Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Human GH pulsatility: an ensemble property regulated by age and gender.

Age and gender impact the full repertoire of neurohormone systems, including most prominently the somatotropic, gonadotropic and lactotropic axes. For example, daily GH production is approximately 2-fold higher in young women than men and varies by 20-fold by sexual developmental status and age. Deconvolution estimates of 24-h GH secretion rates exceed 1200 microg/m2 in adolescents and fall below 60 microg/m2 in aged individuals. The present overview highlights plausible factors driving such lifetime variations in GH availability, i.e., estrogen, aromatizable androgen, hypothalamic peptides and negative feedback by GH and IGF-I. In view of the daunting complexity of potential neuromodulatory signals, we underline the utility of conceptualizing a simplified three-peptide regulatory ensemble of GHRH, GHRP (ghrelin) and somatostatin. The foregoing signals act as individual and conjoint mediators of adaptive GH control. Regulation is enforced at 3-fold complementary time scales, which embrace pulsatile (burst-like), entropic (orderly) and 24-h rhythmic (nycthemeral) modes of GH release. This unifying platform offers a convergent perspective of multivalent control of GH outflow.

Accelerated escape from GH autonegative feedback in midpuberty in males: evidence for time-delimited GH-induced somatostatinergic outflow in adolescent boys.

A single injected pulse of GH inhibits the time-delayed secretion of GH in the adult by way of central mechanisms that drive somatostatin and repress GHRH outflow. The marked amplification of spontaneous GH pulse amplitude in puberty poses an autoregulatory paradox. We postulated that this disparity might reflect unique relief of GH-induced autonegative feedback during this window of development. The present study contrasts GH autonegative feedback in: 1) normal prepubertal boys (PP) (n = 6; Tanner genital stage I, chronologically aged 8 yr, 9 months to 10 yr, 1 month; median bone age 8.5 yr); 2) longitudinally identified midpubertal boys (MP) (n = 6; Tanner genital stages III/IV, aged 12 yr, 6 months to 15 yr, 6 months; median bone age 15 yr); and 3) healthy young men (YM) (n = 6, aged 18-24 yr; bone age >18 yr). Subjects each underwent four randomly ordered tandem peptide infusions on separate mornings while fasting: i.e. 1) saline/saline infused iv bolus at 0830 h and 1030 h; 2) saline/GHRH (0.3 microg/kg i.v. bolus) at the foregoing times; 3) recombinant human (rh) GH (3 microg/kg as a 6-min square-wave i.v. pulse)/saline; and 4) rhGH and GHRH. To monitor GH autofeedback effects, blood samples were obtained every 10 min for 5.5 h beginning at 0800 h (30 min before GH or saline infusion). Serum GH concentrations were quantitated by ultrasensitive chemiluminometry (threshold 0.005 microg/liter). On the day of successive saline/saline infusion, MP boys maintained higher serum concentrations of: 1) GH ( microg/liter), 2.2 +/- 0.25, compared with PP (0.61 +/- 0.10) or YM (0.88 +/- 0.36) (P = 0.011); 2) IGF-I ( micro g/liter), 493 +/- 49 vs. PP (134 +/- 16) and YM (242 +/- 22) (P < 0.001); 3) T (ng/dl), 524 +/- 58 vs. PP (<20) (P < 0.001); and 4) E2 (pg/ml),19 +/- 3 vs. PP (< 10) (P = 0.030) (mean +/- SEM). Consecutive saline/GHRH infusion elicited comparable peak (absolute maximal) serum GH concentrations (micrograms per liter) in the three study groups, i.e. 18 +/- 5.0 (PP), 9.6 +/- 1.7 (MP), and 14 +/- 5.3 (YM) (each P < 0.01 vs. saline; P = NS cohort effect). Injection of rhGH attenuated subsequent GHRH-stimulated peak serum GH concentrations (micrograms per liter) to 7.8 +/- 1.9 (PP), 5.8 +/- 1.2 (MP), and 4.8 +/- 1.1 (YM) (each P < 0.01 vs. saline; P = NS pubertal effect). GH autofeedback reduced non-GHRH-stimulated (basal) serum GH concentrations by 0.74 +/- 0.28 (PP), 5.7 +/- 1.7 (MP) and 1.4 +/- 0.27 (YM) fold, compared with saline (P = 0.016 for MP vs. PP or YM). In addition to greater fractional autoinhibition, MP boys exhibited markedly accentuated postnadir escape (4.6-fold steeper slope) of suppressed GH concentrations (P < 0.001 vs. PP or YM). Linear regression analysis of data from all 18 subjects revealed that the fasting IGF-I concentration negatively predicted fold-autoinhibition of GHRH-stimulated peak GH release (r = -0.847, P = 0.006) and positively forecast fold-autoinhibition of basal GH release (r = +0.869, P < 0.001). In contrast, the kinetics of rhGH did not differ among the three study cohorts. In summary, boys in midpuberty manifest equivalent responsiveness to exogenous GHRH-stimulated GH secretion; heightened susceptibility to rhGH-induced fractional inhibition of endogenous secretagogue-driven GH release, compared with the prepubertal or adult male; and accelerated recovery of GH output after acute autonegative feedback. This novel tripartite mechanism could engender recurrent high-amplitude GH secretory bursts that mark sex hormone-dependent activation of the human somatotropic axis.

