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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 4 1202-1209
Copyright © 1997 by The Endocrine Society

Experimental Studies

Short-Term Effects of Growth Hormone and Insulin-Like Growth Factor I on Cancellous Bone in Rhesus Macaque Monkeys1

D. A. Sass, C. P. Jerome, A. R. Bowman, A. Bennett-Cain, T. A. Ginn, D. LeRoith and S. Epstein

Division of Endocrinology and Metabolism, Department of Medicine, Albert Einstein Medical Center (D.A.S., A.R.B., S.E.), Philadelphia, Pennsylvania 19141; the Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University (C.P.J., A.B.-C., T.A.G.), Winston-Salem, North Carolina 27127; and the Diabetes Branch, National Institutes of Health (D.L.), Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Sol Epstein, M.D., Division of Endocrinology and Metabolism, Albert Einstein Medical Center, 5401 Old York Road, Philadelphia, Pennsylvania 19141.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of our study was to determine the effects of GH and insulin-like growth factor I (IGF-I) administration singly and in combination on vertebral, tibial, and femoral bone in aged female monkeys as well as the various treatment effects on serum hormone levels and osteocalcin gene expression. Twenty-one ovulating female monkeys (rhesus macaque), aged 16–20 yr (5–6 kg), were divided into four groups to receive the following treatment for 7 weeks via Alzet pumps inserted sc: A, eluant (control group); B, recombinant human IGF-I (rhIGF-I; 120 µg/kg·day); C) rhGH (100 µg/kg·day); D, combination of rhIGF-I (120 µg/kg·day) and rhGH (100 µg/kg·day). Serum was assayed serially for glucose, IGF-I, GH, and IGF-binding protein-3 levels. All groups received double labeling with calcein. On the day of death, the primates’ second lumbar vertebrae, tibiae, and femora were carefully dissected, fixed in 70% ethanol, and subjected to histomorphometric analysis. Ribonucleic acid was extracted from contralateral tibiae for the purpose of osteocalcin gene expression analysis. Serum glucose was unaffected by treatment. Serum GH was significantly elevated in groups C and D, whereas serum IGF-I and IGFBP-3 were only significantly increased in group D. Histomorphometric analysis showed no significant differences or trends for bone volume in any treatment group. Bone formation rate, surface and/or bone volume referent were significantly higher in both groups treated with GH (C and D) in tibia and femur, with a similar trend in vertebrae. The increase in bone formation rate was due mainly to a significant increase in mineral apposition rate, but there was also an increase in tibial mineralizing surface by GH by factorial analysis (P < 0.05). There were significant treatment effects on osteoid surface and osteoclastic surface in femur in the combination treatment group vs. the controls. Osteocalcin gene expression analysis supported an enhanced expression in both groups treated with GH. These findings are consistent with a short term effect of GH to increase bone remodeling and predominantly osteoblastic activity in the appendicular skeleton. In contrast, other than an isolated increase in osteoclastic surface in femoral bone, IGF-I, when administered alone, was unable to significantly influence bone formation or resorption activity in this short term study.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE REMODELING is regulated by systemic hormones and growth factors acting in concert to maintain normal bone mass (1). GH is an anabolic hormone with multiple effects on skeletal and nonskeletal tissues. It has both direct and indirect effects on bone and appears to be important not only in the stimulation of linear growth, but also in the regulation of bone formation and normal physiology of adult bone after cessation of the skeletal growth phase (2). In vitro studies have shown that GH increases both the number and the function of osteoblasts (3), whereas in vivo, histomorphometry has provided evidence of augmented bone turnover, with increases in bone formation and, to a lesser extent, bone resorption (4). Human studies have demonstrated a predominantly anabolic action of GH as recombinant human GH (rhGH) treatment stimulates osteoblasts and activates bone remodeling (5).

