Age disrupts androgen receptor-modulated negative feedback in the gonadal axis in healthy men.

Testosterone (T) exerts negative feedback on the hypothalamo-pituitary (GnRH-LH) unit, but the relative roles of the CNS and pituitary are not established. We postulated that relatively greater LH responses to flutamide (brain-permeant antiandrogen) than bicalutamide (brain-impermeant antiandrogen) should reflect greater feedback via CNS than pituitary/peripheral androgen receptor-dependent pathways. To this end, 24 healthy men ages 20-73 yr, BMI 21-32 kg/m2, participated in a prospective, placebo-controlled, randomized, double-blind crossover study of the effects of antiandrogen control of pulsatile, basal, and entropic (pattern regularity) measurements of LH secretion. Analysis of covariance revealed that flutamide but not bicalutamide 1) increased pulsatile LH secretion (P = 0.003), 2) potentiated the age-related abbreviation of LH secretory bursts (P = 0.025), 3) suppressed incremental GnRH-induced LH release (P = 0.015), and 4) decreased the regularity of GnRH-stimulated LH release (P = 0.012). Furthermore, the effect of flutamide exceeded that of bicalutamide in 1) raising mean LH (P = 0.002) and T (P = 0.017) concentrations, 2) accelerating LH pulse frequency (P = 0.013), 3) amplifying total (basal plus pulsatile) LH (P = 0.002) and T (P < 0.001) secretion, 4) shortening LH secretory bursts (P = 0.032), and 5) reducing LH secretory regularity (P < 0.001). Both flutamide and bicalutamide elevated basal (nonpulsatile) LH secretion (P < 0.001). These data suggest the hypothesis that topographically selective androgen receptor pathways mediate brain-predominant and pituitary-dependent feedback mechanisms in healthy men.

Pre- versus postmenopausal age, estradiol, and peptide-secretagogue type determine pulsatile growth hormone secretion in healthy women: studies using submaximal agonist drive and an estrogen clamp.

CONTEXT: GH-releasing peptide (GHRP), GHRH, and somatostatin are physiological regulators of pulsatile GH secretion. HYPOTHESIS: Age, independently of abdominal visceral fat (AVF) and basal (nonpulsatile) GH secretion, damps pulsatile GH secretion driven by physiological (rather than pharmacological) amounts of GHRP and GHRH in an experimentally controlled estradiol (E(2)) milieu. DESIGN AND SETTING: A prospectively randomized, double-blind parallel-cohort study was conducted at an academic medical center. PARTICIPANTS: Community-dwelling healthy premenopausal (PRE, age 24 +/- 0.8 yr, n = 20) and postmenopausal (POST, age 63 +/- 1.8 yr, n = 22) women participated in the study. INTERVENTIONS: Gonadal-axis down-regulation with leuprolide was followed by randomized addback of placebo or transdermal E(2) and separate-day iv bolus injections of a half-maximally stimulatory dose of GHRP-2 or GHRH (each 0.33 mug/kg). ANALYSIS: Three-way analysis of covariance included main factors age, E(2) status, and secretagogue type and covariates AVF and basal GH secretion. RESULTS: Submaximally stimulated pulsatile GH secretion was positively determined by PRE vs. POST age (P < 0.001), E(2) repletion vs. depletion (P = 0.001) and GHRP-2 vs. GHRH stimulation (P < 0.001), after adjustment for AVF and basal secretion. E(2) vs. placebo elevated fasting mean GH concentrations in both PRE and POST women (P = 0.006) but increased basal (nonpulsatile) GH secretion in PRE only (P = 0.002). PRE vs. POST age prolonged GHRH-driven GH secretory bursts by 36% (P = 0.006). CONCLUSION: PRE vs. POST age, E(2) availability, and physiological peptide drive are triple determinants of pulsatile GH secretion independently of abdominal visceral fat and nonpulsatile GH secretion in healthy women.

Aromatase and 5alpha-reductase inhibition during an exogenous testosterone clamp unveils selective sex steroid modulation of somatostatin and growth hormone secretagogue actions in healthy older men.

