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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 4 1557-1562
Copyright © 2000 by The Endocrine Society

Original Studies

Insulin-Like Growth Factor I (IGF-I) Replacement during Growth Hormone Receptor Antagonism Normalizes Serum IGF-Binding Protein-3 and Markers of Bone Formation in Ovariectomized Rhesus Monkeys1

Mark E. Wilson

Yerkes Primate Research Center, Emory University, Lawrenceville, Georgia 30043

Address all correspondence and requests for reprints to: Dr. Mark E. Wilson, Yerkes Primate Research Center, Emory University, 2409 Taylor Lane, Lawrenceville, Georgia 30043. E-mail: markw{at}rmy.emory.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Previous work from this laboratory has shown that the constant sc infusion of insulin-like growth factor I (IGF-I) to normal pituitary monkeys results in a sustained elevation in circulating concentrations of IGF-binding protein-3 (IGFBP-3), whereas the acute administration of IGF-I to monkeys pretreated with a GH receptor antagonist produces a brief, but significant, elevation in serum IGFBP-3. The present study tested the hypothesis that the constant infusion of IGF-I would normalize serum concentrations of IGFBP-3 in females treated with the GH receptor antagonist. To assess the biological significance of these effects, serum levels of the acid-labile subunit (ALS) and biomarkers for bone formation, osteocalcin, and collagen type I C-terminal propeptide, were also examined. Five female rhesus monkeys were studied over 21 consecutive days involving 7 days of baseline, 7 days of treatment with the GH receptor antagonist (1.0 mg/kg·week, sc), and 7 days of treatment with the GH receptor antagonist supplemented with IGF-I (120 µg/kg·day, sc infusion with osmotic minipump). Within 48 h of the initiation of treatment with the GH receptor antagonist, serum IGF-I and IGFBP-3 were decreased by 40% and 18% from baseline, respectively, and levels continued to decline through the remainder of treatment. However, within 48 h of the initiation of IGF-I administration during GH receptor antagonist treatment, both serum IGF-I and IGFBP-3 were elevated and normalized to baseline values. Serum concentrations of ALS were also decreased by GH antagonism, but levels increased in some (n = 2), but not all, subjects upon administration of IGF-I. Size exclusion ultrafiltration indicated that the amount of IGF-I found in the high molecular mass complex (>100 kDa) decreased significantly during GH antagonism, but was similar during the baseline and IGF-I infusion phases. Finally, treatment with the GH receptor antagonist also significantly reduced serum levels of osteocalcin and collagen type I C-terminal propeptide, an effect reversed by the addition of IGF-I. These data support the hypothesis that IGF-I increases serum concentrations of IGFBP-3 when endogenous GH action is compromised and that such treatment produces biologically active IGF-I, as evidenced by normalization of biomarkers for bone formation. These results indicate that IGF-I administration during GH receptor antagonism restores circulating levels of IGFBP-3 and the amount of IGF-I found in the high molecular mass complex to levels observed during baseline conditions. It remains to be determined whether IGF-I directly affects hepatic synthesis and secretion of IGFBP-3 and what role IGF-I has in the direct regulation of ALS in the monkey.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE MAJORITY of insulin-like growth factor I (IGF-I) circulates as a 150-kDa complex, bound to IGF-binding protein-3 (IGFBP-3) and to an acid-labile subunit (ALS), which functions to slow degradation of IGF-I, limiting transport of IGF-I to the extravascular space (1, 2). Furthermore, interaction of IGF-I with its receptor is diminished in the presence of a molar excess of IGFBP-3 (3). However, proteolysis of IGFBP-3 facilitates the release of IGF-I into the interstitial space and interaction with the IGF receptor (1, 2, 3). Indeed, the formation of the 150-kDa ternary complex promotes more growth than produced by the binary complex of IGF-I and IGFBP-3 (4), which, in turn, may be a more effective than IGF-I alone (5). Thus, the presence of bound IGF-I is important for its eventual bioactivity. Like IGF-I, IGFBP-3 (6, 7, 8, 9) and ALS (10) synthesis and release are dependent upon GH. Consequently, serum levels of these are reduced in GH deficiency (4, 11) or GH receptor deficiency (GHRD) (11, 12) and are increased in acromegaly (13). However, IGF-I stimulates IGFBP-3 messenger ribonucleic acid (mRNA) (6, 12) and secretion (14) and decreases IGFBP-3 mRNA degradation in rats (15). Although some evidence in humans suggests that IGF-I may increase serum IGFBP-3 in children with GHRD (16, 17, 18) or diabetes (19), reports generally conclude that IGF-I does not increase (20, 21, 22, 23) and may actually suppress serum IGFBP-3 (24, 25). In contrast to these data from humans, observations of monkeys with undisturbed GH secretion show that acute IGF-I treatment produces a brief, but significant, increase in serum IGFBP-3 (26) and a constant sc infusion (26, 27) or twice daily injection of IGF-I (28) sustains an elevation in serum IGFBP-3. Furthermore, acute IGF-I administration during either GH inhibition with octreotide or antagonism with the GH receptor blocker, B2036-PEG, produces an acute, yet significant, increase in serum IGFBP-3 (29). However, it is not known whether IGF-I administration can sustain an increase in IGFBP-3 when GH secretion or action is disrupted in this animal model.

