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. 2010 Mar 8;24(5):1077–1089. doi: 10.1210/me.2009-0393

Targeted Deletion of Somatotroph Insulin-Like Growth Factor-I Signaling in a Cell-Specific Knockout Mouse Model

Christopher J Romero 1, Yewade Ng 1, Raul M Luque 1, Rhonda D Kineman 1, Linda Koch 1, Jens C Bruning 1, Sally Radovick 1
PMCID: PMC2870932  PMID: 20211984

Abstract

The role of IGF-I in the negative regulation of GH expression and release is demonstrated by in vitro and in vivo models; however, the targets and mechanisms of IGF-I remain unclear. We have developed a cell-specific knockout mouse in which the IGF-I receptor was ablated from the somatotroph in order to validate and characterize IGF-I negative regulation; we termed this the somatotroph IGF-I receptor knockout (SIGFRKO) mouse. The SIGFRKO mice demonstrated increased GH gene expression and secretion as well as increased serum IGF-I. Compensatory changes were noted with decreased GHRH and increased somatostatin mRNA expression levels. SIGFRKO mice had normal linear growth, but by 14 wk of age weighed significantly less than controls. Furthermore, metabolic studies revealed SIGFRKO mice had significantly less fat mass and body percent fat. These data support somatotroph IGF-I negative regulation and suggest that hypothalamic feedback limits the extent of GH release. The SIGFRKO mouse is a model delineating the mechanisms of IGF-I regulation in the hypothalamic-pituitary axis and demonstrates compensatory mechanisms that mediate growth and metabolic function in mammals.

The ablation of the somatotroph IGF-1 receptor demonstrates the importance of IGF-1 in the regulation of growth and metabolism.

GH expression and release are thought to be primarily regulated by the counter-regulatory effects of hypothalamic hormones: GHRH and somatostatin (SRIF). Other factors, however, such as IGF-I, have also been shown to provide feedback regulation to the somatotroph and play a role in negative regulation of target genes in the growth axis. In vitro studies using human pituitary gland tissue demonstrated a direct inhibition of GH release in response to a semipurified preparation of IGFs (1). Further study of IGF-I regulation using recombinant IGF-I also demonstrated its action in suppressing basal GH secretion and decreasing GH mRNA levels in primary rat pituitary cell cultures (2). These findings were supported by data obtained from an established rat somatotroph tumor cell line a decade later, although there are limitations in pituitary cell lines as models of IGF-I negative regulation of GH gene expression (3,4). In vivo studies in rats using a preparation of IGFs showed a decrease in GH secretory episodes thought to be secondary to increased SRIF release (5). More recently, transgenic mouse models that overexpress GH, overexpress IGF-I, or abolish IGF-I production have further confirmed IGF-I’s role in somatotroph feedback (6,7,8,9,10). Despite the overwhelming evidence of IGF-I’s role in negative regulation of the central growth axis, its target(s) site (pituitary, hypothalamus, or both) and mechanism of control remain unclear.

Currently available transgenic and knockout (KO) mouse models used to study the GH axis result in major perturbations at multiple loci and therefore limit insight into specific mechanisms responsible for regulation at target tissues. The goal of our investigation was to study the regulation of GH expression within a model system that maintained the integrity of the hypothalamic-pituitary GH axis, with the single exception of somatotroph IGF-I receptor (IGF-IR) deficiency. Using a Cre/loxP strategy, we developed the somatotroph IGF-IR KO (SIGFRKO) mouse. Findings in this model support the role of IGF-I in GH regulation as demonstrated by increased serum GH and IGF-I levels. Changes in the expression of GHRH and SRIF mRNAs, however, suggest compensatory actions in SIGFRKO mice that limit elevations in these growth factors and consequently their effect on somatic growth. Moreover, SIGFRKO mice were found to have decreased weight gain velocity over time, and metabolic studies demonstrated a reduction in fat deposition. We hypothesize the levels of growth factors in SIGFRKO mice result in a metabolically selective phenotype. Thus, the SIGFRKO mouse is a unique cell-specific KO (cKO) model that supports IGF-I’s role in feedback regulation of the somatotroph and highlights that feedback control is at both the pituitary and hypothalamic level.

Results

Generation and characterization of SIGFRKO mice

To determine the role of somatotroph IGF-I feedback, we developed a cKO mouse model in which the somatotroph IGF-IR was ablated. Using the Cre/loxP strategy, we crossed a female mouse expressing a transgene containing Cre recombinase downstream from the GH promoter (rGHpCre) to male mice containing loxP sites flanking exon 3 of the IGF-IR (IGF-IR flox/flox mouse; Fig. 1, A and B) (11,12). Cre recombinase expression in somatotrophs was expected to result in excision of the floxed exon of the IGF-IR (Fig. 1C). Genotyping of extracted tail DNA from the progeny revealed a band of 350 bp for animals bearing the Cre recombinase gene. PCR products for the IGF-IR floxed allele resulted in bands at 380 and 300 bp for heterozygous floxed animals or a single band at 300 bp for control mice (Fig. 1D). Siblings from this generation were then mated to create the SIGFRKO mouse and control littermates with all possible genotypes. Only female mice carrying the Cre recombinase gene were selected, because during the development of the rGHpCre mouse, Cre was detected in the testis but not in the ovary (11). A previous report demonstrates that expression of Cre in the spermatids could lead to germline recombination; therefore, our matings were designed to avoid this risk in the progeny (13). Females from the F1 progeny, Cre(+)/IGF-IR(flox/Wt), were mated with male siblings, Cre(−)/IGF-IR(flox/Wt). This second mating provided five different control genotypes, as well as the SIGFRKO mouse, Cre(+)/IGF-IR flox/flox.

Figure 1.