Mechanisms of conjoint failure of the somatotropic and gonadal axes in ageing men.

Endogenous growth hormone (GH) production falls by 50% every 7 years and bioavailable testosterone concentrations decline concomitantly by 12-15% every decade in ageing men. Despite this temporal parallelism, the neuroendocrine bases of the somatopause and gonadopause are not known. This knowledge deficit contrasts with the recent unfolding of new insights into the nature of oestrogen-dependent control of the GH-insulin-like growth factor (IGF)1 axis in pre- and postmenopausal women. The present overview examines the postulate that the pathophysiology of somatopause and gonadopause in ageing men is bidirectionally linked. According to this broader thesis, hyposomatotropism accentuates Leydig cell steroidogenic failure and, conversely, progressive androgen deficiency exacerbates the decline in GH-IGF1 output in ageing.

Impact of estradiol supplementation on dual peptidyl drive of GH secretion in postmenopausal women.

As an indirect probe of estrogen-regulated hypothalamic somatostatin restraint, the present study monitors the ability of short-term oral E2 supplementation to modulate GH secretion during combined continuous stimulation by recombinant human GHRH [GHRH-(1-44)-amide] and the potent and selective synthetic GH-releasing peptide, GHRP-2. According to a simplified tripeptidyl model of GH neuroregulation, the effects of estrogen in this dual secretagogue paradigm should mirror alterations in endogenous somatostatinergic signaling. To this end, seven healthy postmenopausal women underwent frequent (10-min) blood sampling for 24 h during simultaneous i.v. infusion of GHRH and GHRP-2 each at a rate of 1 microg/kg x h on d 10 of randomly ordered placebo or 17beta-estradiol (E2) (1 mg orally twice daily) replacement. Serum GH concentrations (n = 280/subject) were assayed by chemiluminescence. The resultant GH time series was evaluated by deconvolution analysis, the approximate entropy statistic, and cosine regression to quantitate pulsatile, entropic (feedback-sensitive), and 24-h rhythmic GH release, respectively. Statistical comparisons revealed that E2 repletion increased the mean (+/- SEM) serum E2 concentration to 222 +/- 26 pg/ml from 16 +/- 1.7 pg/ml during placebo (P < 0.001) and suppressed the serum LH by 48% (P = 0.0033), serum FSH by 64% (P < 0.001), and serum IGF-I by 44% (P = 0.021). Double peptidyl secretagogue stimulation elevated mean 24-h serum GH concentrations to 8.1 +/- 1.0 microg/liter (placebo) and 7.7 +/- 0.89 microg/liter (E2; P = NS) and evoked prominently pulsatile patterns of GH secretion. No primary measure of pulsatile or basal GH release was altered by the disparate sex steroid milieu, i.e. GH secretory burst amplitudes of 0.62 +/- 0.93 (placebo) and 0.72 +/- 0.16 (E2) microg/liter x min, GH pulse frequencies of 27 +/- 1.8 (placebo) and 23 +/- 1.9 (E2) events/24 h, GH half-lives of 12 +/- 0.74 (placebo) and 15 +/- 4.5 (E2) min, and basal (nonpulsatile) GH secretion 70 +/- 22 (placebo) and 57 +/- 18 (E2) ng/liter x min. The approximate entropy (ApEn) of serial GH release [1.297 +/- 0.061 (placebo) and 1.323 +/- 0.06 (E2)] and the mesor (cosine mean), amplitude, and acrophase (time of the maximum) of 24-h rhythmic GH secretion were likewise invariant of estrogen supplementation. Estimated statistical power exceeded 90% for detecting significant (P < 0.05) within-subject changes exceeding 30-50% in the mean serum GH concentration, GH ApEn, or GH mesor. In contrast, ApEn analysis of the evolution of successive GH secretory burst-mass values over 24 h disclosed that E2 replacement disrupts the serial regularity of pulsatile GH output (elevates the ApEn ratio) during combined GHRH/GHRP-2 stimulation (P = 0.004). In summary, short-term elevation of serum E2 concentrations in postmenopausal individuals into the midfollicular phase range observed in young women does not significantly alter 24-h basal, pulsatile, entropic, or nyctohemeral GH secretion monitored under continuous combined drive by GHRH and GHRP-2. As E2 repletion without enforced GHRH/GHRP-2 stimulation augments each of the foregoing regulated facets of GH release, we infer that one or both of the infused peptidyl secretagogues may itself participate in E2's short-term amplification of GH secretion in postmenopausal individuals. Estrogen's disruption of the orderliness of sequential GH pulse-mass values during fixed GHRH/GHRP-2 feedforward would be consistent with a subtle reduction in the release and/or actions of hypothalamic somatostatin or an (unexpected) direct pituitary action of the sex steroid. Whether comparable dynamics mediate the effects of endogenous estrogen on the GH axis in premenopausal women or pubertal girls is not known.