It is not known how IGF-I infusion to normal pituitary monkeys affects serum concentrations of ALS. However, it is assumed that acute or more chronic IGF-I administration would suppress ALS levels given the negative feedback effectiveness of IGF-I on GH secretion (30). Size exclusion chromatography reveals that approximately 85% of IGFBP-3 is found in a molecular mass complex greater than 100 kDa during both baseline and these IGF-I infusion conditions (28). These data suggest that IGF-I has little, if any, impact on GH-dependent regulation of ALS in monkeys under these experimental conditions. On the other hand, IGF-I administration to hypophysectomized rats results in the formation of a high molecular mass complex (200 kDa) of IGF-I/ IGFBP-3, which is absent ALS (4). Although growth in these hypophysectomized animals is enhanced by IGF-I treatment alone, it fails to achieve the level produced when the ternary complex is reestablished by GH or GH given in combination with IGF-I replacement (4).

To better understand how IGF-I may regulate IGFBP-3, the present study tested the hypothesis that circulating concentrations of IGFBP-3 would be suppressed by GH receptor antagonism, but would be normalized upon addition of IGF-I administration. We also measured serum markers for bone formation to determine the biological effectiveness of the treatments. It was expected that the markers for bone formation would be reduced by GH antagonism and restored toward normal by coadministration with IGF-I.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Subjects for this study were young adult, female rhesus monkeys (n = 5) who had been ovariectomized approximately 5 yr earlier as juveniles. Ovariectomized subjects were used to obviate any effects that estradiol could have on the GH-IGF-I axis (29). Animals were housed indoors in pairs under a fixed photoperiod (12 h of light, 12 h of darkness) and temperature (25 C) as described previously (27). The subjects were fed commercial monkey chow (Harland Tekland, Madison, WI) twice daily (0700 and 1500 h) and fresh fruit once daily (1500 h) and had continuous access to water. The protocol was approved by the Emory University institutional animal care and use committee in accordance with all NIH and USDA standards.

Procedures

Each subject was studied during each of the three treatment conditions: baseline (no treatment), GH receptor antagonism (GHx), and GH receptor antagonism plus IGF-I administration (GHx + IGF-I). Each condition lasted 7 days. The GH receptor antagonist (B2036-PEG, Sensus Drug Development Corp., Austin, TX) was administered on the last day of the baseline and GHx conditions at a dose of 1.0 mg/kg, sc. This dosing frequency effectively antagonizes endogenous GH activity for 7 days (29). Thus, in the present study, subjects received two injections of the GH receptor antagonist, 7 days apart. IGF-I administration was achieved by the constant sc infusion of IGF-I (Genentech, Inc., South San Francisco, CA) at a dose of 120 µg/kg·day, which effectively elevates serum IGF-I approximately 80% above baseline values (26). Infusions were accomplished with osmotic minipumps (Alza Corp., Palo Alto, CA) implanted between the scapula while the subjects were anesthetized with Telazol (3 mg/kg, im). Pumps were implanted on the last day of the GHx condition.

