Original Paper Neuroendocrinology 1992 ;56:208—213
Institutes of Endocrinology and Biochemistry, Catholic University School of Medicine, Rome, Italy
Effects of Sex and Age on Pyridostigmine Potentiation of Growth Hormone-Releasing Hormone-Induced Growth Hormone Release
Key Words
Abstract
GH GHRH Pyridostigmine
Previous studies have shown that pyridostigmine (PD) is capable of increasing the growth hormone (GH) response to GH-releasing hormone (GHRH) in young healthy subjects. In order to investigate the influence of age and sex on the PD potentiation of GHRH-induced GH release, we have studied the GH response to GHRH (50 ug i.v.) 1 h after oral adminis tration of placebo or PD (60 mg) in 8 young healthy men (aged 19-28 years) and 8 agematched young women (aged 18-25 years) during the follicular phase of the menstrual cycle, as well as in 8 postmenopausal women (aged 57-62 years) and 8 age-matched elderly men (aged 56-64 years). In the same subjects the effect of PD alone (60 mg p.o.) was also studied. Furthermore, in 6 postmenopausal women and 6 elderly men, the effect of a 30-mg PD oral dose on GH secretion and GH response to GHRH was evaluated with a similar protocol. The GH responses (mean ± SE) to GHRH F placebo were similar in young men (peak 20.1 ± 2 ng/ml, AUC 1,250 ±113 ng/ml/min) and women (peak 29.3 ± 2.3 ng/ml, AUC 1,769 ±305 ng/ml/min). PD 60 mg was capable of significantly increasing the GH response to GHRH in young men (peak 43.5 ± 5 .1 ng/ml, AUC 3,734 ±472 ng/ml/min, p < 0.005) but not in women (peak 39 ±2.3 ng/ml, AUC 2,479 ±205 ng/ml/min). The GH responses to GHRH + placebo were also similar in postmenopausal women (peak 6.2 ± 0.7 ng/ml, AUC 540 ± 80 ng/ml/min) and age-matched men (peak 11.3 ± 0.6 ng/ml, AUC 763 ±73 ng/ml/min) although these responses were significantly decreased when compared to those observed in young individuals. PD 60 mg administration induced a significant increase in GH response to GHRH both in postmenopausal women (peak 27±3.6 ng/ml, AUC 2,224± 251 ng/ml/min. p< 0.001) and elderly men (peak 49.7 ±2.4 ng/ml, AUC 4,557 ± 263 ng/ml/min, p < 0.001 ). The GH increment, however, was greater in elderly men than in postmenopausal women (p < 0.005). In young men, elderly men and postmenopausal women, PD 60 mg potentiated the action of GHRH rather than merely being additive. A similar effect was observed when a 30-mg PD dose was preadministered in elderly men and women. Our data clearly demonstrate a marked in fluence of age and sex on the PD potentiation of the GHRH-stimulated GH secretion. PD significantly potentiated GH responses to GHRH in young men, elderly men and post menopausal women but not in young women.
Received: March 22. 1991 Accepted after revision: November 5, 1991
Prof. Antonino Barbarino Via Diano Marina 9 / 11 1-001(»8 Rome (Italy)
© 1992 S. Karger AG, Basel 0028-3835/92/0562-0208 $ 2.75/0
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Salvatore M. Corsello ° Anna Tofani“ Silvia Della Casa “ Carlo A. Rota “ Rosa Sciuto “ Simonetta Colasanti° Angela Barini1’ Antonino Barbarino “
Materials and Methods Eight young men aged 19-28 years and 8 age-matched young women aged 18-25 years as well as 8 postmenopausal women aged 57-65 years and 8 age-matched elderly men aged 56-64 years par ticipated in the study after giving informed consent. All subjects were nonstressed. AH were within 10% of ideal body weight and were eating their customary diet before and during the interval be tween the tests. None had clinical evidence of endocrine and other disease and all were free of any medication. All young women had regular menstrual cycles. After an overnight fast, in recumbent sub jects, an indwelling cannula was inserted into an antecubital vein at 07.30 h, 30 min before starting the tests. It was used for blood sam pling and GHRH (or saline) injection. Two separate GHRH stimu lation tests were performed in each subject, intravenously injecting a 50-pg bolus dose of GHRH at time 0 (09.00 h) 1 h after oral admin istration of placebo or 60 mg PD, respectively. To study the effect of PD administration alone, in the same subjects a third test was per formed administering orally 60 mg PD a time -60 and injecting 2 ml saline i.v. at time 0. Finally, in 6 postmenopausal women and 6 elderly men, already included in the study group, the same protocol was repeated using a 30-mg PD oral dose. Blood was obtained 60, 30, and 0 min before and 15, 30, 60, 90, and 120 min after GHRH (or saline) administration. In individual subjects the various tests were performed at least at 72-hour intervals, in a blind fashion. In young women, they were performed during the follicular phase of the menstrual cycle. The time of the cycle was established by mea suring estradiol and progesterone levels.
