Impact of Androgens, Growth Hormone, and IGF ... - Wiley Online Library

JOURNAL OF BONE AND MINERAL RESEARCH Volume 22, Number 1, 2007 Published online on October 2, 2006; doi: 10.1359/JBMR.060911 © 2007 American Society for Bone and Mineral Research

Impact of Androgens, Growth Hormone, and IGF-I on Bone and Muscle in Male Mice During Puberty Katrien Venken,1 Sofia Movérare-Skrtic,2 John J Kopchick,3 Karen T Coschigano,3 Claes Ohlsson,2 Steven Boonen,1 Roger Bouillon,1 and Dirk Vanderschueren1

ABSTRACT: The interaction between androgens and GH/IGF-I was studied in male GHR gene disrupted or GHRKO and WT mice during puberty. Androgens stimulate trabecular and cortical bone modeling and increase muscle mass even in the absence of a functional GHR. GHR activation seems to be the main determinant of radial bone expansion, although GH and androgens are both necessary for optimal stimulation of periosteal growth during puberty. Introduction: Growth hormone (GH) is considered to be a major regulator of postnatal skeletal growth, whereas androgens are considered to be a key regulator of male periosteal bone expansion. Moreover, both androgens and GH are essential for the increase in muscle mass during male puberty. Deficiency or resistance to either GH or androgens impairs bone modeling and decreases muscle mass. The aim of the study was to investigate androgen action on bone and muscle during puberty in the presence and absence of a functional GH/insulin-like growth factor (IGF)-I axis. Materials and Methods: Dihydrotestosterone (DHT) or testosterone (T) were administered to orchidectomized (ORX) male GH receptor gene knockout (GHRKO) and corresponding wildtype (WT) mice during late puberty (6–10 weeks of age). Trabecular and cortical bone modeling, cortical strength, body composition, IGF-I in serum, and its expression in liver, muscle, and bone were studied by histomorphometry, pQCT, DXA, radioimmunoassay and RT-PCR, respectively. Results: GH receptor (GHR) inactivation and low serum IGF-I did not affect trabecular bone modeling, because trabecular BMD, bone volume, number, width, and bone turnover were similar in GHRKO and WT mice. The normal trabecular phenotype in GHRKO mice was paralleled by a normal expression of skeletal IGF-I mRNA. ORX decreased trabecular bone volume significantly and to a similar extent in GHRKO and WT mice, whereas DHT and T administration fully prevented trabecular bone loss. Moreover, DHT and T stimulated periosteal bone formation, not only in WT (+100% and +100%, respectively, versus ORX + vehicle [V]; p < 0.05), but also in GHRKO mice (+58% and +89%, respectively, versus ORX + V; p < 0.05), initially characterized by very low periosteal growth. This stimulatory action on periosteal bone resulted in an increase in cortical thickness and occurred without any treatment effect on serum IGF-I or skeletal IGF-I expression. GHRKO mice also had reduced lean body mass and quadriceps muscle weight, along with significantly decreased IGF-I mRNA expression in quadriceps muscle. DHT and T equally stimulated muscle mass in GHRKO and WT mice, without any effect on muscle IGF-I expression. Conclusions: Androgens stimulate trabecular and cortical bone modeling and increase muscle weight independently from either systemic or local IGF-I production. GHR activation seems to be the main determinant of radial bone expansion, although GHR signaling and androgens are both necessary for optimal stimulation of periosteal growth during puberty. J Bone Miner Res 2007;21:72–82. Published online on October 2, 2006; doi: 10.1359/JBMR.060911 Key words: growth hormone receptor gene disrupted or knockout mice, puberty, orchidectomy, androgens, growth hormone, insulin-like growth factor I, trabecular bone, periosteal bone, muscle mass ure.(1,2) The biological effects of GH have been related to the production of insulin-like growth factor-I (IGF-I), of which the GH-dependent hepatic IGF-I expression is the most important.(3) Liver-derived IGF-I acts in an endocrine fashion on target organs, including bone. Although GH action is not exclusively IGF-I dependent, and IGF-I action is not solely GH dependent, their interdependent roles in the

INTRODUCTION

G

ROWTH HORMONE (GH) is a primary regulator of postnatal growth. Inadequate concentrations of, or insufficient response to, GH results in manifest growth fail-

The authors state that they have no conflicts of interest.

1 Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium; 2Center for Bone Research, Department of Internal Medicine, The Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden; 3Edison Biotechnology Institute and Department of Biomedical Sciences, Ohio University, Athens, Ohio, USA.

