Molecular and Cellular Endocrinology 394 (2014) 88–98

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Genetic dissection of IGF1-dependent and -independent effects of permanent GH excess on postnatal growth and organ pathology of mice A. Blutke a,⇑, M.R. Schneider b, I. Renner-Müller b, N. Herbach a, R. Wanke a,1, E. Wolf b,1 a

Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität, Veterinärstr. 13, 80539 Munich, Germany Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, 81377 Munich, Germany b

a r t i c l e

i n f o

Article history: Received 22 February 2014 Received in revised form 7 June 2014 Accepted 3 July 2014 Available online 10 July 2014 Keywords: Growth GH IGF1 Mouse Organ pathology

a b s t r a c t To study insulin-like growth factor 1 (IGF1)-independent effects of permanent growth hormone (GH) excess on body and organ growth and pathology in vivo, hemizygous bovine GH transgenic mice with homozygous disruption of the Igf1 gene (Igf1 / /GH) were generated, and examined in comparison to Igf1 / , Igf1+/ , wild-type (WT), Igf1+/ /GH, and GH mice. GH mice and Igf1+/ /GH mice showed increased serum IGF1 levels and the well-known giant-phenotype of GH transgenic mice. In contrast, the typical dwarf-phenotype of Igf1 / mice was only slightly ameliorated in Igf1 / /GH mice. Similar to GH mice, Igf1 / /GH mice displayed hepatocellular hypertrophy, glomerulosclerosis, and reduced volumes of acidophilic cells in the pituitary gland. However, GH excess associated skin lesions of male GH mice were not observed in Igf1 / /GH mice. Therefore, development of GH excess induced liver-, kidney-, and pituitary gland-alterations in GH transgenic mice is independent of IGF1 whereas GH stimulated body growth depends on IGF1. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Growth factors of the growth hormone (GH)/insulin-like growth factor (IGF)-system regulate major cellular growth and differentiation processes of pre- and postnatal body and organ growth, and are involved in pathological growth and differentiation processes in different disease conditions, such as growth disorders, chronic renal diseases, and cancer (Cingel-Ristic et al., 2004; Tao et al., 2007; Vasylyeva and Ferry, 2007). The GH/IGF system essentially consists of GH, its releasing and inhibiting hormones, two insulin-like growth factors (IGF1 and IGF2), GH- and IGF-receptors (GHR, IGF1R, IGF2R) and at least six different high-affinity IGFbinding proteins (IGFBP1-6), which transport and stabilize IGFs in the circulation, thereby regulating their availability and biological half-life (Butler and Le Roith, 2001; Le Roith et al., 2001). Regulation of somatic growth through the GH/IGF1 system involves common and dissociated, local and systemic, time-/developmental- and tissue/cell type-specific actions of GH and IGF1. Most of the GH-dependent growth processes are thought to be mediated through (GH induced) locally produced IGF1, acting in a paraand autocrine fashion (Butler and Le Roith, 2001; Le Roith et al., ⇑ Corresponding author. Tel.: +49 (0)89 2180 2590; fax: +49 (0)89 2180 2544. 1

E-mail address: [email protected] (A. Blutke). Equal last author contribution.

http://dx.doi.org/10.1016/j.mce.2014.07.002 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

2001; Stratikopoulos et al., 2008), whereas systemic, predominantly liver-derived IGF1 appears to be not inevitably necessary for normal postnatal somatic growth (Le Roith et al., 2001; Yakar et al., 1999). In addition, direct GH actions, indirect GH effects mediated by other growth factors than IGF1, and autonomous, systemic and local GH-independent actions of IGF1 have been demonstrated (Butler and Le Roith, 2001; Le Roith et al., 2001; Liu and LeRoith, 1999; Wang et al., 2004). Adding further complexity to the system, GH and/or IGF1 actions are modulated by the temporal regulation and spatial distribution of GH- and IGF-receptors, multiple level interactions between IGF1 and IGFBPs, different downstream intracellular signal transduction pathways, and various systemic feedback regulations between GH and IGF1 (Vasylyeva and Ferry, 2007; Butler and Le Roith, 2001; Le Roith et al., 2001). Studies investigating the consequences of overexpression, and/or of deletion of single and/or multiple factors of the GH/IGF system, as GH (von Waldthausen et al., 2008; Wanke et al., 1991; Wanke et al., 1992; Wolf et al., 1993; Wolf and Wanke, 1997), GHR (Bartke et al., 2002; Lupu et al., 2001; Zhou et al., 1997), IGF1 (Baker et al., 1993; Powell-Braxton et al., 1993; Quaife et al., 1989), IGF2 (Baker et al., 1993; DeChiara et al., 1990; Moerth et al., 2007), IGFRs (Baker et al., 1993; Liu et al., 1993), and different IGFBPs (Doublier et al., 2000; Hoeflich et al., 2001, 2002, 1999; Wolf et al., 2005) in transgenic or knockout animal models and in in vitro systems, have significantly contributed to our

A. Blutke et al. / Molecular and Cellular Endocrinology 394 (2014) 88–98

understanding of the actions, and highly complicated interactions, of the different components of the GH/IGF-system under physiological and disease conditions (Cingel-Ristic et al., 2004; Butler and Le Roith, 2001; Le Roith et al., 2001). Demonstrating that IGF1 is one of the most important mediators of pre- and postnatal growth processes, Igf1 knockout mice exhibit significantly reduced birth weights, and high perinatal mortalities of up to 95% (Liu and LeRoith, 1999; Powell-Braxton et al., 1993). Despite of elevated endogenous GH concentrations, homo- and heterozygous Igf1 mutant mice show drastically reduced postnatal growth rates, and reach only 30% (Igf1 / ) or, 80% (Igf1+/ ) of the body weight of wild-type controls at two months of age (Le Roith et al., 2001; Powell-Braxton et al., 1993; He et al., 2006). Whereas fetal growth appears to be largely independent of GH (Liu and LeRoith, 1999; Zhou et al., 1997; Baker et al., 1993; Powell-Braxton et al., 1993), postnatal somatic growth depends on IGF1 mediated growth stimulation through GH. Accordingly, even continuous administration of GH fails to stimulate body growth in Igf1 knockout mice (Liu and LeRoith, 1999). The effects of permanently elevated systemic concentrations of (heterologous) GH on body and organ growth, and development of GH excess associated pathological alterations have been investigated extensively in various GH transgenic mouse models (Le Roith et al., 2001; von Waldthausen et al., 2008; Wanke et al., 1991, 1992; Wolf et al., 1993; Quaife et al., 1989; Doi et al., 1990, 1988; Miquet et al., 2008). In GH transgenic mice, GH transgene overexpression induces a significant, 2-fold elevation of (systemic) IGF1 levels, as well as accelerated and absolutely enhanced body growth from the third week of age on (Le Roith et al., 2001; Wanke et al., 1991, 1992), leading to an almost doubled body weight at 15 weeks of age (Le Roith et al., 2001; Hoeflich et al., 2001). Moreover, the growth of several organs is disproportionately increased in GH transgenic mice, and these animals reproducibly develop a series of typical, partially age-related and gender-specific morphological alterations of i.a. the pituitary gland (decreased numbers and volumes of somatotroph cells (Stefaneanu et al., 1993), the liver (sequential development of hypertrophic, pre-neoplastic, and cancerous hepatocellular alterations (Wanke et al., 1991, 1992; Miquet et al., 2008), the kidneys (progressive glomerulosclerosis (Wanke et al., 1991, 2001; Wolf et al., 1993; Doi et al., 1990), and the skin (increased skin area and thickness in male mice (Wanke et al., 1999). However, it is still not completely clarified, which of the disease mechanisms are mediated by secondarily elevated IGF1 levels, and which occur independently of IGF1. To dissociate IGF1-dependent, and IGF1independent effects of systemically elevated GH levels on body/ organ growth, and on morphological organ alterations, the present study examined hemizygous (bovine) growth hormone (GH) transgenic mice with homozygous disruption of the Igf1 gene in comparison to homo- and heterozygous Igf1 knockout mice, wild-type (WT) control mice, GH transgenic mice, and GH transgenic mice with heterozygous knockout of the Igf1 gene. 2. Materials and methods 2.1. Ethics statement All experiments were approved by the author’s institutional committee on animal care, were carried out in accordance with the German Animal Welfare Act, and conformed to international guidelines on the ethical use of animals. All efforts were made to minimize the number of animals used and their suffering. 2.2. Mice The Igf1 mutant mouse line (Powell-Braxton et al., 1993) used in this study was kindly provided by Dr. Lyn Powell-Braxton