Numerous studies indicate a role of insulin-like growth factor I (IGF-I) as a mediator of GH’s actions (6, 7), suggesting important and interdependent effects of these two peptides on skeletal physiology. This synergy is best characterized at the epiphyseal growth plate, where GH directly stimulates differentiation of chondrocyte precursors while indirectly promoting bone growth by enhancing the local production of and responsiveness to IGF-I, which, acting in an autocrine or paracrine fashion, stimulates clonal expansion of differentiating chondrocytes (7). Possibly changes in IGF-binding protein-3 (IGFBP-3), the production of which by osteoblasts is enhanced by GH (8) and which stimulates IGF-I-induced mitogenesis, are related to the positive effect of GH.

IGF-I has been shown in vitro to increase the replication of cells of the osteoblastic lineage, enhance osteoblastic collagen synthesis and matrix apposition rates (MARs) and decrease collagen degradation in calvariae (9, 10). More recently, there is a growing body of evidence supporting the idea that IGF-I stimulates bone resorption by enhanced osteoclastic recruitment (11). Hence, this growth factor seemingly acts on both bone formation and resorption and may couple the two processes. The effects of systemic administration of IGF-I on bone formation in experimental animals has been controversial depending on the dose, frequency, and route of administration, including contrasting effects on trabecular and cortical bone in vivo (12, 13, 14, 15, 16). However, recent work in humans has shown IGF-I to be primarily an anabolic agent on bone (17, 18).

Kupfer et al. (19), in a study involving calorically restricted normal volunteers, suggested that the combination of GH and IGF-I treatment is substantially more anabolic (as demonstrated by increased nitrogen retention) than either agent alone and, moreover, attenuated the hypoglycemic effect of IGF-I. By virtue of the anabolic potential of these two agents, the purpose of the present study was to evaluate the short term effects of GH and IGF-I administration singly and in combination on cancellous bone in vertebra, tibia, and femur of skeletally mature monkeys. To the best of our knowledge, detailed histomorphometry describing the effects of GH and IGF-I on bone of nonhuman primates has not been published to date. Moreover, in an effort to explore our bone histomorphometric findings at the molecular level, we endeavored to elucidate the effects of GH and IGF-I on messenger ribonucleic acid (mRNA) expression of the marker of bone turnover, osteocalcin (or bone Gla protein), which has also not previously been demonstrated by other investigators.

The rhesus macaque (Macaca mulatta) monkey provides a reliable nonhuman model for pathophysiological research of the bone-remodeling system (20, 21, 22, 23). Unlike rats and mice, the monkey has meaningful amounts of Haversian remodeling of cortical bone. Linear growth ends in female macaques at about 5–7 yr of age, with a peak bone mass achieved by age 10 yr (22, 23). Moreover, female macaques have regular monthly menstrual cycles similar to those of women (24); however, age at menopause and even the existence of menopause in the rhesus monkey have not been reliably determined, although a limited study in the area indicated that menopause may occur between age 25–30 yr (25). Thus, animals past peak bone mass age (i.e. between 10–20 yr) are ideal for studies designed to model skeletally mature women with intact ovaries.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Twenty-one ovulating female monkeys (rhesus macaque), aged 16–20 yr and 5–6 kg in weight, were obtained from the Walter Reed Army Institute of Research (Washington DC) and the Veterinary Resources Program (NIH, Bethesda, MD) and were quarantined for 60 days at the animal facility in Poolesville, MD, before experimentation. The animals were housed in individual cages according to a protocol approved by the animal care and use committee of the NIDDK, NIH (Bethesda, MD). Animals were maintained on a 12-h light, 12-h dark cycle, and the temperature was kept constant at 21 C. Monkeys were fed Purina monkey pellets (Ralston-Purina, St. Louis, MO) and fresh fruit with free access to water. All animals used in the study were initially examined by a veterinarian and underwent a comprehensive blood screen to exclude the presence of diseases before inclusion in the study. All experimentations were performed in the Primate Unit of the Veterinary Resources Program in Bethesda.

