0021-972X/90/7106-1481$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 6 Printed in U.S.A.
Growth Hormone (GH) Responsiveness to Combined Administration of Arginine and GH-Releasing Hormone Does not Vary with Age in Man* EZIO GHIGO, STEFANIA GOFFI, MARIO NICOLOSI, EMANUELA ARVAT, FRANCESCA VALENTE, ENRICO MAZZA, MARIA CRISTINA GHIGO, and FRANCO CAMANNI Department of Clinical Physiopathology, Division of Endocrinology, University of Turin, Turin, Italy
ABSTRACT. At present, the mechanism(s) underlying the reduced spontaneous and stimulated GH secretion in aging is still unclear. To obtain new information on this mechanism(s), the GH responses to both single and combined administration of GH-releasing hormone (GHRH; 1 Mg/kg iv) and arginine (ARG; 30 g infused over 30 min), a well known GH secretagogue probably acting via inhibition of hypothalamic somatostatin release, were studied in seven elderly normal subjects and seven young healthy subjects. Basal GH levels were similar in both groups, while insulinlike growth factor-I levels were lower in elderly subjects (76.7 ± 9.2 us. 258.3 ± 29.2 ^g/L; P = 0.01). In aged subjects GHRH induced a GH increase (area under the curve, 314.9 ± 91.9 ng/ L-h) which was lower (P = 0.01) than that in young subjects (709.1 ± 114.4 Mg/Lh). On the other hand, the ARG-induced GH increase in the elderly was not significantly different from
A
N AGE-related decrease in GH secretion has been shown in both animals and man. In normal aging, basal GH levels were reported decreased (1, 2) or unchanged (3, 4), while spontaneous peak amplitude has been shown to be reduced, mainly during sleep (4-7). In agreement with the existence of impaired GH secretion, insulin-like growth factor-I (IGF-I) levels have been shown to be decreased in aged humans (1). At present, the mechanism(s) underlying the reduced GH secretion in aging is still unclear. In animals, evidence for a hypothalamic pathogenesis of this GH hyposecretory state has been presented (8-15). In man, a low somatotroph responsiveness to some stimuli (16, 17) and even to GH-releasing hormone (GHRH) (18-21) has been reported. However, the GHRH-induced GH rise seems to be partly restored by repetitive GHRH pretreatment (20, 21), suggesting an altered GHRH control of
that in young subjects (372.8 ± 81.8 us. 470.6 ± 126.5 ARG potentiated GH responsiveness to GHRH in both elderly (1787.1 ± 226.0 Mg/L-h; P = 0.0001 us. GHRH alone) and young subjects (2113.0 ± 444.3 Mg/L-h; P = 0.001 us. GHRH alone). The potentiating effect of ARG on the GHRH-induced GH response was greater in elderly than in young subjects (1013.0 ± 553.5% us. 237.9 ± 79.1%; P = 0.0001); thus, the GH increase induced by combined administration of ARG and GHRH overlapped in two groups. In conclusion, these results show that, differently from the GHRH-induced GH increase, the somatotroph response to combined administration of ARG and GHRH does not vary with age. Our finding suggests that an increased somatostatinergic activity may underlie the reduced GH secretion in normal aging. (J Clin Endocrinol Metab 7 1 : 1481-1485, 1990)
somatotrophs in aging. Recently, it has been shown that arginine, a well known GH secretagogue (22), clearly potentiates the maximal somatotroph responsiveness to GHRH in man (23, 24), suggesting that this effect is mediated by suppression of endogenous hypothalamic somatostatin release. Based on the foregoing, the aim of this study was to verify the effect of arginine on both basal and GHRHstimulated GH secretion in normal aging.
Subjects and Methods
Received March 13, 1990. Address requests for reprints to: Prof. F. Camanni, Divisione di Endocrinologia, Ospedale Molinette, Corso Polonia 14, 10126 Torino, Italy. * This work was supported by a grant from the Consiglio Nazionale delle Ricerche.