Serum samples (3 mL) were obtained on 5 consecutive days beginning 24 h after the initiation of each condition (designated days 1–5). These samples were obtained at 0800 h, or 60 min after the morning meal. Subjects were well habituated to the handling procedures, so that samples could be collected without anesthetizing the animals (31, 32). All samples were assayed for IGF-I and IGFBP-3. In addition, selected samples were assayed for ALS, osteocalcin, and collagen type I C-terminal propeptide (PICP).

Analyses

Serum IGF-I was determined by RIA using procedures previously described (33). The IGFBPs were denatured by preincubation of the samples with equal volumes of 0.2 mol/L glycine-HCl (pH 3.2) for 48 at 37 C as described previously (33). After removal of the binding proteins, samples were assayed at 0.1–0.5 µL equivalents, which yielded an assay range from 20–5,000 ng/mL. Intra- and interassay coefficients of variation were less than 5% and 11.6% (n = 8 assays), respectively. Serum IGFBP-3 was determined using a commercially available enzyme-linked immunosorbent assay (ELISA; Diagostics Systems Laboratories, Inc., Webster, TX). This assay has a sensitivity of 202 ng/mL and an upper limit of 11,615 ng/mL. Intra- and interassay coefficients of variation were 2.79% and 2.74% (n = 7 assays), respectively. Serum ALS was determined using a commercially available ELISA (Diagostics Systems Laboratories, Inc.). Samples were assayed at 1 and 2 µL equivalents, yielding an assay sensitivity of 0.15 µg/mL. Intra- and interassay coefficients of variation were less than 8.7% and 11.39% (n = 3 assays), respectively. Serum concentrations of osteocalcin were determined with a commercially available RIA (Diagostics Systems Laboratories, Inc.) with a sensitivity of 1.0 ng/mL. All sample measurements of osteocalcin were performed in the same assay. Serum concentrations of PICP were determined with a commercially available RIA (Metra Biosystems, Mountain View, CA) with a sensitivity of 1.0 ng/mL. All sample measurements of PICP were made in the same assay.

As the ELISA for IGFBP-3 detects the intact molecule as well as fragments of IGFBP-3, serum samples from each subject were pooled on days 2 and 4 of each phase and were also subjected to electrophoresis and immunodetection of IGFBP-3 following procedures previously described (28, 34). Photographs of the resulting gel films were scanned (Afga ArtLine) into a Macintosh computer, and the resulting images were digitized (Silk Scientific, Orem, UT), which determines the band density, migration distances, and mol wt of each band based on mol wt standards. Samples from day 5 of each treatment phase were also subjected to size exclusion ultrafiltration following the procedure described previously (35). Samples were diluted 1:4 in 0.05 mol/L sodium phosphate buffer and applied to a unit with a 100-kDa cut-off (Centricon-100, Amicon, Inc., Beverly, MA) and centrifuged at 1000 x g for 30 min. The retentate, containing the ternary complexes, and the filtrate, containing the binary complexes, were assayed for IGF-I as described above.