Figure 1

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Targeting strategy for the development of the somatotroph SIGFRKO mouse model. The Cre/loxP strategy was used to target the somatotroph IGF-IR by breeding rGHp-Cre transgenic mice (A) with mice homozygous for loxP-flanked IGF-IR alleles at exon 3 (C). The wild-type IGF-IR is diagrammed in B. Cre expression in the progeny leads to the deletion of exon 3 as shown in C. PCR genotyping of extracted genomic tail DNA was used to identify the presence of Cre recombinase and IGF-IR floxed alleles. Results from the PCR analysis of the IGF-IR alleles are shown in D; the homozygous floxed allele, IGF-IR flox/flox (380 bp), the heterozygous floxed allele, IGF-IR flox/Wt, and wild-type IGF-IR (300 bp).

Double label immunohistochemistry (IHC) was then performed to confirm the absence of the IGF-IR on somatotrophs. By using primary antibodies directed against GH and the IGF-IR (Fig. 2, A and B), we show the IGF-IR present on the majority of somatotrophs in pituitary sections from control mice as seen in the merged image (Fig. 2C). IGF-IR immunoreactivity was noted to be abundant in both somatotrophs and other pituitary cell types as has been reported in the rodent anterior pituitary (14,15,16). Although immunoreactivity for both GH and the IGF-IR is seen in SIGFRKO mice (Fig. 2, D and E), no IGF-IR immunoreactivity is found on somatotrophs as shown by the lack of yellow color in the merged image (Fig. 2F). Normal mouse pituitary contains approximately 30–40% somatotrophs as reported by previous IHC studies (17); therefore, the presence of GH immunostaining in SIGFRKO mice confirms that the absence of the somatotroph IGF-IR does not affect somatotroph development. This correlates with previous studies of mice with disrupted IGF-I gene, which demonstrated the lack of IGF-I did not affect the development of pituitary cells (18). Multiple sections (n = 3–7) immunostained for GH and IGF-IR from three control and three SIGFRKO mice were analyzed in a blinded fashion to determine differences between groups. Each section was analyzed at ×400 magnification. Control and SIGFRKO mice demonstrated an average percentage of GH immunostaining of 5.88 ± 0.63 and 7.65 ± 0.385%, respectively, per section based on the number of cells per field, which was not statistically significant. In contrast, the percentage of somatotroph cells showing double-label immunostaining in the control vs. SIGFRKO mice (60.28 ± 3.3 vs. 6.43 ± 0.91%, respectively) was significant (P < 0.0001). Furthermore, given the proportion of somatotrophs within the pituitary and the abundance of IGF-IR, we anticipated lower levels of IGF-IR mRNA expression. Figure 2G demonstrates IGF-IR mRNA expression levels measured by quantitative real-time PCR (qRT-PCR) from whole pituitaries of males and females in both control and SIGFRKO mice. Female SIGFRKO mice demonstrated only 57.6% of female control expression levels, whereas male SIGFRKO mice demonstrated 57.3% of male control expression levels (both, P < 0.05).

Figure 2.

Figure 2

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Characterization of the somatotroph SIGFRKO mouse. Double label IHC studies were performed on pituitary sections from control littermates (A–C) and SIGFRKO mice (D–F) using primary antibodies to GH (A and D) and the IGF-IR (B and E). GH positive cells were visualized using FITC secondary antibody (green color) and IGF-IR was visualized using Cy3 (red color). C, In the control merged image, the bright yellow color represents the presence of IGF-IR on somatotrophs (white arrows); however, this is not appreciated in SIGFRKO mice (F). The blue color represents nuclear staining with DAPI. G, qRT-PCR for IGF-IR mRNA expression illustrates a significant reduction in levels of both SIGFRKO female and male mice in comparison with expression of control female and male mice, respectively. The expression values for each group are corrected using expression of 18S. Data are expressed as mean ± sem (n = 5–7). Significance (P < 0.05) was determined by Student’s t test.

Serum GH, GH mRNA expression, serum IGF-I, and IGF binding protein 3 (IGFBP-3) levels in SIGFRKO mice

To determine the somatotroph response to a loss of negative feedback by IGF-I, we obtained sera from 6- to 8-wk-old SIGFRKO and control mice for measurement of GH and IGF-I levels using the Luminex assay system. Considering the pulsatile nature of GH, which could obscure measured differences in GH levels from a single time point, we decided to fast SIGFRKO and control mice overnight and obtain serum under similar stressed conditions the following morning. The mean serum GH in control female mice was 1.74 ± 0.382 ng/ml; however, the mean value in SIGFRKO female mice was significantly higher at 6.81 ± 1.131 ng/ml (P < 0.01; Fig. 3A). The mean GH value in control male mice was 0.71 ± 0.233 ng/ml, whereas the mean value in SIGFRKO male mice was significantly higher at 2.51 ± 0.305 ng/ml (P < 0.01; Fig. 3A). GH studies in rodents have also demonstrated sexual dimorphism with respect to the timing of pulses, the amplitude of GH peaks and total GH levels (19). Both control and SIGFRKO mice demonstrate a sexual dimorphic GH response, which has been previously described in the literature (20).

Figure 3.

Figure 3

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Measurement of circulating serum growth factor levels and GH mRNA expression in SIGFRKO and control mice using Luminex assay system. A, Serum GH levels were measured from serum of fasted postpubertal SIGFRKO mice and compared with matched control mice (n = 9–14/group). Both female and male SIGFRKO mice had significant increases in average serum GH compared with female and male mice, respectively. Average GH levels also demonstrated significant sexual dimorphism between control female and male groups. Sexual dimorphism was also preserved and significant between female and male SIGFRKO mice. B, Correlating to serum GH levels, relative mRNA expression of GH from pituitary tissue was increased in both female and male SIGFRKO mice in comparison with female and male controls, respectively (n = 5–7/group). The expression of 18S was measured to correct for gene expression values. C, Average serum IGF-I levels of SIGFRKO were significantly increased compared with controls for both sexes (n = 9–14/group). D, Serum IGFBP-3 levels were measured in postpubertal SIGFRKO mice and compared with matched control mice (n = 6–8/ group). No significant differences in average serum IGFBP-3 levels were noted between SIGFRKO and control mice for both females and males. All values are shown as mean ± sem. Significance was determined by Student’s t test.

