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Endocrinology Vol. 138, No. 12 5316-5324
Copyright © 1997 by The Endocrine Society

ARTICLES

Recombinant Human Growth Hormone-Binding Protein Fails to Enhance the in Vivo Bioactivity of Human Growth Hormone in Normal Rats1

Marinella Tzanela2, Clemens Wagner3 and Gloria Shaffer Tannenbaum4

Departments of Pediatrics, Neurology and Neurosurgery, McGill University; and the Neuropeptide Physiology Laboratory, McGill University-Montreal Children’s Hospital Research Institute, Montreal, Québec H3H 1P3, Canada

Address all correspondence and requests for reprints to: Dr. Gloria S. Tannenbaum, Neuropeptide Physiology Laboratory, McGill University-Montreal Children’s Hospital Research Institute, 2300 Tupper Street, Montreal, Québec H3H 1P3, Canada. E-mail: mcta{at}musica.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH circulates in the plasma partially bound with a GH-binding protein (GHBP), but the physiological significance of the GHBP and how it affects GH bioactivity in vivo is still unknown. In the present study, we took advantage of the known biological action of exogenous human (h) GH to inhibit endogenous rat (r) pulsatile GH release and examined the effect of combining hGH with recombinant hGHBP on this response in normal rats. Spontaneous 7-h plasma rGH and hGH profiles were obtained from four groups of free-moving adult male rats sc administered either: 1) 200 µg hGH alone; 2) a mixture of 200 µg hGH and 200 µg hGHBP preincubated for 30 min before injection; 3) 200 µg hGHBP alone; or 4) Tris buffer (vehicle) alone. Rats administered the vehicle or hGHBP separately exhibited the typical pulsatile pattern of rGH secretion. Injection of hGH alone resulted in a marked (P < 0.01) suppression of spontaneous rGH pulses for approximately 3.5 h after the injection compared with vehicle-injected controls; during the subsequent 3.5- to 7-h period, recovery of spontaneous rGH peaks was evident. Plasma levels of hGH in these animals reached a peak within 1 h after hGH injection and declined to near undetectable levels by the end of the sampling period. In contrast, the disappearance rate of hGH was markedly slower in rats administered the hGH + hGHBP complex; plasma hGH concentrations at 7 h after injection were 14-fold higher than those in animals administered hGH alone, and hGH was still readily detectable up to 24 h after injection. However, despite the markedly higher levels of hGH persisting throughout the sampling period in complex-injected rats, both the time course of hGH-induced inhibition of rGH and the recovery of spontaneous rGH pulses were similar to those of animals administered hGH alone. Moreover, there were no significant modifications of plasma insulin-like growth factor-1 levels for up to 24 h after injection of the hGH + hGHBP complex. Computer simulations revealed that most of the total hGH observed during the 3.5- to 7-h period was circulating in the bound form. These results demonstrate that, despite hGHBP’s ability to markedly prolong the bioavailability of hGH, precomplexing hGH with hGHBP failed to enhance hGH’s in vivo bioactivity in the inhibition of endogenous pulsatile rGH release. Our findings do not provide support for the concept that the GHBP enhances the bioactivity of GH in vivo, at least over the time course examined here.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DISCOVERY of GH-binding proteins (GHBP) in plasma (1, 2) has added a new complexity to the field of GH regulation and action. The principal GHBP in humans is a 60 kDa glycoprotein with high affinity and specificity but low capacity for GH; the kinetics of the GHBP allow rapid complex formation with GH in vivo, so that under normal basal conditions 50% of plasma GH circulates in bound form (see Ref. 3 for review). The cloning of the GH receptor and the sequencing of GHBP identified the GHBP as the extracellular portion of the GH receptor (4). The GHBP has been found in the blood of humans and several animal species (5, 6, 7); in rats and mice it arises from an alternatively spliced messenger RNA (mRNA) encoding a shortened version of the GH receptor gene transcript (8, 9), whereas in humans and rabbits it results from specific proteolysis of the full length receptor (10).

Despite advances in elucidating the structure and regulation of plasma GHBP, the biological significance of the GHBP for GH action remains obscure. A prominent effect of the GHBP in plasma is its influence on the kinetics of GH. The metabolic clearance of bound GH is about 10-fold lower than that of free GH and the distribution volume is markedly restricted in the rat (11, 12) and guinea pig (13), as the GH + GHBP complex is too large for glomerular filtration and degradation. Thus, it has been postulated that the GHBP may serve an important positive role to augment GH bioactivity by prolonging its tissue bioavailability (14, 15).