Seven young healthy subjects (seven males aged 20-30 yr; mean, 26.1 ± 1.5 yr) and seven elderly normal subjects (seven males aged 68-81 yr; mean, 75.4 ± 2 . 1 yr) were studied. All subjects were within 15% of their ideal body weight and in good health, as evaluated by clinical examination and blood biochemistry. None was taking any medication known to influence GH secretion. Informed consent was obtained from all subjects. All subjects underwent three tests in random order and at least 3 days apart: 1) GHRH-29 (Kabi Vitrum, Stockholm, Sweden; 1 Mg/kg, iv, as a bolus at 0 min), 2) arginine (ARG; 30 g in 100 mL infused from 0-30 min), and 3) GHRH plus ARG.
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GHIGO ETAL.
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All tests were begun between 0800-0830 h after an overnight fast and 30 min after cannulation of a cubital vein of one side, kept patent by slow infusion of 0.9% saline. Blood samples were taken basally at —15 and 0 min and then every 15 min until 90 min. GH was measured in duplicate by a double antibody RIA, using reagents provided by Sorin (Saluggia, Italy). All samples from an individual subject were analyzed together. The sensitivity of the assay was 0.2 Mg/L- The intra- and interassay coefficients of variation were 4.5% and 7.9%, respectively. The GH secretory responses were expressed as either absolute values (micrograms per L) or areas under the curve (AUC; micrograms per L/h) calculated by trapezoidal integration. The percent potentiating effect of arginine on the GH response to GHRH was evaluated as the difference between the AUC response to GHRH with and without ARG and the AUC response to GHRH alone. IGF-I levels were measured in duplicate by RIA, using reagents provided by Nichols Institute Diagnostics (San Juan Capistrano, CA). The sensitivity of this assay was 90 pg/mL. The interassay coefficient of variation was 14%. To avoid interference by binding proteins, all plasma samples were treated with acid-ethanol. IGF-I concentrations were expressed as absolute values (micrograms per L) with reference to a pure recombinant IGF-I preparation. Results were expressed as the mean ± SEM. Statistical analysis of the data was carried out using a nonparametric analysis of variance (Kruskall-Wallis test) and the unpaired Student's t test where applicable.
Results Basal plasma GH levels were similar in elderly and young subjects (0.9 ± 0.2 us. 0.9 ± 0.3 /xg/L)- In aged subjects GHRH induced an increase (peak, 7.9 ± 2.4 ng/ L; AUC, 314.9 ± 91.9 /xg/L-h), which was significantly lower (P < 0.01) than that observed in young subjects (peak, 18.3 ± 2.2 Mg/L; AUC, 709.1 ± 114.4 Mg/L-h; Figs. 1 and 2). On the other hand, the ARG-induced GH increase in the elderly was not significantly different from that in young subjects (peak, 11.1 ± 2.2 us. 13.8 ± 4.6 Afg/L; AUC, 372.8 ± 81.8 us. 470.6 ± 126.5 /zg/L-h; Figs. 1 and 2). ARG potentiated the GH responsiveness to GHRH in both elderly (peak, 44.5 ± 5.2 Mg/L; AUC, 1787.1 ± 226.0 jug/L-h; P = 0.0001 us. GHRH alone) and young subjects (peak, 56.8 ± 12.3 fig/L; AUC, 2113.0 ± 444.3 Mg/L-h; P = 0.001 us. GHRH alone; Figs. 1 and 2). The potentiating effect of ARG on the GH response to GHRH was higher in elderly than in young subjects (1013.0 ± 553.5% us. 237.9 ± 79.1%; P = 0.0001); thus, the GH increase induced by combined administration of ARG and GHRH overlapped in the two groups. IGF-I levels were lower in elderly than in young subjects (76.7 ± 9.2 us. 258.3 ± 29.2 ^ig/L; P = 0.01). A transient facial flushing was observed in eight sub-
JCE & M • 1990 Vol 71 • No 6
jects after GHRH administration. No side-effects were observed after ARG infusion.
