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Effects of 12 Months of GH Treatment on Cortical and Trabecular Bone Content of IGFs and OPG in Adults with Acquired GH Deficiency: A Double-Blind, Randomized, Placebo-Controlled Study

Department of Endocrinology (T.U., J.B.), National University Hospital, N-0027 Oslo, Norway; Medical Research Laboratory M (A.F.), Aarhus University Hospital, DK-8000 Aarhus, Denmark; Department of Endocrinology (T.B.H.), Odense University Hospital, DK-5000 Odense, Denmark; Department of Internal Medicine and Endocrinology (N.V.), Aarhus Kommunehospital, DK-8000 Aarhus, Denmark; and Department of Cell Biology (L.M.), University of Aarhus, DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: Thor Ueland, Department of Endocrinology, National University Hospital, N-0027 Oslo, Norway. E-mail: . thor.ueland{at}klinmed.uio.no

Abstract

To investigate the effects of 12 months of GH treatment on cortical and trabecular bone content of IGFs, iliac crest bone biopsies were obtained from 25 patients with GH deficiency (9 women and 16 men; ages, 21–61 yr; mean, 46 yr) who were randomized to sc injections with GH (2 IU/m²·d) or placebo for 12 months. Levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-3, IGFBP-5, osteocalcin, OPG, RANKL, and total protein were determined in extracts obtained after EDTA and guanidine hydrochloride extraction. Calcium was determined after HCl hydrolysis. Comparing changes during GH or placebo treatment, significant increases were observed during GH substitution for cortical and trabecular bone content of IGF-I [mean difference vs. placebo (mean ± SEM), 97 ± 30 and 72 ± 38%] and OPG (mean difference vs. placebo, 109 ± 59 and 51 ± 19%). Also, a significant decline was found for cortical osteocalcin (mean difference vs. placebo, -49 ± 22%) during GH treatment. In conclusion, our results indicate that long-term GH treatment increases the accumulation of IGF-I and OPG in cortical and trabecular bone in patients with GH deficiency, and this may in turn lead to an increase in bone mass and improved skeletal biomechanical competence.

ADULTS WITH ACQUIRED GH deficiency (GHD) have secondary osteoporosis characterized by reduced bone mass, decreased bone turnover measured by biochemical markers, and increased fracture risk (1, 2, 3). Studies on the impact of GH substitution have yielded conflicting results, probably due to short duration of treatment period (4, 5). Longer studies, with treatment periods of 1 yr or more, have shown significant increases in bone mass and turnover (6, 7, 8). The effects of GH on bone tissue are mediated through a complex interaction of circulating GH, IGFs, IGF binding proteins (IGFBPs), and locally produced IGFs and binding proteins, acting in an autocrine and paracrine way. Although IGF-I and IGFBP-3 are the most important IGFs in the circulation, IGF-II and IGFBP-5 are the predominant IGFs in bone. GH treatment increases serum IGFBP-5 in both GH-deficient children and adults (9, 10), whereas IGF-II seems to be only minimally GH dependent (9), and normal serum levels are reported in patients with acromegaly (11, 12). We have, however, recently demonstrated increased cortical bone content of IGF-I, IGF-II, and IGFBP-5 in acromegalic patients, suggesting that excess GH leads to an increased local production of IGFs (13). To our knowledge, there have been no human studies on the effects of GH substitution on bone matrix composition of IGFs. The aim of the present study was to investigate the effect of long-term treatment with GH on bone matrix composition of IGF system components in patients with GHD acquired in adulthood. In addition, we have measured the newly characterized osteoblast-produced antiresorptive TNF receptor superfamily member, OPG (14).

Patients and Methods

Patients

Twenty-five patients with GHD of at least 12 months duration were included in the study. Their characteristics are detailed in Table 1. Data on calcitropic hormones, biochemical bone markers, bone mineral measurements, and dynamic bone histomorphometry have been published elsewhere (6, 15). Of the original 29 patients, 25 cortical and 23 trabecular paired biopsies (both baseline and 12 months) were available in the present study. GHD was defined as maximal GH peak of less than 10 mg/liter after an insulin tolerance test (blood glucose, <2.0 mmol/liter). Additional hormone replacement was continued unchanged throughout the study (Table 1). No patients had previously been treated with GH. All patients received written and oral information and gave their written consent. The study was approved by the local ethical committee and the National Board of Health (journal no. 91/239). The study was conducted according to the Declaration of Helsinki II and the guidelines of Good Clinical Practice.

