The authors have no conflict of interest.
Longitudinal In Vivo Effects of Growth Hormone Overexpression on Bone in Transgenic Mice†
Article first published online: 15 MAR 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 5, pages 802–810, May 2004
How to Cite
Eckstein, F., Weusten, A., Schmidt, C., Wehr, U., Wanke, R., Rambeck, W., Wolf, E. and Mohan, S. (2004), Longitudinal In Vivo Effects of Growth Hormone Overexpression on Bone in Transgenic Mice. J Bone Miner Res, 19: 802–810. doi: 10.1359/jbmr.040308
- Issue published online: 2 DEC 2009
- Article first published online: 15 MAR 2004
- Manuscript Accepted: 16 JAN 2004
- Manuscript Revised: 11 DEC 2003
- Manuscript Received: 1 AUG 2003
- growth hormone;
- bone turnover;
- bone mineral;
- longitudinal study
In this study we examined the effect of systemic overexpression of GH on bone in transgenic mice longitudinally in vivo over a period of 9 months. We observed substantially increased BMC in GH transgenic mice and a significant reduction in serum osteocalcin. GH effects on bone were strongly dependent on gender and developmental stage.
Introduction: State-of-the-art bone marker and microimaging technology was applied in this longitudinal study to examine bone metabolism, BMC, bone density, and cortical bone structure over the life span of growth hormone (GH) transgenic (tg) mice.
Materials and Methods: Thirty-eight mice from four genetic groups (male, female, tg, and controls) were examined with DXA, and their femur and tibia were examined with peripheral QCT (pQCT). Osteocalcin (formation) and collagen cross-links (resorption) from serum and urine were also measured at postnatal weeks 3, 6, 9, 12, 18, 26, and 38.
Results: GH tg mice displayed a significant increase in body weight (up to 50%) and BMC (up to 90%), but serum osteocalcin was significantly reduced compared with controls. GH tg females (but not males) displayed increased trabecular density over controls up to week 12. In contrast, male (but not female) GH tg mice displayed a higher cortical cross-sectional area than controls. Cortical density was significantly lower in both male and female GH tg mice compared with control mice.
Conclusions: The increase in BMC in GH tg mice is associated with reduced serum osteocalcin levels, indicating that bone turnover may be lower than in the control mice. On a structural level, bone responds to GH excess in a gender-specific manner, with alterations varying substantially between different developmental stages.
Transgenic and knockout mice represent important models for examining the function of genes and gene products in vivo.(1) Such models are very important in the field of bone research, allowing the functional dissection of the complex systems regulating bone metabolism, BMC, bone density, and bone structure. These systems include endocrine, paracrine, autocrine, mechanical, and neural factors.(2–5) Typically, skeletal phenotyping of transgenic mice is performed using static and dynamic bone histomorphometry,(6–8) but these techniques can only be applied ex vivo. Because animals should be used with austerity, analysis is usually confined to one specific developmental stage. If different developmental stages are to be examined, different groups of animals need to be killed at different points in time.(9–11) However, under these conditions, the statistical analysis of longitudinal changes is limited by intersubject variability. A more elegant approach is to study identical animals longitudinally, but this requires noninvasive technology to be used in vivo.
To our knowledge, no previous study has examined the skeletal phenotype of a transgenic mouse model longitudinally throughout various stages of development. Quantification of bone markers permits indirect analysis of bone formation and resorption rates by noninvasive means from serum and urine samples.(12) DXA and peripheral QCT (pQCT) allow for the analysis of body composition (DXA), BMC (DXA), volumetric density (pQCT), and cortical bone structure (pQCT) in mice, with acceptable accuracy and precision.(13–16) The first (general) objective of this study was, therefore, to employ state-of-the-art marker and microimaging methodology to study the skeletal phenotype of a transgenic mouse model longitudinally over the life span of the animals.