GHRH(l-44) was furnished by Bachem, Bubendorf (Switzer land). Vials containing 0.1 mg were diluted in 0.9% saline giving a final concentration of 50 pg/ml. Pyridostigmine bromide (Mestinon), a cholinesterase inhibitor, was furnished by Hoffmann-La Roche, Nutley, N.J., USA. Plasma GH levels were measured in du plicate by RIA, using polyethyleneglycol to separate free and bound hormone, with reagents obtained by Biodata, Milan (Italy). One na nogram of GH corresponded to 2 uU/ml WHO 66/217 standard. The intra-assay and interassay coefficients of variation ranged from 2.5 to 3.9% and from 5.8 to 8.5%, respectively. Plasma concentra tions of estradiol, progesterone and testosterone were measured by RIA, as previously described [13, 14]. All samples from a given study were measured in the same assay. The results are given as mean ± SE. The area under the curve (AUC) was calculated by a trapezoidal method. Analysis of variance and Newman-Keuls test were used for statistical evaluation. The significance level was estab lished at p < 0.05.
Results
In young subjects, 50 ug GHRH administration, after placebo pretreatment, induced a clearcut increase in GH levels without significant differences between sexes. The mean peak plasma GH levels were 20.1 ± 2 ng/ml in men and 29.3 ±2.3 ng/ml in women while the AUC were 1,250± 113 ng/m l/m in in men and 1,769±305 ng/m l/ min in women (fig. 1, 3). In young men, 60 mg PD pretreatment induced a significant enhancement (vs. placebo) of the GH re sponse to GHRH both in terms of peak response (43.5 ± 5.1 ng/ml, p < 0.005) and AUC (3,734 ± 472 ng/ ml/min, p < 0.005). On the contrary, in young women, 60 mg PD pretreatment was not able to significantly en hance the GH response to GHRH (mean peak 39 ± 2.3 ng/ml, AUC 2,479 ± 205 ng/m l/m in; fig. 1, 3). No significant differences in GH response to GHRH, after placebo pretreatment, were also found between postmenopausal women (mean peak 6.2 ±0.7 ng/ml, AUC 540 ±80 ng/m l/m in) and age-matched elderly men (mean peak 11.3 ±0.6 ng/ml, AUC 763 ± 73; fig. 2, 3) although these responses were significantly decreased (p < 0.05) when compared to those observed in young in dividuals. Sixty milligrams PD pretreatment induced a signifi cant increase (vs. placebo) in GH response to GHRH both in postmenopausal women (mean peak 27 ± 3.6 ng/ ml, AUC 2,224 ±251 ng/m l/m in, p < 0.001) and elderly men (mean peak 49.7 ± 2.4 ng/ml, AUC 4,557 ± 263 ng/ ml/min, p < 0.001; fig. 2, 3). No significant differences in GH responses to GHRH plus 60 mg PD were found between young and elderly subjects of both sexes. The
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The influence of age and sex on neuroregulation of growth hormone (GH) secretion is controversial. In pre vious studies, a number of sex differences in GH secretory pattern have been described in several mammalian species [1-8] and are known to be related to the perinatal action of steroid hormones [9, 10]. In man, on the con trary, it is unclear whether sexual steroids are capable of influencing the GH response to GH-releasing hormone (GHRH) as well as the effect of pyridostigmine (PD) on the GHRH-induced GH release. Very recently, Ghigo et al. [ 11 ] have presented data showing that PD is capable of increasing the GH response to GHRH both in young and elderly subjects. However, they did not discriminate be tween male and female subjects in the belief that no dif ferences are present in the neuroregulatory mechanisms governing GH secretion in the two sexes. We have re cently shown that there is a sex difference in the CRH inhibition of the GHRH-induced GH release [12], These data suggest that a gender-related difference in the neuroregulation of GH secretion is present also in the human. We have investigated whether an activation of the cholin ergic tone by PD could show a sex-related difference of the pituitary responsiveness to GHRH in healthy nonobese subjects of different sex and age.
Time, min
Time, min
Fig. 2. Mean ( ± SE) GH responses to GHRH 50 pg ( • ) , GHRH 50 pg + PD 60 mg (O ) and PD 60 mg + in 8 elderly men and 8 postmenopausal women. **p< 0.005, comparing values after GHRH + PD with GHRH + placebo at the reported time.
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+ placebo saline (■ ) *p< 0.05; those after
Fig. 3. Mean (± S E ) GH AUC after PD 60 mg + saline (□ ), GHRH 50 pg + placebo (H ) and GHRH 50 pg + P D 6 0 m g (B ) in 8 young men, 8 young women, 8 elderly men and 8 postmeno pausal women.
GH increament, however, was greater in elderly men than in postmenopausal women (p < 0.005). The pattern of GH response to GHRH after 60 mg PD administration was similar in young men, elderly men and postmeno pausal women in whom GH levels peaked at 60 min and then gradually decreased but remained high until 120 min after GHRH, while in young women GH levels peaked at 30 min and returned to basal values at 90 min (fig. 1, 2). When 60 mg PD plus saline was given, a small in crease in plasma GH was observed in all the groups tested without significant differences between the groups (young men: mean peak 7.3±0.81 ng/ml, AUC 662±79 ng/m l/m in; young women: mean peak 9.1 ± 2 ng/ml, AUC 893±152 ng/m l/m in; elderly men: mean peak 8.3± 1.3 ng/ml, AUC 760±93 ng/ ml/min; elderly women: mean peak 9.4±1.4 ng/ml, AUC 774 ±94 ng/ml/min). Therefore, in young men, elderly men and postmenopausal women, 60 mg PD potentiated the effect of GHRH rather than being merely additive to it (fig. 1-3). Thirty mg PD pretreatment induced a significant enhancement (vs. placebo) of the GH response to GHRH in 6 elderly men (mean peak 22.7 ± 1 ng/ml vs.