BACKGROUND: How endogenous testosterone (Te), 5alpha-dihydrotestosterone (DHT), and estradiol (E(2)) regulate pulsatile GH secretion is not understood. HYPOTHESIS: Conversion of Te to androgenic (Te-->DHT) or estrogenic (Te-->E(2)) 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 5alpha-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 E(2) concentrations by 1.9-fold above placebo/placebo. Te/dutasteride and Te/anastrozole reduced stimulated DHT and E(2) 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 E(2) jointly predict GH responses to the triple stimulus (positively for Te, P = 0.006, and negatively for E(2), 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 E(2) along with abdominal visceral fat determine the selective actions of GH secretagogues and SS.

Estrogen elevates the peak overnight production rate of acylated ghrelin.

CONTEXT: Acylated ghrelin is the putatively bioactive GH secretagogue. HYPOTHESIS: Estradiol (E2) stimulates the synthesis rather than inhibits the metabolic clearance of acylated ghrelin. SETTING: The study took place at an academic medical center. SUBJECTS: Healthy postmenopausal women participated. INTERVENTIONS: Interventions included prospectively randomized, double-blind separate-day iv infusions of saline or five graded doses of ghrelin in estrogen-deficient (n=12) and E2-supplemented (n=8) women. OUTCOMES: Metabolic clearance rate (MCR), volume of distribution, half-life, and secretion rate of acylated ghrelin were assessed. RESULTS: In pilot iv bolus ghrelin infusions, the median half-lives of acylated and total ghrelin were 21 and 36 min (P<0.01), MCRs 58 and 8.1 liters/kg.d (P<0.01), and volumes of distribution of 1.0 and 0.32 liters/kg (P<0.01), respectively. Transdermal E2 supplementation for 3 wk increased peak nighttime acylated ghrelin concentrations from 99+/-12 to 141+/-34 pg/ml (P=0.039). Exposure to E2 did not alter the linear relationships between 1) plasma acylated ghrelin concentration and ghrelin infusion rate (638+/-12 slope units), 2) MCR of acylated ghrelin and ghrelin infusion rate (10+/-2.5 slope units), and 3) MCR and plasma concentration of acylated ghrelin (0.017+/-0.004 slope units). These data predict peak nighttime production rates of acylated ghrelin of 3.8+/-0.9 (E2) and 1.9+/-0.2 (no E2) ng/kg.min (P=0.039). CONCLUSION: Acylated ghrelin has a multifold larger distribution volume and MCR than total ghrelin. An estrogenic milieu augments synthesis and/or acylation of ghrelin peptide without altering its MCR.

Estrogen supplementation selectively enhances hypothalamo-pituitary sensitivity to ghrelin in postmenopausal women.

CONTEXT: Sex-steroid hormones amplify pulsatile GH secretion by unknown mechanisms. Ghrelin is the most potent natural GH secretagogue discovered to date. A plausible unifying postulate is that estradiol (E(2)) enhances hypothalamo-pituitary sensitivity to ghrelin (a physiological effect). The hypothesis is relevant to understanding the basis of hyposomatotropism in aging and other relatively hypogonadal states. OBJECTIVE: Our objective was to test the hypothesis that E(2) supplementation potentiates ghrelin's stimulation of pulsatile GH secretion. SETTING: The study was conducted at an academic medical center. SUBJECTS: Healthy postmenopausal women (n = 20) were included in the study. INTERVENTIONS: Separate-day iv infusions of saline vs. five graded doses of ghrelin were performed in volunteers prospectively randomly assigned to receive (n = 8) or not receive (n = 12) transdermal E(2) for 21 d were performed. MEASURES: GH secretion was estimated by deconvolution analysis and abdominal visceral fat mass determined by computerized axial tomography were calculated. RESULTS: E(2) supplementation augmented ghrelin's stimulation of basal (nonpulsatile) GH secretion by 3.6-fold (P = 0.022), increased GH responses to low-dose ghrelin by 2.9-fold (P = 0.035), did not alter ghrelin efficacy, and elicited more regular patterns of acylated ghrelin concentrations during saline infusion (P = 0.033). Abdominal visceral fat negatively determined responses to ghrelin (R = -0.346; P < 0.005). CONCLUSIONS: Transdermal E(2) supplementation potentiates GH secretion stimulated by physiological but not pharmacological concentrations of acylated ghrelin, and concomitantly regularizes patterns of bioactive ghrelin secretion in postmenopausal women. Accordingly, the estrogen milieu appears to control sensitivity of the hypothalamopituitary unit to acylated ghrelin.

