCK-IGF-II transgene did not alter the body weight at any time point investigated.


Figure 3
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  		FIG. 3. Postnatal body weight development. A, Growth curves showing the body weight gain of [I+/+ IIwt] (n = 5 males and 5 females), [I+/+ IItg] (n = 8 males and 11 females), [I–/– IIwt] (n = 6 males and 7 females), and [I–/– IItg] (n = 13 males and 4 females) animals. Data were corrected to a weighing age of n x 3 by linear interpolation. Asterisks indicate statistically significant (P < 0.05) differences between Igf1 intact PEPCK-IGF-II transgenic and nontransgenic males. B, Three female littermates at the age of 60 d. Genotypes from left to right: [I+/+ IIwt], [I–/– IIwt], and [I–/– IItg].

 
Organ and tissue weights, nose-rump length (NRL)
Absolute organ weights, NRL, and body weight at 60 d of age were significantly affected by the IGF-I status, with higher (P < 0.001) values for Igf1 intact vs. Igf1 mutant mice (Table 2Go). Most of these parameters were also affected by gender: the weights of the heart, lungs, liver, kidneys, pancreas, and carcass (all P < 0.001) as well as NRL (P < 0.01) and body weight (P < 0.001) were significantly greater in males than females. The effect of gender was significantly greater in Igf1 intact than Igf1 mutant mice for the weights of the liver and carcass and body weight, as demonstrated by a significance of the interaction I*II*sex (Table 2Go). The weights of adrenal glands were greater (P < 0.001) in female than male mice. No effect of gender was observed for brain and spleen weights.


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  		TABLE 2. Absolute organ weights, NRL, and body weight in 8-wk-old mice

 
A clear effect (P < 0.01) of the IGF-II status was only observed for kidney weights; however, this effect was gender dependent. Overexpression of IGF-II significantly increased the kidney weights of Igf1 intact (+28.6%) and Igf1 mutant male mice (+32.4%). In contrast, IGF-II overexpression had no effect on kidney weights of Igf1 intact females and only moderately increased (+8.1%) kidney weights in Igf1 mutant females (Table 2Go). There was a moderate effect of IGF-II overexpression on lung weights of Igf1 intact male (+8.3%) and female mice (+10.8%), whereas no effect and a slight decrease of lung weights were observed in IGF-II-overexpressing male and female Igf1 mutant mice, respectively (P < 0.05 for the interaction effect I*II). A significant (P < 0.001) interaction effect of I*II*sex was observed for the absolute weight of the adrenal glands. IGF-II overexpression increased adrenal weights of Igf1 intact females (+43.5%) but caused a moderate reduction (–17.7%) in the adrenal weights of their male counterparts. Conversely, in Igf1 mutant mice IGF-II overexpression resulted in a moderate increase (+16.0%) of adrenal weights in males but a slight decrease (–6.5%) in female mice (Table 2Go).

Evaluation of relative organ weights and body dimensions also revealed significant effects of the IGF-I status (Table 3Go). Relative weights of brain, heart, and spleen were increased (all P < 0.001), whereas relative carcass weight (P < 0.01) and relative NRL (P < 0.001) were reduced in Igf1 mutant mice, compared with their Igf1 intact counterparts. A gender-specific effect was observed for the relative weights of brain (P < 0.001), lungs (P < 0.01), adrenal glands (P < 0.001), carcass (P < 0.05), and relative NRL (P < 0.01), with greater values in female than male mice. In contrast, relative liver and kidney weights were significantly (P < 0.05 and P < 0.001, respectively) increased in male mice, compared with their female counterparts (Table 3Go). IGF-II overexpression had a significant (P < 0.001) effect on relative kidney weight; however, as for the absolute weight of this organ, this effect was observed only in males (P < 0.01 for the interaction effect I*II*sex). Relative kidney weights of IGF-II transgenic male mice were significantly (P < 0.001) increased, compared with male controls, both in the presence (+22.4%) and absence (+29.0%) of IGF-I. A significant effect of the interaction I*II*sex was also observed for the relative weights of adrenal glands (P < 0.01) and spleen (P < 0.05). In males, IGF-II overexpression decreased (–50.0%) relative adrenal weights of Igf1 intact mice, whereas the opposite effect was observed in IGF-I-deficient mice. In female Igf1 intact mice, relative adrenal weights were increased (+66.6%) by IGF-II overexpression, which had no effect on this parameter in IGF-I-deficient females. Relative spleen weight was greater in female than male Igf1 intact mice, independent of the IGF-II status. In Igf1 mutants, relative spleen weight was increased in male vs. female mice, but this difference was eliminated by expression of the IGF-II transgene.


