The Role of Cholinergic Tone in Modulating the Growth Hormone Response Growth Hormone-Releasing Hormone in Normal Man Andrea
Giustina,
Simonetta
Bossoni, Maurizio
Corrado Schettino,
Bodini,
Mauro
and William
Doga,
Angela
Girelli,
Maria
Grazia
to
Buffoli,
B. Wehrenberg
Growth hormone-releasing hormone (GHRH) increases serum GH levels in a dose-dependent manner. 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 if an enhancement of the cholinergic tone was able to influence the dose-response relationship between GHRH and GH in normal adult subjects. Six healthy adult volunteers underwent IO experimental protocols. They were: human GHRH (I-2g)NH,, 1 pg/kg injected as an intravenous (IV) bolus 60 minutes after (a) PD. 120 mg administered orally, or (b) placebo, two tablets administered orally; GHRH, 0.3 bg/kg injected as an IV bolus 60 minutes after (c) PD or (d) placebo; GHRH, 0.1 @g/kg injected as an IV bolus 60 minutes after (e) PD or (f) placebo; GHRH, 0.01 Fg/kg injected as an IV bolus 60 minutes after(g) PD or (h) placebo; saline, 1 mL injected as an IV bolus 60 minutes after (i) PD or (I) placebo. The GH response in placebo-treated subjects was similar after 1 @g/kg and 0.3 Fg/kg GHRH, while the 0.1 pg/kg dose elicited a lower response. The 0.01 pg/kg dose of GHRH did not significantly increase GH levels as compared with saline. After PD. the GH responses to GHRH were greatly enhanced at all doses tested: 1.0, 0.3, and 0.1 kg/kg GHRH all elicited similar GH responses; the GH response to 0.01 pg/kg GHRH was lower, but was still higher than that observed after saline. We conclude that the endogenous somatostatin tone is a significant factor in modulating the dose-response relationship of GHRH-induced GH secretion in normal man. Copyright 0 199 1 by W.8. Saunders Company
G
ROWTH HORMONE (GH) synthesis and secretion are regulated by the hypothalamic peptides GHreleasing hormone (GHRH), which has an excitatory role, and somatostatin, which has an inhibitory role.’ Several studies have shown that human GHRH (l-40)NHz and GHRH (l-44)NH,, and their equipotent synthetic analog GHRH ( l-29)NH2, are able to stimulate GH secretion in a dose-dependent fashion in normal humans.‘.’ There is increasing evidence for an important role of cholinergic neurotransmission in the regulation of GH secretion in normal man. Anticholinergic drugs are known to abolish the GH response to various physiological and pharmacological stimuli.‘,’ 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.’ Experimental studies suggest that the action of PD may be mediated by a decrease in the hypothalamic release of somatostatin.“’ Recently, it has been reported that the simultaneous administration of PD and GHRH causes a biphasic dose-response of GH when various doses of GHRH are tested in different groups of subjects.5 However, it still is not clear what is the role of endogenous somatostatin in determining the dose-response relationship between GHRH and GH. The aim of our study was to investigate the role of somatostatin in modulating the dose-response effect of GHRH on GH secretion in the same normal subjects using PD-mediated enhancement of the central cholinergic tone.
Methods After an overnight fast, each subject was admitted to the Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. A catheter was inserted percutaneously into an antecubital vein and kept patent by a slow saline infusion. After a 30.minute stabilization period, the following treatments were initiated in randomized order: human GHRH (l-29)NH, (Geref. Serono, Italy), 1 &kg in 1 mL of saline injected as an intravenous (IV) bolus 60 minutes after (a) PD (Mestinon, Roche. Switzerland), 120 mg administered orally, or (b) placebo, two tablets administered orally; GHRH, 0.3 kg/kg injected as an IV bolus 60 minutes after (c) PD, 120 mg orally, or (d) placebo, orally; GHRH. 0.1 &kg injected as an IV bolus 60 minutes after (e) PD. 120 mg orally, or (f) placebo, orally; GHRH, 0.01 kg/kg injected as an IV bolus 60 minutes after (g) PD, 120 mg orally, or (h) placebo, orally; saline, I mL injected as an IV bolus 60 minutes after(i) PD. 120 mg orally. or (I) placebo, orally. Blood samples for GH assay were taken at -75, -60 (time of PD or placebo administration), -45, -30, -15, 0 (time of GHRH injection), 15,30,45,60. 90, and 120 minutes. GH secretory responses were expressed as absolute values (kg/L), peak values (&L), and the area under the curve (AUC) for GH secretion (pg/L 180 min). GH secretory responses to each different dose of GHRH were compared with the responses to each of the other four GHRH doses within and between the two treatments (GHRH + placebo or GHRH + PD study). One-way ANOVA was used to compare peak GH values and AUCs. Individual GH values were compared using the ANOVA for repeated measures. P < .OSwas considered statistically significant. All results are expressed as the mean 2 SEM.
