Recombinant human IGF-I does not modify the ACTH and cortisol responses to hCRH and hexarelin, a peptidyl GH secretagogue, in humans

An inhibitory influence of insulin-like growth factor-I (IGF-I) on hypothalamus-pituitary-adrenal (HPA) axis has been hypothesized. In fact, it has been reported that the rhGH (recombinant human GH)-induced IGF-I increase inhibits both cortisol and GH response to MK-0677, a non-peptidyl GH secretagogue in animals. The aim of this study was to further clarify the inhibitory role, if any, of IGF-I on corticotroph function. We studied the effect of rhIGF-I (recombinant human IGF-I; 20 microg/kg s.c. at -180 min) or placebo on the ACTH and cortisol responses to hCRH (human CRH; 2.0 microg/kg i.v. at 0 min) or hexarelin (HEX; 2.0 microg/kg i.v. at 0 min), a peptidyl GHS, in normal young women. The effect of rhIGF-I on the GH response to HEX was also studied. The subjects were six normal young women [age: 26-35 yr; body mass index (BMI): 19-23 kg/m2] in their early follicular phase. The results showed that after s.c. rhIGF-I administration, circulating IGF-I levels increased approximately 77%, peaking at -60 min and persisting similar up to +120 min. The mean ACTH, cortisol and GH concentrations did not change from -180 to 0 min when evaluated after both placebo or rhIGF-I. CRH and HEX induced similar ACTH (peak vs baseline, mean+/-SE: 47.5+/-10.9 vs 21.3+/-3.0 pg/ml and 30.3+/-6.9 vs 19.2+/-3.8 pg/ml, respectively; p<0.04) and cortisol responses (177.5+/-5.4 vs 109.3+/-10.3 microg/l and 149.4+/-12.3 vs 119.8+/-16.4 microg/l, respectively, p<0.04). RhIGF-I pretreatment did not modify the ACTH and cortisol responses to hCRH (46.0+/-13.8 pg/ml and 181.1+/-16.9 microg/l, respectively) as well as those to HEX (28.8+/-5.0 pg/ml and 144.1+/-16.2 microg/l, respectively). On the other hand, the GH response to HEX was clearly reduced by rhIGF-I (23.9+/-4.7 vs 64.7+/-14.8 microg/l, p<0.05). Our findings show that rhIGF-I-induced increase of circulating IGF-I levels exerts negative feedback action on somatotroph secretion, while it does not modify the corticotroph and the adrenal responsiveness to CRH or hexarelin.     

Effects of the combined administration of hexarelin, a synthetic peptidyl GH secretagogue, and hCRH on ACTH, cortisol and GH secretion in patients with Cushing's disease

Hexarelin (HEX) is a peptidyl GH secretagogue (GHS) which markedly stimulates GH release but, like other GHS, possesses also CNS-mediated ACTH- and cortisol-releasing activity. Interestingly, the stimulatory effect of HEX on ACTH and cortisol release is exaggerated and higher than that of hCRH in patients with Cushing's disease (CD). To further clarify the mechanisms by which HEX stimulates the activity of hypothalamo-pituitary-adrenal (HPA) axis in man, in 6 patients with CD (6 women, 38-68 yr old) and in 7 control subjects (CS, 7 women, 22-29 yr old) we studied the effects of HEX (2.0 microg/kg i.v.) and/or hCRH (2.0 microg/kg i.v.) on ACTH and cortisol (F) secretion. The GH responses to HEX alone and combined with hCRH were also studied in all subjects. Basal ACTH and F levels in CD were higher than in CS (66.3+/-5.1 vs 16.5+/-0.6 pg/ml and 217.8+/-18.5 vs 134.4+/-4.6 microg/l, respectively; p<0.02). In CS, the ACTH and F responses to HEX, evaluated as deltaAUC (mean+/-SE: 128.7+/-39.2 pg x min/ml and 328.5+/-93.2 microg x min/l, respectively) were lower, though not significantly, than those after hCRH (375.8+/-128.4 pg x min/ml and 1714.2+/-598.0 microg x min/l, respectively), though the peak ACTH and F responses to both stimuli were similar. The co-administration of HEX and hCRH had an additive effect on both ACTH (1189.6+/-237.2 pg x min/ml) and F secretion (3452.9+/-648.6 microg x min/l). In fact, the ACTH and F responses to HEX+/-hCRH were significantly higher (p<0.01) than those elicited by single stimuli. In CD, HEX induced ACTH and F responses (3603.8+/-970.7 pg x min/ml and 10955.9+/-6184.6 microg x min/l, respectively) clearly higher (p<0.002) than those in CS. The HEX-induced ACTH and F responses in CD were higher, though not significantly, than those recorded after hCRH (1432.7+/-793.5 pg x min/ml and 4832.7+/-2146.5 microg x min/l, respectively). On the other hand, the hCRH-induced ACTH and F responses in CD were similar to those in CS. In CD, the coadministration of HEX and hCRH had an additive effect on ACTH (8035.7+/-1191.1 pg x min/ml) but not on F (10985.4+/-3900.8 microg x min/l) secretion. In fact, the ACTH, but not the F response to HEX+hCRH was significantly higher (p<0.02) than that elicited by single stimuli. In conclusion, the present study demonstrates that in patients with Cushing's disease as well as in subjects control Hexarelin and hCRH have an additive effect on ACTH secretion. Considering that, at least in humans, differently from hCRH, GHS have no interaction with AVP, our present findings further agree with the hypothesis that the ACTH-releasing activity of GHS is, at least partially, independent of CRH-mediated mechanisms.     

