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Ghrelin is a gut hormone best known for its role in regulating appetite and stimulating the secretion of the anabolic hormone growth hormone (GH). However, there is considerable evidence to show wider-ranging biological actions of ghrelin that favour improvements in cellular and systemic metabolism, as well as neuroprotection. Activation of these ghrelin-mediated pathways may alleviate pathogenic processes that are assumed to contribute to accelerated progression of disease in patients with neurodegenerative disease. Here, we provide a brief overview on the history of discoveries that led to the identification of ghrelin. Focussing on the neurodegenerative disease amyotrophic lateral sclerosis (ALS), we also present an overview of emerging evidence that suggests that ghrelin and ghrelin mimetics may serve as potential therapies for the treatment of ALS. Given that ALS is a highly heterogeneous disease, where multiple disease mechanisms contribute to variability in disease onset and rate of disease progression, we speculate that the wide-ranging biological actions of ghrelin might offer therapeutic benefit through modulating multiple disease-relevant processes observed in ALS. Expanding on the well-known actions of ghrelin in regulating food intake and GH secretion, we consider the potential of ghrelin-mediated pathways in improving body weight regulation, metabolism and the anabolic and neuroprotective actions of GH and insulin-like growth factor-1 (IGF-1). This is of clinical significance because loss of body weight, impairments in systemic and cellular metabolism, and reductions in IGF-1 are associated with faster disease progression and worse disease outcome in patients with ALS.

Background: Oral administration of estradiol (E-2) generally increases GH secretion in postmenopausal women. Oral administration of E-2 is associated with a decrease in IGF-1, whereas parenteral or transdermally administered E-2 may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E-2 levels stimulate GH and that P4 modulates E-2-stimulated GH secretion.Study Design: Four parallel groups of randomly assigned postmenopausal women (n = 40). Treatments were saline placebo and oral placebo, saline placebo and oral micronized P4 (3 x 200 mg/d IM), E-2 (5 mg IM) and oral placebo, and E-2 IM and oral micronized P4. Outcome measures were overnight GH secretion (10 hours), stimulated (ghrelin, 0.3 mu g/kg IV bolus) GH secretion, and CT-estimated visceral fat.Results: Intramuscular E-2 administration did not alter nocturnal and ghrelin-stimulated GH secretion. Nocturnal GH secretion was not changed by P4 administration. However, P4 diminished ghrelin-stimulated pulsatile GH release with or without E-2 (average, 7.20 +/- 2.14 and 9.58 +/- 1.97 mu g/L/2 h, respectively; P = 0.045). Respective outcomes for mean GH concentrations and GH peak amplitudes were 0.97 +/- 0.31 and 1.52 mu g/L +/- 0.29 (P = 0.025) and 2.76 +/- 1.04 and 3.95 mu g/L +/- 0.90 (P = 0.031). Ghrelin-stimulated GH secretion correlated negatively with P4 concentration with or without correction for visceral fat area in the regression equation (R = 0.49, P = 0.04, beta = -0.040 +/- 0.016).Conclusions: Low-range physiological E-2 concentrations do not affect spontaneous or ghrelin-stimulated pulsatile GH secretion. Conversely, P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion. The mechanistic aspects and physiological significance of natural P4's regulation of ghrelin-evoked GH secretion require further study.

CONTEXT: Testosterone (T) increases GH secretion in older men with a relative lack of T, in hypogonadal men of all ages, and in patients undergoing sex reassignment. The role of estradiol (E(2)) in men is less well defined. OBJECTIVE: To assess the contribution of aromatization of T to spontaneous nocturnal and stimulated GH secretion. PARTICIPANTS: Four groups of healthy older men (N = 74, age range 57 to 77 years) were studied. The gonadotropic axis was clamped with the gonadotropin-releasing hormone antagonist degarelix. Three groups received T and one group placebo addback. Two T-replaced groups were treated with anastrozole (an aromatase inhibitor) and either placebo or E(2) addback. MAIN OUTCOME MEASURES: Ten-minute GH concentration profiles were quantified by deconvolution analysis, after overnight (2200 to 0800 hours) sampling, and after combined IV injection of GHRH (0.3 µg/kg) and GHRH-2 (0.3 µg/kg) and withdrawal of a 2-hour somatostatin infusion (1 µg/kg/h). RESULTS: E(2) addback during aromatase inhibition increased basal (P = 0.046), pulsatile (P = 0.020), and total (P = 0.018) GH secretion by 60% to 70%. E(2) did not potentiate GH secretory stimuli. Logarithmically transformed pulsatile GH secretion correlated strongly and positively with concurrent E(2) concentrations overall (P = 0.028) and under anastrozole treatment (P = 0.005). CONCLUSION: E(2) administration in older men transdermally stimulates overnight pulsatile GH secretion. The exact site of E(2) action cannot be ascertained from these experiments but may include hypothalamic loci involved in GH regulation, especially because GH secretagogue effects on somatotrope pituitary cells were not affected.

BACKGROUND: In Japan, growth hormone releasing peptide-2 (GHRP-2) is clinically used as a diagnostic agent for growth hormone secretion deficiency, but the therapeutic application of GHRP-2 has not been studied in anorexia nervosa. GHRP-2 reportedly exhibits agonistic action for ghrelin receptor and increases food intake. METHODS: We administered GHRP-2 to a patient with a 20-year history of anorexia nervosa to determine whether GHRP-2 treatment increases food intake and body weight. GHRP-2 was administered before every meal by an intranasal approach for 1 year. RESULTS: Although the patient reported a decreased fear of eating and decreased desire to be thin by our previous treatment, she was unable to increase food intake or body weight because of digestive tract dysfunction. Vomiting after meals caused by delayed gastric emptying and incurable constipation were prolonged, and sub-ileus and hypoglycemia were observed. GHRP-2 increased the feeling of hunger and food intake, decreased early satiety and improved hypoglycemia. The patient's body weight gradually increased by 6.7 kg (from 21.1 kg to 27.8 kg) in 14 months after starting GHRP-2 administration. The fatigability and muscle strength improved, and the physical and mental activities were also increased. No obvious side effects were observed after long-term intranasal administration of GHRP-2. CONCLUSIONS: Patients with a long-term history of eating disorder occasionally recover from the psychological problems such as fear for obesity but remain emaciated. We believe that ghrelin agonists such as GHRP-2 may be promising agents for the effective treatments of severe anorexia nervosa in a chronic condition.