SUBJECTS AND METHODS
Subjects Five male and one female adult healthy volunteers with no history of endocrine or metabolic disease were studied. Their ages ranged Erom 25 to 35 years and their body mass index ranged from 19 to 24 kg/m’. None of the subjects were taking any drugs during the course of the study. Ten different experimental trials were conducted in each of the subjects at no less than 7-day intervals. Metabolism,
Vol40, No 5 (May), 1991: pp 519-523
From the Cattedra di Clinica Medica, Universityof Brescia, Brescia. Italy; and the Department of Health Sciences, Universityof Wisconsin. Milwaukee, WI. Address reprint requests to Andrea Giustina. MD, Cattedra di Clinica Medica. Universitd di Brescia clo 2a Medicina-Spedali Civili, 25125 Brescia, Italy. Copyright 0 1991 by W B. Saunders Company 0026-0495/91/4005-0013$03.OOlO 519
520
GIUSTINA ET AL
GH AUC
b
IaglLXlaomhl 3cao
2000
1
Fig 1. GH response to saline and different doses of GHRH in placebo pretreated normal subjects. (a) Mean k SEM GH levels (pg/L) after: O, 1 pg/kg; 0.0.3 pg/kg; A, 0.1 pg/kg; O,O.Ol pg/kg GHRH (l-29)NH,; and V, after saline alone in six adult healthy subjects. (b) Mean ? SEM GH AUC (pg/L 190 mini 0) after: 1 &kg; 0.3 pg/kg; 0.1 pg/kg; 0.01 pg/kg GHRH (129)NH,; and after saline alone in six adult healthy subjects. lP < .05 Y GHRH. 1 pg/kg; V, P < .05v GHRH, 0.3 Kg/kg; Y, P < .05 v GHRH, 0.1 pg/kg.
Assays A commercial
immunoradiometric kit was used for estimation of serum GH concentrations (Allegro HGH. Nichols Institute, CA; interassay and intraassay coefficients of variation were ?4.6% and 7%. respectively; sensitivity was 0.06 kg/L). All samples from the same patient were measured in a single assay in triplicate. RESULTS
GHRH + Placebo Study GHRH (l-29)NH, had no significant effect on GH release at a dose of 0.01 kg/kg. However, doses of 0.1 p&kg and above significantiy stimulated the release of GH with respect to saline. Figure la illustrates the mean GH values after saline, 0.01, 0.1, 0.3, 1 t&kg GHRH (l-29)NHz. No differences between GH levels after 0.3 and 1 p&kg were observed. Therefore, 0.3 &kg appears to elicit the maximal GH response. The lower dose of 0.1 kg/kg produced a submaximal GH response. A similar pattern of responses was observed when the data were analyzed in terms of GH peaks (Fig 2) and GH AUCs (Fig lb). GH peak values after the different doses of GHRH were obtained in each subject at times ranging from 15 to 90 minutes. In most of the subjects the GH peak occurred at 30 minutes after GHRH injection. No significant differences in the time of GH peak occurrence among the different doses of GHRH tested were observed. GHRH + PD Study PD administration induced a clear-cut increase in plasma GH levels compared with those after placebo (P < .OSfrom 15 to 90 minutes) in saline-treated subjects. Furthermore, pretreatment with PD enhanced mean GH levels with respect to the same dose of GHRH (P < .05 from 15 to 90 minutes for all doses of GHRH). Figure 2a illustrates the mean GH values after PD and saline or 0.01, 0.1, 0.3, or 1 t.@kg GHRH (l-29)NH,. No differences among GH levels after 0.1, 0.3, and 1 ug/kg were observed. Therefore, 0.1
pg./kg elicited the maximal GH response. The lower dose of 0.01 &kg produced a submaximal GH response that was significantly higher than that observed after saline. Interestingly, this dose elicited a similar GH response to that obtained in subjects treated with 1 pg/kg GHRH + placebo. A similar pattern of response was observed when the data were analyzed in terms of GH peaks (Fig 2) and GH AUCs (Fig 3b). GH peak values after GHRH + PD were obtained at times ranging from 15 to 90 minutes. In most of the subjects the GH peak occurred at 30 minutes after
GH peak (IQ/L) 4%
30* T
10 * I$’ OJ r
0
I
,
02
I
,
0.4
GHRH
I
,
0.6
I
,
0.6
,
(
1
dose (pg/kg)
Fig 2. Dose-response relationship between GHRH and peak GH secretion in normal subjects with and without PD pretreatment 60 minutes before GHRH administration. Peak GH levels (mean t SEM; kg/L) plotted against their respective GHRH (l-29)NH, dose (a) after saline: 0.1 Kg/kg: 0.0.3 pg/kg; A, 0.1 pg/kg; and 0.0.01 pg/kg. (b) After PD: 0.1 pg/kg; n , 0.3 pg/kg; A, 0.1 pg/kg; and l , 0.01 pg/kg in six adult healthy subjects. Peak responses usually occurred 30 minutes after GHRH administration. lP < .05 Y PD + GHRH.