Tyr-Ala-Hexarelin, a synthetic octapeptide, possesses the same endocrine activities of Hexarelin and GHRP-2 in humans

Hexarelin (HEX) and GHRP-2 are two synthetic hexapeptides, superanalogs of GHRP-6, belonging to GH secretagogue (GHS) family. GHS act via specific receptors at both the pituitary and the hypothalamic level to stimulate GH release both in animals and in humans. However, GHS also possess significant PRL- and ACTH/cortisol-releasing activity. Tyr-Ala-HEX as well as Tyr-Ala-GHRP-6 are, in turn, synthetic octapeptides generally used to perform binding studies because of their easy iodination. However, their endocrine activities have never been studied in humans. To clarify the endocrine activities of Tyr-Ala-HEX, in 7 young adult volunteers we compared the effects of the maximal effective dose of HEX (2.0 microg/kg i.v.) or GHRP-2 (2.0 microg/kg i.v.) with the same one of Tyr-Ala-HEX on GH, PRL, ACTH and cortisol levels. Basal GH, PRL, ACTH and cortisol levels in all testing sessions were similar. The administration of placebo did not modify hormonal levels. HEX and GHRP-2 administration induced the well known strong GH response (Cmax, mean+/-SE: 77.3+/-6.0 and 74.1+/-12.1 microg/l; AUC, mean+/-SE: 2596.7+/-251.1 and 2480.0+/-343.6 microg*min/l). These responses were similar to that induced by Tyr-Ala-HEX (63.7+/-18.5 microg/l; 1986.6+/-622.4 microg*min/l). Moreover, HEX, GHRP-2 and Tyr-Ala-HEX had the same significant stimulatory effect on PRL (14.9+/-2.5, 12.3+/-2.0 and 10.0+/-1.5 microg/l; 497.8+/-61.8, 480.4+/-66.9 and 415.8+/-58.5 microg*min/l), ACTH (48.0+/-10.1, 51.4+/-10.6 and 44.9+/-12.2 pg/ml; 1531.6+/-235.7, 1586.7+/-277.0 and 1338.1+/-164.5 pg*min/ml) and cortisol (179.9+/-10.0, 181.2+/-14.1 and 149.7+/-20.1 microg/l; 8465.0+/-406.6, 8689.2+/-788.1 and 6295.2+/-797.0 microg*min/l). Also the mean Tmax of the endocrine responses to HEX, GHRP-2 and Tyr-Ala-HEX were similar. In conclusion, the present results demonstrate that in humans Tyr-Ala-HEX is a GH secretagogue as potent as HEX and GHRP-2, two GHRP-6 superanalogs. Tyr-Ala-HEX also shares with HEX and GHRP-2 the same PRL- ACTH- and cortisol-releasing activity.     

Endocrine activities of alexamorelin (Ala-His-d-2-methyl-Trp-Ala-Trp-d-Phe-Lys-NH2), a synthetic GH secretagogue, in humans

OBJECTIVE: Peptidyl and non-peptidyl synthetic GH secretagogues (GHS) possess significant GH-, prolactin (PRL)- and ACTH/cortisol-releasing activity after i.v. and even p.o. administration, acting via specific hypothalamo-pituitary receptors in both animals and humans. The hexapeptide hexarelin (HEX) is a paradigmatic GHS whose activities have been widely studied in humans. The heptapeptide Ala-His-d-2-methyl-Trp-Ala-Trp-d-Phe-Lys-NH(2) (alexamorelin, ALEX) is a new synthetic molecule which inhibits GHS binding in vitro, but its endocrine activity has never been studied in humans. DESIGN: In six young adults we studied the effects of 1.0 and 2.0 microgram/kg i.v. ALEX or HEX on GH, PRL, ACTH, cortisol and aldosterone levels and those of 20mg p.o. ( approximately 300 microgram/kg) on GH levels. RESULTS: Basal GH, PRL, ACTH, cortisol and aldosterone levels in all testing sessions were similar. ALEX and HEX (1.0 and 2.0 microgram/kg i.v.) induced the same dose-dependent increase of GH and PRL levels. Both ALEX and HEX induced a dose-dependent increase of ACTH and cortisol levels. The ACTH and cortisol responses to the highest ALEX dose were significantly higher than those after HEX. Aldosterone levels significantly increased after both i.v. ALEX doses, but not after HEX. The GH response to 20mg p.o. ALEX was higher, though not significantly, than that to the same HEX dose. CONCLUSION: ALEX, a new GHS, shows the same GH-releasing activity as HEX. On the other hand, ALEX seems endowed with an ACTH-releasing activity more marked than that of HEX; this evidence could explain the significant increase of aldosterone levels after its i.v. administration.     

Interaction between glucagon and hexarelin, a peptidyl GH secretagogue, on somatotroph and corticotroph secretion in humans