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  		TABLE 3. Relative organ weights and NRL in 8-wk-old mice

 
Bone parameters
Geometric and structural bone parameters are shown in Table 4Go. At the femoral metaphysis, a predominantly trabecular bone site, the pQCT analyses showed slightly higher total volumetric BMD in male and female IGF-I-deficient mice, compared with control littermates with intact Igf1 alleles. In contrast, trabecular BMD was strongly reduced by 52 and 37% in male and female Igf1 knockout mice, respectively. Total cross-sectional area and total BMC of the bone slices were reduced by approximately 65–70% in male and female IGF-I-deficient mice. Female mice had generally higher total but not trabecular BMD and lower cross-sectional area, compared with male mice at the distal femoral metaphysis. At the femoral shaft, IGF-I-deficient mice showed a pronounced reduction in bone size and total BMC but unchanged total volumetric BMD, compared with Igf1 wild-type mice. Similar to the distal metaphysis, bone size of the shaft was reduced in female relative to male mice. At both the distal femoral metaphysis and the femoral shaft, three-way factorial ANOVA revealed significant interactions between sex and IGF-I, i.e. the bone phenotype of male Igf1 knockout mice was more severe than that of female Igf1 knockouts relative to sex-matched wild-type controls. However, most importantly, the presence or absence of the IGF-II transgene failed to show any significant effect on any of the geometric or BMD parameters in wild-type mice or mice lacking IGF-I. The pQCT findings were confirmed by bone histology. Femurs of IGF-I-deficient mice were much smaller in size and showed less cancellous bone together with reduced cortical bone thickness in comparison with Igf1 wild-type controls (data not shown). Taken together, an increase of the circulating levels of IGF-II did not affect skeletal parameters, in the presence or absence of IGF-I.


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  		TABLE 4. BMD and geometric variables of the femur in 8-wk-old mice

 
Signal transduction in the kidney
To study the marked stimulatory effect of IGF-II on kidney weight in male but not female mice in more detail, we investigated signal transduction mechanisms in the kidneys of both sexes. As an established candidate for both kidney growth and sex-specific effects, we selected the activation status of p38 MAPK as a starting point for our signal transduction studies. In Igf1 knockout mice, the level of p38 MAPK phosphorylation was significantly (P < 0.01) higher in males than females. Overexpression of IGF-II significantly (P < 0.005) reduced phosphorylation of p38 MAPK at Thr180/Tyr182 in male but not female mice (Fig. 4AGo). In male Igf1 intact mice, the level of p38 MAPK activation was significantly (P < 0.01) lower than in IgfI knockout mice (Fig. 4BGo). There was a trend of further reduction of p38 MAPK phosphorylation by IGF-II overexpression in Igf1 wild-type males. Because the IGF-I receptor mediates activation of additional pathways, we studied activation of p42/44 MAPK and PDK1 in the kidneys. In contrast to p38 MAPK, activation of p42/44 MAPK or PDK1 was similar in female and male IgfI knockout mice. Overexpression of IGF-II reduced activation of p42/44 MAPK in male but not female IgfI knockout mice (P < 0.05; data not shown). In Igf1 knockout mice, expression of total p38 MAPK and ß-actin was not affected by gender or the IGF-II transgene (Fig. 4AGo). In male IgfI wild-type mice, expression of ß-actin was significantly (P < 0.05) weaker, compared with IgfI knockout mice, whereas total p38 MAPK was similar in all groups (Fig. 4BGo).


