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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2649-2659
Copyright © 2000 by The Endocrine Society

From the Clinical Research Centers

Oral Estradiol Administration Modulates Continuous Intravenous Growth Hormone (GH)-Releasing Peptide-2-Driven GH Secretion in Postmenopausal Women1

N. Shah2, W. S. Evans, C. Y. Bowers and J. D. Veldhuis

Division of Endocrinology, Department of Internal Medicine (N.S., W.S.E., J.D.V.), General Clinical Research Center, National Science Foundation Center for Biological Timing, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0202; and Division of Endocrinology and Metabolism, Department of Internal Medicine (C.Y.B.), Tulane University Medical Center, New Orleans, Louisiana 70112-2699

Address correspondence and requests for reprints to: J. D. Veldhuis, Division of Endocrinology, Department of Internal Medicine, P.O. Box 800202, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0202. E-mail: JDV{at}Virginia.Edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Exactly how estradiol (E2) regulates the human GH-insulin-like growth factor I axis is not known. Here, we explore the impact of oral E2 supplementation on the stimulatory actions of a potent and specific synthetic GH-releasing peptide (GHRP), GHRP-2. To this end, we studied 10 healthy postmenopausal women following the administration of placebo or 17ß-estradiol (1 mg twice daily orally) for 7–12 days in a prospectively randomized, double-blind, within-subject crossover design. To drive GH secretion via the GHRP-receptor/effector pathway, we infused GHRP-2 (1 µg/kg·h) or saline continuously iv for 24 h. Deconvolution analysis was used to quantitate the separate basal and pulsatile modes of GH secretion based on 24-h serum GH concentrations profiles collected at 10-min intervals and assayed by chemiluminescence. As complementary (nonpulsatile) measures, we used the approximate entropy (ApEn) statistic and cosine regression to define feedback-dependent and circadian-related changes, respectively. E2 administration amplified the mass of GH secreted per burst by 1.9-fold over placebo, 24-h GHRP-2 infusion by 7.0-fold, and, the two agonists together by 8.8-fold (P < 10-14). Intravenous GHRP-2 infusion augmented the basal (nonpulsatile) rate of GH secretion by 4.4-fold (P < 10-4). E2 treatment had no effect alone, but doubled the stimulatory effect of GHRP-2, on basal GH secretion. Neither E2 nor GHRP-2 influenced 24-h GH pulse frequency, interburst interval, half-life or pulse duration. Combined E2 and GHRP-2 elevated the ApEn of GH secretory profiles significantly above control, thereby indicating a marked alteration of within-axis feedback control (P = 0.00033). Dual stimulation with E2 and GHRP-2 also synergistically increased the amplitude (by 11-fold, P < 10-11) and the mesor (by 10-fold, P < 10-10) of the 24-h GH rhythm. Infusion of GHRP-2 advanced the GH acrophase (time of daily maximum of GH release) by 8.75 h, whereas combined treatment with E2 and GHRP-2 normalized the acrophase. Cross-correlation analysis showed that GHRP-2 infusion (but not E2 administration) significantly synchronized paired 24-h serum GH concentration profiles (P < 10-3).

In summary, short-term oral E2 replacement in post-menopausal women strongly modulates the actions of a synthetic hexapeptide GH secretagogue on three quantifiable modes of GH secretion [i.e. 1) basal (nonpulsatile) GH release; 2) feedback-dependent ApEn; and 3) the mesor, amplitude and timing of the 24-h GH rhythm]. Moreover, a continuous GHRP-2 stimulus also synchronizes inter diem GH secretory patterns. The present pharmacological study, thus, offers a further framework for exploring the nature of the interactions of E2 with the GHRP-receptor/effector pathway in the aging and/or gonadoprival human.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SYNTHETIC GH-releasing oligopeptides (GHRPs) (1, 2) and sex hormones (3, 4, 5, 6, 7) are potent individual agonists of GH secretion. However, few studies have delineated the nature of neuroregulatory interactions between these two prominent and distinct classes of GH stimuli (8). The GHRP-receptor/effector pathway is of interest following the successive in vitro identification of potent oligopeptide secretagogues of GH from 1977–1980 (9, 10, 11), cloning of hypothalamo-pituitary receptors for GHRPs in 1996 (12, 13), and the isolation in 1999 of an endogenous peptidyl ligand of the GHRP receptor, which specifically stimulates GH secretion in the rat and also circulates in human plasma (14).