OBJECTIVE: Glucagon administration stimulates both somatotroph and corticotroph secretion in humans, although this happens only if glucagon is administered by the intramuscular route and not by the intravenous route. On the other hand, GH secretagogues (GHS) strongly stimulate GH and also possess ACTH-releasing activity. DESIGN AND METHODS: To clarify the mechanisms underlying the stimulatory effects of both glucagon and GHS on somatotroph and corticotroph secretion, we studied the GH, ACTH and cortisol responses to glucagon (GLU, 0.017 mg/kg i.m.) and Hexarelin, a peptidyl GHS (HEX, 2.0 microg/kg i.v.) given alone or in combination in 6 normal young volunteers (females, aged 26-32 years, body mass index 19.7-22.5 kg/m). RESULTS: GLU administration elicited a clear increase in GH (peak vs baseline, mean+/-S.E.M.: 11.6+/-3.4 vs 3. 3+/-0.7 microg/l, P<0.02), ACTH (11.6+/-3.3 vs 4.1+/-0.3 pmol/l, P<0. 02) and cortisol (613.5+/-65.6 vs 436.9+/-19.3 nmol/l, P<0.05) levels. HEX induced a marked increase in GH levels (55.7+/-19.8 vs 3. 7+/-1.9 microg/l, P<0.005) and also significant ACTH (5.7+/-1.1 vs 3. 4+/-0.6 pmol/l, P<0.01) and cortisol (400.2+/-31.4 vs 363.4+/-32.2 nmol/l, P<0.05) responses. The GH area under the curve (AUC) after HEX was clearly higher than after GLU (1637.3+/-494.0 vs 479.1+/-115. 7 microg/l/120 min, P<0.04) while HEX and GLU coadministration had a true synergistic effect on GH release (3243.8+/-687.5 microg/l/120 min, P<0.02). The ACTH and cortisol AUCs after HEX were lower (P<0. 02) than those after GLU (208.3+/-41.3 vs 426.3+/-80.9 pmol/l/120 min and 18 874.5+/-1626.1 vs 28 338.5+/-2430.7 nmol/l/120 min respectively). The combined administration of HEX and GLU had an effect which was less than additive on both ACTH (564.02+/-76.5 pmol/l/120 min) and cortisol (35 424.6+/-5548.1 nmol/l/120 min) secretion. CONCLUSIONS: These results show that the intramuscular administration of glucagon releases less GH but more ACTH and cortisol than Hexarelin. The combined administration of glucagon and Hexarelin has a true synergistic effect on somatotroph secretion but a less than additive effect on corticotroph secretion; these findings suggest that these stimuli act via different mechanisms to stimulate somatotrophs while they could have a common action on the hypothalamo-pituitary-adrenal axis.     

Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone

An endogenous ligand for the GH secretagogue-receptor (GHS-receptor) has recently been isolated, from both the rat and the human stomach, and named ghrelin. It is a 28-amino-acid peptide showing a unique structure with an n-octanoyl ester at its third serine residue, which is essential for its potent stimulatory activity on somatotroph secretion. In fact, it has been demonstrated that ghrelin specifically stimulates GH secretion from both rat pituitary cells in culture and rats in vivo. The aim of the present study was to test the GH-releasing activity of ghrelin in humans and to compare it with that of GHRH and hexarelin (HEX), a nonnatural peptidyl GHS, which possesses strong GH-releasing activity but also significantly stimulates PRL, ACTH, and cortisol secretion. To clarify the mechanisms of action underlying the GH-releasing activity of ghrelin in humans, its interaction with GHRH and HEX was also studied. Seven normal young volunteers (7 men; 24-32 yr old; body mass index, 20-24 kg/m(2)) were studied. All subjects underwent the administration of ghrelin, HEX, and GHRH-29 (1.0 microg/kg i.v. at 0 min) as well as placebo (2 mL isotonic saline i.v. at 0 min). Six subjects also underwent the combined administration of ghrelin and GHRH or HEX. Blood samples were taken every 15 min from -15 up to +180 min. GH levels were assayed at each time point in all sessions; PRL, ACTH, cortisol, and aldosterone levels were also assayed after administration of ghrelin and/or HEX. Ghrelin administration induced a prompt and marked increase in circulating GH levels (Cmax, mean +/- SEM, 92.1 +/- 16.7 microg/L; area under the curve, 1894.9 +/- 347.8 microg/L.h). The GH response to ghrelin was clearly higher (P < 0.01) than the one recorded after GHRH (26.7 +/- 8.7 microg/L; 619.6 +/- 174.4 microg/L.h) and even significantly higher (P < 0.05) than after HEX (68.4 +/- 14.7 microg/L; 1546.9 +/- 380.0 microg/L x h). Ghrelin administration also induced an increase in PRL, ACTH, and cortisol levels; these responses were higher (P < 0.05) than those elicited by HEX. A significant increase in aldosterone levels was recorded after ghrelin but not after HEX. The endocrine responses to ghrelin were not modified by the coadministration of HEX. On the other hand, the coadministration of ghrelin and GHRH had a real synergistical effect (P < 0.05) on GH secretion (133.6 +/- 22.5 microg/L; 3374.3 +/- 617.3 microg/L x h). In conclusion, ghrelin, a natural ligand of GHS-receptor, exerts a strong stimulatory effect on GH secretion in humans, releasing more GH than GHRH and even more than a nonnatural GHS such as HEX. Ghrelin, as well as HEX, also stimulates lactotroph and corticotroph secretion. Ghrelin shows no interaction with HEX, whereas it has a synergistical effect with GHRH on GH secretion. Thus, ghrelin is a new hormone playing a major role in the control of somatotroph secretion in humans, and its effects are imitated by nonnatural GHS.     

Alprazolam, a benzodiazepine, blunts but does not abolish the ACTH and cortisol response to hexarelin, a GHRP, in obese patients

GH secretagogues (GHS) act on specific receptors at the pituitary and hypothalamic level and possess potent GH-releasing activity but also stimulate prolactin (PRL), ACTH and cortisol (F) secretion. However, hyperactivity of the HPA axis in obesity has been reported. The objective of this study was to clarify the endocrine activity of GHS in obesity. In nine obese patients (obese OB), 9 F, age, (34.8 +/- 3.7 y, body mass index (BMI), 35.0 +/- 2.2 kg/m2; WHR, 0.9 +/- 0.02), 14 controls (normal subjects, NS), 14 F, 30.4 +/- 0.9 y, 20.0 +/- 0.4 kg/m2), we studied the ACTH, F and GH responses to hexarelin (HEX, 2.0 microg/kg), a peptidyl GHS, alone and preceded by alprazolam (ALP, 0.02 mg/kg), and a benzodiazepine which has an inhibitory effect on corticotroph secretion. The HEX-induced ACTH response in OB was higher than that in n.s., but this difference did not attain statistical significance. In n.s. the HEX-induced ACTH response was abolished by ALP (P < 0.03) which, however, only blunted that in OB (P < 0.02). The GH response to HEX in OB was lower (P < 0.02) than that in n.s.. ALP blunted the GH response to HEX in n.s. (P < 0.03) while it did not modify that in OB. The GABAergic activation by alprazolam abolishes the ACTH response to hexarelin in normal subjects, while it only blunts that in obese subjects. Moreover, alprazolam blunts the GH response to hexarelin in normal but not in obese subjects. Thus, obese patients show partial refractoriness to the inhibitory effect of alprazolam on both corticotroph and somatotroph function.     