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ACTION OF ANDROGENS, GH, AND IGF-I ON MALE BONE AND MUSCLE regulation of normal growth have been well established.(4) The GH receptor (GHR) gene disrupted or knockout (GHRKO) mouse model is a model for the absence of GH signaling,(5) and in accordance to the human GH insensitivity syndrome (also called Laron syndrome), characterized by severe postnatal growth failure, elevated GH levels and greatly decreased serum IGF-I concentrations.(6) Beside the well-known effects of GH on longitudinal skeletal growth, both mice and men with inactivation of GHR signaling also have decreased cross-sectional bone area and reduced cortical thickness,(7–10) which is considered to be related to the low serum IGF-I concentrations.(11,12) In addition to the well-documented role of the GH/IGF-I axis, androgens also play an essential role in male pubertal growth and radial bone expansion.(13,14) A greater enlargement of the bone diameter and greater increase in cortical thickness, resulting from an accelerated periosteal bone apposition, is characteristic for male pubertal growth in comparison with females. This effect has traditionally been attributed to the direct stimulatory action of testosterone (T) on periosteal bone through the androgen receptor (AR).(15,16) Additionally, androgen resistance abolishes the typical male bone phenotype as a female-like bone structure is observed in rodents and humans with functionally inactive ARs.(17–20) Interestingly, inhibition of IGF-I action in mice, either directly(21,22) or indirectly through inactivation of the GHR,(6,8) also disrupts the skeletal sexual dimorphism. Moreover, both androgens and GH must be present for normal pubertal growth to occur because pubertal growth is attenuated in patients with either GH deficiency/resistance or hypogonadism.(23) In this context, the male bone phenotype seems to require a functional GH/ IGF-I axis and an interaction between androgens and GH/ IGF-I may therefore provide a mechanism for the regulation of male skeletal growth. In addition to the endocrine control of pubertal skeletal growth, overall body growth also regulates skeletal growth as a result of increased mechanical loading imposed on the skeleton.(24) The increase in body weight and muscle mass during puberty is associated with increased loading and, in turn, bone deformation or strain, which ultimately results in increased bone modeling and (micro)architectural adaptations of bone tissue.(25) As a consequence, the increase in bone mass and muscle mass are closely linked during growth.(26) The greater periosteal apposition observed during male pubertal growth confers greater resistance to bending and hence greater bone strength.(27) Both androgens and GH are essential for the increase of muscle mass during growth and the maintenance of muscle into adulthood, because both GH deficiency/resistance and hypogonadism are associated with decreased muscle mass and reduced muscle strength.(28,29) Whether or how androgens and GH/IGF-I interact with respect to muscle size, BMD, bone geometry, and bone strength remains unclear. In this study, the impact of loss of GHR signaling on trabecular and cortical bone and body composition during puberty was investigated in GHRKO and corresponding wildtype (WT) mice. In addition, we evaluated the effects of orchidectomy (ORX) and replacement therapy with DHT or T on trabecular and cortical bone and muscle in

73

GHRKO and corresponding WT mice to study androgen action in the presence and absence of a functional GH/ IGF-I axis during male puberty.

MATERIALS AND METHODS Animals Homozygous GHRKO mice were generated by disruption of the GHR/BP gene by homologous recombination, as previously described.(6) Their genetic background was 129Ola/Balb/c. Genotyping was performed using PCR amplification.(30) WT littermates were used as controls. Mice were housed in conventional conditions: 12-h light/dark cycle, standard diet (1% calcium, 0.76% phosphate), and water ad libitum.

