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.

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.

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.

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).

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.

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).

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.

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.

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.

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.

Summary of findings in the SIGFRKO mouse model.