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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2008 Dec 16;94(3):973–981. doi: 10.1210/jc.2008-2108

Aromatase and 5α-Reductase Inhibition during an Exogenous Testosterone Clamp Unveils Selective Sex Steroid Modulation of Somatostatin and Growth Hormone Secretagogue Actions in Healthy Older Men

Johannes D Veldhuis 1, Kristi L Mielke 1, Mihaela Cosma 1, Cacia Soares-Welch 1, Remberto Paulo 1, John M Miles 1, Cyril Y Bowers 1
PMCID: PMC2681279  PMID: 19088159

Abstract

Background: How endogenous testosterone (Te), 5α-dihydrotestosterone (DHT), and estradiol (E2) regulate pulsatile GH secretion is not understood.

Hypothesis: Conversion of Te to androgenic (Te→DHT) or estrogenic (Te→E2) products directs GH secretion.

Subjects and Location: Healthy older men (N = 42, ages 50–79 yr) participated at an academic medical center.

Methods: We inhibited 5α-reduction with dutasteride and aromatization with anastrozole during a pharmacological Te clamp and infused somatostatin (SS), GHRH, GH-releasing peptide-2 (GHRP-2), and l-arginine/GHRH/GHRP-2 (triple stimulus) to modulate GH secretion.

Endpoints: Deconvolution-estimated basal and pulsatile GH secretion was assessed.

Results: Administration of Te/placebo elevated Te by 2.8-fold, DHT by 2.6-fold, and E2 concentrations by 1.9-fold above placebo/placebo. Te/dutasteride and Te/anastrozole reduced stimulated DHT and E2 by 89 and 86%, respectively. Stepwise forward-selection regression analysis revealed that 1) Te positively determines mean (P = 0.017) and peak (P < 0.001) GH concentrations, basal GH secretion (P = 0.015), and pulsatile GH secretion stimulated by GHRP-2 (P < 0.001); 2) Te and E2 jointly predict GH responses to the triple stimulus (positively for Te, P = 0.006, and negatively for E2, P = 0.031); and 3) DHT correlates positively with pulsatile GH secretion during SS infusion (P = 0.011). These effects persisted when abdominal visceral fat was included in the regression.

Conclusion: The present outcomes suggest a tetrapartite model of GH regulation in men, in which systemic concentrations of Te, DHT, and E2 along with abdominal visceral fat determine the selective actions of GH secretagogues and SS.

Systemic concentrations of androgenic and estrogenic sex steroids along with abdominal visceral-fat mass jointly determine GH responses to peptidyl secretagogues and (exogenous) somatostatin.

GH is secreted by the anterior pituitary gland in bursts driven by interactions among endogenous secretagogues and somatostatin (SS) (1). GHRH, a 40- and 44-amino-acid hypothalamic peptide, is a proximate agonist of GH secretion and somatotroph cell growth (2). Ghrelin, a 28-amino-acid Ser3-octanoylated GH-releasing peptide (GHRP) of gastro-pancreatico-hypothalamo-pituitary origin, and synthetic GHRPs stimulate GH secretion and synergize with GHRH (1,3,4,5,6,7,8,9,10). Both peptide signals are important physiologically, because mutations of the human GHRH or ghrelin receptor and transgenic silencing of the murine pituitary GHRH or hypothalamic ghrelin receptor reduce GH secretion, IGF-I concentrations, and/or stature (7,11,12). SS, a 14- and 28-amino-acid hypothalamic peptide, is a noncompetitive inhibitor of GH release (8). All three, GHRH, GHRP, and SS, govern pulsatile GH secretion (1,2).

