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. 2009 Apr 7;94(7):2544–2550. doi: 10.1210/jc.2008-2767

Factors Other than Sex Steroids Modulate GHRH and GHRP-2 Efficacies in Men: Evaluation Using a GnRH Agonist/Testosterone Clamp

Johannes D Veldhuis 1, Cyril Y Bowers 1
PMCID: PMC2708950  PMID: 19351731

Abstract

Background: Sex steroids are prominent regulators of pulsatile GH secretion.

Hypothesis: An experimentally controlled sex-steroid milieu will permit detection of nonsteroidal factors that determine GH secretion.

Subjects: Eleven young (age, 24 ± 0.99 yr) and 11 older (64 ± 2.4 yr) men participated in the study.

Location: The study was conducted at a tertiary medical center.

Methods: The study consisted of GnRH-agonist down-regulation of the gonadal axis followed by fixed-dose testosterone (T) replacement (leuprolide/T clamp) and consecutive infusion of l-arginine and GHRH or GH-releasing peptide-2 (GHRP-2) to quantify peptide-secretagogue efficacies.

Outcomes: The experimental leuprolide/T clamp yielded statistically age-comparable total, bioavailable, and free T and estradiol (E2) concentrations. In this controlled milieu, sequential l-arginine/GHRH infusion stimulated 1.4-fold more (P = 0.021) and l-arginine/GHRP-2 1.3-fold more (P = 0.045) GH release in young than older men. Abdominal visceral fat (AVF) correlated negatively with both GHRH (P = 0.0006; R2 = 0.39) and GHRP-2 (R2 = 0.29) efficacy, whereas IGF-I positively predicted the same endpoints (R2 = 0.25 to 0.30). In multivariate analysis, AVF emerged as a dominant negative determinant of GHRH efficacy (P = 0.002; R2 = 0.41) and IGF-I as a primary positive determinant of GHRP-2 efficacy (P = 0.007; R2 = 0.31).

Conclusion: During fixed T/E2 availability, AVF contributes 41% of the GH-response variability to maximal GHRH drive, whereas IGF-I accounts for 31% of that for GHRP-2. Accordingly, a statistically equalized sex-steroid milieu permits dissection of age-independent and T/E2-independent modulators of GHRH and GHRP efficacy in men.

During experimentally fixed sex-steroid availability, visceral fat contributes 41% of the GH-response variability to maximal GHRH drive, whereas IGF-I accounts for 31% of that for GHRP-2.

The production of GH varies markedly across the human life span (1). Puberty is marked by multifold augmentation of pulsatile GH secretion, primarily driven by sex-steroid actions on the hypothalamo-pituitary unit (2,3). Conversely, chronic hypogonadism decreases GH secretion by about 50% (1,3). However, daily GH output begins to decline in young adulthood before androgen or estrogen concentrations change significantly (4) and then decreases more markedly after midlife (5,6,7,8,9). These data suggest that age and sex steroids constitute nonexclusive regulators of GH secretion. Indeed, factors such as body composition, physical exercise, feedback by GH and IGF-I, cytokines, free fatty acids, l-thyroxine, and glucose also influence GH production (1,3,10).

Administration of testosterone (T) doubles GH secretion in individuals with delayed puberty, anorchia, primary Leydig-cell failure, hypogonadotropic hypogonadism, Klinefelter’s syndrome, orchidectomy, or radiation injury to the testes (11,12,13,14,15,16,17,18,19,20,21,22). The effects of T supplementation in healthy older adults are less well studied. Transdermal T delivery for 4–6 wk did not stimulate GH secretion in two studies in older men (23,24). Intramuscular administration of T for 26 wk, which resulted in physiological T concentrations of 523–699 ng/dl, augmented overnight GH secretion by 2.0-fold in aging volunteers (25). Higher parenteral doses of T also amplified pulsatile GH secretion by 1.8- to 3.1-fold within 10 d in aging men (25,26,27,28,29). Assuming that the sex-steroid milieu modulates pulsatile GH secretion in men, then distinguishing regulation by nonsteroidal factors in healthy cohorts with disparate T and estradiol (E2) concentrations becomes more difficult.