Several experimental observations point toward a possible interaction between sex-steroid hormones and GHRP-like secretagogues in the human. First, molecular analysis of the human GHRP-receptor gene promoter has identified a putative hemi-estrogen-responsive cis-DNA element (15). Although transactivation of the GHRP-receptor gene by estrogen has not been established, in prepubertal girls oral administration of ethinyl estradiol for 3 days nearly doubles acute GHRP (Hexarelin)-stimulated GH release (16). In addition, investigations of the efficacy of GHRP across the human lifespan have disclosed maximal GH secretion during the sex-steroid-rich milieu of mid-to-late puberty (17, 18). More recently, a dose-response analysis of the actions of GHRP-2 in postmenopausal women revealed that oral estradiol supplementation for 5–12 days enhances both the potency and efficacy of acute GHRP-2 stimulation of GH secretion (19). Nonetheless, in two other clinical studies, GH secretion after a single iv pulse of Hexarelin or GHRP-6, respectively, was invariant of gender (20) or menstrual cycle stage (20). In another report, 3 months of low-dose (0.05 mg daily) transdermal 17ß-estradiol supplementation in postmenopausal women did alter the effect of single-bolus injection of Hexarelin on acute GH secretion.

As a complementary strategy to explore the nature of the putative interaction of estrogen with GHRP-driven GH secretion, we here use a continuous iv GHRP stimulus. We combine unvarying GHRP-2 drive with frequent (10-min) blood sampling over 24 h to monitor discrete facets of regulated GH secretion in healthy postmenopausal women. To enhance statistical power, we used a within-subject randomized crossover design to replace oral E2 or placebo. GHRP-2 was selected as the most potent specific GHRP-receptor agonist available for investigations in the human (21, 22). Because 24-h GH secretion is governed physiologically by basal (nonpulsatile), pulsatile, entropic (the feedback-dependent orderliness of secretory patterns), and daily rhythmic (circadian-like) control, this intensive 24-h experimental paradigm allowed us to quantitate the single and interactive impact of E2 and/or GHRP-2 on each of these distinct modes of GH neuroregulation. This is relevant because pulsatile and basal GH secretion probably mirror changes in GHRH and somatostatin release, entropic GH release serves as a barometer of GH-insulin-like growth factor I (IGF-I) feedback activity, and 24-h rhythmic GH production reflects the dual input of circadian (CNS) and rest-activity-sleep signals (8).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical protocol

Ten healthy postmenopausal women (mean age, 60 ± 2.4 yr; absolute range, 48–74 yr) with onset of clinical menopause at least 2 yr earlier provided written informed consent approved by the Human Investigation Committee of the University of Virginia Health Sciences Center. The (mean ± SEM) body mass index was 27 ± 1.2 kg/m2 (median, 26.3). Volunteers had received no estrogen replacement therapy for at least 4 weeks prior to study, and each had a normal medical history and physical examination, unremarkable screening biochemical tests of hematological, renal, metabolic, endocrine and hepatic function. There was no acute illness, chronic disease, psychiatric disorder, recent use of medications (within five biological half-lives), transmeridian travel (within 10 days), or significant weight change (2 kg or more within 10 days).

Estrogen (E2, 1 mg 17ß-estradiol orally twice daily) or placebo was administered in a prospective, randomly assigned, double-blind, within-subject crossover manner for 7–12 days before each of the four separate and randomly ordered infusion studies. The latter were separated by at least 72 h (i.e. saline vs. GHRP-2).

Volunteers were admitted to the General Clinical Research Center the evening before study to allow overnight adaptation. At least 4 (and up to 6) weeks separated consecutive admissions. The next day subjects received a weight-maintaining diet consisting of three isocaloric meals provided at 0800, 1200, and 1700 h. Each meal contained 55% carbohydrate, 30% fat, and 15% protein. Blood samples (1.5 mL) were withdrawn at 10-min intervals for 24 h beginning at 0800 h via an indwelling catheter placed in a forearm vein at least 1 h before. Concurrently, volunteers received either iv saline (300 mL/day) or continuous GHRP-2 (1 µg/kg·h) infusions. The order of infusions was randomized.