On the other hand, it has been demonstrated in vitro, in several cell lines, that GHBP can inhibit the binding of GH to its receptor, thereby presumably diminishing its biological action. Thus, GHBP competes with tissue GH receptors for GH binding in a dose-dependent fashion in human, rabbit, and female rat liver (2, 16, 17), in rat adipocytes (2, 16), and in IM-9 human lymphocytes (14, 18). The GHBP also inhibits GH-stimulated adipogenesis of 3T3-F442A preadipocytes (14), GH bioactivity in Nb2 lymphoma cells (18, 19), and insulin-like growth factor 1 (IGF-1) production by cultured human fibroblasts (16). The net effect of these two opposing actions of the GHBP for normal GH physiology is not known.

At the present time, there is a paucity of data on GHBP’s effects on the bioactivity of GH in vivo. In one recent study (20), chronic coadministration of recombinant human (h) GHBP with recombinant hGH was shown to enhance the growth-promoting and IGF-1 producing activity of hGH in GH-deficient hypophysectomized and dwarf (dw/dw) rats, although in two previous reports (21, 22) it failed to do so. In general, these authors (20, 22) interpreted their data to indicate that the effect of the GHBP on prolonging GH bioavailability was dominant over the effect of competition with hepatic GH receptors. However, all of these studies were carried out in GH-deficient animal models. To further explore the fundamental question of GHBP’s role in the regulation of GH bioactivity under normal physiological conditions, in the present study we took advantage of the known biological action of exogenous hGH to inhibit endogenous rat (r) GH secretion (23), and examined the effect of combining hGH with recombinant hGHBP on this response in normal conscious rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental procedure
Adult male Sprague-Dawley rats (290–375 g) were obtained from Charles River Canada (St. Constant, Québec, Canada) and individually housed on a 12-h light, 12-h dark cycle (lights on, 0600–1800 h) in a temperature (22 ± 1 C)- and humidity-controlled room. Purina rat chow (Ralston Purina, St. Louis, MO) and tap water were available ad libitum. Chronic intracardiac venous cannulae were implanted under sodium pentobarbitol (50 mg/kg, ip) anesthesia, using a previously described technique (24). After surgery, the animals were placed directly in isolation test chambers, with food and water available ad libitum until body weight returned to preoperative levels (usually within 7 days). During this period, the rats were handled daily and were habituated to sc punctures into the dorsum to minimize any possible stress associated with handling on the day of the test. On the test day, food was removed 1.5 h before the start of sampling and returned at the end.

We first documented the effects of a single sc injection of recombinant hGH (Somatotropin 5 mg/vial, code no. GO72A-49032AX; Genentech, South San Francisco, CA) on endogenous spontaneous rGH release. One group of free-moving chronically cannulated rats (n = 7) was sc administered 200 µg hGH, freshly dissolved in 0.4 ml Tris buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, pH 7.5), at 0900 h after removal of the first blood sample, whereas a second group (n = 8) served as control and received 0.4 ml Tris buffer alone at the same time point.

We then compared the effects of hGH injected alone with those of hGH complexed with nonglycosylated recombinant hGHBP on spontaneous rGH release. The GHBP (molecular mass: 28 kDa) was kindly provided by Dr. Ross Clark and the late Dr. Michael Cronin, Genentech, South San Francisco, CA (lot no. 16589–76). It was produced in Escherichia coli, binds indistinguishably from full-length recombinant GHBP in vitro and in vivo and corresponds to a naturally occurring splice variant of the GH receptor (25). Mixtures of hGH and hGHBP were preincubated in Tris buffer at room temperature for 30 min before injection. In one group of rats (n = 5), the complex of 200 µg hGH with 200 µg hGHBP, in a total volume of 0.4 ml, was sc administered at 0900 h, whereas another group (n = 7) received hGHBP (200 µg/0.4 ml) alone at the same time point. Blood samples (0.4 ml) were withdrawn every 15 min over a 7-h sampling period (0900–1600 h) from all animals. An additional blood sample was obtained the next morning at 0900 h, i.e. 24 h post treatment.

All blood samples were immediately centrifuged, and the plasma was separated and stored at -20 C for subsequent assay of rGH, hGH, and IGF-1. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample.

All animal-based procedures were approved by the McGill University Animal Care Committee.