E2 supplementation selectively relieves GH's autonegative feedback on GH-releasing peptide-2-stimulated GH secretion.

Female gender confers resistance to GH autonegative feedback in the adult rat, thereby suggesting gonadal or estrogenic modulation of autoregulation of the somatotropic axis. Here we test the clinical hypothesis that short-term E2 replacement in ovariprival women reduces GH's repression of spontaneous, GHRH-, and GH-releasing peptide (GHRP)-stimulated GH secretion. To this end, we appraised GH autoinhibition in nine healthy postmenopausal volunteers during a prospective, randomly ordered supplementation with placebo vs. E [1 mg micronized 17 beta-E2 orally twice daily for 6-23 d]. The GH autofeedback paradigm consisted of a 6-min pulsed i.v. infusion of recombinant human GH (10 microg/kg square-wave injection) or saline (control) followed by i.v. bolus GHRH (1 microg/kg), GHRP-2 (1 microg/kg), or saline 2 h later. Blood was sampled every 10 min and serum GH concentrations were measured by chemiluminescence. Poststimulus GH release was quantitated by multiparameter deconvolution analysis using published biexponential kinetics and by the incremental peak serum GH concentration response (maximal poststimulus value minus prepeak nadir). Outcomes were analyzed on the logarithmic scale by mixed-effects ANOVA at a multiple-comparison type I error rate of 0.05. E2 supplementation increased the (mean +/- SEM) serum E2 concentration from 43 +/- 1.8 (control) to 121 +/- 4 pg/ml (E2) (158 +/- 6.6 to 440 +/- 15 pmol/liter; P < 0.001), lowered the 0800 h (preinfusion) serum IGF-I concentration from 127 +/- 7.7 to 73 +/- 3.6 microg/liter (P < 0.01), and amplified spontaneous pulsatile GH production from 7.5 +/- 1.1 to 13 +/- 2.3 microg/liter per 6 h (P = 0.020). In the absence of exogenously imposed GH autofeedback, E2 replacement enhanced the stimulatory effect of GHRP-2 on incremental peak GH release by 1.58-fold [95% confidence interval, 1.2- to 2.1-fold] (P = 0.0034) but did not alter the action of GHRH (0.83-fold [0.62- to 1.1-fold]). In the E2-deficient state, bolus GH infusion significantly inhibited subsequent spontaneous, GHRH-, and GHRP-induced incremental peak GH responses by, respectively, 33% (1-55%; P = 0.044 vs. saline), 79% (68-86%; P < 0.0001), and 54% (32-69%; P = 0.0002). E2 repletion failed to influence GH autofeedback on either spontaneous or GHRH-stimulated incremental peak GH output. In contrast, E2 replenishment augmented the GHRP-2-stimulated incremental peak GH response in the face of GH autoinhibition by 1.7-fold (1.2- to 2.5-fold; P = 0.009). Mechanistically, the latter effect of E2 mirrored its enhancement of GH-repressed/GHRP-2-stimulated GH secretory pulse mass, which rose by 1.5-fold (0.95- to 2.5-fold over placebo; P = 0.078). In summary, the present clinical investigation documents the ability of short-term oral E2 supplementation in postmenopausal women to selectively rescue GHRP-2 (but not spontaneous or GHRH)-stimulated GH secretion from autonegative feedback. The secretagogue specificity of E's relief of GH autoinhibition suggests that this sex steroid may enhance activity of the hypothalamopituitary GHRP-receptor/effector pathway.

Growth hormone-releasing peptide-2 stimulates GH secretion in GH-deficient patients with mutated GH-releasing hormone receptor.

GH-releasing peptides (GHRPs) are synthetic peptides that bind to specific receptors and thereby stimulate the secretion of pituitary GH. In vivo it is uncertain whether these peptides act directly on somatotroph cells or indirectly via release of GHRH from the hypothalamus. In this study we compared the pituitary hormone response to GHRP-2 in 11 individuals with isolated GH deficiency (GHD) due to a homozygous mutation of the GHRH receptor (GHRH-R) gene and in 8 normal unrelated controls. Basal serum GH levels were lower in the GHD group compared with controls [0.11 +/- 0.11 (range, <0.04 to 0.38) vs. 0.59 +/- 0.76 microg/L (range, 0.04-2.12 microg/L); P = 0.052]. After GHRP-2 administration there was a 4.5-fold increase in serum GH relative to baseline values in the GHD group (0.49 +/- 0.41 vs. 0.11 +/- 0.11 microg/L; P = 0.002), which was significantly less than the 79-fold increase in the control group (46.8 +/- 17.6 vs. 0.59 +/- 0.76 microg/L; P = 0.008). Basal and post-GHRP-2 serum levels of ACTH, cortisol, and PRL were similar in both groups. Basal levels of serum TSH were significantly higher in the GHD group than in the control group (3.23 +/- 2.21 vs. 1.37 +/- 0.34 microIU/mL; P = 0.003). TSH levels in both groups did not change after GHRP-2 administration. These results suggest that an intact GHRH signaling system is not an absolute requirement for GHRP-2 action on GH secretion and that GHRP-2 has a GHRH-independent effect on pituitary somatotroph cells.

