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Original Studies |
Department of Endocrinology, National University Hospital (T.U., J.B., O.D.), N-0027 Oslo, Norway; the Department of Endocrinology, Odense University Hospital (T.B.H., E.N.E.), DK-5000 Odense, Denmark; and the Department of Cell Biology, University of Aarhus (L.M.), and the Department of Endocrinology (K.B.) and the Medical Research Laboratory M (A.F.), Aarhus University Hospital, DK-8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Dr. Thor Ueland, Department of Endocrinology, National University Hospital, N-0027 Oslo, Norway.
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Abstract |
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Introduction |
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The effects of GH on bone tissue are mediated through a complex interaction of circulating GH, insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and locally produced IGFs and IGFBPs acting in auto- and paracrine manners (7). IGF-I and IGF-II have similar biological activities, although IGF-II is the most abundant in bone tissue (8). The IGF system has an important role in regulation of bone cell metabolism. IGFs affect the proliferation and differentiation of bone cells (9, 10) and stimulate cell activity and collagen synthesis. Moreover, IGFs and their binding proteins may be released by bone resorption to act as local determinants of site-specific coupled bone formation (11).
The aim of the present study was to investigate cortical bone composition and the role of the IGF system and osteocalcin content in cortical bone from acromegalic patients compared with those in bone from controls.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study comprised 15 patients (3 women and 12 men), aged 21–64 yr (mean, 45.6 yr), with active acromegaly and 25 age- and sex-matched controls (8 women and 17 men), aged 22–66 yr (mean, 44.6 yr). Activity was defined as nonsuppressible serum levels of GH (>5 mU/L) by oral glucose tolerance test. From each patient, an iliac crest bone biopsy was obtained under local anesthesia, using a modified Bordier trephine (id, 7 mm) from the standard site 2 cm below the iliac crest and 2 cm behind the anterior superior iliac spine. The specimens were frozen at -20 C immediately after removal. Still frozen, the biopsies were later sawed carefully to remove the cortical bone. Anterior-superior parts from the iliac crest were obtained in the same fashion from the controls at necropsy early after death. The controls had died in the hospital or due to accident. None of the individuals had disease interfering with bone metabolism. The study was approved by the local ethical committee and conducted according to the Declaration of Helsinki II.
Preparations of bone specimens
The cortical bone specimens were washed with water to remove soft tissue and blood. Samples were defatted in trichloroethylene for 6 days (changed every second day) at 4 C and dried by immersion in ethanol-ether (1:1, vol/vol). 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 pore size sieve and stored at -40 C until use.
Extraction procedures
For determining IGF-I, IGF-II, IGFBP-3, IGFBP-5, and total protein contents, 15 mg bone powder were extracted once with 1.5 mL 0.5 mol/L ammonium ethylenediamine tetraacetate containing the protease inhibitors benzamidine (5 mmol/L), 6-aminocaproic acid (10 mmol/L), p-hydroxymercuribenzoic acid (100 µmol/L), and phenylmethylsulfonylfluoride (30 µmol/L), pH 6.2, and once with 1.5 mL 4 mol/L 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 dialyzed (mol wt cut-off, 3500; Spectrapor, Spectrum Medical Industries, Los Angeles, CA) on an orbital shaker for 2 days at 4 C against 80 mL 0.02 mol/L acetic acid, lyophilized in a Speed-Vac concentrator (Savant Instruments, Hicksville, NY), and stored at -40 C until assayed.
For the determination of calcium and osteocalcin, 5 mg bone powder were hydrolyzed in 0.5 mL 6 mol/L HCl for 1 week at 4 C by rotation. The hydrolyzed samples were stored at -40 C after the addition of 1 mL 3 mol/L NaOH.
RIAs for IGFBP-3, IGF-I, and IGF-II
The lyophilized ammonium and guanidine extracts were redissolved in 1.0 mL 0.02 mol/L acetic acid and used to determine the cortical contents of IGF-I, IGF-II, IGFBP-3, and total protein.