Statistics

Data were expressed as the mean ± SEM. The effect of treatments were evaluated with ANOVA for repeated measures, with differences between specific time points assessed with the Newman-Keuls post-hoc test (GB-Stat version 6.5 for the Macintosh, Scolari Software, Thousand Oaks, CA). The analysis of treatment effects on serum IGF-I and IGFBP-3 had power estimates of 95% or more ({alpha} = 0.05), whereas the statistical analysis of the markers of bone formation had power estimates between 70–80%. If the assumption for the homogeneity of variance was violated, the nonparametric Friedman statistic for related samples was used followed by the Wilcoxon signed rank test to determine differences at specific time points. Regression models were used to evaluate the linear relationship among specific variables. All statistical tests with P <= 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Serum concentrations of IGF-I were suppressed significantly by GHx administration but were normalized to baseline levels once IGF-I treatment was initiated (F2, 8 = 7.21; Fig. 1Go). Serum IGF-I was decreased within 24 h of GHx administration, and levels continued to decline significantly through day 5 of treatment (Newman-Keuls tests). Upon initiation of IGF-I infusion, levels returned to baseline within 24 h, where they remained throughout the GHx + IGF-I condition (Newman-Keuls tests). The percent changes in serum IGF-I from baseline on day 5 of GHx and day 5 of GHx + IGF-I were -78 ± 4% and 4 ± 28%, respectively.



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  		Figure 1. Mean ± SEM serum concentrations of IGF-I (top) and IGFBP-3 (bottom) during baseline (open bar), GH receptor antagonism (GHx; shaded bar), and GHx + IGF-I (black bar). By post-hoc analyses with P <= 0.05: *, baseline, GHx + IGF-I vs. GHx; a, baseline vs. GHx; b, baseline and GHx vs. GHx + IGF-I.

 
Serum concentrations of IGFBP-3 also were significantly suppressed by GHx and were normalized upon initiation of IGF-I treatment (F2, 8 = 7.07; Fig. 1Go). Unlike serum IGF-I, levels of IGFBP-3 were still similar to baseline values 1 day after GHx administration, but declined significantly thereafter through day 5 of the GHx condition (Newman-Keuls tests). Upon the initiation of IGF-I treatment, serum IGFBP-3 was increased significantly above values observed during both the GHx treatment phase as well as during baseline (Newman-Keuls tests). These treatment differences are reflected in the percent change in serum IGFBP-3 from baseline on day 5 of GHx and day 5 of GHx + IGF-I (-30 ± 5% and 21 ± 15%, respectively).

The immunoblot analysis of serum collected on days 2 and 4 of each treatment phase also showed that IGFBP-3 fragments were reduced by GHx and restored toward baseline with the addition of IGF-I (Fig. 2Go). Digital analysis of the resulting images revealed that the percent change from baseline in the area of the band migrating to 42 kDa was reduced by 59% during GHx treatment, but was increased by 15% above baseline during IGF-I treatment. The percent change in the area of the band migrating to 31 kDa was reduced from baseline by 35% during GHx and by 14% during IGF-I replacement. Thus, although still reduced compared to baseline, the intensity of the 31-kDa band approached that observed during baseline with the addition of IGF-I.



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  		Figure 2. Immunoblot of IGFBP-3 fragments after SDS-PAGE of serum pools at 48 and 96 h during each baseline (base), GH receptor antagonism (GHx), and GHx + IGF-I (Igf) treatment. Molecular masses (kilodaltons) are shown at the left.

 
Serum concentrations of ALS were also decreased by GHx, but the response to IGF-I administration varied among the individual subjects (Fig. 3Go). The percent change from day 5 of baseline to day 5 of GHx was -36 ± 10%, with all females showing a decline. In contrast, serum ALS levels during day 5 of the GHx + IGF-I condition remained suppressed in three subjects (-38% ± 12 of baseline values), but were increased in two subjects (+86 ± 7% of baseline). Subsequent regression analysis revealed that the percent change from baseline values during GHx in ALS (i.e. a decrease) was not related to the change in either IGF-I (r = 0.43) or IGFBP-3 (0.43). In contrast, the percent change from baseline in serum ALS during the IGF-I replacement phase was significantly related to the changes in IGF-I (r = 0.96) and IGFBP-3 (r = 0.92). Stated another way, those subjects that did not show an increase in ALS upon addition of IGF-I replacement had a smaller increase in serum IGF-I during this phase. Multiple regression analysis revealed that the change in serum IGF-I resulting from IGF-I infusion significantly predicted the change in ALS (r = 0.96; t = 5.07). Changes in IGFBP-3 did not contribute significantly to the equation given the significant correlation between the change in IGF-I and IGFBP-3 during both GHx (r = 0.93) and the IGF-I replacement phase (r = 0.99). Indeed, multiple regression analyses revealed that the change in IGF-I significantly predicts the change in IGFBP-3 during both GHx (r = 0.99; t = 14.28) and IGF-I replacement condition (r = 0.93; t = 4.45), with ALS not contributing significantly to the equation. Thus, the magnitude of the change from baseline in IGFBP-3 and ALS was related to the change in IGF-I induced by the treatments.