Corsello/Tofani/Della Casa/Rota/ Sciuto/Colasanti/Barini/Barbarino
Sex, Age and GH Secretion
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Fig. 1. Mean ( ± SE) GH responses to GHRH 50 pg + placebo ( • ) , GHRH 50 pg + PD 60 mg (O ) and PD 60 mg + saline (■ ) in 8 young men and 8 young women. *p< 0.05; **p< 0.005, com paring values after GHRH -I- PD with those after GHRH + placebo at the reported time.
Discussion
Our present findings confirm that there is no sex dif ference in GH responsiveness to an effective submaximal dose of GHRH in age-matched young subjects as well as in elderly individuals. There is, however, a significant agedependent decline in GH responses in both sexes. The lack of differences in GHRH-induced GH secretion in young male and female subjects is in agreement with the results by Gelato et al. [15] but in contrast with data by other authors [16, 17]. Lang et al. [16] reported that the responsiveness of GH to GHRH is significantly greater in premenopausal women than in age-matched men. Smals et al. [17], on the contrary, showed an increased GH re sponsiveness to GHRH in men compared to women. These discrepant findings are difficult to explain. In fact, the differences in the GHRH dose administered do not seem a possible explanation because it has been found by different authors that doses of GHRH between 50 and 500 pg/subject induced similar increases in plasma GH levels [15, 18, 19], In agreement with most of the data from the literature we found that the magnitude of GH response to GHRH was significantly lower in older subjects than in young in dividuals [11, 16, 20, 21], GH responses to GHRH were similar in magnitude in elderly men and women. The diminished pituitary responsiveness to GHRH with aging can be related to an increased somatostatin tone or to changes intrinsic to the pituitary gland. Our findings,
Fig. 4. Mean ( ± SE) GH AUC after PD 30 mg + saline (□ ), GHRH 50 |ig + placebo ( M ) and GHRH 50 pg + PD 30 mg ( ■ ) in 6 elderly men and 6 post menopausal women.
however, are in favor of an increased somatostatin activ ity, as discussed below. In our present paper we have shown that in young sub jects the modulation of cholinergic neurotransmission of GH response to GHRH is sex-related. Administration of 60 mg PD potentiated the GH response to GHRH in young male subjects. On the contrary, in young women during the follicular phase of the menstrual cycle plasma GH responses to GHRH were not potentiated by admin istration of PD. These findings suggest that there is a sexrelated difference of the somatostatinergic tone, since it has been shown that the activation of cholinergic system by PD positively influences GH secretion by inhibiting hypothalamic somatostatin release [22, 23]. Since PD potentiated GH response to GHRH in young male but not female subjects, it is conceivable that different cholin ergic and/or somatostatinergic activities are present in the two sexes during the reproductive period of life, pos sibly as a consequence of the different gonadal steroid en vironment. In particular, young male subjects could present a low cholinergic activity which induces a very high somatostat inergic tone. On the contrary, in young premenopausal women cholinergic activity that controls GH secretion seems to be considerably high and this induces a low so matostatinergic tone. In this latter situation, the further inhibition of samatostatin release induced by PD would not be able to potentiate the GH response to GHRH. Our data show that in normal elderly subjects (both men and women) combined administration of PD and GHRH induced a GH response which is significantly higher than that observed after GHRH plus placebo, as similarly seen in young men. Furthermore GH responses to GHRH plus PD in young and older subjects were simi lar. These results are partially at variance with those of
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11.2± 0.8 ng/ml, p < 0.001; AUC 1,768± 142 n g/m l/ min vs. 752 ± 121 ng/m l/m in, p < 0.005) and 6 post menopausal women (mean peak 18.7 ±4.4 ng/ml vs, 6.1 ±1 ng/ml, p < 0 .0 5 ; AUC 1,566 + 393 ng/m l/m in vs. 538 ± 108 ng/m l/m in, p < 0.005). When compared to the response to 30 mg PD alone (men: mean peak 2.7±0.9 ng/ml, AUC 248±60 ng/m l/m in; women: mean peak 2.4 ± 1.2 ng/ml, AUC 205 ±98 ng/m l/m in), the efTect of this dose of PD analogously appeared to be synergistic to that of GHRH (fig. 4). Estradiol and progesterone plasma levels in women were according to the follicular phase of the menstrual cycle (65 ± 5.3 pg/ml and 0.8 ± 0.1 ng/ml, respectively). These hormones were very low in all postmenopausal women (< 30 pg/ml and < 0.2 ng/ml, respectively). Plasma testosterone levels were 6.2 ± 0.3 ng/ml, in young men and 3.9 ± 0.2 ng/ml in elderly men. Side effects after PD administration such as abdominal cramps and nausea were observed especially in young women.