We also compared relative expression levels of GH mRNA (Fig. 3B). Consistent with the changes seen in the serum levels, SIGFRKO female mice had a 2.58-fold increase in GH mRNA levels when compared with control littermates (P < 0.01), whereas SIGFRKO male mice had a 2.7-fold increase in relative expression of GH mRNA when compared with control littermates (P < 0.01).

Because GH is the major regulator of IGF-I production, serum IGF-I levels from the same SIGFRKO and control mice were analyzed. The mean IGF-I level in control female mice was 113.19 ± 18.68 ng/ml, whereas SIGFRKO female mice had a significantly higher mean level of 232.45 ± 37.23 ng/ml (P < 0.01; Fig. 3C). In control male mice, the mean IGF-I level was 113.61 ± 19.95 ng/ml vs. a significantly higher level in the SIGFRKO male mice of 188.62 ± 26.95 ng/ml (P < 0.05; Fig. 3C).

Circulating IGF-I has been shown to circulate in the serum in a ternary complex bound with IGFBP-3 and the acid labile subunit (ALS), which has been reported to stabilize IGF-I (21). An ELISA was used to measure whether changes in IGFBP-3 levels differ between SIGFRKO mice and controls. No significant differences were noted between groups for both females and males (Fig. 3D).

Determination of ALS levels

Western blot analysis of serum using antibodies directed against ALS or actin (as control) revealed higher levels in the SIGFRKO mice when compared with control littermates (Fig. 4). These data correlate with the regulation of ALS by the elevated GH levels (22,23).

Figure 4.

Figure 4

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Western blot analysis for ALS. A, Immunoblotting for ALS demonstrated increased levels in both female and male SIGFRKO mice compared with control mice. B, The intensity of each band, based on an equal volume, was measured to further demonstrate an increased level of ALS in SIGFRKO mice compared with controls.

Postnatal growth of SIGFRKO mice

Body weight and length measurements (nose to anus) of mice were recorded starting from 1–2 d after birth to 28 wk of age (Fig. 5, A–D). At weaning (21–25 d), no significant differences in weight or length were found between SIGFRKO and control mice. After 7 wk of life, however, SIGFRKO mice were gaining weight at a lower velocity compared with control animals. This difference in weight gain became significant after 14 wk of age in both female and male SIGFRKO mice. At 14 wk of life, control female mice had an average weight of 23.15 ± 0.82 g, whereas the SIGFRKO female mice had an average weight of 21.19 ± 0.42 g (P < 0.05; Fig. 5A). The average weight of male control mice at 14 wk was 32.96 ± 0.918 g, whereas SIGFRKO male mice had an average weight of 30.06 ± 1.03 g (P < 0.05; Fig. 5B). The significant difference in SIGFRKO male and female weight compared with control mice continued through 28 wk of life. At 25 wk of life, female control and SIGFRKO mice weighed 27.35 ± 0.754 vs. 24.10 ± 0.603 g, respectively (P < 0.01). Male control and SIGFRKO mice at 25 wk of age weighed 44.03 ± 1.724 vs. 36.41 ± 1.796 g, respectively (P < 0.01). At 28 wk of life, female control mice weighed 30.58 ± 1.44 g vs. female SIGFRKO, 26.03 g ± 0.800 (P < 0.01); and male control mice weighed 47.32 ± 1.09 g vs. SIGFRKO, 37.03 ± 3.01 g (P < 0.01). No significant difference was noted between the lengths of SIGFRKO and control mice at all the measured time points (Fig. 5, C and D).

Figure 5.

Figure 5

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Measurements of SIGFRKO and control animals. Mice were weighed (A and B) and length (nose to anus) recorded (C and D) at various week intervals. A and B, SIGFRKO female and male mice were noted to have significantly less weight at wk 14, 25, and 28 compared with control mice (n = 10–14). C and D, No significant differences seen for length between SIGFRKO female and male mice compared with control at any time point (n = 10–14). *, P < 0.05; **, P < 0.01. Data is graphed as mean ± sem. Significance was determined using one-way ANOVA.

The calculated weight/length ratio was plotted for SIGFRKO and control mice at each time point to account for subtle differences in measurements. Significance in the weight/length ratio was appreciated at wk 25 and 28 between the SIGFRKO and control animals (data not shown). At wk 25, female control mice had an average ratio of 3.121 ± 0.085 vs. female SIGFRKO mice, which had an average ratio of 2.655 ± 0.048 (P < 0.01). At wk 28, female control mice had an average ratio of 3.421 ± 0.159 vs. female SIGFRKO mice, which had an average ratio of 2.882 ± 0.090 (P < 0.01). In male mice, the wk 25 average ratio in control mice was 4.653 ± 0.179 vs. male SIGFRKO mice having a ratio of 3.861 ± 0.169 (P < 0.01). Finally, at wk 28, male control mice had an average ratio of 4.967 ± 0.121 vs. male SIGFRKO having a ratio of 3.942 ± 0.293 (P < 0.01).