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  		Figure 3. Mean ± SEM serum concentrations of ALS throughout the study period. Samples corresponding to each treatment phase are indicated at top of the panel.

 
The size exclusion ultrafiltration analysis of samples obtained at the end of each treatment phase revealed that GH receptor antagonism resulted in a significant decrease in IGF-I found in the ternary complex (i.e. complexes >100 kDa), but replacement with IGF-I during GH receptor antagonism restored the proportion of IGF-I found in the high molecular mass complex to baseline values (F2, 8 = 10.09). The proportion of IGF-I found in the high molecular mass complex was similar during baseline (96.0 ± 0.5%) and IGF-I supplementation (96.8 ± 0.5%), but was significantly decreased during GH antagonism (93.2 ± 0.8%; post-hoc tests). In terms of actual concentrations of IGF-I found in this high molecular mass complex, values during baseline (302 ± 29 ng/mL) and IGF-I supplementation (293 ± 66 ng/mL) were greater than those observed during GH antagonism (63 ± 10 ng/mL). Despite this significant decrease in the high molecular mass complex during GH receptor antagonism, these data indicate that more than 90% of IGF-I in circulation is found in complexes more than 100 kDa as opposed to lower molecular mass complexes or free IGF-I.

These changes in the IGF-I axis resulting from GHx and subsequent IGF-I replacement were associated with significant changes in biomarkers for bone formation (Fig. 4Go). Concentrations of PICP declined significantly over the course of the three treatment conditions (F2, 8 = 9.03). Post-hoc analyses indicated that PICP values on treatment day 3 of the GHx + IGF-I phase were significantly lower than those on all other days sampled. Values observed after 5 days of IGF-I treatment were indistinguishable from those observed during baseline. Although concentrations during the GHx phase were 9 ± 4% lower than baseline values, differences were not significant. Serum levels of osteocalcin show a similar pattern, but given the individual variation in absolute values differences were only significant using the nonparametric Friedman test ({chi}2 = 12.22). Post-hoc (Wilcoxon matched pair tests) indicated that levels of osteocalcin on day 3 of the GHx + IGF-I treatment phase were significantly lower than values from the day preceding and following this sample as well as from day 3 of baseline. Again, levels during GHx were 11 ± 4% lower than during baseline, but differences were not significant. Finally, osteocalcin concentrations after 5 days of IGF-I treatment were also not different from baseline values.



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  		Figure 4. Mean ± SEM serum concentrations of PICP (open symbol) and osteocalcin (closed symbol) during baseline, GH receptor antagonism (GHx), and GHx + IGF-I treatments. By post-hoc tests, P <= 0.05: a, day vs. all others; b, day vs. baseline day 3, GHx day 4, and GHx + IGF-I day 5.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The reduction in serum concentrations of IGF-I and IGFBP-3 induced by administration of the GH receptor antagonist were reversed by the addition of IGF-I to the treatment regimen. Although serum ALS was also reduced by the GH receptor antagonist, the response in ALS to IGF-I varied among the individual subjects. This diminution in the IGF-I axis by the GH receptor antagonist was associated with a delayed decrease in serum markers for bone formations, an effect reversed after 5 days of IGF-I administration.