Ghigo et al. [11], who found that PD is able to potentiate the blunted GH response to GHRH in elderly subjects but with a response significantly lower than that seen in young subjects. However these authors have recently re ported that arginine, a substance whose GH-releasing mechanism seems to be the same as that of PD, increases the GH response to GHRH with no difference between young and elderly subjects [24]. Our findings are there fore in favor of a change in cholinergic and/or somatostatinergic activities with aging, while ruling out an intrin sic secretory hypoactivity of somatotropes. Postmenopau sal women present after PD and GHRH administration a pattern of GH response to GHRH similar, although somewhat lower, than that observed in young and elderly men. The observation that in elderly men and women PD significantly potentiates the GH response to GHRH as seen in young male subjects suggests that sex steroids have an influence in the neuroregulation of GH secretion. This evidence is more pronounced in women in whom a PD potentiation of GHRH-induced GH response was seen only when estradiol plasma levels were less than 30 pg/ml, as in our postmenopausal women. Our data also confirm that in the three groups in which 60 mg PD significantly enhanced the GH response to GHRH, the combined administration of PD and GHRH
induced a GH release that greatly exceeded the sum of the responses to each agent given alone. We have recently re ported that this synergistic effect of PD is still evident when a 30-mg dose is administered in young men but not in women [25], In the present study, the effect of 30 mg PD has been evaluated also in elderly subjects. Our re sults show that in both male and female elderly subjects, this small dose of PD is capable of inducing a significant enhancement of the GH response to GHRH in a syner gistic way. Recent studies have shown that human GH improves many aspects of anabolic function in the elderly [26], Therefore, on the basis of our results, it may be supposed that a combined treatment with PD plus GHRH, restor ing somatotroph function in the elderly, may improve their quality of life. However the still debated problems about the route and frequency of administration of thera peutical doses of GHRH as well as the possible side ef fects of PD represent a limitation to this hypothetical use. In conclusion, our data clearly demonstrate a marked influence of sex and age on the PD potentiation of GH response to GHRH. A significant potentiation of this re sponse is present in young and elderly men as well as postmenopausal women. PD had no effect in young women during the follicular phase of the cycle.
1 Saunders A, Terry LC, Audet J, Brazeau P, Martin JB: Dynamic studies of growth hor mone and prolactin secretion in the female rat. Neuroendocrinology I976;21: 193—203. 2 Eden S: Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 1979;105:555-560. 3 Plotsky PM, Vale W: Patterns of growth hor mone-releasing factor and somatostatin secre tion into the hypophysial-portal circulation of the rat. Science 1985;230:461-463. 4 Ferland L, Labrie F, Jobin M, Arimura A, Schally AV: Physiological role of somatostatin in the control of growth hormone and thyro tropin secretion. Biochem Biophys Res Com mun 1976;68:149-154. 5 Terry LC, Martin JB: The effects of lateral hy pothalamic-medial forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats: Evidence for a dual regulatory mechanism. Endocrinology 1981;109:622-627. 6 Clark RG, Robinson ICA: Growth hormone responses to multiple injections of a fragment of human growth hormone-releasing factor in conscious male and female rats. J Endocrinol 1985;106:281-286.
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7 Ono M, Masunaga S, Miki N: The secretory pattern of growth hormone-releasing factor in female rat. 70th Annua Meet Endocr Soc, New Orleans, 1988, abstr No 385. 8 Maiter DM, Gabriel SM. Koenig J I, Russel WE, Martin JB: Sexual differentiation of growth hormone feedback effects on hypotha lamic growth hormone-releasing hormone and somatostatin. Neurocndocrinology 1990; 51:174-180. 9 Jansson JO. Frohman LA: Differential effects of neonatal and adult androgen exposure on the growth hormone secretory pattern in male rats. Endocrinology 1987;120:1551-1557. 10 Gorsky RA: Effects of androgen exposure on the perinatal animal brain. Ann Intern Med 1982;96:488-492. 11 Ghigo E, Golfi S, Arvat E, Nicolosi M, Procopio M, Bellone J, Impériale E, Mazza E, Baracchi G, Camanni F: Pyridostigmine partially restores the GH responsiveness to GHRH in normal aging. Acta Endocrinol (Copenh) 1990;123:169-174.
12 Barbarino A, Corsello SM, Della Casa S, Tofani A, Sciuto R, Rota CA, Bollanti L, Barini A: Corticotropin-releasing hormone inhibi tion of growth hormone-releasing hormoneinduced growth hormone release in man. J Clin Endocrinol Metab 1990;71:1368-1374. 13 Barbarino A, De Marinis L, Anile C, Menini E, Merlini G, Maira G: Dopaminergic mecha nisms regulating prolactin secretion in pa tients with prolactin secreting pituitary adenoma. Long term studies after selective transsphenoidal surgery. Metabolism 1982; 31:1100-1104. 14 De Marinis L, Mancini A, Calabrô F. Massari M, Torlontano M, Barbarino A: Differential effect of a dopaminergic drug (piribcdil) on pituitary hormone release in normal men and women. Acta Endocrinol (Copenh) 1983; 104:385-389. 15 Gelato MC, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR: Dose-response rela tionship for the effects of growth hormone-releasing factor-( 1—44)-NHi in young adult men and women. J Clin Endocrinol Metab 1984; 59:197-202.
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Sex, Age and GH Secretion
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References
20 Shibasaki T, Shaizume K, Nakahara M, Masuda A, Jibiki K, Demura H. Wakabayashi I, Ling N: Age-related changes in plasma growth hormone response to growth hor mone-releasing factor in man. J Clin Endocri nol Metab 1984;58:212-214. 21 lovino M, Monteleone P, Steardo L: Repeti tive growth hormone-releasing hormone ad ministration restores the attenuated growth hormone response to GH-releasing hormone testing in normal aging. J Clin Endocrinol Metab 1989;69:910-913. 22 Locatelli V, Torsello A. Radaclli M, Ghigo E, Massara F, Muller EE: Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone-releas ing hormone in the rat: Evidence for media tion by somatostatin. J Endocrinol 1986; 111:271-278.