Experimental design At 6 weeks of age, male WT and homozygous GHRKO mice were randomly divided into groups. A baseline group (BASE) was killed at the beginning of the experiment. The other mice were either sham-operated (SHAM) or ORX. ORX mice were treated for 4 weeks with vehicle (V), dihydrotestosterone (DHT; Fluka), or testosterone (T; Serva), by subcutaneous silastic implants (Silclear Tubing; Degania Silicone, Jordan Valley, Israel) in the cervical region.(31) Vehicle animals received empty implants. Eight animals were included in each group. Body weight was measured weekly. All mice received two intraperitoneal injections of the fluorochrome calcein at a 5-day interval and were killed 1 day after the second injection. At death, serum was collected, stored at –20°C, and used for osteocalcin and IGF-I measurements. Left femurs and tibias were dissected and used to perform pQCT and histomorphometric analyses. Efficacy of ORX and DHT and T replacement was verified by measurement of seminal vesicle wet weight immediately after death. Mean seminal vesicle weights of SHAM WT and GHRKO mice expressed per gram body weight was 5.9 ± 0.4 and 6.8 ± 0.6 mg/g, respectively. ORX reduced the mean seminal vesicle weight to 0.5 ± 0.1 mg/g for WT and 0.4 ± 0.1 mg/g for GHRKO mice (p < 0.001). Mean seminal vesicle weights were 7.2 ± 0.4 and 11.2 ± 0.7 mg/g for the DHT-treated WT and GHRKO groups, respectively, and 8.4 ± 0.3 and 13.0 ± 0.7 mg/g for the Ttreated WT and GHRKO groups, respectively, which reflects a slightly supraphysiological dose in androgen-treated mice. The ethical committee of the Katholieke Universiteit Leuven approved all experimental procedures.

Histomorphometric analysis Left femurs and tibias were immersed in Burckhardt’s fixative (24 h, 4°C), transferred to 100% ethanol, and embedded in methylmethacrylate. Longitudinal sections of the undecalcified tibia were cut at 4 ␮m thickness, using a rotation microtome (RM 2155 Autocut; Leica, Heidelberg, Germany) with tungsten carbide blade. Sections were stained by a modified Goldner technique and subjected to static histomorphometry. Measurements were performed in the secondary spongiosa of at least three Goldner-stained sections, as previously described.(18,32) In each section,

74 three consecutive fields were measured along the vertical axis of the central metaphysis, starting at a fixed distance of the growth plate. Trabecular width and trabecular number were calculated according to the parallel plate model developed by Parfitt et al.(33) Cross-sections of the undecalcified femur perpendicular to the long axis were prepared at 200 ␮m thickness in the mid-diaphyseal region using the contact-point precision band saw (Exakt, Norderstedt, Germany). Sections were ground to a final thickness of 25 ␮m using a grinding system (Exakt) and were left unstained. Three sections in the middiaphyseal region were measured by fluorescence microscopy, and the bone formation rate (BFR/B.Pm., ␮m2/␮m/ day) was assessed at both the endocortical and periosteal bone surfaces. The BFR was obtained by the product of mineral apposition rate (MAR) and mineralizing perimeter per bone perimeter (Min.Pm./B.Pm., %). The mineralizing perimeter was calculated as follows: Min.Pm. ⳱ (dL + [sL/ 2])/B.Pm., where dL represents the length of the double labels and sL represents the length of single labels along the entire endocortical or periosteal bone surfaces. The MAR (␮m/day) was calculated as the mean width of double labels, divided by interlabel time (5 days). Min.Pm. is a measure of osteoblast number and MAR of osteoblast activity. All measurements were performed with a Kontron Image Analyzing computer (KS400 3.00; Kontron Bildanalyze, Munich, Germany) and a Zeiss microscope with drawing attachment. Specific software was developed in collaboration with the manufacturer. Histomorphometric parameters are reported according to the recommended American Society for Bone and Mineral Research nomenclature.(33)

pQCT Trabecular and cortical volumetric BMC and BMD, bone geometry, and strength strain index (SSI) of the left femur were assessed ex vivo by pQCT using the Stratec XCT Research M+ densitometer (Norland Medical Systems, Fort Atkinson, WI, USA). Slices of 0.2 mm thickness were scanned using a voxel size of 0.070 mm. Three slices were taken 2 mm from the distal end of the femur, using contmode 1, peelmode 20, and a density threshold of 280 mg/cm3. The trabecular bone region was defined by setting an inner threshold corresponding to 30% of the total crosssectional area. These metaphyseal scans were performed to measure trabecular BMC and BMD, which are expressed as the mean ± SE of the three slices. One scan was taken 7 mm for WT and 4 mm for GHRKO from the distal end of the femur (an area containing only cortical bone) using separation mode 1 and density threshold of 710 mg/cm3. This mid-diaphyseal scan was performed to determine cortical BMC, BMD, cortical thickness, cortical area, endocortical and periosteal perimeters, and SSI.

Whole body DXA Body composition was analyzed in vivo by DXA (PIXImus densitometer; Lunar Corp., Madison, WI, USA), using ultra-high resolution (0.18 × 0.18 pixels, resolution of 1.6 line pairs/mm) and software version 1.45. DXA was performed at the start and the end of the experimental period.