Figure 4
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  		FIG. 4. Evaluation of renal p38 MAPK activity. A, Expression of IGF-II impairs activation of p38 MAPK in male but not female Igf1 knockout mice. B, In male Igf1 knockout mice activation of p38 MAPK was much higher if compared with Igf1 wild-type mice. Western immunoblot analyses using phospho-specific antibodies were performed and evaluated as described in Materials and Methods. The graphs represent LSMs and SEs of LSMs (n ≥ 5 per group). Different superscripts indicate significant differences (P < 0.05). Below the graph, representative Western immunoblots detecting phosphorylated or total p38 MAPK and ß-actin are shown. wt, Wild-type; tg, transgenic.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a two-step mating scheme, we generated mice lacking IGF-I and overexpressing IGF-II under the control of a PEPCK promoter with considerably increased levels of circulating IGF-II in the postnatal period. On a genetic background of 75% NMRI, about 50% of the IGF-I-deficient mice survived. It has previously been shown that the survival rate of Igf1 knockout mice strongly depends on their genetic background (13, 14). The relatively large proportion of surviving IGF-I-deficient outbred mice is most likely due to positive effects of heterozygosity (hybrid vigor) (35).

IGF-I was undetectable by RIA in serum from 60-d-old [I–/– IIwt] and [I–/– IItg] mice. Animals harboring the PEPCK-IGF-II transgene displayed a significant increase in serum IGF-II concentrations but reduced circulating IGF-I levels when compared with their wild-type counterparts. The negative correlation between serum IGF-II and IGF-I levels is most likely due to competition of the IGF peptides for IGFBPs and rapid clearance of free IGFs (27). The six IGFBPs (IGFBP-1 to -6) are expressed in a tissue-specific manner and act as modulators of IGF activity (reviewed in Ref. 36). Western ligand blot analysis of serum samples from [I+/+ IIwt], [I+/+ IItg], [I–/– IIwt], and [I–/– IItg] animals revealed that the levels of 30- and 32-kDa IGFBPs (most likely IGFBP-1 and IGFBP-2) and the 39- to 43-kDa IGFBP-3 double band were strongly reduced in [I–/– IIwt] mice, but their levels were partially restored by IGF-II overexpression in [I–/– IItg] mice. Whereas IGF-II may exert this effect by directly influencing the expression of these IGFBPs, a possible alternative explanation would be regulation of IGFBP levels by a posttranslational mechanism, e.g. by influencing their half-life in the circulation.

To investigate the effects of postnatally elevated IGF-II on body weight gain, we recorded the body weight of selected litters regularly, starting from d 3 to 4 after birth. In agreement with the initial description of Igf1 knockout mice (12, 13, 14), IGF-I deficiency resulted in a marked decrease in postnatal growth. This phenotype was not altered by expression of the PEPCK-IGF-II transgene, demonstrating that substantial levels of circulating IGF-II in the postnatal period do not rescue the dwarfism of IGF-I-deficient mice. In contrast to the Igf1 mutant mice, IGF-II overexpression slightly increased the body weight gain of Igf1 intact male mice, compared with their nontransgenic counterparts. The difference was significant in growing mice, but the final body weight was not different between the two groups. This observation indicates that IGF-I and IGF-II may have additive effects on postnatal overall body weight gain, but the effect of IGF-II decreases with age. In view of the high contribution to total body weight and the slightly increased weight at autopsy (+5.3%; see Table 2Go), the carcass is probably the compartment that reacted to the combined stimulus of IGF-I and IGF-II, leading to a transient overall increase in body weight of PEPCK-IGF-II transgenic males. Such an effect was not seen in female mice, most likely due to the relatively low expression level of the PEPCK-IGF-II transgene in the set of mice investigated.