521
CHOLINERGIC TONE AND GH SECRETION
a
GH
b
AUC
trJBILX18Omin) 3000
T
l-
. :
. i
EL lzB_ OH RH O.Olpg(Kg
PD
Fig 3.
GH response to saline and different doses of GHRH in PD (120 mg, orally) pretreated normal subjects. (a) Mean 2 SEM GH levels (pg/L) and g, after PD alone in six aduft heafthy subjects. (b) Mean 2 SEM GH AUC (pg/L 190 min; a) after: 1 pg/kg; 0.3 pg/kg; 0.1 @g/kg; 0.01 pg/kg GHRH (l-29)NH,; and after PD alone in six adult healthy subjects, lP < .05 Y GHRH. 1 &kg; VP < .05 v GHRH, 0.3 Kg/kg; xP < .05 v GHRH, 0.1 Pg/kg; AP < .05 Y GHRH, 0.01 Pg/kg.
after: 0, 1 kg/kg; n . 0.3 pglkg; A. 0.1 pg/kg; +, 0.01 pgfkg GHRH (l-29)NH,;
GHRH injection. PD pretreatment did not shift the time of the GH peak response. All subjects experienced facial flushing after 1 and 0.3 ug/kg GHRH IV injection. The lower GHRH doses and PD did not cause overt side effects in any subjects. DISCUSSION
Our data confirm that GHRH (l-29)NH, elicits GH secretion in a dose-dependent fashion.4,5 Moreover, our data demonstrate that this dose-response relationship is significantly enhanced by PD administration. Dose-response studies for the different hypothalamic releasing hormones have been performed.2-5~‘1.”The aims of these studies were to establish an appropriate dose to be used in clinical practice, to establish the maximal secretory capacity of the pituitary for each hormone, and to establish the potency of native releasing factors and analogues. GHRH-elicited GH secretion has been demonstrated to be dose-dependent in normal humans. Studies with human GHRH (l-40)NH,,* GHRH (l-44)NH,,’ or with their equipotent synthetic analog GHRH (1-29)NH,,4’S disclosed that a dose of 0.3 Fg/kg is able to elicit maximal GH responses. The GH response to GHRH at doses of 0.1 &kg or lower is submaximal. These studies have led to the conclusion that the maximal somatotrope secretory capacity is represented by the GH peak response obtained after the administration of GHRH at a dose of 0.3 @kg or higher. Recently, several in vivo studies have investigated the
role of somatostatin tone in modulating the GH response to exogenous GHRH. It has been shown that this is an important factor in modulating the pituitary GH response to GHRH.‘.14 For example, in the rat, passive immunization with somatostatin antibodies” has shown that substances known to inhibit the GH response to GHRH, such as glucocorticoids, may act through increased somatostatin secretion.‘” In man, direct tools to investigate somatostatin tone are not available. However, there is increasing evidence that somatostatin release may be primarily under cholinergic control.“‘.” The acetyl-cholinesterase inhibitor PD increases the central nervous system cholinergic tone and is hypothesized to cause a decrease in the release of somatostatin by the hypothalamus.8-‘0 The administration of PD is known to restore the GH response to GHRH in normal subjects after administration of substances that are hypothesized to increase somatostatin secretion, such as glucose,” exogenous GH,19 and glucocorticoids~” and in patients with spontaneously elevated somatostatin, ie, obese subjects.ZZ.Z3It has also been reported that PD causes an enhancement of the GH response to very high doses of GHRH.’ Our data show that, in placebo-treated subjects, the administration of 0.3 and 1.0 kg/kg GHRH (l-29)NH: elicited similar GH responses in normal adult subjects. The GH response to 0.1 ng/kg GHRH was submaximal, while the response to 0.01 t&kg GHRH did not significantly increase GH levels as compared with saline-treated sub-
GIUSTINA
522
jects. After pretreatment of the subjects with PD, a maximal GH response to GHRH was obtained at a dose of 0.1 ug/kg. In addition, this response was significantly greater than the maximal response observed in placebo-treated subjects. Moreover, the lowest dose of GHRH (0.01 kg/kg), ineffective when evaluated following placebo pretreatment, resulted in a significant GH response after PD pretreatment. Interestingly, the GH response to 0.01 u&kg GHRH after PD was similar to the maximal GH responses to 1.0 ugikg GHRH after placebo. Plotting GH peak concentrations versus the GHRH dose, we observed that PD pretreatment shifted the response curve up and to the left (Fig 2). Thus, both the maximum response and the sensivity to GHRH were increased in the PD-treated subjects. In our experiment, we did not find a biphasic doseresponse curve following PD treatment as previously described.’ This discrepancy may be explained by the fact that independent groups of subjects were studied in the earlier
ET AL
report, whereas we used the same subjects. Moreover, the doses of GHRH were not normalized for the body weight in the earlier study.’ Our data support the concept that dose-response studies with GHRH in man lead to variable results concerning the maximal pituitary GH response. This variability might be due to variable endogenous somatostatin tone. Our findings strongly support this, since PD pretreatment, which enhances cholinergic tone and consequently is thought to decrease somatostatin, significantly enhances the GH response to GHRH. These conclusions can be explained by a lower sensitivity of GHRH receptors or by decreased responsivity of the synthetic/secretory somatotropc functions in the presence of somatostatin.” We conclude that somatostatin tone is a significant factor in modulating the dose-response secretion in man.
