Ghrelin as a treatment for amyotrophic lateral sclerosis.

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.

Modulating Effects of Progesterone on Spontaneous Nocturnal and Ghrelin-Induced GH Secretion in Postmenopausal Women.

BACKGROUND: Oral administration of estradiol (E2) generally increases GH secretion in postmenopausal women. Oral administration of E2 is associated with a decrease in IGF-1, whereas parenteral or transdermally administered E2 may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E2 levels stimulate GH and that P4 modulates E2-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 × 200 mg/d IM), E2 (5 mg IM) and oral placebo, and E2 IM and oral micronized P4. Outcome measures were overnight GH secretion (10 hours), stimulated (ghrelin, 0.3 µg/kg IV bolus) GH secretion, and CT-estimated visceral fat. RESULTS: Intramuscular E2 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 E2 (average, 7.20 ± 2.14 and 9.58 ± 1.97 µg/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 µg/L ± 0.29 (P = 0.025) and 2.76 ± 1.04 and 3.95 µg/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, ß = -0.040 ± 0.016). CONCLUSIONS: Low-range physiological E2 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.

Aromatized Estrogens Amplify Nocturnal Growth Hormone Secretion in Testosterone-Replaced Older Hypogonadal Men.

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 (E2) 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 E2 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: E2 addback during aromatase inhibition increased basal (P = 0.046), pulsatile (P = 0.020), and total (P = 0.018) GH secretion by 60% to 70%. E2 did not potentiate GH secretory stimuli. Logarithmically transformed pulsatile GH secretion correlated strongly and positively with concurrent E2 concentrations overall (P = 0.028) and under anastrozole treatment (P = 0.005). Conclusion: E2 administration in older men transdermally stimulates overnight pulsatile GH secretion. The exact site of E2 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.

Effects of Toremifene, a Selective Estrogen Receptor Modulator, on Spontaneous and Stimulated GH Secretion, IGF-I, and IGF-Binding Proteins in Healthy Elderly Subjects.

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.

Endogenous Estrogen Regulates Somatostatin-Induced Rebound GH Secretion in Postmenopausal Women.

BACKGROUND: Systemic concentrations of T, estradiol (E2), GH, IGF-1, and IGF binding protein-3 decline in healthy aging individuals. Conversely, T and E2 stimulate GH and IGF-1 production in hypogonadal patients. HYPOTHESIS: Because E2 stimulates GH secretion, putatively via the nuclear estrogen receptor-α and E2 and GH fall with menopause, we postulated that diminished endogenous E2 contributes to low GH output in older women. LOCATION: The study was conducted at the Mayo Center for Clinical and Translational Science. STUDY DESIGN: This was a randomized, double-blind, controlled study in 60 healthy postmenopausal women treated with the following: 1) double placebo; 2) anastrozole, a potent inhibitor of aromatase-enzyme activity, which mediates E2 synthesis from T; and/or 3) fulvestrant, a selective estrogen receptor-α antagonist. METHODS: GH pulse generation was quantified by frequent GH sampling before and after short-term iv somatostatin infusion, thought to induce hypothalamic GHRH-mediated rebound-like GH secretion. RESULTS: On anastrozole, E2 fell from 3.1 ± 0.35 pg/mL to 0.36 ± 0.04 pg/mL, and estrone from 13 ± 1.4 pg/mL to 1.9 ± 0.01 pg/mL (P < .001) by mass spectrometry. Estrogen values were unchanged by fulvestrant. T concentrations did not change. One-hour peak GH rebound after somatostatin infusion declined markedly during both estrogen-deprivation schedules (P < .001). Mean (150 min) maximal GH rebound decreased comparably (P < .001). Measures of GH rebound correlated negatively with computed tomography-estimated abdominal visceral fat (all P < .05). CONCLUSION: These data suggest a previously unrecognized dependence of hypothalamo-pituitary GH regulation on low levels of endogenous estrogen after menopause.

One-year intranasal application of growth hormone releasing peptide-2 improves body weight and hypoglycemia in a severely emaciated anorexia nervosa patient.

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.