Compensatory changes in GHRH, SRIF, and GHRH receptor (GHRH-R) expression

It is unclear whether IGF-I feedback is predominantly at the level of the pituitary somatotroph, hypothalamus, or both (24,25,26). To determine hypothalamic changes in response to loss of pituitary IGF-I feedback, GHRH and SRIF mRNA levels from hypothalamic tissue were measured using qRT-PCR. The expression of hypothalamic GHRH in female SIGFRKO mice was 51.3% of control female levels (P < 0.05). In the male SIGFRKO mice, GHRH expression was only 58.5% of control male levels (P < 0.05; Fig. 6A). Conversely, significant increases in SRIF levels were present in both male and female SIGFRKO mice (Fig. 6B). Female SIGFRKO mice had a 1.35-fold increase over female control levels (P < 0.05), whereas male SIGFRKO mice had a 1.86-fold increase in SRIF when compared with male control levels (P < 0.05). These changes in hypothalamic expression reported in a single in vivo model system are unique, because the alterations in GHRH and SRIF in the setting of elevated IGF-I have been reported only in separate experiments (24,27). We also measured a significant increase in pituitary GHRH-R mRNA in both female and male SIGFRKO mice (Fig. 6C). Female SIGFRKO mice had a 1.42-fold increase when compared with control levels (P < 0.05), whereas male SIGFRKO mice had a 2.69-fold increase when compared with control levels (P < 0.01). Finally, given the increased SRIF expression, we also measured expression levels of the pituitary SRIF receptor subtypes 2 and 5, which are considered the predominant receptors in the pituitary (28,29,30). Analysis of their mRNA expression levels in SIGFRKO and control mice found no significant differences in either female or male mice (Fig. 6, D and E, respectively).

Figure 6.

Figure 6

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Relative levels of mRNA expression of genes expressed in the hypothalamus and pituitary. In order determine compensatory changes in the hypothalamic-pituitary axis of SIGFRKO mice, RNA was extracted from isolated pituitary and hypothalamic tissue from both SIGFRKO and control littermates. Specific primers were selected for several genes, and qRT-PCR was used to measure relative expression levels. A, In the hypothalamus, GHRH mRNA levels for SIGFRKO female and male mice were significantly less than expression levels in female and male control mice, respectively (n = 5–7/group). B, Conversely, SRIF expression in SIGFRKO female and male mice was increased significantly compared with female and male controls, respectively (n = 5–6/group). C, In the pituitary, SIGFRKO female and male mice were found to have a significant increase fold difference in the expression of GHRH-R mRNA compared with control female and male littermates, respectively (n = 5). D and E, The expression of SRIF receptor subtypes 2 and 5, the predominant subtypes noted on the somatotroph, were also measured, and no significance in expression levels between groups was found (n = 5–7/group). All data expressed as mean ± sem. The expression of 18S was used to correct for calculated counts for each gene. Significance was determined using Student’s t test.

Elevated GH levels in older adult SIGFRKO mice are maintained

Although serum GH levels are expected to decline with age, we hypothesized that the disruption in the GH/IGF-I axis of the SIGFRKO mouse may potentially obviate or delay this decline. In addition, the elevation of growth factors may be acting in a metabolically selective manner because both SIGFRKO female and male mice continued to gain less weight when compared with control littermates through 28 wk of age (Fig. 5, A and B, respectively). Mice between 28 and 32 wk of age were fasted overnight as in the previous experiment, and serum was obtained in the morning for analysis of serum GH levels using the Luminex assay system (Fig. 7). SIGFRKO female mice had an average serum GH of 3.34 ± 0.42 ng/ml, which continued to be significantly elevated when compared with the average of control female levels, 1.65 ± 0.30 ng/ml (P < 0.05). SIGFRKO male mice had an average serum GH of 2.68 ± 0.88 ng/ml vs. male control mice that had an average of 0.70 ± 0.12 ng/ml (P < 0.05). The data indicate that, despite a decline in serum GH levels overtime, the loss of IGF-I signaling to the somatotroph continued to result in higher GH levels in SIGFRKO mice compared with levels seen in control animals. The data also indicate sexual dimorphism of serum GH appears preserved in the control animals (P < 0.01); however, despite higher GH levels in the female SIGFRKO mice to the male SIGFRKO, the differences were not significantly different.

Figure 7.

Figure 7

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Serum GH levels in older SIGFRKO and control mice. Serum GH levels were measured from serum of fasted SIGFRKO mice and compared with matched control mice (n = 10–12/group) at ages 28–32 wk of life. Both female and male SIGFRKO mice continued to have significant rises in GH levels compared with female and male control mice. The average GH levels between female and male control mice indicated preservation of sexual dimorphism and were significantly different. Significant sexual dimorphism was not appreciated in the SIGFRKO mice at this age, although SIGFRKO female mice did have a greater average than SIGFRKO male mice. Data are expressed as mean ± sem. Significance was determined using Student’s t test.

Organ weights in SIGFRKO mice

Elevations of GH and IGF-I have been associated with organomegaly (9,31). Thus individual organs were dissected and weighed to determine differences between SIGFRKO vs. control mice (Fig. 8). No significant differences were noted in the average weights for brain, heart, lungs, and kidneys. The average weight for liver in SIGFRKO was 1.67 ± 0.04 vs. 1.52 ± 0.04 g in control mice (P < 0.05). Furthermore, the average weight of the spleen in the SIGFRKO mice (0.142 ± 0.01 g) was also greater than the average weight in control mice (0.099 ± 0.002 g; P < 0.01). These changes most likely reflect the modest increases in serum GH and IGF-I.

Figure 8.

Figure 8

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Organ weights in SIGFRKO and control mice. Average weights of brain, heart, lungs, liver, spleen, and kidney were performed at time of dissection to compare control vs. SIGFRKO mice (n = 4–7).