Treatment groups

After the acclimatization period, the monkeys were randomly divided into four groups to receive the following treatment for 7 weeks via slow release pumps: group A (control group) received eluant (n = 5), group B received rhIGF-I (120 µg/kg; n = 6), group C received rhGH (100 µg/kg; n = 5), and group D received rhIGF-I (120 µg/kg) and rhGH (100 µg/kg; n = 5). For the purpose of drug administration, 2-mL Alzet pumps (Alzet Corp., Palo Alto, CA) were inserted sc into the dorsal thoracic region of all monkeys under ketamine HCl anesthesia (15 mg/kg; 1 mol/L). After 4 weeks, pumps were replaced on the contralateral side. After an additional 3 weeks, the animals were killed using sodium pentobarbital, given iv. Disposal of carcasses was performed according to NIH protocols. Ketamine was used for anesthesia during all procedures, and penicillin (30,000 U/kg) was administered im after every surgical procedure.

Hormone administration

rhIGF-I was supplied in a concentration of 38 mg/mL (gift from Genentech, South San Francisco, CA) and administered at a rate of 120 µg/kg·day. The stock solution was diluted in a solution containing 5.84 mg/mL NaCl, 50 mmol/L sodium acetate buffer, 9 mg/mL benzyl alcohol, and 2 mg/mL polysorbate 20, pH 5.4. rhGH was supplied in a concentration of 5 mg/mL (gift from Genentech) and injected at a rate of 100 µg/kg·day. The stock solution was diluted in a solution containing 10 mmol/L Na citrate, 2.5 mg/mL phenol, 8.77 mg/mL NaCl, and 2 mg/mL Tween-20, pH 6.0. rhIGF-I administration required a single Alzet pump, whereas rhGH required two pumps and combination therapy a total of three pumps per monkey.

Serum measurements

While under ketamine anesthesia, at baseline, and after 4 and 7 weeks of treatment, blood was taken, and serum was separated by centrifugation and stored at -20 C until assayed for later serial determinations of glucose, IGF-I, IGF-binding protein-3 (IGFBP-3), and GH levels. Serum glucose was measured by a Monarch clinical chemistry machine (Genentech), and IGFBP-3 was determined by an in-house enzyme-linked immunosorbent assay technique (Genentech). hGH was measured in the serum by enzyme-linked immunosorbent assay, the validity of which has been previously reported (26). Serum IGF-I was measured by RIA after acid-ethanol extraction (27).

Histological techniques

Three weeks and 1 week before death, all groups received double labeling with calcein (10 mg/kg, iv; interlabel time period, 14 days) to enable subsequent histomorphometric determination of dynamic parameters of bone remodeling. On the day of death, the second lumbar vertebral bodies, femur, and tibia of all monkeys were carefully dissected, fixed in 70% ethanol, and subjected to hisomorphometric analysis.

Preparation

Vertebral bodies were measured from end plate to end plate ({ell} mm). Using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL), 0.2 x {ell} mm were cut from each end of each vertebra. Tibiae were cut longitudinally through the tibial plateau in the frontal plane into 2-mm slabs. The central slab was selected for embedment. Femora were cut transversely between the femoral neck and lesser trochanter. A second cut was made 1 cm below the first to create a block for embedment. All blocks were embedded in methyl methacrylate/dibutyl pthalate and sectioned at 8 µm using a sledge microtome (28). Sections were cut from the cranial end of vertebrae and the proximal end of femoral blocks. All sections were mounted unstained for fluorescent label measurement and stained with toluidine blue for histological surface feature measurements.

Histomorphometry

Sections were viewed at x100 magnification for point and intersection counting and at x200 for measurement of interlabel width. Label measurements were made using every other line of a Merz eyepiece sampling grid, whereas surface histology measurements were made using every line. The system was calibrated by measuring total grid width with a stage micrometer. The basic technique and working formulae for the grid have previously been described (29). Intersection and point counts were collected using a custom program written in QBasic (Microsoft, Seattle, WA). Interlabel widths were measured as orthogonal intercepts at grid-line/label intersections using Bioquant Intro software (R&M Biometrics, Nashville, TN). The raw data collected and formulae for derived values are listed in Table 1Go. Abbreviations, terminology, and formulas are based on the standardized nomenclature for bone histomorphometry (30).