VENKEN ET AL.

Assays Serum osteocalcin was measured by an in-house radioimmunoassay (RIA).(34) After acid-ethanol extraction, serum IGF-I concentrations were measured by an in-house RIA(35,36) in the presence of an excess of IGF-II (25 ng/ tube).

RT-PCR analysis Total RNA was prepared from liver, quadriceps muscle, and femur (n ⳱ 6–8/group), using TriZol Reagent (Life Technologies). RT-PCR analysis was performed with the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Stockholm, Sweden), using probes labeled with the reporter fluorescent dye FAM. The sequence for the specific IGF-I probe was 5⬘-TTCAACAAGCCCACAGGCTAT-3⬘ and for the IGF-I primers was forward primer (FP), 5⬘-GCTCTTCAGTTCGTGTGTGGAC-3⬘, and reverse primer (RP), 5⬘-CATCTCCAGTCTCCTCAGATC3⬘. Predesigned primers and a probe labeled with the reporter fluorescent dye VIC, specific for 18S rRNA, were included in the reactions as an internal standard. The oligonucleotide primers and probes were purchased from PE Applied Biosystems. The cDNA was amplified using the following conditions: 1 cycle at 50°C for 2 minutes and 95°C for 1 minute, followed by 40 cycles at 95°C for 15 s and 60°C for 1 minute. The mRNA amount of each gene was calculated using the ‘‘standard curve method’’ (multiplex reaction, following the instructions in User Bulletin 2; PE Applied Biosystems) and adjusted for the expression of 18S rRNA.

Statistical analysis Statistical analysis of data were performed using NCSS software (Kaysville, UT, USA). One-way ANOVA, followed by Fisher’s least significant difference multiple comparison test, and t-test were performed to assess significance of difference between groups of the same genotype and between respective WT and GHRKO groups. p < 0.05 was accepted as significant. For cortical BMD, cortical thickness, cortical area, and SSI, analysis of covariance (ANCOVA) models were used to test whether a difference between V-treated and androgen-treated ORX mice exists also after correction for femur length and DXA-derived lean body mass. This was done within the group of knockout and WT mice separately. p < 0.05 was accepted as significant. Data are represented as mean ± SE.

RESULTS Trabecular phenotype and effects of T and DHT on trabecular bone in GHRKO mice Trabecular BMC was significantly lower in GHRKO mice compared with WT mice (Table 1). Interestingly, trabecular bone volume (bone area per total area; B.Ar./ T.Ar., %) and BMD were similar in SHAM GHRKO and WT mice (Fig. 1A; Table 1). Trabecular microarchitecture was characterized by an equal number and normal size (width) of trabeculae in both genotypes (Figs. 1B and 1C). ORX significantly decreased trabecular bone volume and

116 ± 13 85 ± 11* (+26%) 1.067 ± 0.041* (+29%) 0.387 ± 0.027*¶ (+49%) 1239 ± 9* (+9%) 986 ± 15*¶ (+11%) 215 ± 4* (+21%) 124 ± 3*¶ (+39%) 0.86 ± 0.03* (+16%) 0.39 ± 0.02*¶ (+11%) 3.33 ± 0.07* (−6%) 2.93 ± 0.09*¶ (−14%) 4.68 ± 0.08 3.68 ± 0.07¶ 0.382 ± 0.022* (+22%) 0.111 ± 0.006*¶ (+32%)

123 ± 7 67 ± 10

0.827 ± 0.022 0.259 ± 0.012

1134 ± 8 891 ± 6¶

177 ± 2 89 ± 3¶

0.73 ± 0.02 0.29 ± 0.01¶

3.56 ± 0.06 3.42 ± 0.09¶

4.67 ± 0.07 3.65 ± 0.09¶

0.313 ± 0.015 0.084 ± 0.006¶

0.128 ± 0.019 0.044 ± 0.007¶

SHAM

CORTICAL BONE PARAMETERS

0.148 ± 0.013 0.040 ± 0.007¶

AND

15.6 ± 0.1* (+5%) 11.5 ± 0.2¶

BASE

TRABECULAR

14.8 ± 0.2 11.3 ± 0.1¶

AND

AS

BY

pQCT IN THE

214 ± 4* (+21%) 134 ± 4*‡¶ (+51%)

202 ± 4*† (+14%) 122 ± 5*¶ (+37%)