Estrogen-like potentiation of ghrelin-stimulated GH secretion by fulvestrant, a putatively selective ER antagonist, in postmenopausal women.

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/m(2)). 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).

Estradiol regulates GH-releasing peptide's interactions with GH-releasing hormone and somatostatin in postmenopausal women.

OBJECTIVE: Estrogen stimulates pulsatile secretion of GH, via mechanisms that are largely unknown. An untested hypothesis is that estradiol (E2) drives GH secretion by amplifying interactions among GH-releasing hormone (GHRH), somatostatin (SS), and GH-releasing peptide (GHRP). DESIGN: The design comprised double-blind randomized prospective administration of transdermal E2 vs placebo to healthy postmenopausal women (n=24) followed by pulsatile GHRH or SS infusions for 13 h overnight with or without continuous GHRP2 stimulation. METHODS: End points were mean concentrations, deconvolved secretion, and approximate entropy (ApEn; a regularity measure) of GH. RESULTS: By generalized ANOVA models, it was observed that E2 vs placebo supplementation: i) augmented mean (13-h) GH concentrations (P=0.023), GHRH-induced pulsatile GH secretion over the first 3 h (P=0.0085) and pulsatile GH secretion over the next 10 h (P=0.054); ii) increased GHRP-modulated (P=0.022) and SS-modulated (P<0.001) GH ApEn; and iii) did not amplify GHRH/GHRP synergy during pulsatile GH secretion. By linear regression, E2 concentrations were found to be positively correlated with GH secretion during GHRP2 infusion (P=0.022), whereas BMI was found to be negatively correlated with GH secretion during GHRH (P=0.006) and combined GHRH/GHRP (P=0.015) stimulation. E2 and BMI jointly determined triple (combined l-arginine, GHRH, and GHRP2) stimulation of GH secretion after saline (R²=0.44 and P=0.003) and pulsatile GHRH (R²=0.39 and P=0.013) infusions. CONCLUSION: In summary, in postmenopausal women, E2 supplementation augments the amount (mass) and alters the pattern (regularity) of GH secretion via interactions among GHRH, SS, GHRP, and BMI. These outcomes introduce a more complex model of E2 supplementation in coordinating GH secretion in aging women.

Short-term estradiol supplementation potentiates low-dose ghrelin action in the presence of GHRH or somatostatin in older women.

CONTEXT: Ghrelin is a potent gastric-derived GH-releasing peptide. How ghrelin interacts with sex steroids, GHRH, and somatostatin (SS) is not known. OBJECTIVE: 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 17ß-estradiol (E2) (n=12) at the Center for Translational Science Activities in a randomized double-blind prospective study. METHODS: 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. RESULTS: 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. SUMMARY: 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.

Differential pulsatile secretagogue control of GH secretion in healthy men.

Pulsatile growth hormone (GH) secretion putatively reflects integrated regulation by GH-releasing hormone (GHRH), somatostatin (SST), and GH-releasing peptide (GHRP). GHRH and SST secretion is itself pulsatile. However, how GHRH and SST pulses act along with GHRP to jointly determine pulsatile GH secretion is unclear. Moreover, how testosterone (T) modulates such interactions is unknown. These queries were assessed in a prospectively randomized, placebo-controlled double-blind cohort comprising 26 healthy older men randomized to testosterone (T) vs. placebo supplementation. Pulses of GHRH, SST, or saline were infused intravenously at 90-min intervals for 13 h, along with either continuous saline or ghrelin analog (GHRP-2). The train of pulses was followed by a triple stimulus (combined l-arginine, GHRH, and GHRP-2) to estimate near-maximal GH secretion over a final 3 h. Testosterone vs. placebo supplementation doubled pulsatile GH secretion during GHRH pulses combined with continuous saline (GHRH/saline) (P < 0.01). Pulsatile GH secretion correlated positively with T concentrations (270-1,170 ng/dl) in the 26 men during saline pulses/saline (P = 0.015, R(2) = 0.24), GHRH pulses/saline (P = 0.020, R(2) = 0.22), and combined GHRH pulses/GHRP-2 (P = 0.016, R(2) = 0.25) infusions. Basal nonpulsatile GH secretion correlated with T during saline pulses/GHRP-2 drive (P = 0.020, R(2) = 0.16). By regression analysis, pulsatile GH secretion varied negatively with body mass index (BMI) during saline/GHRP-2 infusion (P = 0.001, R(2) = 0.36), as well as after the triple stimulus preceded by GHRH/GHRP-2 (P = 0.013, R(2) = 0.23). Mean (10-h) GH concentrations under GHRP-2 were predicted jointly by estradiol (positively) and BMI (negatively) (P < 0.001, R(2) = 0.520). These data indicate that estradiol, T, and BMI control pulsatile secretagogue-specific GH-regulatory mechanisms in older men.