Interactions of growth hormone secretagogues and growth hormone-releasing hormone/somatostatin.

The class of novel synthetic compounds termed growth hormone secretagogues (GHSs) act in the hypothalamus through, as yet, unknown pathways. We performed physiologic and histochemical studies to further understand how the GHS system interacts with the well-established somatostatin (SRIF)/growth hormone-releasing hormone (GHRH) neuroendocrine system for regulating pulsatile GH secretion. Comparison of the GH-releasing activities of the hexapeptide growth hormone-releasing peptide-6 (GHRP-6) and GHRH administered intravenously to conscious adult male rats showed that the pattern of GH responsiveness to GHRP-6 was markedly time-dependent, similar to that observed with GHRH. Immunoneutralization of endogenous SRIF reversed the blunted GH response to GHRP-6 at trough times, suggesting that GHRP-6 neither disrupts nor inhibits the cyclical release of endogenous hypothalamic SRIF. By striking contrast, passive immunization with anti-GHRH serum virtually obliterated the GH responses to GHRP-6, irrespective of the time of administration. These findings suggest that the GHSs do not act by altering SRIF release but, rather, stimulate GH release via GHRH-dependent pathways. Our dual chromogenic and autoradiographic in situ hybridization experiments revealed that a subpopulation of GHRH mRNA-containing neurons in the arcuate (Arc) nucleus and ventromedial nucleus (VMN) of the hypothalamus expressed the GHS receptor (GHS-R) gene. These results provide strong anatomic evidence that GHSs may directly stimulate GHRH release into hypophyseal portal blood, and thereby influence GH secretion, through interaction with the GHS-R on GHRH- containing neurons. Altogether, these findings support the notion that an additional neuroendocrine pathway may exist to regulate pulsatile GH secretion, possibly through the influence of the newly discovered GHS natural peptide, ghrelin.

Interactive regulation of postmenopausal growth hormone insulin-like growth factor axis by estrogen and growth hormone-releasing peptide-2.

Estrogen is the proximate sex steroid sustaining GH secretion throughout the human life span in both sexes. However, very little is known about the specific neuroendocrine mechanisms by which estrogen activates and maintains GH secretion in the young or aging human. The identification of somatostatin in 1973 as a key negative peptidyl regulator of the GH axis and the discovery of GH-releasing hormone (GHRH) in 1982 as a dominant feedforward agonist of GH secretion provided an initial basic science foundation for exploring sex-steroid control of the GH-IGF-1 axis. Although GH-releasing peptides (GHRPs) were first recognized in 1977-1981, subsequent cloning of hypothalamopituitary receptors transducing potent secretagogue actions of GHRPs in 1996 and of an endogenous ligand for this effector pathway in 1999 now extend the framework for examining the mechanisms of estrogen-driven GH secretion in aging. Herein, we review several novel and multifaceted interactions in postmenopausal women between estrogen and GHRP-2. We combine these observations into a simplified construct of GH-axis neuroregulation comprising the somatostatin, GHRH, and GHRP effector pathways, as well as GH and IGF-1 autofeedback. We suggest the thesis that estrogen controls the interfaces among these pivotal regulatory peptides in hyposomatotropic postmenopausal individuals.

Growth hormone/insulin-like growth factor-1 response to acute and chronic growth hormone-releasing peptide-2, growth hormone-releasing hormone 1-44NH2 and in combination in older men and women with decreased growth hormone secretion.

To better appreciate the interactions of GHRP-2 and GHRH 1-44NH2 on the release of GH in normal adult men and women with decreased GH secretion and low serum IGF-1 levels, a series of acute and chronic studies have been performed (n = 5 men, 5 women). The acute iv bolus GH responses of these subjects to the two peptides alone and together suggest that the decreased GH secretion may be primarily due to a deficiency of the natural endogenous GHRP, ghrelin, rather than a decreased secretion of endogenous GHRH or excess secretion of SRIF. To determine whether the low GH response to GHRH was due to a limited capacity of pituitary to release GH, higher dosages of GHRP-2 alone were administered. At a dose of 1 microg/kg GHRP-2 the GH response was essentially the same as that elicited by 1 microg/kg GHRH + 0.1 microg/kg GHRP-2 while the GH response to 10 microg/kg GHRP-2 sc was about twice as high in both men and women. Although these subjects have a limited pituitary capacity to release GH, which is also an indication of decreased GH secretion in the presence of low serum IGF-1 levels, this alone would not explain the low GH response to GHRH. Furthermore, the finding that a low dose of 0.1 microg/kg GHRP-2 augments the GH response to 1 microg/kg GHRH is strongly against an excess secretion of SRIF. Twenty-four hour profiles of GH secretion during placebo, GHRP-2, and various doses of GHRH alone and together with GHRP-2 were studied. In addition, 1 microg/kg/h GHRP-2 was infused continuously sc to these subjects for 30 d. The normal pulsatile secretion of GH as well as the serum IGF-1 level was increased after 24 h and remained elevated for 30 d. With a deficiency of endogenous GHRH, the GH response of GHRP-2 would be little to none, while in subjects with a deficiency of the natural GHRP, the GH response to GHRH would be more attenuated. Thus, in chronic deficiency the GH response would be expected to depend on the degree of the capacity of the pituitary to release GH as well as the type(s) of hormonal deficiency.