Assays

Serum GH concentrations were measured in duplicate in the 145 samples obtained from each 24-h study session in one run by an automated ultrasensitive GH chemiluminescence assay (modified Nichols Luma Tag hGH assay; sensitivity, 0.005 µg/L), as described previously (23, 24). Human recombinant GH (22 kDa) was used as assay standard. The median intra- and interassay coefficients of variation were, respectively, less than 6.5 and less than 8.5%. Serum E2, cortisol, TSH, LH, FSH, and PRL concentrations were assayed in duplicate by RIA or chemiluminescence-based assays of a 24-h serum pool prepared from each sampling session by combining 20-µL aliquots of serum from each of 145 samples thawed at the time of GH assay (23). Plasma IGF-I concentrations were measured in two separate samples collected at the beginning and end of each 24-h study session (at 0800 h), to evaluate any incremental rise in IGF-I across the 24-h infusions.

Deconvolution analysis

Multiparameter deconvolution analysis was used to quantitate pulsatile GH secretion and estimate its half-life (25). Daily pulsatile GH secretion is the product of secretory burst frequency and the mean mass of GH released per pulse. Basal GH secretion represents the calculated time-invariant interpulse component of the release profile. Total secretion is the sum of the pulsatile and basal components. Secretory pulse identification required that GH secretory burst amplitudes and the basal GH secretion rate exceed 95% statistical confidence intervals (26). The analyst was blinded to the randomization scheme.

Cosine regression

The 24-h rhythm of serum GH concentrations was evaluated by cosinor analysis, as described earlier. Ninety-five percent statistical confidence intervals were determined individually for the fitted (cosine) amplitude (50% of the nadir-zenith difference), mesor (cosine mean) and acrophase (time of maximum). In addition, the 24-h rhythms of deconvolution-calculated GH secretory burst mass and interpulse intervals were evaluated in each of the four sessions in the 10 women considered as a group (27).

Approximate entropy (ApEn)

ApEn was used as a scale- and model-independent statistic, which is complementary to cosine fitting and deconvolution analysis (28). ApEn quantifies the serial orderliness of hormone measurements (29, 30). Normalized ApEn parameters of m = 1 (series length) and r = 20% (threshold) of the intraseries SD were used, as previously validated for 24-h GH time series (31, 32, 33). This statistic is, thus, designated ApEn (1, 20%). The normalized ApEn statistic applied here has good replicability for series of this length. Increased ApEn (at equal series lengths and similar parameter values, as used here) indicates greater irregularity secretory of patterns, as reported for GH profiles in acromegaly (32), as well as for GH release in the female compared with the male (33, 34, 35).

Cross-correlation analysis

Cross-correlation analysis with lag was applied, as described previously (36). We tested the null hypothesis that serum GH concentrations in the four randomized study sessions are uncorrelated within individuals. A protected P value of less than 0.01 was used for significance testing to limit false positive errors in assessing several time lags.

Statistical analyses

Because of nonnormality, analytically derived parameters were logarithmically transformed and then compared among the four treatment groups by one-way ANOVA (26). Duncan’s new multiple-range test was applied post hoc to contrast the values of two or more means. Paired Student’s t testing was applied with Bonferroni correction where noted. Mean and integrated (24-h) serum GH concentrations were evaluated without logarithmic transformation.

Data are presented as the mean ± SEM or as box-and-whisker plots (median, interquartile, and range). When multiple parameters were assessed, statistical significance was construed for P less than 0.01 to limit type I statistical errors.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pooled (24-h) serum E2 concentrations rose significantly from 15 ± 1.3 pg/mL during placebo to 365 ± 17 pg/mL at the end of 7–12 days of oral 17ß-estradiol (E2) administration.