Pulsatile GH secretion, which constitutes about 85% of total daily GH production, declines markedly in aging due to selective attenuation of GH pulse size (13,14,15). The factors determining this adaptation are unknown but may include an age-related decrease in sex steroid availability, an increase in abdominal visceral fat (AVF), and/or primary age-related neural impairment (2,16,17,18,19,20,21). Testosterone (Te) and estradiol (E2) stimulate pulsatile GH production by augmenting the size of (mass of GH secreted in) each burst; conversely, factors associated with AVF in some manner reduce GH burst mass (20,22,23). In postmenopausal women, E2 administration amplifies the amount of GH secreted per burst, enhances hypothalamo-pituitary responsivity to GHRP, potentiates stimulation by GHRH, and attenuates inhibition by SS (9,24,25,26). In contrast, how Te regulates the actions of GHRP, GHRH, and SS in men is either controversial or unknown. In the human and animals, all three potentiating, inhibitory, and neutral effects of Te on GH responses to ghrelin/GHRP stimulation have been reported (27,28,29). Conversely, neither Te deprivation nor Te repletion seems to alter the efficacy of maximal GHRH drive (30,31,32). No clinical studies exist on whether Te availability modulates SS’s suppression of GH secretion. Conflicting data, where data exist, may reflect the capability of Te to act by way of distinct steroidogenic products, 5α-dihydrotestosterone (DHT) and E2, on separate nuclear-receptor pathways (33,34), which mediate putatively opposing effects on the hypothalamic secretion and pituitary actions of GHRH and SS (1,2,35).

The present study examines sex steroid modulation of GH secretion in a novel paradigm comprising selective blockade of 5α-reduction using dutasteride and of aromatization using anastrozole. A fixed dose of Te was administered concurrently to obviate potentially confounding feedback adjustments in the hypothalamo-pituitary-testicular axis induced by the steroidogenic inhibitors. Healthy older men were studied, because high Te concentrations augment 24-h pulsatile GH secretion in aging but not young individuals (20).

Subjects and Methods

Inhibitors

Dutasteride was obtained from GlaxoSmithKline (Research Triangle Park, NC) and anastrozole from AstraZeneca (Wilmington, DE) under investigator-initiated new drug registration numbers assigned by the U.S. Food and Drug Administration (36,37).

Subjects

The protocol was approved by the Mayo Institutional Review Board, reviewed by the U.S. Food and Drug Administration, and performed according to the Helsinki declaration. Volunteers underwent medical screening as outpatients, including history and physical examination. Inclusion criteria were normal hepatic, renal, hematological, metabolic, and endocrine function (TSH, prolactin, LH, FSH, and total Te). Reasons for exclusion were concurrent use of neuroactive medications or sex hormones, diabetes mellitus, untreated thyroxine deficiency, systemic or organ-level disease, abnormal baseline biochemical data, drug or alcohol abuse, hemoglobin less than 12.8 g/dl, history of thrombotic arterial disease (stroke, transient ischemic attack, myocardial infarction, and angina), congestive heart failure, prostatic disease, prostate-specific antigen of 4 ng/liter or higher, and unwillingness to provide written informed consent.

Diet

To limit nutritional confounds, a standardized meal (10 kcal/kg of 50% carbohydrate, 20% protein, and 30% fat) was served at 1800 h the night before study. Subjects then remained fasting, alcohol abstinent, and caffeine free until 1300 h the next day.

Body composition measurement

Volunteers had a single-slice computerized tomogram of the abdomen at L3–L4 to quantify the cross-sectional area (square centimeters) of visceral adipose tissue, as described earlier (38).

Hormone assays

GH concentrations were determined in duplicate by automated ultrasensitive two-site immunoenzymatic chemiluminescence assay (Beckman Instruments, Chaska, MN), exactly as described (39).

E2, Te, and DHT were measured by tandem liquid-chromatography ion-spray mass spectrometry, exactly as described (39).

IGF-binding protein-1 (IGFBP-1), IGFBP-3, and total IGF-I concentrations were quantified by immunoradiometric assays (Diagnostic Systems Laboratories, Webster, TX) as described (40).

Deconvolution analysis

Each GH concentration time series was analyzed using a recently developed deconvolution method (41). The automated Matlab program first detrends the data and normalizes concentrations to the unit interval (0, 1). Second, multiple successive potential pulse-time sets, each containing one fewer burst, are created by a smoothing process (a nonlinear adaptation of the heat-diffusion equation). Third, a maximum-likelihood estimation method calculates all secretion and elimination rates simultaneously for each candidate pulse-time set. The deconvolution model specifies basal secretion (β0), two half-lives (α1, α2), secretory-burst mass (η0, η1), random effects on burst mass (σA), procedural/measurement error (σε), and a three-parameter flexible γ-secretory-burst waveform (β1, β2, β3). Here, α1 was fixed at 3.5 min and α2 at 20.9 min with the slow fraction representing 63% of total decay (42). Thus, eight parameters are estimated. After all candidate pulse-time sets are deconvolved, model selection is performed using the Akaike information criterion. The parameters (and units) are basal and pulsatile secretion rates (micrograms per liter per hour), secretory-burst mass (micrograms per liter), and secretory-burst shape (mode of waveform or time delay in minutes to maximal secretion after burst onset).