Short-term estradiol supplementation augments growth hormone (GH) secretory responsiveness to dose-varying GH-releasing peptide infusions in healthy postmenopausal women.

Estrogen is a prominent stimulus to GH secretion throughout the human life span, albeit via neuroendocrine mechanisms that are incompletely defined. Here, we test the hypothesis that estradiol replacement in postmenopausal women enhances the responsiveness of the hypothalamo-pituitary unit to the GH-releasing effect of GH-releasing peptide-2 (GHRP-2). GHRP-2 is a potent and selective synthetic hexapeptide capable of activating an endogenous GHRP receptor/effector pathway, for which a (3)Ser-octanoylated 28-amino acid ligand was cloned recently. To examine this postulate, we studied 10 healthy estrogen-withdrawn postmenopausal women, who were given oral placebo or estrogen supplementation [1 mg micronized 17 beta-estradiol (E(2)) twice daily for 7-15 days] in a patient-blinded, prospective, randomized, and within-subject cross-over design. The GH-releasing actions of five semilogarithmically increasing doses of GHRP-2 (absolute range, 0.03-3 microg/kg by bolus iv infusion) vs. saline were evaluated by frequent blood sampling on separate days in the morning while fasting. Serum GH concentrations were determined in blood sampled every 10 min using an ultrasensitive chemiluminescence assay and analyzed by multiparameter deconvolution to calculate the summed mass of GH secreted during the 2-h interval after bolus GHRP-2 infusion. Logarithmically transformed secretory responses were compared across the different dosages of infused GHRP-2 by two-way repeated measures ANOVA. Estradiol replacement increased the global mean (+/-SEM) serum E(2) concentration from 15 +/- 0.8 to 470 +/- 17 pg/mL (55 +/- 2.9 to 1725 +/- 62 pmol/L; P = 0.004) and lowered insulin-like growth factor I levels by approximately 27% (P = 0.087). Administration of E(2) elevated the geometric mean basal (saline-infused) GH secretory burst mass by 2.1-fold (95% confidence interval, 1.4- to 3.1-fold) compared with placebo ingestion (geometric mean ratios; P < 0.001). E(2) exposure enhanced the efficacy of the highest GHRP-2 dose tested (3 microg/kg) by 2.1-fold (1.3- to 3.3-fold; P = 0.010). Compared with the effect of placebo and saline, E(2) combined with the highest dose of GHRP-2 stimulated GH secretory burst mass by a total of 31-fold (24- to 41-fold; P < 0.001). Random coefficient regression analysis of the relationship between the logarithm of GHRP-2 dose and GH secretory burst mass revealed that E(2) significantly augmented the amount of GH secreted per unit GHRP-2 dose (E(2), 16.6 +/- 1.8 slope units; placebo, 10.1 +/- 1.4 slope units; P = 0.03). Although the global mean endogenous GH half-life did not differ between the E(2) and placebo sessions (E(2), 18 +/- 0.6 min; placebo, 17 +/- 0.5 min), GH half-life varied directly with dose of GHRP-2 (and, hence, the mean serum GH concentration) in both the E(2) and placebo sessions (test of zero slope hypothesis, P = 0.0018). The deconvolved GH secretory burst peaked within 8-13 min of the bolus iv injection of GHRP-2, and this latency was not altered by E(2). Based on a mixed effects analysis of covariance model, GHRP-2 dose and E(2), but not the plasma insulin-like growth factor I concentration, determined the magnitude of the GH secretory response (P < 0.001). We conclude that short-term oral E(2) repletion in postmenopausal women selectively augments GH secretory pulse mass, enhances the steepness of the GHRP-2 dose-GH secretory response relationship (greater sensitivity), and heightens the maximal GH secretory response to the highest dose of GHRP-2 tested (greater efficacy). These data point to a facilitative interaction between E(2) and the GHRP receptor/effector pathway in driving the mass of GH secreted per burst.

Continuous 24-hour intravenous infusion of recombinant human growth hormone (GH)-releasing hormone-(1-44)-amide augments pulsatile, entropic, and daily rhythmic GH secretion in postmenopausal women equally in the estrogen-withdrawn and estrogen-supplemented states.