In line with previous studies (16, 37, 38), we found a generalized reduction in bone size together with cancellous bone osteopenia and reduced cortical thickness in IGF-I-deficient mice. Our study demonstrates for the first time that elevated circulating levels of IGF-II fail to exert any significant effect on the skeleton in IGF-I-deficient mice. The IGF system is thought to play an important role in bone physiology. In fact, the IGFs are profusely produced by osteoblasts and are the most abundant growth factors stored in bone (39). Smaller bones and delayed ossification were among the first alterations observed in Igf1 knockout mice (12, 13, 14). Further studies revealed, among other effects, a reduction in the size of hypertrophic chondrocytes in the femur (1), tibial bone formation rate (16), and femoral BMD (37). In accordance with these data, overexpression of IGF-I in osteoblasts increased peak BMD (40). Recently Wang et al. (38) compared the size and growth plate parameters in homozygous IGF-I-deficient and homozygous GH receptor (Ghr) knockout mice. Animals lacking GHR displayed a stronger reduction in bone size and more severe effects in the growth plate, compared with the alterations observed in mice lacking IGF-I. Based on previous observations that IGF-II is expressed in the germinal and proliferative zones of the growth plate, the authors hypothesized that the effects of GH on chondrocyte generation and proliferation may be mediated by GH-induced IGF-II expression. In fact, the authors were able to measure increased levels of IGF-II in the growth plate of Igf1 knockout mice and reduced levels of this growth factor in GHR-deficient animals (38). Although we did not evaluate growth plate parameters, femoral bone length was identical in [I–/– IItg] and [I–/– IIwt] mice, strongly arguing against this hypothesis. Nevertheless, it is still possible that the growth-promoting effects of IGF-II in the growth plate are mediated exclusively by locally produced IGF-II, which may not be elevated in our experimental model. At least for IGF-I, however, the circulating growth factor is important for normal bone growth. In double acid-labile subunit/liver-specific IGF-I-deficient mice, which have only 10–15% of normal serum IGF-I levels, linear bone growth is impaired as measured by various skeletal parameters (41).

Organ weight measurements confirmed and extended previous findings of differences between Igf1 intact and mutant mice (42). However, the focus of the present study was on the question whether elevated IGF-II levels can stimulate organ growth in the absence of IGF-I. Interestingly, IGF-II overexpression caused a marked increase in absolute (+28.6 and +32.4%; P < 0.01) and relative kidney weights (+22.4 and +29.0%; P < 0.001) in male Igf1 intact and IGF-I-deficient mice, whereas only moderate changes in kidney weight were observed in their female counterparts. For the particular set of female Igf1 intact PEPCK-IGF-II transgenic mice investigated in the present study, this may be due to the fact that they exhibited relatively low serum IGF-II levels (Table 1Go) and also 8-fold lower renal transgene-specific mRNA levels, compared with their male counterparts (data not shown). In the group of Igf1 mutant mice, PEPCK-IGF-II transgenic females also displayed lower serum IGF-II levels than their male counterparts; however, circulating IGF-II was 6-fold increased, compared with the respective controls. Furthermore, serum IGF-II levels of [I–/– IItg] females were in the same range as those of [I+/+ IItg] males, which exhibited markedly increased kidney growth. These findings suggest a gender-specific, IGF-I independent effect of IGF-II on kidney growth.

In the context of altered kidney weight, the genetic background (75% NMRI) of the mice investigated in this study requires specific attention. NMRI mice are known to exhibit a relatively high incidence of glomerulonephritis (43, 44), which might interfere with organ growth regulation by IGFs. However, in these studies glomerulonephritis was found only in animals much older than those investigated in our study. Moreover, our own studies of GH transgenic mice and nontransgenic littermate controls on NMRI background (45, 46), which included thorough pathological analyses of the kidneys, did not reveal glomerulonephritis in controls younger than 6 months. Therefore, it is very unlikely that the growth-stimulating effect of IGF-II overexpression on the kidney observed in the present study is related to the NMRI-specific high incidence of glomerulonephritis.