Day 1 (preinfusion, 0800 h) serum IGF-I concentrations declined significantly from 172 ± 24 µg/L (placebo) to 137 ± 13 µg/L (E2) on the saline infusion days and from 170 ± 17 µg/L (placebo) to 145 ± 14 µg/L (E2) on the GHRP-2 infusion days (P < 10-2 paired comparisons). On day 2 after placebo pretreatment, the 24-h continuous iv GHRP-2 infusion increased the serum IGF-I concentration significantly to 269 ± 16 µg/L (P = 0.0018 interventional effect by ANOVA) (Fig. 1Go). On day 2 after E2 pretreatment, the GHRP-2 infusion elevated IGF-I levels to a lesser degree to 187 ± 13 µg/L. This still represented a significant rise over the corresponding basal value (P = 0.017). The day 2 control (post-saline) infusion sessions had mean 0800-h serum IGF-I levels of 175 ± 16 µg/L (control) and 125 ± 13 µg/L (E2). Neither of these values differed from the corresponding day 1 mean.



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  		Figure 1. Serum IGF-I concentrations (µg/L) measured twice in relation to each intervention [i.e. at 0800 h on day 1 (preinfusion) and again at 0800 h on day 2 (after 24-h infusion)]. Data are the mean ± SEM for 10 postmenopausal women, who received placebo (control) or oral E2 for 7–12 days before undergoing a 24-h iv infusion of saline or GHRP-2 (1 µg/kg·h) in randomly assigned order. P values are Bonferonni-corrected differences by paired Student’s t testing of pre- and postinfusion means (0800 h day 1 vs. day 2) for each intervention. Different alphabetic superscripts denote significantly different means for values of day 2 among the four interventions (P < 10-6 by ANOVA).

 
During placebo ingestion, infusion of GHRP-2 amplified basal (nonpulsatile) GH secretion by 4.4-fold (P < 10-4) and GH secretory burst mass by 7.0-fold (P < 10-11) (Table 1Go). Prior administration of E2 enhanced the stimulation of GHRP-2 on basal GH secretion by a further 2.0-fold, without additionally elevating GH burst mass. E2 replacement alone did not alter basal GH secretion, but stimulated GH secretory burst mass. Neither GHRP-2 nor E2 influenced GH interburst interval, frequency, half-life, or calculated pulse half-duration (P = 0.054 only for the interburst interval comparison between GHRP-2 and E2 only) (Table 1Go).


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  		Table 1. Measures of the GH-IGF-I axis in 10 women treated with placebo or E2 orally for 7–12 days and administered GHRP-2 or saline infusions for 24 h

 
For all interventions, pulsatile GH secretion constituted the major mode of 24-h GH release [i.e., 88 ± 1.4% (control), 87 ± 2.5% (E2), 92 ± 1.9% (GHRP-2), and 90 ± 2.5% (combined agonists) of total GH secretion was pulsatile (P = NS)].

Daily pulsatile (P < 10-14) GH release and total (pulsatile plus basal, P < 10-13) GH secretion were strongly stimulated by GHRP-2 and/or E2 (Table 1Go). As summarized further in Table 1Go, mean (24-h) serum GH concentrations mirrored these values. Combined E2 and GHRP did not increase these measures over values achieved by GHRP-2 infusion alone. Statistical power analyses suggested more than 85% statistical power for detecting at least a 30% or greater (paired Student’s t test) treatment interaction at P less than 0.0125 (Bonferroni corrected).

The ApEn of 24-h GH release profiles increased significantly during combined administration of E2 and GHRP-2 over placebo/saline (P = 0.00033) (Fig. 2Go).



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  		Figure 2. Box-and-whisker plots of ApEn (1, 20%) values, a measure of the disorderliness of GH release patterns, in 10 postmenopausal women. Higher ApEn signifies greater pattern irregularity (see Materials and Methods for definition). Means with no shared alphabetic superscripts are significantly different.