How estrogen amplifies GH secretion in the human is not known. The present study tests the clinical hypothesis that estradiol modulates the stimulatory actions of a primary GH feedforward signal, GHRH. To this end, we investigated the ability of short-term (7- to 12-day) supplementation with oral estradiol vs. placebo to modulate basal, pulsatile, entropic, and 24-h rhythmic GH secretion driven by a continuous iv infusion of recombinant human GHRH-(1--44)-amide vs. saline in nine healthy postmenopausal women. Volunteers underwent concurrent blood sampling every 10 min for 24 h on four occasions in a prospectively randomized, single blind, within-subject cross-over design (placebo/saline, placebo/GHRH, estradiol/saline, estradiol/GHRH). Intensively sampled serum GH concentrations were quantitated by ultrasensitive chemiluminescence assay. Basal, pulsatile, entropic (feedback-sensitive), and 24-h rhythmic modes of GH secretion were appraised by deconvolution analysis, the approximate entropy (ApEn) statistic, and cosine regression, respectively. ANOVA revealed that continuous iv infusion of GHRH in the estrogen-withdrawn (control) milieu 1) amplified individual basal (P = 0.00011) and pulsatile (P < 10(-13)) GH secretion rates by 12- and 11-fold, respectively; 2) augmented GH secretory burst mass and amplitude each by 10-fold (P < 10(-11)), without altering GH secretory burst frequency, duration, or half-life; 3) increased the disorderliness (ApEn) of GH release patterns (P = 0.0000002); 4) elevated the mesor (cosine mean) and amplitude of the 24-h rhythm in serum GH concentrations by nearly 30-fold (both P < 10(-12)); 5) induced a phase advance in the clocktime of the GH zenith (P = 0.021); and 6) evoked a new 24-h rhythm in GH secretory burst mass with a maximum at 0018 h GH (P < 10(-3)), while damping the mesor of the 24-h rhythm in GH interpulse intervals (P < 0.025). Estradiol supplementation alone 1) increased the 24-h mean and integrated serum GH concentration (P = 0.047); 2) augmented GH secretory burst mass (P: = 0.025) without influencing pulse frequency, duration, half-life, or basal secretion; 2) stimulated more irregular patterns of GH release (higher ApEn; P = 0.012); and 3) elevated the 24-h rhythmic GH mesor (P = 0.0005), but not amplitude. Notably, combined stimulation of the GH axis with GHRH-(1--44)-amide and estradiol exerted no further effect beyond that evoked by GHRH alone, except for normalizing the acrophase of 24-h GH rhythmic release and elevating the postinfusion plasma insulin-like growth factor I concentration (P = 0.016). Unexpectedly, the two GHRH-infused serum GH concentration profiles monitored after placebo and estradiol pretreatment showed strongly nonrandom synchrony with a 20- to 30-min lag (P < 0.001). In summary, the present clinical investigations unmask a 3-fold (pulsatile, entropic, and daily rhythmic) similitude between the neuroregulatory actions of estradiol and GHRH in healthy postmenopausal women. However, GHRH infusion was multifold more effectual than estradiol, and only GHRH elevated nonpulsatile (basal) GH secretion, shifted the GH acrophase, and synchronized GH profiles. Given the nonadditive nature of the joint effects of estradiol and GHRH on pulsatile and entropic GH release, we hypothesize that estrogen amplifies GH secretion in part by enhancing endogenous GHRH release or actions. In addition, the distinctive ability of GHRH (but not estradiol) to increase basal (nonpulsatile) GH secretion, shift the GH acrophase and synchronize GH output patterns identifies certain divergent hypothalamo-pituitary actions of these two major GH secretagogues.

Generation of growth hormone pulsatility in women: evidence against somatostatin withdrawal as pulse initiator.

To test whether endogenous hypothalamic somatostatin (SRIH) fluctuations are playing a role in the generation of growth hormone (GH) pulses, continuous subcutaneous octreotide infusion (16 microg/h) was used to create constant supraphysiological somatostatinergic tone. Six healthy postmenopausal women (age 67 +/- 3 yr, body mass index 24.7 +/- 1.2 kg/m(2)) were studied during normal saline and octreotide infusion providing stable plasma octreotide levels of 2,567 +/- 37 pg/ml. Blood samples were obtained every 10 min for 24 h, and plasma GH was measured with a sensitive chemiluminometric assay. Octreotide infusion suppressed 24-h mean GH by 84 +/- 3% (P = 0.00026), GH pulse amplitude by 90 +/- 3% (P = 0.00031), and trough GH by 54 +/- 5% (P = 0.0012), whereas GH pulse frequency remained unchanged. The response of GH to GH-releasing hormone (GHRH) was not suppressed, and the GH response to GH-releasing peptide-6 (GHRP-6) was unaffected. We conclude that, in women, periodic declines in hypothalamic SRIH secretion are not the driving force of endogenous GH pulses, which are most likely due to episodic release of GHRH and/or the endogenous GHRP-like ligand.

Synergy of L-arginine and growth hormone (GH)-releasing peptide-2 on GH release: influence of gender.