Evaluation of metabolic phenotype in SIGFRKO mice

We subsequently performed a series of metabolic studies on older SIGFRKO mice to investigate the impact of elevated serum GH and IGF-I. The attenuated weight gain noted in SIGFRKO mice, suggested a potential metabolically advantageous phenotype vs. control mice. Because GH has been shown in both humans and animal models to play an important metabolic role in the deposition of fat and lean mass, we investigated the body composition of SIGFRKO mice. An Echo magnetic resonance imaging (EchoMRI) Quantitative Magnetic Resonance Body Composition Analyzer was used to directly measure the amount of total body fat, lean mass, free water, and total body water in both SIGFRKO and control mice. The average amount of fat was significantly lower in female SIGFRKO mice compared with control mice (5.94 ± 0.71 vs. 9.32 ± 1.06 g; P < 0.01) (Fig. 9A). This was also true for SIGFRKO male mice when compared with control male mice (11.00 ± 1.51 vs. 16.27 ± 0.94 g; P < 0.01). The measurement of percent body fat between animal groups correlated with the differences in fat mass. SIGFRKO female mice had an average of 20.31 ± 1.88% body fat vs. control female mice, which had 33.00 ± 1.88% (P < 0.01; Fig. 9B). SIGFRKO male mice had an average of 24.19 ± 2.15% body fat vs. control male mice that had 33.36 ± 1.43% (P < 0.01). Measurements of lean body mass, however, were not different between SIGFRKO and control female mice (19.56 ± 0.38 vs. 19.50 ± 0.44 g; Fig. 9C). No differences in lean mass were found between male SIGFRKO and control mice (26.10 ± 1.33 vs. 23.26 ± 0.79 g). No differences in free water were appreciated between SIGFRKO and control female or male groups (data not shown). Finally, no differences in total water content were noted between SIGFRKO and control littermates. SIGFRKO and control female mice had average values of 14.52 ± 0.32 vs. 14.67 ± 0.26 g, respectively, whereas SIGFRKO and control males had values of 19.16 ± 0.74 vs. 17.46 ± 0.36 g, respectively.

Figure 9.

Figure 9

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EchoMRI quantitative magnetic resonance body composition analyzer data of older SIGFRKO and control mice. SIGFRKO and control mice aged 28–32 wk were measured for total body fat, lean body mass, fat percentage, total water, and free water (n = 4–8/group). A, A significant difference in the average fat mass between control and SIGFRKO mice was measured. Both female and male SIGFRKO mice measured with less fat mass then control. B, These findings correlated with a significantly lower percentage of body fat in both SIGFRKO female and male mice when compared with female and male control mice, respectively. C, No significant differences, however, were found lean body mass was seen between the SIGFRKO and control mice. Furthermore, no significant differences were noted for free water and total water content between SIGFRKO and control mice (data not shown). Each body parameter measurement was done in triplicate and data are expressed as mean ± sem. Significance was determined using Student’s t test.

It has been shown in both humans and mouse models that elevated serum GH can lead to insulin resistance (32,33). Given the decreased level of fat deposition in SIGFRKO, which we attribute to the modest elevations of GH, both glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed on SIGFRKO and control mice at 18 wk of life (Fig. 10, A and B). No significant differences were seen in the average fasting blood glucose (BG) levels between SIGFRKO and control mice before the administration of glucose (122.0 ± 3.7 and 122.5 ± 13.2 mg/dl, respectively). In addition, no significant differences in BG levels were noted between SIGFRKO and control mice during the GTT (Fig. 10A). Consistent with the previous experiment, no significant differences in fasting BG levels were seen between SIGFRKO and control mice before insulin administration (135.3 ± 13.3 and 117.8 ± 10.2 mg/dl, respectively). There were no differences in BG levels between SIGFRKO and control mice in response to insulin administration at all time points (Fig. 10B).

Figure 10.

Figure 10

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GTT and ITT. A, GTT. Glucose levels were checked after fasting followed by an ip injection of glucose into control and SIGFRKO mice (n = 6–8). Glucose levels were measured at time points noted. B, ITT. Glucose levels were checked at time 0 followed by ip injection of insulin into control and SIGFRKO mice (n = 6–8). Glucose levels were checked at noted time points. No statistical difference in GTT and ITT was detected between groups.

Discussion

We have developed a new mouse model to study the significance of IGF-I negative regulation on the GH axis. Although GH gene expression is regulated by IGF-I in vitro, limited in vivo studies have been performed to document the physiological relevance of this regulatory event. SIGFRKO mice demonstrate higher levels of serum GH and IGF-I, and pituitary GH mRNA levels. Thus the SIGFRKO mouse model proves in vivo that IGF-I feedback directly on the somatotroph is a physiologically important regulatory pathway. By comparison with other mouse models with various disruptions in the IGF-I signaling pathway, the SIGFRKO model also demonstrates the relative importance of pituitary vs. hypothalamic feedback. Unexpectedly, the modest elevations of GH and IGF-I did not increase linear growth, but did decrease fat mass compared with control animals. The lack of difference in IGFBP-3 correlates with studies in transgenic mice overexpressing of IGF-I, which had no differences in levels compared with controls, whereas the apparent increases in ALS correlate with the elevated serum GH levels (34). We postulate that modest elevations of serum GH were sufficient to dissociate changes in metabolism from effects on linear growth leading to a selective metabolic phenotype in SIGFRKO mice.

There are ample studies demonstrating IGF-I regulation of GH gene expression and release (3,5,24,25). However, the global effects of these genetic manipulations present limitations to understanding cell-specific feedback mechanisms. Previous work in rodents demonstrated an abundance of IGF-IRs within the somatotroph as well as other pituitary cell types (14,16). Our IHC studies confirmed abundant expression of the IGF-IR throughout the pituitary in the SIGFRKO, except in the somatotroph; and comparable GH immunostaining demonstrated that the loss of somatotroph IGF-IR expression does not impact somatotroph development.