 
E2 and GHRP-2 administration individually enhanced the amplitude of the 24-h rhythm in serum GH concentrations by 2.0- and 6.7-fold, respectively. Combined E2 and GHRP-2 administration increased the amplitude supraadditively to 11-fold over basal (P < 10-9) (Fig. 3AGo). E2 and GHRP-2 alone augmented the 24-h cosine mean (mesor) by 1.5- and 4.9-fold, respectively. Their combination did so synergistically by a total of 10-fold over basal (control) (P < 10-15) (Fig. 3BGo). The mean acrophase (time of maximal 24-h rhythm in the serum GH concentration) among the four treatment groups differed significantly (P = 0.0022) [i.e. control, 0447 h (±45 min); E2, 0414 h (±37 min); GHRP-2, 2000 h (±115 min); and combined agonist exposure, 0145 h (±77 min)] (Fig. 3CGo).



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  		Figure 3. Box-and-whisker plots of the amplitude (A), mesor (B), and acrophase (C) values of 24-h serum concentration GH rhythms. Means with no shared alphabetic superscripts are significantly different.

 
Cosine regression was applied separately to assess possible 24-h rhythms of deconvolution-calculated GH secretory burst mass and interburst interval values, as distinct from serum GH concentration (27). As summarized statistically in Table 2Go, E2 and GHRP-2 each augmented the mesor of the daily variation in GH burst mass, but their combined effect was not additive. GHRP-2 infusion (but not E2) heightened the amplitude of the 24-h rhythm in GH burst mass and also abolished the 24-h rhythm in GH secretory burst frequency (reciprocal of interburst interval). The diurnal rhythmicity of the GH interburst interval was normalized by dual stimulation with E2 and GHRP-2.


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  		Table 2. Cosinor analyses of 24-h rhythms in deconvolution-calculated GH secretory burst mass and interpulse intervals

 
Table 3Go summarizes the impact of E2 and/or GHRP-2 administration on daily pooled serum LH, FSH, PRL, and cortisol concentrations. E2 lowered LH and FSH and elevated PRL levels. Whether infused after placebo or E2 pretreatment, GHRP-2 had no effect on any of these hormones.


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  		Table 3. Impact of oral E2 replacement on serum hormone concentration

 
Fig. 4Go shows individual 24-h profiles of serum GH concentrations and corresponding calculated GH secretion rates plotted against clocktime (h) in two women during each of the four study sessions.




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  		Figure 4. A, Individual profiles of serum GH concentrations measured by chemiluminescence assay in blood sampled every 10 min for 24 h as plotted against clocktime (h). B, Corresponding logarithmic plots of deconvolution-calculated GH secretion rates over time. Arrows denote significant pulses. The data illustrate observations in 2 (of 10) postmenopausal women, each of whom underwent all four interventions in randomly assigned order.

 
Given a visual impression of concordant GH profiles in the two separate (randomly ordered) iv GHRP-2 infusion sessions (i.e. GHRP-2 infusion without vs. with E2 pretreatment), we evaluated the cross-correlation between 24-h serum GH concentration measurements in these and other paired sessions (i.e. placebo/E2, placebo/GHRP-2, E2/GHRP-2, and GHRP-2/E2 plus GHRP-2) (Fig. 5Go). Only the paired GHRP-2 sessions (GHRP-2 alone vs. GHRP-2 combined with E2) were significantly positively cross-correlated (P < 0.001).



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  		Figure 5. Linear cross-correlation coefficients (y-axis) at various time lags (x-axis) to quantitate pairwise synchrony between serial serum GH concentrations in the indicated paired study sessions. Within-subject synchrony of 24-h serum GH profiles was evaluated between control, E2, GHRP-2 infusion, and combined intervention sessions. P values apply to the group of 10 women. A positive lag denotes that GH concentrations in the first-named series lead those in the second, and vice versa. Data are the median and range (n = 10) of cross-correlation coefficients at each lag. The horizontal continuous lines denote the expected median zero (random) correlation predicted by the null hypothesis that serial GH values are unrelated on separate days.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present clinical experiments reveal distinctive and contrasting neuroregulatory actions of oral E2 and continuous stimulation of 24-h iv GHRP-2 on the GH-IGF-I axis in postmenopausal women. First, constant iv infusion of GHRP-2 elevated basal (nonpulsatile) GH secretion by 4.4-fold, whereas oral E2 supplementation exerted no effect. Second, GHRP-2 augmented GH pulse amplitude by 8.4-fold, whereas E2 did so by 1.8-fold. Third, GHRP-2 increased the mesor of the 24-h rhythm in serum GH concentrations by 4.9-fold and its amplitude by 6.7-fold, while advancing the acrophase by 8.75 h. The last time-shift may reflect prompt stimulation of GH secretion, even at the outset of the infusion and/or CNS actions of this secretagogue (37). In contrast, E2 replacement enhanced the mesor and amplitude of 24-h rhythmic GH release by 1.5- and 2.0-fold, respectively, and did not shift the GH acrophase. Lastly, GHRP-2 infusion over 24 h significantly stimulated serum IGF-I concentrations. Although E2, as expected, lowered preinfusion (basal) serum IGF-I concentrations significantly, it exerted no further effect during the 24-h saline infusion.