23 Torsello A, Panzeri G, Cermenati P, Caroleo MC, Ghigo E, Camanni F, Müller EE, Loca telli V: Involvement of the somatostatin and cholinergic systems in the mechanism of growth hormone autofeedback regulation in the rat. J Endocrinol 1988; 117:273-281. 24 Ghigo E, Goffi S, Arvat E, Imperiale E, Boffano GM. Valetta MR, Maz.za E, Santi I, Magliona A, Boccuzzi G, Camanni F: Effects of arginine and pyridostigmine on GHRH in duced GH release in aging. Evidence for an impairment in the central cholinergic func tion. J Endocrinol Invest I99l;6(suppl 4): 127. 25 Barbarino A, Corsello SM, Tofani A, Sciuto R, Della Casa S. Rota CA, Barini A: Sexual dimorphism in pyridostigmine potentiation of GHRH-induced GH release in humans. J Clin Endocrinol Metab 1991;73:75-78. 26 Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L. Rudman I, Mattson DE: Effects of human growth hormone in men over 60 years old. N Engl J Med 1990;323:1-6.
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16 Lang I, Schemthaner G, Pietschmann P, Kurtz R, Stephenson JM, Tempi H: Effects of sex and aging on growth hormone response to growth hormone-releasing hormone in healthy individuals. J Clin Endocrinol Mctab 1987:65:535-540. 17 Smals AEM, Pieters GFFM, Smals AGH, Benraad TJ. Van Laarhoven J, Kloppenborg PWC: Sex difference in human growth hor mone (GH) response to intravenous human pancreatic GH-releasing hormone adminis tration in young adults. J Clin Endocrinol Metab 1986;62:336-340. 18 Vance ML, Borges JLC, Kaiser DL, Evans WS, Furlanetto R, Thominel JL, Frohman LA, Rogol AD, McLeod RM, Bloom S: Human pancreatic tumor growth hormone-re leasing factor: Dose-response relationships in normal men. J Clin Endocrinol Metab 1984; 58:838-842. 19 Penalva A, Muruais C, Casanueva FF, Dieguez C: Effect of enhancement of endogenous cholinergic tone with pyridostigmine on the dose-response relationships of growth hor mone-releasing hormone-induced GH secre tion in normal subjects. J Clin Endocrinol Metab 1990;70:324-327.
Institutes of Endocrinology and Biochemistry, Catholic University School of Medicine, Rome, Italy
Effects of Sex and Age on Pyridostigmine Potentiation of Growth Hormone-Releasing Hormone-Induced Growth Hormone Release
Key Words
Abstract
GH GHRH Pyridostigmine
Previous studies have shown that pyridostigmine (PD) is capable of increasing the growth hormone (GH) response to GH-releasing hormone (GHRH) in young healthy subjects. In order to investigate the influence of age and sex on the PD potentiation of GHRH-induced GH release, we have studied the GH response to GHRH (50 ug i.v.) 1 h after oral adminis tration of placebo or PD (60 mg) in 8 young healthy men (aged 19-28 years) and 8 agematched young women (aged 18-25 years) during the follicular phase of the menstrual cycle, as well as in 8 postmenopausal women (aged 57-62 years) and 8 age-matched elderly men (aged 56-64 years). In the same subjects the effect of PD alone (60 mg p.o.) was also studied. Furthermore, in 6 postmenopausal women and 6 elderly men, the effect of a 30-mg PD oral dose on GH secretion and GH response to GHRH was evaluated with a similar protocol. The GH responses (mean ± SE) to GHRH F placebo were similar in young men (peak 20.1 ± 2 ng/ml, AUC 1,250 ±113 ng/ml/min) and women (peak 29.3 ± 2.3 ng/ml, AUC 1,769 ±305 ng/ml/min). PD 60 mg was capable of significantly increasing the GH response to GHRH in young men (peak 43.5 ± 5 .1 ng/ml, AUC 3,734 ±472 ng/ml/min, p < 0.005) but not in women (peak 39 ±2.3 ng/ml, AUC 2,479 ±205 ng/ml/min). The GH responses to GHRH + placebo were also similar in postmenopausal women (peak 6.2 ± 0.7 ng/ml, AUC 540 ± 80 ng/ml/min) and age-matched men (peak 11.3 ± 0.6 ng/ml, AUC 763 ±73 ng/ml/min) although these responses were significantly decreased when compared to those observed in young individuals. PD 60 mg administration induced a significant increase in GH response to GHRH both in postmenopausal women (peak 27±3.6 ng/ml, AUC 2,224± 251 ng/ml/min. p< 0.001) and elderly men (peak 49.7 ±2.4 ng/ml, AUC 4,557 ± 263 ng/ml/min, p < 0.001 ). The GH increment, however, was greater in elderly men than in postmenopausal women (p < 0.005). In young men, elderly men and postmenopausal women, PD 60 mg potentiated the action of GHRH rather than merely being additive. A similar effect was observed when a 30-mg PD dose was preadministered in elderly men and women. Our data clearly demonstrate a marked in fluence of age and sex on the PD potentiation of the GHRH-stimulated GH secretion. PD significantly potentiated GH responses to GHRH in young men, elderly men and post menopausal women but not in young women.