Clinical protocol

This was a prospective, double-blind, randomized placebo-controlled parallel-cohort outpatient study. Randomization was into four groups: 1) placebo/placebo (Pl/Pl); 2) Te/Pl; 3) Te/dutasteride (Te/Dut), an inhibitor of 5α-reductase type 1 and type 2; and 4) Te/anastrozole (Te/Ana), a selective aromatase inhibitor.

Specific interventions included 1) Pl/Pl, im saline 1.0 ml weekly times three injections and oral placebo once daily times 21 d; 2) Te/Pl, enanthate 200 mg im weekly times three injections and oral placebo (as above); 3) Te/Dut im and oral dutasteride 1 mg once daily times 21 d; and 4) Te/Ana im and oral anastrozole 2 mg once daily times 21 d.

Secretagogue infusions were scheduled in the Clinical Research Unit in prospectively randomized order on any four nonconsecutive mornings within the 15-d window defined by d 9–24 after the first Te injection (d 1). Volunteers arrived at or before 0600 h for iv catheter placement and stayed until 1300 h. A blood sample was obtained at 0700 h for baseline hormone measurements. The GH stimulation/inhibition protocols were 1) saline (30 ml/h) from 0800–0930 h to establish a baseline; 2) somatostatin-14 (5 μg/m2 · h continuously) 1000–1300 h; 3) GHRH (0.15 μg/kg bolus) at 1000 h; 4) GHRP-2 (3.0 μg/kg bolus) at 1000 h; and 5) l-arginine (30 gm iv 0930–1000 h) followed by a combined bolus of GHRP-2 and GHRH (above) at 1000 h, hereafter referred to as a triple stimulus.

At the peptide doses chosen, E2 supplementation potentiates stimulation by GHRP and GHRH and mutes inhibition by SS in women (9,24,25,26). The triple stimulus was used to estimate maximal somatotroph secretory capacity.

Blood sampling

Repetitive blood sampling (1.5 ml every 10 min) was begun at 0800 h and continued for 5 h. Ambulation was permitted to the lavatory. Sleep was disallowed during sampling. Lunch was offered at 1300 h before discharge from the unit.

Statistical assessment

The primary null hypothesis was that dutasteride and anastrozole do not modify mean fasting GH concentrations during fixed Te supplementation. One-way ANOVA with four categorical factors was used to test this conjecture, using the mean of the four saline-infused baseline (90-min) intervals in each subject. Given a 1.6 ± 0.44 (sd)-fold expected stimulatory effect of Te above placebo on mean GH concentrations in older men (20,28), statistical power to detect a 30% treatment effect when 42 individuals are studied would exceed 90% by unpaired Student’s t test at two-sided Bonferroni-adjusted P ≤ 0.01.

The secondary hypothesis was that sex steroid milieu determines pulsatile GH secretory responses to distinct peptidyl effectors, viz. SS repression and GHRH, GHRP-2, and triple stimulation. This postulate was addressed by two-way analysis of covariance (using the mean prestimulus GH concentration as a covariate) in a 4 × 4 factorial design: four sex steroid interventions (categorical variables) and four secretagogues (repeated measures within subject). Data were log-transformed to limit dispersion of residual variance. Post hoc comparisons were made by Fisher’s least significantly different test. Based on a sample size of 37 subjects, statistical power exceeded 90% to detect a 30% effect of sex hormone milieu by unpaired Student’s t test at P ≤ 0.01 for the least-expected contrast of a 0.50 ± 0.13 (sd)-fold inhibitory effect of SS (9,24,25,26).

A corollary postulate was that systemic Te, DHT, or E2 concentration or AVF determines GH secretion. This postulate was addressed by stepwise forward-selection multivariate linear regression analysis at α = 0.050 (n = 42 subjects) using Systat version 11.0 (San Jose, CA).