Combining oral E2 replacement with the continuous iv GHRP-2 stimulus doubled the basal GH secretion rate beyond that stimulated by GHRP-2 alone (total increase, 9-fold). Heightened basal GH release may be significant to the metabolic responses of certain target tissues that depend particularly on the interpulse serum GH concentration (e.g. hepatic but not muscle IGF-I gene expression) (8). The synergistic stimulation of basal GH secretion was highly specific because E2 and GHRP-2 together did not alter GH secretory burst mass, duration, interval or frequency, or the half-life of GH.

Oral E2 supplementation and iv GHRP-2 infusion also increased the amplitude and mesor of 24-h rhythmic GH release supraadditively (by a total of more than 10-fold). In addition, whereas an unvarying iv GHRP-2 stimulus begun at 0800 h markedly displaced the daily GH acrophase (by 8.75 h), cotreatment with estrogen normalized this value to 0145 h. Concomitant oral E2 administration and iv GHRP-2 infusion also reinstated the physiological circadian-like variation in GH secretory burst frequency, which was abolished by continuous GHRP-2 infusion alone. A nonvarying GH pulse frequency across 24 h during constant GHRP-2 stimulation may reflect the unvarying GHRP-2 stimulus in this pharmacological paradigm or indicate that GHRP-2 exerts CNS effects to drive a nearly constant frequency of GH pulse generation.

Combined administration of E2 and GHRP-2 maximally heightened the disorderliness of 24-h GH release patterns over baseline, as reflected by a marked rise in the value of the ApEn measure. This probably denotes altered feedback control within the GH-IGF-I axis (29, 30, 32, 34, 38). Thus, the present experiments identify not only quantitatively synergistic (basal and 24-h rhythmic) effects of, but also feedback-dependent responses to, joint agonism by estrogen and GHRP-2.

The oral E2 replacement regimen used here stimulates the amplitude and mass of GH secreted per pulse (and per diem) and increases the serum GH concentration peak height by 1.8- to 2.3-fold in postmenopausal women (39, 40). Serum E2 concentrations approximate those observed in the late follicular or preovulatory phase of the normal menstrual cycle (4, 6, 41, 42, 43), but are less than values achieved in typical ovulation-induction regimens (42). In both contexts, serum GH concentrations rise to the degree recorded here. This E2 replacement regimen sustains a stable and consistent increase in serum GH concentrations (within 8.5–13%) in any given individual for 6–16 days, whereas LH and FSH fall and PRL rises slightly (39, 44).