Context: Estrogens amplify spontaneous and stimulated growth hormone (GH) secretion, whereas they diminish GH-dependent insulin-like growth factor (IGF)-I in a dose-dependent manner. Selective estrogen receptor modulators (SERMs), including tamoxifen and toremifene, are widely adjunctively used in breast and prostate cancer. Although some endocrine effects of tamoxifen are known, few data are available for toremifene. Objective: To explore sex-dependent effects of toremifene on spontaneous 10-hour overnight GH secretion, followed by GH-releasing hormone–ghrelin stimulation. Additionally, effects on IGF-I, its binding proteins, and sex hormone–binding globulin (SHBG) were quantified. Participants and Design: Twenty men and 20 women, within an allowable age range of 50 to 80 years, volunteered for this double-blind, placebo-controlled prospective crossover study. Ten-minute blood sampling was done for 10 hours overnight and then for 2 hours after combined GH-releasing hormone–ghrelin injection. Main Outcome Measures: Pulsatile GH and stimulated GH secretion, and fasting levels of IGF-I, IGF-binding protein (IGFBP)1, IGFBP3, and SHBG. Results: Toremifene did not enhance pulsatile or stimulated GH secretion, but decreased IGF-I by 20% in men and women. IGFBP3 was unchanged, whereas while IGFBP1 and SHBG increased in both sexes to a similar extent. Conclusions: The expected rise in spontaneous and stimulated GH secretion under the diminished negative feedback restraint of powered IGF-I favors a central inhibitory antiestrogenic effect of toremifene. Estrogenic effects of toremifene on the liver were present, as evidenced by increased IGFBP1 and SHBG levels. Men and women responded to this SERM comparably., The SERM toremifene acts in a sex-independent way. The effect on liver proteins are estrogenic, but its central effects on the hypothalamic/somatotrope system are antiestrogenic.

Context: Hyposomatotropism in healthy aging women reflects in part physiological estrogen (estradiol [E2]) depletion associated with menopause. Objective and Design: The purpose of this study was to test the hypothesis that low concentrations of endogenous E2 after menopause continue to drive GH secretion. Setting: The study was performed at the Mayo Center for Clinical and Translational Science. Participants: The participants were 24 postmenopausal women (aged 50–77 years with body mass index of 19–32 kg/m2). Interventions: This was a randomized, double-blind, placebo-controlled, parallel-cohort treatment study with placebo (PL) (n = 14) or the antiestrogen fulvestrant (FUL) (n = 10) for 3 weeks, followed by infusion of l-arginine with saline, GHRH, ghrelin, or both peptide secretagogues. Outcomes: GH concentrations were measured over 6 hours with 10-minute sampling and mass spectrometry measures of testosterone, E2, and estrone. Results: Concentrations of testosterone, E2, estrone, SHBG, IGF-I, LH, and FSH were not influenced by antiestrogen treatment. In contrast, GH rose from 0.096 ± 0.018 (PL) to 0.23 ± 0.063 μg/L (FUL, P = .033), and IGF-I binding protein type 3 (IGFBP-3) from 3.6 ± 0.18 to 4.0 ± 2.0 mg/L (P = .041). Conversely, prolactin fell from 7.1 ± 0.69 (PL) to 5.5 ± 0.57 μg/L (FUL) (P = .05), and IGF-I binding protein type 1 (IGFBP-1) fell from 44 ± 9.4 to 27 ± 4.3 μg/L (P = .048). Moreover, FUL vs PL potentiated mean GH responses to l-arginine/saline (P = .007), l-arginine/ghrelin (P = .008), and l-arginine/GHRH + ghrelin (P = .031), but not l-arginine/GHRH. Conclusion: The potent antiestrogen, FUL, amplifies fasting and secretagogue-driven GH secretion and IGFBP-3 concentrations in postmenopausal women without altering SHBG or sex steroid levels. FUL also suppresses prolactin and IGFBP-1, without altering IGF-I. Thus, a major antiestrogen mediates 3 actions of estrogen: agonism (GH), neutral effects (sex steroids), and estrogen antagonism (prolactin and IGFBP-1).

Ghrelin is a potent gastric-derived GH-releasing peptide. How ghrelin interacts with sex steroids, GHRH, and somatostatin (SS) is not known.Our objective was to test the hypotheses that ghrelin's interactions with GHRH (synergistic) and SS (disinhibitory) are ghrelin dose-dependent and amplified by estrogen. SUBJECTS, SETTING, AND DESIGN: Healthy postmenopausal women were treated with placebo (n=12) or 17β-estradiol (E2) (n=12) at the Center for Translational Science Activities in a randomized double-blind prospective study.Ghrelin dose-dependence was assessed by nonlinear curve fitting of the relationship between deconvolved GH secretory-burst mass and 5 randomly ordered ghrelin doses (0, 0.03, 0.135, 0.6, and 2.7 μg/kg bolus iv) during saline, GHRH, and SS infusion.Under placebo, neither GHRH nor SS altered the ED50 of ghrelin (range 0.64-0.67 μg/kg). Under E2 (median E2 88 pg/mL), the ED50 of ghrelin declined in the presence of GHRH to 0.52 μg/kg. In contrast, the efficacy of ghrelin rose markedly during GHRH vs saline exposure with and without E2: placebo and saline 52±1.0 vs GHRH 173±3.8 μg/L; and E2 and saline 56±0.90 vs GHRH 174±3.7 μg/L. Sensitivity to ghrelin was similar under all conditions.Short-term E2 supplementation in postmenopausal women reduces the ED50 (increases the potency) of ghrelin when GHRH is present, without altering ghrelin efficacy (maximal effect) or hypothalamo-pituitary sensitivity (slope of dose response) to ghrelin. The data suggest possible physiological interactions among sex steroids (endogenous), ghrelin, and GHRH during E2 replacement in postmenopausal women.

To test the postulate that sex difference, sex steroids, and peptidyl secretagogues control GH autofeedback, 11 healthy postmenopausal women and 14 older men were each given 1) a single iv pulse of GH to enforce negative feedback and 2) continuous iv infusion of saline vs. combined GHRH/GHRP-2 to drive feedback escape during pharmacological estradiol (E2; women) or testosterone (T; men) supplementation vs. placebo in a double-blind, prospectively randomized crossover design. By three-way ANCOVA, sex difference, sex hormone treatment, peptide stimulation, and placebo/saline responses (covariate) controlled total (integrated) GH recovery during feedback (each P < 0.001). Both sex steroid milieu ( P = 0.019) and dual-peptide stimulation ( P < 0.001) determined nadir (maximally feedback-suppressed) GH concentrations. E2/T exposure elevated nadir GH concentrations during saline infusion ( P = 0.003), whereas dual-peptide infusion did so independently of T/E2 and sex difference ( P = 0.001). All three of sex difference ( P = 0.001), sex steroid treatment ( P = 0.005), and double-peptide stimulation ( P < 0.001) augmented recovery of peak (maximally feedback-escaped) GH concentrations. Peak GH responses to dual-peptidyl agonists were greater in women than in men ( P = 0.016). E2/T augmented peak GH recovery during saline infusion ( P < 0.001). Approximate entropy analysis corroborated independent effects of sex steroid treatment ( P = 0.012) and peptide infusion ( P < 0.001) on GH regularity. In summary, sex difference, sex steroid supplementation, and combined peptide drive influence nadir, peak, and entropic measurements of GH release under controlled negative feedback. To the degree that the pharmacological sex steroid, GH, and dual-peptide clamps provide prephysiological regulatory insights, these outcomes suggest major determinants of pulsatile GH secretion in the feedback domain.