As a transgenic model, we selected growth hormone (GH) transgenic (tg) mice.(17) These mice exhibit supra-physiological serum levels of GH and insulin-like growth factor-I (IGF-I)(14,18) and substantially increased body weight.(17,19,20) We have previously shown that adult GH tg mice display a disproportionate skeletal gigantism(21,22) and a substantial increase in BMC compared with control animals.(14) Interestingly, trabecular bone volume fraction (BV/TV) was found increased in female, but not male, tg animals.(14)
In human pathophysiology, increased serum levels of GH are observed in states of neoplastic hypersecretion of the pituitary gland. GH excess is known to lead to gigantism before, and to acromegaly after, closure of the bone epiphyses. Reports on the bone mineral status in patients with GH hypersecretion have been inconsistent. While some studies have reported an elevation of BMC and bone density at various skeletal sites, others have reported a reduction of the same parameters.(23–30) Reasons for these contradictory findings include (1) confounding by variable degrees of hypogonadism; (2) variations in the location of bone measurements (trabecular or cortical sites); and (3) variation in measurement technique (i.e., pQCT or DXA). In addition, GH has also been studied as a therapeutic agent in osteoporosis. In contrast to commonly used antiresorptive agents,(31) GH has the potential to increase bone formation.(32,33) Coincidental epidemiologic evidence has indicated that high IGF-I concentration is associated with an increased risk of cancer,(34–37) and therefore, GH has been viewed with skepticism. However, recent findings indicate that GH may be a safe and effective drug, particularly in combination with antiresorptive agents.(38–40)
Although previous studies have investigated the effects of GH overexpression on selected bone parameters and age groups,(9,14,21,41-43) to date there has not been a comprehensive longitudinal study examining the effects of GH overexpression on both cortical and trabecular bone and on biochemical markers of bone turnover in male and female mice in vivo. Based on what is known about GH effects on bone metabolism, we asked the following key questions in this study.
- 1Is the anabolic effect of GH on bone dependent on age and/or sex of animals?
- 1What are the effects of GH overexpression on biochemical markers of bone turnover?
- 1Does GH overexpression produce differential effects on cortical versus trabecular bone?
MATERIALS AND METHODS
Measurements were performed in mice that were 3, 6, 9, 12, 18, 26, and 38 weeks of age. Because there were difficulties in obtaining DXA and pQCT measurements in parallel during one anesthetic session, two sets of animals were examined longitudinally. Set 1 (DXA and bone markers) was comprised of 38 animals of NMRI background (10 tg females [tg F]; 10 non-tg female controls [con F]; 11 tg males [tg M]; 7 non-tg male controls [con M]). All but one animal (tg F) survived up to week 26, and eight animals died between weeks 26 and 38. Set 2 (pQCT) consisted of 34 animals of the same background (8 tg F; 9 con F; 8 tg M; 9 con M), 6 of which died between weeks 18 and 26, and 8 between weeks 26 and 38. GH overexpression was achieved by a transgene in which the expression of bovine (b)GH was controlled by the mouse metallothionein I (MT) promoter.(14,18) The genotype was determined by PCR analysis as described previously.(44) Previous studies have shown that MT-bGH tg mice display serum bGH levels in the range of 100-200 ng/ml, whereas baseline values in controls corresponded to 10 ng/ml(14,18); no significant differences were observed in bGH serum levels between genders.(14,18). Serum IGF-I levels were found to be elevated ∼1.5-fold in both male and female MT-bGH tg animals compared with controls.(13)
Bone marker analysis
A bone formation marker (serum osteocalcin) was measured from serum samples by specific radioimmunoassay (RIA), using mouse osteocalcin as tracer and standard (Biomedical Technologies, Stoughton, MA, USA). This test has an interassay variability of <8%.(45) Two bone resorption markers (pyridinoline [PYD] and deoxypyridinoline [DPD]) were measured from urine samples by isocratic, reversed-phase, ion-pair HPLC.(46,47) The concentrations of PYD and DPD were obtained by comparison with an external standard and were corrected for urinary creatinine. Creatinine was determined by a quantitative colorimetric assay (Metra Biosystems, Mountain View, CA, USA). The overall reproducibility of the assay is 4% for PYD and 11% for DPD. Note that urine samples in animals 3-9 weeks of age were too small for individual analysis, and samples had to be pooled from several animals.