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  		Table 1. Histomorphometric measurements and calculations

 
In all sites, central cancellous tissue was measured using the following sampling protocols. For lumbar vertebrae, measurements were made in a region anterior to the level of transverse processes (>2 mm anterior to the spinal canal), approximately 4.5 mm wide by 2.5 mm deep (mean area measured, 11.9 mm2). In tibiae, cancellous tissue in the metaphysis, more than 3 mm below the subchondral bone, was measured in a sampling region approximately 9.6 mm wide by 3.2 mm deep, excluding any tissue less than 1.2 mm from the periosteal surface (mean area measured, 30.3 mm2). In femora, all cancellous tissue was measured, except that within 400 µm of the endocortical surface (mean area measured, 50.3 mm2).

Gene expression analysis

Total cellular RNA from monkey tibial metaphyses devoid of marrow was isolated and analyzed according to the method of Nemeth et al. (31). Left tibiae were rapidly dissected immediately preceding death and frozen in liquid nitrogen. Tibiae in each group were pooled, and the bones were crushed under liquid nitrogen using a mortar and pestle. The resultant granular powder was then homogenized in a 5 mol/L guanidium isothiocyanate solution and thereafter exposed to low speed centrifugation and filtration of the supernatant. The resulting solution was centrifuged at 32,000 rpm in a SW 41 Ti rotor (Beckman, Palo Alto, CA) through a 5.6 mol/L cesium chloride density gradient. RNA pellets were resuspended in TE-SDS (10 mmol/L Tris-Cl, pH 8.0; 1 mmol/L ethylenediamine tetraacetate, pH 8.0; and 0.1% SDS) solution and precipitated from 0.25 mol/L sodium acetate with ethanol. Five micrograms of total RNA per group together with one lane containing 5 µg total RNA from untreated rat tibial bone (as control) were electrophoresed through a 1.2% agarose and formaldehyde denaturing gel and thereafter transferred onto a nylon membrane and fixed by UV cross-linking. RNA integrity was verified by ethidium bromide staining of 28S and 18S ribosomal RNAs.

Northern blots were performed by hybridization with a 60-mer oligonucleotide that was complementary to the mRNA sequence encoding amino acids 11–30 of the rat osteocalcin protein (32) (gift from Dr. G. Hendy, Montreal, Canada). The probe was end labeled with [{gamma}-32P]ATP (Amersham, Arlington Heights, IL) using T4 polynucleotide kinase (Sigma). The nylon membrane was prehybridized for 4 h at 42 C in 50% formamide, 5 x Denhardt’s solution, 200 µg/mL denatured salmon sperm DNA, 1% SDS, 0.1 mol/L sodium phosphate buffer, and 5 x SSC (standard saline citrate). The hybridization fluid was the same as that used for the prehybridization, but with the addition of 0.1 g/mL dextran sulfate and 1 x 106 cpm/mL radioactively labeled probe. After hybridizing overnight at 42 C, the membrane was washed twice at 37 C with 1 x SSC and 0.1% SDS solution. While still wet, the membranes were wrapped and exposed to autoradiographic film in cassettes with intensifying screens at -70 C. Radioactivity was stripped from the membranes, which were then reprobed with complementary DNA for the mouse ß-actin gene (33), and labeled, employing standard riboprobe techniques, with [{alpha}-32P]CTP (Amersham). ß-Actin was used as an internal standard for determining the relative sample loading efficiency. The signal intensities were quantified by a GS-670 imaging densitometer (Bio-Rad Melville, NY). A ratio of signal intensity for osteocalcin mRNA vs. ß-actin mRNA was calculated.