effect of GHRH
on GH
REFERENCES 1. Wehrenberg WB, Ling N, Bohlen P. et al: Physiological roles of somatocrinin and somatostatin in the regulation of growth hormone secretion. Biochem Biophys Res Commun 109:562-567. 1982 2. Vance ML, Borges JL. Kaiser DL. et al: Human pancreatic tumor growth hormone-releasing factor: Dose-response relationships in normal man. J Clin Endocrinol Metab 58:838-844,1984 3. Gelato MC, Pescovitz OH, Cassorla F, et al: Dose-response relationships for the effects of growth hormone-releasing factor-( l44)NH in young adult men and women. J Clin Endocrinol Metab 59:197-201, 1984 4. Grossman A, Savage MO, Lytras N, et al: Responses to analogues of growth hormone-releasing hormone in normal subjects and in growth hormone deficient children and young adults. Clin Endocrinol21:321-330, 1984 5. Penalva A, Muruais C, Casanueva FF, et al: Effect of enhancement of endogenous cholinergic tone with pyridostigmine on the dose-response relationships of growth hormone (GH)releasing hormone-induced GH secretion in normal subjects. J Clin Endocrinol Metab 70:324-327, 1990 6. Massara F, Ghigo E, Goffi S, et al: Blockade of hp-GRF 40 induced GH release in normal men by a cholinergic muscarinic antagonist. J Clin Endocrinol Metab 59:1025-1026, 1984 7. Casanueva FF, Villanueva L, Cabranes JA, et al: Cholinergic mediation of GH secretion elicited by arginine, clonidine and physical exercise in man. J Clin Endocrinol Metab 59:526-530,1984 8. Ghigo E, Mazza E, Imperiale E. et al: Growth hormone responses to pyridostigmine in normal adults and in normal and short children. Clin Endocrinol27:669-673, 1987 9. Giustina A, Bodini C, Bossoni S, et al: Effects of calcitonin on GH response to pyridostigmine in combination with hGHRH (l-29)NH, in normal adult subjects. Clin Endocrinol 33:345-380, 1990 10. Locatelli V, Torsello A, Redaelli M. et al: Cholinergic agonist and antagonist drugs modulate in the rat the growth hormone response to growth hormone releasing hormone. Evidence for mediation by somatostatin. J Endocrinol 111:271-278, 1986 11. Orth DN, Jackson RV, DeCherney GS. et al: Effect of synthetic ovine corticotropin-releasing factor. Dose response of plasma adrenocorticotropin and cortisol. J Clin Invest 71:587-591, 1983
I?. Hsueh AJW, Adashi EY, Tucker E, et al: Relative potencies of gonadotropin-releasing hormone agonists and antagonists on ovarian and pituitary functions. Endocrinology 112:689-695. 1983 13. Vance ML, Evans WS, Kaiser DL. et al: The effect of intravenous. subcutaneous, and intranasal GH-RH analog, (Nle27)GHRH(I-29)NH:, on growth hormone secretion in normal men: Dose response relationships. Clin Pharmacol Ther 40:627-633, 1986 14. Davies RR, Turner SJ, Orskow H, et al: The interaction of human pancreatic growth hormone releasing factor 1-44 with somatostatin in vivo in normal man. Clin Endocrinol 23:271-276. 1985 15. Wehrenberg WB, Brazeau P, Luben R, et al: A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology 36:489-491. 1983 16. Wehrenberg WB. Janowski BA, Piering AW. et al: Glucocorticoids: Potent inhibitors and stimulators of growth hormone secretion. Endocrinology 126:3200-3203. 1990 17. Richardson SB, Hollander CS, D’Eletto R, et al: Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology 107:122-129, 1980 18. Penalva A. Burguera B, Casabiell X, et al: Activation of cholinergic neurotransmission by pyridostigmine reverses the inhibitory effect of hyperglycaemia on growth hormone releasing hormone induced growth hormone secretion in man: Suggesting that glucose acts through hypothalamic release of somatostatin. Neuroendocrinology 49:55 l-554, 1989 19. Ross RJM. Tsagarakis S. Grossman A, et al: GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: Studies with pyridostigmine and GHRH. Clin Endocrinol27:727-733, 1987 20. Giustina A, Doga M, Bodini C. et al: Acute effects of cortisone acetate on growth hormone response to growth hormonereleasing hormone in normal adult subjects. Acta Endocrinol 122:206-210. 1990 21. Giustina A, Girelli A, Doga M. et al: Pyridostigmine blocks the inhibitory effect of glucocorticoids on growth hormone reieasing hormone-stimulated growth hormone secretion in man. J Clin Endocrinol Metab 71:580-584. 1990 22. Williams T, Berelowitz M, Joffee SN, et al: Impaired growth hormone responses to growth hormone-releasing factor in obesity.