Hormone assays
Plasma rGH, hGH, and IGF-1 concentrations were measured in duplicate by double antibody RIA, using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, MD). For rGH, the averaged plasma rGH values are reported in terms of the rGH reference preparation (rGH RP-2). The standard curve was linear between 0.62–320 ng/ml; the least detectable concentration of plasma rGH under the conditions used was 1.2 ng/ml. The intra and interassay coefficients of variation were 7.8% and 14.7%, respectively, for duplicate samples of pooled plasma containing a mean GH concentration of 9.1 ng/ml. For hGH, the averaged plasma hGH values are reported in terms of the hGH reference preparation (hGH RP-1). The standard curve was linear between 1.0–100 ng/ml and the intra and interassay coefficients of variation were 7.3% and 9.5%, respectively, for duplicate samples of pooled plasma containing a mean hGH concentration of 3.3 ng/ml. Both free and bound hGH are measured as immunoreactive hGH (26). The degree of cross-reactivity of hGH and rGH in the two immunoassays was less than 2%.

Plasma IGF-1 concentrations were measured in duplicate by double antibody RIA using a modification (27) of previously described methods (28, 29). To decrease the interference of IGF binding proteins in the assay, the samples were prepared by acid-ethanol extraction followed by cryoprecipitation. The IGF-l/SmC rabbit antiserum (UB3–189) was obtained from the NIDDK Hormone Distribution Program (Bethesda, MD; gift of Drs. L. Underwood and J. Van Wyk). Recombinant human IGF-1 (Eli Lilly, Indianapolis, IN) was iodinated by the chloramine-T method. The reference preparation was a pool of extracted serum from adult male Sprague-Dawley rats that corresponded to 1 U/ml and the averaged plasma IGF-1 values are reported in terms of this standard. The standard curve was linear between 0.01–8 U/ml. All samples were run in a single assay.

Computer simulations of plasma concentrations of total, bound, and free hGH over time
In the experiment where the hGH + hGHBP complex was injected, we formulated a simple model to deconvolute the time course of total hGH in the plasma into free hGH and bound hGH (hGH.hGHBP). The simulations were based on the following assumptions: 1) the initial sc injection constitutes a depot of both free hGH and hGH.hGHBP based on the equilibrium constant for the binding between hGH and hGHBP (1, 15); 2) irreversible diffusion of hGH and hGH.hGHBP into the circulating blood; 3) irreversible degradation of lost hGH and hGH.hGHBP; 4) the stoichiometry for the binding between hGH and hGHBP is predominantly 1:1 (30); 5) equal binding and dissociation rate constants for hGH and hGHBP in the rat as in human (in the depot as well as in the circulating blood); 6) equal distribution volume of hGH and hGHBP in the rat; 7) the clearance rate of free hGH is the same in the presence or absence of hGHBP (clearance includes binding of hGH to rat GH receptors); and 8) a 10-fold lower clearance rate for hGHBP and hGH.hGHBP than for hGH (12).

Similar diffusion rate constants for hGH and hGHBP in the tissue were assumed due to the similar size of the molecules, whereas for the hGH + hGHBP complex we assumed a lower diffusion rate constant.

The following differential equations were formulated: In the sc depot:

(1)

(2)

(3)
In the circulating blood:

(4)

(5)

(6)
where [hGH.hGHBPd], [hGHBPd], [hGHd] and [hGH.hGHBP], [hGHBP], [hGH] denote the concentrations of bound hGH, hGHBP, and free hGH in the depot and in the circulating blood, respectively. Moreover, kd1 and kd2 are the diffusion rate constants of hGH and hGH.hGHBP, respectively, whereas kloss1 and kloss2 represent the rate constants of the loss of hGH and hGH.hGHBP, respectively. The rate constants, kon = 2.47 x 107 M-1·min-1 and koff = 0.037 min-1, for the dissociation process are taken from Veldhuis et al. (15). The clearance rates of hGH and hGH.hGHBP are kt1 and kt2, respectively. The rate constants for hGHBP alone are either kd1 and kloss1 for the diffusion processes or kt2 for the clearance. The volume of the injection is V1, whereas the volume of the circulating blood is denoted by V2. The numerical integration was performed via the Euler-algorithm. The initial conditions are determined by the association constant Ka = kon/koff. The percentage of free hGH can be calculated by the equation

(7)
where a = hGH/hGHtot

The quantities hGHtot and hGHBPtot denote the total amount of hGH and hGHBP in the sample, respectively.

