E. Imperiale

Arginine potentiates the GHRH- but not the pyridostigmine-induced GH secretion in normal short children. Further evidence for a somatostatin suppressing effect of arginine.

To investigate the mechanism underlying the GH‐releasing effect of arginine (ARG), we studied the interactions of ARG (0.5 g/kg infused i. v. over 30 min) with GHRH (1 μg/kg i. v.) and with pyridostigmine (PD, 60 mg orally) on GH secretion in 15 children and adolescents with familial short stature (5.1‐15.4 years). In a group of eight subjects ARG induced a GH increase not statistically different to that observed after GHRH (peak, mean±SEM: 38.0±10.4 vs 64.0±14.4 mU/1). The combined administration of ARG and GHRH led to GH levels (101±15.2 mU/1) higher than those observed after GHRH (P < 0.025) or ARG alone (P < 0.001) and overlapping with those recorded after combined PD and GHRH administration (111±22.4 mU/1). In the other seven subjects, ARG and PD administration induced a similar GH response either when administered alone (25.2±13.6 and 27.8±4.0 mU/1, respectively) or in combination (33.8±5.4 mU/1). In conclusion, our results show that in children ARG administration potentiates GHRH‐ but not PD‐induced GH increase. These findings agree with the hypothesis that the GH‐releasing effect of both ARG and PD is mediated via the same mechanism, namely, by suppression of endogeneous somatostatin release. Combined administration of either ARG or PD with GHRH has a similar striking GH‐releasing effect which is clearly higher than that of GHRH alone.

Effect of cholinergic enhancement by pyridostigmine on growth hormone secretion in obese adults and children

In obesity the reduced growth hormone (GH) responses to several provocative stimuli including growth hormone-releasing hormone (GHRH) indicate a diminished somatotroph responsiveness but do not distinguish between primary pituitary and hypothalamic pathogenesis. However, it has been shown that the cholinergic system positively influences Gh secretion likely by modulating somatostatin release in a negative way. Thus, the effect of cholinergic activity enhancement by pyridostigmine (PD), an acetylcholinesterase inhibitor, on both basal and GHRH-induced GH secretion was studied in 14 obese subjects (eight adults and six children). Eighteen nonobese subjects (seven adults and 11 children) were studied as controls. In obese subjects the GHRH-induced GH increase was lower than in controls (peak, mean +/- SEM, adults, 9.2 +/- 2.7 v 16.8 +/- 5.7 ng/mL; children, 8.0 +/- 0.8 v 20.3 +/- 4.6 ng/mL) attaining statistical significance only in children group (P less than .02). The PD-induced GH response in the two obese groups was similar to that observed in relative controls (adults, 5.3 +/- 1.0 v 7.4 +/- 1.7 ng/mL; children, 9.6 +/- 1.6 v 13.3 +/- 1.4 ng/mL). PD clearly potentiated the GH response to GHRH in obese subjects, both adults (P less than .05 v GHRH alone) and children (P less than .0005 v GHRH alone). However, the GH responses to PD + GHRH was significantly reduced in obese subjects compared with controls (adults, 18.1 +/- 2.2 v 42.7 +/- 10.7 ng/mL, P less than .05; children, 28.3 +/- 4.5 v 58.2 +/- 7.7 ng/mL, P less than .01). In conclusion, PD is able to potentiate the blunted GH responses to GHRH in obese adults and children, inducing a GH increase similar to that observed after GHRH alone in normal subjects. This finding suggests that an alteration of somatostatinergic tone could be involved in the reduced GH secretion in obesity.

Effect of the potentiation of cholinergic activity on the variability in individual GH response to GH-releasing hormone

In man the GH response to GHRH is highly variable and some normal subjects may be completely unresponsive to the neuropeptide. On the other hand, the potentiation of cholinergic activity by pyridostigmine (PD), a cholinesterase inhibitor, increases the GH response to GHRH, probably by inhibiting somatostatin release. The aim of this study was to assess the existence of intrain-dividual variability in the GH response to GHRH and verify the effects of PD treatment on inter- and intraindividual variability. Twenty normal adults (17 M and 3 F) and 10 normal prepubertal children (9 M and 1 F) underwent 2–5 administrations of 1 µg/kg GHRH on different days. Seven adults and all children also underwent 1–5 other tests in which GHRH was preceded (60 min before) by oral PD (120 mg in adults and 60 mg in children). The GH responses to GHRH were highly variable, not only within subjects but also in the same subject on different occasions (peak range; adults: 0.4–49.0 ng/ml; children: 2.4–50.0 ng/ml). PD always markedly increased the GH response to GHRH, even unmasking this response in 3 adults and 4 children hyporesponsive to the neuropeptide alone. However the variability in the GH response was still present (adults: 27.2–108.5 ng/ml; children: 25.0–144.0 ng/ml), though reduced (adults: p = 0.0005; children: p = 0.0204). These data indicate that: i. A great inter- and intraindividual variability in the GH response to GHRH is present. ii. PD always potentiates this response, reducing the variability and abolishing false negative responses. The variability in the GH response to GHRH may be due, at least in part, to a different somatostatinergic tone that can be blunted by the enhancement of the cholinergic function.

