0021-972X/90/7102-0580$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 3 Printed in U.S.A.
Pyridostigmine Blocks the Inhibitory Effect of Glucocorticoids on Growth Hormone-Releasing Hormone Stimulated Growth Hormone Secretion in Normal Man ANDREA GIUSTINA, ANGELA GIRELLI, MAURO DOGA, CORRADO BODINI, SIMONETTA BOSSONI, GIUSEPPE ROMANELLI, AND WILLIAM B. WEHRENBERG Cattedra di Clinica Medica, University of Brescia, Italy; and Department of Health Sciences (W.B. W.), University of Wisconsin, Milwaukee, Wisconsin 53201
injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Mean GH levels, peak GH levels, and GH area under the curves (AUCs) were significantly lower after GHRH + cortisone as compared to GHRH alone. However, these parameters were not significantly different after PD + GHRH + cortisone when compared to PD + GHRH and after PD + cortisone when compared to PD alone. We conclude that acute administration of pharmacological amounts of glucocorticoids cannot inhibit the GH response to PD alone or in combination with GHRH. Thus, we hypothesize that the inhibitory action of glucocorticoids on the GH response to GHRH in man may be mediated by an enhancement of either somatostatin release by the hypothalamus or somatostatin action on the pituitary. (J Clin Endocrinol Metab 7 1 : 580-584, 1990)
ABSTRACT. Glucocorticoids have been shown to inhibit GH secretion in normal man when acutely and chronically administered in pharmacological amounts. Pyridostigmine (PD), an acetylcholinesterase inhibitor, is able to elicit GH secretion when administered alone and to enhance the GH response to GHRH in normal subjects probably via a decrease in the hypothalamic release of somatostatin. The aim of the present study was to investigate the influence of glucocorticoids on the GH response to PD administered either alone or in combination with GHRH in normal adult subjects. Six healthy adult volunteers underwent six experimental protocols. They received 1) human (h) GHRH(1-29)NH2, 100 ng injected as an iv bolus; 2) cortisone acetate, 50 mg administered orally (po) 60 min before an hGHRH iv bolus injection; 3) PD, 120 mg administered po, 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus
G
H SYNTHESIS and secretion are regulated by the hypothalamic peptides GHRH, which was an excitatory role, and somatostatin, which has an inhibitory role. Numerous secretagogues affect GH release by influencing GHRH and/or somatostatin secretion (1). The role played by glucocorticoids in the regulation of GH secretion is still unclear. Normal subjects after administration of supraphysiological doses of glucocorticoids and patients with Cushing's disease have blunted GH responses to various pharmacological stimuli (2-6). However, glucocorticoids are also known to enhance GH release by cultured human pituitary somatotropes (7), and to increase GH gene transcription (8) and GHRH receptor synthesis (9). In rats the GH response to GHRH seems to depend on the concentrations of circulating glucocorticoids (10). Moreover, GH deficiency in patients with idiopathic adrenocorticotropin deficiency resolves during glucocorticoid replacement (11).
Recently, studies performed in the rat have suggested that the contradictory evidence obtained from in vitro and in vivo studies regarding the effects of glucocorticoids on GH secretion is due to the enhancement of hypothalamic somatostatin release by glucocorticoids in vivo (12). There is increasing evidence for an important stimulatory role of cholinergic neurotransmission in the regulation of GH secretion in normal man. PD, an acetylcholinesterase inhibitor, is able to elicit GH secretion when given alone (13) and to enhance the GH response to GHRH in normal subjects (14). Experimental studies suggest that the action of PD may be mediated by a decrease in the hypothalamic release of somatostatin (15). The aim of our study was to investigate the acute effects of glucocorticoids on the GH response to PD alone or combined with GHRH in normal adult subjects.
Received January 16,1990. Address correspondence and requests for reprints to: Dr. Andrea Giustina, Cattedra di Clinica Medica, Universita' di Brescia c/o 2a Medicina-Spedali Civili, 25125 Brescia, Italy.