To determine the rate constants of free hGH, we performed a fit procedure for the experiment where only hGH was injected. Based on the same model as above, but without hGHBP, the differential equation system can be solved analytically, which yields the equation for the time course of free hGH in the circulating blood

(8)
where A0 denotes the initial concentration of hGH in the depot, and kd1, kloss1 and kt1 are the diffusion rate constant, the rate constant of the loss of hGH and the clearance rate constant, respectively. The initial sample contained 200 µg hGH in a volume V1 = 445 µl, which yields for A0 = 449,438 ng/ml. The volume of the circulating blood V2 is estimated as 15 ml.

Statistical analyses
One- and two-way ANOVAs, followed by Duncan’s or Scheffé’s test for multiple comparisons, and Student’s t tests for paired and unpaired data, as appropriate, were used for statistical comparisons between and within experimental groups. The Pearson product-moment correlation coefficient was used to evaluate the degree of relation between hGH and rGH concentrations. The integrated area under the rGH and hGH response curves was calculated by the linear trapezoidal method. The results are expressed as the mean ± SE. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of hGHBP + hGH complex on plasma hGH concentrations
The mean plasma hGH levels achieved over the 7-h sampling period in rats injected with either hGH alone or with hGH complexed with hGHBP are shown in Fig. 1Go. Animals treated with hGH alone (n = 7) exhibited a significant rise of plasma hGH levels within 15 min after injection; hGH concentrations in plasma reached a mean peak of 247.7 ± 18.1 ng/ml at 60 min after the injection and declined to near undetectable levels by the end of the sampling period. In contrast, animals injected with the hGH + hGHBP complex (n = 5) exhibited a slower rise of plasma hGH levels; plasma hGH concentrations were significantly (P < 0.05) lower at 15 and 30 min post treatment when compared with those of rats administered hGH alone. The mean peak hGH level (283 ± 35.4 ng/ml) achieved in this group, at 105 min after injection, was not significantly different from that observed after the injection of hGH alone, and plasma hGH concentrations remained similar between the two groups from 45–135 min post injection. Subsequently, however, hGH levels in the hGH + hGHBP complex-injected animals exhibited a much slower disappearance from the circulation than that observed in rats administered hGH alone; plasma hGH concentrations remained markedly elevated for the remainder of the sampling period and, at 7 h post treatment, were 14-fold higher than those in animals administered hGH alone (144.4 ± 29.6 vs. 10.3 ± 1.4 ng/ml; P < 0.001).



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  		Figure 1. Mean plasma hGH concentrations over the 7-h experimental period in normal rats sc injected at 0900 h with the hGH (200 µg) + hGHBP (200 µg) complex compared with those of rats given hGH (200 µg) alone. Whereas both groups of animals exhibited a similar peak in plasma hGH levels, the disappearance of hGH from the circulation was markedly slower in rats administered hGH preincubated with hGHBP than that of rats administered hGH alone. Values are the mean ± SE; the number of animals in each group is shown in parentheses. a, P < 0.05 or less compared with animals administered hGH alone at the same time point.

 
Figure 2Go summarizes the time course of effects of the coinjection of hGH with hGHBP on plasma hGH concentrations. During the initial 3.5 h of the experiment, the integrated area under the hGH response curve (AUC) was similar in the two groups of animals (573.7 ± 39.2 and 683.0 ± 27.0 ng/ml·h in hGH- and hGH + hGHBP-treated rats, respectively). In contrast, during the 3.5- to 7-h period of the experiment, the hGH AUC in hGH + hGHBP-injected animals was almost 5-fold higher than that of animals injected with hGH alone (632.2 ± 117.5 vs. 130.3 ± 23.5 ng/ml·h; P < 0.001). Moreover, at 24 h after the injections, plasma hGH levels were still 6-fold higher in animals administered the hGH + hGHBP complex than in rats given hGH alone (16.5 ± 3.0 vs. 2.7 ± 0.7 ng/ml; P < 0.001).



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  		Figure 2. Comparison of the time course of effects of the sc injection of the hGH + hGHBP complex vs. hGH alone on mean area under the hGH response curve over the 0- to 3.5-h and 3.5- to 7-h periods, and on mean plasma hGH concentrations at 24 h, after injection. Each bar represents the mean ± SE; the number of animals in each group is shown in parentheses. a, P < 0.001 compared with rats administered hGH alone during the same time period.