Statistical analysis

Statistical analyses were performed using the statistics package SPSS for Windows Release 6.0 (SPSS, Chicago, IL). Data were transformed if necessary to meet assumptions of normality and equal variance, then subjected to ANOVA. Overall significance for biochemical data was determined using repeated measures ANOVA in the context of a 2 x 2 factorial analysis. The two between-group factors were GH, which had two levels (i.e. absent and present), and IGF-I, which had two levels (i.e. absent and present). Time was the repeated measures factor in all analyses. Significance between the presence or absence of a particular drug over time was determined with a priori pairwise polynomial contrasts (34). Similarly, the bone histomorphometric data were analyzed by a single time point factorial analysis (ANOVA) using a priori pairwise simple contrasts to determine whether significance existed between drug groups. Throughout all analyses, P < 0.05 (P < 0.05) was considered a significant difference. All values are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum levels ( Figs. 1–3GoGoGo)

The biochemical data from this study were described in more extensive detail previously (35). Fasting glucose levels were normal throughout the experiment (data not shown). Serum GH was significantly elevated in the two groups treated with rhGH, groups C and D (P < 0.01 vs. control). The combination treatment group exhibited a significant elevation in serum IGF-I levels at weeks 4 and 7 (P < 0.01 vs. control). By factorial analysis, rhIGF-I exerted a significant effect at week 4 (P < 0.05), whereas rhGH had an independent effect at week 7 (P < 0.01). There was an indication that continued treatment with rhIGF-I alone may have led to a fall in serum IGF-I from weeks 4–7. Serum IGFBP-3 levels were affected in a similar manner as serum IGF-I levels. rhGH and rhIGF-I increased IGFBP-3, with the combination treatment group having the highest levels.



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  		Figure 1. Serum GH in control (•) and IGF-I-treated ({blacktriangleup}), GH-treated ({blacktriangledown}), and GH/IGF-I-treated ({blacksquare}) monkeys measured at baseline and weeks 4 and 7, as detailed in Materials and Methods. All values are the mean ± SEM. **, P < 0.01 vs. placebo-treated control.

 


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  		Figure 2. Serum IGF-I in control (•) and IGF-I-treated ({blacktriangleup}), GH-treated ({blacktriangledown}), and GH/IGF-I-treated ({blacksquare}) monkeys measured at baseline and weeks 4 and 7, as detailed in Materials and Methods. All values are the mean ± SEM. **, P < 0.01 vs. placebo-treated control.

 


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  		Figure 3. Serum IGFBP-3 in control (•) and IGF-I-treated ({blacktriangleup}), GH-treated ({blacktriangledown}), and GH/IGF-I-treated ({blacksquare}) monkeys measured at baseline and weeks 4 and 7, as detailed in Materials and Methods. All values are the mean ± SEM. *, P < 0.05 vs. placebo-treated control.

 
Histomorphometry (Tables 2–5GoGoGoGo)

The group treated with IGF-I alone showed no significant differences from controls for any of the variables. There were no significant differences or trends for structural variables (bone volume and bone surface/bone volume) for any treatment group. The bone formation rate (BFR), the surface and/or bone volume referents were significantly higher in GH and IGF/GH groups in tibiae and femora, with a similar trend in vertebrae. The increase in BFR was due mainly to a significant increase in MAR, but there was also an increase in tibial mineralizing surface caused by GH as determined by factorial analysis (P < 0.05). There was a significant effect of GH on the vertebral and femoral parameters of MAR, BFR/bone surface, and BFR/bone volume (P < 0.05), whereas in tibial bone, the effect of GH on these histomorphometric indexes was even more significant (P < 0.01; Table 5Go). With regard to the markers of bone resorption, there was a significant treatment effect on osteoclastic surface in femur in the combination treatment group vs. that in controls, which by factorial analysis was predominantly due to a GH effect (P < 0.05). The tibia showed somewhat similar trends, but no statistical significance. Besides an isolated effect on femoral osteoclastic surface, there was no independent effect of IGF-I, nor was there a GH by IGF-I interaction for all of the measured parameters at the three skeletal sites.