Despite demonstrating direct effects of IGF-I signaling on both GH gene expression and release, SIGFRKO mice also demonstrate compensatory hypothalamic changes resulting in decreased GHRH and increased SRIF mRNA levels. The changes in SRIF correlate with previous in vivo studies that demonstrated increased SRIF secretion after the administration of recombinant IGF-I (35). Another proposed mechanism for IGF-I regulation of the somatotroph is through modulation of pituitary GHRH-R expression (36,37). These studies are limited, however, because changes in hypothalamic releasing hormone levels might also indirectly affect GHRH-R levels. The preserved hypothalamic feedback in the SIGFRKO model suggests that changes in GHRH-R mRNA expression may not simply be the direct result of loss of pituitary IGF-I signaling.

The SIGFRKO mice displayed normal linear growth despite modest increases in both serum GH and IGF-I levels. The reports regarding skeletal growth in transgenic mice overexpressing IGF-I are controversial (9,22). Furthermore, although the physiologic changes in the SIGFRKO mice parallel transgenic mice overexpressing GH, certainly the excess linear growth and weight in these models is attributable to a far greater increase in GH levels. Table 1 summarizes the hormone levels and phenotypes among several mouse models exhibiting perturbations in the growth axis. Of note, IGF-I and the IGF-IR are clearly important in development and viability; however, alterations in body length and weight are not necessarily intuitive from the changes in growth factor levels (38). The disruption of the growth axis in the SIGFRKO mouse is less dramatic than other models in Table 1 and further emphasizes that regulation of growth is multifactorial. Accordingly, the changes in hypothalamic gene expression, secondary to increased IGF-I feedback, act as a compensatory mechanism to limit GH synthesis and secretion to a level that does not promote excess linear growth.

Table 1.

Comparison of data from existing mouse models with genetic manipulation of the GH/IGF-I axis

Mouse model system Body weight Body length Serum GH Serum IGF-I Notes Refs.
GH overexpression ↑↑ ↑ ↑↑ ↑↑ 6
IGF-I overexpression ↑ ND/↑ ↓ ↑ 9,22
GH deficient ↓ ↓ ↓↓ ↓↓ 48
IGF-I gene KO ↓ ↓ NR (presumably ↑↑) BLD Variable perinatal survival 38
IGF-I (liver-specific) gene KO ND ND ↑↑ ↓ 10
IGF-I receptor gene KO ↓↓ ↓↓ NR NR Postnatal death 38
IGFBP-3 overexpression ↓ ↓ NR ↑ Intrauterine/postnatal growth retardation 49
Somatotroph IGF-I receptor gene KO ↓ ND ↑ ↑ Weight divergence after 7 wk
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ND, No difference compared with wild type; NR, not reported; BLD, below level of detection.

Nevertheless, there was a notable difference in weight in the SIGFRKO mice that persisted after 14 wk of age. It has been recognized that increased weight gain in transgenic mice does not necessarily indicate an obese phenotype as noted by Palmer et al. (31). This study reported that the bovine GH transgenic mouse, despite increased body weights, had significant decrease in both fat mass and percentage fat at 4 months in males and 6 months in females (31). This protective effect against obesity by elevated serum GH was also recognized in a liver-specific IGF-I gene deletion mouse model, when fat mass accumulation was compared with control mice at 13 months of age (39).

GH is known to have effects, not only on postnatal body length, but also on body composition. Classically, GH deficiency presents with short stature, an increase in fat mass, and a decrease in lean body mass (40). This association of GH deficiency with obesity in humans has also been demonstrated in studies in the lit/lit mice, which are GH-deficient (41). Conversely, patients with excess GH (acromegaly), accompanied by excess IGF-I, present with increased body water and lean body mass as well as a reduction in body fat (32). Therefore, the modest elevation of GH in SIGFRKO mice may be sufficient to dissociate the metabolic effects from the linear growth effects of GH. Although we anticipated insulin resistance in the SIGFRKO mice, no differences were demonstrated in the GTT and ITT. We believe that subtle abnormalities in insulin function and sensitivity are not detected by these studies. The balance of serum GH and IGF-I in the SIGFRKO mice secondary to compensatory actions of counter regulatory hormones such as GHRH and SRIF result in a unique metabolic phenotype, limiting the amount of fat deposition over time and maintaining a stable lean mass.

The differences in the magnitude of serum GH levels between SIGFRKO male and female mice indicate sexual dimorphism in secretion. Previous work in mice, using intraatrial catheters to serially measure GH levels, clearly demonstrates sexual dimorphism in both the pulsatility and level of GH between male and female animals (20). Because GH pulses are highly variable in mice, a random single serum measurement would not be an accurate representation of serum levels or allow detection of subtle differences in GH levels. Thus, we performed serum collection under similar conditions and at the same time point to minimize variability between genotypes. In addition, it is noted that studies in mice with insulin-induced hypoglycemia demonstrated no altered changes in hypothalamic expression of GHRH and SRIF mRNA levels (42). We therefore believe that alterations in hypothalamic and pituitary gene expression in our mice are not induced by the experimental design. Finally, we speculate that the magnitude of difference between male and female animals may be secondary to sex hormone differences.

In conclusion, studies in SIGFRKO mice document not only the role of IGF-IR in pituitary regulation but also its regulatory effects on the hypothalamus. These data demonstrate that effects on GH gene expression and serum GH levels appear to be direct and indirect as evidenced by changes in hypothalamic mRNA and pituitary GHRH-R mRNA. This novel cKO mouse model has provided a functional role for the IGF-IR on the somatotroph while maintaining the integrity of other feedback loops (Fig. 11). The SIGFRKO mouse also offers an in vivo system to study mechanisms of IGF-I negative regulation that ultimately affect transcriptional control of GH gene expression in the pituitary. Future studies may help to delineate the mechanisms by which GH and IGF-I affects various metabolic parameters and whether it is feasible to dissociate the favorable from detrimental effects of these growth factors. Understanding the role and mechanisms of IGF-I feedback on GH regulation will also provide insight into the pathogenesis of growth disorders, as well as its potential role in states of metabolic dysregulation.

Figure 11.