To our knowledge, GHRP (present data) and GHRH (45) are the only peptidyl secretagogues demonstrated to stimulate calculated basal (nonpulsatile) GH secretion in the human. Basal GH levels could not be evaluated until recently, because 25–93% of interpulse serum GH concentrations in fed and awake humans remained undetectable, at least before the development of ultrahigh sensitivity chemiluminescent, immunofluorometric, and enzyme-linked immunoassay methods (23, 24, 46, 47, 48). Moreover, basal (interpeak) GH secretion per se is a derived value that requires deconvolution-based correction for the otherwise confounding effects of: 1) variable hormone half-life, and 2) unequal pulse sizes preceding different nadirs (25, 26, 49). To address these technical issues, we combined an ultrasensitive GH chemiluminescence-based assay (with a threshold of 0.005 µg/L rh 22 kDa GH) with deconvolution-based partitioning of the serum GH concentration profile into its analytically separate basal, pulsatile, and half-life components. This analysis disclosed that GHRP-2 individually stimulates, and combined GHRP-2 and E2 synergistically elevate, basal GH secretion. Notably, E2 alone does not actually stimulate basal GH secretion but amplifies GH secretory burst mass, thereby indirectly increasing the subsequent interpeak serum GH concentrations (39, 49). The neuroendocrine mechanisms that sustain low basal rates of GH secretion in the human are not known, albeit recent data indicate that this mode of secretion is somatostatin suppressible (44, 46, 50, 51). Basal GH secretion is of interest because it may exert important metabolic effects on certain target tissues (52, 53) and correlates strongly with elevated IGF-I levels in some states of pathological GH excess [e.g. acromegaly (32)].

Estrogen and GHRP-2 each augmented the 24-h GH production rate by selectively amplifying GH secretory burst amplitude and mass. This neuroendocrine phenotype also unfolds physiologically in normal puberty in boys and girls (48, 54). Pulsatile GH secretion accounted for ~90% of total daily GH secretion here. Assuming a nominal GH distribution volume of 4 L in these women [i.e. ~7% body weight, which is not influenced by E2 (55)], then we estimate that GHRP-2 without or with E2 replacement stimulates the daily secretion of ~600 µg GH under these conditions in postmenopausal women. This observed output of GH is that of an early postpubertal girl (48), and about one half that achieved in mid-to-late puberty in boys (54). The precise degree of GH axis stimulation likely depends, in part, on the type, route, dose, and/or duration of estrogen delivery, as well as the GHRP stimulus and its delivery mode (e.g. continuous vs. bolus) (8). Despite this marked response, maximal somatotrope secretory capacity might not have been attained here by GHRP-2 (1 µg/kg·h) infusion, because E2 can enhance further the GH-releasing effect of even 3 µg/kg GHRP-2 (19). The latter dose, in turn, is consistently more effective than 1 µg/kg at least acutely, as also observed in the nonhuman primate (56).

Neither E2 nor GHRP-2 altered 24-h GH secretory burst frequency or interpulse interval. In contrast, iv somatostatin infusion, and, conversely, short-term fasting and sleep (both of which putatively relieve somatostatinergic activity), do influence detectable GH pulse frequency (46, 47, 57). If somatostatin normally represses the frequency of GH peaks, then the ability of combined E2 and GHRP-2 agonism to stimulate pulsatile GH secretion markedly (here by 8.5-fold) without accelerating GH peak frequency further could indicate that these secretagogues do not further attenuate the inhibition of somatostatinergic on GH pulse generation, at least in postmenopausal individuals.

Coadministration of GHRP-2 and E2 amplified GH secretory pulse mass without changing the apparent half-duration of underlying secretory events. Burst half-duration is invariant of or increases slightly across early human puberty, whereas GH peak amplitude rises severalfold (48, 54, 58). In contrast, pulsatile iv infusion of GHRH for 3 days augmented GH burst mass, while reciprocally (by 3- to 5-fold) shrinking secretory pulse duration (45). Thus, the dynamics of pituitary GH release driven by continuous GHRP-2 and pulsatile GHRH are remarkably different. Assuming that the hypothalamic release of somatostatin terminates a GH pulse (8), then the unchanging duration of GHRP-2-stimulated GH pulses could signify that GHRP-2 elicits less hypothalamic somatostatin release than a bolus GHRH stimulus. Indeed, an acute GHRP stimulus does not evoke somatostatin secretion in vivo in sheep (59, 60). Moreover, GHRPs can antagonize the actions of available somatostatin (8), which could also contribute to maintaining a normal rather than abbreviated GH pulse duration. For example, GHRP is more effective than GHRH in overcoming GH autofeedback (61), which is putatively mediated via hypothalamic somatostatin release (62, 63).