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  		Table 2. Histomorphometry of vertebral bone (LV2)

 

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  		Table 3. Histomorphometry of tibial bone

 

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  		Table 4. Histomorphometry of femoral bone

 

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  		Table 5. Factorial analysis of histomorphometric data

 
Osteocalcin gene expression ( Figs. 4–6GoGoGo)

The data depicted in Figs. 4–6GoGoGo represent the mean of several runs. The osteocalcin mRNA levels in the four monkey groups after densitometry (using ß-actin as an internal control) indicate that osteocalcin expression is greatest in the group treated with GH alone, whereas the steady state levels of osteocalcin mRNA in the combination treatment group were also up-regulated relative to those in the control group (treated with eluant). The group receiving IGF-I by itself had marginally elevated levels compared with control values. The lane consisting of untreated rat tibial RNA demonstrated the strongest osteocalcin expression.



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  		Figure 4. Photograph of ethidium bromide staining of a 1.2% agarose formaldehyde denaturing gel consisting of 5 µg total RNA/lane used to indicate RNA integrity and to verify equal loading. Groups are as follows: untreated rat tibial bone RNA (control); A, eluant (monkey control); B, IGF-I; C, GH; and D, GH/IGF-I.

 


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  		Figure 5. Autoradiographic expression of osteocalcin mRNA in monkey tibiae at week 7 generated by Northern blot analysis. The nylon membrane was hybridized to a [{gamma}-32P]ATP 5'-end-labeled 60-mer oligonucleotide known to be complementary to rat osteocalcin RNA. The signals are in a position compatible with the migration of a 600-nucleotide RNA molecule. Groups are as follows: untreated rat tibial bone RNA (as control); A, eluant (monkey control); B, IGF-I; C, GH; and D, GH/IGF-I.

 


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  		Figure 6. Osteocalcin/ß-actin mRNA ratio of volume OD/mm2. After hybridization with osteocalcin, blots were stripped and rehybridized with [{alpha}-32P]CTP-labeled complementary DNA insert for mouse ß-actin, which was used as an internal control. The autoradiograph was analyzed by a GS-670 imaging densitometer (Bio-Rad). Groups are as follows: untreated rat tibial bone RNA (as control); A, eluant (monkey control); B, IGF-I; C, GH; D, GH/IGF-I. SE bars reflect the fact that this histogram represents the mean of several runs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has characterized the bone histomorphometry and osteocalcin gene expression in aged female rhesus macaques treated with a short 7-week course of rhGH and rhIGF-I, singly and in combination. In addition to histomorphometric analysis, we explored the trend of serum levels of the anabolic agents employed in this study.

Serum IGF-I in the group treated with rhIGF-I declined from weeks 4–7, although still remained higher than that in the placebo group. This trend has been observed previously with IGF-I administration to female rhesus monkeys (36). As expected, GH increased IGF-I levels, with values in the combination treatment group being significantly elevated. In the present study, the group treated with GH alone exhibited the most marked effects on bone formation in tibia and femur, even more so than the combination treatment group. GH has been shown to stimulate local production of IGF-I, which acts to promote tissue growth in a paracrine/autocrine fashion (37). Seeing that the group receiving GH/IGF-I had much higher serum IGF-I levels than the group treated with GH alone, it would seem that serum IGF-I levels do not completely account for and may not be an accurate index of IGF-I tissue effects (38). The lack of a close relationship between serum IGF-I levels and linear growth has also been observed in GH-deficient children treated long term with GHRH (39) and may be related at least in part to the paracrine effects of IGF-I on bone growth.