Gonadal status and body mass index jointly determine growth hormone (GH)-releasing hormone/GH-releasing peptide synergy in healthy men.

CONTEXT: Sex steroid hormones potentiate whereas increased body mass index (BMI) represses GH secretion. Whether sex steroids modify the negative effect of BMI on secretagogue-induced GH secretion in men is not known. The issue is important in designing GH-stimulation regimens that are relatively insensitive to both gonadal status and adiposity. OBJECTIVE: Our objective was to compare the relationships between BMI and peptide-stimulated GH secretion in men with normal and reduced testosterone and estradiol availability. SETTING: The study was performed at an academic medical center. SUBJECTS: Healthy young men were included in the study. INTERVENTIONS: Randomized separate-day iv infusion of saline and/or maximally effective doses of L-arginine/GHRH, L-arginine/GH-releasing peptide (GHRP)-2, and GHRH/GHRP-2 in eugonadal (n=12) and experimentally hypogonadal (n=10) men was performed. OUTCOMES: Regression of paired secretagogue-induced GH responses on BMI was determined. RESULTS: In eugonadal men, peak GH concentrations correlated negatively with BMI. In particular, BMI accounted for only 38% of the response variability after L-arginine/GHRH (P=0.0165), but 62% after GHRH/GHRP-2 (P=0.0012) and 65% after L-arginine/GHRP-2 (P=0.00075). In contrast, in hypogonadal men, GH responses were uncorrelated with BMI. The negative effects of BMI on peak GH responses in eugonadal and hypogonadal states differed most markedly after stimulation with GHRH/GHRP-2 (P=0.0019). This contrast was corroborated using integrated GH responses (P=0.0007). CONCLUSIONS: Short-term experimental gonadal sex hormone depletion attenuates dual secretagogue-stimulated GH secretion in lean young men. The inhibitory effect of relative adiposity on GH secretion appears to predominate over that of acute sex steroid withdrawal.

Short-term testosterone supplementation does not activate GH and IGF-I production in postmenopausal women.

BACKGROUND: Testosterone (Te), oestradiol, GH and IGF-I concentrations fall in postmenopausal women. OBJECTIVE: To test the effect of increased Te availability on impoverished GH/IGF-I secretion in this context. DESIGN/PATIENTS: Placebo (Pl) and a low vs. high dose of Te were administered transdermally for 7 days to each of 15 healthy older women (ages 48-81 years) in a randomized, double-blind crossover design. MEASUREMENTS: Frequent blood sampling, GHRP-2 (Growth hormone releasing peptide-2) infusion, and deconvolution analysis were used to assess GH secretion. RESULTS: Graded Te supplementation increased serum Te concentrations (nmol/l) from 0.87 +/- 0.06 [Pl] to 5 +/- 1 [low] and 7.3 +/- 1.6 [high] (P < 0.001), but did not affect: (i) pulsatile GH secretion (microg/l/12 h; conversion factor: 1 microg/l = 0.33 mU/l), 29 +/- 7.2, 32 +/- 5.6 and 27 +/- 4.9; (ii) GH regularity (approximate entropy), 0.52 +/- 0.05, 0.57 +/- 0.05 and 0.56 +/- 0.05; (iii) IGF-I concentrations (microg/l; conversion factor: 1 microg/l = 0.131 nmol/l), 126 +/- 13, 135 +/- 15 and 141 +/- 13; (iv) IGFBP-3 (mg/l), 3.7 +/- 0.2, 3.6 +/- 0.1 and 3.7 +/- 0.2; (v) IGFBP-1 (microg/l), 41 +/- 2.3, 44 +/- 3.8 and 44 +/- 3.2; and (vi) the mass of GH secreted (microg/l/3 h) after GHRP-2 injection, 123 +/- 27, 146 +/- 32 and 147 +/- 38. CONCLUSION: Up to 8-fold elevation of Te concentrations for 1 week in postmenopausal women does not detectably amplify GH secretion or action, thus indicating that low androgen availability is not a proximate acute mediator of hyposomatotropism in postmenopausal individuals.