Effects of cortistatin-14 and somatostatin-14 on the endocrine response to hexarelin in humans

Cortistatin (CST)-14, a neuropeptide with high structural homology with somatostatine (SS)-14, binds all SS receptor subtypes but also shows activities not shared by SS. CST and SS are often co-expressed in the same neurons but are regulated by different stimuli. Moreover, CST, but not SS, also binds the GH secretagogue (GHS) receptor. We compared the effects of CST-14 and SS-14 (2.0 microg/kg/h i.v. from -30 to +90 min) on the endocrine response to hexarelin (HEX, 1.0 microg/kg i.v. at 0 min), a synthetic GHS, in 6 normal volunteers [age (mean+/-SEM): 28.7+/-2.9 yr; body mass index: 23.4+/-0.8 kg/m2]. GH, PRL, ACTH, cortisol, insulin and glucose levels were measured at each time point. CST-14 inhibited spontaneous GH secretion [delta-areas under curves (-AUC): -83.57+/-44.8 vs 2.3+/-2.7 microg/l/h, p<0.01] to the same extent of SS-14 (-186.1+/-162.9 microg/l/h, p<0.01). CST-14 as well as SS-14 also inhibited insulin secretion (p<0.05). The GH response to HEX was similarly inhibited by either CST-14 (AUC: 3814.1+/-924.2 vs 1212.9+/-379.8 microg/l/h, p<0.05) or SS-14 (720.9+/-158.6 microg/l/h, p<0.05). HEX significantly increased PRL, ACTH and cortisol levels but these responses were not modified by either CST-14 or SS-14. The effects of CST-14 and SS-14 on insulin and glucose levels were not modified by HEX. In conclusion, this study shows that CST-14 inhibits the GH response to HEX to the same extent of SS-14. Like SS-14, CST-14 also inhibits insulin secretion but both do not modify the stimulatory effects of HEX on lactotroph and corticotroph secretion. Thus, CST-14 exerts full SS-14 activity in humans.     

The relationship of saline-induced changes in vasopressin secretion to basal and corticotropin-releasing hormone-stimulated adrenocorticotropin and cortisol secretion in man

To investigate the effect of endogenous arginine vasopressin (AVP) on ACTH secretion, normal subjects were given infusions of either hypertonic saline (HS) or isotonic saline (NS) combined with human corticotropin-releasing hormone (CRH) or placebo. Basal plasma AVP was 2.3 +/- 0.3 (+/- SE) pg/ml, did not change with NS treatment, and rose to 5.4 +/- 0.6 pg/ml during HS infusion (P less than 0.01). Both basal and CRH-stimulated plasma ACTH and cortisol concentrations increased during HS infusion. Peak plasma ACTH and cortisol levels were 11.4 +/- 1.5 pg/ml and 8.6 +/- 0.8 micrograms/dl, respectively, during the HS (plus placebo) infusion. During the NS (plus placebo) infusion, plasma ACTH and cortisol gradually declined to 6.8 +/- 0.5 pg/ml and 2.6 +/- 0.4 micrograms/dl. The timing of the rise in ACTH during the HS infusion paralleled the rise in AVP. When an iv dose of 1 microgram/kg CRH was administered during the saline infusions, peak plasma ACTH and cortisol levels were 27.7 +/- 6.3 pg/ml and 17.5 +/- 1.0 micrograms/dl, respectively, during the HS infusion and 15.6 +/- 1.7 pg/ml and 13.4 +/- 1.2 micrograms/dl during the NS infusion. When the areas under the hormone response curves were compared, CRH stimulated ACTH and cortisol secretion to a greater extent than did HS (P less than 0.05). The hormonal stimulation due to combined CRH and hypertonic saline was greater than that attributable to either factor alone (P less than 0.025), but was not different than the sum of the effects of the individual factors. These results indicate that increases in endogenous AVP produced by HS are associated with increases in both basal and CRH-stimulated ACTH and cortisol release. The effect of HS appears to be additive to but not consistently synergistic with the effect of CRH.     

Inhibition of ACTH response to oral and intravenous metyrapone by antiserotoninergic treatment in man.

Plasma ACTH levels after oral and iv metyrapone administration were studied in 7 and 5 healthy women respectively both under basal conditions and after a 4-day treatment with metergoline, a specific antiserotoninergic agent. In 3 additional women, the effects of methysergide, another antiserotoninergic drug, on the plasma ACTH rise induced by oral metyrapone, were evaluated. A significant lowering of the plasma ACTH levels attained after either oral or iv metyrapone was observed following metergoline administration: 149+/-64.3 vs 239+/-49.1 pg/ml (mean peak values), P less than 0.05 in the oral test and 331+/-19.7 vs 221+/-19.5 pg/ml, P less than 0.02 in the iv test. The fall of plasma cortisol caused by metyrapone was comparable before and after the antiserotoninergic treatment. An interference of metergoline in the ACTH radioimmunoassay was also excluded. After metergoline administration, a slight reduction in the baseline plasma ACTH values was noted: 79+/-7.7 vs 67+/-7.7 pg/ml (NS). A decrease, however not statistically significant, of the metyrapone-induced plasma ACTH elevation occured after methysergide administration: 421+/-150.7 vs 344+/-135.1 pg/ml. These results can be interpreted as indicating that antiserotoninergic treatment caused an inhibition of hypophysial ACTH release in response to metyrapone. Caution is recommended, however, before concluding, on the basis of these findings, that serotonin as such plays a physiological stimulating role on ACTH secretion.     