We test the hypotheses that 1) growth hormone (GH)-releasing peptide-2 (G) synergizes with L-arginine (A), a compound putatively achieving selective somatostatin withdrawal and 2) gender modulates this synergy on GH secretion. To these ends, 18 young healthy volunteers (9 men and 9 early follicular phase women) each received separate morning intravenous infusions of saline (S) or A (30 g over 30 min) or G (1 microg/kg) or both, in randomly assigned order. Blood was sampled at 10-min intervals for later chemiluminescence assay of serum GH concentrations. Analysis of covariance revealed that the preinjection (basal) serum GH concentrations significantly determined secretagogue responsiveness and that sex (P = 0.02) and stimulus type (P < 0.001) determined the slope of this relationship. Nested ANOVA applied to log-transformed measures of GH release showed that gender determines 1) basal rates of GH secretion, 2) the magnitude of the GH secretory response to A, 3) the rapidity of attaining the GH maximum, and 4) the magnitude or fold (but not absolute) elevation in GH secretion above preinjection basal, as driven by the combination of A and G. In contrast, the emergence of the G and A synergy is sex independent. We conclude that gender modulates key facets of basal and A/G-stimulated GH secretion in young adults.

Synergy of L-arginine and GHRP-2 stimulation of growth hormone in men and women: modulation by exercise.

We investigated the ability of exercise, a multipathway, potent, physiological stimulus for GH release, to alter the synergistic interaction of L-arginine (A) and GH-related peptide (GHRP)-2 (G) observed at rest and the ability of gender to further modulate this putative interaction. Subjects (9 men and 9 early follicular phase women) completed 30 min of constant load aerobic exercise in combination with intravenous infusions of saline (S), A (30 g over 30 min), G (1 microg/kg bolus), or both (AG) in separate study sessions in randomly assigned order. Measures of GH release were logarithmically transformed for statistical analysis. Similar to rest, exercise maintained the rank order (AG > G > A > S) of effective stimulation of GH release for the key response measures in men or women, a gender disparity in the time to reach the maximal serum GH concentration, the calculated endogenous GH half-life, and the observed effect of preinfusion (basal) serum GH concentrations on determining secretagogue responsiveness. Exercise potentiated the individual stimulatory actions of A and G, while blunting the relative magnitude of the synergistic (supra-additive) interaction observed at rest. We infer from the present data that 1) exercise is likely to induce release of both GHRH and somatostatin, 2) L-arginine may facilitate the effect of exercise by limiting somatostatin release, 3) GHRP-2 could further enhance the stimulatory impact of exercise by opposing central actions of somatostatin and/or heightening endogenous GHRH release, and 4) gender strongly controls the relative but not absolute magnitude of A/G synergy both at rest and after exercise.

Oral estradiol administration modulates continuous intravenous growth hormone (GH)-releasing peptide-2-driven GH secretion in postmenopausal women.

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 17beta-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 microg/kg x 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.

Growth hormone responses during strenuous exercise: the role of GH-releasing hormone and GH-releasing peptide-2.

PURPOSE AND METHODS: This study was designed to investigate the role of two effective releasers of growth hormone (GH): GHRH and GHRP-2 during exercise (EX). Eight healthy male subjects (ages: 22 +/- 1.2 (mean +/- SD) yr, BMI: 22.5 +/- 2.2 kg x m(-2)) were exposed to maximally stimulating dose of 100 microg GHRH iv, and 200 microg GHRP-2 iv, during incremental EX on a cycle ergometer to exhaustion. GH responses after EX alone were compared with the responses after the combined administration of the same EX plus GHRH, EX plus GHRP-2, and EX plus GHRH plus GHRP-2. Blood samples were obtained in the fasted state at intervals for 2 h postexercise and the area under the GH response curve (AUC) was calculated by trapezoidal integration. RESULTS: Significant differences (P < 0.003) were observed between the AUCs after administration of EX alone (mean +/- SEM): 2,324 +/- 312 microg x L(-1) 120 min, after EX plus GHRH: 6,952 +/- 1,083, after EX plus GHRP-2: 14,674 +/- 2,210, and after the combination EX plus GHRH plus GHRP-2: 17,673 +/- 1,670. However, AUCs after each combination did not differ significantly from those after arithmetical addition of each separate stimulus, indicating that the mechanisms of the respective stimuli do not interact. Linear regression analysis on mean GH responses between 20 and 30 min after the start of EX showed that EX alone and GHRH alone explain about 59% (adj. R2) of the GH response to the combination EX plus GHRH. The ratio of the respective regression coefficients (GHRH vs EX) was about 2:1 (instead of 1:1), indicating that EX seems to potentiate the activity of GHRH. GHRH alone and EX alone also explained about 74% of the response to the combination EX plus GHRP-2. In the latter response, a synergistic action of GHRP-2 on GHRH could be observed. CONCLUSIONS: The data indicate that under strenuous EX conditions, endogenous GHRH activity causes a further increase of GH release. A GHRP-2 mediated mechanism in the central neuroendocrine regulation acts as a "booster," possibly by stimulating the effects of GHRH and/or an unknown hypothalamic factor, as well as by stimulating the pituitary GH release directly.

A paradoxical gender dissociation within the growth hormone/insulin-like growth factor I axis during protracted critical illness.