DesignWhile androgens and estrogens control glucocorticoid secretion in animal models, how the sex-steroid milieu determines cortisol secretion in humans is less clear. To address this issue, cortisol was measured in archival sera obtained at 10-min intervals for 5 h in 42 healthy men administered double placebo, placebo and testosterone, testosterone and dutasteride (to block 5α-reductases type I and type II), or testosterone and anastrozole (to block aromatase) in a double-blind, placebo-controlled, prospectively randomized design.MethodsSubjects received i.v. injection of saline, GHRH, GH-releasing peptide-2 (GHRP-2), somatostatin (SS), and GHRP-2/GHRH/l-arginine (triple stimulus) each on separate mornings fasting. Outcomes comprised cortisol concentrations, pulsatile cortisol secretion, and relationships with age or abdominal visceral fat (AVF).ResultsBy ANCOVA, baseline (saline-infused) cortisol concentrations (nmol/l) did not differ among the sex-steroid milieus (overall mean 364±14). In contrast, stimulated peak cortisol concentrations were strongly determined by secretagogue type (PGHRP-2 (616±42)>saline=SS=GHRH (grand mean 420±21). After GHRP-2 injection, pulsatile cortisol secretion increased with age (R2=0.16,P=0.012). After the triple stimulus, pulsatile cortisol secretion correlated i) inversely with serum 5α-dihydrotestosterone (DHT) concentrations (R2=0.53,P=0.026) and ii) directly with computerized tomography-estimated AVF (R2=0.11,P=0.038).ConclusionAge, DHT concentrations, AVF, and secretagogue type influence pulsatile cortisol secretion at least in men. Further studies should be performed to assess ACTH secretion and native ghrelin action in defined sex-steroid milieus.

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 (E2) 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 E2 and separate-day iv bolus injections of a half-maximally stimulatory dose of GHRP-2 or GHRH (each 0.33 μg/kg). Analysis: Three-way analysis of covariance included main factors age, E2 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), E2 repletion vs. depletion (P = 0.001) and GHRP-2 vs. GHRH stimulation (P < 0.001), after adjustment for AVF and basal secretion. E2 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, E2 availability, and physiological peptide drive are triple determinants of pulsatile GH secretion independently of abdominal visceral fat and nonpulsatile GH secretion in healthy women.

GH-releasing peptide-2 (GHRP-2) is a synthetic peptide that increases circulating GH and IGF-I levels. It also binds to CD36, a scavenger receptor for oxidized low-density lipoprotein (OxLDL), and may prevent cellular uptake of this proatherogenic complex. To determine its potential antiatherogenic effects, GHRP-2 (20 microg twice daily) was administered sc to ApoE(-/-) mice for 12 wk. GHRP-2 increased circulating IGF-I 1.2- to 1.6-fold and decreased circulating interferon-gamma by 66%. Although GHRP-2 did not alter atherosclerotic plaque area, it decreased aortic production of superoxide as assessed by dihydroethidium staining. GHRP-2 decreased aortic gene expression of 12/15-lipoxygenase by 92% and reduced the aortic expression of interferon-gamma and macrophage migration inhibitory factor. In cultured aortic smooth muscle cells, GHRP-2 prevented the OxLDL-induced generation of peroxides, down-regulation of IGF-I receptor, and apoptosis. In macrophages, GHRP-2 reduced lipid accumulation with OxLDL exposure. In summary, GHRP-2 exerts antioxidant effects in vivo and in vitro but does not reduce plaque burden. The lack of an antiatherogenic effect may be due to GH-dependent effects in vivo, thereby blunting the effect of increased IGF-I.

BackgroundSomatostatin (SS), GHRH, GH-releasing peptide (GHRP), and the sex-steroid milieu regulate GH secretion.ObjectiveTo test whether GHRH and GHRP remain effective secretagogs in the face of short-term hypogonadism.DesignProspective, randomized double-blind.MethodsHealthy young men (n=24) received a GnRH agonist twice 3 weeks apart followed by placebo (n=13, Pl) or testosterone (n=11, testosterone) addback. Subjects were then given consecutive i.v. infusions of l-arginine (to restrain SS outflow) and a maximally effective dose of GHRH or GHRP-2 (to test corresponding secretagog pathways).ResultsGH secretion stimulated by l-arginine/GHRH and by l-arginine/GHRP-2 was unaffected by combined testosterone/estradiol (E2) depletion. The low testosterone/E2 milieu decreased basal (nonpulsatile) GH secretion (P=0.038), without altering fasting pulsatile GH secretion or IGF1 or IGF-binding protein (IGFBP)-3 concentrations. IGFBP-1 (PP=0.017) correlated negatively with fasting basal GH secretion. By contrast, IGF1 (P=0.0012) and IGFBP-3 (P=0.015) correlated positively with fasting pulsatile GH secretion. AVF (P=0.0024) was a negative determinant, and IGF1 a positive determinant (P=0.018), of GHRH-driven GH pulses. Responses to GHRP-2 were unrelated to any of these factors.Conclusionl-arginine/GHRP-2 appears to be an especially robust stimulus of GH secretion, since efficacy is unmodified by profound short-term hypogonadism, a range of AVF estimates, and a spectrum of IGF1, IGFBP-1, and IGFBP-3 concentrations. Whether robustness also applies to chronic hypogonadism is not known.

Background: Sex steroids are prominent regulators of pulsatile GH secretion. Hypothesis: An experimentally controlled sex-steroid milieu will permit detection of nonsteroidal factors that determine GH secretion. Subjects: Eleven young (age, 24 ± 0.99 yr) and 11 older (64 ± 2.4 yr) men participated in the study. Location: The study was conducted at a tertiary medical center. Methods: The study consisted of GnRH-agonist down-regulation of the gonadal axis followed by fixed-dose testosterone (T) replacement (leuprolide/T clamp) and consecutive infusion of l-arginine and GHRH or GH-releasing peptide-2 (GHRP-2) to quantify peptide-secretagogue efficacies. Outcomes: The experimental leuprolide/T clamp yielded statistically age-comparable total, bioavailable, and free T and estradiol (E2) concentrations. In this controlled milieu, sequential l-arginine/GHRH infusion stimulated 1.4-fold more (P = 0.021) and l-arginine/GHRP-2 1.3-fold more (P = 0.045) GH release in young than older men. Abdominal visceral fat (AVF) correlated negatively with both GHRH (P = 0.0006; R2 = 0.39) and GHRP-2 (R2 = 0.29) efficacy, whereas IGF-I positively predicted the same endpoints (R2 = 0.25 to 0.30). In multivariate analysis, AVF emerged as a dominant negative determinant of GHRH efficacy (P = 0.002; R2 = 0.41) and IGF-I as a primary positive determinant of GHRP-2 efficacy (P = 0.007; R2 = 0.31). Conclusion: During fixed T/E2 availability, AVF contributes 41% of the GH-response variability to maximal GHRH drive, whereas IGF-I accounts for 31% of that for GHRP-2. Accordingly, a statistically equalized sex-steroid milieu permits dissection of age-independent and T/E2-independent modulators of GHRH and GHRP efficacy in men.

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.