0.356 ± 0.014 0.100 ± 0.011¶

4.71 ± 0.05 3.65 ± 0.08¶

3.44 ± 0.05 2.99 ± 0.06*¶ (−13%)

0.399 ± 0.025* (+27%) 0.114 ± 0.006*¶ (+35%)

4.77 ± 0.09 3.75 ± 0.07¶

3.44 ± 0.09 3.06 ± 0.08*¶ (−11%)

0.88 ± 0.03* (+24%) 0.38 ± 0.02*¶ (+14%)

1228 ± 10* (+8%) 956 ± 14*¶ (+7%)

1209 ± 8*† (+6%) 936 ± 18*†¶ (+5%)

0.83 ± 0.03* (+12%) 0.35 ± 0.03*¶ (+2%)

1.080 ± 0.036* (+31%) 0.361 ± 0.019*¶ (+39%)

0.396 ± 0.024* (+27%) 0.110 ± 0.008*¶ (+31%)

4.74 ± 0.10 3.86 ± 0.06*†‡¶ (+6%)

3.36 ± 0.10 3.14 ± 0.05*¶ (−8%)

0.89 ± 0.03* (+20%) 0.38 ± 0.02*¶ (+25%)

221 ± 4*‡ (+24%) 136 ± 3*†‡¶ (+53%)

1254 ± 9*‡ (+11%) 954 ± 8*¶ (+7%)

1.121 ± 0.032*‡ (+36%) 0.359 ± 0.019*¶ (+39%)

129 ± 17‡ 163 ± 17*†‡§ (+143%)

0.129 ± 0.016‡ 0.096 ± 0.011*†‡§ (+140%)

0.133 ± 0.018‡ 0.070 ± 0.006*†‡¶ (+75%) 131 ± 18‡ 124 ± 10*†‡ (+85%)

15.5 ± 0.2* (+5%) 11.8 ± 0.1¶

ORX + T

15.7 ± 0.1* (+6%) 11.7 ± 0.2¶

ORX + DHT

DIFFERENT EXPERIMENTAL GROUPS COMPARED WITH BASELINE

0.999 ± 0.028* (+21%) 0.328 ± 0.033*¶ (+27%)

13 ± 2*† (−90%) 39 ± 6†¶

0.013 ± 0.002*† (−91%) 0.021 ± 0.003†¶

15.8 ± 0.2* (+7%) 11.4 ± 0.2¶

ORX + V

ASSESSED

Values are mean ± SE. Six-week-old mice were randomly divided in groups. A baseline group (BASE) was killed at the start of the experiment (6 weeks of age). The other mice were SHAM or ORX. ORX mice were treated with V, DHT, or T for 4 weeks. Significant changes from baseline are indicated in parentheses as percentage increase or decrease. (n ⳱ 6–8 mice/group). * p < 0.05 vs. respective BASE group. † p < 0.05 vs. respective SHAM group. ‡ p < 0.05 vs. respective ORX + V group. § p < 0.05 vs. respective ORX + DHT group. ¶ p < 0.05 vs. respective WT group.

Femur length (mm) WT GHRKO Trab. BMC (mg) WT GHRKO Trab. BMD (mg/cm3) WT GHRKO Cort. BMC (mg) WT GHRKO Cort. BMD (mg/cm3) WT GHRKO Cort. thickness (␮m) WT GHRKO Cort. area (mm2) WT GHRKO Ec. Pm.(mm) WT GHRKO Ps. Pm.(mm) WT GHRKO SSI (mm3) WT GHRKO

TABLE 1. BONE LENGTH

ACTION OF ANDROGENS, GH, AND IGF-I ON MALE BONE AND MUSCLE 75

76

VENKEN ET AL.

FIG. 2. (A) Serum osteocalcin (ng/ml) and (B) osteoid perimeter per bone perimeter (O.Pm./B.Pm., %) in SHAM or ORX WT and GHRKO mice. ORX mice were treated with V, DHT, or T. Mice were SHAM or ORX at 6 weeks of age and were treated for 4 weeks. ap < 0.05 vs. SHAM, bp < 0.05 vs. ORX + V.

FIG. 1. (A) Trabecular bone volume (B.Ar./T.Ar, %), (B) trabecular (trab.) number (mm−1), and (C) trabecular width (␮m) in SHAM or ORX WT and GHRKO mice. ORX mice were treated with V, DHT, or T. Mice were SHAM or ORX at 6 weeks of age and were treated for 4 weeks. ap < 0.05 vs. SHAM; bp < 0.05 vs. ORX + V.