These data support previous observations from monkeys with undisturbed GH secretion (26, 27, 28) and indicate that the constant sc infusion of IGF-I produces a sustained elevation in serum concentrations of IGFBP-3 when endogenous GH action is blocked. Indeed, the individual change in IGFBP-3 from baseline resulting from GH receptor antagonism and subsequent IGF-I replacement was predicted from the change in IGF-I. This effect could be due to either an IGF-I- induced increase in IGFBP-3 synthesis and secretion or a decrease in IGFBP-3 degradation. IGF-I replacement to hypophysectomized rats normalizes hepatic IGFBP-3 mRNA (9) and increases circulating concentrations of IGFBP-3 in GH-deficient rats (14). Furthermore, IGF-I and insulin stimulate IGFBP-3 mRNA and secretion from the hepatic nonparenchymal, Kupffer cells, but only when these cells are cocultured with hepatocytes (36). In addition, there is evidence from rats that IGF-I slows IGFBP-3 mRNA degradation (15).

These data contrast with the view that IGFBP-3 synthesis and secretion are exclusively GH dependent (2, 7) as the overriding conclusion from studies of IGF-I replacement to patients with GHRD is a failure of IGF-I to increase IGFBP-3 concentrations (20, 21, 22, 23, 24, 25). Nevertheless, other studies do suggest that circulating IGFBP-3 is enhanced by IGF-I therapy in these clinical situations (16, 17, 18). It is not known what accounts for the unequivocal increase in IGFBP-3 by IGF-I in the present monkey model. Although it is possible that the facilitating effects of IGF-I are receptor mediated, IGF-I may simply slow degradation of IGFBP-3 from the circulation. This hypothesis is supported by the observation that an IGF-I analog that has normal affinity for the IGF receptor but reduced affinity for the IGFBPs does not increase serum IGFBP-3 (37). Finally, it would seem that multiple factors may regulate IGFBP-3 production and contribute to its circulating concentrations. The GH receptor antagonist used in the present study effectively reduces IGF-I levels by almost 80%, with values approaching 50 ng/mL with the assay system used in the present study. In contrast, serum IGFBP-3 during GH antagonism was reduced only 30% to values in the range of 2200 ng/mL. Furthermore, immunoblot analysis of IGFBP-3 revealed reduced, yet prominent, bands at 42 and 31 kDa during GH receptor antagonism. It is possible that an even larger dose of B2036-PEG would have diminished IGFBP-3 levels further. On the other hand, some other factor(s) appears to be maintaining IGFBP-3 synthesis, secretion, and stability during GH receptor antagonism. Given the differences in methodologies and assay systems used to investigate the regulation of the IGFBPs by IGF-I (38), systematic studies can only rectify these apparent species differences and provide a unifying understanding of IGFBP-3 regulation.

Administration of the GH receptor antagonist significantly reduced serum concentrations of ALS after 5 days of treatment, supporting the idea that ALS is GH dependent (11, 12, 13). However, unlike IGFBP-3, the effects of IGF-I replacement on ALS concentrations during GH receptor antagonism were equivocal. In some cases ALS levels were increased by IGF-I; in others levels remained suppressed, a pattern not unlike that seen in GH receptor-deficient children receiving IGF-I therapy (11, 17). Interestingly, the magnitude of the change in ALS during IGF-I replacement in the present study was positively related to the change in IGF-I. Although other experimental models (4, 10, 14) support clinical observations of the dependence of ALS secretion on GH (11, 12, 13), the observation that IGF-I increased ALS concentrations in some subjects in the present study is similar to a brief report in children with GHRD (16). Furthermore, high concentrations of IGF-I increase ALS mRNA in rat hepatocyte cultures, whereas GH and insulin dose dependently stimulate ALS gene expression (36). The assay employed in the present study uses a polyclonal antibody to human ALS. Although we were able to detect dose (i.e. volume) responses (binding changes) using monkey serum, ALS values were at the low end of the assay system. Furthermore, a more rigorous sampling schedule across longer treatment periods would more fully define the precise timing of the response to GH receptor antagonism and replacement with IGF-I as well as the magnitude of this response. Additional studies of the hormonal regulation of ALS in monkeys are needed to elucidate how IGF-I affects circulating forms of ALS.