Received: March 22. 1991 Accepted after revision: November 5, 1991
Prof. Antonino Barbarino Via Diano Marina 9 / 11 1-001(»8 Rome (Italy)
© 1992 S. Karger AG, Basel 0028-3835/92/0562-0208 $ 2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 1:24:28 AM
Salvatore M. Corsello ° Anna Tofani“ Silvia Della Casa “ Carlo A. Rota “ Rosa Sciuto “ Simonetta Colasanti° Angela Barini1’ Antonino Barbarino “
Materials and Methods Eight young men aged 19-28 years and 8 age-matched young women aged 18-25 years as well as 8 postmenopausal women aged 57-65 years and 8 age-matched elderly men aged 56-64 years par ticipated in the study after giving informed consent. All subjects were nonstressed. AH were within 10% of ideal body weight and were eating their customary diet before and during the interval be tween the tests. None had clinical evidence of endocrine and other disease and all were free of any medication. All young women had regular menstrual cycles. After an overnight fast, in recumbent sub jects, an indwelling cannula was inserted into an antecubital vein at 07.30 h, 30 min before starting the tests. It was used for blood sam pling and GHRH (or saline) injection. Two separate GHRH stimu lation tests were performed in each subject, intravenously injecting a 50-pg bolus dose of GHRH at time 0 (09.00 h) 1 h after oral admin istration of placebo or 60 mg PD, respectively. To study the effect of PD administration alone, in the same subjects a third test was per formed administering orally 60 mg PD a time -60 and injecting 2 ml saline i.v. at time 0. Finally, in 6 postmenopausal women and 6 elderly men, already included in the study group, the same protocol was repeated using a 30-mg PD oral dose. Blood was obtained 60, 30, and 0 min before and 15, 30, 60, 90, and 120 min after GHRH (or saline) administration. In individual subjects the various tests were performed at least at 72-hour intervals, in a blind fashion. In young women, they were performed during the follicular phase of the menstrual cycle. The time of the cycle was established by mea suring estradiol and progesterone levels.
GHRH(l-44) was furnished by Bachem, Bubendorf (Switzer land). Vials containing 0.1 mg were diluted in 0.9% saline giving a final concentration of 50 pg/ml. Pyridostigmine bromide (Mestinon), a cholinesterase inhibitor, was furnished by Hoffmann-La Roche, Nutley, N.J., USA. Plasma GH levels were measured in du plicate by RIA, using polyethyleneglycol to separate free and bound hormone, with reagents obtained by Biodata, Milan (Italy). One na nogram of GH corresponded to 2 uU/ml WHO 66/217 standard. The intra-assay and interassay coefficients of variation ranged from 2.5 to 3.9% and from 5.8 to 8.5%, respectively. Plasma concentra tions of estradiol, progesterone and testosterone were measured by RIA, as previously described [13, 14]. All samples from a given study were measured in the same assay. The results are given as mean ± SE. The area under the curve (AUC) was calculated by a trapezoidal method. Analysis of variance and Newman-Keuls test were used for statistical evaluation. The significance level was estab lished at p < 0.05.
Results
In young subjects, 50 ug GHRH administration, after placebo pretreatment, induced a clearcut increase in GH levels without significant differences between sexes. The mean peak plasma GH levels were 20.1 ± 2 ng/ml in men and 29.3 ±2.3 ng/ml in women while the AUC were 1,250± 113 ng/m l/m in in men and 1,769±305 ng/m l/ min in women (fig. 1, 3). In young men, 60 mg PD pretreatment induced a significant enhancement (vs. placebo) of the GH re sponse to GHRH both in terms of peak response (43.5 ± 5.1 ng/ml, p < 0.005) and AUC (3,734 ± 472 ng/ ml/min, p < 0.005). On the contrary, in young women, 60 mg PD pretreatment was not able to significantly en hance the GH response to GHRH (mean peak 39 ± 2.3 ng/ml, AUC 2,479 ± 205 ng/m l/m in; fig. 1, 3). No significant differences in GH response to GHRH, after placebo pretreatment, were also found between postmenopausal women (mean peak 6.2 ±0.7 ng/ml, AUC 540 ±80 ng/m l/m in) and age-matched elderly men (mean peak 11.3 ±0.6 ng/ml, AUC 763 ± 73; fig. 2, 3) although these responses were significantly decreased (p < 0.05) when compared to those observed in young in dividuals. Sixty milligrams PD pretreatment induced a signifi cant increase (vs. placebo) in GH response to GHRH both in postmenopausal women (mean peak 27 ± 3.6 ng/ ml, AUC 2,224 ±251 ng/m l/m in, p < 0.001) and elderly men (mean peak 49.7 ± 2.4 ng/ml, AUC 4,557 ± 263 ng/ ml/min, p < 0.001; fig. 2, 3). No significant differences in GH responses to GHRH plus 60 mg PD were found between young and elderly subjects of both sexes. The
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The influence of age and sex on neuroregulation of growth hormone (GH) secretion is controversial. In pre vious studies, a number of sex differences in GH secretory pattern have been described in several mammalian species [1-8] and are known to be related to the perinatal action of steroid hormones [9, 10]. In man, on the con trary, it is unclear whether sexual steroids are capable of influencing the GH response to GH-releasing hormone (GHRH) as well as the effect of pyridostigmine (PD) on the GHRH-induced GH release. Very recently, Ghigo et al. [ 11 ] have presented data showing that PD is capable of increasing the GH response to GHRH both in young and elderly subjects. However, they did not discriminate be tween male and female subjects in the belief that no dif ferences are present in the neuroregulatory mechanisms governing GH secretion in the two sexes. We have re cently shown that there is a sex difference in the CRH inhibition of the GHRH-induced GH release [12], These data suggest that a gender-related difference in the neuroregulation of GH secretion is present also in the human. We have investigated whether an activation of the cholin ergic tone by PD could show a sex-related difference of the pituitary responsiveness to GHRH in healthy nonobese subjects of different sex and age.