The present study uses a paradigm of short-term gonadal-axis down-regulation and controlled T repletion to equalize the sex-steroid milieu in young and older men. The goal was to elucidate nonsteroidal modulators of peptide-secretagogue efficacy. This paradigm unmasked distinct effects of age, IGF-I, and abdominal visceral fat (AVF) on secretagogue-regulated GH secretion.

Subjects and Methods

Volunteers were 22 healthy unmedicated men ages 24 ± 1.0 yr (n = 11) and 64 ± 2.4 yr (n = 11). Subjects provided written informed consent approved by the Mayo Institutional Review Board. The protocol was reviewed by the U.S. Food and Drug Administration under investigator-initiated new drug numbers for GH-releasing peptide-2 (GHRP-2) and GHRH. Exclusion criteria were exposure to psychotropic or neuroactive drugs within five biological half-lives; body mass index (BMI) less than 18 or more than 32.5 kg/m2; anemia (hemoglobin <12.8%); drug or alcohol abuse, psychosis, depression, mania or severe anxiety; acute or chronic organ-system disease; use of T, other anabolic steroids, or glucocorticoids; endocrinopathy, other than primary thyroidal failure receiving replacement; nightshift work or recent transmeridian travel (exceeding three time zones within 7 d of admission); acute weight change (loss or gain of >2 kg in 6 wk); allergy to administered peptides; and unwillingness to provide written informed consent. Each subject had an unremarkable medical history and physical examination and normal screening laboratory tests of hepatic, renal, endocrine, metabolic, and hematological function. The men reported normal sexual development and function. Volunteers maintained a consistent daily schedule of arising between 0630 and 0830 h, worked 35–45 h/wk, consumed no more than 3 ounces of alcohol per week, had no history of illicit drug use, and had an unchanged exercise pattern and stable weight during the study interval. None participated in competitive endurance sports or had chronic or acute pain or inflammation.

Protocol

The study design was parallel-cohort, double-blind, and prospectively randomized. Volunteers received two consecutive injections of depot leuprolide acetate (3.75 mg im, 3 wk apart) to deplete systemic T and E2 concentrations, followed by T addback (T enanthate 200 mg im weekly, a total of three times) beginning on the day of the second leuprolide injection (day zero). Secretagogue infusions were scheduled in random order 10–18 d after the first T injection. Each subject was studied twice in the Clinical Translational Research Unit (CRU) at least 72 h apart on separate mornings after an overnight fast.

Subjects were admitted to the CRU at 0630 h on the day of secretagogue infusion. To limit nutritional confounds, a constant meal (vegetarian or nonvegetarian) was given to ingest at 1800 h the night before the CRU study. This comprised 8 kcal/kg distributed as 50% carbohydrate, 20% protein, and 30% fat. Volunteers then remained fasting, alcohol-abstinent, and caffeine-free overnight and during sampling.

In the CRU, iv catheters were placed in contralateral forearm veins at 0700 h to allow simultaneous infusion of secretagogues and blood sampling every 10 min, beginning at 0800 h. Sampling consisted of a 3-h baseline (saline infusion) in each session before iv infusion of: 1) l-arginine 30 g over 30 min, followed by 1 μg/kg bolus GHRH (GEREF; Serono, Norwalk, MA); and 2) l-arginine 30 g over 30 min, followed by 3 μg/kg bolus GHRP-2 (Takeda Pharmaceuticals, Deerfield, IL). These doses of l-arginine and peptides are maximally stimulatory in adults (30,31). l-Arginine was used to limit differences in somatostatin inhibition, because this amino acid appears to antagonize GH autofeedback-induced somatostatin outflow (32,33).

Blood was also withdrawn at 0800 h for later assay of serum E2, T, LH, FSH, IGF-I, IGF binding-protein-3 (IGFBP-3), IGFBP-1, and SHBG concentrations. Lunch was provided after sampling, before discharge from the CRU.