Subjects
Subjects and Methods We studied five male and one female adult volunteers with no history of endocrine or metabolic disease. Their ages ranged 580
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2016. at 10:37 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOIDS, PYRIDOSTIGMINE, AND GH SECRETION from 24-29 yr and their body mass index ranged from 19-24 kg/m2. None of the subjects were taking any drugs during the course of the study. Six different experimental trials were conducted, at no less than 7-day intervals, in each of the subjects. Methods After an overnight fast each subject was admitted to the Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. An antecubital vein catheter was inserted percutaneously and kept patent by a slow saline infusion. After a 30-min stabilization period the following treatments were initiated in randomized order 1) human GHRH (129) NH2 (hGHRH, Serono, Italy), 100 ng in 1 ml saline injected as an iv bolus; 2) Cortisone acetate (Cortone, Merck Sharp & Dohme, Rahway, NJ), 50 mg administered po 60 min before an hGHRH iv bolus injection; 3) PD (Mestinon, Roche, Switzerland), 120 mg administered po 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Blood samples for GH, cortisol and glucose assays were taken at —75, —60 (time of PD and cortisone acetate administration), -45, -30, -15, 0 (time of GHRH injection), 15, 30, 45, 60, 90, 120 min. GH secretory responses were expressed as absolute values (Mg/L), peak values (/zg/L) and AUC (/ig/L X 180 min) calculated by trapezoid integration. Statistical comparison of the results was made with the twotailed Student's t test. A P < 0.05 was considered statistically significant. All results are expressed as mean ± SEM. Assays Commercial RIA kits were used for estimation of cortisol (Corti-Cote, Beckton-Dickson,Orangeburg, NY) and GH (Lepetit, Italy, double antibody polyclonal RIA kit; inter and intraassay coefficients of variation ±5.4% and 2.3%, respectively, sensitivity limit of the assay 0.1 fig/L). Blood glucose
25.
*
*
The kinetics of the GH responses to the various stimuli are illustrated in Figs. 1-3. Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were significantly lower after GHRH + cortisone than after GHRH alone (Fig. 1). Peak GH concentrations occurred between 15 and 90 min after GHRH injection. Peak values averaged 7.3 ± 0.8 /ug/L after cortisone + GHRH and were significantly lower than the peak values observed after GHRH alone (25.1 ± 1.8 /zg/L, P < 0.001). Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were not significantly different after PD + GHRH and after PD + cortisone + GHRH (Fig. 2). The mean GH peak was 39.8 ± 3.3 ^g/L after PD + cortisone + GHRH and was similar to the peak value observed after PD + GHRH, 42.7 ± 4.3 /*g/L. Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were similar after PD + cortisone and after PD alone (Fig. 3). The mean GH peak was 10.3 ± 1 Mg/L after PD + cortisone and was not significantly different from the peak value found after PD alone, 11 ± 1.2 Mg/L. All subjects receiving cortisone acetate at —60 min had significantly elevated cortisol levels from -45 to 120 min of the experiment as compared to control treated subjects (Fig. 4). The effects of cortisone acetate treatment on plasma cortisol did not differ between experiments. Plasma glucose levels did not show significant differences between the studies. All subjects experienced facial flushing after GHRH injection. Cortisone and PD did not cause overt side effects in any subject.
Discussion Our data confirm that acute administration of pharmacological doses of glucocorticoids is able to inhibit the 1500. *
•
*
*
*
E o
20.
2 900.
_J
In 3 X
Results
• GHRH+CORT
* * *
-.
was measured by the gluco-oxidase method (Beckman II glucose analyzer). All samples from the same patient were determined in a single assay in triplicate.
a GHRH
30_
581
X
15.
of 600.
o
u
10J
3 < X
5.
300.
o o.
OJ
-75 -60 -45 -30 -IS 0
15 30 45 60
90
GHRH
GHRH+CORT
Time(min)
FlG. 1. a, Mean ± SEM GH levels (/zg/L): D, after GHRH alone; • , after GHRH + cortisone acetate, b, Mean GH AUCs after GHRH alone; M after GHRH + cortisone acetate in six normal adult subjects. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
X 180 min): D,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2016. at 10:37 For personal use only. No other uses without permission. . All rights reserved.
582
JCE & M • 1990 Vol 71 • No 3
GIUSTINA ET AL. AGHRH*PD AGHRH+PD
SO-
5000-,
•CORT 4000-
3000.
Vol. 71, No. 3 Printed in U.S.A.