Figure 11

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Summary of findings in the SIGFRKO mouse model.

Materials and Methods

Generation, genotyping, and breeding of the SIGFRKO mouse

C57BL/6J (B6) mice expressing a heterozygous Cre gene in the GH promoter were crossed with mice homozygous for the floxed IGF-IR gene (11,12). Only female mice that expressed Cre recombinase were used in mating because authors reported the presence of Cre recombinase in the male testis (11). The rGHpCre transgene was identified by PCR on genomic tail DNA using oligonucleotides that amplify a 300-bp product (5′-ACGACCAAGTGACAGCAATGCTGT-3′ and 5′-CGGTGCTAACCAGCGTTTTCGTTC-3′). Reactions proceeded for 30 cycles of denaturation at 94 C for 15 sec, annealing at 60 C for 45 sec and extension at 72 C for 45 sec, with a final extension at 72 C for 7 min. The IGF-IR floxed gene was also identified by PCR using oligonucleotides that amplify a 380-bp product spanning exon 3 of the IGF-IR gene (5′-TCCCTCAGGCTTCATCCGCAA-3′ and 5′-CTTCAGCTTTGCAGGTGCACG-3′). Reactions proceeded for 25 cycles of denaturation at 95 C for 30 sec, annealing at 59 C for 30 sec and extension at 72 C for 40 sec with a final extension at 72 C for 7 min. All reactions were performed under standard conditions using 100 ng of genomic DNA, 0.5 pmol/μl primers, 2.5 mm MgCl2, and 0.02 U/μl Taq DNA polymerase per reaction.

Auxological and hormonal studies

A microtattooing strategy was used to label pups at 0–2 d of life (Animal Tattoo Ink; Ketchum Manufacturing, Inc., Brockville, Ontario, Canada). Total body length (naso-anal) was recorded with steel vernier calipers, and body weight was obtained on a single electronic scale. Blood from mice at 6–8 wk of life was obtained via a nonterminal vein bleed after a 12–14 h overnight fast. Blood collection was also done with animals at 28–32 wk of age. Serum was collected after centrifugation and stored at −20 C; 20 μl of serum were analyzed for GH using the xMap technology (Millipore, Billerica, MA). A standard curve was generated using 5-fold serial dilutions of the GH standard cocktail provided by the vendor. Standards and samples were incubated with the antibody coated beads on a microplate shaker overnight at 4 C and washed three times using a vacuum manifold apparatus. Detection antibody was then added to the wells and incubated on a microplate shaker at room temperature for 30 min. Streptavidin-phycoerythrin solution was then added for an additional 30-min incubation at room temperature and placed on a microplate shaker. Plates were then washed three times, and sheath fluid was added to each well. Beads were resuspended microplate shaker for 5 min. Plates were then read on the Luminex 200IS System with xPonent software. Data were analyzed with a 5-parametelogistic curve fitting. The limit of detection for the assay for GH was 12 pg/ml; the interassay coefficient variance was less than 9.5%, and the intraassay coefficient of variance was less than 15%. Measurement of serum IGF-I required a 5 μl serum also using the xMap technology (Millipore). A standard curve was generated as above, and the limit of detection for the assay for IGF-I was 3.2 pg/ml. The interassay variation was less than 10%, and intraassay variation less than 5%. The assay procedure and data analysis were as described above using IGF-I detection antibody beads.

IGFBP-3 levels were measured on serum collected from mice as described. The levels were determined using the mouse/rat IGFBP-3 ELISA kit (Mediagnost, Reutlingen, Germany). Serum was diluted 1:301 with dilution buffer, and 100 μl were pipetted into wells. Each sample was assayed in duplicate. After 1-h incubation, wells were washed and antibody conjugate was added for an additional 1-h incubation at room temperature. Wells were washed again, and enzyme conjugate was added for a 15-min incubation. A 3,3′,5,5′-tetramethylbenzidine-substrate solution was added after a third washing and followed by the addition of stop solution. Measurement of the absorbance was performed at 450 nm using a Bio-Rad microplate reader (Model 680XR; Bio-Rad, Hercules, CA). A standard curve was created using a four parametric logistic curve. The inter- and intraassay variation coefficients were less than 8.4 and 4.4%, respectively. Average values were plotted using GraphPad Prism software.

Western blot analysis for ALS

Serum from SIGFRKO and control littermates was diluted 1:5 in PBS solution, separated on a 10% acrylamide gel and transferred to nitrocellulose. The membrane was blocked using Tris-buffered saline with Tween 20 (TBST) containing 5% fat-free dry milk powder for 1 h at room temperature. The membrane was then incubated in TBST (5% milk) containing either the ALS antibody (1:1000 dilution, AF1436; R&D Systems, Minneapolis, MN) or actin antibody (1:10,000, MAB1502; Millipore, Temecula, CA) at 4 C overnight. After washing with TBST, the membrane was incubated in TBST (5% milk) containing the appropriate secondary antibody (1:5000 dilution). The membrane was washed in TBST, developed for 5 min using enhanced chemiluminescence solution and exposed to x-ray film. The intensity of each band was measured using the ChemiDoc XR5 system (Bio-Rad) along with Quantity One software (version 4.6).