Glucagon stimulates GH secretion after intramuscular but not intravenous administration. Evidence against the assumption that glucagon per se has a GH-releasing activity

In order to verify the true GH-releasing effect of glucagon and to explain the mechanism underlying this effect, we studied the effect of glucagon (GLU, 1 mg) administered either iv or im on both basal and GHRH (1 μg/kg)- induced GH rise in 48 normal short children and adolescents. Moreover, the in vitro effect of GLU on rat anterior pituitary cells was studied. Intravenous administration of GLU induced no significant GH rise. On the other hand, im GLU administration induced a clearcut GH increase (mean ± SE GH peak after GLU vs placebo = 25.7±3.9 vs 10.1±3.6 μg/L, p<0.01). Intravenous administration of GLU failed to modify the GHRH-induced GH rise either when coadministered with the neurohormone (35.2±4.1 vs 34.1±6.0 μg/L) or when given 60 min earlier (20.2±5.8 vs 21.1±8.3 μg/L). Differently from iv GLU, im GLU strikingly potentiated the GH response to GHRH given 90 min later (57.5±6.3 vs 24.7±9.1 μ/L, p<0.01). Mean plasma glucose levels increased 30 min after GLU, administered either iv or im, and returned to basal levels 60 min later. GH secretion from dispersed rat pituitary cells was unaffected by incubation with GLU (10−10-10−4 mol/L). Incubation of the cells with 10−7 mol/L GHRH induced instead a clear-cut stimulation of GH release. In conclusion, our data demonstrate that glucagon perse has not GH-releasing activity as indicated by its uneffectiveness to release GH in vitro and after intravenous administration. The mechanisms underlying the GH increase which follows intramuscular administration of glucagon are not clear.

GROWTH HORMONE RESPONSES TO PYRIDOSTIGMINE IN NORMAL ADULTS AND IN NORMAL AND SHORT CHILDREN

There is evidence indicating that the cholinergic system positively modulates GH release probably by inhibiting somatostatinergic tone. In the present study, the effects of cholinergic enhancement by pyridostigmine, (PD), a cholin‐esterases inhibitor, on GH release in normal adults (n= 14) (NA) and in both normal (n = 5) (NC) and short children (n = 19) (SC) with familial short stature (n =7) or constitutional growth delay (n= 12) were studied. In SC the insulin hypoglycaemia (IH)‐induced GH increase was also studied. In both NC and SC 60 mg orally PD induced a significant GH increase with mean peak at 90 min (mean ± SEM 11.0 ± 2.2 ng/ml in NC and 11.2 ± 2.3 ng/ml in SC). The GH areas under response curve (AUC) were 379.3 ± 76.6 and 327.8 ±43.2 ng/ml/h in NC and SC respectively. In NA 120 mg orally PD induced a significant GH increase with mean peak at 120 min (5.1 ± 11 ng/ml) which was significantly lower (P < 0.05) than that observed in both NC and SC. This statistical difference was strengthened by evaluating AUC (NA: 205.6 ± 33.7 ng/ml/h, P < 0.05 vs NC and SC). The correlation of drug dosage with body area ruled out that this difference could be related to the different PD dose in adults and children. In SC, IH induced a GH increase significantly lower than that observed after PD (GH peak 7.8 ± 0.6 vs 16.4 ± 1.9 ng/ml P<0.001). Our data show that cholinergic enhancement by PD induces a higher GH increase in both normal and short children than that observed in adults. This could be related to a variation of somatostatinergic tone. The higher PD‐induced GH response compared to the IH‐induced one indicates that PD test may be considered a more potent GH provocative stimulus than insulin hypoglycaemia.