History to the discovery of ghrelin.

The most important initial historical time points in the development of the enlarging ghrelin system were 1973, 1976, 1982, 1984, 1990, 1996, 1998, and 1999. At these respective times, the following occurred sequentially: isolation of somatostatin, discovery of unnatural growth-hormone-releasing peptides (GHRPs), isolation of growth-hormone-releasing hormone (GHRH), hypothesis of a new natural GHRP different from GHRH, GHRP+GHRH synergism in humans, discovery of the growth hormone secretagogue GHS/GHRP receptor, cloning of the receptor, and finally, isolation and identification of the new natural endogenous GHRP ghrelin. To understand the pharmacology and probably also the physiological regulation of growth hormone (GH) secretion, an important finding was that GHRP increased pulsatile GH secretion in children as well as normal younger and older men and women. This requires endogenous GHRH secretion, even though GHRP alone substantially releases GH from the pituitary in vitro without the addition of GHRH. Unnatural GHRP gave rise to natural GHRP ghrelin because of many talented researchers worldwide. GHRP was first envisioned to be an analog of GHRH but, from comparison of the activity of GHRH and GHRPs between 1982 and1984, it was hypothesized to reflect the activity of a new hormone regulator of GH secretion yet to be isolated and identified. Intravenous bolus GHRP releases more GH than GHRH in humans, but the reverse occurs in vitro. GHRPs are pleiotropic peptides with major effects on GH, nutrition, and metabolism, especially as an additional hormone in combination with GHRH as a new regulator of pulsatile GH secretion. The first indication of pleiotropism was an increase of food intake by GHRP. A major reason for the prolonged initial interest in the GHRPs has been its similar, yet different and complementary, action with GHRH on GH regulation and secretion. Particularly noteworthy is the variable chemistry of the GHRPs. They consist of three major chemical classes, including peptides, partial peptides, and nonpeptides, and all probably act via the same receptor and cellular mechanisms. Generally, most GHRPs have been active by all routes of administration, intravenously (iv), subcutaneously (sc), orally, intranasally, and intracerebroventricularly (icv), which supports their possible broad future clinical utility. From evolutionary studies starting with the zebrafish, the natural receptor and hormone have been present for hundreds of years, underscoring the fundamental evolutionary and functional importance of the ghrelin system. GHRPs were well established to act directly on both the hypothalamus and pituitary several years before the GHS receptor assay (Howard et al., 1996; Smith et al., 1996; Van der Ploeg et al., 1998). Finally, the ghrelin chemical isolation and identification was accomplished surprisingly from the stomach, which is the major site but not the only site, for example, the hypothalamus (Bowers, 2005; Kojima et al., 1999; Sato et al., 2005). Ghrelin was isolated and identified by Kojima and Kangawa et al. in 1999. A primary action of GHRPs continues to concern GH secretion and regulation, but increasingly this has included direct and indirect effects on nutrition and metabolism as well as a variety of other actions which may be pharmacological and/or physiological. Possible continuing and expanding roles of this new hormonal receptor include the central nervous system as well as the cardiovascular, renal, gastrointestinal, pancreatic, immunological, and anti-inflammatory systems. Our basic and clinical studies have mainly involved effects on GH regulation and secretion and this relationship to metabolism. So far in our studies, the actions of GHRPs and ghrelin on GH secretion and regulation in rats and probably in humans have generally been the same. A current objective is the incorporation of ghrelin into the diffuse endocrine hormonal system especially via GH.