Complementary secretagogue pairs unmask prominent gender-related contrasts in mechanisms of growth hormone pulse renewal in young adults.

The present study examines the thesis that pulsatile GH secretion is controlled simultaneously by three principal signals; viz., GHRH, GH-releasing peptide (GHRP, ghrelin), and somatostatin (SS). According to this ensemble notion, no single regulatory peptide acts alone or can be interpreted in isolation. Therefore, to investigate gender-specific control of pulsatile GH secretion, we designed dual-effector stimulation paradigms in eight young men and six women as follows: 1) L-arginine/GHRH (to clamp low SS and high GHRH input); 2) L-arginine/GHRP-2 (to clamp low SS and high GHRP drive); 3) GHRH/GHRP-2 (to clamp high GHRH and high GHRP feedforward); vs. 4) saline (unclamped). Statistical comparisons revealed that: 1) fasting pulsatile GH secretion was 7.6-fold higher in women than men (P < 0.001); 2) L-arginine/GHRH and L-arginine/GHRP-2 evoked, respectively, 4.6- and 2.2-fold greater burst-like GH release in women than men (P < 0.001 and P = 0.015); and 3) GHRH/GHRP-2 elicited comparable GH secretion by gender. In the combined cohorts, estradiol concentrations positively predicted responses to L-arginine/GHRP-2 (r2= 0.49, P = 0.005), whereas testosterone negatively predicted those to L-arginine/GHRH (r2= 0.56, P = 0.002). Based upon a simplified biomathematical model of three-peptide control, the current outcomes suggest that women maintain greater GHRH potency, GHRP efficacy, and opposing SS outflow than men. This inference upholds recent clinical precedence and yields valid predictions of sex differences in self-renewable GH pulsatility.

A randomized placebo-controlled trial of short-term graded transdermal estradiol in healthy gonadotropin-releasing hormone agonist-suppressed pre- and postmenopausal women: effects on serum markers of bone turnover, insulin-like growth factor-I, and osteoclastogenic mediators.

The acute effects of estradiol on procollagen type 1 formation in pre- and postmenopausal women are controversial. Twenty-three premenopausal women and 13 postmenopausal women received two consecutive im injections of 3.75 mg leuprolide acetate 3 wk apart to block endogenous ovarian steroidogenesis. Transdermal estradiol therapy commenced on the night of the second leuprolide injection in all subjects, except five pre- and two postmenopausal women who were randomized to receive placebo patches. Estradiol therapy was applied incrementally, with each dose of 0.05, 0.10, 0.15, and 0.20 mg/d administered for 4 consecutive days, to mimic the estradiol changes typifying the follicular phase of the menstrual cycle. Blood aminoterminal propeptide of type I procollagen (PINP), intact osteocalcin (OC), carboxyterminal telopeptide of type I collagen (CTx), IGF-I, and estradiol were measured before and at the end of each estradiol increment. Potential mediators such as osteoprotegerin and receptor activator of nuclear factor-kappaB ligand (RANKL) were also measured. Despite comparable increases in serum estradiol, PINP increased more in postmenopausal compared with premenopausal women (between-group P = 0.03) and occurred at a time when CTx and OC did not change. CTx and IGF-I changed minimally and inconsistently, whereas OC, RANKL, and osteoprotegerin were stable. Repeated measures linear regression disclosed a significant negative association between increases in estradiol and PINP in premenopausal women (P = 0.0006) only. This suggests that lower dose estradiol should greatly increase PINP. Analogous regressions also showed significant negative relationships between changes in estradiol and RANKL in both pre- (P = 0.04) and postmenopausal (P = 0.002) women. Changes in serum markers of bone formation (PINP or OC) did not correlate with those of IGF-I. We conclude that lower dose estradiol rapidly increases osteoblastic collagen synthesis in women at a time when collagen degradation is stable and that this response differs between pre- and postmenopausal women. The effect of estradiol on bone formation does not appear to be mediated by IGF-I. In contrast, RANKL is likely to mediate the effect of estradiol on osteoclastogenesis.