CHOLINERGIC TONE AND GH SECRETION
A pituitary defect reversed with weight reduction. N Engl J Med 311:1403-1407,1984 23. Cordido F. Casanueva FF, Dieguez C: Cholinergic receptor activation by pyridostigmine restored growth hormone responsiveness after growth hormone-releasing hormone administration in obese subjects: Evidence for hypothalamicsomatostatinergicpartic-
523
ipation in the blunted GH release of obesity. J Clin Endocrinol Metab 68:290-293, 1989 24. Vale W, Vaughan J, Yamamoto G. et al: Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethasone on GH secretion in vitro. Endocrinology 112:1553-1555, 1983
Giustina,
Simonetta
Bossoni, Maurizio
Corrado Schettino,
Bodini,
Mauro
and William
Doga,
Angela
Girelli,
Maria
Grazia
to
Buffoli,
B. Wehrenberg
Growth hormone-releasing hormone (GHRH) increases serum GH levels in a dose-dependent manner. 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 if an enhancement of the cholinergic tone was able to influence the dose-response relationship between GHRH and GH in normal adult subjects. Six healthy adult volunteers underwent IO experimental protocols. They were: human GHRH (I-2g)NH,, 1 pg/kg injected as an intravenous (IV) bolus 60 minutes after (a) PD. 120 mg administered orally, or (b) placebo, two tablets administered orally; GHRH, 0.3 bg/kg injected as an IV bolus 60 minutes after (c) PD or (d) placebo; GHRH, 0.1 @g/kg injected as an IV bolus 60 minutes after (e) PD or (f) placebo; GHRH, 0.01 Fg/kg injected as an IV bolus 60 minutes after(g) PD or (h) placebo; saline, 1 mL injected as an IV bolus 60 minutes after (i) PD or (I) placebo. The GH response in placebo-treated subjects was similar after 1 @g/kg and 0.3 Fg/kg GHRH, while the 0.1 pg/kg dose elicited a lower response. The 0.01 pg/kg dose of GHRH did not significantly increase GH levels as compared with saline. After PD. the GH responses to GHRH were greatly enhanced at all doses tested: 1.0, 0.3, and 0.1 kg/kg GHRH all elicited similar GH responses; the GH response to 0.01 pg/kg GHRH was lower, but was still higher than that observed after saline. We conclude that the endogenous somatostatin tone is a significant factor in modulating the dose-response relationship of GHRH-induced GH secretion in normal man. Copyright 0 199 1 by W.8. Saunders Company
G
ROWTH HORMONE (GH) synthesis and secretion are regulated by the hypothalamic peptides GHreleasing hormone (GHRH), which has an excitatory role, and somatostatin, which has an inhibitory role.’ Several studies have shown that human GHRH (l-40)NHz and GHRH (l-44)NH,, and their equipotent synthetic analog GHRH ( l-29)NH2, are able to stimulate GH secretion in a dose-dependent fashion in normal humans.‘.’ There is increasing evidence for an important role of cholinergic neurotransmission in the regulation of GH secretion in normal man. Anticholinergic drugs are known to abolish the GH response to various physiological and pharmacological stimuli.‘,’ 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.’ Experimental studies suggest that the action of PD may be mediated by a decrease in the hypothalamic release of somatostatin.“’ Recently, it has been reported that the simultaneous administration of PD and GHRH causes a biphasic dose-response of GH when various doses of GHRH are tested in different groups of subjects.5 However, it still is not clear what is the role of endogenous somatostatin in determining the dose-response relationship between GHRH and GH. The aim of our study was to investigate the role of somatostatin in modulating the dose-response effect of GHRH on GH secretion in the same normal subjects using PD-mediated enhancement of the central cholinergic tone.