In vivo imaging methods
Animals were anesthetized as described previously.(16) The body weight was measured using a laboratory scale (PM 100; Mettler, Gieβen, Germany). Total body BMC and body composition (% fat)(14) were determined using a high-resolution DXA scanner (Sabre pDEXA; Stratec Medizintechnik, Pforzheim, Germany).(13–15) We have shown previously that there exists a highly linear relationship between DXA and ash analysis in mice using this system,(14,15) although the BMC is (systematically) underestimated. To test the accuracy of DXA in small mice (10.9-19.6 g at week 3 in this study), we examined 8 mice weighing 8.8 ± 0.55 g, 8 mice weighing 21.2 ± 1.4 g, and 13 mice weighing 35.5 ± 3.3 g ex vivo. Using an automatic adaptable threshold, we achieved a high linear relationship between DXA and ash analysis (SEE = 5.8%/6.6%/5.4%), with the underestimation being similar in these three weight groups (−44%/−30%/−35%). This threshold was used throughout the study. The precision of BMC measurements was reported to range from 13% (11 mg) in 10-g mice to 3.5% (35 mg) in 40-g mice.(13)
pQCT was performed with a dedicated small animal scanner (pQCT-M Research; Stratec).(15,16) At week 3, the limbs of the mice were too small to be positioned in the scanner. At week 6, measurements were feasible, but aligning the limb perpendicular to the scanner gantry was still difficult. Measurements at week 6 must therefore be interpreted with some caution. From week 9 onward, appropriate positioning and satisfactory precision was achieved ex vivo and in vivo.(16) Peelmode (PM) 2 (600 mg/cm3) was used to separate trabecular from the (sub)cortical bone at the femoral metaphysis, and analysis of cortical bone structure was performed as described previously.(16)
A Student's t-test was used to perform three comparisons between genetic groups (1: con M versus con F; 2: tg F versus con F; 3: tg M versus con M) at each time point. Error levels were set to p < 0.05 for a single comparison without correction for multiple testing, p < 0.0166 to indicate statistical significance at a 5% global error level (three tests being performed in parallel), and p < 0.0033 to indicate statistical significance at a 1% global error level. pQCT data at week 38 were not tested because of the relatively low number of animals at that time point.
BMC, body weight, and body composition
Tg F mice displayed a significantly higher body weight than con F mice from week 3 onward, and tg M mice had higher body weights than con M mice from week 6 onward (Table 1). Figure 1 shows that the body weight gain is greater in tg F compared with tg M mice. The BMC was elevated in tg animals from week 6, the gain being greater in female compared with male mice. Only in tg F did the gain in BMC exceeded the gain in body weight from week 9 onward (Fig. 2). The relative (%) fat tissue content was substantially higher in both male and female tg animals compared with controls at weeks 6-12 (Table 1).
The body weight and BMC increased continuously from weeks 3 to 26 in all groups. Con M and con F displayed an increase in BMC up to week 38, whereas a plateau (or a decrease) was observed from weeks 26 to 38 in tg F and tg M mice (Table 1). The relative (%) BMC increased from weeks 3 to 18 in all genetic groups. Figure 2 shows that the BMC relative to body weight was increased by 25% in tg F compared with control female mice at 18 weeks of age and 10% in tg M compared with control male mice. The relative fat tissue content displayed a much stronger increase from weeks 3 to 18 in tg animals compared with controls. At week 38, however, the percent fat tissue content became lower in tg mice than in controls.
Metaphyseal bone properties (distal femur)
Tg F and tg M displayed larger bone cross-sectional areas (CSA) than con F and con M at the distal femoral epiphyses (Table 2). Trabecular bone density was elevated in tg F versus con F between weeks 6 and 12, but was similar in both groups at weeks 26-38 (Fig. 3). In tg M mice, trabecular density was elevated compared with con M mice only at week 6 (Table 2; Fig. 3). Subcortical density was not significantly different between transgenic and control animals.
The metaphyseal CSA displayed smaller values at week 9 compared with week 6, likely because scans were taken obliquely in the smaller week 6 animals as a result of the short lower limbs. From week 9, the CSA increased in all groups, particularly in tg M mice (Table 2). Trabecular density decreased in all groups between weeks 6 and 9, but remained relatively constant thereafter. However, in tg F mice, trabecular density dropped considerably from weeks 12 to 26, and in tg M from weeks 26 to 38 (Fig. 3). Subcortical density increased in all groups from weeks 6 to 12 but remained relatively constant thereafter (Table 2).
Cortical bone properties (tibial midshaft)
Tg F and con F animals displayed similar total CSA as well as cortical CSA at the tibial midshaft (Table 3; Fig. 4). Cortical density was, however, significantly lower in tg F than in con F mice (Table 3). In contrast, tg M mice displayed a substantially higher total CSA and cortical CSA (Table 3; Fig. 4) than con M mice, although this increase was not larger than that of body weight. Cortical density was reduced in tg M versus con M mice from weeks 12 to 38 (Table 3).
The cortical CSA displayed a steady increase in most groups until week 26 (Table 3). The total CSA, in contrast, remained relatively constant over the entire life span (except for tg M, which continued to expand up to week 38; Table 3). The cortical density increased substantially from weeks 6 to 12 but was relatively constant thereafter (Table 3).