BMD in both GHRKO and WT mice (p < 0.001; Fig. 1A; Table 1). Trabecular bone loss was explained by a reduction in the number of trabeculae (Fig. 1B), whereas trabecular width remained unchanged (Fig. 1C). The ORX-induced changes in trabecular microstructure were associated with an increased bone turnover rate, as reflected by the increased serum osteocalcin levels (Fig. 2A), enhanced osteoid perimeter (Fig. 2B), and enhanced endocortical BFR and MAR (Table 2). Administration of DHT or T com-

pletely prevented the loss of trabecular bone even in the absence of a functional GHR (Fig. 1A; Table 1). Both androgens therefore preserved the number of trabeculae but did not affect the trabecular width (Figs. 1B and 1C). This bone-sparing effect was further evidenced by a suppression of serum osteocalcin levels and reduction in osteoid perimeter (Figs. 2A and 2B). Additionally, the suppression of the endocortical BFR in both GHRKO and WT mice, resulting from a reduction of the endocortical MAR, further completed the picture of the bone-sparing action of DHT and T, which was clearly independent from the presence of a functional GHR (Table 2). Longitudinal growth, as assessed by femur length, was significantly reduced in GHRKO mice, but remained unchanged in response to androgen withdrawal and replacement in both GHRKO and WT (Table 1).

Cortical phenotype and the effects of T and DHT on cortical bone in GHRKO mice In a previous study,(12) we showed that the periosteal BFR, as measured by dynamic histomorphometric analysis, was dramatically reduced (−76%, p < 0.001) in GHRKO mice, resulting in nearly 50% decreases of static parameters of bone size, such as cross-sectional area and cortical thick-

ACTION OF ANDROGENS, GH, AND IGF-I ON MALE BONE AND MUSCLE TABLE 2. DYNAMIC HISTOMORPHOMETRIC PARAMETERS SHAM Ec.Min.Pm./B.Pm. (%) WT GHRKO Ec.MAR (␮m/day) WT GHRKO Ec.BFR/B.Pm. (␮m2/␮m/day) WT GHRKO Ps.Min.Pm./B.Pm. (%) WT GHRKO Ps.MAR (␮m/day) WT GHRKO Ps.BFR/B.Pm (␮m2/␮m/day) WT GHRKO

OF

77

RADIAL BONE GROWTH

ORX + V

ORX + DHT

ORX + T

69.4 ± 7.6 94.9 ± 1.5‡

83.4 ± 2.1 95.8 ± 1.6‡

73.4 ± 4.1 89.7 ± 2.1†

70.3 ± 8.6 88.0 ± 2.8†

3.31 ± 0.59 3.46 ± 0.82

4.08 ± 0.89 7.46 ± 1.05*‡

2.02 ± 0.19† 4.60 ± 0.65†‡

1.77 ± 0.39† 3.35 ± 1.32†

2.74 ± 0.60 3.36 ± 0.81

3.76 ± 0.90 7.37 ± 1.06*‡

1.47 ± 0.14† 4.21 ± 0.70†‡

1.47 ± 0.39† 3.19 ± 1.33†

39.3 ± 6.3 15.5 ± 4.1‡

21.0 ± 4.6* 21.2 ± 5.5

36.9 ± 3.9† 30.3 ± 3.0*

37.5 ± 3.2† 36.4 ± 6.0*†

1.06 ± 0.07 0.88 ± 0.04‡

0.89 ± 0.08 0.78 ± 0.10

1.05 ± 0.02 0.86 ± 0.05‡

1.06 ± 0.08 0.86 ± 0.10‡

0.47 ± 0.10 0.13 ± 0.04‡

0.21 ± 0.05* 0.19 ± 0.05

0.43 ± 0.05† 0.30 ± 0.04*‡

0.43 ± 0.06† 0.36 ± 0.07*†

Values are mean ± SE. Six-week-old mice were SHAM or ORX. ORX mice were treated with V, DHT, or T for 4 weeks. * p < 0.05 vs. respective SHAM group. † p < 0.05 vs. respective ORX + V group. ‡ p < 0.05 vs. respective WT group (n ⳱ 6–8 mice/group). Ec, endocortical bone surface; Ps, periosteal bone surface.

ness. Moreover, the decrease in radial bone expansion was highly correlated with the lowered serum IGF-I concentrations.(12) We hereby confirm the dramatic cortical phenotype in GHRKO mice associated with low serum IGF-I concentrations (