Methods After an overnight fast, each subject was admitted to the Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. A catheter was inserted percutaneously into an antecubital vein and kept patent by a slow saline infusion. After a 30.minute stabilization period, the following treatments were initiated in randomized order: human GHRH (l-29)NH, (Geref. Serono, Italy), 1 &kg in 1 mL of saline injected as an intravenous (IV) bolus 60 minutes after (a) PD (Mestinon, Roche. Switzerland), 120 mg administered orally, or (b) placebo, two tablets administered orally; GHRH, 0.3 kg/kg injected as an IV bolus 60 minutes after (c) PD, 120 mg orally, or (d) placebo, orally; GHRH. 0.1 &kg injected as an IV bolus 60 minutes after (e) PD. 120 mg orally, or (f) placebo, orally; GHRH, 0.01 kg/kg injected as an IV bolus 60 minutes after (g) PD, 120 mg orally, or (h) placebo, orally; saline, I mL injected as an IV bolus 60 minutes after(i) PD. 120 mg orally. or (I) placebo, orally. Blood samples for GH assay were taken at -75, -60 (time of PD or placebo administration), -45, -30, -15, 0 (time of GHRH injection), 15,30,45,60. 90, and 120 minutes. GH secretory responses were expressed as absolute values (kg/L), peak values (&L), and the area under the curve (AUC) for GH secretion (pg/L 180 min). GH secretory responses to each different dose of GHRH were compared with the responses to each of the other four GHRH doses within and between the two treatments (GHRH + placebo or GHRH + PD study). One-way ANOVA was used to compare peak GH values and AUCs. Individual GH values were compared using the ANOVA for repeated measures. P < .OSwas considered statistically significant. All results are expressed as the mean 2 SEM.
SUBJECTS AND METHODS
Subjects Five male and one female adult healthy volunteers with no history of endocrine or metabolic disease were studied. Their ages ranged Erom 25 to 35 years and their body mass index ranged from 19 to 24 kg/m’. None of the subjects were taking any drugs during the course of the study. Ten different experimental trials were conducted in each of the subjects at no less than 7-day intervals. Metabolism,
Vol40, No 5 (May), 1991: pp 519-523
From the Cattedra di Clinica Medica, Universityof Brescia, Brescia. Italy; and the Department of Health Sciences, Universityof Wisconsin. Milwaukee, WI. Address reprint requests to Andrea Giustina. MD, Cattedra di Clinica Medica. Universitd di Brescia clo 2a Medicina-Spedali Civili, 25125 Brescia, Italy. Copyright 0 1991 by W B. Saunders Company 0026-0495/91/4005-0013$03.OOlO 519
520
GIUSTINA ET AL
GH AUC
b
IaglLXlaomhl 3cao
2000
1
Fig 1. GH response to saline and different doses of GHRH in placebo pretreated normal subjects. (a) Mean k SEM GH levels (pg/L) after: O, 1 pg/kg; 0.0.3 pg/kg; A, 0.1 pg/kg; O,O.Ol pg/kg GHRH (l-29)NH,; and V, after saline alone in six adult healthy subjects. (b) Mean ? SEM GH AUC (pg/L 190 mini 0) after: 1 &kg; 0.3 pg/kg; 0.1 pg/kg; 0.01 pg/kg GHRH (129)NH,; and after saline alone in six adult healthy subjects. lP < .05 Y GHRH. 1 pg/kg; V, P < .05v GHRH, 0.3 Kg/kg; Y, P < .05 v GHRH, 0.1 pg/kg.
Assays A commercial
immunoradiometric kit was used for estimation of serum GH concentrations (Allegro HGH. Nichols Institute, CA; interassay and intraassay coefficients of variation were ?4.6% and 7%. respectively; sensitivity was 0.06 kg/L). All samples from the same patient were measured in a single assay in triplicate. RESULTS
GHRH + Placebo Study GHRH (l-29)NH, had no significant effect on GH release at a dose of 0.01 kg/kg. However, doses of 0.1 p&kg and above significantiy stimulated the release of GH with respect to saline. Figure la illustrates the mean GH values after saline, 0.01, 0.1, 0.3, 1 t&kg GHRH (l-29)NHz. No differences between GH levels after 0.3 and 1 p&kg were observed. Therefore, 0.3 &kg appears to elicit the maximal GH response. The lower dose of 0.1 kg/kg produced a submaximal GH response. A similar pattern of responses was observed when the data were analyzed in terms of GH peaks (Fig 2) and GH AUCs (Fig lb). GH peak values after the different doses of GHRH were obtained in each subject at times ranging from 15 to 90 minutes. In most of the subjects the GH peak occurred at 30 minutes after GHRH injection. No significant differences in the time of GH peak occurrence among the different doses of GHRH tested were observed. GHRH + PD Study PD administration induced a clear-cut increase in plasma GH levels compared with those after placebo (P < .OSfrom 15 to 90 minutes) in saline-treated subjects. Furthermore, pretreatment with PD enhanced mean GH levels with respect to the same dose of GHRH (P < .05 from 15 to 90 minutes for all doses of GHRH). Figure 2a illustrates the mean GH values after PD and saline or 0.01, 0.1, 0.3, or 1 t.@kg GHRH (l-29)NH,. No differences among GH levels after 0.1, 0.3, and 1 ug/kg were observed. Therefore, 0.1
pg./kg elicited the maximal GH response. The lower dose of 0.01 &kg produced a submaximal GH response that was significantly higher than that observed after saline. Interestingly, this dose elicited a similar GH response to that obtained in subjects treated with 1 pg/kg GHRH + placebo. A similar pattern of response was observed when the data were analyzed in terms of GH peaks (Fig 2) and GH AUCs (Fig 3b). GH peak values after GHRH + PD were obtained at times ranging from 15 to 90 minutes. In most of the subjects the GH peak occurred at 30 minutes after
GH peak (IQ/L) 4%
30* T
10 * I$’ OJ r
0
I
,
02
I
,
0.4
GHRH
I
,
0.6
I
,
0.6
,
(
1
dose (pg/kg)
Fig 2. Dose-response relationship between GHRH and peak GH secretion in normal subjects with and without PD pretreatment 60 minutes before GHRH administration. Peak GH levels (mean t SEM; kg/L) plotted against their respective GHRH (l-29)NH, dose (a) after saline: 0.1 Kg/kg: 0.0.3 pg/kg; A, 0.1 pg/kg; and 0.0.01 pg/kg. (b) After PD: 0.1 pg/kg; n , 0.3 pg/kg; A, 0.1 pg/kg; and l , 0.01 pg/kg in six adult healthy subjects. Peak responses usually occurred 30 minutes after GHRH administration. lP < .05 Y PD + GHRH.