Results

Table 1 shows subject characteristics, which did not differ with respect to age, body mass index, or AVF. Screening hormone concentrations also did not differ (not shown). After pharmacological Te administration, Te, E2, and DHT concentrations rose by 2.8-fold, 1.9-fold, and 2.6-fold, respectively, compared with Pl/Pl (Table 1). Combined Te/Dut administration did not affect E2 or Te concentrations but reduced DHT values by 89% compared with Te/Pl and by 70% compared with Pl/Pl (both P < 0.001). Combined Te/Ana treatment did not alter DHT or Te concentrations but decreased E2 values by 86% compared with Te/Pl and by 74 and 89% compared with Pl/Pl and Te/Dut, respectively (each P < 0.001). Bioavailable and free Te concentrations changed analogously, thereby yielding four distinct sex steroid milieus.

Table 1.

Subject characteristics, sex steroid, and IGF-I concentrations on day of study

Pl/Pl (n = 11) Pl/Te (n = 14) Te/Dut (n = 9) Te/Ana (n = 8) ANOVA P value
Subject characteristics
 Age (yr) 62 ± 2.8 (63) 62 ± 2.1 (61) 63 ± 2.7 (63) 64 ± 2.5 (64) NS
 BMI (kg/m2) 26 ± 1.4 (26) 26 ± 0.60 (26) 27 ± 1.1 (27) 28 ± 1.3 (27) NS
 AVF (cm2) 168 ± 28 (152) 160 ± 16 (158) 176 ± 39 (126) 231 ± 57 (212) NS
Sex steroid concentrations
 DHT (pg/ml) 278 ± 28a (286) 724 ± 90a (657) 83 ± 8.5a (90) 936 ± 72a (872) <0.001
 E2 (pg/ml) 23 ± 2.1a (24) 44 ± 6.28 (42) 67 ± 13a (57) 6.0 ± 0.87a (5.0) <0.001
 Te (ng/dl) 528 ± 42a (496) 1475 ± 186a (1238) 1392 ± 209a (1248) 1337 ± 141a (1211) <0.001
 SHBG (nmol/liter) 30 + 2.4 (29) 35 ± 3.1 (33) 38 ± 5.7 (39) 30 ± 3.0 (30) 0.28
 IGF-I (μg/liter) 218 ± 22 (203) 227 ± 26 (196) 261 ± 37 (281) 203 ± 21 (187) 0.78
 IGFBP-1 (μg/liter) 21 ± 3.6 (22) 20 ± 2.5 (19) 23 ± 2.3 (24) 28 ± 7.1 (20) 0.68
 IGFBP-3 (μg/liter) 3329 ± 176 (3481) 3149 ± 151 (3311) 3417 ± 258 (3434) 3030 ± 97 (3086) 0.60
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Data are the mean ± sem (median). Preintervention sex steroid concentrations at 0800 h were DHT 272 ± 50 pg/ml, E2 25 ± 4 pg/ml, and Te 426 ± 37 ng/dl. Multiply DHT by 0.0345, E2 by 3.67, and Te by 0.0347 to obtain values in nanomoles per liter, picomoles per liter, and nanomoles per liter, respectively. NS denotes P > 0.05. BMI, Body mass index. 

a

Different superscript letters denote significantly different means within each row. 

Mean (± sem) 5-h GH concentration profiles in all 42 men are depicted in Fig. 1. Visual inspection suggested a descending rank order of stimulated GH secretion of triple stimulus more than GHRP-2 more than GHRH more than baseline (prestimulus) more than SS.

Figure 1.

Figure 1

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Mean (± sem) serum GH concentration profiles obtained by sampling blood every 10 min for 1.5 h before and 3.5 h after injecting SS or the indicated secretagogues in healthy older men. The number of subjects in each study group is denoted by N.

One-way ANOVA revealed that inhibitor type controlled baseline (prestimulus) mean GH concentrations (P = 0.011). Post hoc comparisons indicated that GH concentrations were higher after both Te/Pl and Te/Dut than after Pl/Pl (P = 0.041 and P = 0.015, respectively) and analogously for both Te/Pl and Te/Dut vs. Te/Ana (P = 0.021 and P = 0.008, respectively) (Fig. 2). Thus, Te/Ana reduces both Te/Pl- and Te/Dut-stimulated mean GH concentrations. Corresponding outcomes with respect to baseline (unstimulated) peak GH concentrations were P = 0.007 (overall treatment effect), Te/Dut more than Pl/Pl (P = 0.015), Te/Pl more than Pl (P = 0.060), Te/Pl more than Te/Ana (P = 0.012), and Te/Dut more than Te/Ana (P = 0.003).