Time, min
Time, min
Fig. 2. Mean ( ± SE) GH responses to GHRH 50 pg ( • ) , GHRH 50 pg + PD 60 mg (O ) and PD 60 mg + in 8 elderly men and 8 postmenopausal women. **p< 0.005, comparing values after GHRH + PD with GHRH + placebo at the reported time.
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+ placebo saline (■ ) *p< 0.05; those after
Fig. 3. Mean (± S E ) GH AUC after PD 60 mg + saline (□ ), GHRH 50 pg + placebo (H ) and GHRH 50 pg + P D 6 0 m g (B ) in 8 young men, 8 young women, 8 elderly men and 8 postmeno pausal women.
GH increament, however, was greater in elderly men than in postmenopausal women (p < 0.005). The pattern of GH response to GHRH after 60 mg PD administration was similar in young men, elderly men and postmeno pausal women in whom GH levels peaked at 60 min and then gradually decreased but remained high until 120 min after GHRH, while in young women GH levels peaked at 30 min and returned to basal values at 90 min (fig. 1, 2). When 60 mg PD plus saline was given, a small in crease in plasma GH was observed in all the groups tested without significant differences between the groups (young men: mean peak 7.3±0.81 ng/ml, AUC 662±79 ng/m l/m in; young women: mean peak 9.1 ± 2 ng/ml, AUC 893±152 ng/m l/m in; elderly men: mean peak 8.3± 1.3 ng/ml, AUC 760±93 ng/ ml/min; elderly women: mean peak 9.4±1.4 ng/ml, AUC 774 ±94 ng/ml/min). Therefore, in young men, elderly men and postmenopausal women, 60 mg PD potentiated the effect of GHRH rather than being merely additive to it (fig. 1-3). Thirty mg PD pretreatment induced a significant enhancement (vs. placebo) of the GH response to GHRH in 6 elderly men (mean peak 22.7 ± 1 ng/ml vs.
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Fig. 1. Mean ( ± SE) GH responses to GHRH 50 pg + placebo ( • ) , GHRH 50 pg + PD 60 mg (O ) and PD 60 mg + saline (■ ) in 8 young men and 8 young women. *p< 0.05; **p< 0.005, com paring values after GHRH -I- PD with those after GHRH + placebo at the reported time.
Discussion
Our present findings confirm that there is no sex dif ference in GH responsiveness to an effective submaximal dose of GHRH in age-matched young subjects as well as in elderly individuals. There is, however, a significant agedependent decline in GH responses in both sexes. The lack of differences in GHRH-induced GH secretion in young male and female subjects is in agreement with the results by Gelato et al. [15] but in contrast with data by other authors [16, 17]. Lang et al. [16] reported that the responsiveness of GH to GHRH is significantly greater in premenopausal women than in age-matched men. Smals et al. [17], on the contrary, showed an increased GH re sponsiveness to GHRH in men compared to women. These discrepant findings are difficult to explain. In fact, the differences in the GHRH dose administered do not seem a possible explanation because it has been found by different authors that doses of GHRH between 50 and 500 pg/subject induced similar increases in plasma GH levels [15, 18, 19], In agreement with most of the data from the literature we found that the magnitude of GH response to GHRH was significantly lower in older subjects than in young in dividuals [11, 16, 20, 21], GH responses to GHRH were similar in magnitude in elderly men and women. The diminished pituitary responsiveness to GHRH with aging can be related to an increased somatostatin tone or to changes intrinsic to the pituitary gland. Our findings,
Fig. 4. Mean ( ± SE) GH AUC after PD 30 mg + saline (□ ), GHRH 50 |ig + placebo ( M ) and GHRH 50 pg + PD 30 mg ( ■ ) in 6 elderly men and 6 post menopausal women.
however, are in favor of an increased somatostatin activ ity, as discussed below. In our present paper we have shown that in young sub jects the modulation of cholinergic neurotransmission of GH response to GHRH is sex-related. Administration of 60 mg PD potentiated the GH response to GHRH in young male subjects. On the contrary, in young women during the follicular phase of the menstrual cycle plasma GH responses to GHRH were not potentiated by admin istration of PD. These findings suggest that there is a sexrelated difference of the somatostatinergic tone, since it has been shown that the activation of cholinergic system by PD positively influences GH secretion by inhibiting hypothalamic somatostatin release [22, 23]. Since PD potentiated GH response to GHRH in young male but not female subjects, it is conceivable that different cholin ergic and/or somatostatinergic activities are present in the two sexes during the reproductive period of life, pos sibly as a consequence of the different gonadal steroid en vironment. In particular, young male subjects could present a low cholinergic activity which induces a very high somatostat inergic tone. On the contrary, in young premenopausal women cholinergic activity that controls GH secretion seems to be considerably high and this induces a low so matostatinergic tone. In this latter situation, the further inhibition of samatostatin release induced by PD would not be able to potentiate the GH response to GHRH. Our data show that in normal elderly subjects (both men and women) combined administration of PD and GHRH induced a GH response which is significantly higher than that observed after GHRH plus placebo, as similarly seen in young men. Furthermore GH responses to GHRH plus PD in young and older subjects were simi lar. These results are partially at variance with those of
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11.2± 0.8 ng/ml, p < 0.001; AUC 1,768± 142 n g/m l/ min vs. 752 ± 121 ng/m l/m in, p < 0.005) and 6 post menopausal women (mean peak 18.7 ±4.4 ng/ml vs, 6.1 ±1 ng/ml, p < 0 .0 5 ; AUC 1,566 + 393 ng/m l/m in vs. 538 ± 108 ng/m l/m in, p < 0.005). When compared to the response to 30 mg PD alone (men: mean peak 2.7±0.9 ng/ml, AUC 248±60 ng/m l/m in; women: mean peak 2.4 ± 1.2 ng/ml, AUC 205 ±98 ng/m l/m in), the efTect of this dose of PD analogously appeared to be synergistic to that of GHRH (fig. 4). Estradiol and progesterone plasma levels in women were according to the follicular phase of the menstrual cycle (65 ± 5.3 pg/ml and 0.8 ± 0.1 ng/ml, respectively). These hormones were very low in all postmenopausal women (< 30 pg/ml and < 0.2 ng/ml, respectively). Plasma testosterone levels were 6.2 ± 0.3 ng/ml, in young men and 3.9 ± 0.2 ng/ml in elderly men. Side effects after PD administration such as abdominal cramps and nausea were observed especially in young women.