The study was double-blind, randomized, and placebo-controlled, with a duration of 12 months. Patients were stratified according to sex, age above or below 50 yr, and pituitary disease (Cushing/non-Cushing). After randomization, patients were assigned GH (Norditropin; Novo Nordisk A/S, Gentofte, Denmark), administered as daily sc self-injections at bedtime in a dosage of 2.0 IU/m²·d or placebo preparations administered in a similar fashion. From each patient, fasting blood samples and an iliac crest bone biopsy were obtained under local anesthesia at baseline, after 6 months (only serum) and after 12 months, using a modified Bordier trephine (inner diameter, 9 mm) from the standard site 2 cm below the iliac crest and 2 cm behind the anterior superior iliac spine. The samples were frozen at -40 C immediately after removal. Still frozen, the biopsies were later sawed carefully to divide cortical and trabecular bone.

Extraction procedures

Bone samples were treated as described previously (13). Briefly, the cortical and trabecular bone specimens were washed with water to remove soft tissue and blood. Samples were defatted in trichloroethylene for 6 d (changed every second day) at 4 C and dried by immersion in ethanol/ether (1:1). The samples were pulverized in a liquid nitrogen-cooled mortar and pestle, adding liquid nitrogen as necessary to keep the samples frozen. The pulverized bone particles were then passed through an 84-µm sieve and stored at -40 C until use. For determining IGF-I, IGF-II, IGFBP-3, IGFBP-5, osteocalcin, OPG, RANKL, and total protein contents, 15 mg of bone powder was extracted once with 1.5 ml 0.5 mol/liter ammonium EDTA containing the following protease inhibitors: benzamidine, 5 mmol/liter; 6-aminocaproic acid, 10 mmol/liter; p-hydroxymercuribenzoic acid, 100 mmol/liter; and phenylmethylsulfonyl fluoride, 30 mmol/liter (pH 6.2); and once with 1.5 ml 4 mol/liter guanidinium-HCl (pH 7.4) containing the same protease inhibitors. Extractions were carried out for 18 h at 4 C by rotation. After extraction, the solution was centrifuged (12,000 x g for 30 min), and the supernatant was separated from the remaining bone residues. Supernatants from both extractions were combined and desalted in Sephadex PD-10 columns (Amersham Pharmacia Biotech, Uppsala, Sweden), lyophilized in a Speed Vac Concentrator (Savant Instruments, Hicksville, NY), and stored at -40 C until assayed.

For the determination of calcium, 5 mg of bone powder was hydrolyzed in 0.5 ml 6 mol/liter HCl for 1 wk at 4 C by rotation. The hydrolyzed samples were stored at -40 C after the addition of 1 ml 3 mol/liter NaOH.

Assays for IGFBP-3, IGF-I, IGF-II, osteocalcin, OPG, and RANKL

The lyophilized ammonium and guanidine extracts were redissolved in 1.0 ml 0.02 mol/liter acetic acid and used to determine the cortical contents of IGF-I, IGF-II, IGFBP-3, osteocalcin, and total protein by commercial assays as previously described (13). Briefly, IGF-I and IGFBP-3 were analyzed using RIAs from Nichols Institute Diagnostics (Nijmegen, The Netherlands). IGF-II was analyzed using an immunoradiometric assay from Diagnostics Systems Laboratories, Inc. (Webster, TX). Before assay of IGF-I and IGF-II, IGFBPs were separated by acid gel filtration (16). Osteocalcin was measured by RIA using a kit from DiaSorin, Inc. (Stillwater, MN). This is a single site assay and has been used and validated with bone extracts previously (17). Samples were lyophilizied and reconstituted in a BSA-borate buffer (boric acid, 10 mmol/liter; 1% BSA; NaOH, 25 mmol/liter; EDTA, 25 mmol/liter, pH 8.5). The kit has a sensitivity of 0.2 ng/ml and intra-assay variations of less than 10%. Bone matrix levels of OPG were quantified by enzyme immunoassay using matched commercially available antibodies (R&D Systems, Minneapolis, MN). We have recently validated this assay for serum samples (18). Briefly, wells were coated overnight with monoclonal mouse antihuman OPG antibody (clone 69127.11; 1 µg/ml in sterile PBS). Standard was recombinant OPG (15.6–2000 pg/ml). Subsequent steps included biotinylated polyclonal goat antihuman OPG (100 ng/ml), streptavidin horseradish peroxidase (1:200, R&D Systems), and tetramethylbenzidine as substrate (R&D Systems). Intra-assay variation for bone homogenates was 5% (n = 8), and sensitivity was 25 pg/ml. The mean recovery of two samples (after extraction) spiked with different concentrations of recombinant OPG was 101%, range 73–130%. Serial dilution of a sample (1:1–1:8) gave a mean ± SD coefficient of variation percentage of 100 ± 11% between measured and expected values. To evaluate the extraction efficiency for bone matrix OPG, 10 cortical bone specimens were extracted with two additional guanidinium-HCl extractions, and all supernatants were analyzed separately. The extraction efficiency after the two extractions used in the study was 89 ± 3%. Also, there was no correlation with bone mineral density of the spine or neck of the donors of these bone specimens, as measured by dual-energy x-ray absorptiometry, indicating that extraction efficiency was independent of bone density (data not shown). RANKL was measured as described by Kotake et al. (19), except that the antihuman soluble RANKL (rabbit) was biotinylated (PeproTech, London, UK), without use of secondary antibodies but with streptavidin horseradish peroxidase (1:200; R&D Systems) and tetramethylbenzidine as substrate (R&D Systems). Intra-assay variation for bone homogenates spiked with recombinant RANKL was 19%, whereas the detection limit was 70 pg/ml. Recovery of one sample (after extraction) spiked with different concentrations of recombinant RANKL was 95% (92–99%). Calcium was determined by a colorometric method (20) with a kit from Roche Molecular Biochemicals GmbH (Mannheim, Germany). Protein concentration was determined with Bio-Rad protein assay (Bio-Rad Laboratories, Inc. GmbH, Munich, Germany). µ-Albumin was determined immunonephelometrically with a Behring Nephelometer Analyzer (Behring, Marburg, Germany) and with reagents from the manufacturer. The detection limit was 2 µg/ml. Growth factor, osteocalcin, and OPG concentrations were related to total protein content.