Discussion The present data confirm the age-dependent reduction of GH responsiveness to GHRH (18-21), but show that the GH responsiveness to combined administration of ARG and GHRH in the elderly strictly overlaps with that in young subjects. Interestingly, in contrast to other stimuli (16, 17), the GH response to ARG alone is not different in elderly and young subjects. This evidence is in agreement with a previous report (3). Our findings show that GH secretory capacity of somatotroph cells to an acute stimulus is preserved in normal aged humans. In aged man, the pituitary content of GH was found to be unchanged by some researchers (25), but not by others (26). In animals, the pituitary GH content was generally reported to be preserved (10, 11, 14). Moreover, in uitro the somatotroph responsiveness to GHRH was reported to be unchanged in aged rats by some researchers (10, 11, 14), but not by others (27, 28). The mechanism of action by which ARG stimulates GH secretion is still unclear. In rats, ARG failed to influence both basal and GHRH-induced GH secretion from anterior pituitary in uitro (23). This finding seems to rule out a direct effect of ARG at the pituitary level. However, in old rats a decreased pituitary sensitivity to GHRH has been reported (27, 28), and the possibility that ARG influences this mechanism cannot be excluded. Actually, there is more evidence favoring the hypothesis that the GH hyposecretory state, which connotes normal aging, is due to hypothalamic impairment. A decreased hypothalamic GHRH content in old rats has been reported (29, 30). Moreover, in old dogs prolonged treatment with clonidine, an «2-adrenergic agonist known to stimulate GH secretion via stimulation of endogenous GHRH release, leads to a GH increase similar to that in young dogs (15). In further agreement with the involvement of an altered GHRH control in aging, the GH response to GHRH in humans has been shown to be fully (20) or partly (21) restored by repetitive GHRH administration. However, the possibility that ARG acts at the hypothalamic level by stimulating endogenous GHRH release seems unlikely. In fact, ARG is able to strikingly potentiate the maximal somatotroph responsiveness to GHRH (23, 24). Moreover, differently from L-dopa, ornithine, the active form of arginine, is unable to modify plasma GHRH levels in humans (31). The more plausible mechanism by which ARG is so effective on GH secretion even in normal aging consists of inhibition of hypothalamic somatostatin release. Favoring a somatostatin-mediated mechanism of action for arginine are the following findings. 1) As mentioned
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ARG ON GHRH-INDUCED GH RISE IN AGING
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YOUNG
FIG. 1. Plasma GH levels (mean ± SEM) after GHRH, ARG, and ARG plus GHRH in normal young and elderly subjects.
ELDERLY
70
70
60
60
50
50
•> 40
40
X O 30 m
30
M
20
E
JS 20 a.
10
10
0
0 15
-15
30
46
60
75
90
-15
15
30
45
60
75 90
Time(min) 2800CHI YOUNG
2400- • i
ELDERLY
2000D)
FIG. 2. Plasma GH AUCs (mean ± SEM) after GHRH, ARG, and ARG plus GHRH in normal young and elderly subjects.
O jj -^ O E iS
1600
p < 0.01
1200 800
Q.
400-
oGHRH
above, this amino acid potentiates the GH response to the maximal GHRH dose (23, 24). 2) The potentiating effect of ARG on GHRH-induced GH secretion strictly overlaps with that of pyridostigmine (24), a cholinergic drug that inhibits cholinesterases, probably acting via somatostatin inhibition (32-37). 3) The combined administration of ARG and pyridostigmine has no additive effect on GH secretion in normal short children (24), suggesting their common mechanism of action. 4) In contrast to GHRH, ARG induced a pronounced GH rise even when preceded by prior GHRH stimulation of GH secretion (38). The marked potentiating effect of ARG on the blunted GHRH-induced GH rise in normal aging may be due to
•
ARQ
ARQ*QHRH
inhibition of a somatostatin hypertone. This hypothesis agrees with evidence that in old rats both somatostatin antiserum and pilocarpine, a muscarinic cholinergic agonist, increase GH levels and antagonize the impaired GHRH-induced GH secretion (9, 12, 13). Although the hypothalamic somatostatin content has been reported to be increased (14, 39) or decreased (40, 41), other data showed an increase of fractional efflux of somatostatin in old rats (14, 42). In conclusion, our results show that, differently from the GH response to GHRH alone, the GH response to combined administration of arginine and GHRH does not vary with age, suggesting the presence of an increased somatostatinergic activity in aging.
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GHIGO ETAL.
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Acknowledgments The authors wish to thank Dr. J. Bellone, M. Procopio, M. R. Valetto, and Prof. G. Baracchi for their cooperation in performing this study.