89

(Genentech Inc., South San Francisco, USA), and bred in our facility for more than 10 generations into an NMRI outbred stock (Charles River, Germany). Genotyping of Igf1 mutant mice was performed by PCR, as previously described (Powell-Braxton et al., 1993). Transgenic mice expressing bovine growth hormone gene (GH) under the transcriptional control of the rat PEPCK-promoter were maintained on NMRI background and genotyped as described earlier (Hoeflich et al., 2001). Due to the infertility of homozygous Igf1 knockout mice, two mating steps were performed to generate homozygous Igf1 mutant GH transgenic mice. First, female Igf1+/ mice were mated with male hemizygous GH transgenic mice (GH). In a second step, male Igf1+/ /GH offspring were mated with female Igf1+/ mice to obtain the following six genotypes: Igf1 / , Igf1 / /GH, Igf1+/ , Igf1+/ /GH, WT, and GH mice. Animals were maintained under specified pathogen-free conditions in a closed barrier system and had free access to a standard rodent maintenance diet containing 19.3% raw protein (XP), 3.4% raw fat (XL), 5.0% raw fiber, 13.0 MJ/kg ME and 14.8 g/MJ XP/ME (V1536, Ssniff, Soest, Germany) and tap water. 2.3. Perinatal mortality To investigate the perinatal mortality of offspring descending from mating of male Igf1+/ /GH and female Igf+/ mice, the genotypes of 67 stillborn/perinatally deceased pups of 31 l with 310 pups were determined by PCR (Powell-Braxton et al., 1993; Hoeflich et al., 2001). 2.4. Body and organ growth Body weight of selected litters was recorded twice weekly, starting at day 21 post partum to 11 weeks of age. One cohort of mice was sacrificed at 16 weeks of age (n = male/female): Igf1 / (7/4); Igf1 / /GH (5/4); WT (5/5); GH (6/5); Igf1+/ (5/5); Igf1+/ /GH (7/4). After determination of body weight, mice were killed by cervico-cranial dislocation. Subsequently, blood samples were obtained by cardiac puncture and serum was separated by centrifugation. Organs and fat deposits were dissected, blotted dry on tissue paper, and weighed to the nearest milligram. After removal of internal organs and skin, the carcass (without head and tail) was weighed to the nearest 0.1 g. The skin was weighed after removal of subcutaneous fat, carefully spread on a plastic transparency film (hair on top) and traced with a waterproof pen. The skin area was planimetrically determined (Wanke et al., 1999), using a semi-automated image analysis system (Videoplan, Zeiss-Kontron, Eching, Germany). For examination of kidney and pituitary alterations, another cohort of mice was sacrificed at 11 weeks of age (n = male/female): Igf1 / (4/3); Igf1 / / GH (3/4); WT (4/6); GH (4/4); Igf1+/ (5/7); Igf1+/ /GH (5/6). These mice were killed by cervico-cranial dislocation and immediately perfused with neutrally buffered 2.5% glutaraldehyde solution through the heart, as previously described (Wanke et al., 2001; Herbach et al., 2009). After immersion fixation in 2.5% glutaraldehyde solution (>48 h), pituitary and kidneys were removed, blotted dry, and weighed. Pituitary gland volumes were calculated from their specific weight, determined by the principle of Archimedes (Scherle, 1970). 2.5. Serum IGF1 levels IGF1 levels were determined in serum samples of 16-week-old mice, using the mouse IGF1 DuoSet ELISA Development kit (Cat. DY791, R&D Systems Europe, Ltd., UK), according to the manufacturer’s instructions.

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2.6. Immunoblot analysis

2.8. Statistical analyses and data presentation

Detection of GH in randomly taken serum samples of 16-weekold Igf1 / mice (4 male and 4 female mice), Igf1 / /GH mice (3 male and 3 female mice) and WT mice (3 male and 3 female mice) was performed by Western blot analyses. Each 2 ll serum and a 1 ng marker-sample of recombinant murine GH (LOT# AFP10783B) were subjected to immunoblot analysis after denaturation under reducing conditions. Proteins were separated on 12% SDS–PAGE gels and transferred to Immobilon-P membranes (Millipore, Eschborn, Germany). The membranes were stained using Ponceau red according to standard procedures, to verify equal loading and proper protein transfer. Membranes were blocked (5% dry milk and 1% Tween 20 in Tris-buffered saline) and incubated with a polyclonal rabbit anti rat GH-antibody (LOT# AFP5672099Rb, dilution 1:20,000) overnight at 4 °C. The murine GH standard and the rabbit anti rat GH-antibody were kindly provided by A. F. Parlow, National Hormone & Peptide Program, Torrance, California, USA. After three washings in Tris-buffered saline containing 1% Tween 20, the membranes were incubated with horseradish peroxidasecoupled goat-anti-rabbit IgG (1:2000, Cell Signaling No. 7074; New England Biolabs) for 1 h at room temperature. Bound antibodies were visualized using ECL reagent (RPN2106; GE Healthcare Amersham Biosciences, Freiburg, Germany). The detection limit of this assay was 0.5 ng GH, corresponding to 250 ng GH/ml serum.

All data are presented as means ± SD. Data were analyzed by one-way ANOVA, taking the effects of genetic group and sex into account. Differences were tested for statistical significance, using LSD post hoc tests (IBM SPSS Statistics, Vers. 18). P-values 6 0.05 were considered significant. For description of IGF1-dependent and -independent GH excess effects, the tables, graphs and figures presented in the main paper show the data of Igf1 / vs. Igf1 / /GH mice and WT vs. GH mice. The data of Igf1+/ mice and of Igf1+/ /GH mice are presented in the Supplemental online material.