 
Effects of hGHBP + hGH complex on hGH-induced inhibition of spontaneous rGH release
Figure 3Go illustrates individual, representative 7-h plasma rGH and concurrent hGH profiles in rats sc administered 200 µg hGH + 200 µg hGHBP, in comparison to those of rats given either 200 µg hGH alone or Tris buffer and/or 200 µg GHBP alone. Tris buffer-injected control animals (Fig. 3AGo, left panel) showed the typical pulsatile pattern of rGH secretion characteristic of the male rat (24) with two major episodes of rGH secretion evident during the 7-h sampling period (approximately at 1100–1230 h and 1400–1530 h). The administration of 200 µg hGHBP alone did not significantly alter this ultradian rhythm of endogenous rGH secretion (Fig. 3AGo, right panel). Injection of 200 µg hGH alone resulted in a marked suppression of spontaneous rGH pulses which lasted for up to 3.9 ± 0.2 h after the injection; subsequently there was some recovery of spontaneous rGH secretion (Fig. 3BGo). Rats administered the hGH + hGHBP complex exhibited an initial suppression of spontaneous rGH release in the face of high plasma hGH concentrations (Fig. 3CGo), similar to that observed in animals given hGH alone. However, despite the persistence of dramatically higher hGH levels in the hGH + hGHBP complex-injected group, the time course of hGH-induced inhibition of endogenous rGH release (lasting up to 4.5 ± 0.2 h after the injections) was similar in both groups and was followed by a similar recovery of spontaneous rGH secretory bursts (Fig. 3CGo).



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  		Figure 3. Individual representative 7-h plasma rGH profiles in Tris-, hGHBP-, hGH- and hGH + hGHBP-treated animals. Concurrent hGH concentrations in plasma are also shown. Rats sc administered the Tris buffer vehicle (A, left panel) or 200 µg hGHBP alone (A, right panel) exhibited the typical pulsatile pattern of rGH secretion. Injection of 200 µg hGH alone (B) resulted in a suppression of spontaneous rGH pulses for approximately 3.5 h; during the subsequent 3.5 h, recovery of spontaneous GH peaks was evident. Despite the persistence of elevated plasma hGH levels (C), the inhibitory effect of hGH on endogenous pulsatile rGH release was not prolonged by the coadministration of hGHBP; a similar recovery of spontaneous rGH secretory bursts was evident. Arrows indicate sc injections at 0900 h.

 
Comparison of the effects of the different treatments on both GH peak amplitude and GH AUC in the four groups of rats is shown in Fig. 4Go. ANOVA revealed that both parameters were significantly suppressed during the first 3.5 h after treatment (peak amplitude: F = 9.16, P < 0.01; AUC: F = 19.92, P < 0.01). When compared with their respective controls, there was a similar degree (3.5-fold) of suppression in rGH peak amplitude (the highest rGH value during this time period) in animals injected with hGH alone (63.9 ± 29.1 vs. 221.0 ± 36.3 ng/ml in Tris-treated animals; P < 0.01) compared with those injected with the hGH + hGHBP complex (54.8 ± 16.0 vs. 184.0 ± 10.5 ng/ml in rats administered hGHBP alone; P < 0.001). During this period, the rGH AUC was also similarly suppressed in hGH-treated (39.7 ± 16.3 vs. 208.4 ± 26.0 ng/ml·h in Tris-injected; P < 0.01) and in hGH + hGHBP-treated rats (42.7 ± 15.3 vs. 169.4 ± 11.7 ng/ml·h in hGHBP-injected; P < 0.01). During the second 3.5 h of the sampling period (i.e. 3.5–7 h), a recovery of both rGH peak amplitude and rGH AUC was observed, and again this was similar in both hGH- and hGH + hGHBP-treated animals (Fig. 4Go). Neither the rGH peak amplitude (F = 1.75; P > 0.1) nor the rGH AUC (F = 2.5; P > 0.05) were significantly different across groups during this time period, although both parameters tended to be decreased in the hGH + hGHBP complex-injected rats by comparison with those rats given hGH alone.



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  		Figure 4. Summary of the time course of effects of the hGH + hGHBP complex on rGH peak amplitude and rGH AUC in the four experimental groups. Each bar represents the mean ± SE; the number of animals in each group is shown in parentheses. a, P < 0.01 or less compared with the respective control groups.