IHC of the pituitary

Animals were anesthetized with ketamine/xylazine and perfused using 4% cold paraformaldehyde and after removal transferred to 10% formalin. Pituitaries were washed twice with 1× PBS and transferred to 70% ethanol overnight at 4 C. After the dehydration, the pituitary was processed for paraffin embedding and 6-μm sections were cut. Pituitary sections were deparaffinized and serially rehydrated in ethanol. After antigen retrieval with 3% horse serum/1× PBS solution, somatotrophs were labeled using a guinea-pig antirat GH primary antibody (1:500 dilution; A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program), and IGF-IR’s were labeled with a rabbit-antimouse primary antibody [1:5000 dilution, IGF-IRβ (C-20), sc-713; Santa Cruz Biotechnology, Inc., Santa Cruz, CA]. After incubation for 16 h at 4 C, cyanine-3 (Cy3) and fluorescein isothiocyanate (FITC) conjugated secondary antibodies were applied to the sections followed by 1-h incubation at room temperature. Sections were then washed with 1× PBS and mounted with Vectashield mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI). Slides were viewed using a fluorescence inverted microscope (Zeiss Axioskop 2; Zeiss, Thornwood, NY) equipped with a charged-coupled device digital camera for image capture and processing with Axiovision (Zeiss) software. Images were captured using appropriate fluorescence filters for DAPI, FITC, and Cy3 detection. Overlay images of different fluorescence signals were generated using Axiovision software. Magnification shown is ×400 using ×40 oil immersion objective.

Animal care

All the animal procedures were performed according to the Johns Hopkins University protocol approved by the Animal Care and Use Committee.

Quantitative RT-PCR for hypothalamic and pituitary gene expression

Total RNA was obtained from hypothalamic and pituitary tissue by Trizol (Invitrogen, Carlsbad, CA) extractions as previously described (43,44); 2 μg of RNA were reverse transcribed (iScript cDNA Synthesis kit; Bio-Rad) to produce cDNA. cDNA obtained from 50 ng of total RNA was used in each reaction; 25-μl PCRs were performed using the IQ SybrGreen supermix (Bio-Rad). Reactions were measured using the MyiQ qRT-PCR machine (Bio-Rad). Primer sets for IGF-IR (sense 5′-CGACGTATGAGAACTTCATGC-3′ and antisense 5′-CACATGGTGACAATTGAACTCC-3′), GH (sense 5′-CCTCAGCAGGATTTTCACCA-3′ and antisense 5′-CTTGAGGATCTGCCCAACAC-3′), GHRH-R (sense 5′-ACCCGTATCCTCTGCTTGCT-3′ and antisense 5′-AGGTGTTGTTGGTCCCCTC T-3′), somatostatin receptor 2 (sense 5′-TGATCCTCACC TATGCCAACA-3′ and antisense 5′-CTGC CTTGACCAAGCAAAGA-3′), somatostatin receptor 5 (sense 5′-ACCCCCTGCTCTATGGCTTT-3′ and antisense 5′-GCTCTATGGCA TCTGCATCCT-3′), GHRH (sense 5′-TGCCATCTT CACCACCAAC-3′ and antisense 5′-TCAT CTGCT TGTC CTCTGTCC-3′), SRIF (sense 5′-TCTGCATCGTCCTGGCTTT-3′ and antisense 5′-CTTGGCCAGTTCCTGT TTCC-3′), and a ribosomal 18S control (sense 5′-TGGTTGATCCTGCCAGTAG-3′ and antisense 5′-CGACCAAAGGAACCAT AACT-3′) were used (45,46,47). PCR conditions were optimized to generate more than 95% PCR efficiency, and only those reactions between 95 and 105% efficiency were included in subsequent analysis. Cycle threshold (Ct) was obtained for each sample. A corrected Ct (ΔCt) was calculated by subtracting the 18S Ct from the unknown sample Ct for each sample. Relative differences from the control sample were then calculated by using the formula: fold change = 2^(control ΔCt − sample ΔCt).

Organ weights

Mice were killed at 35–38 wk of life by anesthesia followed by cervical dislocation. Tissues, including brain, heart, lungs, liver, spleen, and kidney, were collected and immediately weighed.

Metabolic studies

Body composition studies

EchoMRI-100 QNMR system (Echo Medical System, Houston, TX) was used to measure whole body composition parameters in SIGFRKO and control mice between 28 and 32 wk of age. Direct measurements were taken in vivo of total body fat, lean mass, free water, and total body water. Measurements were performed in triplicate.

GTT and ITT

Adult SIGFRKO and control male mice (17–19 wk of life) were fasted overnight. BG levels were measured from tail blood using the OneTouch Ultra glucometer (LifeScan, Inc., Milipitas, CA). During the GTT, mice were given an ip injection of a 20% glucose solution equivalent to 2 g/kg body weight. Mouse BG levels were measured at 10, 20, 30, 60, 90, and 120 min. During the ITT, mice were given an ip injection of insulin (0.8 U/kg) after recording fasting BG level. Glucose levels were measured at 10, 20, 30, 60, 90, and 120 min.

Acknowledgments

We thank Mehboob Hussain and Jimmy Song for their expertise with IHC and use of the fluorescence inverted microscope; Tameeka Williams and Katie Brothers for assistance in mouse blood collections; and Fredric Wondisford, Andrew Wolfe, and Sara Divall for their critique of this manuscript.

Footnotes

This work was supported by National Institutes of Health Grants T32DK07751 (to S.R.) and 1F32DK081280-01 (to C.J.R.). Additional support was provided by the Veteran Affairs Merit and National Institute on Aging Grant 5621AG031465VA (to R.D.K.) and the Spanish of Ministry of Science and Innovation Grants JC2008-00220 and RYC-2007-00186 (to R.M.L.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 8, 2010

Abbreviations: ALS, Acid labile subunit; BG, blood glucose; cKO, cell-specific KO; Ct, cycle threshold; Cy3, cyanine-3; DAPI, 4′,6′-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; GHRH-R, GHRH receptor; GTT, glucose tolerance test; IGFBP-3, IGF binding protein 3; IGF-IR, IGF-I receptor; IHC, immunohistochemistry; ITT, insulin tolerance test; KO, knockout; MRI, magnetic resonance imaging; qRT-PCR, quantitative real-time PCR; rGHpCre, Cre recombinase downstream from the GH promoter; SIGFRKO, IGF-IR KO; SRIF, somatostatin; TBST, Tris-buffered saline with Tween 20.

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