To get insight into the mechanisms of stimulation of kidney growth by IGF-II in male mice, we performed an analysis of the activity state of candidate signaling molecules in the IGF signal transduction pathway. In general, the IGF system controls two MAPK pathways (p42/44 MAPK and p38 MAPK) and the phosphatidylinositol 3-kinase pathway (47). Regarding the kidney, activation of the p42/44 MAPK has been implicated with organ development, whereas p38 MAPK is thought to be directly relevant for growth regulation (48). The effect of IGFs on renal p38 MAPK phosphorylation in vivo has, to our knowledge, not been investigated so far. Thus, our study provides the first evidence for a sex-dependent regulatory role of IGFs to modulate p38 MAPK phosphorylation in the kidney and demonstrates a clear association with kidney size. In male mice, the level of renal p38 MAPK activation was significantly (P < 0.01) lower in IgfI intact than IGF-I-deficient animals, suggesting a negative regulatory effect of IGFs on p38 MAPK activation in the kidney. This is in line with the fact that overexpression of IGF-II significantly reduced p38 MAPK phosphorylation in Igf1 mutants and as a tendency further reduced the already low level of phosphorylated p38 MAPK in Igf1 intact mice. Interestingly, we observed a negative correlation between the level of renal p38 MAPK phosphorylation and kidney weight. These findings contrast with findings in other cell types or tissues, such as smooth muscle cells (49) or keloids (50), in which activation of p38 MAPK has been observed in association with cell proliferation or extracellular matrix protein production. However, our data are in line with the observation that specific inhibition of p38 MAPK in rats with remnant kidneys, a model of slowly progressive renal failure, resulted in augmentation of renal hypertrophy due to induction of tubular cell proliferation and inhibition of apoptosis (51). Thus, reduced renal p38 MAPK activity in male mice overexpressing IGF-II is likely to contribute to the markedly increased kidney weight in these groups. Tissue-specific effects of p38 MAPK deserve further investigation and clarification.

In contrast to male mice, no effect of IGF-II overexpression on renal p38 MAPK activation and kidney growth was observed in female mice. For the set of Igf1 intact females investigated, a potential explanation could be the relatively low circulation IGF-II levels and the low level of PEPCK-IGF-II transgene expression in the kidney. However, this was not the case for IGF-I-deficient, PEPCK-IGF-II transgenic females, which exhibited substantial levels of IGF-II in the circulation. These findings argue for a sex-dependent effect of IGF-II to modulate p38 MAPK activity and stimulate kidney growth, at least in IGF-I-deficient mice. The mechanism of down-regulation of p38 MAPK activity by IGF-II in male mice is not clear and deserves further investigation. Sex-dependent differences in the activity of p38 MAPK activity have been described in several other organ systems, including liver (52) and heart (53). Our observation of reduced activation of p42/44 MAPK and PDK1 in male but not female Igf1 knockout mice overexpressing IGF-II might represent a mechanism antagonizing the marked stimulation of kidney growth associated with inhibition of the p38 MAPK.

In summary, our findings support the notion that, despite a high degree of structural homology and a common signaling receptor, IGF-I and -II have distinct functions. Our genetic model demonstrates that substantial circulating levels of IGF-II in the postnatal period cannot rescue body and skeletal growth deficits of IGF-I-deficient mice. However, IGF-II partially restored circulating levels of specific IGFBPs and exhibited a remarkable male-specific stimulatory effect on kidney growth, which appears to involve inhibition of p38 MAPK activity. Thus, our data show that IGF-I and IGF-II can, possibly in concert with other regulatory systems such as sex steroids, initiate specific signaling pathways after activation of the IGF1R. Little attention has been given to this issue by researchers so far, but this exact point may provide important clues for further resolving the IGF-II enigma.


   Acknowledgments
 
We thank Petra Renner, Tanja Mittmann, and Antonia Martin (animal care); Petra Demleitner, Steffen Schiller, and Olga Fettscher (molecular biology) for excellent technical assistance; and Karin Weber (University of Tübingen) for performing the IGF-I and -II RIAs.