 
Relationship between plasma hGH and rGH concentrations
There was a significant inverse relationship between plasma rGH and hGH levels over the 7-h experimental period in animals administered hGH alone (r = -0.66; P < 0.01). However, no significant relationship was found between hGH and rGH concentrations in those rats injected with the hGH + hGHBP complex.

Effects of hGHBP + hGH complex on plasma IGF-1 concentrations
Mean plasma IGF-1 levels, measured at baseline and at 3.5, 6, 7, and 24 h post treatment in the four experimental groups, are shown in Table 1Go. Statistical analysis using two-way ANOVA revealed no significant differences between the groups at any time point (F = 2.3; P > 0.05).


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  		Table 1. Effects of the hGH + hGHBP complex on plasma IGF-1 concentrations

 
Computer simulations of time course of total, bound, and free plasma hGH concentrations
The initial condition in the depot was determined by Eq 7Go. The prepared sample contained 200 µg hGH and 200 µg hGHBP in a volume of 445 µl. Given the molecular masses of hGH and hGHBP used in this study, 22 kDa and 28 kDa, respectively, the calculated percentage of free hGH in the depot was 21.4% whereas the amount bound (hGH.hGHBP) was 78.6%. The number of hGHBP molecules in the depot was negligibly small at the beginning.

In the experiment where only hGH was injected, the fit procedures provided the following values for the parameters: diffusion rate constant, kd1 = 7.31 x 10-4 min-1, the rate constant for the loss of hGH, kloss1 = 1.83 x 10-2 min-1, and the clearance rate for hGH, kt1 = 1.14 x 10-2 min-1. Based on the assumptions presented in Materials and Methods and the rate constants obtained above, the remaining parameters for the simulation were determined as follows: kd2 = 4.1 x 10-4 min-1; kloss2 = 1.12 x 10-2 min-1; kt2 = 1.14 x 10-3 min-1, whereby kd2 and kloss2 were used to fit the data points.

Figure 5Go shows the simulations of the time course of total, bound, and free plasma hGH concentrations evoked by the sc injection of a sample that contained free and bound hGH in a 21.4/78.6 ratio. The maximal amount of free hGH (90 ng/ml) was observed 80 min after injection; by 3.5 h, the calculated amount of free hGH declined to 60 ng/ml and was only 28 ng/ml by 7 h. The maximal amount of bound hGH (183 ng/ml) occurred at 160 min after injection. During the first 3.5 h of the simulation where hGH + hGHBP was injected, the free hGH AUC was 252 ng/ml·h, whereas in the second 3.5-h period it was reduced by approximately 50% (143.5 ng/ml·h), as obtained by numerical integration.



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  		Figure 5. Simulations of the time course of total hGH, free hGH, and hGH bound to hGHBP in the plasma after a sc injection of a sample that contained free and bound hGH in a 21.4/78.4 ratio. The open circles represent the data points of measured total plasma hGH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The inhibitory effect of exogenous GH on its own endogenous release is well established in both rats (31, 32, 33) and humans (34, 35). In recent studies (23, 36), we have taken advantage of the lack of cross-reactivity between immunoassayable rat and human GH, and also of the capability of hGH to bind rat somatogenic receptors (37) and exhibit bioactivity in this animal, to characterize the time course of GH negative feedback in the rat. The present results obtained in animals sc administered hGH alone have confirmed our previous findings. Spontaneous pulsatile rGH release was inhibited for approximately 4 h after a single sc injection of 200 µg hGH, when compared with Tris buffer-injected controls, and this inhibition occurred in parallel with the increase of hGH concentrations in the blood; when circulating hGH levels fell, recovery of endogenous rGH secretion was evident. A significant inverse relationship between plasma rGH and hGH levels was observed over the 7-h period of observation.

Whereas rats administered the hGH + hGHBP complex exhibited a similar peak in plasma hGH levels, the sc administration of hGH preincubated with hGHBP, in a 1:1 (wt:wt) ratio, caused a markedly slower disappearance of hGH from the circulation. Plasma hGH concentrations at 7 h after injection of the complex were 14-fold higher than the hGH levels achieved at the same time point after the administration of hGH alone, and hGH was still readily detectable in blood 24 h after injection. These findings are largely in agreement with previous in vivo studies (11, 12, 13), which have shown that administration of hGH in combination with hGHBP markedly reduces the clearance rate of hGH. Restriction of the access of hGH to tissue sites of catabolism, such as the proximal renal tubule in the kidney and to GH receptor-mediated cellular uptake, especially in the liver, is presumably the reason for the low degradation rate of complexed hGH (11).