Figure 2.

Figure 2

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Mean prestimulus GH concentrations in the four study groups. Post hoc testing (arrows) was by Fisher’s least significantly different test. Data are the mean ± sem (N as indicated).

Two-way analysis of covariance demonstrated that secretagogue type (P < 0.001) and inhibitor type (P = 0.030) individually determined stimulated/repressed peak GH concentrations (overall P < 0.001; covariate P < 0.001; interaction P > 0.3) (Fig. 3). The peak GH response in the cohort treated with Te/Ana was significantly lower than that in both Te/Pl (P = 0.007) and Te/Dut (P = 0.009) but not Pl/Pl. All peptidyl regulators differed in effect from the prestimulus saline control and from one another (P < 0.001).

Figure 3.

Figure 3

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Secretagogue-induced and SS-inhibited peak GH concentrations compared with prestimulus (saline) values. Data were subjected to two-way ANOVA (overall P < 0.001; n = 42 men). Means with no shared letters are significantly different. Thus, A differs from B but not from AB.

Deconvolution analysis was applied to assess sex steroid regulation of basal pulsatile GH secretion. One-way ANOVA disclosed a significant effect of inhibitor treatment on prestimulus basal (nonpulsatile) GH secretion, which was higher after Te/Pl than after Te/Ana administration (P = 0.032) (Table 2).

Table 2.

Mean GH concentrations, secretory-burst mode, and secretion rates

Pl/Pl (n = 11) Te/Pl (n = 14) Te/Dut (n = 9) Te/Ana (n = 8)
Pre-bolus mean GH concentration (μg/liter)
 GHRH 0.26 ± 0.22 0.33 ± 0.18 0.32 ± 0.22 0.17 + 0.090
 GHRP-2 0.18 ± 0.13 0.57 ± 0.27 0.76 ± 0.35 0.23 ± 0.38
 Triple stimulus 0.31 ± 0.16 0.49 ± 0.32 0.53 ± 0.30 0.20 ± 0.16
 SS 0.077 ± 0.080 0.31 ± 0.16 0.52 ± 0.35 0.14 + 0.14
Secretory-burst mode (min)
 GHRH 17 ± 1.7 19 ± 1.9 22 ± 2.0 13 ± 2.3
 GHRP-2 16 ± 1.0 16 ± 0.9 20 ± 2.9 14 ± 1.9
 Triple stimulus 18 ± 3.2 14 ± 1.6 15 ± 3.1 14 ± 1.2
 SS 19 ± 5.5 21 ± 3.9 18 ± 2.2 8.4 + 3.4
Basal GH secretion rate (μg/liter/h)
 GHRH 0.070 ± 0.090 0.049 ± 0.023 0.057 + 0.054 0.039 ± 0.063
 GHRP-2 0.072 ± 0.13 0.18 ± 0.14 0.032 ± 0.054 0.024 ± 0.056
 Triple stimulus 0.16 ± 0.26 0.23 ± 0.37 0.38 ± 0.33 0.13 ± 0.33
 SS 0.055 ± 0.025 0.041 ± 0.033 0.022 ± 0.014 0.019 ± 0.031
Pulsatile GH secretion rate (μg/liter/h)
 GHRH 0.68 ± 0.42 0.91 ± 0.24 1.1 ± 0.35 0.44 ± 0.92
 GHRP-2 3.1 ± 1.2 3.6 ± 3.3 3.3 ± 1.4 3.1 ± 1.90
 Triple stimulus 18 ± 3.5 19 ± 4.5 22 ± 3.7 17 ± 7.60
 SS 0.089 ± 0.12 0.36 ± 0.18 0.69 ± 0.31 0.083 + 0.36
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Data are the geometric mean ± sem (n = 42 men). 