α1-blocker doxazosin improves peripheral insulin sensitivity in diabetic hypertensive patients

The antihypertensive doxazosin is a selective alpha 1-adrenoceptor-blocking drug whose favorable impact on lipid metabolism is well known. A single-blind placebo-controlled crossover study was designed to determine whether antihypertensive treatment with doxazosin affects insulin sensitivity in diabetic, mildly hypertensive, non-obese patients. Twelve subjects (diastolic blood pressure, 98 +/- 1.5 mm Hg; body mass index, 25 +/- 0.6 kg/m2; hemoglobin A1c [HbA1c], 7.6% +/- 0.4%) who were not taking drugs and were treating diabetes only by diet were randomly assigned to placebo treatment for 6 weeks and then to doxazosin for the same period, or vice versa. The doxazosin dose (maximum, 12 mg/d) was increased to achieve a normotensive blood pressure (final diastolic pressure, 85 +/- 2 mm Hg, P < .05). A euglycemic (100 +/- 4 mg/dL) hyperinsulinemic (61 +/- 6 microU/mL) glucose clamp was performed at baseline and at the end of both placebo and doxazosin administration. Hepatic glucose production was measured by the isotope dilution technique using 3H-glucose. Body weights and HbA1c did not vary during the entire study. The basal mean glucose uptake and the insulin sensitivity index (2.3 +/- 0.3 mg/kg/min and 4 +/- 0.5 mg/kg/min per U/L x 100) remained unchanged during placebo administration (2.5 +/- 0.4 and 4 +/- 0.6, NS), but significantly increased during doxazosin treatment (3.3 +/- 0.4 and 5.6 +/- 0.7, P < .05). Hepatic glucose production showed no modification during both placebo and doxazosin. These data provide evidence that doxazosin improves insulin sensitivity in diabetic hypertensive patients, mainly through peripheral effects.

A new test for the diagnosis of growth hormone deficiency due to primary pituitary impairment: combined administration of pyridostigmine and growth hormone-releasing hormone

The diagnosis of growth hormone (GH) deficiency (GHD) is currently based on failure to increase plasma GH levels to an arbitrary cutoff point of 7 or 10 μg/l in response to two provocative stimuli. False negative responses to these tests, however, frequently occur thus reducing their diagnostic reliability. The aim of this study was to assess a combination of pyridostigmine (PD) and GH-releasing hormone (GHRH) (60 mg oral PD 60 min before 1 μg/Kg GHRH iv) as a reliable test probing pituitary somatotropic function. In fact PD, an acetylcholinesterase inhibitor, strikingly potentiates GH response to GHRH likely by inhibiting somatostatin release. The combination PD + GHRH was tested in normal children and adolescents (NS, n = 27) and in a large group of short children classified as having familial short stature (FSS, n = 24), constitutional growth delay (CGD, n = 34) and GH deficiency (organic, oGHD, n = 6; idiopathic, iGHD, n = 10). In all groups results obtained by PD + GHRH were compared with those obtained by testing with GHRH, Clonidine (CLON) and PD alone and by studying spontaneous nocturnal GH secretion over 8 hours. Assuming 7 μg/l as minimum normal GH peak, a positive response occurred in only 18/24, 11/12 and 12/13 NS for GHRH, CLON, and PD, respectively. In contrast even assuming a minimum normal GH peak as high as 20 μg/l, PD + GHRH induced a positive response in 27/27 NS all having a nocturnal GH mean concentration (MC) ≥ 3 ug/l. Therefore PD + GHRH test gave no false negative responses and this was true not only in NS but even in all FSS and CGD having a GH MC ≥ 3 μg/l. On the other hand, PD + GHRH induced a negative GH response in all oGHD and in 8/10 iGHD patients. In the remaining two iGHD patients, PD + GHRH demonstrated a normal pituitary GH reserve in spite of a GH MC < 3 μg/l and low IGF-I level, thus pointing to a hypothalamic pathogenesis for the GHD. Considering FSS and CGD children having a GH MC < 3 μg/l, PD + GHRH showed a primary pituitary GH deficiency in 3/12 CGD with low plasma IGF-I levels. In conclusion, in slowly growing children PD + GHRH test is the most reliable provocative test for the diagnosis of primary pituitary GH deficiency being capable to discriminate between an unequivocally normal and impaired somatotropic function. The study of spontaneous GH secretion is mandatory only for those patients having low height velocity but a normal GH response to PD + GHRH in order to look for the existence of a GHD due to hypothalamic dysfunction.

A neuroendocrinological approach to evidence an impairment of central cholinergic function in aging.