AVF was estimated by computerized tomography at the L3-L4 interspace, exactly as described earlier (34).

Remuneration

Study participants received institutional review board-approved reimbursement for time spent in the outpatient and inpatient visits.

Hormone assays

GH concentrations were determined in duplicate by automated ultrasensitive double-monoclonal immunoenzymatic, magnetic particle-capture, chemiluminescence assay using 22-kDa recombinant human GH as assay standard (Sanofi Diagnostics Pasteur Access, Chaska, MN). Sensitivity is 0.010 μg/liter (defined as 3 sd values above the zero-dose tube). Interassay coefficients of variation (CV) were 7.9 and 6.3% at GH concentrations of 3.4 and 12 μg/liter, respectively. Intraassay CV were 4.9% at 1.1 μg/liter and 4.5% at 20 μg/liter. No values decreased to less than 0.020 μg/liter. Cross-reactivity with 20-kDa GH is less than 5% on a molar basis.

E2 and T concentrations were measured by tandem liquid-chromatography ion-spray mass spectrometry. For E2, intraassay CV were 18, 3.8, and 7.2% at concentrations of 3.5, 40, and 297 pg/ml (multiply by 3.68 for pmol/liter). Interassay CV were 8.1, 4.7, and 4.9% at 16, 31, and 119 pg/ml, respectively. For T, the analytic range is 7–2000 ng/dl (multiply by 0.0347 for nmol/liter) for a 0.1 ml volume. Intraassay CV were 3.3, 2.8, 2.2, and 2.0% at T concentrations of 16, 64, 184, and 927 ng/dl, respectively. Corresponding interassay CV were 5.1, 3.8, 3.7, and 2.8%. Free and bioavailable T were calculated, as described in the Appendix of Ref. 35.

Albumin, LH, FSH, prolactin, and SHBG were assayed as described (35). IGFBP-1, IGFBP-3, and total IGF-I concentrations were measured by immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX) (26,36). Interassay CV for IGF-I were 9% at 64 μg/liter and 6.2% at 157 μg/liter. Intraassay CV were 3.4% at 9.4, 3% at 55, and 1.5% at 264 μg/liter.

Statistical analysis

Two-way ANOVA (2 × 2 factorial design) in a repeated-measures design was used to examine the individual and interactive effects of age (two factors) and secretagogue type (two factors) on: 1) basal (nonpulsatile) saline-infused GH secretion (μg/liter · 3 h); and 2) secretagogue-stimulated pulsatile GH secretion (μg/liter · 3 h). Post hoc contrasts were made via Tukey’s honestly significantly different test (37). Linear regression analysis was applied to examine the relationship between GH responses and IGF-I, IGFBP-3, or IGFBP-1 concentrations and AVF. An unpaired two-tailed Student’s t test was used to compare baseline hormone concentrations in the two age groups.

Stepwise forward-selection multivariate linear regression analysis was applied to data for the combined cohorts using Systat (Systat Software, Point Richmond, CA) (37). Data are presented as the mean ± sem. Experiment-wise, P < 0.05 was construed as statistically significant.

Statistical power analysis

Data from 16 studies in hypogonadal young or eugonadal older males indicated that the mean-weighted effect size of parenteral T supplementation to increase GH secretion was 2.4 sd values (12,13,14,15,17,19,20,21,25,26,27,28,29,38,39). Power analysis assumed that nonsex-steroidal factors can exert an effect 50% of this magnitude (1.2 sd). If comparisons are made via a one-tailed unpaired Student’s t test at P ≤ 0.05, then studying 23 subjects would achieve more than 99% power to detect a 50% effect size.