IGFBP-3 was measured using a commercial RIA kit from Nichols Institute Diagnostics (Nijmegen, The Netherlands). The following modification was made: samples were lyophilized and redissolved in assay buffer, giving a dilution of 1:5 before assay. This kit has a sensitivity of 0.25 ng/mL, an intrassay variation of less than 10%, and no detectable cross-reactivity with IGFBP-1, IGFBP-2, IGF-I, or IGF-II.
IGF-I and IGF-II were measured by RIA and immunoradiometric assay (IRMA) after separation from IGFBPs by acid-gel filtration (12). IGF-I was measured by a commercial kit from Nichols Institute Diagnostics. Samples were lyophilized and redissolved in assay buffer before analysis. This kit has a sensitivity of 0.06 µg/L, an intraassay variation of less than 10%, and no cross-reactivity with IGF-II.
IGF-II was measured using a commercial IRMA kit from Diagnostics Systems Laboratories, Inc. (Webster, TX). Samples were lyophilized and redissolved in phosphate buffer before analysis. This kit has a sensitivity of 0.13 µg/L, an intraassay variation of less than 10%, and no cross-reactivity with IGFBP-2. IGFBP-3, IGFBP-4, or IGFBP-5.
Western ligand blotting (WLB) of cortical bone extracts
Cortical bone extracts (5 µg protein on each lane) were
subjected to WLB to attain a semiquantitative estimate of IGFBP-5
levels. SDS-PAGE (10% polyacrylamide) and WLB were performed under
nonreducing conditions as previously described (13). The
electrophoresed proteins were transferred by electroelution onto
nitrocellulose paper (Schleicher & Schuell, Inc., Munich,
Germany), and membranes were incubated overnight at 4 C with
approximately 500,000 cpm [125I]IGF-I (SA, 2000 Ci/mmol)
in 10 mL 10 mmol/L Tris-HCl buffer containing 1% BSA and 0.1% Tween
(pH 7.4). Membranes were washed with phosphate-buffered saline, and
after drying overnight, the nitrocellulose sheets were autoradiographed
with Kodak X-AR film (Eastman Kodak Co., Rochester, NY)
and exposed to DuPont-New England Nuclear enhancing
screens (Boston, MA) at -80 C for 3–7 days. The specificity of the
IGFBP bands was ensured by competitive coincubation with unlabeled
IGF-I purchased from Bachem (Bubendorf, Switzerland). WLB yielded one
major 28-kDa IGFBP and in some samples a faint 38-/42-kDa (doublet);
the doublet band corresponded to the acid-stable IGF-binding subunit of
IGFBP-3 (14) (Fig. 1
). To confirm the
identity of the major band (28 kDa) in cortical extracts as IGFBP-5,
extracts were analyzed by immunoprecipitation followed by SDS-PAGE and
WLB as described above. Cortical extracts were incubated at 4 C
overnight with a specific human IGFBP-5 antibody (Austral Biologicals,
San Ramon, CA) in a dilution of 1:250. Precipitation was achieved by
the addition of 250 µL solid phase coated cellulose suspension
(Sac-Cel, IDS, Tyne and Wear, UK) followed by incubation at 37 C for
1 h. Bound antibody was separated by centrifugation at 2800 rpm
for 10 min. The precipitated proteins were washed (three times) by the
addition of 1 mL phosphate-buffered saline, 0.25% BSA, and 0.1%
Tween-20; centrifuged at 2800 rpm for 10 min before resuspension in 100
µL gel loading buffer; and exposed to SDS-PAGE and WLB.
Immunoprecipitation with the IGFBP-5 antibody revealed a single 28-kDa
band (data not shown). 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 (Shimadzu Europe, Gmbh, Duisburg, Germany). The relative
densities of the bands were measured as arbitrary absorbance units (AU)
per mm2.
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To evaluate our methodology, nine samples were extracted in duplicate. Two additional extractions with guanidinium-HCl were performed, and supernatants from each extraction were kept separate and analyzed for IGF-I and IGFBP-3 contents as described above. The mean coefficients of variation for duplicate extracted samples were 12.3% and 8.8% for IGFBP-3 and IGF-I, respectively. The extraction efficiencies after the two extractions used in the study were 89 ± 2% and 99 ± 1% for IGF-I and IGFBP-3, respectively.