   Footnotes
 
Current address for C.M.: Department of Comparative Medicine, GSF-National Research Centre for Environment and Health, Neuherberg, Germany.

Current address for R.G.E.: Institute of Pathophysiology, Department of Natural Sciences, University of Veterinary Medicine, 1210 Vienna, Austria.

This work was supported in part by the Bundesministerium für Bildung und Forschung, Nationales Genomforschungsnetz (BMBF-NGFN, PMM-S31T06) and the Deutsche Forschungsgemeinschaft (DFG, GRK1029).

Disclosure Statement: The authors have nothing to declare.

First Published Online September 28, 2006

1 C.M. and M.R.S. contributed equally to this study. Back

Abbreviations: BMC, Bone mineral content; BMD, bone mineral density; CV, coefficient of variation; GHR, GH receptor; IGFR, IGF receptor; IGFBP, IGF binding protein; LSM, least squares means; NRL, nose-rump length; PDK, phosphoinositide-dependent kinase; PEPCK, phosphoenolpyruvate carboxykinase; pQCT, peripheral quantitative computed tomography.

Received March 24, 2006.

Accepted for publication September 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A 2001 Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 229:141–162[CrossRef][Medline]
  2. Stewart CE, Rotwein P 1996 Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76:1005–1026[Abstract/Free Full Text]
  3. D’Ercole AJ, Applewhite GT, Underwood LE 1980 Evidence that somatomedin is synthesized by multiple tissues in the fetus. Dev Biol 75:315–328[CrossRef][Medline]
  4. Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Tornell J, Isaksson OG, Jansson JO, Ohlsson C 1999 Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 96:7088–7092[Abstract/Free Full Text]
  5. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, LeRoith D 1999 Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:7324–7329[Abstract/Free Full Text]
  6. Mathews LS, Norstedt G, Palmiter RD 1986 Regulation of insulin-like growth factor I gene expression by growth hormone. Proc Natl Acad Sci USA 83:9343–9347[Abstract/Free Full Text]
  7. Rotwein P 1991 Structure, evolution, expression and regulation of insulin-like growth factors I and II. Growth Factors 5:3–18[Medline]
  8. LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143–163[CrossRef][Medline]
  9. Blakesley VA, Scrimgeour A, Esposito D, Le Roith D 1996 Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev 7:153–159[CrossRef][Medline]
  10. Kornfeld S 1992 Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors. Annu Rev Biochem 61:307–330[CrossRef][Medline]
  11. Efstratiadis A 1998 Genetics of mouse growth. Int J Dev Biol 42:955–976[Medline]
  12. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  13. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59–72[Medline]
  14. Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart TA 1993 IGF-I is required for normal embryonic growth in mice. Genes Dev 7:2609–2617[Abstract/Free Full Text]
  15. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A 1996 Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 10:903–918[Abstract]
  16. Bikle D, Majumdar S, Laib A, Powell-Braxton L, Rosen C, Beamer W, Nauman E, Leary C, Halloran B 2001 The skeletal structure of insulin-like growth factor I-deficient mice. J Bone Miner Res 16:2320–2329[CrossRef][Medline]
  17. Woods KA, Camacho-Hubner C, Savage MO, Clark AJ 1996 Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med 335:1363–1367[Free Full Text]