Results

Baseline (preintervention, screening) subject characteristics included comparable AVF and BMI in the two age groups (see Supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Fasting concentrations of IGF-I (P = 0.005), IGFBP-3 (P = 0.001), SHBG (P = 0.023), albumin (P < 0.001), and FSH (P < 0.01) differed by age. Leuprolide plus T addback increased concentrations of total T, bioavailable T, free T, and E2 comparably by age (P > 0.10, age effect) (Fig. 1). The FSH concentration was higher after leuprolide/T in older than young men (P = 0.003) (Supplemental Table 1). Compared with baseline, concentrations of prolactin rose (absolute increment) in older but not young men given leuprolide/T (Supplemental Table 2). LH, FSH, SHBG, and albumin concentrations fell, whereas T and E2 rose in both cohorts compared with preintervention.

Figure 1.

Figure 1

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Impact of age and leuprolide/T clamp on total and bioavailable T concentrations (left) and E2 and free T concentrations (right) in 11 young and 11 older men studied at baseline (preintervention) and after 3 wk of leuprolide down-regulation of the gonadal axis with T addback. Data are the mean ± sem. P values denote the results of ANOVA. Means with unshared alphabetic superscripts differ significantly by post hoc testing.

Consecutive iv infusion of l-arginine over 30 min, followed by bolus iv injection of a maximally stimulatory dose of GHRH or GHRP-2, induced marked GH release (Fig. 2A). Pulsatile GH secretion after successive l-arginine/GHRH stimulation was 21-fold, and that after l-arginine/GHRP-2 was 56-fold, higher than baseline unstimulated values in young men (both P < 0.0001). By comparison, GHRH and GHRP-2-stimulated GH responses were only 8.6-fold (P = 0.031 vs. young) and 24-fold (P = 0.055 vs. young) baseline values, respectively, in older men. In absolute terms, pulsatile GH secretion (μg/liter · 3 h) was also significantly greater in young men than in older men after maximal GHRH (P = 0.021) and GHRP-2 (P = 0.045) stimulation (Fig. 2B). In contrast, there was no age difference in unstimulated pulsatile GH secretion assessed during saline infusion.

Figure 2.

Figure 2

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A, Time course of 10-min GH concentrations in 11 young and 11 older men evaluated under a leuprolide/T clamp during iv saline infusion (clock time, 0800–1100 h) followed by consecutive iv infusion of l-arginine over 30 min and GHRH (left) or GHRP-2 (right) as maximally effective bolus doses immediately thereafter. Data are the mean ± sem. Open circles denote data from young men, and closed circles denote data from older individuals. B, Deconvolution-calculated pulsatile GH secretion during saline infusion and stimulated by consecutive infusions of l-arginine/GHRH or l-arginine/GHRP-2 in 11 young and 11 older men (light and dark columns, respectively). Data are the mean ± sem. P values were estimated by an unpaired two-tailed Student’s t test. Observations were made under a combined leuprolide/T clamp.

The utility of the sex-steroid clamp was affirmed by the absence of any significant associations between experimentally controlled total, bioavailable, or free T or total E2 concentrations and: 1) pulsatile GH responses to sequential-secretagogue infusions; 2) unstimulated (saline-infused) pulsatile GH secretion; or 3) basal (nonpulsatile) GH secretion (viz., P values ≥ 0.14 and R2 values ≤ 0.12). As summarized in Supplemental Table 3, preplanned regression analyses showed that AVF explained more than two fifths of the variability in l-arginine/GHRH action (P = 0.006; R2 = 0.45) and nearly one third of that for l-arginine/GHRP-2 (P = 0.012; R2 = 0.29) (Fig. 3, left). IGF-I was a direct correlate of the efficacies of GHRH (P = 0.026; R2 = 0.25) and GHRP-2 (P = 0.013; R2 = 0.30), accounting for at least one fourth of the variation in GH responses (Fig. 3, right).

Figure 3.

Figure 3

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Left, Linear regression of pulsatile GH secretion on AVF during stimulation with l-arginine/GHRH (top) or l-arginine/GHRP-2 (bottom). The single value marked 320 is replotted in the top left panel but was not a statistical outlier. Right, Regression of pulsatile GH secretion (y-axis) stimulated by l-arginine/GHRH or l-arginine/GHRP-2 on IGF-I concentrations (x-axis) in young and older men.