To evaluate whether growth factors were lost during extraction, dialysis, or acid-gel filtration, four samples were spiked with recombinant IGF-I, and two samples were spiked with IGFBP-3 and assayed according to normal protocol. The mean recoveries of samples spiked with recombinant IGF-I and IGFBP-3 were 112% (118%, 109%, 106%, and 117%) and 97% (72% and 121%), respectively.
To rule out the influence of time delay for necropsy on growth factor levels in controls, three bone samples where divided into two portions each. One was frozen immediately, and the other was left at 2–8 C for 2 days and then frozen. Samples were defatted and extracted as described, and levels of IGF-I and IGFBP-3 were measured. In the time delay experiment, we observed an increase of 6.4 ± 5.1% in IGFBP-3; the coefficient of variation between basal samples (0 days) and samples left for 2 days was 4.3%. The corresponding values for IGF-I were an increase of 9.7 ± 9.1% and a coefficient of variation of 6.1%.
Assay for osteocalcin
Osteocalcin was measured by IRMA with a commercial kit from Incstar Corp. (Stillwater, MN). The assay measures intact osteocalcin-(1–49). Samples were lyophilized and reconstituted in a BSA-borate buffer (10 mmol/L boric acid, 1% BSA, 25 mmol/L NaOH, and 25 mmol/L ethylenediamine tetraacetate, pH 8.5). Samples were diluted 1:16, as suggested by the manufacturer, because fragment interference gave poor dilution linearity. The kit has a sensitivity of 0.2 ng/mL, and an intraassay variation of less than 10%.
Calcium was determined by a colorometric method (15), using a Cobas Mira autoanalyzer (F. Hoffmann-La Roche Ltd., Basel, Switzerland) with a kit from Boehringer Mannheim GmbH (Mannheim, Germany).
Protein concentration was determined with Bio-Rad protein assay (Bio-Rad Laboratories, Inc. GmbH, Munich, Germany).
Statistical analysis
Comparisons between variables in the two groups were performed by 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 < 0.05. A ratio between IGF-I and IGF-II was generated by converting their cortical bone contents from mass equivalents to molar quantities, using molecular masses of 7.7 and 7.5 kDa, respectively.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, the
cortical bone contents of IGF-I and IGF-II were significantly elevated
in the acromegalic patients. The most pronounced increase was seen in
IGF-I, with a concentration twice that found in normal controls
(0.84 ± 0.10 vs. 0.44 ± 0.03 ng/mg dry bone;
P < 0.001). The concentration of IGF-II was 2.28
± 0.32 ng/mg dry bone compared with 1.58 ± 0.14 in controls
(P < 0.05).
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The major 28-kDa IGFBP detected by WLB (IGFBP-5) was significantly
increased (115%) in cortical bone extracts from acromegalic patients
compared with that from controls (278 ± 53 vs.
129 ± 26 AU/mm2; P < 0.01; Table 1
).
The concentrations of total protein and osteocalcin were not significantly different in the two groups.
The calcium content of cortical bone was slightly, but insignificantly, higher in patients compared with controls (3%; P < 0.09).
The ratio between IGF-I and IGF-II showed that cortical bone from acromegalics and controls contained 3.2 and 4.1 times, respectively, more IGF-II than IGF-I. The mean differences were insignificant (P = 0.12).
The age-related changes in cortical content are shown in Table 2. There was a significant age-related
decrease in IGF-I in the patients when expressed per mg bone (r =
-0.79; P < 0.001) and per mg protein (r =
-0.85; P < 0.001). There were no relationships
between age and IGF-II in acromegalics or between age and IGFs in the
controls. A significant age-related increase was observed for IGFBP-3
in acromegalics when expressed per mg bone (r = 0.56;
P < 0.03). However, the relationship was insignificant
when expressed per mg protein.
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No age-related changes were observed in the cortical bone content of calcium. However, a significantly age-dependent increase in protein was observed for the patient population.