Two-way ANOVA of pulsatile GH secretion rates (micrograms per liter per hour) disclosed significant individual effects of treatment group (Pl/Pl, Te/Pl, Te/Dut, and Te/Ana) and peptide effector (three secretagogues and SS) (P = 0.024 treatment; P < 0.001 effector; P < 0.001 overall; interaction P > 0.30) (Table 2). Responses to all four peptidyl effectors (GHRH, GHRP-2, triple stimulus, and SS) differed from one another (n = 42; P < 0.001). Pulsatile GH secretion was significantly lower in the Te/Ana compared with both the Te/Pl (P = 0.017) and Te/Dut (P = 0.006) cohorts (Fig. 4). Moreover, pulsatile GH secretion during SS infusion differed markedly by sex steroid condition: Te/Pl more than Pl/Pl (P = 0.025), Te/Dut more than Pl/Pl (P = 0.003), Te/Pl more than Te/Ana (P = 0.034), and Te/Dut more than Te/Ana (P = 0.005). Te/Ana did not differ from Pl/Pl, and Te/Pl was similar to Te/Dut.

Figure 4.

Figure 4

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Influence of administration of Te and selective steroidogenic inhibitors on peptide-modulated pulsatile GH secretion. Data are presented as in Fig. 3.

GH secretory-burst mode (time delay from onset of a burst to maximal secretion) was influenced by sex-steroid milieu (P < 0.001) and not by peptide (P = 0.345) (Table 2). When all peptidyl effectors were considered together, the mode observed after Te/Ana treatment (mean 13 ± 2 min) was less than that during each of Pl/Pl, Te/Pl, and Te/Dut (grand mean 18.6 ± 1.3 min, P < 0.001). During SS infusion, the GH secretory-burst mode associated with Te/Ana administration was shorter than that for Pl/Pl, Te/Pl, and Te/Dut (each P < 0.001) (Fig. 5). After GHRH stimulation, the Te/Ana mode (8.4 ± 3 min) was also less than that for Te/Dut (22 ± 2 min, P = 0.022), with a similar trend for Te/Ana less than Te/Pl (P = 0.055).

Figure 5.

Figure 5

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Reduction of GH secretory-burst mode (time delay from burst onset to maximal secretion) by Te/Ana for all peptidyl effectors combined (top) and for SS and GHRH individually (bottom). See data presentation in Fig. 3.

Multivariate stepwise forward-selection regression analysis revealed that Te, independently of DHT or E2, was a strongly positive determinant of 1) baseline (prestimulus) mean GH concentrations (P = 0.017; R2 = 0.141); 2) baseline peak GH concentrations (P = 0.011; R2 = 0.164) (not shown); 3) basal (nonpulsatile) GH secretion (P = 0.015; R2 = 0.149); pulsatile GH secretion during SS infusion (P = 0.016; R2 = 0.14); and 5) pulsatile GH secretion stimulated by GHRP-2 (P = 0.001; R2 = 0.243) (Fig. 6). In addition, Te and E2 jointly determined pulsatile GH secretion in response to the triple stimulus (Te positively, P = 0.006, and E2 negatively, P = 0.031; overall P = 0.013; R2 = 0.210). E2 had no independent effect on basal or pulsatile GH release but positively predicted prestimulus peak GH concentrations (P = 0.0083; R2 = 0.170) and IGF-I concentrations (P = 0.0057; R2 = 0.184) (Fig. 7A). DHT concentrations correlated negatively with GHRH-stimulated GH secretory-burst mode (P = 0.020; R2 = 0.138) and positively with pulsatile GH secretion after SS administration (P = 0.011; R2 = 0.159) (Fig. 7B).

Figure 6.

Figure 6

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Linear regression of principal GH secretion measures on total Te concentrations. A boxed symbol denotes a significant regression outlier (P < 0.001 Studentized t residual). R2 denotes the square of the correlation coefficient.

Figure 7.

Figure 7

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Linear correlation between E2 concentrations and GH-IGF-I measures (A) and between DHT concentrations and GH secretion (B). See Fig. 6 legend.