The capacity of IGF-I to increase serum IGFBP-3 and ALS may impact the biological effectiveness of IGF-I replacement in GH-depleted conditions. Formation of the binary and ternary complexes not only slows IGF-I degradation, but, through proteolysis of IGFBP-3, facilitates the release of IGF-I into the interstitial space and interaction with the IGF receptor (1, 2, 3). Low mol wt complexes, i.e. those without ALS, are capable of stimulating growth in rat models (39). Results from ultrafiltration analysis in the present study indicated that the proportion of IGF-I found in the high mol wt complex was significantly reduced by GH receptor antagonism, corresponding to a significant reduction in circulating IGFBP-3 and ALS. Despite this decrease, more than 90% of the remaining IGF-I in circulation was nevertheless found in the high mol wt complex. Thus, even during GH receptor antagonism, the small amount of IGF-I that was available (~63 ng/mL) was found in the complex that delays IGF-I degradation, prolonging its potential for biological activity.

In the present study GH antagonism decreased and the addition of IGF-I increased serum markers of bone formation by osteoblasts, PICP and osteocalcin. Interestingly, the decrease in these biomarkers was not statistically significant until day 10 of GH receptor antagonist treatment, corresponding to day 3 of the combined antagonist-IGF-I replacement phase. However, these biomarkers had returned to baseline by day 5 of the combined treatment regimen. IGF-I administration to humans increases serum levels of osteocalcin (40, 41) and PICP (40, 41, 42, 43), and this increase occurs within 2 days of IGF-I administration in some studies (40) and after 4 days in others (41, 43). In the present study resources were not available to monitor daily levels of these biomarkers to determine precisely when treatments were effective. However, it is evident that the IGF-I-induced increase in these markers occurred between days 2 and 5 of the IGF-I replacement phase. Thus, the data indicate that IGF-I administration can promote bone formation when endogenous GH activity is blocked and support numerous studies in hypophysectomized rats (39) and children receiving GH-releasing hormone (22, 44, 45) that systemic IGF-I increases bone growth. As IGFBP-3 concentrations are in molar excess of IGF-I concentrations despite GH receptor deficiency, it appears that exogenously administered IGF-I readily forms the binary and ternary complexes. A critical point not addressed by the present analysis is how the variability in these markers of bone formation in response to treatment is accounted for by the response of IGFBP-3 and, perhaps, ALS to IGF-I therapy. Understanding the mechanisms involved in this interindividual variation may better define potential treatment strategies for IGF-I replacement.

In summary, the present study indicates that the GH receptor antagonist, B2036-PEG, significantly reduces circulating concentrations of IGF, IGFBP-3, and ALS as well as markers of bone formation. Despite these reductions, the majority of IGF-I in circulation appears to be found in the ternary complex. Upon subsequent replacement therapy with IGF-I, IGFBP-3 levels are normalized within 1 day of treatment, and markers of bone formation are normalized within 5 days of treatment. In contrast, the response of ALS to IGF-I replacement was variable. Taken together, these data suggest that the synthesis, secretion, and stability of the components of the ternary complex are not exclusively dependent on GH in this monkey model.


   Acknowledgments
 
The technical assistance of Susie Lackey and Mara Lindsley is greatly appreciated. The antibody used in the IGF-I assay was a gift from the National Hormone and Pituitary Program, USDA, NIDDK, and Dr. A. F. Parlow (University of California-Los Angeles Medical Center). The author is most appreciative to Genentech, Inc., for the gift of the IGF-I and Sensus Drug Development Corp. for the gift of the GH receptor antagonist. All assays were performed in the Yerkes Assay Services Laboratory.


   Footnotes
 
1 This work was supported by NIH Grants HD-16305 and in part RR-00165 and an award from Sensus Drug Development Corp. The Yerkes Research Center is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Back

Received September 10, 1999.

Revised December 14, 1999.

Accepted December 20, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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