Endocrine responses to ghrelin in adult patients with isolated childhood-onset growth hormone deficiency

OBJECTIVE: Ghrelin, a 28 amino acid acylated peptide, is a natural ligand of the GH secretagogues (GHS) receptor (GHS-R), which is specific for synthetic GHS. Similar to synthetic GHS, ghrelin strongly stimulates GH secretion but also displays significant stimulatory effects on lactotroph and corticotroph secretion. It has been hypothesized that isolated GH deficiency (GHD) could reflect hypothalamic impairment that would theoretically involve defect in ghrelin activity. PATIENTS: In the present study, we verified the effects of ghrelin (1 microg/kg i.v.) on GH, PRL, ACTH and cortisol levels in adult patients with isolated severe GHD [five males and one female, age (mean +/- SEM) 24.7 +/- 2.6 years, BMI 25.7 +/- 2.7 kg/m2]. In all patients, the GH response to insulin-induced hypoglycaemia (ITT, 0.1 IU regular insulin i.v.) and GH releasing hormone (GHRH) (1 microg/kg i.v.) + arginine (ARG, 0.5 g/kg i.v.) was also studied. The hormonal responses in GHD were compared with those in age-matched normal subjects (NS, seven males, age 28.6 +/- 2.9 years, BMI 22.1 +/- 0.8 kg/m2). RESULTS: IGF-I levels in GHD were markedly lower than in NS (69.8 +/- 11.3 vs. 167.9 +/- 19.2 microg/l, P < 0.003). Ghrelin administration induced significant increase in GH, PRL, ACTH and cortisol levels in all GHD. In GHD, the GH response to ghrelin was higher (P < 0.05) than that to GHRH + ARG, which, in turn, was higher (P < 0.05) than that to ITT (9.2 +/- 4.1 vs. 5.3 +/- 1.7 vs. 1.4 +/- 0.4 microg/l). These GH (1 microg/l = 2 mU/l) responses in GHD were markedly lower (P < 0.0001) than those in NS (ghrelin vs. GHRH + ARG vs. ITT 92.1 +/- 16.7 vs. 65.3 +/- 8.9 vs. 17.7 +/- 3.5 microg/l). In GHD, the highest individual peak GH response to ghrelin was markedly lower than the lowest peak GH response in NS (28.5 vs. 42.9 microg/l). GHD and NS showed overlapping PRL (1 microg/l = 32 mU/l) (10.0 +/- 1.4 vs. 14.9 +/- 2.2 microg/l), ACTH (22.3 +/- 5.3 vs. 18.7 +/- 4.6 pmol/l) and cortisol responses (598.1 +/- 52.4 vs. 486.9 +/- 38.9 nmol/l). CONCLUSIONS: This study shows that ghrelin is one of the most powerful provocative stimuli of GH secretion, even in those patients with isolated severe GHD. In this condition, however, the somatotroph response is markedly reduced while the lactotroph and corticotroph responsiveness to ghrelin is fully preserved, indicating that this endocrine activity is fully independent of mechanisms underlying the GH-releasing effect. These results do not support the hypothesis that ghrelin deficiency is a major cause of isolated GH deficiency but suggest that ghrelin might represent a reliable provocative test to evaluate the maximal GH secretory capacity provided that appropriate cut-off limits are assumed.     

Impact of two or three daily subcutaneous injections of hexarelin, a synthetic growth hormone (GH) secretagogue, on 24-h GH, prolactin, adrenocorticotropin and cortisol secretion in humans

OBJECTIVE: To extend the insights on the action of GH secretagogues (GHS) on pituitary function, we studied the impact of intermittent daily s.c. administration of a peptidyl GHS, hexarelin (HEX), on 24-h GH, PRL, ACTH and cortisol release in healthy volunteers. DESIGN: We investigated the impact of two or three times daily s.c. administration of a short-acting peptidyl GHS, the hexapeptide HEX (1.5 microg/kg) on 24-h GH, PRL, ACTH and cortisol secretion (sampling every 20 min) in six normal young men. To monitor possible down-regulation, the effect of 1 microg/kg i.v. HEX at the end of each 24-h sampling period was studied. METHODS: Multi-parameter deconvolution analysis was used to quantitate pulsatile GH, PRL, ACTH and cortisol secretion and estimate the corresponding hormone half-lives. Complementary to deconvolution analysis, approximate entropy was used as a scale- and model-independent statistic to quantify the serial orderliness or pattern regularity of hormone measurements. RESULTS: Mean and integrated (24-h) serum GH concentrations were increased from baseline values to the same extent by two and three HEX injections. Both HEX schedules equally increased GH secretory burst mass (but not burst frequency), mean daily GH production rate, GH half-life and irregularity of GH release patterns. No change occurred in the secretion of IGF-I, PRL, ACTH and cortisol. Intravenous HEX at the end of each spontaneous 24-h profile induced a significant rise in GH, PRL, ACTH and cortisol. Prior HEX administration blunted the GH response, abolished that of ACTH and cortisol and did not modify the PRL increase. CONCLUSIONS: The study showed that two or three daily s.c. injections of HEX augmented 24-h GH secretion equally, amplifying selectively GH secretory pulse mass without altering lactotroph and corticotroph secretion. IGF-I levels were not modified by these 1-day HEX treatment schedules.     