The syntheses of five partial retro-inverso luteinizing hormone-releasing hormone (LH-RH)analogs, [g-Tyr5, m-Gly6]LH-RH, [g-Tyr5-r-Gly6, R,S-m-Leu7]LH-RH, [g-p-Glu1, m-His2]LH-RH, [g-p-Glu1 -r-D-His-R,S-m-Trp3]LH-RH, and [g-Pro9- propionyl-des-Gly10]LH-RH, have been accomplished by solution methods. The choice of sequence to be reversed was based on suggested biodegradation mechanisms of LH-RH. A (gem)-diamino alkylidene residue, which was produced via Curtius rearrangement of a peptide segment, and a 2-substituted malonyl residue mark the initiating and terminating site, respectively, of the reversed sequence.

A series of antagonists of the luteinizing hormone releasing hormone (LHRH) with substitutions in position 5 and/or 6 that included acylated lysine or p-aminophenylalanine were synthesized, characterized and tested for antiovulatory activity (AOA) in rats, and histamine releasing activity. Some of these antagonists were considerably more soluble at neutral pH than antagonists like Antide. Of 37 new antagonists, the best physico-chemical and biological properties were found for the following two analogs: NAcDNal-DCpa-DPal-Ser-PicLys-D(PicSar)Lys- Leu-ILys-Pro-DAlaNH2 (named Sartide) and NAcDNal-DCpa-DPal-Ser-Tyr-D(PicSar)Lys-Leu-IL ys-Pro-DAlaNH2. They are both soluble in water, inhibit ovulation completely at 0.5 microgram per rat, and have ED50 values for histamine release of about 30 micrograms/mL.

N-Ac-D-O-phenyltyrosine was synthesized via the corresponding azlactone. Resolution of the DL methyl esters was achieved by Subtilisin Carlsberg. Treatment with palladium(II) acetate in trifluoroacetic acid converted N-Ac-D-O-phenyltyrosine into N-Ac-D-3-(2-dibenzofuranyl)alanine. These two amino acids were incorporated instead of N-Ac-D-2-Nal into position 1 of the LHRH-antagonist (N-Ac-D-2-Nal1, D-p-ClPhe2, D-3-Pal3, c-PzACAla5, D-PicLys6, ILys8,D-Ala10)-LHRH. The more rigid N-Ac-D-3-(2-dibenzofuranyl)alanine was structurally more effective than N-Ac-D-O-phenyltyrosine; the AOAs for the corresponding analogs were 82 and 38%, respectively, at 0.5 micrograms. Replacement of c-PzACAla in position 5 by O-phenyltyrosine significantly decreased potency.

Acylated ghrelin is the putatively bioactive GH secretagogue.Estradiol (E2) stimulates the synthesis rather than inhibits the metabolic clearance of acylated ghrelin.The study took place at an academic medical center.Healthy postmenopausal women participated.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.Metabolic clearance rate (MCR), volume of distribution, half-life, and secretion rate of acylated ghrelin were assessed.In pilot iv bolus ghrelin infusions, the median half-lives of acylated and total ghrelin were 21 and 36 min (P0.01), MCRs 58 and 8.1 liters/kg.d (P0.01), and volumes of distribution of 1.0 and 0.32 liters/kg (P0.01), 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).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.

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 (E2) 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 E2 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 E2 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: E2 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 E2 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.

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.Our objective was to compare the relationships between BMI and peptide-stimulated GH secretion in men with normal and reduced testosterone and estradiol availability.The study was performed at an academic medical center.Healthy young men were included in the study.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.Regression of paired secretagogue-induced GH responses on BMI was determined.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).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.

Ghrelin is a 28-amino-acid Ser(3)-octanoylated peptide, and CRH is a 41-amino-acid peptide, both of which stimulate ACTH secretion. In principle, actions of these agonists could be subject to inhibitory modulation by hypothalamic somatostatin (SS).Our objective was to test the hypothesis that endogenous SS restrains ghrelin and CRH-stimulated ACTH secretion, thereby linking all three, ghrelin, CRH, and SS, with ACTH secretion.We conducted a randomized, double-blind, placebo-controlled, crossover interventional study at an academic medical center.Ten healthy postmenopausal women participated in the study.Interventions included iv injection of saline, ghrelin, human CRH, or both after an infusion of saline vs. l-arginine to putatively inhibit SS outflow (eight visits per subject).ACTH concentrations quantified by repetitive blood sampling and immunochemiluminometry.Infusion of ghrelin induced peak ACTH concentrations [median (range)] of 21 (17-28) compared with 16 (11-20) ng/liter after saline (P = 0.037). CRH and l-arginine infusion evoked ACTH peaks of 23 (14-48) and 31 (21-286) ng/liter, respectively (P = 0.037 and P = 0.005 vs. saline). l-Arginine enhanced stimulation by ghrelin by 1.43-fold (P = 0.028) and that by CRH by 1.91-fold (P = 0.005). Triple stimulation with ghrelin, CRH, and l-arginine potentiated the effect of combined ghrelin/CRH by 1.45-fold (P = 0.028). Downstream cortisol responses mimicked those of ACTH but were time delayed.The present outcomes indicate that the peptide ensemble comprising ghrelin, CRH, and SS (inferred by l-arginine infusion) can regulate ACTH and cortisol secretion in healthy adults.

Models of physiological systems facilitate rational experimental design, inference, and prediction. A recent construct of regulated growth hormone (GH) secretion interlinks the actions of GH-releasing hormone (GHRH), somatostatin (SRIF), and GH secretagogues (GHS) with GH feedback in the rat (Farhy LS, Veldhuis JD. Am J Physiol Regul Integr Comp Physiol 288: R1649–R1663, 2005). In contrast, no comparable formalism exists to explicate GH dynamics in any other species. The present analyses explore whether a unifying model structure can represent species- and sex-defined distinctions in the human and rodent. The consensus principle that GHRH and GHS synergize in vivo but not in vitro was explicable by assuming that GHS 1) evokes GHRH release from the brain, 2) opposes inhibition by SRIF both in the hypothalamus and on the pituitary gland, and 3) stimulates pituitary GH release directly and additively with GHRH. The gender-selective principle that GH pulses are larger and more irregular in women than men was conferrable by way of 4) higher GHRH potency and 5) greater GHS efficacy. The overall construct predicts GHRH/GHS synergy in the human only in the presence of SRIF when the brain-pituitary nexus is intact, larger and more irregular GH pulses in women, and observed gender differences in feedback by GH and the single and paired actions of GHRH, GHS, and SRIF. The proposed model platform should enhance the framing and interpretation of novel clinical hypotheses and create a basis for interspecies generalization of GH-axis regulation.