We then addressed the question whether hGH’s in vivo bioactivity was maintained and/or prolonged in the face of this apparent marked increase in bioavailability. We first showed that hGHBP given alone did not alter the typical pulsatile pattern of rat GH secretion; this finding was not unexpected because human GHBP does not bind to rat GH (20). Rats administered hGHBP in combination with hGH exhibited a suppression of spontaneous rGH pulses in the first 3.5 h after the injection similar to that observed in animals administered hGH alone. However, during the subsequent 3.5 h of the experiment, the sustained high plasma hGH concentrations, which were similar to those levels achieved during the first 3.5-h period, failed to induce a significant suppression of endogenous rGH release. Both the time course of hGH-induced inhibition of rGH, and the recovery of spontaneous rGH pulses during the 3.5- to 7-h period, in hGH + hGHBP complex-treated rats were not significantly different from those of animals administered hGH alone, although it should be noted that their plasma rGH levels tended to be lower, an effect perhaps related to the persisting hGH concentration during this period. There was no significant correlation between rGH and hGH levels in this group of rats.

Moreover, measurement of plasma IGF-1 levels as a marker of another GH-dependent biological response, i.e. IGF-1 production, revealed no significant modification of plasma IGF-1 levels, at any time point examined, in animals injected with the hGH + hGHBP complex despite the prolongation of the higher circulating hGH concentrations for up to 24 h after injection. Admittedly, absence of an effect of the hGH + hGHBP complex on this parameter should not be overemphasized because injection of hGH alone did not significantly increase plasma IGF-1 levels. Albeit, taken together, these findings do not support the thesis that the GHBP enhances GH’s bioactivity in vivo, at least over the time course examined here.

Because the assay used for determining hGH concentration in rat plasma measures both free and bound hGH, and only free hGH is presumed to be bioactive, a deconvolution of the measured total hGH concentration into free and bound hGH was performed to provide a basis for interpreting our results. The preincubation of hGH with hGHBP in a 1:1 (wt:wt) ratio leads to an approximately 1:4 (mol:mol) ratio of free and bound hGH due to the molecular weights of hGH and hGHBP used in this study and the high binding affinity of hGHBP to hGH. Thus, approximately 22% of the total amount of hGH is initially unbound in the depot after sc injection.

The diffusion process into the circulating blood resulted in a peak of free hGH (90 ng/ml) in the blood stream after 80 min, amplified by some release of hGH from the binding protein, and in an AUC of free hGH in the first 3.5 h of the simulation equivalent to 252 ng/ml·h. This amount of free hGH obtained in the computer simulations likely provides a good explanation for the suppression of endogenous rGH release observed during this time period; indeed, doses of exogenous hGH 2- to 3-fold lower than those used here have been shown to effectively suppress endogenous GH pulses in the male rat (32, 33). In addition, considering the restricted distribution volume of hGHBP (11, 12), the simulations likely show an underestimation of the free hGH in the first 3.5-h period, and an overestimation of free hGH in the second 3.5 h due to the higher clearance rate of free hGH compared with bound hGH. During the 3.5- to 7-h period, after depletion of the depot, the time course of free hGH is governed by the capacity of the binding protein, the dynamics of the dissociation, and the clearance. The amount of free hGH declined to 28 ng/ml at 7 h. In fact, the calculated AUC of free hGH between 3.5 and 7 h (143.5 ng/ml·h) was similar to the value obtained in the fit procedure for the experiment, where only hGH was administered (133 ng/ml·h; a value which failed to inhibit endogenous GH release in our model). Thus, most of the total hGH during this observation period was circulating in the bound form and therefore had no access to its receptor. Again, this finding provides a good explanation for the lack of hGH bioactivity observed in the second half of the experiment in which the hGH + hGHBP complex was injected.