The endocrine response to acute ghrelin administration is blunted in patients with anorexia nervosa, a ghrelin hypersecretory state

OBJECTIVE: Ghrelin, a gastric-derived natural ligand of the GH secretagogue (GHS)-receptor (GHS-R), strongly stimulates GH secretion but also possesses other neuroendocrine actions, stimulates food intake and modulates the endocrine pancreas and energy homeostasis. Ghrelin secretion is negatively modulated by food intake. Similarly, glucose and also insulin probably exert an inhibitory effect on ghrelin secretion. Fasting ghrelin levels are reduced in obesity, elevated in anorexia nervosa and restored by weight recovery. The chronic elevation of circulating ghrelin levels in anorexia suggested the hypothesis of an alteration of the sensitivity to the orexigenic action of ghrelin in this condition. The aim of this study was to define the endocrine actions of ghrelin in patients with anorexia nervosa. DESIGN: We enrolled nine women with anorexia nervosa of restricter type [AN; age (mean +/- SEM) 24.2 +/- 1.8 years; body mass index (BMI) 14.7 +/- 0.4 kg/m2] and seven normal young women in their early follicular phase as control group (NW; age 30.6 +/- 3.1 years; BMI 20.3 +/- 0.5 kg/m2). MEASUREMENTS: In all the subjects we studied the GH, PRL, ACTH, cortisol, insulin and glucose responses to acute ghrelin administration (1.0 microg/kg as i.v. bolus). The GH response to GHRH (1.0 microg/kg as i.v. bolus) and basal ghrelin and IGF-I levels were also evaluated in all the subjects. RESULTS: Basal morning ghrelin and GH levels in AN (643.6 +/- 21.3 ng/l and 10.4 +/- 0.5 microg/l, respectively) were higher (P < 0.05) than in NW (233.5 +/- 14.2 ng/l and 0.7 +/- 0.7 microg/l, respectively). However, IGF-I levels in AN (145.3 +/- 10.9 microg/l) were lower (P < 0.05) than in NW (325.4 +/- 12.6 microg/l). The GH response to GHRH in AN was higher (P < 0.05) than that in NW, but in AN the GH response to ghrelin was lower (P < 0.05) than that in NW. In AN and NW ghrelin also induced similar increases (P < 0.05) in PRL, ACTH and cortisol levels. Ghrelin administration was followed by significant increase in glucose levels in NW (P < 0.05) but not in AN. CONCLUSIONS: This study demonstrates that anorexia nervosa, a clinical condition of ghrelin hypersecretion, shows a specific reduction in the GH response to ghrelin, despite the hyper-responsiveness to GHRH administration. The impaired GH response to ghrelin in anorexia nervosa agrees with previous evidence of blunted GH response to synthetic GH secretagogues and could reflect desensitization of the GHS receptor induced by the chronic elevation of ghrelin levels in this pathological state.     

Acute cardiovascular and hormonal effects of GH and hexarelin, a synthetic GH-releasing peptide, in humans.

Reduced cardiac mass and performances are present in GH deficiency and are counteracted by rhGH replacement. GH and IGF-I possess specific myocardial receptors and have been reported able to exert an acute inotropic effect. Synthetic GH secretagogues (GHS) possess specific pituitary and hypothalamic but even myocardial receptors. In 7 male volunteers, we studied cardiac performance by radionuclide angiocardiography after iv administration of rhGH or hexarelin (HEX), a peptidyl GHS. The administration of rhGH or HEX increased circulating GH levels to the same extent (AUC: 1594.6+/-88.1 vs 1739.3+/-262.2 microg/l/min for 90 min) while aldosterone and catecholamine levels did not change; HEX, but not rhGH, significantly increased cortisol levels. Left ventricular ejection fraction (LVEF), mean blood pressure (MBP) and heart rate (HR) were unaffected by rhGH (62.4+/-2.1 vs 62.1+/-2.3%, 90.6+/-3.4 vs 92.0+/-2.5 mm Hg, 62.3+/-1.8 vs 66.7+/-2.7 bpm). HEX increased LVEF (70.7+/-3.0 vs 64.0+/-1.5%, p<0.03) without significant changes in MBP and HR (92.8+/-4.7 vs 92.4+/-3.2 mm Hg, 63.1+/-2.1 vs 67.0+/-2.9 bpm). LVEF significantly raised at 15 min, peaked at 30 min and lasted up to 60 min after HEX. These findings suggest that in man, the acute administration of Hexarelin exerts a short-lasting, positive inotropic effect. This effect seems GH-independent and might be mediated by specific GHS myocardial receptors.     

Pituitary-adrenal responses to CRH and insulin hypoglycemia in patients with idiopathic GH deficiency

It is well known that the activation of the hypothalamus-pituitary-adrenal axis (HPA) by insulin-induced hypoglycemia (IIH) is more potent and multifactorial than that caused by CRH administration. In this study we compared the clinical value of both tests in assessing the integrity of the HPA system. Plasma ACTH and cortisol responses to oCRH (1 microgram/kg iv) and IIH (insulin 0.1 U/kg iv, glycemia less than 40 mg/dl) were compared in 15 patients with idiopathic GH deficiency. The CRH-induced mean ACTH response was lower, but not significantly, in patients than in controls (peak: 8.8 +/- 1.7 vs 13.4 +/- 2.2 pmol/l), while the mean cortisol response was significantly lower than in normals (peak: 585.7 +/- 49.5 vs 764.5 +/- 52.2 nmol/l, p less than 0.005). Plasma ACTH and cortisol responses to IIH were significantly lower than in normal subjects (peak: 22.3 +/- 5.3 vs 35.8 +/- 5.2 pmol/l, p less than 0.05 and peak: 566.5 +/- 55 vs 803 +/- 38.5 nmol/l, p less than 0.02, respectively). Both in controls and in patients the CRH-induced mean ACTH response was significantly lower (p less than 0.02) than that after insulin, while cortisol peaks were not different. In conclusion, in patients with GH deficiency the impairment of ACTH secretion is not evident in basal condition, but it is disclosed after appropriate dynamic tests. It is confirmed that insulin hypoglycemia is a more potent stimulus than CRH for ACTH release.     

ACUTE DEXAMETHASONE SUPPRESSION OF ACTH SECRETION STIMULATED BY HUMAN CORTICOTROPHIN RELEASING HORMONE, AVP AND HYPOGLYCAEMIA

In order to obtain more insight into the mechanisms regulating endogenous ACTH secretion in humans we studied the inhibitory effect of acute i.v. dexamethasone administration on ACTH release under various conditions. Six male volunteers were subjected to six different protocols. After combined i.v. injection of 100 micrograms corticotrophin releasing hormone (CRH) and 100 micrograms growth hormone releasing hormone (GRH) there was the expected rise in ACTH (area under the curve, 1053 +/- 204 (SE) (pmol/l) min) and cortisol (59788 +/- 10098 (nmol/l) min) rise which was suppressed by prior i.v. injection of 2 mg dexamethasone (ACTH: 444 +/- 63 (pmol/l) min; cortisol: 28528 +/- 2152 (nmol/l) min). Insulin hypoglycaemia (IH) led to a more pronounced ACTH and cortisol rise compared with CRH (6307 +/- 817 (pmol/l) min and 82080 +/- 21934 (nmol/l) min, respectively) which was not completely suppressed by prior pretreatment with dexamethasone (ACTH, 580 +/- 103 (pmol/l) min; cortisol: 55649 +/- 5821 (nmol/l) min). Combined AVP/CRH injection (10 IU/100 micrograms) after pretreatment with dexamethasone (344 +/- 41 (pmol/l) min for ACTH; 32832 +/- 3173 (nmol/l) min for cortisol) could not reproduce the ACTH secretion following IH after pretreatment with dexamethasone (579 +/- 103 (pmol/l) min for ACTH and 55649 +/- 5821 (nmol/l) min for cortisol). In all subjects a saline control with 2 mg dexamethasone was performed. These findings confirm the acute inhibitory effect of glucocorticoids on CRH-stimulated ACTH secretion. Since CRH-induced ACTH secretion is almost completely abolished by administration of dexamethasone the ACTH rise following IH after dexamethasone can not be mediated by endogenous CRH alone. Moreover, since the addition of AVP to CRH (after dexamethasone suppression) could not reproduce the ACTH rise during IH after dexamethasone pretreatment, an additional, yet unknown factor stimulating ACTH secretion may be involved. In the same protocols, no significant difference could be observed comparing IH and GRH induced GH secretion (4948 +/- 1172 (mU/l) min vs 3596 +/- 820 (mU/l) min, NS); furthermore, in contrast to results obtained by chronic steroid administration, acute i.v. dexamethasone pretreatment did not affect IH or GRH-induced GH secretion (4110 +/- 666 (mU/l) min vs 2916 +/- 462 (mU/l) min, NS). The GRH-stimulated GH secretion (3596 +/- 820 (mU/l) min) was not suppressed by prior intravenous treatment with dexamethasone (2916 +/- 504 (mU/l) min, NS).     

Effect of metyrapone pretreatment on adrenocorticotropin secretion induced by corticotropin-releasing hormone in normal subjects and patients with Cushing's disease

To examine the influence of endogenous cortisol on the ACTH response to CRH, we compared ACTH secretion during CRH tests before and after metyrapone administration in 9 normal subjects and 12 patients with Cushing's disease. The administration of 4.5 g metyrapone (750 mg, orally, every 4 h) resulted in a decrease in basal (pre-CRH) plasma cortisol levels and an increase in basal plasma ACTH levels in both normal subjects and Cushing's patients. The pretreatment with metyrapone significantly blunted the increase in plasma cortisol levels and markedly enhanced ACTH secretion after iv injection of 100 micrograms human CRH. The peak ACTH levels during CRH test before and after metyrapone administration were 8 +/- 1 and 58 +/- 8 pmol/L, respectively, in normal subjects (P less than 0.01) and 26 +/- 5 and 50 +/- 11 pmol/L, respectively, in Cushing's patients (P less than 0.05). Although the basal and peak ACTH levels as well as delta ACTH (peak ACTH - basal ACTH) during the CRH test before metyrapone administration were significantly higher in Cushing's disease patients than in normal subjects (P less than 0.01), no such difference was observed between the 2 groups after metyrapone administration. The results clearly indicate that the endogenous cortisol levels greatly influence the ACTH response to CRH, and that the CRH test as commonly performed does not allow a correct evaluation of potential responsiveness of normal pituitaries and Cushing's adenomas to CRH.     

Prolactin-releasing peptide releases corticotropin-releasing hormone and increases plasma adrenocorticotropin via the paraventricular nucleus of the hypothalamus

Intracerebroventricular (ICV) injection of prolactin-releasing peptide (PrRP) is known to increase plasma adrenocorticotropin (ACTH) and cause c-fos expression in the hypothalamic paraventricular nucleus (PVN). We hypothesize that this is the site at which PrRP acts to increase plasma ACTH. We have used ICV injection and direct intranuclear injection of PrRP into the PVN to investigate the sites important in the stimulation of ACTH release in vivo. To investigate the mechanism of action by which PrRP increases ACTH, we have used primary culture of pituitary cells and measured neuropeptide release from in vitro hypothalamic incubations. ICV administration of PrRP increased plasma ACTH 10 min post-injection (PrRP 5 nmol 81.0 +/- 23.5 pg/ml vs. saline 16.8 +/- 14.1 pg/ml, p < 0.05). Intra-PVN injection of PrRP increased ACTH 5 min post-injection (PrRP 1 nmol 22.9 +/- 5.0 pg/ml vs. saline 10.3 +/- 1.4 pg/ml, p < 0.05). This effect continued until 40 min post-injection (PrRP 1 nmol 9.9 +/- 1.5 pg/ml vs. saline 6.2 +/- 0.5 pg/ml, p < 0.05). In vitro PrRP (1-100 nmol/l) did not effect basal or corticotropin-releasing hormone (CRH)-stimulated ACTH release from dispersed anterior pituitary cells. PrRP increased hypothalamic release of CRH (PrRP 100 nmol/l 1.4 +/- 0.2 nmol/explant vs. the basal 1.1 +/- 0.2 nmol/explant, p < 0.05) but not arginine vasopressin. PrRP also stimulated neuropeptide Y release (PrRP 100 nmol/l 56.5 +/- 11.8 pmol/explant vs. basal 24.0 +/- 1.9 pmol/explant, p < 0.01), a neuropeptide known to stimulate the hypothalamo-pituitary-adrenal axis. Our data suggest that in vitro PrRP does not have a direct action on the corticotrope but increases plasma ACTH via the PVN and this effect involves the release of hypothalamic neuropeptides including CRH and neuropeptide Y.     

Evidence that cortisol inhibits basal adrenocorticotropin secretion in the sheep fetus by 0.70 gestation

To ascertain if reductions in fetal plasma cortisol cause increases in fetal plasma ACTH, we treated pregnant ewes or their fetuses with aminoglutethimide (10 mg/kg BW) and metyrapone (20 mg/kg BW) and measured the hormonal responses with RIAs. When given to fetuses (n = 9) at 0.90 +/- 0.01 gestation (term-145 days), the steroid synthesis inhibitors reduced fetal plasma cortisol from 35.1 +/- 11.9 to 18.5 +/- 6.2 ng/ml (P less than 0.01) and plasma ACTH increased from 37 +/- 7 to 189 +/- 74 pg/ml (P less than 0.02). Thus, late in gestation cortisol from the fetal adrenal suppresses basal fetal ACTH secretion. Blockade of steroid biosynthesis in pregnant ewes carrying intact fetuses at 0.76 +/- 0.02 gestation (n = 11) or adrenalectomized fetuses at 0.81 +/- 0.01 gestation (n = 6) also reduced cortisol and increased ACTH in fetal plasma. In intact fetuses cortisol declined from 9.4 +/- 2.0 to 3.6 +/- 0.9 ng/ml (P less than 0.05), and ACTH increased from 46 +/- 8 to 183 +/- 67 (P less than 0.01); cortisol declined in adrenalectomized fetuses from 2.1 +/- 0.4 to 1.1 +/- 0.3 ng/ml (P less than 0.01), and ACTH increased from 106 +/- 13 to 400 +/- 104 pg/ml (P less than 0.01). Cortisol infusions into intact and adrenalectomized fetuses prevented both the decline in steroid concentration caused by the biosynthesis inhibitors given to the ewe and the increase in fetal plasma ACTH concentration. These data indicate that reductions in plasma cortisol in adrenalectomized fetuses or intact fetuses at a time in development when the fetal adrenal produces little cortisol cause compensatory increases in fetal plasma ACTH concentration. The simplest explanation for these observations is that from approximately 0.70 gestation, basal fetal ACTH secretion is tonically inhibited by cortisol circulating in fetal plasma. This cortisol can originate from sources other than the fetal adrenal.     

The GH-releasing effect of ghrelin,a natural GH secretagogue,is only blunted by the infusion of exogenous somatostatin in humans

OBJECTIVE: Ghrelin, a 28-amino-acid peptide purified from the stomach and showing a unique structure with an n-octanoyl ester at the serine 3 residue, is a natural ligand of the GH secretagogue (GHS) receptor (GHS-R). Ghrelin strongly stimulates GH secretion in both animals and humans, showing a synergistic effect with GH-releasing hormone (GHRH) but no interaction with synthetic GHS. However, the activity of ghrelin as well as that of non-natural GHS is not fully specific for GH; ghrelin also induces a stimulatory effect on lactotroph and corticotroph secretion, at least in humans. DESIGN: To further clarify the mechanisms underlying the GH-releasing activity of this natural GHS, we studied the effects of somatostatin (SS, 2.0 microg/kg/h from -30 to +90 min) on the endocrine responses to ghrelin (1.0 microg/kg i.v. at 0 min) in seven normal young male volunteers [age (mean +/- SEM) 28.6 +/- 2.9 years; body mass index (BMI) 22.1 +/- 0.8 kg/m2]. In the same subjects, the effect of SS on the GH response to GHRH (1.0 microm/kg i.v. at 0 min) was also studied. MEASUREMENTS: Blood samples were taken every 15 min from -30 up to +120 min. GH levels were assayed at each time point in all sessions; PRL, ACTH and cortisol levels were assayed after ghrelin administration alone and during SS infusion. RESULTS: The GH response to ghrelin (hAUC0'-->120' 2695.0 +/- 492.6 microg min/l) was higher (P < 0.01) than that after GHRH (757.1 +/- 44.1 microg min/l). SS infusion almost abolished the GH response to GHRH (177.0 +/- 37.7 microg min/l, P < 0.01); the GH response to ghrelin was inhibited by SS (993.8 +/- 248.5 microg min/l, P < 0.01) but GH levels remained higher (P < 0.05) than with GHRH. Ghrelin induced significant increases in PRL, ACTH and cortisol levels and these responses were not modified by SS. CONCLUSIONS: Ghrelin, a natural GHS-R ligand, exerts a strong stimulatory effect on GH secretion in humans and this effect is only blunted by an exogenous somatostatin dose which almost abolishes the GH response to GHRH. The stimulatory effect of ghrelin on lactotroph and corticotroph secretion is refractory to exogenous somatostatin, indicating that these effects occur through pathways independent of somatostatinergic influence.