Inclusion of AVF in multivariate analysis abolished the effect of E2 alone on baseline peak GH concentrations but did not alter the joint effects of Te (positive) and E2 (negative) on the triple GH stimulus. AVF independently predicted inhibition of 1) mean baseline (prestimulus) GH concentration (P = 0.012); 2) pulsatile GH secretion during SS repression (P = 0.017); and 3/4) pulsatile GH secretion stimulated by GHRH (P = 0.003) and the triple stimulus (P = 0.036) but not GHRP-2 (P = 0.538) (Fig. 8). During inhibition by SS, AVF (regression coefficient −0.006) and DHT (regression coefficient +0.002) together determined pulsatile GH secretion (respective partial P values P = 0.005 and P = 0.003; overall P = 0.001; R2 = 0.323). After triple stimulation, AVF negatively (P = 0.018), Te positively (P = 0.005), and E2 negatively (P = 0.018) jointly predicted pulsatile GH secretion (overall P = 0.003; R2 = 0.315).

Figure 8.

Figure 8

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Linear regression of key GH secretion parameters on AVF determined by computed tomography scan. See Fig. 6 legend.

Discussion

The present investigation demonstrates that systemic concentrations of Te, E2, and DHT and AVF are strong determinants of the amount of GH secreted in the baseline fasting state as well as in response to GHRH and GHRP-2 stimulation, SS inhibition, and administration of triple secretagogues (a putative test of maximal somatotroph secretory capacity). The paradigm used to elucidate these relationships created a wide range of Te, E2, and DHT concentrations measured by tandem ion-spray mass spectrometry, wherein Te/Pl served to stimulate GH secretion, as reported earlier in older men (20). Pivotal findings were that high systemic Te concentrations no longer elevate baseline (unstimulated) fasting and SS-suppressed GH concentrations when E2 is experimentally lowered by 86% but continue to do so when DHT is reduced by 89%. Furthermore, Te concentrations positively determined fasting mean and peak GH concentrations, basal (nonpulsatile) GH secretion, and pulsatile GH secretion driven by GHRP-2 alone and inhibited by SS. E2 concentrations positively predicted baseline (prestimulus) peak GH concentrations and fasting IGF-I concentrations. DHT concentrations correlated negatively with the duration of GHRH-stimulated GH secretory bursts and positively with pulsatile GH secretion during inhibition with SS. Moreover, there was a reciprocal relationship between AVF and DHT concentrations in determining pulsatile GH secretion during SS suppression as well as between Te and E2 concentrations in modulating the GH response to triple-agonist stimulation. Accordingly, exogenous Te’s stimulation of GH secretion in older men requires an increment in E2 but not DHT production. Moreover, systemic Te, E2, and DHT concentrations are individual predictors of selected facets of regulated GH secretion.

Hypothalamo-pituitary aromatization of Te may explain its concentration-dependent potentiation of the GHRP-2 stimulus and relief of GH inhibition by SS. In this regard, E2 supplementation in women mutes suppression of GH release by SS, attenuates GH autofeedback on GHRP stimulation, and amplifies GH responses to GHRH and GHRP (10,26,38,43). Unexpectedly, whereas Te concentrations correlated positively, concomitant E2 concentrations correlated negatively with the GH response to triple stimulation. Inclusion of AVF as a third independent variable in the regression model did not vitiate the reciprocal Te/E2 relationship. Beyond stimulatory actions (1,34,44,45,46), E2 can exert inhibitory effects, viz. induce hypothalamic SS expression, activate the IGF-I receptor, decrease pituitary GH and GHRH-receptor gene expression, inhibit appetitive stimulation by ghrelin, and augment pituitary SS-receptor subtype 2 expression (35,44,46,47,48,49). The E2 dose-response relationships for such effects are not known. A plausible hypothesis is that higher concentrations transduce inhibitory and lower concentrations stimulatory effects of E2 on the GH axis.

Previous studies indicate that nonsteroidal estrogen receptor (ER) and androgen receptor (AR) blockers decrease and increase GH secretion, respectively (18,19,21). However, the ER antagonist tamoxifen exerts intrinsic estrogenic effects and inhibits fasting GH secretion and the GH response to a pharmacological GHRH stimulus in a low-estrogen milieu (50,51). Flutamide elevates endogenous Te and E2 concentrations, one or both of which changes could increase GH secretion (1). Moreover, pharmacological amounts of DHT suppress GH, Te, and E2 concentrations (31,31). In this case, the decline in endogenous Te and E2 may mediate the fall in GH concentrations (1). The present paradigm avoids these limitations by administering highly selective steroidogenic inhibitors with no intrinsic agonist effects along with a fixed dose of Te sufficient to restrict gonadal-axis feedback adjustments.