Ghigo et al. [11], who found that PD is able to potentiate the blunted GH response to GHRH in elderly subjects but with a response significantly lower than that seen in young subjects. However these authors have recently re ported that arginine, a substance whose GH-releasing mechanism seems to be the same as that of PD, increases the GH response to GHRH with no difference between young and elderly subjects [24]. Our findings are there fore in favor of a change in cholinergic and/or somatostatinergic activities with aging, while ruling out an intrin sic secretory hypoactivity of somatotropes. Postmenopau sal women present after PD and GHRH administration a pattern of GH response to GHRH similar, although somewhat lower, than that observed in young and elderly men. The observation that in elderly men and women PD significantly potentiates the GH response to GHRH as seen in young male subjects suggests that sex steroids have an influence in the neuroregulation of GH secretion. This evidence is more pronounced in women in whom a PD potentiation of GHRH-induced GH response was seen only when estradiol plasma levels were less than 30 pg/ml, as in our postmenopausal women. Our data also confirm that in the three groups in which 60 mg PD significantly enhanced the GH response to GHRH, the combined administration of PD and GHRH
induced a GH release that greatly exceeded the sum of the responses to each agent given alone. We have recently re ported that this synergistic effect of PD is still evident when a 30-mg dose is administered in young men but not in women [25], In the present study, the effect of 30 mg PD has been evaluated also in elderly subjects. Our re sults show that in both male and female elderly subjects, this small dose of PD is capable of inducing a significant enhancement of the GH response to GHRH in a syner gistic way. Recent studies have shown that human GH improves many aspects of anabolic function in the elderly [26], Therefore, on the basis of our results, it may be supposed that a combined treatment with PD plus GHRH, restor ing somatotroph function in the elderly, may improve their quality of life. However the still debated problems about the route and frequency of administration of thera peutical doses of GHRH as well as the possible side ef fects of PD represent a limitation to this hypothetical use. In conclusion, our data clearly demonstrate a marked influence of sex and age on the PD potentiation of GH response to GHRH. A significant potentiation of this re sponse is present in young and elderly men as well as postmenopausal women. PD had no effect in young women during the follicular phase of the cycle.
1 Saunders A, Terry LC, Audet J, Brazeau P, Martin JB: Dynamic studies of growth hor mone and prolactin secretion in the female rat. Neuroendocrinology I976;21: 193—203. 2 Eden S: Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 1979;105:555-560. 3 Plotsky PM, Vale W: Patterns of growth hor mone-releasing factor and somatostatin secre tion into the hypophysial-portal circulation of the rat. Science 1985;230:461-463. 4 Ferland L, Labrie F, Jobin M, Arimura A, Schally AV: Physiological role of somatostatin in the control of growth hormone and thyro tropin secretion. Biochem Biophys Res Com mun 1976;68:149-154. 5 Terry LC, Martin JB: The effects of lateral hy pothalamic-medial forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats: Evidence for a dual regulatory mechanism. Endocrinology 1981;109:622-627. 6 Clark RG, Robinson ICA: Growth hormone responses to multiple injections of a fragment of human growth hormone-releasing factor in conscious male and female rats. J Endocrinol 1985;106:281-286.
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7 Ono M, Masunaga S, Miki N: The secretory pattern of growth hormone-releasing factor in female rat. 70th Annua Meet Endocr Soc, New Orleans, 1988, abstr No 385. 8 Maiter DM, Gabriel SM. Koenig J I, Russel WE, Martin JB: Sexual differentiation of growth hormone feedback effects on hypotha lamic growth hormone-releasing hormone and somatostatin. Neurocndocrinology 1990; 51:174-180. 9 Jansson JO. Frohman LA: Differential effects of neonatal and adult androgen exposure on the growth hormone secretory pattern in male rats. Endocrinology 1987;120:1551-1557. 10 Gorsky RA: Effects of androgen exposure on the perinatal animal brain. Ann Intern Med 1982;96:488-492. 11 Ghigo E, Golfi S, Arvat E, Nicolosi M, Procopio M, Bellone J, Impériale E, Mazza E, Baracchi G, Camanni F: Pyridostigmine partially restores the GH responsiveness to GHRH in normal aging. Acta Endocrinol (Copenh) 1990;123:169-174.
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