2.7. Histology and morphometric analyses Histological examination of the liver, kidneys, and skin was performed on hematoxylin and eosin (H&E)- and Giemsa-stained sections of glycol methacrylate/methyl methacrylate (GMA/MMA) embedded tissue samples (Hermanns et al., 1981). A semi-quantitative glomerulosclerosis index was assessed in a blinded fashion, i.e. without knowledge of the genotype, using silver-methenamine stained GMA/MMA sections, as previously described (Herbach et al., 2009; el Nahas et al., 1991). Per case, at least 100 glomerular cross section profiles, sampled according to the unbiased counting rule (Gundersen and Jensen, 1987) were evaluated. For morphometric analysis of cutaneous alterations, two paired, orthogonally aligned lumbar skin samples per case were taken as described previously (Wanke et al., 1999), fixed in 4% buffered formaldehyde (>24 h) and vertically embedded in GMA/MMA. The thicknesses of skin, dermis and epidermis were determined in HE-stained GMA/MMA sections, using an automated stereology system (VIS-Visiopharm Integrator SystemÒ Version 3.4.1.0 with newCASTÒ software, Visiopharm A/S, Hørsholm, Denmark) at 100 (skin/dermis), respectively at 400 magnification (epidermis), as the mean of ten equidistant interfollicular measurements (skin/dermal thickness: 200 lm distance; epidermis: 20 lm distance) per section (19 ± 3 measurements per case). Pituitary glands were completely embedded in paraffin, and entirely sectioned at 3 lm thickness. The volume fraction of the pars distalis in the pituitary gland (VV(p.dist./hypophysis)) was calculated from the cumulative section areas of the pars distalis and the entire pituitary gland, planimetrically determined at 100 magnification in every 20th HE-stained section (23 ± 8 sections per case). The volume fractions of acidophilic cells (summarizing somatotroph and lactotroph cells) in the pars distalis (VV(acid./p.dist.)) were calculated from their section profile areas, determined by point-counting (598 ± 7 points per case) (Howard and Reed, 2004) in each six randomly sampled test fields (area: 10,000 lm2) of the pars distalis. The total volumes of the pars distalis (V(p.dist.)) and of the acidophilic cells in the pars distalis (V(acid.)) were calculated from their respective volume fractions and the total pituitary gland volume.

3. Results 3.1. Perinatal mortality The genotype frequencies in offspring descending from mating of male Igf1+/ /GH and female Igf1+/ mice (Igf1 / and Igf1 / /GH, Igf1+/ and Igf1+/ /GH, WT and GH mice) followed the expected Mendelian distribution, with a gender ratio of 1:1.1 (male:female). 85% of all homozygous Igf1 knockout mice were born dead, or died within 12 h after birth, whereas the perinatal mortality of mice carrying at least one intact Igf1 allele was 2.5%. 3.2. Serum IGF1 levels IGF1 was not detectable in serum samples from Igf1 / and Igf1 / /GH mice. Corresponding to the expression of the GH transgene, the serum IGF1 levels in 16-week-old GH and Igf1+/ /GH mice were significantly higher, as compared to WT, or Igf1+/ mice, respectively, with GH mice exhibiting the highest IGF1 levels of all investigated genotypes (Fig. 1B, Supplemental Fig. 1). 3.3. Immunoblot analysis In Western blot analyses, intense bands at 22 kDa (referring to GH) were detected in all serum samples of Igf1 / /GH mice, whereas the GH levels in serum samples taken from WT mice were consistently beyond the detection limit of 0.5 ng GH. In serum specimen of Igf1 / mice, distinct GH bands were occasionally detectable, displaying a considerably weaker intensity as compared to those of Igf1 / /GH mice (as exemplarily shown in Supplemental Fig. 7). 3.4. Body and organ growth During the entire observation period (week 3–11), GH mice exhibited the highest body weights of all investigated genotypes. Igf1 / and Igf1 / /GH mice consistently exhibited significantly lower body weights, as compared to mice of all other genotypes (Fig. 1A, C and D). At eleven weeks of age, the body weights of Igf1 / mice reached 42% (male mice) and 35% (female mice) of the body weights of sex-matched WT controls, whereas Igf1 / /GH mice reached 53% (male mice) and 63% (female mice) of WT control body weights. Compared to sex-matched Igf1 / mice, Igf1 / /GH mice displayed significantly higher body weights from week five (female mice) or week seven (male mice) onwards (Fig. 1C and D). The body weight development data of Igf1+/ and Igf1+/ /GH mice are shown in Supplemental Fig. 2. At 16 weeks of age, GH mice displayed significantly higher body weights and absolute weights of the carcass, liver, kidneys, heart, lungs, pancreas, spleen, and skin, than WT mice (Tables 1 and 3). The same effects of GH transgene expression on body and organ weights were observed in Igf1+/ /GH vs. Igf1+/ mice (Supplemental Tables 1 and 3). In Igf1 knockout mice, GH transgene expression

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Fig. 1. A. Body size comparison of female Igf1 / , Igf1 / /GH, wild-type (WT), and GH mice at 16 weeks of age. Bar = 1 cm. B. Serum IGF1 concentrations at 16 weeks of age. IGF1 levels of Igf1 / and Igf1 / /GH mice were below the detection limit of the assay (nd). C, D. Development of body weight (week 3–11) of male (C) and female (D) Igf1 / , Igf1 / /GH, WT, and GH mice. B–D. Numbers of investigated animals are given in brackets. Statistically significant differences (p 6 0.05) between groups are indicated by different superscripts.

Table 1 Body weight, body length, absolute and relative carcass and organ weights at 16 weeks of age. Genotype, n [m/f] Body weight [g] Nose-rump length [cm] Carcass [g] % of body weight Liver [g] % of body weight Kidneya [mg] % of body weight Heart [mg] % of body weight Lung [mg] % of body weight Pancreas [mg] % of body weight Spleen [mg] % of body weight Fatb [g] % of body weight a b c d e

Igf1

/

[7/4] c,e

Igf1 c,d,e

18.5 ± 1.8 /13.4 ± 2.5 8.2 ± 0.4c,d,e/7.6 ± 0.4c,d,e 6.0 ± 0.7e/4.4 ± 0.4e 32.3 ± 3.6d,e/34.0 ± 6.6e 1.3 ± 0.2d,e/1.3 ± 0.3 7.0 ± 1.4c,d,e/9.8 ± 1.7c,e 261 ± 56e/166 ± 34d,e 1.42 ± 0.30/1.24 ± 0.08 142 ± 17d,e/127 ± 40 0.77 ± 0.11c,d,e/0.96 ± 0.34c,e 99 ± 11e/94 ± 5e 0.54 ± 0.09c/0.72 ± 0.11c,d 137 ± 23e/123 ± 24e 0.75 ± 0.15/0.92 ± 0.13e 76 ± 1d,e/77 ± 12 0.41 ± 0.06c,d/0.59 ± 0.01c,e 1.0 ± 0.3d/1.2 ± 0.2d 5.7 ± 2.1c,d/8.6 ± 0.7c,e

/

/GH [5/4]