Histomorphometry revealed no significant effects on bone mass and structure after 7 weeks. This was not surprising after such a short treatment period, and our primary intention was, rather, to explore the dynamic bone effects of GH and IGF-I in a skeletally mature animal that has already completed its growth phase. The increases in mineralizing surface in tibia and femur indicate a rise in activation frequency, as by factorial analysis this parameter is significantly increased by GH, but not by IGF-I (P = 0.044 and P = 0.050 for tibia and femur, respectively). This increased activation frequency may explain the increase in MAR, as the stimulation of new remodeling sites by the treatment would be more or less coherent and thus in the early rapid phase of mineral apposition at bone-forming sites. Therefore, the average MARs would be expected to be higher in those animals that responded to the treatment.

The dose of IGF-I employed in our study is regarded as an intermediate level dose for the avoidance of hypoglycemia. The optimal dose required to stimulate bone formation is not known, nor is it known whether the threshold for stimulating bone formation is below the threshold for producing symptoms. Ebeling et al. (18) found that within the dose range of 30–180 µg/kg·day, hypoglycemia did not occur; however, side-effects, such as cardiovascular effects and weight gain, did occur at doses of 120 and 180 µg/kg·day. Although one may contend that the failure of IGF-I to achieve a response in this study may be due to a toxic effect of dose, the fact that serum IGF-I levels in the IGF-I group actually decreased below those of GH and GH/IGF-I between weeks 4–7 would tend to contradict this idea.

The tibial and femoral bone results suggest that GH and IGF-I have similar effects on bone resorption markers, with a more pronounced effect by both hormones on osteoclast function in femur than in tibia. A study focusing on bone turnover in elderly females found that both GH and high doses of IGF-I activate bone-remodeling osteons (17). By contrast, low dose IGF-I may directly increase osteoblast function, with only a minimal increase in bone resorption (17). We employed a relatively high dose of IGF-I and, not surprisingly, noticed a significant stimulatory effect on femoral bone resorption.

As alluded to in the introduction, we are the first to evaluate the effects of GH and IGF-I on the expression of osteocalcin. The enhanced gene expression in the groups treated with GH and GH/IGF-I is in accordance with our histomorphometric findings of increased bone turnover.

We have demonstrated by detailed histomorphometric analysis at three skeletal sites a significant stimulatory effect of GH in increasing activation frequency. In contrast, other than an isolated significant effect on osteoclastic surface in femoral bone, IGF-I alone had no significant effect, compared to placebo, on bone histomorphometry, and when added to the GH treatment regimen did not further enhance the effects of GH alone. Our study parallels another short term trial in human subjects, in which a 7-day course of rhGH in pharmacological dosage activated bone remodeling and stimulated osteoblasts (5). In that study, the effects of GH on the biochemical markers of bone formation, namely osteocalcin and alkaline phosphatase, were sustained for as long as 6 months posttreatment, whereas hydroxyproline, a bone resorption marker, was initially elevated, but normalized within 2–4 weeks.

Having studied the effects of GH and IGF-I on cancellous bone only in this experiment, future work should explore the hormonal effects on cortical bone in these nonhuman primates. Moreover, a more prolonged treatment period than that adopted for the present study may be more prudent, especially if the treatment effects on bone mass and structure in addition to the dynamic effects on bone turnover are to be investigated.


   Acknowledgments
 
The authors extend their sincere thanks to Genentech, Inc. (South San Francisco, CA), for kindly donating rhGH and rhIGF-I for this study.


   Footnotes
 
1 This work formed the basis of poster presentations and abstracts at the American Society for Bone and Mineral Research Annual Conference in Baltimore, MD (September 1995) and at the International Conference of Endocrinology in San Francisco, CA (June 1996). Back

Received September 6, 1996.

Revised December 18, 1996.

Accepted January 13, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. Ohlsson, B.-A. Bengtsson, O. G. P. Isaksson, T. T. Andreassen, and M. C. Slootweg
Growth Hormone and Bone
Endocr. Rev., February 1, 1998; 19(1): 55 - 79.
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