521
CHOLINERGIC TONE AND GH SECRETION
a
GH
b
AUC
trJBILX18Omin) 3000
T
l-
. :
. i
EL lzB_ OH RH O.Olpg(Kg
PD
Fig 3.
GH response to saline and different doses of GHRH in PD (120 mg, orally) pretreated normal subjects. (a) Mean 2 SEM GH levels (pg/L) and g, after PD alone in six aduft heafthy subjects. (b) Mean 2 SEM GH AUC (pg/L 190 min; a) after: 1 pg/kg; 0.3 pg/kg; 0.1 @g/kg; 0.01 pg/kg GHRH (l-29)NH,; and after PD alone in six adult healthy subjects, lP < .05 Y GHRH. 1 &kg; VP < .05 v GHRH, 0.3 Kg/kg; xP < .05 v GHRH, 0.1 Pg/kg; AP < .05 Y GHRH, 0.01 Pg/kg.
after: 0, 1 kg/kg; n . 0.3 pglkg; A. 0.1 pg/kg; +, 0.01 pgfkg GHRH (l-29)NH,;
GHRH injection. PD pretreatment did not shift the time of the GH peak response. All subjects experienced facial flushing after 1 and 0.3 ug/kg GHRH IV injection. The lower GHRH doses and PD did not cause overt side effects in any subjects. DISCUSSION
Our data confirm that GHRH (l-29)NH, elicits GH secretion in a dose-dependent fashion.4,5 Moreover, our data demonstrate that this dose-response relationship is significantly enhanced by PD administration. Dose-response studies for the different hypothalamic releasing hormones have been performed.2-5~‘1.”The aims of these studies were to establish an appropriate dose to be used in clinical practice, to establish the maximal secretory capacity of the pituitary for each hormone, and to establish the potency of native releasing factors and analogues. GHRH-elicited GH secretion has been demonstrated to be dose-dependent in normal humans. Studies with human GHRH (l-40)NH,,* GHRH (l-44)NH,,’ or with their equipotent synthetic analog GHRH (1-29)NH,,4’S disclosed that a dose of 0.3 Fg/kg is able to elicit maximal GH responses. The GH response to GHRH at doses of 0.1 &kg or lower is submaximal. These studies have led to the conclusion that the maximal somatotrope secretory capacity is represented by the GH peak response obtained after the administration of GHRH at a dose of 0.3 @kg or higher. Recently, several in vivo studies have investigated the
role of somatostatin tone in modulating the GH response to exogenous GHRH. It has been shown that this is an important factor in modulating the pituitary GH response to GHRH.‘.14 For example, in the rat, passive immunization with somatostatin antibodies” has shown that substances known to inhibit the GH response to GHRH, such as glucocorticoids, may act through increased somatostatin secretion.‘” In man, direct tools to investigate somatostatin tone are not available. However, there is increasing evidence that somatostatin release may be primarily under cholinergic control.“‘.” The acetyl-cholinesterase inhibitor PD increases the central nervous system cholinergic tone and is hypothesized to cause a decrease in the release of somatostatin by the hypothalamus.8-‘0 The administration of PD is known to restore the GH response to GHRH in normal subjects after administration of substances that are hypothesized to increase somatostatin secretion, such as glucose,” exogenous GH,19 and glucocorticoids~” and in patients with spontaneously elevated somatostatin, ie, obese subjects.ZZ.Z3It has also been reported that PD causes an enhancement of the GH response to very high doses of GHRH.’ Our data show that, in placebo-treated subjects, the administration of 0.3 and 1.0 kg/kg GHRH (l-29)NH: elicited similar GH responses in normal adult subjects. The GH response to 0.1 ng/kg GHRH was submaximal, while the response to 0.01 t&kg GHRH did not significantly increase GH levels as compared with saline-treated sub-
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jects. After pretreatment of the subjects with PD, a maximal GH response to GHRH was obtained at a dose of 0.1 ug/kg. In addition, this response was significantly greater than the maximal response observed in placebo-treated subjects. Moreover, the lowest dose of GHRH (0.01 kg/kg), ineffective when evaluated following placebo pretreatment, resulted in a significant GH response after PD pretreatment. Interestingly, the GH response to 0.01 u&kg GHRH after PD was similar to the maximal GH responses to 1.0 ugikg GHRH after placebo. Plotting GH peak concentrations versus the GHRH dose, we observed that PD pretreatment shifted the response curve up and to the left (Fig 2). Thus, both the maximum response and the sensivity to GHRH were increased in the PD-treated subjects. In our experiment, we did not find a biphasic doseresponse curve following PD treatment as previously described.’ This discrepancy may be explained by the fact that independent groups of subjects were studied in the earlier