Unstimulated fasting pulsatile GH secretion was not significantly associated with age, AVF, IGF-I, or IGFBP-3 under the leuprolide/T clamp. In contrast, unstimulated, fasting basal (nonpulsatile) GH secretion was strongly positively related to IGFBP-1 concentrations (P < 0.001; R2 = 0.64), which explained almost two thirds of the variability in this measure. Conversely, AVF correlated negatively with basal GH release (P = 0.025; R2 = 0.23). Multivariate regression showed that basal GH release was best accounted for statistically by IGFBP-1 (P < 0.001) and IGF-I (P = 0.0095) considered together, both having positive associations (Fig. 4).

Figure 4.

Figure 4

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Three-dimensional plot of positive relationships between IGF-I and IGFBP-I (both independent variables) on basal GH secretion (dependent variable) in 22 men studied under a combined leuprolide/T clamp. Young and older subjects are designated by open and closed circles, respectively. Overall multivariate R2 was 0.72 (P < 0.0001), with individual partial P values and corresponding slopes as noted.

The number of GH pulses was invariant of T, E2, or any nonsex-steroidal correlates. The mode of l-arginine/GHRH-stimulated GH secretory bursts was positively but weakly influenced by E2 concentrations (R2 = 0.20) and AVF (R2 = 0.20) [both P < 0.05] (Fig. 5). A longer mode signifies a greater time delay to attain maximal GH secretion within GHRH-induced secretory bursts. These relationships did not apply to l-arginine/GHRP-2-stimuated bursts.

Figure 5.

Figure 5

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Correlation between GH secretory-burst mode (time delay from onset of secretory burst to maximal release) and AVF (independent variable, top panel) or E2 concentrations (independent variable, bottom panel) in 22 men consecutively infused with l-arginine and GHRH. Volunteers were studied under a combined leuprolide/T clamp. The single solid circle enclosed by a box in the bottom panel was a statistical outlier by Studentized residuals (P < 0.001).

Discussion

Implementation of a clinical paradigm comprising an experimentally controlled sex-steroid milieu in young and older men unveiled strong nonsteroidal modulators of pulsatile GH secretion driven by GHRH and GHRP in a putatively reduced-somatostatin milieu. In particular, in a fixed T and E2 environment, age attenuated maximal stimulation by GHRH by 40% and that by GHRP-2 by 30%.

Consecutive iv infusions of l-arginine, an inhibitor of GH autonegative feedback via somatostatin (32,33,40), and GHRH (1 μg/kg) or GHRP-2 (3 μg/kg) should interrogate maximal pituitary responsiveness to (viz., should define the efficacy of) these peptidyl secretagogues. Under this assumption, age and AVF were individual (univariate) determinants and AVF was a powerful aggregate (multivariate) determinant of GHRH efficacy. The last finding indicates that activity of the GHRH receptor-effector pathway is restrained by factor(s) associated with visceral adiposity, independently of age, sex-steroid availability, IGFBP-1, IGFBP-3, or IGF-I concentrations. Cytokines and adipokines, such as TNF-α and IL-6, are able to inhibit GH secretion in laboratory models (1,3). However, whether these or other cytokines mediate the negative effect of AVF on GHRH action is unknown.

AVF was a negative predictor of GHRP efficacy as well. AVF accounted for about 30% of the variability in GHRP-2 efficacy in the 22 men studied (P = 0.012). In contrast to GHRH, pulsatile GH responses to GHRP-2 were not significantly affected by age or IGFBP-3. Like GHRH, GHRP-2 stimulation was positively related to IGF-I concentrations, which accounted for 30% of GH response variability (P = 0.013). In multivariate analysis, IGF-I emerged as the dominant positive correlate of GHRP-2 efficacy (P = 0.007). This novel outcome could plausibly mean that higher hypothalamopituitary responses to GHRP in part reflect greater GHRH release from the hypothalamus, which in turn predicts greater fasting GH pulsatility and IGF-I gene induction via STAT5b signaling (41). A reverse association, in which higher IGF-I concentrations elevate GHRP efficacy, would be more difficult to postulate. IGF-I and IGFBP-3 pathways were involved selectively because peptide-secretagogue efficacies bore no relationship to IGFBP-1.