  18. Bonapace G, Concolino D, Formicola S, Strisciuglio P 2003 A novel mutation in a patient with insulin-like growth factor 1 (IGF1) deficiency. J Med Genet 40:913–917[Free Full Text]
  19. Walenkamp MJ, Karperien M, Pereira AM, Hilhorst-Hofstee Y, van Doorn J, Chen JW, Mohan S, Denley A, Forbes B, van Duyvenvoorde HA, van Thiel SW, Sluimers CA, Bax JJ, de Laat JA, Breuning MB, Romijn JA, Wit JM 2005 Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab 90:2855–2864[Abstract/Free Full Text]
  20. DeChiara TM, Efstratiadis A, Robertson EJ 1990 A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345:78–80[CrossRef][Medline]
  21. Mathews LS, Hammer RE, Behringer RR, D’Ercole AJ, Bell GI, Brinster RL, Palmiter RD 1988 Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123:2827–2833[Abstract]
  22. Behringer RR, Lewin TM, Quaife CJ, Palmiter RD, Brinster RL, D’Ercole AJ 1990 Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology 127:1033–1040[Abstract]
  23. Schoenle E, Zapf J, Hauri C, Steiner T, Froesch ER 1985 Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomized rats. Acta Endocrinol (Copenh) 108:167–174[Medline]
  24. Wilson DM, Thomas JA, Hamm Jr TE, Wyche J, Hintz RL, Rosenfeld RG 1987 Transplantation of insulin-like growth factor-II-secreting tumors into nude rodents. Endocrinology 120:1896–1901[Abstract]
  25. Wolf E, Hoeflich A, Lahm H 1998 What is the function of IGF-II in postnatal life? Answers from transgenic mouse models. Growth Horm IGF Res 8:185–193[CrossRef][Medline]
  26. Holly JM 1998 The IGF-II enigma. Growth Horm IGF Res 8:183–184[CrossRef][Medline]
  27. Wolf E, Kramer R, Blum WF, Foll J, Brem G 1994 Consequences of postnatally elevated insulin-like growth factor-II in transgenic mice: endocrine changes and effects on body and organ growth. Endocrinology 135:1877–1886[Abstract]
  28. Lahm H, Gittner K, Krebs O, Sprague L, Deml E, Oesterle D, Hoeflich A, Wanke R, Wolf E 2002 Diethylnitrosamine induces long-lasting re-expression of insulin-like growth factor II during early stages of liver carcinogenesis in mice. Growth Horm IGF Res 12:69–79[CrossRef][Medline]
  29. Hoeflich A, Weber MM, Fisch T, Nedbal S, Fottner C, Elmlinger MW, Wanke R, Wolf E 2002 Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo. FASEB J 16:1721–1731[Abstract/Free Full Text]
  30. Hoeflich A, Nedbal S, Blum WF, Erhard M, Lahm H, Brem G, Kolb HJ, Wanke R, Wolf E 2001 Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2. Endocrinology 142:1889–1898[Abstract/Free Full Text]
  31. Blum WF, Breier BH 1994 Radioimmunoassays for IGFs and IGFBPs. Growth Regul 4(Suppl 1):11–19
  32. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143[CrossRef][Medline]
  33. Erben RG 1997 Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 45:307–313[Abstract/Free Full Text]
  34. Schenk R, Olah A, Herrmann W 1984 Preparation of calcified tissues for light microscopy. In: Dickson G, ed. Methods of calcified tissue preparation. Amsterdam: Elsevier; 1–56
  35. Linder CC, Davisson MT 2004 Strains, stocks and mutant mice. In: Hedrich H, ed. The laboratory mouse. Amsterdam: Elsevier; 25–44
  36. Wolf E, Schneider MR, Zhou R, Fisch TM, Herbach N, Dahlhoff M, Wanke R, Hoeflich A 2005 Functional consequences of IGFBP excess-lessons from transgenic mice. Pediatr Nephrol 20:269–278[CrossRef][Medline]
  37. Mohan S, Richman C, Guo R, Amaar Y, Donahue LR, Wergedal J, Baylink DJ 2003 Insulin-like growth factor regulates peak bone mineral density in mice by both growth hormone-dependent and -independent mechanisms. Endocrinology 144:929–936[Abstract/Free Full Text]