Ghrelin is a 28-amino acid, Ser(3)-octanoylated peptide that stimulates GH secretion in vivo and in vitro. Beyond the capability of ghrelin to synergize with GHRH, little is known about multipeptide modulation of ghrelin's actions in humans.The objective of this study was to test the hypothesis that ghrelin can stimulate GH secretion in the absence or presence of somatostatin withdrawal (induced by l-arginine infusion) and stress-like drive by CRH.This was a randomized, double-blind, placebo-controlled, cross-over interventional study.This study was performed at an academic medical center.Nine healthy postmenopausal women not receiving sex hormones were studied.Subjects were given an iv infusion of saline and/or l-arginine or human CRH, followed by a bolus iv injection of ghrelin.The outcome measures were pulsatile GH secretion quantified by repetitive blood sampling, immunochemiluminometry, and deconvolution analysis.Consecutive saline/ghrelin infusion increased pulsatile GH secretion from 2.7 +/- 1.0 (saline/saline; mean +/- sem) to 20 +/- 5.0 microg/liter.3 h (P0.01). The magnitude of the effect of l-arginine/saline was comparable at 20 +/- 4.5 microg/liter.3 h (P0.01). In contrast, sequential l-arginine/ghrelin evoked true synergy of GH release (93 +/- 14 microg/liter.3 h; P = 0.003 vs. l-arginine alone and P = 0.008 vs. ghrelin alone). Human CRH did not affect GH responses to saline/saline (3.9 +/- 1.1 microg/liter.3 h), saline/ghrelin (19 +/- 3.3 microg/liter.3 h), l-arginine/saline (16 +/- 2.7 microg/liter.3 h), or l-arginine/ghrelin (90 +/- 13 microg/liter.3 h).Assuming that l-arginine reduces somatostatin outflow, we infer that ghrelin can activate hypothalamo-pituitary pathways that are both dependent upon and independent of somatostatinergic restraint even in the face of a strong stress-related signal.

Aging reduces the size (mass) of GH secretory bursts and thereby reduces total GH secretion. Experimental data indicate that high-amplitude GH pulses are evoked by reversible cycles of GH-induced negative feedback. Whether aging impairs autofeedback is unknown.The objective of this study is to assess whether age attenuates and IGF-I potentiates negative feedback by a near-physiological pulse of GH.In a university setting, 17 healthy men ages 19-71 yr each underwent four randomly ordered infusion studies on separate mornings fasting.Intravenous injection of a pulse of: 1) saline or 2) recombinant human (rh) GH to impose controlled negative feedback, followed in 2 h by a bolus of 3) saline or (iv) the ghrelin analog GHRP-2 to overcome feedback inhibition.The impact of age and IGF-I concentrations on GH autofeedback was assessed by regression analysis.Percentage feedback inhibition correlated negatively with: 1) age after consecutive rh GH/saline infusion (R(2) = 0.42, P = 0.005) at any IGF-I concentration; and 2) total IGF-I concentrations after rh GH/GHRP-2 infusion (R(2) = 0.40, P = 0.009) at any age. In contrast, sex-steroid concentrations and body mass index were unrelated to degree of autoinhibition.Increased age in healthy men predicts impaired GH autofeedback, which may contribute to attenuated renewal of high-amplitude GH pulses. Conversely, higher IGF-I concentrations in young men forecast accentuated GH autoinhibition, which may drive prominent GH pulses.

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.

The present study tests the hypothesis that a high dose of testosterone (Te) drives GH and IGF-I production, in part, by blunting autonegative feedback by the end-product peptide. To this end, we infused saline or recombinant human IGF-I (10 microg/kg.h iv for 6 h) in seven healthy men ages 51-72 yr after administration of placebo (Pl) and Te in randomized order. GH release was quantitated fasting before and after injection of GHRH (1 microg/kg). Statistical analyses disclosed that Te vs. Pl: 1) increased the mean concentration of GH from 0.15 +/- 0.045 to 0.48 +/- 0.11 microg/liter (P = 0.007) and IGF-I from 108 +/- 5.0 to 124 +/- 4.1 (P = 0.047) without altering GHRH-induced GH release; 2) elevated the GH nadir from 0.13 +/- 0.03 to 0.23 +/- 0.06 microg/liter (P < 0.05) in the control session and from 0.06 +/- 0.02 to 0.14 +/- 0.04 microg/liter (P = 0.038) during IGF-I infusion; 3) augmented GHRH-stimulated GH release from 3.0 +/- 0.56 (Pl) to 3.7 +/- 0.52 microg/liter (Te) (P < 0.05) during IGF-I infusion; and 4) did not influence estimated IGF-I kinetics. In summary, supplementation of a high dose of Te in middle-aged and older men attenuates IGF-I feedback-dependent inhibition of nadir and peak GH secretion. Both effects of Te differ from those reported recently for estradiol in postmenopausal women. Accordingly, we postulate that Te and estrogen modulate IGF-I negative feedback differentially.

GHRP-2 is a synthetic agonist of ghrelin, the newly-discovered gut peptide which binds to the growth hormone (GH) secretagogue receptor. Ghrelin has two major effects, stimulating both GH secretion and appetite/meal initiation. GHRP-2 has been extensively studied for its utility as a growth hormone secretagogue (GHS). Animal studies have shown its effect on food intake. However, whether GHRP-2 can also stimulate appetite in humans when administered acutely is not known. We subcutaneously infused 7 lean, healthy males with GHRP-2 (1 microg/kg/h) or saline for 270 minutes and then measured their intake of an ad libitum, buffet-style meal. Similar to what has been reported for ghrelin administration, our subjects ate 35.9 +/- 10.9% more when infused with GHRP-2 vs. saline, with every subject increasing their intake even when calculated per kg body weight (136.0 +/- 13.0 kJ/kg [32.5 +/- 3.1 kcal/kg] vs. 101.3 +/- 10.5 kJ/kg [24.2 +/- 2.5 kcal/kg], p = 0.008). The macronutrient composition of consumed food was not different between conditions. As expected, serum GH levels rose significantly during GHRP-2 infusion (AUC 5550 +/- 1090 microg/L/240 min vs. 412 +/- 161 microg/L/240 min, p = 0.003). These data are the first to demonstrate that GHRP-2, like ghrelin, increases food intake, suggesting that GHRP-2 is a valuable tool for investigating ghrelin effects on eating behavior in humans.

GH secretagogues (GHS) are synthetic ghrelin receptor agonists that stimulate GH secretion. It is not clear whether they act predominantly by stimulating the secretion of hypothalamic growth hormone-releasing hormone (GHRH), or directly on the somatotrope cells. In addition, it is not known whether combined treatment with GHRH and GHS has synergistic effects on growth. To address these questions, we used the GH-deficient GHRH knock out (GHRHKO) mouse model, which has severe somatotrope cell hypoplasia. We treated GHRHKO mice for 5 weeks (from week 1 to week 6 of age) with the GHRH analogue JI-38 alone, or in combination with a GHS (GHRP-2), and at the end of the treatment we examined their response to an acute stimulus with GHRP-2 or GHRP-2 plus JI-38. We used placebo-treated GHRHKO mice and animals heterozygous for the GHRHKO allele as controls. Animals treated with JI-38+GHRP-2 reached higher body length and weight than animals treated with JI-38 alone. All the animals receiving JI-38 (with or without GHRP-2) showed similar correction of somatotrope cell hypoplasia. None of the GHRHKO animals showed a serum GH response to the acute stimulation with GHRP-2 alone, while both treated groups responded to the combined test with JI-38 + GHRP-2. These data demonstrate that in GHRHKO mice, GHRP-2 has a growth-stimulating effect that augments the response induced by JI-38. In addition, the presence of GHRH seems necessary for the stimulation of GH secretion by GHRP-2.