ET AL
report, whereas we used the same subjects. Moreover, the doses of GHRH were not normalized for the body weight in the earlier study.’ Our data support the concept that dose-response studies with GHRH in man lead to variable results concerning the maximal pituitary GH response. This variability might be due to variable endogenous somatostatin tone. Our findings strongly support this, since PD pretreatment, which enhances cholinergic tone and consequently is thought to decrease somatostatin, significantly enhances the GH response to GHRH. These conclusions can be explained by a lower sensitivity of GHRH receptors or by decreased responsivity of the synthetic/secretory somatotropc functions in the presence of somatostatin.” We conclude that somatostatin tone is a significant factor in modulating the dose-response secretion in man.
effect of GHRH
on GH
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I?. Hsueh AJW, Adashi EY, Tucker E, et al: Relative potencies of gonadotropin-releasing hormone agonists and antagonists on ovarian and pituitary functions. Endocrinology 112:689-695. 1983 13. Vance ML, Evans WS, Kaiser DL. et al: The effect of intravenous. subcutaneous, and intranasal GH-RH analog, (Nle27)GHRH(I-29)NH:, on growth hormone secretion in normal men: Dose response relationships. Clin Pharmacol Ther 40:627-633, 1986 14. Davies RR, Turner SJ, Orskow H, et al: The interaction of human pancreatic growth hormone releasing factor 1-44 with somatostatin in vivo in normal man. Clin Endocrinol 23:271-276. 1985 15. Wehrenberg WB, Brazeau P, Luben R, et al: A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology 36:489-491. 1983 16. Wehrenberg WB. Janowski BA, Piering AW. et al: Glucocorticoids: Potent inhibitors and stimulators of growth hormone secretion. Endocrinology 126:3200-3203. 1990 17. Richardson SB, Hollander CS, D’Eletto R, et al: Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology 107:122-129, 1980 18. Penalva A. Burguera B, Casabiell X, et al: Activation of cholinergic neurotransmission by pyridostigmine reverses the inhibitory effect of hyperglycaemia on growth hormone releasing hormone induced growth hormone secretion in man: Suggesting that glucose acts through hypothalamic release of somatostatin. Neuroendocrinology 49:55 l-554, 1989 19. Ross RJM. Tsagarakis S. Grossman A, et al: GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: Studies with pyridostigmine and GHRH. Clin Endocrinol27:727-733, 1987 20. Giustina A, Doga M, Bodini C. et al: Acute effects of cortisone acetate on growth hormone response to growth hormonereleasing hormone in normal adult subjects. Acta Endocrinol 122:206-210. 1990 21. Giustina A, Girelli A, Doga M. et al: Pyridostigmine blocks the inhibitory effect of glucocorticoids on growth hormone reieasing hormone-stimulated growth hormone secretion in man. J Clin Endocrinol Metab 71:580-584. 1990 22. Williams T, Berelowitz M, Joffee SN, et al: Impaired growth hormone responses to growth hormone-releasing factor in obesity.
CHOLINERGIC TONE AND GH SECRETION
A pituitary defect reversed with weight reduction. N Engl J Med 311:1403-1407,1984 23. Cordido F. Casanueva FF, Dieguez C: Cholinergic receptor activation by pyridostigmine restored growth hormone responsiveness after growth hormone-releasing hormone administration in obese subjects: Evidence for hypothalamicsomatostatinergicpartic-
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ipation in the blunted GH release of obesity. J Clin Endocrinol Metab 68:290-293, 1989 24. Vale W, Vaughan J, Yamamoto G. et al: Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethasone on GH secretion in vitro. Endocrinology 112:1553-1555, 1983