Western ligand blotting (WLB) of cortical bone extracts

Cortical and trabecular bone extracts (5 µg protein on each lane) were subjected to WLB to attain a semiquantitative estimate of IGFBP-5 levels as previously described (13). Briefly, SDS-PAGE (10% polyacrylamide) and WLB were performed under nonreducing conditions (21). The identity of IGFBP-5 as the major band (28 kDa) in cortical extracts was confirmed by immunoprecipitation with a specific human IGFBP-5 antibody (13). No detectable IGFBPs could be demonstrated when immunoprecipitation was performed with specific IGFBP-1, IGFBP-2, or IGFBP-4 antibodies (data not shown). Autoradiograms were quantified by densitometry using a Shimadzu CS-9001 PC dual wavelength flying spot scanner. The relative densities of the bands were measured as arbitrary absorbance units per square millimeter.

Statistical analysis

Effects of GH treatment were tested by comparing changes in the measured parameters from baseline to 12 months in the two groups with the Mann-Whitney rank-sum test for unpaired data. Relationships between variables were tested using Spearman’s rank-correlation test, and the level of significance was set at P less than 0.05.

Results

Key demographic data were similar in the two treatment groups (Table 1). Data in the text are given as mean ± SEM. No significant treatment effects were found for cortical or trabecular bone contents of calcium or protein. Significant treatment effects were detected for bone matrix IGF-I increasing during GH substitution in both cortical (mean difference vs. placebo, 97 ± 30%; P = 0.002) and trabecular bone (mean difference vs. placebo, 72 ± 38%; P = 0.011). In contrast, no significant differences between the placebo and GH groups were found for trabecular or cortical bone contents of IGF-II, IGFBP-3, or IGFBP-5. A significant decline in cortical osteocalcin (mean difference vs. placebo, -49 ± 22%; P = 0.019) was observed after treatment with GH. Finally, a significant treatment effect was observed for both cortical (mean difference vs. placebo, 109 ± 59%; P = 0.003) and trabecular (mean difference vs. placebo, 51 ± 19%; P = 0.009) bone contents of OPG, increasing in both compartments compared with placebo, in which no change was found. We were not able to detect RANKL in the bone extracts, most probably because the assay was not sensitive enough to measure the protein. Thus, the concentration of RANKL in our bone samples is less than 0.5 pg/mg dry bone. Because GH also increases circulating IGF-I, we determined albumin in 10 of our bone extracts with the highest IGF-I content, as a marker of blood contamination. Albumin was undetectable in all samples, and on the basis of the albumin/IGF-I ratio and concentration in blood we calculated that our extracts have less than 0.2%.