Body composition exhibits marked variations across the early human lifetime. The precise physiological mechanisms that drive such developmental adaptations are difficult to establish. This clinical challenge reflects an array of potentially confounding factors, such as marked intersubject differences in tissue compartments; the incremental nature of longitudinal intrasubject variations in body composition; technical limitations in quantitating the unobserved mass of mineral, fat, water, and muscle ad seriatim; and the multifold contributions of genetic, dietary, environmental, hormonal, nutritional, and behavioral signals to physical and sexual maturation. From an endocrine perspective (reviewed here), gonadal sex steroids and GH/IGF-I constitute prime determinants of evolving body composition. The present critical review examines hormonal regulation of body composition in infancy, childhood, and puberty.

The primary cause of waning GH and IGF-I concentrations in healthy aging adults is not established. To test the postulate that age influences negative feedback by IGF-I in a secretagogue-specific fashion, 17 normal men (nine young and eight older) each completed eight randomly ordered injections of placebo or recombinant human (rh) IGF-I (20 microg/kg sc), followed by saline/rest, aerobic exercise, GHRH (1 microg/kg iv bolus), or GH-releasing peptide-2 (1 microg/kg iv bolus) stimulation. GH secretion was monitored by sampling blood every 10 min for 7 h, high-sensitivity immunochemiluminometric assay, and deconvolution analysis conditioned on prior pulse-onset times and biexponential kinetics. Analysis of covariance showed that age (P = 0.028), secretagogue (P < 0.001), and rhIGF-I (P < 0.005) individually determine pulsatile GH secretion and exhibit a strong 3-fold interaction (P < 10(-5)). Post hoc comparisons revealed that elderly subjects manifest less IGF-I inhibition of a maximal GHRH stimulus (P = 0.013 vs. young), blunted initial IGF-I suppression of fasting GH release (P = 0.038), and impaired IGF-I feedback on the regularity of GH secretion (P = 0.023). Age stratum did not influence peak IGF-I and nadir GH concentrations or rhIGF-I-induced inhibition of GH secretion stimulated by exercise or GH-releasing peptide-2. In summary, experimental elevation of IGF-I concentrations unmasks reduced rhIGF-I-dependent feedback inhibition of fasting and GHRH-stimulated GH secretion in healthy older men, indicating that aging selectively modulates the autoinhibition process.

The present study tests the postulate that testosterone (Te) stimulates GH secretion, in part, by attenuating autonegative feedback. To this end, 13 healthy men (ages 43-71 yr) received three consecutive weekly im injections of placebo (Pl) (n = 7) or Te (200 mg) (n = 6) in a prospectively randomized, double-blind, parallel-cohort design. An iv pulse of saline or recombinant human (rh)GH (3 micro g/kg.6 min) was infused 2 h before bolus saline or GH-releasing peptide (GHRP)-2 (1 micro g/kg) in the fasting state. Blood was withdrawn every 10 min, GH concentrations were quantitated by chemiluminometry, secretion was determined by deconvolution analysis, and outcomes were compared by ANOVA. After Pl, rhGH suppressed basal, pulsatile, and GHRP-2-stimulated GH secretion by 2.6-, 2.4-, and 2.1-fold, respectively (each P < 0.03), and truncated GHRP-2-stimulated GH secretory bursts (P < 0.005). Compared with Pl, Te: 1) stimulated basal and pulsatile GH secretion by 1.9 and 2.4-fold (P < 0.01 and P < 0.02), respectively; 2) reduced feedback on basal GH secretion (P < 0.01); 3) blunted GHRP-2-stimulation by 1.9-fold (P < 0.01); and 4) facilitated initial recovery of rhGH-suppressed GH concentrations (P < 0.005). The foregoing actions were selective, inasmuch as Te did not relieve autoinhibition of pulsatile GH secretion. In summary, short-term Te supplementation decreases rhGH-imposed negative feedback on basal GH secretion and enhances early escape of GH from autoinhibition. In principle, such actions could potentiate the renewal of high-amplitude pulses of GH in androgen-replete individuals.

To test the clinical hypothesis that an estrogen-enriched milieu enhances GHRH action, we administered placebo (Pl) and estradiol-17β (E2) orally for 23 d to six postmenopausal women in a prospectively randomized, double-masked, within-subject crossover design with 6 wk intervening. The GHRH stimulation protocol entailed consecutive iv infusion of l-arginine and a single iv pulse of saline or one of five randomly ordered doses of recombinant human GHRH-1,44-amide (0.03, 0.1, 0.3, 1.0, or 3.0 μg/kg) in a total of 12 separate morning, fasting sessions. GH secretion was monitored by sampling blood every 10 min for 6 h; chemiluminescence assay of GH concentrations; deconvolution analysis of stimulated GH release; and nonlinear dose-response reconstruction. Supplementation with E2, compared with Pl: 1) increased (mean ± sem) E2 concentrations from 18 ± 3 (Pl) to 164 ± 12 pg/ml (to convert to picomoles per liter, multiply by 3.57) (P < 0.001); 2) decreased IGF-I concentrations from 181 ± 14 to 120 ± 11 μg/liter (P < 0.01); 3) elevated mean GH concentrations from 0.27 ± 0.06 to 0.59 ± 0.08 μg/liter (P = 0.014); 4) potentiated GH secretion stimulated by l-arginine alone by 1.43-fold (P = 0.012); 5) reduced the ED50 of GHRH from 0.27 ± 0.02 to 0.13 ± 0.01 μg/kg (P < 0.01), denoting enhanced GHRH potency; and 6) heightened the maximal slope of the dose-response function from 1.1 ± 0.1 to 1.4 ± 0.05 [(μg/liter) (μg/kg)−1] (P < 0.05), signifying augmented pituitary sensitivity. The foregoing facilitative mechanisms were specific because E2 replacement did alter maximal l-arginine/GHRH-induced GH secretion, indicating unchanged secretagogue efficacy. In conclusion, inasmuch as E2 also attenuates inhibition of GH secretion by infused somatostatin and potentiates stimulation of GH secretion by GH-releasing peptide-2, we postulate that estrogenic steroids drive pulsatile GH production in part via mechanisms that include all three of GHRH, somatostatin, and putatively GH-releasing peptide/ghrelin signaling.