Sex steroids, GHRH, somatostatin, IGF-I, and IGFBP-1 modulate ghrelin's dose-dependent drive of pulsatile GH secretion in healthy older men.

CONTEXT: Ghrelin is a potent endogenous stimulator of GH secretion. However, clinical factors that regulate ghrelin dose-responsiveness are incompletely defined. OBJECTIVE: The aim of the study was to test the multipathway hypothesis that testosterone (T) and estradiol, GHRH, and somatostatin (SS) jointly modulate ghrelin's action. DESIGN/PARTICIPANTS/SETTING: Healthy older men (n = 21) participated in a double-blind, prospectively randomized, placebo (Pl)-controlled study in a Clinical Translational Research Center. INTERVENTIONS: To create a range of sex-steroid milieus, men received leuprolide + Pl (n = 10) or leuprolide + T addback (n = 11). Sixteen to 21 d later, subjects received three separate randomly ordered overnight constant i.v. infusions of saline, GHRH, and SS. Interactions between the peptide clamp and ghrelin were tested by superimposed injections of four randomly ordered bolus i.v. doses of ghrelin (0.03, 0.135, 0.60, and 2.7 µg/kg). GH was measured every 10 min, and GH responses were assessed by nonlinear dose-response analysis. Linear associations were assessed by stepwise regression. OUTCOME MEASURES/RESULTS: The descending numerical order of ghrelin efficacy (maximal GH secretory-burst mass; micrograms/liter) was 107 (GHRH + Pl), 104 (GHRH + T), 73 (saline + T), 73 (SS + T), 60 (saline + Pl), and 52 (SS + Pl) [means], wherein SS + T exceeded SS + Pl. GHRH and IGF binding protein-1 augmented, whereas IGF-I attenuated ghrelin potency. Age and IGF-I decreased ghrelin/GHRH synergy. Ghrelin sensitivity was independent of interventions. CONCLUSIONS: These studies introduce composite regulatory effects of sex hormones, GHRH, SS, IGF binding protein-1, and IGF-I on ghrelin dose-responsiveness, suggesting multipathway modulation of GH-secretagogue action.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient little mice.

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormone-releasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/lit mice, which represent a model of GH deficiency arising from mutated growth hormone-releasing hormone-receptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormonereleasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormonereceptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Regulated recovery of pulsatile growth hormone secretion from negative feedback: a preclinical investigation.

Although stimulatory (feedforward) and inhibitory (feedback) dynamics jointly control neurohormone secretion, the factors that supervise feedback restraint are poorly understood. To parse the regulation of growth hormone (GH) escape from negative feedback, 25 healthy men and women were studied eight times each during an experimental GH feedback clamp. The clamp comprised combined bolus infusion of GH or saline and continuous stimulation by saline GH-releasing hormone (GHRH), GHRP-2, or both peptides after randomly ordered supplementation with placebo (both sexes) vs. E(2) (estrogen; women) and T (testosterone; men). Endpoints were GH pulsatility and entropy (a model-free measure of feedback quenching). Gender determined recovery of pulsatile GH secretion from negative feedback in all four secretagog regimens (0.003 ≤ P ≤ 0.017 for women>men). Peptidyl secretagog controlled the mass, number, and duration of feedback-inhibited GH secretory bursts (each, P < 0.001). E(2)/T administration potentiated both pulsatile (P = 0.006) and entropic (P < 0.001) modes of GH recovery. IGF-I positively predicted the escape of GH secretory burst number and mode (P = 0.022), whereas body mass index negatively forecast GH secretory burst number and mass (P = 0.005). The composite of gender, body mass index, E(2), IGF-I, and peptidyl secretagog strongly regulates the escape of pulsatile and entropic GH secretion from autonegative feedback. The ensemble factors identified in this preclinical investigation enlarge the dynamic model of GH control in humans.

Gender, sex-steroid, and secretagogue-selective recovery from growth hormone-induced feedback in older women and men.