The inference that systemic sex steroid concentrations and AVF modulate peptide-selective control of pulsatile GH secretion assumes that 1) volunteers were randomly assigned to the interventional groups (done prospectively), 2) the steroidogenic inhibitors used do not exert effects independently of modifying sex steroid concentrations (36,37), and 3) the effects of Te, DHT, and E2 on the GH axis can be approximated as linear functions of their concentrations. Under these assumptions, when AVF was included in regressions, Te remained a strong positive determinant of GHRP- and triple agonist-stimulated, and DHT a significant positive correlate of SS-suppressed, pulsatile GH secretion. In contrast, the positive effect of E2 on baseline peak GH concentrations was eliminated. In addition, AVF alone was a negative predictor of fasting GH concentrations, basal GH secretion, and both GHRH-stimulated and SS-suppressed pulsatile GH secretion. Thus, a suitable model of GH regulation must include tetrapartite interactions among sex steroids, peptidyl secretagogues, SS, and AVF.

DHT stimulates GHRH outflow in laboratory animals (1,2). If the same is true in the human, then elevated GHRH outflow could explain the positive correlation between DHT concentrations and pulsatile GH secretion observed after SS infusion. In addition, DHT and E2 can both induce SS outflow under some conditions (1,2). In the current study, higher DHT concentrations and SS infusion prolonged, whereas Te/Ana treatment and l-arginine/GHRH/GHRP-2 abbreviated, the duration of GH secretory bursts. A parsimonious hypothesis is that DHT, SS, and E2 extend, whereas GHRP reduces, secretory-burst duration. An ER-α antagonist and combined GHRH/GHRP-2 stimulation both shorten GH secretory bursts in women (52). The waveform of secretory bursts provides a window into the somatotroph secretory process, which requires the effectual fusion of secretory vesicles with membrane pores (53).

In principle, whether Te acts via E2 or DHT would reflect relative activities of aromatase and 5α-reductase enzymes and availabilities of ER and AR (1). ER-α is expressed in GHRH neurons and AR in SS neurons (33,54). ER-β is also identifiable in the brain and pituitary gland (55). Whereas the role of DHT metabolites in regulating the human GH axis is unknown, 5α-androstane-3α,17β-diol can activate the ER-β pathway in the corticotropic axis and enhance GH release in some species (56,57). Thus, differences in steroid biotransformation may contribute to earlier conflicting inferences about the effects of Te on GH secretion (see introductory section). For example, seven studies that inferred the capability, unlike one study that described the failure, of Te to stimulate GH secretion in older or hypogonadal men reported an increase in systemic E2 concentrations (20,21,23,28,58,59,60,61).

Caveats include limitations in generalizing inferences from a relatively small cohort of 42 men (840 h of GH sampling), the possibility that selective inhibitors of steroid biotransformation could exert unexpected effects, and the need to explore ultimately the full dose-response range of peptidyl effectors and sex steroid concentrations. Although a schedule of 3 wk of pharmacological Te and inhibitor exposure was adequate to alter GH secretion in older men, how more prolonged physiological changes in Te, DHT, and E2 concentrations regulate GH secretion is not yet known.

In conclusion, individual sex steroid concentrations, type of peptidyl effector, and abdominal visceral adiposity jointly determine basal and pulsatile GH secretion and the waveform of GH secretory bursts in healthy older men, indicating the need for more complex models of GH regulation.

Acknowledgments

We thank Kay Nevinger and 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 (CTSA) Grant Number 1 UL 1 RR024150 to the Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD) and R01 NIA AG29362 and AG19695 from the National Institutes of Health (Bethesda, MD).

Disclosure Statement: Authors have nothing to declare.

First Published Online December 16, 2008

Abbreviations: AR, Androgen receptor; AVF, abdominal visceral fat; DHT, 5α-dihydrotestosterone; E2, estradiol; ER, estrogen receptor; GHRP-2, GH-releasing peptide-2; IGFBP-1, IGF-binding protein-1; Pl/Pl, placebo/placebo; SS, somatostatin; Te, testosterone; Te/Ana, Te/anastrozole; Te/Dut, Te/dutasteride.

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