Pyridostigmine Blocks the Inhibitory Effect of Glucocorticoids on Growth Hormone-Releasing Hormone Stimulated Growth Hormone Secretion in Normal Man ANDREA GIUSTINA, ANGELA GIRELLI, MAURO DOGA, CORRADO BODINI, SIMONETTA BOSSONI, GIUSEPPE ROMANELLI, AND WILLIAM B. WEHRENBERG Cattedra di Clinica Medica, University of Brescia, Italy; and Department of Health Sciences (W.B. W.), University of Wisconsin, Milwaukee, Wisconsin 53201
injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Mean GH levels, peak GH levels, and GH area under the curves (AUCs) were significantly lower after GHRH + cortisone as compared to GHRH alone. However, these parameters were not significantly different after PD + GHRH + cortisone when compared to PD + GHRH and after PD + cortisone when compared to PD alone. We conclude that acute administration of pharmacological amounts of glucocorticoids cannot inhibit the GH response to PD alone or in combination with GHRH. Thus, we hypothesize that the inhibitory action of glucocorticoids on the GH response to GHRH in man may be mediated by an enhancement of either somatostatin release by the hypothalamus or somatostatin action on the pituitary. (J Clin Endocrinol Metab 7 1 : 580-584, 1990)
ABSTRACT. Glucocorticoids have been shown to inhibit GH secretion in normal man when acutely and chronically administered in pharmacological amounts. Pyridostigmine (PD), an acetylcholinesterase inhibitor, is able to elicit GH secretion when administered alone and to enhance the GH response to GHRH in normal subjects probably via a decrease in the hypothalamic release of somatostatin. The aim of the present study was to investigate the influence of glucocorticoids on the GH response to PD administered either alone or in combination with GHRH in normal adult subjects. Six healthy adult volunteers underwent six experimental protocols. They received 1) human (h) GHRH(1-29)NH2, 100 ng injected as an iv bolus; 2) cortisone acetate, 50 mg administered orally (po) 60 min before an hGHRH iv bolus injection; 3) PD, 120 mg administered po, 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus
G
H SYNTHESIS and secretion are regulated by the hypothalamic peptides GHRH, which was an excitatory role, and somatostatin, which has an inhibitory role. Numerous secretagogues affect GH release by influencing GHRH and/or somatostatin secretion (1). The role played by glucocorticoids in the regulation of GH secretion is still unclear. Normal subjects after administration of supraphysiological doses of glucocorticoids and patients with Cushing's disease have blunted GH responses to various pharmacological stimuli (2-6). However, glucocorticoids are also known to enhance GH release by cultured human pituitary somatotropes (7), and to increase GH gene transcription (8) and GHRH receptor synthesis (9). In rats the GH response to GHRH seems to depend on the concentrations of circulating glucocorticoids (10). Moreover, GH deficiency in patients with idiopathic adrenocorticotropin deficiency resolves during glucocorticoid replacement (11).
Recently, studies performed in the rat have suggested that the contradictory evidence obtained from in vitro and in vivo studies regarding the effects of glucocorticoids on GH secretion is due to the enhancement of hypothalamic somatostatin release by glucocorticoids in vivo (12). There is increasing evidence for an important stimulatory role of cholinergic neurotransmission in the regulation of GH secretion in normal man. PD, an acetylcholinesterase inhibitor, is able to elicit GH secretion when given alone (13) and to enhance the GH response to GHRH in normal subjects (14). Experimental studies suggest that the action of PD may be mediated by a decrease in the hypothalamic release of somatostatin (15). The aim of our study was to investigate the acute effects of glucocorticoids on the GH response to PD alone or combined with GHRH in normal adult subjects.
Received January 16,1990. Address correspondence and requests for reprints to: Dr. Andrea Giustina, Cattedra di Clinica Medica, Universita' di Brescia c/o 2a Medicina-Spedali Civili, 25125 Brescia, Italy.