Serum osteocalcin (OC) levels were significantly lower in tg F compared with con F mice from weeks 3 to 18. The difference between tg M and con M mice was, however, only significant at week 3 (Table 4; Fig. 5). PYD and DPD tended to be reduced in tg F and tg M versus con F and con M mice, respectively, but the difference did not attain statistical significance (Table 4). Note, however, that no statistical testing was performed for weeks 3-9, where differences were most striking, because analyses had to be pooled from several animals within each group.
OC levels decreased continuously from weeks 3 to 26 in all genetic groups, and these leveled out between weeks 26 and 38. Resorption markers declined dramatically from weeks 3 to 9 and leveled out (or decreased slightly) from weeks 12 to 38 (Table 4).
In this study, we evaluated the effect of ubiquitous overexpression of GH under the control of the mouse metallothionein promoter on musculoskeletal changes during the first 9 months of life. The salient findings of this study are as follows: (1) GH overexpression increases both bone size and bone mass, two major determinants of bone strength; (2) the magnitude of GH effects on target tissues depends on both developmental stage and sex of animals; (3) GH overexpression decreases serum levels of bone turnover markers; (4) GH overexpression increases fat content in both males and females; and (5) the decrease in cortical density in female tg mice cannot be explained by lower mechanical stress caused by higher CSA.
MT-bGH tg mice are known to display a variety of pathological alterations, with kidney and liver lesions being consistently found in these animals. Renal alterations are characterized by progressive glomerulosclerosis with secondary tubulo-interstitial changes and represent the primary cause of shortening of life span. Mean life expectancies correspond to 11 months in MT-bGH-tg mice versus 20 months in con mice.(18,19) For this reason, the study was terminated after 9 months.
In previous studies, it was found that GH-deficient mice exhibit significant decreases in both bone size and bone density, and GH treatment caused a significant increase in both of these parameters.(48) Consistent with these findings, we observed that transgenic overexpression of GH increased both bone size and bone mass. However, the magnitude of GH effect on these parameters varied considerably in males versus females. For example, total body BMC adjusted for body weight was increased maximally by 25% in female transgenic mice compared with corresponding control mice versus 10% in male tg mice compared with control male mice. Because GH overexpression increases body weight and because male and female mice exhibit differences in body weight, we used total body BMC adjusted for body weight as a measure of bone mass changes in response to GH overexpression. In contrast to the greater increase in total body BMC adjusted for body weight in female transgenic mice, the magnitude of GH-induced increase in bone size was greater in male transgenic mice compared with female transgenic mice. In this regard, total cross-sectional area at the femoral metaphysis was increased by 44% and 66%, respectively, in the female and male transgenic mice compared with corresponding control mice. Furthermore, the total cross-sectional area at the mid-diaphysis of the tibia was increased significantly in male but not female transgenic mice. Thus, these data suggest that GH effects on bone mass and bone size are influenced differently in male and female mice.
In terms of the mechanisms that contribute to the gender difference in the amount and spatial distribution of bone in transgenic mice overexpressing GH, the differences in the interaction between androgens and estrogens with GH are likely to play an important role. In this regard, Yeh et al.(49) have shown that combined treatment of GH plus 17β-estradiol resulted in an additive increase in cancellous bone mass compared with GH or 17β-estradiol treatment alone in the ovariectomized rat with hypophysectomy. In contrast, testosterone but not GH administration significantly increased femoral BMC or BMD in orchiectomized rats, whereas GH treatment caused a significant increase in periosteal bone formation rate and cortical bone area.(50) Furthermore, Kim et al.(51) have recently shown that periosteal growth is independently and additively stimulated by androgens and GH in males and inhibited by estrogens and stimulated by GH in females. Thus, androgens and estrogens may interact differently with GH in regulating sexual dimorphic changes in the amount of bone in the cortical and trabecular compartments. The precise molecular mechanisms that contribute to differential interaction between sex hormones and GH remain to be established.
Surprisingly, we found that GH overexpression had little or no effect on body weight or total body BMC at 3 weeks of age either in male or female transgenic mice compared with corresponding control mice. In contrast, both body weight and total body BMC were increased by 60% at 9 weeks of age in GH transgenic female mice compared with control mice. Thus, these data show that GH overexpression produced much greater effects on body weight and BMC during pubertal and postpubertal growth period compared with during the prepubertal growth period. Consistent with these data, it was previously found that the magnitude of BMD deficit in GH-deficient lit/lit mice was only 8% at day 23 compared with 32% at day 56.(52) Furthermore, it was found that GH administration during prepubertal growth period produced a far smaller increase in body weight and skeletal parameters compared with during pubertal and postpubertal growth periods in GH-deficient lit/lit mice (<20% versus 70%). The potential mechanisms that contribute to growth period-dependent GH response in target tissues remain to be established. In this regard, Choi and Waxman(53) have shown that certain liver enriched transcription factors necessary for mediating GH effects are absent in prepubertal rats. Future understanding of the molecular mechanisms that contribute to differences in GH sensitivity in bone and other tissues during various growth periods may lead to better understanding of the molecular pathways for GH action.