A hypothalamic pathogenesis for the reduced GH secretion in aging has been reported for both animal and man. To further address this issue we studied in 31 elderly normal subjects (6 males and 25 females, aged 66–90 yr) and in 22 young healthy controls (13 males and 9 females, aged 20–35 yr) the GH responses to GHRH test (GHRH29, 1 μg/kg iv as a bolus at 0 min) alone and combined with pyridostigmine, a cholinesterase inhibitor (PD, 120 mg po 60 min before GHRH), or with arginine (ARG, 30 g in 100 ml infused from 0 to 30 min). Serum IGF-I levels were lower in elderly than in young subjects (mean±SE: 86.9±7.2 vs 288.7±22.1 μg/L, p<0.01). The GHRH-induced GH increase was lower in elderly than in young subjects (p<0.01). PD increased the GH response to GHRH in both groups (p<0.001), but in elderly subjects this response persisted lower (p<0.0001) than that ob-served in young adults. Also ARG coadministration potentiated the GHRH-induced GH release in both groups (p<0.0001) but in this case the elderly’s responses overlapped with the young’s. The GH in-crease observed after combined administration of ARG and GHRH was higher (p<0.0001) than that elicited by PD plus GHRH in elderly but not in young subjects. Analyzing individual GH responses, a GH peak below the limit of normality for young adults was observed in 19 (61.3%) elderly subjects after PD plus GHRH administration while ARG plus GHRH test elicited a normal GH peak in all but one. Taking into account that the GH-releasing effect of both PD and ARG is likely mediated by inhibition of hypothalamic somatostatin release, our data show that in elderly subjects the acutely releasable GH pool is preserved and give support to the hypothesis that a somatostatin hypertone underlies the reduced GH secretion of aged individuals. PD but not ARG fails to potentiate the GH response to GHRH in about 60% of aged subjects. These findings are suggestive for an impairment of the hypothalamic cholinergic system which, in turn, would be responsible for somatostatin hyperactivity and GH hyposecretion.

Cholinergic Enhancement by Pyridostigmine Potentiates Spontaneous Diurnal but not Nocturnal Growth Hormone Secretion in Short Children

It has been shown that enhanced cholinergic tone induced by pyridostigmine (PD) increases both basal and GHRH-stimulated GH levels in both adults and children. In this study the effects of PD (60 mg orally) on GH secretion were studied both in the morning (from 8.00 to 12.00) and in the night (from 23.00 to 3.00) in 7 short children previously shown as having a normal spontaneous nocturnal GH secretion. In the morning, PD induced a GH increase higher than saline (peak, mean +/- SEM: 17.4 +/- 3.4 vs. 5.5 +/- 3.0 ng/ml, p less than 0.02; area under curve (AUC): 360.8 +/- 71.4 vs. 109.4 +/- 44.7 ng/ml/h, p less than 0.01). In the night, no difference was observed between GH secretion after PD (peak: 16.7 +/- 2.4 ng/ml; AUC: 468.2 +/- 95.5 ng/ml/h) and saline (peak: 16.0 +/- 2.7 ng/ml; AUC: 409.1 +/- 97.7 ng/ml/h). Spontaneous GH secretion was higher during the night than in the morning (p less than 0.02) whereas nocturnal GH secretion overlapped with that in the morning after PD. The ability of PD to increase GH secretion during the morning but not GH hypersecretion occurring at night implies that the cholinergic tone in the central nervous system areas controlling GH secretion is already maximally stimulated at night. Since, reportedly, the cholinergic system negatively modulates somatostatin secretion, presence of a physiologically reduced somatostatinergic tone may be envisaged at night.

Pyridostigmine Potentiates L-Dopa- but not Arginine- and Galanin-Induced Growth Hormone Secretion in Children

The coadministration of growth hormone (GH) secretagogues can provide insight into the neuroregulation of GH secretion. The GH response to L-dopa (125, 250 and 500 mg orally for body weights less than 15 kg, between 15 and 30 kg and greater than 30 kg, respectively), arginine (Arg; 0.5 g/kg infused intravenously over 30 min) and galanin (GAL; 15 micrograms/kg infused intravenously over 60 min) when administered alone or combined with pyridostigmine (PD; 60 mg orally), a cholinergic agonist that likely acts via inhibition of endogenous somatostatin secretion, was studied in children with familial short stature. The GH-releasing effect of PD was also evaluated. In 8 children, PD and L-dopa when administered alone induced an equivalent GH rise (area under the response curve, mean +/- SEM: 241.4 +/- 31.1 vs. 202.9 +/- 38.6 micrograms/l/h) while their coadministration had an additive effect (435.4 +/- 41.4 micrograms/l/h; p less than 0.02 vs. PD and L-dopa alone). On the contrary, in other 8 children, PD and Arg induced similar GH increases either when administered alone (394.2 +/- 68.5 vs. 405.8 +/- 103.9 micrograms/l/h) or in combination (535.8 +/- 97.3 micrograms/l/h). GH increases almost superimposable were also observed when PD and GAL were administered alone (405.2 +/- 72.3 vs. 412.6 +/- 94.1 micrograms/l/h) or in combination (537.9 +/- 139.0 micrograms/l/h) in other 7 children. These data show that the enhancement of the cholinergic activity by PD increases the L-dopa-induced GH release but fails to modify both Arg- and GAL-induced GH release in short children.(ABSTRACT TRUNCATED AT 250 WORDS)