Using a continuous subcutaneous octreotide infusion to create constant supraphysiological somatostatinergic tone, we have previously shown that growth hormone (GH) pulse generation in women is independent of endogenous somatostatin (SRIH) declines. Generalization of these results to men is problematic, because GH regulation is sexually dimorphic. We have therefore studied nine healthy young men (age 26 ± 6 yr, body mass index 23.3 ± 1.2 kg/m2) during normal saline and octreotide infusion (8.4 μg/h) that provided stable plasma octreotide levels (764.5 ± 11.6 pg/ml). GH was measured in blood samples obtained every 10 min for 24 h. Octreotide suppressed 24-h mean GH by 52 ± 13% ( P = 0.016), GH pulse amplitude by 47 ± 12% ( P = 0.012), and trough GH by 39 ± 12% ( P = 0.030), whereas GH pulse frequency and the diurnal rhythm of GH secretion remained essentially unchanged. The response of GH to GH-releasing hormone (GHRH) was suppressed by 38 ± 15% ( P = 0.012), but the GH response to GH-releasing peptide-2 was unaffected. We conclude that, in men as in women, declines in hypothalamic SRIH secretion are not required for pulse generation and are not the cause of the nocturnal augmentation of GH secretion. We propose that GH pulses are driven primarily by GHRH, whereas ghrelin might be responsible for the diurnal rhythm of GH.

Technical, genetic, and clinical developments have unveiled a burgeoning array of novel effectors of GH secretion. The present appraisal of central neuroregulatory components of the somatotropic axis highlights a simplifying concept of ensemble control by the final common peptides, GH-releasing hormone (GHRH), GH-releasing peptide(s) (GHRP, ghrelin), and somatostatin. These potent signals act individually, antagonistically, and synergistically to direct pulsatile GH secretion. GHRH, GHRP/ghrelin, and somatostatin further adapt to autonegative feedback by GH and IGF-I. Estradiol modulates the impact of each of the primary peptidyl inputs; viz.: (i) enhances submaximally effective feedforward by discrete pulses of (injected) recombinant human GHRH-1,44-amide (as defined by increased agonistic potency and pituitary sensitivity); (ii) potentiates the submaximally stimulatory effects of GHRP-2, a hexapeptidyl mimetic of ghrelin; (iii) blunts dose-dependent inhibition of fasting GH secretion by somatostatin- 14; and (iv) relieves rhGH-enforced negative feedback on GHRP-2 (but not on basal, exercise, or GHRH)-stimulated GH secretion. The foregoing estrogenic activities collectively augment GH secretory burst mass by amplifying feedforward (via both GHRH and GHRP) and attenuating feedback (imposed by somatostatin and GH). Whether testosterone fully mimics the foregoing mechanistic actions of estradiol is not known. In conclusion, the present conceptual platform of tri-peptide-directed feedforward and GH/IGF-I-mediated feedback should aid in unraveling some of the complex regulatory dynamics targeted by sex-steroid hormones.

A decline in somatic function with aging in women is associated with a decrease in GH release and a loss of estrogen after menopause. As an initial step to establish a monkey model for the neuroendocrine mechanisms underlying somatopause and menopause, we have conducted three experiments in unrestrained aged (25.7-yr-old) and young (5.4-yr-old) female rhesus monkeys. GH release was pulsatile, and mean GH release and pulse amplitude were significantly lower in aged monkeys than in young monkeys. Injection of GHRH alone, GH-releasing peptide-2 alone, or the combination of both induced an increase in GH release in both age groups. The mean LH level, pulse amplitude, and baseline LH levels were significantly higher in aged animals than in young animals. Both estrogen and IGF-I levels were lower in aged than young monkeys. These results suggest that in female rhesus monkeys 1) there is a clear decline in circulating GH and IGF-I levels with aging; 2) GHRH and GH-releasing peptide-2 stimulate GH release synergistically; and 3) circulating LH levels increase as estrogen decreases with aging. These results indicate that the rhesus monkey is an excellent model for studies of the neuroendocrine mechanisms of aging. (J Clin Endocrinol Metab 87: 5160 –5167, 2002)

Objective Central hyposomatotrophism, hypothyroidism and hypogonadism are present concomitantly in men with prolonged critical illness. This study evaluated the impact of combined treatment with GH-releasing peptide-2 (GHRP-2), TRH and GnRH for 5 days compared with GHRP-2 + TRH and with GHRP-2 alone. Patients and design Thirty-three men with prolonged critical illness participated at baseline compared to 50 age- and body mass index (BMI)-matched controls. Patients were randomly assigned to 5 days of placebo (n = 7), GHRP-2 (1 microg/kg/h; n = 9), GHRP-2 + TRH infusion (1 + 1 microg/kg/h; n = 9) or pulsatile GnRH (0.1 microg/kg every 90 min) together with GHRP-2 + TRH infusion (n = 8). Measurements GH, TSH and LH secretion were quantified by deconvolution analysis of serum concentration time series obtained by sampling every 20 min from 2100 to 0600 h at baseline and on nights 1 and 5 of treatment. Serum concentrations of IGF-I, IGFBPs, thyroid hormones, gonadal and adrenal steroids, proinflammatory cytokines and selected metabolic and inflammation markers were measured daily. Results Patients revealed suppressed pulsatile GH, TSH and LH secretion in the face of low serum concentrations of IGF-I, IGFBP-3 and the acid-labile subunit (ALS) (P 80% from day 1 through day 3 (all P = 0.05). Ureagenesis was reduced by GHRP-2 + TRH + GnRH (P = 0.01) and by GHRP-2 + TRH (P = 0.009) but not by GHRP-2 alone. Serum OC levels were increased only by GHRP-2 + TRH + GnRH (P = 0.03), with a trend for GHRP-2 + TRH (P = 0.09), but not by GHRP-2 alone. On day 5, serum lactate levels and WBC count were increased by GHRP-2 infused alone and in combination with TRH but not by GHRP-2 + TRH + GnRH. Conclusions Coadministration of GHRP-2, TRH and GnRH reactivated the GH, TSH and LH axes in prolonged critically ill men and evoked beneficial metabolic effects which were absent with GHRP-2 infusion alone and only partially present with GHRP-2 + TRH. These data underline the importance of correcting the multiple hormonal deficits in patients with prolonged critical illness to counteract the hypercatabolic state.

High doses of GH, used to induce anabolism in prolonged critically ill patients, unexpectedly increased mortality. To further explore underlying mechanisms, a valid animal model is needed. Such a model is presented in this study. Seven days after arterial and venous cannulae placement, male New Zealand White rabbits were randomly allocated to a control or a critically ill group. To induce prolonged critical illness, a template controlled 15% deep dermal burn injury was imposed under combined general and regional (paravertebral) anesthesia. Subsequently, critically ill rabbits received supplemental analgesia and were parenterally fed with glucose, insulin, amino acids, and lipids. On d 1 and d 8 after randomization, acute and chronic spontaneous hormonal profiles of GH, TSH, and PRL secretion were obtained by sampling blood every 15 min for 7 h. Furthermore, GH, TSH, and PRL responses to an iv bolus of GH-releasing peptide 2 (GHRP-2) + TRH were documented on d 0, 1, and 8. Hemodynamic status and biochemical parameters were evaluated on d 0, 1, 3, 5, and 8, after which animals were killed and relative wet weight and water content of organs was determined. Compared with controls, critically ill animals exhibited transient metabolic acidosis on d 1 and weight loss, organ wasting, systolic hypertension, and pronounced anemia on d 8. On d 1, pulsatile GH secretion doubled in the critically ill animals compared with controls, and decreased again on d 8 in the presence of low plasma IGF-I concentrations from d 1 to d 8. GH responses to GHRP-2 + TRH were elevated on d 1 and increased further on d 8 in the critically ill animals. Mean TSH concentrations were identical in both groups on d 1 and 8, in the face of dramatically suppressed plasma T(4) and T(3) concentrations in the critically ill animals. PRL secretion was impaired in the critically ill animals exclusively on d 8. TSH and PRL responses to GHRP-2 and TRH were increased only on d 1. In conclusion, this rabbit model of acute and prolonged critical illness reveals several of the clinical, biochemical, and endocrine manifestations of the human counterpart.