On the other hand, a recent in vivo study (20) indicates that chronic administration of GHBP precomplexed with GH can enhance GH’s growth-promoting and IGF-1 generating activity in hypophysectomized rats. These results, however, may not reflect the role of GHBP under normal physiological conditions as hypophysectomy, per se, can markedly alter hepatic somatogenic receptors in male and female rats (37, 38), rendering them more sensitive to GH than normal rats. In fact, in this same study (20), when a second animal model of inadequate growth was used, i.e. the dwarf dw/dw rat that does produce a minimal amount of GH (39), plasma IGF-1 concentrations were not altered by the codelivery of GH with GHBP over a 7-day period, and the weight gain was less than that observed in hypophysectomized rats. A similar lack of effect of chronic administration (over 6 days) of hGHBP in various combinations with hGH on liver IGF-1 mRNA transcripts and on body weight gain in dw/dw male rats has also been reported (22). Another possible explanation for the differences observed between the study of Clark et al. (20) and the present one may be related to their use of female rats, as compared with males here, because a sexual dimorphism in serum GHBP (40) and an influence of gonadal steroids on GHBP (41) in the rat have been described. Finally, the different modes of GHBP administration, i.e. chronically over 7 days (20) vs. acutely in the present study, must be taken into account in any comparisons across studies.

One might argue that the lack of effect of hGHBP on hGH action observed in the present study could be the result of GH receptor down-regulation due to the continuous 7-h exposure to high levels of hGH in hGH + hGHBP-treated rats. However, whereas a single GH injection causes an acute down-regulation of liver GH receptors (42), continuous GH delivery results in increased, not decreased, GH binding to liver membranes (43, 44). Moreover, it has been clearly demonstrated in vivo that infusions of hGH for 6 h in conscious male and female rats continue to effectively inhibit the spontaneous rGH secretory bursts (33). These results argue against the induction of GH receptor down-regulation by prolonged GH exposure as a potential interpretation of our results; they further point out the complexity of the system and the need to consider temporal questions in evaluating the biological activity of the GHBP.

It is of interest to note, in this regard, that continuous infusion of GH in vivo in the rat results in increased GHBP levels and GH binding sites yet attenuates growth rate and IGF-1 production, when compared with that produced by intermittent GH injections (43, 44, 45). These authors interpreted their findings to indicate that the higher GHBP levels may be competing with the GH receptor for GH binding and thereby inhibit GH bioactivity by decreasing hormone availability for the target cells. In the human, there is an inverse relationship between GH release and GHBP concentrations under normal physiological conditions (46), and a recent study indicates that serum GHBP levels are not related to stature (47). Indeed, in a familial syndrome of short stature, the presence of very high concentrations of plasma GHBP and GH does not increase GH responsiveness in that these individuals exhibit growth failure and partial GH resistance, suggesting decreased access to tissue GH receptors (48). In GH-deficient children, either infused or daily injected with GH for 6 months, the increase in GHBP levels was more pronounced with continuous than with daily GH treatment, yet the growth rate was identical in both groups; there was no correlation between IGF-1 and GHBP, suggesting that the increase in GHBP was not related to the growth-promoting effect of GH (49). The respective roles of bound and free GH in growth promotion remain to be determined.

In conclusion, the results of the present study in normal rats demonstrate that: 1) the hGHBP markedly prolongs the clearance of hGH from the circulation in vivo; 2) despite hGHBP’s ability to prolong the bioavailability of hGH, precomplexing hGH with hGHBP failed to enhance hGH’s in vivo bioactivity in the inhibition of endogenous pulsatile rGH release and also failed to stimulate IGF-1 levels. Our findings do not provide support for the concept that the GHBP can enhance GH bioactivity in vivo, at least over the time course examined here. Clearly, further studies are needed to provide a better understanding of the physiological significance of the GHBP for GH regulation and action.


   Acknowledgments
 
We thank Wendy Gurd and Martine Lapointe for skillful technical assistance and Julie Temko for expert secretarial help. We are grateful to Drs. Gerhard Baumann and Marie-Catherine Postel-Vinay for their interest in this work and helpful discussions. The generous provision of hGH and hGHBP by Genentech, Inc., and the continuing supply of GH and IGF-1 RIA materials by the NIDDK Hormone Distribution Program, are gratefully acknowledged.


   Footnotes
 
1 This work was supported by Grant MT-6837 (to G.S.T.) from the Medical Research Council of Canada. Back

2 Supported by a grant (to G.S.T.) from the Fonds de la recherche en santé du Québec. Present address: Department of Pathophysiology, Medical School, University of Athens, M. Asias 75, 11527, Athens, Greece. Back

3 Recipient of postdoctoral fellowship awards from The Swiss National Foundation and the Ciba-Geigy-Jubilaeumsstiftung. Back

4 Chercheur de Carrière of the Fonds de la recherche en santé de Québec. Back

Received June 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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