CONTEXT: GH negatively regulates its own secretion. How gender, sex steroids, and secretagogues modulate GH autofeedback is not known. HYPOTHESIS/OBJECTIVE: Supplementation with sex steroids and/or a peptidyl secretagogue will enhance the escape of GH from autoinhibition, thus framing a mechanism for amplifying pulsatile GH secretion. SUBJECTS AND SETTING: Ten healthy postmenopausal women and 10 comparably aged men participated at the Clinical-Translational Science Unit. DESIGN/INTERVENTIONS: Randomly ordered, double-blind, prospective crossover treatment with placebo vs. testosterone (men) or placebo vs. estradiol (women). Autofeedback was imposed by an iv pulse of GH. Recovery of feedback inhibition was quantified during constant infusion of saline, GHRH, or GH-releasing peptide-2 (three peptide categories). OUTCOMES/RESULTS: During negative feedback, total (integrated) GH recovery depended upon gender (P = 0.017), sex hormone (P < 0.001), and peptide category (P < 0.001). Mechanistic analysis revealed that feedback-suppressed nadir GH concentrations were determined by sex-steroid treatment (P = 0.018) but not by gender (P = 0.444). Peak GH escape was controlled by both treatment (P = 0.004) and gender (P = 0.003). Nadir GH and peak GH during feedback were enhanced by GHRH or GHRP-2 (P < 0.001 for both). Gender × peptide (P = 0.012 for nadir GH), treatment × peptide (P < 0.001 total and peak GH), and gender × treatment (P = 0.017 nadir GH) regulated GH recovery interactively. CONCLUSION: Gender, sex-steroid supplementation, and secretagogue type confer distinct feedback-rescuing effects, introducing a new level of complexity in the control of pulsatile GH regulation.

Complex regulation of GH autofeedback under dual-peptide drive: studies under a pharmacological GH and sex steroid clamp.

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 (E(2); 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. E(2)/T exposure elevated nadir GH concentrations during saline infusion (P = 0.003), whereas dual-peptide infusion did so independently of T/E(2) 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). E(2)/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.

Integrating GHS into the Ghrelin System.

Oligopeptide derivatives of metenkephalin were found to stimulate growth-hormone (GH) release directly by pituitary somatotrope cells in vitro in 1977. Members of this class of peptides and nonpeptidyl mimetics are referred to as GH secretagogues (GHSs). A specific guanosine triphosphatate-binding protein-associated heptahelical transmembrane receptor for GHS was cloned in 1996. An endogenous ligand for the GHS receptor, acylghrelin, was identified in 1999. Expression of ghrelin and homonymous receptor occurs in the brain, pituitary gland, stomach, endothelium/vascular smooth muscle, pancreas, placenta, intestine, heart, bone, and other tissues. Principal actions of this peptidergic system include stimulation of GH release via combined hypothalamopituitary mechanisms, orexigenesis (appetitive enhancement), insulinostasis (inhibition of insulin secretion), cardiovascular effects (decreased mean arterial pressure and vasodilation), stimulation of gastric motility and acid secretion, adipogenesis with repression of fat oxidation, and antiapoptosis (antagonism of endothelial, neuronal, and cardiomyocyte death). The array of known and proposed interactions of ghrelin with key metabolic signals makes ghrelin and its receptor prime targets for drug development.

Secretagogue type, sex-steroid milieu, and abdominal visceral adiposity individually determine secretagogue-stimulated cortisol secretion.

DESIGN: While 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 5alpha-reductases type I and type II), or testosterone and anastrozole (to block aromatase) in a double-blind, placebo-controlled, prospectively randomized design. METHODS: Subjects 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). RESULTS: By 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 (P<0.001) as follows: triple stimulus (868+/-27)>GHRP-2 (616+/-42)>saline=SS=GHRH (grand mean 420+/-21). After GHRP-2 injection, pulsatile cortisol secretion increased with age (R(2)=0.16, P=0.012). After the triple stimulus, pulsatile cortisol secretion correlated i) inversely with serum 5alpha-dihydrotestosterone (DHT) concentrations (R(2)=0.53, P=0.026) and ii) directly with computerized tomography-estimated AVF (R(2)=0.11, P=0.038). CONCLUSION: Age, 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.

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

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