Discussion

GHD serves as a model in which the effect of chronic GH on skeletal metabolism can be studied. Depending on the culture conditions, GH stimulates the proliferation of osteoblasts in vitro and increases osteoblastic production of IGFs. However, few efforts have been made to quantitate the effects of GH on the accumulation of growth factors in bone, possibly representing the in vivo situation. Our study demonstrates, for the first time, that 12 months of GH replacement therapy increases cortical as well as trabecular bone content of IGF-I and OPG in a homogenous patient population with GHD acquired in adulthood.

We have recently demonstrated that acromegalic patients, with excess GH and circulating IGF-I and increased bone mass, at least in the appendicular skeleton, are characterized by significantly elevated cortical bone matrix concentration of IGF-I, IGF-II, and IGFBP-5 (13). The present study supports and extends these findings and shows that GH, at much lower doses than that seen in acromegalic patients, has pronounced effects on the accumulation of IGF-I in bone. Recent findings in mice with a liver-specific knockout of the IGF-I gene have challenged the original somatomedin hypothesis and propose that GH mediates somatic growth via local, not circulating IGF-I production, acting in a paracrine/autocrine fashion (22, 23). Based on these findings, our data indicate that exogenous GH increases bone mineral density primarily through local up-regulation of IGF-I. However, other explanations may explain the phenotype in these knockout mice. Circulating IGF-I was reduced by 75% in these animals, although free IGF-I levels were similar in knockouts and controls. We failed to detect treatment effects on IGF-II and IGFBP-5 levels in this study, although increased cortical bone levels of both proteins were detected in 11 of 13 patients receiving GH. This could be due to random variation over time because increases were also seen in the placebo group, but on the basis of our finding of increased cortical levels of IGF-II and IGFBP-5 in acromegalics, this study probably lacked sufficient power to detect any changes in these parameters. Our finding that GH treatment significantly decreases cortical bone contents of osteocalcin is surprising because both GH and IGF-I increase osteoblastic production of osteocalcin. Moreover, within the treatment group, the increase in serum osteocalcin was significantly correlated with a decrease in cortical osteocalcin (r = -0.59; P < 0.05). Thus, our findings could indicate that GH/IGF increases mobilization of osteocalcin from the extracellular matrix to serum, probably related to increased bone turnover. GH and IGF-I may stimulate osteoclastic activity by different mechanisms. Functionally active GH and type I IGF receptors have been reported on osteoclasts (24), and in vitro studies on rabbit bone cells indicate that the effects of GH on osteoclastic resorption are mediated via local IGF secretion by osteoblasts (25). Also, both GH and IGF-I may increase the production of bone resorptive cytokines by osteoblast-like cells and mononuclear cells (26, 27, 28).

A major finding in the present study was that 12-month GH treatment significantly increased both cortical and trabecular bone content of OPG, with a more prominent effect in the cortical envelope. We have recently shown that serum OPG levels were normal in patients with acromegaly, as well as GHD (18). However, serum levels may not necessarily reflect the cytokine levels in the bone microenvironment. On the basis of relatively low levels found in this study, a bone-specific increase in OPG after GH replacement may not be detectable in serum due to production in many tissues and/or changed clearance. In vitro, no effect of GH or IGF-I on OPG mRNA expression in human osteoblasts could be demonstrated. Thus, our findings could represent a compensatory response to increased bone turnover, as found in serum of osteoporotic postmenopausal women (29), or it could be secondary to effects of other OPG-regulating cytokines not accounted for in vitro. On the basis of the stimulatory effect of IGF-I on monocyte and macrophage TNF production and TNF effect on the production of both OPG and RANKL (26, 30), it is possible that the enhanced OPG levels may represent enhanced activity and turnover in the OPG system. To fully evaluate the OPG system in humans, RANKL should be determined. Unfortunately, the assay system used in this study was not sensitive enough to detect RANKL in our bone specimens.

We conclude that 12 months of GH substitution increases cortical and trabecular bone contents of IGF-I and OPG, with a more pronounced effect in cortical bone. This may in turn lead to increased bone mass and biomechanical competence.

View larger version (48K): [in this window] [in a new window]   Figure 1. Cortical and trabecular bone contents of calcium (a), protein (b), IGFs (c–f), osteocalcin (g), and OPG (h) in response to treatment with GH or placebo administration in 25 patients with GHD. Data are expressed per milligram of dry bone (a and b) or total protein (c–h) and are given as mean ± SEM at baseline and after 12 months of treatment. For each parameter, data are also shown as percentage change (mean ± SEM) during GH treatment and placebo. AU, Arbitrary absorbance units; BL, baseline. *, P < 0.05; **, P < 0.01.

Footnotes

Abbreviations: GHD, GH deficiency; WLB, Western ligand blotting.

Received May 31, 2001.

Accepted February 15, 2002.

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