22.8 ± 2.1/20.6 ± 3.6d 8.7 ± 0.4d/9.0 ± 0.7d 6.3 ± 0.6/6.0 ± 0.6 27.8 ± 1.0d/28.6 ± 3.9 1.9 ± 0.4d/1.8 ± 0.3 8.3 ± 1.6d/8.7 ± 0.2 280 ± 31/238 ± 52 1.23 ± 0.06/1.15 ± 0.07 222 ± 34d/187 ± 21 0.99 ± 0.24d/0.92 ± 0.13 132 ± 13/118 ± 33 0.59 ± 0.08/0.57 ± 0.07d 196 ± 36/209 ± 65 0.86 ± 0.11/1.00 ± 0.16 126 ± 37/127 ± 43 0.55 ± 0.15d/0.61 ± 0.16 2.8 ± 0.7d/2.2 ± 0.5d 12.1 ± 2.7d/10.8 ± 3.6

WT [5/5]

GH [6/5]

41.2 ± 4.6c,d,e/35.0 ± 2.9c,d,e 11.0 ± 0.5d,e/11.3 ± 0.3d,e 17.7 ± 2.1d,e/16.0 ± 1.4d,e 41.6 ± 3.4c,d,e/45.9 ± 4.2c,e,d 2.1 ± 0.3c,d,e/1.6 ± 0.2c,e 5.0 ± 0.6d,e/4.4 ± 0.2d,e 589 ± 64d,e/409 ± 45d,e 1.43 ± 0.15/1.17 ± 0.07 235 ± 36c,d,e/171 ± 35c,d 0.58 ± 0.12e/0.49 ± 0.11e 216 ± 26d,e/222 ± 26d,e 0.53 ± 0.08 c/0.64 ± 0.06 c 366 ± 73c,d,e/240 ± 76c,d,e 0.89 ± 0.15d/0.69 ± 0.22d,e 131 ± 31d,e/129 ± 16d 0.31 ± 0.04d/0.37 ± 0.05d,e 1.8 ± 0.5/2.0 ± 0.2d 4.2 ± 0.8/5.8 ± 0.5e

72.7 ± 6.5c,d/63.4 ± 2.6c,d 14.2 ± 0.4d/14.2 ± 0.3d 24.6 ± 2.1 d/24.6 ± 2.4d 33.8 ± 1.9c,d/38.9 ± 3.2c,d 4.8 ± 0.4d/4.3 ± 0.2d 6.6 ± 0.4d/6.9 ± 0.4d 1101 ± 333c,d/747 ± 74c,d 1.52 ± 0.46c/1.18 ± 0.10c 406 ± 70d/373 ± 60d 0.56 ± 0.11/0.59 ± 0.11 393 ± 65d/380 ± 40d 0.54 ± 0.09/0.60 ± 0.08 884 ± 179 c,d/604 ± 95c,d 1.23 ± 0.28c,d/0.95 ± 0.13c,d 374 ± 57d/424 ± 70d 0.52 ± 0.07c,d/0.67 ± 0.10c,d 2.3 ± 0.5c/3.4 ± 2.5c,d 3.2 ± 0.7/5.3 ± 3.9

Cumulative weight of both kidneys. Abdominal and subcutaneous fat. Indicated significant (p 6 0.05) differences: Male vs. female mice of identical genotype. Igf1 / vs. Igf1 / /GH mice and WT vs. GH mice (per gender). Igf1 / vs. WT mice (per gender).

caused a significant increase of the absolute and relative liver and heart weights of male Igf1 / /GH vs. Igf1 / mice, and of the

absolute fat weights (abdominal and subcutaneous fat tissue) in both genders.

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Table 2 Morphometric and quantitative stereological data of the pituitary gland at 11 weeks of age. Genotype, n [m/f] Body weight (BW) [g] Brain weight [mg] % of BW Pituitary weight [mg] ‰ of BW Pituitary volume [mm3] Pars distalis VV(p.dist./hypophysis) V(p.dist) [mm3] Acidophilic cells Vv(acid./p.dist.) V(acid.) [mm3]

Igf1

/

[4/3]

Igf1

b,c

b,c

/

/GH [3/4]

WT [4/6]

b

b,c

GH [4/4] a,b,c

14.4 ± 1.3 /11.8 ± 0.5 292 ± 46c/308 ± 11c 2.02 ± 0.19a,b,c/2.61 ± 0.05a,b,c 1.35 ± 0.21c/1.23 ± 0.06c 0.09 ± 0.01b,c/0.10 ± 0.00b,c 1.43 ± 0.22c/1.31 ± 0.06c

21.5 ± 0.9 /18.4 ± 2.1 297 ± 51/329 ± 27 1.39 ± 0.29a,b/1.79 ± 0.21 1.23 ± 0.10/1.10 ± 0.00 0.06 ± 0.01b/0.06 ± 0.01b 1.31 ± 0.12/1.17 ± 0.00

0.70 ± 0.06 a,b,c/0.63 ± 0.04 a,b,c 1.01 ± 0.23b,c/0.82 ± 0.09b,c 0.58 ± 0.04b,c/0.58 ± 0.02b,c 0.58 ± 0.10b,c/0.47 ± 0.07b,c

0.43 ± 0.02b/0.45 ± 0.03b 0.56 ± 0.03b/0.52 ± 0.03b 0.31 ± 0.02a,b/0.40 ± 0.02a,b 0.17 ± 0.07b/0.21 ± 0.02b

a,b

a,b,c

39.6 ± 3.7 /33.5 ± 4.7 485 ± 20c/501 ± 20c a,b,c 1.23 ± 0.1 /1.51 ± 0.18 a,b,c 2.68 ± 0.22 a,b,c/4.03 ± 0.29 a,b,c 0.07 ± 0.01a,b,c/0.12 ± 0.02a,b,c 2.81 ± 0.20 a,b,c/4.28 ± 0.31 a,b,c

69.3 ± 6.8a,b/51.1 ± 7.2a,b 493 ± 56/520 ± 42 0.71 ± 0.09a,b/1.02 ± 0.07a,b 1.98 ± 0.30 a,b/3.58 ± 0.26 a,b 0.03 ± 0.01 a,b/0.07 ± 0.01a,b 2.09 ± 0.32 a,b/3.79 ± 0.28 a,b

0.77 ± 0.03b,c/0.79 ± 0.02b,c 2.17 ± 0.11a,b,c/3.37 ± 0.26a,b,c 0.63 ± 0.02a,b,c/0.49 ± 0.05a,b,c 1.36 ± 0.07a,b,c/1.64 ± 0.17a,b,c

0.49 ± 0.04 a,b/0.72 ± 0.04 a,b 1.02 ± 0.20a,b/2.71 ± 0.13a,b 0.39 ± 0.02b/0.43 ± 0.02b 0.40 ± 0.08a,b/1.17 ± 0.04a,b

Indicated significant (p 6 0.05) differences: a Male vs. female mice of identical genotype. b Igf1 / vs. Igf1 / /GH and WT vs. GH mice (per gender). c Igf1 / vs. WT mice (per gender).