Subjects
Subjects and Methods We studied five male and one female adult volunteers with no history of endocrine or metabolic disease. Their ages ranged 580
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2016. at 10:37 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOIDS, PYRIDOSTIGMINE, AND GH SECRETION from 24-29 yr and their body mass index ranged from 19-24 kg/m2. None of the subjects were taking any drugs during the course of the study. Six different experimental trials were conducted, at no less than 7-day intervals, in each of the subjects. Methods After an overnight fast each subject was admitted to the Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. An antecubital vein catheter was inserted percutaneously and kept patent by a slow saline infusion. After a 30-min stabilization period the following treatments were initiated in randomized order 1) human GHRH (129) NH2 (hGHRH, Serono, Italy), 100 ng in 1 ml saline injected as an iv bolus; 2) Cortisone acetate (Cortone, Merck Sharp & Dohme, Rahway, NJ), 50 mg administered po 60 min before an hGHRH iv bolus injection; 3) PD (Mestinon, Roche, Switzerland), 120 mg administered po 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Blood samples for GH, cortisol and glucose assays were taken at —75, —60 (time of PD and cortisone acetate administration), -45, -30, -15, 0 (time of GHRH injection), 15, 30, 45, 60, 90, 120 min. GH secretory responses were expressed as absolute values (Mg/L), peak values (/zg/L) and AUC (/ig/L X 180 min) calculated by trapezoid integration. Statistical comparison of the results was made with the twotailed Student's t test. A P < 0.05 was considered statistically significant. All results are expressed as mean ± SEM. Assays Commercial RIA kits were used for estimation of cortisol (Corti-Cote, Beckton-Dickson,Orangeburg, NY) and GH (Lepetit, Italy, double antibody polyclonal RIA kit; inter and intraassay coefficients of variation ±5.4% and 2.3%, respectively, sensitivity limit of the assay 0.1 fig/L). Blood glucose
25.
*
*
The kinetics of the GH responses to the various stimuli are illustrated in Figs. 1-3. Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were significantly lower after GHRH + cortisone than after GHRH alone (Fig. 1). Peak GH concentrations occurred between 15 and 90 min after GHRH injection. Peak values averaged 7.3 ± 0.8 /ug/L after cortisone + GHRH and were significantly lower than the peak values observed after GHRH alone (25.1 ± 1.8 /zg/L, P < 0.001). Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were not significantly different after PD + GHRH and after PD + cortisone + GHRH (Fig. 2). The mean GH peak was 39.8 ± 3.3 ^g/L after PD + cortisone + GHRH and was similar to the peak value observed after PD + GHRH, 42.7 ± 4.3 /*g/L. Mean absolute GH levels at times 15, 30, 45, 60, and 90 min and GH AUC were similar after PD + cortisone and after PD alone (Fig. 3). The mean GH peak was 10.3 ± 1 Mg/L after PD + cortisone and was not significantly different from the peak value found after PD alone, 11 ± 1.2 Mg/L. All subjects receiving cortisone acetate at —60 min had significantly elevated cortisol levels from -45 to 120 min of the experiment as compared to control treated subjects (Fig. 4). The effects of cortisone acetate treatment on plasma cortisol did not differ between experiments. Plasma glucose levels did not show significant differences between the studies. All subjects experienced facial flushing after GHRH injection. Cortisone and PD did not cause overt side effects in any subject.
Discussion Our data confirm that acute administration of pharmacological doses of glucocorticoids is able to inhibit the 1500. *
•
*
*
*
E o
20.
2 900.
_J
In 3 X
Results
• GHRH+CORT
* * *
-.
was measured by the gluco-oxidase method (Beckman II glucose analyzer). All samples from the same patient were determined in a single assay in triplicate.
a GHRH
30_
581
X
15.
of 600.
o
u
10J
3 < X
5.
300.
o o.
OJ
-75 -60 -45 -30 -IS 0
15 30 45 60
90
GHRH
GHRH+CORT
Time(min)
FlG. 1. a, Mean ± SEM GH levels (/zg/L): D, after GHRH alone; • , after GHRH + cortisone acetate, b, Mean GH AUCs after GHRH alone; M after GHRH + cortisone acetate in six normal adult subjects. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
X 180 min): D,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2016. at 10:37 For personal use only. No other uses without permission. . All rights reserved.
582
JCE & M • 1990 Vol 71 • No 3
GIUSTINA ET AL. AGHRH*PD AGHRH+PD
SO-
5000-,
•CORT 4000-
3000.