The daunting regulatory complexity of neurotransmitter control of the mammalian somatotropic (GH) axis requires an initially simplified focus on distal interactive peptidyl pathways that converge o...

Background In Japan, growth hormone releasing peptide-2 (GHRP-2) is clinically used as a diagnostic agent for growth hormone secretion deficiency, but the therapeutic application of GHRP-2 has not been studied in anorexia nervosa. GHRP-2 reportedly exhibits agonistic action for ghrelin receptor and increases food intake. Methods We administered GHRP-2 to a patient with a 20-year history of anorexia nervosa to determine whether GHRP-2 treatment increases food intake and body weight. GHRP-2 was administered before every meal by an intranasal approach for 1 year. Results Although the patient reported a decreased fear of eating and decreased desire to be thin by our previous treatment, she was unable to increase food intake or body weight because of digestive tract dysfunction. Vomiting after meals caused by delayed gastric emptying and incurable constipation were prolonged, and sub-ileus and hypoglycemia were observed. GHRP-2 increased the feeling of hunger and food intake, decreased early satiety and improved hypoglycemia. The patient's body weight gradually increased by 6.7 kg (from 21.1 kg to 27.8 kg) in 14 months after starting GHRP-2 administration. The fatigability and muscle strength improved, and the physical and mental activities were also increased. No obvious side effects were observed after long-term intranasal administration of GHRP-2. Conclusions Patients with a long-term history of eating disorder occasionally recover from the psychological problems such as fear for obesity but remain emaciated. We believe that ghrelin agonists such as GHRP-2 may be promising agents for the effective treatments of severe anorexia nervosa in a chronic condition.

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

Unnatural GH-releasing peptide (GHRP) finally has evolved into the highly probable natural GHRP designated ghrelin. The beginning was in 1976, the receptor in 1996, and, at last, the endogenous hormone in 1999 (1–3). Despite the artificiality of the unnatural GHRPs, results of many talented investigators worldwide are responsible for sustaining the interest in the GHRP/GH secretagogue (GHS) system. The recent isolation and cloning of both rat and human ghrelin certainly are major milestones in the GHRP story. Ghrelin is a novel molecule with a molecular weight of 3314, which is very possibly more readily biodegradable than GHRH and, thus, may have been the reason GHRH was isolated first. It is a relatively charged, linear, non-C-terminal amidated 28 amino acid peptide with a significant number of chemical functional groups. Unique and a first in mammalian peptides is the posttranslation addition of a straight chain octanoyl group covalently linked to the hydroxyl group of Ser (3) via an ester linkage. The n-octanoyl group adds a hydrophobic property to the N terminus that may facilitate entry and distribution in the brain. Without octanoylation, ghrelin is biologically inactive. As do unnatural GHRPs, natural ghrelin binds with high affinity and specificity to the 7 transmembrane G protein-coupled receptor. To reveal the interrelationships of the chemistry of ghrelin and the many chemically diverse small unnatural peptidyl, partial peptidyl, and nonpeptidyl GHRP/GHSs should impart new understanding and dimension to the structure-activity relationship of peptides (4). Seemingly it is a strong precedent for the feasibility of developing bioactive small molecules that mimic the action of larger peptides. More important, in the future this may even include GHRH 1–44NH2. In addition, a limitation of the purely gene approach for the discovery of biologically active peptides is underscored by the chemical structure of ghrelin. Involvement in the regulation of GH secretion is the way the GHRP story began and mainly has proceeded, but over time other actions have become of special interest. The recent outstanding accomplishment of Kojima et al. (3) on the isolation and identification of ghrelin and secretion of ghrelin primarily from the fundus of the stomach in addition to the hypothalamus has allowed new and intriguing dimensions to arise on the possible overall physiological role of the ghrelin/GHRP system. The dual action on GH secretion and food intake in conjunction with the dual anatomical localization of ghrelin in the stomach and hypothalamus present an immediate question about the interdependency of these actions and the site of origin. After intracerebroventricular (icv) ghrelin administration to rats, Date et al. (5) demonstrated that GH release was dose-relatedly increased, including even low dosages of ghrelin. The effectiveness of the GHRPs and now ghrelin on increased food intake in rats and mice has been well established (6–8). After a single icv injection of ghrelin or neuropeptide Y (NPY) to rats, Wren et al. (7) found a similar effect of the two peptides on food intake that was prolonged in that the effect was sustained over 24 h. In this study, icv GHRP-6 also increased food intake but of shorter duration; however, this may be related to differences in peptide potency and dosage. After a single ip injection of ghrelin or GHRP to satiated rats, food intake was immediately increased during the first hour. As the authors stated, the orexigenic activity of ghrelin after peripheral administration was considerably important in that other hypothalamic peptides that alter food intake are ineffective by this route of administration. Despite the findings that GHRP/ghrelin receptors are coexpressed in NPY neurons, GHRP stimulates c-fos in these neurons, and that NPY is a potent orexigenic peptide, it remains questionable whether the GHRP/ghrelin effect on food intake is mediated via NPY (7–9). Particularly relevant, Kamegai et al. (10) reported that icv ghrelin increased the expression of the orexigenic agouti-related protein rather than NPY in the hypothalamic arcuate nucleus. The recent report by Takaya et al. (11) entitled “Ghrelin strongly stimulates GH release in humans” demonstrates the high potency and the extraordinary amount of GH released by ghrelin, which is also characteristic of the GHRP/GHSs. The amount of GH released is much greater than that induced by maximal dosages of GHRH, indicating a distinctive pharmacological action. In these studies in normal young men, the peak GH rise to iv bolus dosages of 0.2, 1, and 5 mg/kg was 43, 81, and 107, respectively, whereas the GH area under the curve was 2451, 6217, and 9581, respectively. Essentially, the same GH responses have been obtained with GHRP-2 at comparable iv bolus dosages in normal young Received October 25, 2000. Revision received December 8, 2000. Accepted December 20, 2000. Address correspondence and requests for reprints to: Cyril Y. Bowers, M.D., Endocrine Section, Tulane University School of Medicine, 1430 Tulane Avenue, SL53, New Orleans, Louisiana 70112-2699. 0021-972X/01/$03.00/0 Vol. 86, No. 4 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright © 2001 by The Endocrine Society

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 μg/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/m2) 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.

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.

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 3Ser-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 17β-estradiol (E2) 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 μg/kg by bolus iv infusion) vs. saline were evaluated by frequent blood s...

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.

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 μg/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.

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 μg/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.