Table 3 Morphometric data of the skin at 16 weeks of age. Genotype, n [m/f] Skin weight [g] % of body weight (BW) Skin area [cm2] Skin area/metabolic BWa Skin thickness [lm] Epidermis thickness [lm] Dermis thickness [lm]

Igf1

/

[7/4] d

Igf1 d

3.2 ± 0.3 /1.8 ± 0.4 17.5 ± 1.8b/13.3 ± 2.3b 82 ± 6b,c,d/65 ± 13b,c 11.8 ± 0.8/11.7 ± 1.3d 466 ± 45b/203 ± 26 b,d 8.4 ± 0.4/8.0 ± 0.4 294 ± 29b/103 ± 10b

/

/GH [5/4]

4.6 ± 1.0/3.2 ± 0.4 20.3 ± 4.9b/15.9 ± 1.5b 100 ± 17b,c/84 ± 7b,c 12.5 ± 1.8b/11.2 ± 0.6b 502 ± 65 b/235 ± 30b 8.0 ± 0.5/8.5 ± 0.9 308 ± 30b/93 ± 7b

WT [5/5] c,d

GH [6/5] c,d

6.7 ± 1.0 /5.0 ± 0.6 16.2 ± 1.6c/14.2 ± 0.9 138 ± 9b,c,d/110 ± 6b,c,d 11.5 ± 0.3b/10.3 ± 0.6b,d 548 ± 102b,c/426 ± 127b,d 8.3 ± 0.2c/8.7 ± 0.4 317 ± 49b,c/138 ± 29b

16.1 ± 3.7b,c/8.7 ± 1.9b,c 22.0 ± 3.8b,c/13.7 ± 2.7b 210 ± 23b,c/177 ± 10b,c 12.0 ± 0.7/11.1 ± 0.9 789 ± 195b,c/373 ± 54b 9.4 ± 1.7c/9.2 ± 1.4 702 ± 156b,c/137 ± 16b

Indicated significant (p 6 0.05) differences: a Metabolic body weight = body weight2/3. b Male vs. female mice of identical genotype. c Igf1 / vs. Igf1 / /GH, Igf1+/ vs. Igf1+/ /GH, and WT vs. GH (per gender). d Igf1 / vs. WT mice (per gender).

3.5. Morphological findings 3.5.1. Brain weight and pituitary alterations In 11-week-old mice, the relative brain weights (% of body weight) were significantly decreased in GH transgenic vs. nontransgenic mice (Igf1 / /GH vs. Igf1 / and GH vs. WT), as well as in WT vs. Igf1 / mice (Table 2). Eleven-week-old GH mice displayed significantly lower absolute and relative pituitary weights and volumes, as compared to WT mice, whereas Igf1 / /GH mice displayed only significantly lower relative pituitary weights (‰ of body weight) as compared to Igf1 / mice. Histologically, the acidophilic cells in the pars distalis of GH transgenic mice consistently displayed increased nucleocytoplasmic ratios, as compared to non GH transgenic animals (Fig. 2). The relative weights of the pituitary gland, the volume fractions of the pars distalis in the pituitary gland (VV(p.dist./hypophysis)), the total volumes of the pars distalis (V(p.dist.)), the volume fractions of acidophilic cells in the pars distalis (VV(acid./p.dist.)), and the total volumes of the acidophilic cells in the pars distalis (V(acid.)), were significantly decreased in Igf1 / /GH vs. Igf1 / mice, as well as in GH vs. WT mice (Table 2, Fig. 2). The pituitary gland data of Igf1+/ and Igf1+/ /GH mice are shown Supplemental Table 2 and Fig. 3.

3.5.2. Liver alterations Grossly, the livers of mice of all investigated GH transgenic genotypes (Igf1 / /GH, Igf1+/ /GH, and GH mice) occasionally displayed indistinctly delimited, liver-colored, nodular changes of up to 8 mm in diameter within the liver parenchyma. Whereas Igf1 / , Igf1+/ , and WT mice exhibited a regular hepatic histology,

Igf1 / /GH, Igf1+/ /GH, and GH mice consistently exhibited histopathological liver alterations, including centri- to panlobular hepatocellular and nuclear hypertrophy and pleomorphy, frequent nuclear pseudo-inclusions, and multifocal single-cell necroses with granulocytic resorptive inflammatory reaction (Fig. 3, Supplemental Fig. 4). Corresponding to the macroscopic findings, occasionally also hepatocellular adenomas with peripheral compression of adjacent liver parenchyma were observed in GH transgenic mice (Fig. 3). 3.5.3. Kidney alterations Histopathological kidney alterations were present in mice of all investigated GH transgenic genotypes, but not in Igf1 / , Igf1+/ , and WT mice. Renal changes comprised of glomerulosclerosis, interstitial fibrosis, nephron atrophy and presence of tubular protein casts, indicative for proteinuria. Accordingly, Igf1 / /GH, Igf1+/ /GH, and GH mice exhibited significantly higher glomerulosclerosis indices than Igf1 / , Igf1+/ , and WT mice, respectively (Fig. 4, Supplemental Fig. 5). 3.5.4. Skin alterations The absolute skin weights were significantly increased in male and female GH vs. WT mice, but not in Igf1 / /GH vs. Igf1 / mice. In male mice, the relative skin weights (referenced to body weight) were significantly increased in GH vs. WT mice, but not in Igf1 / / GH vs. Igf1 / mice. The absolute skin areas were significantly increased in GH vs. WT and in Igf1 / /GH vs. Igf1 / mice. Additionally, male Igf1 / , Igf1 / /GH, WT, and GH mice consistently displayed significantly higher absolute skin areas, as compared to (genotype matched)

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Fig. 2. Left: Histology of the pars distalis of the adenohypophysis of male mice of the indicated groups at 11 weeks of age. The cross-section profile areas of acidophilic (red) cells (arrows) in the pars distalis of Igf1 / /GH and GH mice are reduced, as compared to Igf1 / and WT mice. Paraffin sections, HE staining. Bars = 10 lm. Right: The absolute volumes of acidophilic cells in the pars distalis are significantly decreased in Igf1 / /GH vs. Igf1 / mice and in GH vs. WT mice. Numbers of investigated animals are given in brackets. Statistically significant differences (p 6 0.05) between groups of the same sex are indicated by different superscripts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Liver histology of Igf1 / mice, Igf1 / /GH mice, wild-type (WT) mice and GH mice at 16 weeks of age. GH mice and Igf1 / /GH mice exhibit centri- to panlobular hepatocellular and nuclear hypertrophy and pleomorphy, nuclear pseudo-inclusions (arrow), and multifocal single-cell necroses (asterisks) with resorptive inflammatory reaction. Hepatocellular adenoma (#) with peripheral compression of adjacent liver parenchyma (à). Paraffin sections, HE staining. Bars = 50 lm.