  38. Wang J, Zhou J, Cheng CM, Kopchick JJ, Bondy CA 2004 Evidence supporting dual, IGF-I-independent and IGF-I-dependent, roles for GH in promoting longitudinal bone growth. J Endocrinol 180:247–255[Abstract]
  39. Yakar S, Rosen CJ 2003 From mouse to man: redefining the role of insulin-like growth factor-I in the acquisition of bone mass. Exp Biol Med (Maywood) 228:245–252[Abstract/Free Full Text]
  40. Zhao G, Monier-Faugere MC, Langub MC, Geng Z, Nakayama T, Pike JW, Chernausek SD, Rosen CJ, Donahue LR, Malluche HH, Fagin JA, Clemens TL 2000 Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation. Endocrinology 141:2674–2682[Abstract/Free Full Text]
  41. Yakar S, Rosen CJ, Beamer WG, Ackert-Bicknell CL, Wu Y, Liu JL, Ooi GT, Setser J, Frystyk J, Boisclair YR, LeRoith D 2002 Circulating levels of IGF-1 directly regulate bone growth and density. J Clin Invest 110:771–781[CrossRef][Medline]
  42. Wang J, Zhou J, Powell-Braxton L, Bondy C 1999 Effects of Igf1 gene deletion on postnatal growth patterns. Endocrinology 140:3391–3394[Abstract/Free Full Text]
  43. Deerberg F, Muller-Peddinghaus R 1970 [Comparative studies on the occurrence of spontaneous diseases and spontaneous deaths in conventional and specific germ-free female NMRI-HAN mice]. Z Versuchstierkd 12:341–347[Medline]
  44. Deerberg F, Gleichmann H 1980 The occurrence of spontaneous immune-complex glomerulonephritis in Han:NMRI mice. Contrib Nephrol 19:88–94[Medline]
  45. Brem G, Wanke R, Wolf E, Buchmuller T, Muller M, Brenig B, Hermanns W 1989 Multiple consequences of human growth hormone expression in transgenic mice. Mol Biol Med 6:531–547[Medline]
  46. Wanke R, Wolf E, Brem G, Hermanns W 1996 Physiology and pathology of growth: studies in GH transgenic mice. J Anim Breed Genet 113:445–456
  47. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A 2001 The somatomedin hypothesis: 2001. Endocr Rev 22:53–74[Abstract/Free Full Text]
  48. Hida M, Omori S, Awazu M 2002 ERK and p38 MAP kinase are required for rat renal development. Kidney Int 61:1252–1262[CrossRef][Medline]
  49. Kuemmerle JF, Zhou H 2002 Insulin-like growth factor-binding protein-5 (IGFBP-5) stimulates growth and IGF-I secretion in human intestinal smooth muscle by Ras-dependent activation of p38 MAP kinase and Erk1/2 pathways. J Biol Chem 277:20563–20571[Abstract/Free Full Text]
  50. Daian T, Ohtsuru A, Rogounovitch T, Ishihara H, Hirano A, Akiyama-Uchida Y, Saenko V, Fujii T, Yamashita S 2003 Insulin-like growth factor-I enhances transforming growth factor-ß-induced extracellular matrix protein production through the P38/activating transcription factor-2 signaling pathway in keloid fibroblasts. J Invest Dermatol 120:956–962[CrossRef][Medline]
  51. Ohashi R, Nakagawa T, Watanabe S, Kanellis J, Almirez RG, Schreiner GF, Johnson RJ 2004 Inhibition of p38 mitogen-activated protein kinase augments progression of remnant kidney model by activating the ERK pathway. Am J Pathol 164:477–485[Abstract/Free Full Text]
  52. Vilatoba M, Eckstein C, Bilbao G, Frennete L, Eckhoff DE, Contreras JL 2005 17ß-estradiol differentially activates mitogen-activated protein-kinases and improves survival following reperfusion injury of reduced-size liver in mice. Transplant Proc 37:399–403[CrossRef][Medline]
  53. Wang M, Tsai BM, Reiger KM, Brown JW, Meldrum DR 2006 17ß-Estradiol decreases p38 MAPK-mediated myocardial inflammation and dysfunction following acute ischemia. J Mol Cell Cardiol 40:205–212[CrossRef][Medline]



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