E2 and GHRP-2 together elevated the ApEn of 24-h GH profiles remarkably compared with control values. Elevated ApEn denotes greater relative disorderliness of the pituitary secretory process, as reported in midpuberty just prior to maximal height velocity in boys, in women compared with men, in estrogen-treated (and testosterone-treated) children, and in estrogen-replaced postmenopausal women (33, 34, 38, 39, 64, 65). More disorderly GH secretory patterns are believed to signify feedback changes within the GH-IGF-I axis (38), presumptively due to altered interactions among GH, IGF-I, somatostatin, and/or GHRH signaling (8). A less likely conjecture is that E2 and GHRP-2 jointly trigger more disorderly GH release by direct effects on the pituitary gland.

Estrogen and GHRP-2 supraadditively amplified the amplitude and mesor (cosine mean) of the 24-h GH rhythm, but exerted opposing effects on the physiological acrophase. GHRP-2 advanced the timing of the GH maximum, probably because of its strong stimulatory action even at the outset of the iv infusion, and/or effects on CNS regulatory centers that are coupled to sleep-wake/activity and/or circadian rhythms (66).

E2 and GHRP-2 also differentially affected the 24-h rhythms in GH pulse mass and GH interpulse intervals. These unique rhythms presumptively require CNS integration of circadian and ultradian inputs (8). In particular, GHRP-2 increased the diurnal variation in GH secretory burst mass, while eliminating the daily rhythm in GH burst frequency. Increased nyctohemeral rhythmicity of GH pulse mass would argue against marked down-regulation of hypothalamo-pituitary responsiveness to GHRP-2, although the latter was inferred in another study using a different GHRP agonist and dose in younger adults (67). Abolition of the 24-h variation in GH pulse frequency, on the other hand, might indicate that GHRP-2 opposes a putative 24-h rhythm in hypothalamic somatostatinergic activity (45, 68, 69). This notion would be consistent with the ability of GHRPs to antagonize certain other CNS actions of somatostatin (8, 21, 63). Alternatively, the repression of GHRP on the daily rhythm in GH interpulse intervals could reflect strong and time-invariant potentiation of endogenous pulsatile GHRH release and/or actions, because GHRPs stimulate GHRH secretion and synergize with GHRH (17, 59, 60, 70, 71, 72). Notably, coadministration of E2 and GHRP-2 normalized 24-h rhythmic variation in GH pulse frequency, further consistent with our hypothesis of joint CNS actions of these complementary agonists.

GHRPs stimulate ACTH, cortisol, and PRL secretion at least acutely (18, 37). However, 24-h continuous iv GHRP-2 infusion did not alter daily (pooled) serum cortisol or PRL concentrations. Cortisol was also unchanged by 1 or 2 months of treatment of elderly or obese adults with an orally active, nonpeptidyl analog of GHRP (MK-0677) (73, 74).

Visual inspection of paired 24-h serum GH concentration profiles suggested possible inter diem synchronous GH release in the two GHRP-2 infusion sessions. Cross-correlation analysis confirmed this inference. E2 replacement did not induce or alter such synchrony. Synchronous GH release under GHRP-2 drive could reflect the activity of putative underlying GHRH and/or somatostatin rhythmicities in the human, or far less likely indicate imposed coordination of pituitary somatotrope secretion. Additional investigations will be important to establish the biological significance of and mechanisms subserving such synchrony.


   Acknowledgments
 
We thank Patsy Craig for skillful preparation of the manuscript; Paula P. Azimi for deconvolution analysis, data management, and graphics; Ginger Bauler for performance of the RIAs, immunoradiometric assays, and chemiluminescence assays; and Sandra Jackson and the expert nursing staff at the University of Virginia General Clinical Research Center for conducting the clinical research protocols.


   Footnotes
 
1 Supported in part by NIH Grant MO1-RR00847 (to the General Clinical Research Center of the University of Virginia Health Sciences Center), the National Science Foundation Center for Biological Timing (Grant DIR89-20162), National Institute on Aging Grant RO1-AG14799-01 (to J.D.V.), and a NIH Clinical Research Scholar’s Award (to N.S.). Back

2 Present address: Omni Healthcare, 95 Bulldog Boulevard, Sheridan Building, Suite 101, Melbourne, Florida 32903. Back

Received December 2, 1999.

Revised March 24, 2000.

Accepted April 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals