J. Ramunni

Age-related Variations in the Neuroendocrine Control, More Than Impaired Receptor Sensitivity, Cause The Reduction in the GH-releasing Activity of GHRPs in Human Aging

The mechanisms underlying the reduction in the GH-releasing activity of GHRPs in aging are still unclear. Aim of our study was to verify in man whether age-related impairment of the neurohormonal control of GH secretion and/or receptor alterations are involved in the reduced GH response to GHRPs in aging. To this goal, in 16 normal elderly subjects (E, 66–81 yr) and 12 young controls (Y, 24–28 yr) we studied the effects of 1.0, 2.0 and 3.0 μg/kg iv Hexarelin (HEX), a synthetic hexapeptide, or GHRH, as well as the interaction among HEX (2.0 μg/kg), GHRH (2.0 μg/kg) and arginine (ARG, 0.5 gr/kg) on GH secretion. In Y the GH response to increasing doses of HEX (1.0 vs. 2.0 vs. 3.0 μg/kg; AUC0;v–120 ± SEM: 1728.4 ± 406.4 vs. 2265.9 ± 298.4 vs. 2934.3 ± 482.2 μg//L/h, p < 0.05 for 1.0 vs. 2.0 μg/kg) and GHRH (649.6 ± 111.4 vs. 792.2 ± 117.6 vs. 1402.6 ± 363.0 μg/L/h) showed a progressive increase. Two μg/kg HEX and 1 μg/kg GHRH were the maximal effective doses. Similarly, in E the GH response to increasing doses of HEX (336.7 ± 50.0 vs. 742.8 ± 157.9 vs. 1205.1 ± 178.1 μg/L/h, p < 0.05 for 1.0 vs. 2 μg/kg, p < 0.001 for 1.0 vs. 3.0 μg/kg and p < 0.03 for 2.0 vs. 3.0 μg/kg) and GHRH (183.8 ± 27.3 vs. 260.9 ± 17.3 vs. 356.1 ± 46.3 μg/L/h, p < 0.005 for 1.0 vs. 3.0 μg/kg and p < 0.05 for 2.0 vs. 3.0 μg/kg) showed a progressive increase. In E the GH response to 3 μg/kg HEX or GHRH were clearly higher than those to 2 μg/kg. However, at each dose the GH responses to HEX or GHRH in E were lower (p < 0.05) than those in Y. In Y the GH response to HEX + GHRH was synergistical (4259.2 ± 308.0 μg/L/h, p < 0.05). ARG strikingly potentiated the GHRH-induced GH rise (2640.8 ± 273.6 μg/L/h, p < 0.01) but not the HEX-induced one (2371.7 ± 387.2 μg/L/h) as well as the synergistical effect of HEX and GHRH (4009.1 ± 360.8 μg/L/h). In E the GH response to HEX and GHRH was still synergistical (1947.7 ± 306.0 μg/L/h, p < 0.05) but these responses were lower than those in young (p < 0.01). On the other hand, in E ARG restored the GH response to GHRH (1858.9 ± 172.8 μg/L/h, p < 0.01) and even those to HEX (2069.5 ± 528.7 μg/L/h, p < 0.01) and HEX + GHRH (4406.0 ± 1079.2 μg/L/h, p < 0.05). Our present results indicate that the impairment of GHRP and GHRH receptor activity may have a role in the reduction of the somatotrope responsiveness in aging. However, the age-related reduction in the GH-releasing activity of GHRPs seems mainly dependent on age-related variations in the neural control, i.e. concomitant GHRH hypoactivity and somatostatinergic hyperactivity.

Relationships between dehydroepiandrosterone‐sulphate and anthropometric, metabolic and hormonal variables in a large cohort of obese women

The aim of the present study was to measure dehydroepiandrosterone‐sulphate (DHEA‐S) levels in obesity and assess the relationships between DHEA‐S and anthropometric, metabolic and hormonal variables.

Effects of Dexamethasone and Alprazolam, a Benzodiazepine, on the Stimulatory Effect of Hexarelin, a Synthetic GHRP, on ACTH, Cortisol and GH Secretion in Humans

Hexarelin (HEX) is a synthetic GHRP which acts on specific receptors at both the pituitary and the hypothalamic level to stimulate GH release both in animals and in humans. Like other GHRPs, HEX possesses also acute ACTH and cortisol-releasing activity similar to that of hCRH. The mechanisms underlying the stimulatory effect of GHRPs on hypothalamo-pituitary-adrenal (HPA) axis are still unclear, although a CNS-mediated action has been demonstrated. In 6 normal healthy young women (26–34 years) we studied the effects on ACTH and cortisol secretion of HEX (2.0 µg/kg i.v. at 0 min) alone and preceded by dexamethasone (DEXA, 1 mg p.o. at 23.00 h on the previous night) or alprazolam (ALP, 0.02 mg/kg p.o. at –90 min), a benzodiazepine which binds to GABA receptors and possesses CRH-mediated inhibitory activity on HPA axis. ACTH and cortisol secretion after saline administration as well as the GH response to HEX alone and preceded by DEXA or ALP were also studied. HEX administration elicited an increase in ACTH (peak vs. baseline, mean ± SEM: 28.0 ± 6.7 vs. 11.7 ± 2.2 pg/ml, p < 0.05) and cortisol secretion (162.6 ± 15.0 vs. 137.7 ± 12.6 µg/l, p < 0.05). DEXA pretreatment strongly inhibited basal ACTH (3.2 ± 0.7 pg/ml, p < 0.01) and cortisol levels (11.3 ± 2.5 µg/l, p < 0.001) and abolished the ACTH and cortisol responses to HEX (3.6 ± 0.9 pg/ml, p < 0.01 and 10.7 ± 2.0 µg/l, p < 0.001), respectively. On the other hand, ALP pretreatment did not significantly modify basal ACTH (7.9 ± 2.0 pg/ml) and cortisol levels (127.6 ± 14.5 µg/l) but abolished the HEX-induced ACTH and cortisol secretions (8.6 ± 2.4 pg/ml, p < 0.05 and 111.0 ± 6.0 µg/l, p < 0.05), respectively. ACTH and cortisol levels after HEX when preceded by ALP overlapped with those recorded during saline. HEX induced a clear GH response (peak at 15 min vs. baseline: 65.5 ± 20.5 vs. 2.2 ± 0.7 µg/l, p < 0.03) which was blunted by ALP (peak at 15 min: 21.5 ± 5.5 µg/l, p < 0.05) while it was not modified by DEXA pretreatment (78.7 ± 7.6 µg/l). In conclusion, our present data demonstrate that the ACTH- and cortisol-releasing effect of HEX is abolished by either dexamethasone or alprazolam, a benzodiazepine, which is even able to blunt the GH-releasing activity of the hexapeptide. These findings suggest that, in physiological conditions, the stimulatory effect of GHRPs on HPA axis is sensitive to the negative glucocorticoid feedback and could be mediated by GABAergic mechanisms; the latter seem also involved in the GH-releasing activity of GHRPs.

Hexarelin, a Synthetic Growth-Hormone Releasing Peptide, Shows No Interaction with Corticotropin-Releasing Hormone and Vasopressin on Adrenocorticotropin and Cortisol Secretion in Humans

Hexarelin (HEX) is a synthetic growth-hormone-releasing peptide (GHRP) which acts via specific receptors at both the pituitary and the hypothalamic level to stimulate GH release both in animals and in man. Like other GHRPs, HEX possesses also significant prolactin- and adrenocorticotropin (ACTH) cortisol-releasing activity, but the mechanisms underlying these effects are even less clear. To clarify the mechanisms by which HEX stimulates the pituitary-adrenal axis in man, in 7 healthy young volunteers we studied the effects of HEX (2.0 microg/kg i.v.) and/or human corticotropin-releasing hormone (hCRH; 2.0 microg/kg i.v.) and/or arginine vasopressin (AVP; 0.17 U/kg i.m.) on ACTH and cortisol secretion. The GH responses to HEX alone and combined with hCRH and/or AVP were also studied. HEX increased ACTH and cortisol secretion (peak, mean +/- SEM: 26.3 +/- 5.1 vs. 15.8 +/- 3.1 pg/ml and 145.0 +/- 11.4 vs. 131.7 +/- 11.7 microg/l, p < 0.01, respectively) to levels overlapping with those induced by AVP (27.9 +/- 6.1 vs. 13.1 +/- 3.5 pg/ml and 167.6 +/- 16.2 vs. 113.3 +/- 9.4 microg/l, p < 0.01, respectively) and similar to those elicited by hCRH (28.1 +/- 4.6 vs. 17.4 +/- 3.1 pg/ml and 182.7 +/- 22.8 vs. 114.8 +/- 12.3 microg/l, p < 0.02, respectively). The ACTH but not the cortisol response to hCRH was higher (p < 0.02) than those to HEX when evaluated as area under the curve. The co-administration of HEX and AVP had no significant interaction on ACTH and cortisol peak levels (40.7 micro 5.3 pg/ml and 168.8 +/- 13.5 microg/l, respectively). On the other hand, the co-administration of HEX and hCRH had a less than additive effect on ACTH and cortisol secretion (53.3 +/- 11.2 pg/ml and 204.0 +/- 13.7 microg/l, respectively). CRH and AVP had a true synergistic effect on ACTH (104.9 +/- 14.2 pg/ml, p < 0.01) and an additive effect on cortisol secretion (281.3 +/- 10.8 microg/l, p < 0.02). HEX did not modify the effect of CRH + AVP on both ACTH (135.5 +/- 22.0 pg/ml) and cortisol secretion (261.1 +/- 13.2 microg/l). The GH response to HEX (55.7 +/- 19.8 vs. 2.7 +/- 1.9 microg/l, p < 0.005) was unaffected by the administration of CRH alone (53.5 +/- 21.0 microg/l) and/or AVP co-administration (60.2 +/- 21.2 and 45.9 +/- 10.6 microg/l, respectively). In conclusion, the results of this study demonstrate that GHRPs, beside their well-known GH-releasing activity, possess a remarkable ACTH-releasing activity, overlapping with that of AVP and similar to that of hCRH, two neurohormones which are known to play the major role in the control of the pituitary-adrenal axis. It is noteworthy that HEX shows no synergistic effect with either AVP or hCRH which, on the other hand, truly synergize. This evidence suggests the hypothesis that the ACTH-releasing activity of GHRPs could be, at least partially, independent of both CRH- and AVP-mediated actions in humans.

Mechanisms underlying the negative growth hormone (GH) autofeedback on the GH-releasing effect of hexarelin in man

The growth hormone (GH) response to GH-releasing hormone (GHRH) is strongly inhibited by previous administration of recombinant human GH (rhGH), likely as a consequence of a somatostatin-mediated GH negative autofeedback. Hexarelin (HEX), a synthetic hexapeptide belonging to the GH-releasing peptide (GHRP) family, possesses a GH-releasing activity greater than that of GHRH both in animals and in man. The mechanism of action of GHRPs is yet to be completely clarified, although concomitant actions at the pituitary and hypothalamic level have been hypothesized. To further clarify the mechanisms of action underlying the GH-releasing activity of HEX, in six normal young volunteers we studied the effects of rhGH (2 U intravenously [IV]) on the GH response either to GHRH (2 microg/kg IV) or to HEX (2 microg/kg IV) alone or combined with GHRH and/or pyridostigmine ([PD], 120 mg orally). The GH-releasing effect of HEX was higher than that of GHRH (area under the curve [AUC], 2,200.8 +/- 256.9 v 792.2 +/- 117.6 microg/L/h, P < .001), whereas combined administration of the two substances induced a true synergistic effect, with GH release after HEX plus GHRH (4,259.2 +/- 308.0 microg/L/h) being higher (P < .02) than the arithmetic sum of the GH increases induced by each compound separately administered. After rhGH administration, the GH-releasing effect of HEX was blunted (1,468.9 +/- 193.7 microg/L/h, P < .04; inhibition of 32.1%), whereas that of GHRH was nearly abolished (102.0 +/- 7.8 microg/L/h, P < .02; inhibition of 86.1%). The GH response to combined administration of HEX and GHRH was also blunted by the previous rhGH bolus (3,070.6 +/- 481.8 microg/L/h, P < .02; inhibition of 26.7%). PD did not modify the GH-releasing effect of HEX either alone (2,456.8 +/- 317.5 microg/L/h) or combined with GHRH (4,009.1 +/- 360.8 microg/L/h). rhGH was again able to blunt the GH response to HEX combined with PD (1,619.3 +/- 237.9 microg/L/h, P < .02), but failed to modify the GH response to HEX combined with GHRH and PD (4,548.4 +/- 698.0 microg/L/h). In conclusion, these results demonstrate that rhGH administration only blunts the GH-releasing activity of HEX, but abolishes that of GHRH. The blunting effect of rhGH on the GH response to HEX is probably mediated by a concomitant reduction in the activity of GHRH-secreting neurons and an increase of somatostatinergic tone.

The IGF-I response to very low rhGH doses is preserved in human ageing.

The activity of the GH/IGF‐I axis varies during life and is clearly reduced in the elderly. In fact, GH, IGF‐I and IGFBP‐3 levels in older people are clearly reduced and similar to those observed in patients with GH deficiency. The declining activity of the GH/IGF‐I axis with advancing age may contribute to changes in body composition, structure, function and metabolism. In fact, treatment with pharmacological doses of rhGH restored plasma IGF‐I levels, increased lean body mass and muscle strength while decreased adipose tissue mass in healthy elderly subjects. At present it is unclear whether peripheral GH sensitivity is preserved in aging. To clarify this point, we aimed to verify the effect of both single dose and short term treatment with very low rhGH doses on the IGF‐I levels in normal elderly subjects. Normal young adults were studied as controls.

GH/IGF-I axis in anorexia nervosa

Patients with anorexia nervosa (AN) may develop multiple endocrine abnormalities, including amenorrhea, hyperactivity of the hypothalamus-pituitary-adrenal axis, hypothyroidism and particular changes in the activity of the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Exaggerated GH secretion and reduced IGF-I levels are usually found in AN, as well as in conditions of malnutrition and malabsorption, insulin-dependent diabetes mellitus, liver cirrhosis and catabolic states. In AN, GH hypersecretion at least partially reflects malnutrition-induced peripheral GH resistance, which leads to reduced IGF-I synthesis and release; this implies an impairment of the negative IGF-I feedback action on GH secretion. On the other hand, primary alterations in the neural control of GH secretion cannot be ruled out. The neuroendocrine alterations include enhanced somatotroph responsiveness to growth hormone releasing hormone (GHRH) and impaired GH response to most central nervous system-mediated stimuli. Particular resistance to cholinergic manipulation has also been demonstrated, thus suggesting a somewhat specific alteration in the somatostatin (SS)-mediated cholinergic influence on GH secretion. Moreover, paradoxical GH responses to glucose load, thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) have also been reported. The effect of reduced leptin levels on GH hypersecretion in AN is still unclear, but ghrelin (the gastric hormone that is a natural ligand of the GH secretagogue receptor and strongly stimulates somatotroph secretion) is thought to play a major role. Regardless of the supposed central and peripheral alterations, it has to be emphasised that the activity of the GH/IGF-I axis in AN is generally restored by nutritional and stable weight gain. It therefore reflects an impaired nutritional state and cannot be considered a primary hallmark of the disease.

The activity of GH/IGF-I axis in anorexia nervosa and in obesity: A comparison with normal subjects and patients with hypopituitarism or critical illness

GH/IGF-I axis activity changes have been reported both in anorexia nervosa (AN) and in obesity (OB). AN is characterized by GH hypersecretion and very low IGF-I levels as a result of undernutrition and acquired peripheral GH resistance. On the other hand OB is a GH hyposecretory state but IGF-I levels are generally preserved. The activity of GH/IGF-I axis in AN and OB has never been directly compared with that of other pathophysiological condi tions such as hypopituitarism and critical illness in which a reduction of both GH and IGFsecretion has been demonstrated. To this aim, we evaluated IGF-I levels and both basal and GHRH (1 μg/kg) IV-induced GH secretion in 20 female patients with anorexia nervosa (mean age: 19.1±0.8 years) and in 15 female and 5 male patients with simple obesity (mean age 39.0±3.0 years). We then compared the results with those of hypopituitaric patients with severe GH deficiency (GHD), including 10 female and 10 male patients (mean age: 32.0±2.1 years), and with 4 female and 7 male patients with critical illness (CRI) following multiple trau ma 72 hours after ICU admission (mean age: 59.2±1.2 years). Twenty-six normal subjects (NS including 14 female and 12 male patients (mean age: 37.8±3.7 years) were studied as controls Basal IGF-I levels in AN patients (93.5±11 μg/L) were lower (p<0.001) than in the NS (201.7±13.5 μg/L) and OB (194.5±28.6 μg/L), which, in turn, were similar. IGF-I levels in AN patients were lower than in CRI patients (162.8±17.4 μg/L) and higher than in GHD patients (76.7±13.5 μg/L) but these differences did not attain statistical significance. Basal GH levels in AN patients (7.6±2.5 μg/L) were higher (p<0.001) than in NS (1.8±0.3 μg/L), OB patients (1.1±0.5 μg/L), CRI patients (1.8±0.5 μg/L) and GHD patients (0.3±0.1 μg/L), which were the lowest (p<0.01). The GHRH-induced GH rise in AN patients (AUC: 2032.9±253.5 μg/L/h) was three fold higher (p<0.001) than in NS (662.1±80.3 μg/L). On the other hand in OB (332.4±74.7 μg/L/h) the GH response to GHRH was similar to that in CRI (199.6±98.8 μg/L/h); both were clearly higher (p<0.01) than in GHD patients (25.1±5.2 μg/L/h) but lower (p<0.01) than in NS These findings demonstrate that in AN patients, in spite of a clear increase of both basal and GHRH-induced GH secretion, IGF-I synthesis and release are as markedly impaired as in patients with panhypopituitarism and severe GHD. On the other hand in OB and in CRI, IGFsynthesis and release are preserved despite marked impairment to GHRH-induced GH secre tion. These results reinforce the major role of nutrition in conditioning the activity of GH/IGFaxis in different patho-physiological states.

ALPRAZOLAM, A BENZODIAZEPINE, DOES NOT MODIFY THE ACTH AND CORTISOL RESPONSE TO HCRH AND AVP, BUT BLUNTS THE CORTISOL RESPONSE TO ACTH IN HUMANS

Alprazolam (AL), a benzodiazepine which activates gamma-amino butyrric acid (GA-BA)-ergic receptors, exerts a clear inhibitory effect on the activity of the hypothalamo-pitu-itary-adrenal (HPA) axis and is able to markedly reduce the ACTH response to metyrapone-induced inhibition of glucocorticoid feedback. It has been suggested that its inhibitory action could be regulated by CRH or AVP mediated mechanisms. However, the effect of benzodi-azepines on the HPA response to CRH or AVP is contradictory. It has been shown that benzodi-azepines have specific receptors on the adrenal gland but it is unclear if they mediate biological effects in humans. In order to further clarify the mechanisms underlying the inhibitory effect of benzodiazepine on HPA axis in humans, we studied the effect of AL (0.02 mg/kg po at −90 min) or placebo in 7 healthy young volunteers (7 female, age: 26–34 yr; wt: 50–58 kg, BMI 20–22 kg/m2) on: 1) the ACTH and cortisol responses to hCRH (2.0 μg/kg iv at 0 min) or AVP (0.17 U/kg im at 0 min); 2) the cortisol, aldosterone and DHEA responses to ACTH 1–24 (0.06 and 250 μg iv at 0 and 60 min, respectively). After placebo, the ACTH and cortisol responses to hCRH (peaks, mean±SE: 29.8± 4.4 pg/ml and 199.3±19.6 μg/l) were similar to those recorded after AVP (31.7±6.5 pg/ml and 164.8±18.0 μg/l); the cortisol response to 0.06 μg ACTH (190.4±11.8 μg/l) was similar to that recorded after hCRH and AVP but lower (p<0.01) than that after 250 μg ACTH (260.6±17.4 μg/l). AL did not modify the ACTH response to both hCRH (42.5±7.1 pg/ml) and AVP (33.3±2.7 pg/ml), which even showed a trend toward increase. AL also failed to significantly modify the cortisol response to both hCRH (156.3±12.7 μg/l) and AVP (119.4±14.5 μg/l), which, on the other hand, showed a trend toward decrease. The cortisol peaks after 0.06 μg ACTH were significantly reduced (p<0.02) by AL pre-treatment (115.0±7.7 μg/l) which, in turn, did not modify the cortisol response to the subsequent ACTH bolus (214.7±16.6 μg/l). The DHEA and aldos-terone responses to all the ACTH doses were not significantly modified by AL. In conclusion, these data show that the HPA response to AVP as well as to hCRH is refractory to the inhibitory effect of AL which, in turn, blunts the cortisol response to low ACTH dose. These findings suggest that both CRH- and AVP-mediated mechanisms could underlie the CNS-mediated inhibitory effect of AL on HPA axis; in the meantime, these results suggest that benzodiazepines could also act on adrenal gland by blunting the sensitivity of the fasciculata zone to ACTH.

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 sec-retagogue (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 ώg/kg iv) and/or hCRH (2.0 ώg/kg iv) 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 ώg/l, respectively; p<0.02). In CS, the ACTH and F responses to HEX, evaluated as △AUC (mean±SE: 128.7±39.2 pg*min/ml and 328.5±93.2 ώg*min/l, respectively) were lower, though not significantly, than those after hCRH (375.8±128.4 pg*min/ml and 1714.2±598.0 ώg*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*min/ml) and F secretion (3452.9±648.6 ώg*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*min/ml and 10955.9±6184.6 ώg*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*min/ml and 4832.7±2146.5 ώg*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*min/ml) but not on F (10985.4 ±3900.8 ώg*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.