In this study, we found that serum and urinary levels of bone turnover markers were less in GH transgenic mice compared with corresponding control mice, thus suggesting that GH overexpression may lead to a decrease of bone turnover. However, these findings clearly differ from those obtained when administering GH in elderly subjects, where GH increases bone turnover.(54) This discrepancy may be because of differences in GH effect on bone turnover in growing mice versus elderly individuals or because of differences between mice and humans. One must also take into account that serum markers only provide indirect evidence of the bone formation and resorption rates. Because this study was designed as an in vivo experiment, no histomorphometric analyses were included for studying bone formation rate and osteoclast surface directly. The lack of direct histomorphometric data represents a limitation of this study. These analyses should be performed in future studies to confirm whether or not the increase in BMC in MT-bGH-tg mice is indeed accompanied by reduced bone turnover.
It is now fairly well established that, in addition to its effects on the skeleton, GH influences lean body mass and body fat. Numerous clinical studies have shown that lean body mass increases, whereas percent body fat decreases, in GH-treated GH-deficient adults compared with corresponding controls.(55) Consistent with these data, it was found that the percent body fat was greater in GH-deficient lit/lit mice compared with control mice and that GH treatment decreased percent body fat.(48) In contrast to these findings, we found that percent body fat was significantly increased in GH transgenic female and male mice compared with corresponding control mice. The reason for the greater percent body fat in GH transgenic mice can only be speculated at this time. In this regard, it is possible that GH effect on body fat may differ depending on whether it is administered continuously versus intermittently. Also, the effect of GH on body fat may be dependent on the dose of GH. Further studies are needed to evaluate the cause for the observed increase in percent body fat in this transgenic mouse model overexpressing GH.
The decrease in cortical density in GH tg mice observed in this study was similar to what was found previously. In this regard, Tseng and Goldstein(9) hypothesized that a reduction in cortical tissue properties (density and mechanical strength) did not represent a primary biological perturbation but was caused indirectly by an increase in bone cross-sectional area (CSA) in young mice and corresponding decrease in mechanical strain of cortical bone. In our study, however, the reduction in cortical tissue was similar in female and male tg mice, although tg male mice but not tg female mice showed an increase in CSA. Furthermore, the reduction in cortical density occurred earlier in tg female mice compared with tg male mice. Although analyses of cortical density with pQCT must be interpreted with some caution,(56) our finding of reduced cortical density is consistent with (1) previous observations by Sandstedt et al.(42) using ash analyses in tibial cortical bone (MT-bGH tg mice with systemic overexpression); (2) μCT analysis of femoral cortical bone by Tseng et al.(43) (male osteocalcin promoter-hGH tg mice with osteoblast-specific, local overexpression; and (3) our previous μCT analysis of the entire femur in MT-bGH tg mice with systemic overexpression of bGH.(14) It is important to note that the decrease in cortical density in this study was observed despite a significant increase in cortical thickness in GH tg males (data not shown), whereas partial volume effects(56) would affect the measurements in opposite direction. Taken together, these findings provide evidence that, at least in females, reduction in cortical density does not represent a secondary (adaptational) process in GH tg mice, but may represent a primary biological “perturbation.” Although the concept provided by Tseng and Goldstein(9) is intriguing, it seems difficult to dissect the complex skeletal changes observed with systemic overexpression of GH mice into primary biological and subsequent mechanical effects, given the differences between tg M and tg F mice and given the complex (mechanical and biological) interactions at different hierarchical levels of the skeletal tissue.
We thank Petra Renner, Ingrid Renner-Müller, and Thomas Fisch for valuable help with animal care and with collecting serum and urine samples. We thank Andreas Hoeflich for helping with the PCR analysis. The longitudinal DXA measurements were obtained as part of the doctoral thesis of AW, which will be submitted to the Medical Faculty of the Ludwig-Maximilians-Universität München. The work was supported by German Research Society Grant DFG EC 159/6-1 and National Institutes of Health Grant AR 048139 (SM).
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