Female gender appears to protect against adverse outcome from prolonged critical illness, a condition characterized by blunted and disorderly GH secretion and impaired anabolism. As a sexual dimorphism in the GH secretory pattern of healthy humans and rodents determines gender differences in metabolism, we here compared GH secretion and responsiveness to GH secretagogues in male and female protracted critically ill patients. GH secretion was quantified by deconvolution analysis and approximate entropy estimates of 9-h nocturnal time series in 9 male and 9 female patients matched for age (mean +/- SD, 67+/-11 and 67+/-15 yr), body mass index, severity and duration of illness, feeding, and medication. Serum concentrations of PRL, TSH, cortisol, and sex steroids were measured concomitantly. Serum levels of GH-binding protein, insulin-like growth factor I (IGF-I), IGF-binding proteins (IGFBPs), and PRL were compared with those of 50 male and 50 female community-living control subjects matched for age and body mass index. In a second study, GH responses to GHRH (1 microg/kg), GH-releasing peptide-2 (GHRP-2; 1 microg/ kg) and GHRH plus GHRP-2 (1 and 1 microg/kg) were examined in comparable, carefully matched male (n = 15) and female (n = 15) patients. Despite identical mean serum GH concentrations, total GH output, GH half-life, and number of GH pulses, critically ill men paradoxically presented with less pulsatile (mean +/- SD pulsatile GH fraction, 39+/-14% vs. 67+/-20%; P = 0.002) and more disorderly (approximate entropy, 0.946+/-0.113 vs. 0.805+/-0.147; P = 0.02) GH secretion than women. Serum IGF-I, IGFBP-3, and acid-labile subunit (ALS) levels were low in patients compared with controls, with male patients revealing lower IGF-I (P = 0.01) and ALS (P = 0.005) concentrations than female patients. Correspondingly, circulating IGF-I and ALS levels correlated positively with pulsatile (but not with nonpulsatile) GH secretion. Circulating levels of GH-binding protein and IGFBP-1, -2, and -6 were higher in patients than controls, without a detectable gender difference. In female patients, PRL levels were 3-fold higher, and TSH and cortisol tended to be higher than levels in males. In both genders, estrogen levels were more than 3-fold higher than normal, and testosterone (2.25+/-1.94 vs. 0.97+/-0.39 nmol/L; P = 0.03) and dehydroepiandrosterone sulfate concentrations were low. In male patients, low testosterone levels were related to reduced GH pulse amplitude (r = 0.91; P = 0.0008). GH responses to GHRH were relatively low and equal in critically ill men and women (7.3+/-9.4 vs. 7.8+/-4.1 microg/L; P = 0.99). GH responses to GHRP-2 in women (93+/-38 microg/L) were supranormal and higher (P<0.0001) than those in men (28+/-16 microg/L). Combining GHRH with GHRP-2 nullified this gender difference (77+/-58 in men vs. 120+/-69 microg/L in women; P = 0.4). In conclusion, a paradoxical gender dissociation within the GH/ IGF-I axis is evident in protracted critical illness, with men showing greater loss of pulsatility and regularity within the GH secretory pattern than women (despite indistinguishable total GH output) and concomitantly lower IGF-I and ALS levels. Less endogenous GHRH action in severely ill men compared with women, possibly due to profound hypoandrogenism, accompanying loss of the putative endogenous GHRP-like ligand action with prolonged stress in both genders may explain these novel findings.

Evaluation of Orntide microspheres in a rat animal model and correlation to in vitro release profiles.

Orntide acetate, a novel luteinizing hormone-releasing hormone (LHRH) antagonist, was prepared and evaluated in vivo in 30-day and 120-day sustained delivery formulations using a rat animal model. Orntide poly(d,l-lactide-co-glycolide) (PLGA) and poly(d,l- lactide) (PLA) microspheres were prepared by a dispersion method and administered subcutaneously in a liquid vehicle to rats at 2.2 mg Orntide/kg of body weight (30-day forms) or 8.8 mg Orntide/kg (120-day forms). Serum levels of Orntide and testosterone were monitored by radioimmunoassays, and a dose-response study at 4 doses (3, 2.25, 1.5, and 1.75 mg Orntide/kg) was conducted to determine the effective dose of Orntide. Microspheres with diameters between 3.9 and 14 micron were prepared. The onset and duration of testosterone suppression varied for different microsphere formulations and were influenced both by polymer properties and by microsphere characteristics. Microspheres prepared with 50:50 and 75:25 copolymers effectively sustained peptide release for 14 to 28 days, whereas an 85:15 copolymer and the PLA microspheres extended the pharmacological response for more than 120 days. Increase in drug load generally accelerated peptide release from the microspheres, resulting in higher initial serum levels of Orntide and shorter duration of the release. In general, apparent release was faster in vivo than under in vitro conditions. Orntide microspheres effectively suppressed testosterone in rats, providing rapid onset of release and extended periods of chemical castration. Testosterone suppression occurred immediately after microsphere administration without the initial elevation seen with LHRH superagonists.