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Discussion |
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Serum levels of IGF-II have been found to be normal in acromegaly (19, 20). The bone content of IGF-II is markedly higher than that of IGF-I (8), as confirmed in this study. Moreover, the IGF-II content in cortical bone was markedly increased in our patients. The biological activity of IGF-II in bone is, however, 4–7 times less than that of IGF-I (21). Whether the increase in IGF-II in bone is related to systemic alterations in GH metabolism or is due to local alterations is unknown.
IGFBP-3 is also produced in bone tissue secondary to GH and IGF-I stimulation, and its local production is regulated by calciotropic hormones (22). IGFBP-3 enhances the effect of IGF-I on bone (23) and may, in addition, increase the half-life of IGFs by binding and thus protecting the growth factors for later release into the local environment for biological action (8, 24). We did not find significantly increased levels of IGFBP-3 in cortical bone from acromegalic patients in this study. Recent studies have shown that IGFBP-5 is the most prominent binding protein in bone (11), and its expression is enhanced by IGFs and GH (25). IGFBP-5 has a high affinity for hydroxyapatite and may act to fixate IGFs to the skeletal matrix (11). We found significantly increased levels of IGFBP-5 in acromegalic patients, which may result in increased binding capacity and adherence of more IGFs to the bone matrix. In vitro studies on mouse osteoblasts (26) and human bone cells (27) have shown that IGFBP-5 can increase and potentiate the effects of IGF-stimulated bone cell proliferation, and that it also may modulate its effects via an IGF-independent mechanism. In addition, osteoblasts possess IGFBP-5-specific binding sites (28), sustaining the concept that IGFBP-5 might mediate the anabolic effects of GH on bone formation. As IGF-II is only minimally GH dependent, the elevated levels of the protein found in this study may be due to the increased binding capacity and growth-potentiating effects provided by elevated levels of IGFBP-5. In support of this, a strong relationship between IGFBP-5 and IGFs and osteocalcin was observed in the patients, but not in controls.
Osteocalcin is the major noncollagenous protein in bone and is implicated in the recruitment and differentiation of bone-resorptive cells. The protein is produced by osteoblasts and binds to hydroxyapatite (29). Serum levels of osteocalcin have been found to be normal in acromegalics (30), whereas other serum markers of bone turnover have been found to be increased (3, 4). However, the serum levels of osteocalcin in acromegalics may be explained by the increased renal blood flow secondary to increased GH and IGF-I levels and thereby the increased renal clearance of the protein (31, 32). Surprisingly, a study on the osteocalcin knockout mouse (33) suggests that the absence of osteocalcin leads to increased bone formation without impairing bone resorption or mineralization. This implies that the significance of the bone content of osteocalcin is virtually unknown.
IGF-I was negatively correlated to age in the patient population, but not in the control group. In accordance with recent studies (34, 35), the female subgroup of the controls showed an age-related decrease in IGF-I when expressed per mg protein (r = -0.75; P = 0.031). The age-related changes might reflect the age-dependent decrease in IGF-I, also found in the circulation. As the local bone content of IGF-I seems to be related to the regional bone mineral content (36), the results are in accordance with an age-dependent decrease in cortical bone volume and mineral content. IGF-II was not correlated to age in either of the groups, as also found by others (11), whereas IGFBP-3 was positively age related in patients when expressed per mg bone.
Acromegaly is characterized by appendicular skeletal growth leading to increased dimensions of bones (37). Total body calcium is increased in acromegalics (5), and osteodensitometric investigations have shown normal or increased bone mineral densities (38, 39, 40). We found an insignificantly increased calcium content of cortical bone, expressed per bone weight unit. Thus, based on this result, total bone mineral content should be increased, with a normal or slightly increased density.
In conclusion, this study shows, for the first time, increased levels of growth factors in cortical bone from acromegalics. This is, presumably, a reflection of local production, secondary to a chronic systemic excess of GH and IGF-I.
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Acknowledgments |
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Footnotes |
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Received December 9, 1997.
Revised July 17, 1998.
Revised September 16, 1998.
Accepted September 22, 1998.
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References |
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