In multivariate regression analysis, IGFBP-1 (P < 0.001) and IGFBP-3 (P = 0.007) together explained 72% of the intersubject variability in basal GH release and accounted fully for the individual negative effects of AVF (P = 0.025) and BMI (P = 0.008). The physiological basis for the joint correlation is not clear. Indeed, very little is known in general about the regulation of basal GH secretion, except that E2 in women and T in men may be stimulatory (3). Albeit potentially regulated, basal GH secretion contributes less than 15% of total GH secretion in healthy adults.

The mode (time delay to maximal GH secretion within) of GHRH-induced GH secretory bursts was positively influenced by E2 concentrations and AVF. The first relationship had been inferred in women given E2 replacement (42), which effect was blocked by an estrogen receptor-α antagonist (43). The notion that increased AVF prolongs GH-secretory bursts has not been suggested previously. If affirmed in further studies, delayed maximal GH release could mean that factor(s) associated with AVF impair vectorial delivery, membrane docking, priming, fusion, and/or exocytosis of GH-containing secretory vesicles (44). Restriction of the effect to GHRH stimulation could mean that somatostatin is involved, inasmuch as GHRP can partially antagonize central inhibition by somatostatin (45).

Supraphysiological (but not statistically significantly higher) total T values in some (three) older men and one young man resulted in greater variance about the mean. Higher total T corresponded with higher SHBG levels in these individuals. Since bioavailable and free T were also nonsignificantly higher in the same subjects, one could postulate a relatively lower metabolic clearance rate (or distribution volume) of T in these men. Nonetheless, regression of GH secretion on any of total, bioavailable, or free T was not significant, indicating that GH stimulation in the T-concentration range attained by the leuprolide/T clamp is controlled by age, AVF, and IGF-I-related proteins, rather than by inequalities in T concentrations.

Caveats include the relatively small cohort size (n = 22), attainment of supraphysiological T concentrations in some subjects, a somewhat brief (3-h) interval of baseline sampling before secretagogue infusion, and the possible existence of other nonsteroidal regulators not yet detected. The full dose-response range of T in driving GH secretion is not yet known. However, indirect estimates in one analysis suggested that physiological adult T concentrations (430 ng/dl) are sufficient to stimulate GH secretion one half maximally, and that supraphysiological T concentrations do not stimulate GH further (46).

In summary, a clinical model of experimentally fixed T and E2 availability establishes that age exerts negative effects on both GHRH and GHRP-2 efficacy independently of the short-term sex-steroid milieu. According to multivariate analyses, the negative effect of age on GHRH efficacy was attributable to increasing AVF, whereas the negative effect of age on GHRP-2 efficacy was explicable by declining IGF-I concentrations in older individuals. Further basic laboratory and clinical investigations will be required to ascertain the biochemical mechanisms that subserve these relationships.

Supplementary Material

[Supplemental Data]
jc.2008-2767_index.html (1.2KB, html)

Acknowledgments

We thank Donna Scott for support of manuscript preparation, Ashley Bryant for data analysis and graphics, the Mayo Immunochemical Laboratory for assay assistance, and the Mayo research nursing staff for implementing the protocol.

Footnotes

This work was supported in part via the Center for Translational Science Activities Grant 1 UL 1 RR024150 to the Mayo Clinic and Foundation, by the National Center for Research Resources (Rockville, MD), and by National Institutes of Health Grant R01 NIA AG19695.

Disclosure Summary: The authors have nothing to declare.

First Published Online April 7, 2009

Abbreviations: AVF, Abdominal visceral fat; CRU, Clinical-Translational Research Unit; CV, coefficients of variation; E2, estradiol; GHRP, GH-releasing peptide; IGFBP, IGF binding-protein; T, testosterone.

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