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Fig. 4. Left: Kidney histology of 11-week-old female mice of the different genotypes. Igf1 / /GH and GH mice display glomerulosclerosis (arrows), interstitial fibrosis, and tubular protein casts (arrowheads). GMA/MMA sections, HE staining. Bars = 100 lm. Right: Igf1 / /GH and GH mice exhibit significantly increased glomerulosclerosis indices, as compared to Igf1 / and WT mice. Numbers of investigated animals are given in brackets. Statistically significant differences (p 6 0.05) between groups within sex are indicated by different superscripts.

female mice. The relative skin areas (referenced to the metabolic body weight), however, were neither significantly elevated in GH vs. WT mice, nor in Igf1 / /GH vs. Igf1 / mice (Table 3). Histologically, a severe thickening of the dermis accompanied by augmentation of dermal collagen fibers and replacement of subcutaneous fat tissue by collagen was observed in male GH mice, but not in female GH mice, or in male and female Igf1 / , Igf1 / /GH, and WT mice (Fig. 5). Igf1 / and Igf1 / /GH mice of both genders exhibited increased subcutaneous fat tissue (Fig. 5). In all investigated genotypes, the thicknesses of the skin and the dermis were significantly higher in male mice than in female mice of the same genotype. In male GH mice, the thicknesses of the skin and dermis were significantly increased as compared to male WT mice. However, GH transgene expression did not cause an increase skin or dermal thickness in male Igf1 / /GH vs. Igf1 / mice (Table 3, Fig. 5). Gross- and histopathological examination revealed no evidence for other advanced skin lesions, as epidermal proliferation, subcutaneous edema, inflammatory or ulcerative alterations in any of the examined genotypes. The skin data of Igf1+/ and Igf1+/ /GH mice are shown in Supplemental Table 3 and Fig. 6.

4. Discussion 4.1. General considerations The present study used a panel of GH transgenic  Igf1 mutant mice to genetically dissect IGF1-dependent and -independent effects of permanent GH excess on postnatal growth and organ pathology in mice. The Igf1 mutant and GH transgenic mouse lines used in this study represent well characterized model systems, frequently used in GH/IGF research (Wolf et al., 1993; Powell-Braxton et al., 1993; Moerth et al., 2007; Hoeflich et al., 2001, 2002; Wanke et al., 1999; McGrane et al., 1988). In PEPCK-bGH transgenic mice, the bGH transgene is primarily expressed in the kidney and the liver, with endocrine and metabolic factors capable to modulate its transcription (McGrane et al., 1988). However, the general effects of GH excess on stimulation of body and organ growth and on development of a typical spectrum of organ lesions in GH transgenic mice uniformly occur in different transgenic mouse lines with different genetic backgrounds expressing different GH transgenes (human or bovine GH) under transcriptional control

Fig. 5. Left: Skin histology of 16-week-old male Igf1 / , Igf1 / /GH, WT and GH mice. The skin of male GH mice is severely thickened, with augmentation of dermal collagen fibers and replacement of subcutaneous fat tissue (asterisk) by subdermal collagen. Male Igf1 / and Igf1 / /GH mice display increased subcutaneous fat (asterisks), but unaltered dermal thickness (dashed lines), as compared to WT mice. GMA/MMA sections, HE staining. Bars = 500 lm. Right: GH transgene expression leads to significant thickening of the dermis only in male GH mice, but not in Igf1 / /GH mice. Numbers of investigated animals are given in brackets. Statistically significant differences (p 6 0.05) between groups within sex are indicated by different superscripts.

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of different promoters (PEPCK-, metallothionein-, or chicken betaactin-promoters) with variably elevated circulating GH levels (von Waldthausen et al., 2008; Wolf et al., 1993), as well as in transgenic mice overexpressing GH-releasing hormone (Doi et al., 1988), and in rats bearing tumor-secreting GH (Kawaguchi et al., 1991). Therefore, the phenotypic GH excess associated effects observed in Igf1 / /GH mice can be considered as a consequence of permanently elevated GH levels. Due to the low perinatal survival rates of Igf1 / and Igf1 / /GH mice (see Section 4.2) a relatively limited number (n = 34) of IGF1 deficient animals (range: 3–7 per genotype, sex and age-group) was examined in the present study. Notwithstanding this, the investigated group sizes were sufficient to reveal significant robust effects of long-term GH excess on growth and pathology in Igf1 knockout mice. 4.2. Perinatal mortality Depending on the respective genetic background and the targeted region in the Igf1 gene, investigations of Igf1 knockout mice are generally hindered by their high perinatal mortality rates (Liu and LeRoith, 1999; Lupu et al., 2001; Liu et al., 1993). Confirming the fundamental role of IGF1 for normal fetal development, 85% of all homozygous Igf1 knockout mice (Igf1 / and Igf1 / /GH mice) died perinatally, which is within the range of perinatal mortality rates reported by other investigators of IGF1 deficient mice (Liu and LeRoith, 1999; Powell-Braxton et al., 1993; Liu et al., 1993). 4.3. Serum IGF1 and GH abundance The results of the GH and IGF1 analyses of random serum samples indicate that, as in GH mice (Wanke et al., 1992; Wolf et al., 1993; Srivastava et al., 2002), expression of the bovine GH transgene also led to permanent elevation of heterologous GH serum levels in Igf1 / /GH mice, as compared to WT mice, or Igf1 / mice. However, in contrast to GH mice which exhibit significantly elevated circulating IGF1 levels (Le Roith et al., 2001; Wanke et al., 1992; Srivastava et al., 2002; Palmiter et al., 1983), IGF1 was undetectable in serum of Igf1 / /GH mice. Reflecting the effect of Igf1 haploinsufficiency on elevation of systemic (predominantly liver derived) IGF1 levels by GH overabundance (Le Roith et al., 2001), the serum IGF1 levels in Igf1+/ /GH mice were significantly lower than in GH mice, but still significantly higher than in Igf1+/ mice (Supplemental Fig. 1). By immunoblot analysis, elevated serum levels of endogenous GH were detectable in some, but not all samples of Igf1 / mice (as compared to WT mice). Elevation of serum GH levels has also previously been reported in mice with liverspecific or ubiquitous inactivation of the Igf1 gene (Yakar et al., 1999; Liu and LeRoith, 1999; Sjogren et al., 1999), likely indicating a partial or complete lack of feedback inhibition of GH secretion by IGF1. 4.4. Body growth The growth curve analyses generally confirmed the essential role of GH stimulated IGF1 in postnatal growth. In GH vs. WT mice, GH excess exhibited a significant body weight promoting effect already before the third week of age, indicating a significant role of GH for body growth already in the pre-weaning period. At 11 weeks of age, GH mice displayed approximately 1.9-fold increased body weights, as compared to WT mice. In heterozygously Igf1 deleted mice, the growth stimulatory effect of GH transgene expression was comparably smaller, yet accounting for an approximately 1.6-fold body weight increase in Igf1+/ /GH vs. Igf1+/ mice. Analogously, the body weight differences of 16-week-old GH and Igf1+/ /GH mice accorded well to their serum IGF1 levels. Corresponding to a primarily IGF1-dependent body growth, both

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Igf1 / and Igf1 / /GH mice consistently displayed significantly lower body weights than mice with one or two intact Igf1 alleles. Nevertheless, also in the complete absence of IGF1, GH overexpression induced a significant body weight increase in Igf1 / /GH vs. Igf1 / mice. This GH excess associated effect however, was smaller and occurred later than in mice with intact Igf1 alleles. Significant effects of GH overabundance on body weight increase in Igf1 / /GH vs. Igf1 / mice became evident at week 7 in male, and at week 5 in female Igf1 / /GH vs. sex-matched Igf1 / mice. Female Igf1 / /GH vs. Igf1 / mice displayed significantly increased body weights, rising from + 52% at week 5, to +82% at week 11, then slightly decreasing to +54% at week 16. In contrast, male Igf1 / /GH mice displayed a less marked and transient elevation of body weights between weeks 7 (+23%) and 11 (+27%), whereas at week 16, no significant body weight differences were present, as compared to male Igf1 / mice. This might be explained by the significantly, on average 46% (week 7–11) higher body weights of male Igf1 / mice, as compared to female Igf1 / mice. Correspondingly, Igf1 deletion has previously been reported to result in gender-specific growth deficiencies, with the greatest reduction of body, tissue, and organ growth occurring in female mice (Moerth et al., 2007). In summary, these data show, that GH overabundance is capable to (temporarily) significantly stimulate body weight gain in the complete absence of IGF1 in a gender-specific manner, but fails to effectively rescue the overall dwarf phenotype of IGF1 deficient mice. A previous study reported the ineffectiveness of a six-week treatment with recombinant human GH (rhGH) to stimulate body weight gain of Igf1 knockout mice (Liu and LeRoith, 1999). In this study, mice were finally investigated at the age of 8 weeks. Since the body weight differences between male Igf1 / and Igf1 / /GH mice of the present study just started to reach statistical significance at the age of 7 weeks, the findings of the two studies cannot be directly compared and are thus not contradictory. In addition, the highly immunogenic nature of hGH in mice, associated with the risk of neutralizing antibody formation, has to be taken into account in treatment studies, whereas GH transgenic mice facilitate long-term investigations of chronic GH excess in the absence of immunological complications. 4.4.1. Organ growth and pathology In line with the previously reported effects of GH overabundance (and consequently elevated IGF1 levels) on organ and tissue growth (Le Roith et al., 2001; Wanke et al., 1991), the absolute weights of the carcass, liver, kidneys, heart, lungs, pancreas, spleen, and skin were consistently significantly increased in 16-week-old GH vs. WT and Igf1+/ /GH vs. Igf1+/ mice. In Igf1 / /GH vs. Igf1 / mice, however, significant differences were only observed in the liver and heart weights of male, and the body fat weights of male and female mice. The differences of absolute and relative brain weights in Igf1 / vs. Igf1 / /GH, WT vs. GH, and Igf1 / vs. WT mice are in line with previously published results (Beck et al., 1995). Permanent GH-overabundance in GH transgenic mice reproducibly induces development of characteristic alterations of i.a. the pituitary gland (Stefaneanu et al., 1993), the liver (Wanke et al., 1991; Wolf et al., 1993), the kidneys (Wanke et al., 1991, 2001; Wolf et al., 1993) and the skin (Wanke et al., 1999). Therefore, advanced histological and morphometrical analyses were performed to investigate the effects of an IGF1 deletion on development of GH excess associated lesions in these organs. 4.4.2. Pituitary gland In agreement with the previously reported effects of GH overabundance on morphologic alterations of the pituitary, GH mice and Igf1 / /GH mice displayed significantly reduced relative pituitary weights, absolute volumes of the pars distalis and of

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acidophilic cells, as compared to WT and Igf1 / mice. Reflecting a negative feedback regulation of permanently elevated systemic GH/IGF1 levels on somatotroph function and morphology, bovine GH transgenic mice exhibit significantly decreased pituitary weights (Stefaneanu et al., 1993) and markedly reduced sizes and numbers of somatotrophs (Stefaneanu et al., 1999), accompanied by drastically reduced pituitary levels of endogenous Gh mRNA (Iida et al., 2004). The hypoplasia of somatotrophs in GH transgenic mice is thought to be induced by diminished GH-releasing hormone receptor (GHRHR) signaling, mediated through a negative feedback of increased circulating IGF1 concentrations at the pituitary and hypothalamus, and by hypothalamic feedback of elevated circulating GH levels (Stefaneanu et al., 1993, 1989; Iida et al., 2004; Giustina and Veldhuis, 1998; Romero et al., 2010). Since GH has been demonstrated to exert also a direct feed-back regulation on pituitary somatotroph morphology and function in states of reduced GH-signaling (Asa et al., 2000), in reverse, a direct hypoplastic effect of excess GH on acidiphilic cells in the pituitary of GH transgenic mice could be speculated, as well. Although Igf1 knockout mice, as well as liver-specific Igf1 deleted mice, and mice with a somatotroph-specific disruption of Igf1r display increased somatotroph Gh mRNA- and endogenous GH protein levels (Stefaneanu et al., 1999; Romero et al., 2010; Lembo et al., 1996; Wallenius et al., 2001), the size of somatotrophs in adult Igf1 knockout mice is not increased vs. wild-type controls, indicating that in the absence of IGF1, somatotrophs do not reach their normal size (Stefaneanu et al., 1999). The significant reduction of the total volume of acidophilic cells in the pituitaries of Igf1 / /GH vs. Igf1 / mice thus provides evidence for an IGF1-independent feed-back of enhanced GH overabundance on acidophilic cell mass reduction. 4.4.3. Liver The growth stimulatory effect of GH on the liver appears to be most likely independent of IGF1 (Liu and LeRoith, 1999; Wanke et al., 1991, 1992; Wolf et al., 1993; Quaife et al., 1989; Miquet et al., 2008; Pennisi et al., 2004), since Igf1 knockout mice with elevated endogenous GH secretion also exhibit significantly increased liver weights, as compared to wild-type mice (Liu and LeRoith, 1999). Similarly, administration of rhGH to IGF1 deficient mice induces a significant enlargement of the liver (Liu and LeRoith, 1999), whereas IGF1 transgenic mice exhibit unaltered liver weights (Quaife et al., 1989), and IGF1 transgenic GH deficient mice display even reduced liver weights compared to wild-types (Behringer et al., 1990). Correspondingly, in the present study male Igf1 / /GH mice displayed significantly higher absolute (+46%) and relative (+19%) liver weights than male Igf1 / mice. Along with stimulation of liver growth, a typical, age-dependent spectrum of hypertrophic, hyperplastic, preneoplastic and cancerous hepatic lesions is regularly observed in aged GH transgenic mice (Wanke et al., 1991; Wolf et al., 1993; Quaife et al., 1989; Miquet et al., 2008, 2013), with hepatic up-regulation of several signaling mediators involved in cell growth, proliferation, survival, and migration, as well as dysregulation of oncogenic pathways (Miquet et al., 2008, 2013). Since hepatocytes do not express IGF1-receptors (McElduff et al., 1988), activation of signaling pathways involved in the pathogenesis of GH excess associated hepatic lesions could be due to a direct activation of hepatocellular GH-receptors (Bartke et al., 2002; Quaife et al., 1989), or might be indirectly mediated via paracrine actions of hepatocyte-derived IGF1 on IGF1R expressing non-parenchymal liver cells (Miquet et al., 2008). The results of the present study, where corresponding histopathological hepatic alterations (hepatocellular/nuclear hypertrophy and pleomorphy, necrosis and hepatic adenoma) were regularly observed in GH transgenic mice with intact, heterozygously or homozygously deleted Igf1 alleles, therefore

demonstrate, that the typical liver changes associated with permanent GH overabundance in GH transgenic mice do also develop if IGF1 is reduced or completely absent. 4.4.4. Skin In contrast to male (intact) GH mice, male Igf1 / /GH mice did not display significantly increased skin thicknesses, as compared to non GH transgenic male mice of the corresponding Igf1 genotype. As reported earlier, development of the typical skin alterations associated with permanent GH excess is only observable in male GH transgenic mice with intact gonads (Wanke et al., 1999). In contrast, male Igf1 knockout mice are infertile, display gonadal lesions and drastically reduced plasma testosterone levels (Baker et al., 1996). Since skin lesions in Igf1 transgenic mice do also not resemble those seen in GH transgenic mice (Quaife et al., 1989), the absence of typical GH excess-associated skin lesions in male Igf1 / /GH mice might rather be a consequence of decreased levels of sex hormones, than a result of abolished IGF1 signaling. Since the skin alterations seen in male GH transgenic NMRI mice (Wanke et al., 1999) are also observed in male GH transgenic mice with different genetic backgrounds as FVB/N, and B6NMRI/F1 (own unpublished observations), occurrence of these lesions appears to primarily depend on permanent GH-overabundance, rather than on the genetic background of GH transgenic mice. 4.4.5. Kidney Induction of overall renal growth and age-related development of progressive glomerulosclerosis, finally leading to terminal renal failure, represent almost ‘‘classical’’ alterations associated with permanent GH overabundance in various GH transgenic mouse lines (von Waldthausen et al., 2008; Wanke et al., 1991, 2001; Wolf et al., 1993; Doi et al., 1990, 1988; Yang et al., 1993). The formal pathogenesis of progressive glomerulosclerosis in GH transgenic mice is initiated by development of progressive glomerular hypertrophy with endothelial and mesangial cell hyperplasia and matrix expansion, consequently leading to establishment of podocyte lesions with subsequent impairment of the glomerular filtration barrier, albuminuria, glomerular hyalinosis, synechia formation between the glomerulus and the Bowman’s capsule, and subsequent tubulointerstitial inflammation, nephron atrophy and fibrosis (Wanke et al., 2001). However, a clear differentiation of GH and IGF1 actions on the glomerulus promoting glomerular hypertrophy and glomerulosclerosis is complicated, since both GHR, and IGF1R are simultaneously expressed by different glomerular cell types (Vasylyeva and Ferry, 2007; Bridgewater et al., 2008; Doi et al., 2000; Karl et al., 2005; Reddy et al., 2007; Tack et al., 2002), and both IGF1 transgenic, and GH transgenic mice display increased glomerular volumes and mesangial hypertrophy. However, IGF1 transgenic mice exhibit significantly smaller dimensions of glomerular hypertrophy than GH transgenic mice and do not develop glomerulosclerosis (Doi et al., 1990, 1988). Therefore, elevated systemic IGF1 levels, and probably (GH-stimulated) locally produced, autocrine active mesangial glomerular IGF1 (Karl et al., 2005; Tack et al., 2002) do contribute to the development of glomerular hypertrophy, but these IGF1 effects are obviously not sufficient to induce marked glomerulosclerotic lesions in the absence of concurrently elevated systemic GH levels (Doi et al., 1990). In the present study, significant increases of kidney weights were only observable in GH transgenic mice with one or two intact Igf1 alleles, whereas glomerulosclerotic kidney alterations were consistently also present in Igf1 / /GH mice. These findings show that GH and IGF1 differentially affect renal growth, and prove that development of glomerulosclerosis in GH transgenic mice can occur independently of IGF1. Since Igf1 / /GH (and Igf1+/ /GH) mice displayed lower glomerulosclerosis indices than GH mice, IGF1 apparently augments the degree of severity of GH

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excess-induced glomerulosclerosis in GH transgenic mice. Similarly, GH transgenic mice with decreased IGF1 levels, attained by a dietary protein restriction, were reported to exhibit lower glomerulosclerosis indices as compared to GH transgenic mice with high IGF1 levels (Doi et al., 2001). Given the essential involvement of the GH/IGF1 system in the development of glomerulosclerotic alterations in diverse entities of kidney diseases (Grunenwald et al., 2011; Kumar et al., 2011), our next studies will use Igf1 / / GH mice to further elucidate different IGF1- and GH-mediated glomerular growth effects involved in the pathogenesis of progressive glomerulosclerosis in GH transgenic mice. 4.5. Conclusion In summary, our genetic model facilitated dissection of IGF1dependent and -independent effects of permanent GH excess on postnatal growth and organ pathology of mice. Acknowledgments The authors thank Petra Renner and Tanja Mittmann for animal care, and Lisa Pichl and Heike Sperling for excellent technical and experimental assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mce.2014.07.002. References Asa, S.L., Coschigano, K.T., Bellush, L., Kopchick, J.J., Ezzat, S., 2000. Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic mice and GH receptor-deficient mice. Am. J. Pathol. 156, 1009–1015. Baker, J., Liu, J.P., Robertson, E.J., Efstratiadis, A., 1993. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75, 73–82. Baker, J., Hardy, M.P., Zhou, J., Bondy, C., Lupu, F., et al., 1996. Effects of an Igf1 gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. Bartke, A., Chandrashekar, V., Bailey, B., Zaczek, D., Turyn, D., 2002. Consequences of growth hormone (GH) overexpression and GH resistance. Neuropeptides 36, 201–208. Beck, K.D., Powell-Braxton, L., Widmer, H.R., Valverde, J., Hefti, F., 1995. Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons. Neuron 14, 717–730. Behringer, R.R., Lewin, T.M., Quaife, C.J., Palmiter, R.D., Brinster, R.L., et al., 1990. Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology 127, 1033–1040. Bridgewater, D.J., Dionne, J.M., Butt, M.J., Pin, C.L., Matsell, D.G., 2008. The role of the type I insulin-like growth factor receptor (IGF-IR) in glomerular integrity. Growth Horm. IGF Res. 18, 26–37. Butler, A.A., Le Roith, D., 2001. Control of growth by the somatropic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63, 141–164. Cingel-Ristic, V., Flyvbjerg, A., Drop, S.L., 2004. The physiological and pathophysiological roles of the GH/IGF-axis in the kidney: lessons from experimental rodent models. Growth Horm. IGF Res. 14, 418–430. DeChiara, T.M., Efstratiadis, A., Robertson, E.J., 1990. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345, 78–80. Doi, T., Striker, L.J., Quaife, C., Conti, F.G., Palmiter, R., et al., 1988. Progressive glomerulosclerosis develops in transgenic mice chronically expressing growth hormone and growth hormone releasing factor but not in those expressing insulinlike growth factor-1. Am. J. Pathol. 131, 398–403. Doi, T., Striker, L.J., Gibson, C.C., Agodoa, L.Y., Brinster, R.L., et al., 1990. Glomerular lesions in mice transgenic for growth hormone and insulinlike growth factor-I. I. Relationship between increased glomerular size and mesangial sclerosis. Am. J. Pathol. 137, 541–552. Doi, S.Q., Jacot, T.A., Sellitti, D.F., Hirszel, P., Hirata, M.H., et al., 2000. Growth hormone increases inducible nitric oxide synthase expression in mesangial cells. J. Am. Soc. Nephrol. 11, 1419–1425. Doi, S.Q., Rasaiah, S., Tack, I., Mysore, J., Kopchick, J.J., et al., 2001. Low-protein diet suppresses serum insulin-like growth factor-1 and decelerates the progression of growth hormone-induced glomerulosclerosis. Am. J. Nephrol. 21, 331–339.

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