References 1. Rudman D, Kutner MH, Rogers M, Lubin MF, Fleming GA, Bain RP. Impaired growth hormone secretion in the adult population. J Clin Invest. 1981;67:1361-9. 2. Zadik Z, Chalew SA, McCarter RJ, Meistas M, Kowarski AA. The influence of age on the 24-hour integrated concentration of growth hormone in normal individuals. J Clin Endocrinol Metab. 1985;60:513-6. 3. Dudl RJ, Ensinck JW, Palmer HE, Williams RH. Effect of age on growth hormone secretion in man. J Clin Endocrinol Metab. 1973;37:ll-6. 4. Vermeulen A. Nyctohemeral growth hormone profiles in young and aged men: correlation with somatomedin-C levels. J Clin Endocrinol Metab. 1987;64:884-8. 5. Finkelstein JW, Roffwarg HP, Boyar RM, Kream J, Hellman L. Age-related change in the twenty-four hour spontaneous secretion of growth hormone. J Clin Endocrinol Metab. 1972;35:665-70. 6. Carlson HE, Gillin JC, Gorden P, Snyder F. Absence of sleeprelated growth hormone peaks in aged normal subjects and in acromegaly. J Clin Endocrinol Metab. 1972;34:1102-5. 7. Ho KY, Evans WS, Blizzard RM, et al. Effect of sex and age on the 24-hour secretory profile of GH secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab. 1987;64:51-8. 8. Burek CL, Frohman LA. Growth hormone synthesis by rat pituitaries in vitro. Endocrinology. 1970;86:1361-7. 9. Sonntag WE, Forman LJ, Miki N, et al. Effects of CNS active drugs and somatostatin antiserum on growth hormone release in young and old male rats. Neuroendocrinology. 1981;33:73-8. 10. Sonntag WE, Hylka VW, Meites J. Impaired ability of old male rats to secrete growth hormone in vivo but not in vitro in response to hpGRF-(l-44). Endocrinology. 1983;113:2305-7. 11. Weherenberg WB, Ling N. The absence of an age-related change in the pituitary response to growth hormone-releasing factor in rats. Neuroendocrinology. 1983;37:463-6. 12. Locatelli V, Arimura A, Torsello A, Cella SG, Muller EE. Somatostatin antiserum antagonized the impaired ability of hpGRF-40 to stimulate growth hormone release in old unanesthetized male rats. Neuroendocrinol Lett. 1984;5:261-6. 13. Locatelli V, Cella SG, Cermenati P, Panzeri G, Sellan R, Muller EE. Defective growth hormone (GH) secretion in aging mammals: contribution of central and peripheral inhibitory influences and of GH-releasing hormone. In: Valenti G, ed. Psychoneuroendocrinology of aging: basic and clinical aspects. Fidia Res Ser. Padova: Liviana Press; 1988; 16:61-7. 14. Ge F, Tsagarakis S, Rees LH, Besser GM, Grossman A. Relationship between growth hormone-releasing hormone and somatostatin in the rat: effects of age and sex on content and in-vitro release from hypothalamic explants. J Endocrinol. 1989;123:53-8. 15. Cella SG, Moiraghi V, Minuto F, et al. Prolonged fasting or clonidine can restore the defective growth hormone secretion in old dogs. Acta Endocrinol (Copenh) 1989;121:177-84. 16. Muggeo M, Fedele D, Tiengo A, Molinari M, Crepaldi G. Human growth hormone and cortisol responses to insulin stimulation in aging. J Gerontol. 1975;30:546-50. 17. Gil-Ad I, Gurewitz R, Marcovici 0, Rosenfeld J, Laron Z. Effects of aging on human plasma growth hormone response to clonidine. Mech Age Dev. 1984;27:97-100.
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18. Shibasaki T, Shizume K, Nakahara M, et al. Age-related changes in plasma growth hormone response to growth hormone-releasing factor in man. J Clin Endocrinol Metab. 1984;58:212-4. 19. Lang I, Schernthaner G, Pietschmann P, Kurz R, Stephenson JM, Tempi H. Effects of sex and age on growth hormone response to growth hormone-releasing hormone in healthy individuals. J Clin Endocrinol Metab. 1987;65:535-40. 20. Iovino M, Monteleone P, Steardo L. Repetitive growth hormonereleasing hormone administration restores the attenuated growth hormone (GH) response to GH-releasing hormone testing in normal aging. J Clin Endocrinol Metab. 1989;69:910-3. 21. Franchimont P, Urbain-Choffray D, Lambelin P, Fontaine MA, Frangin G, Reginster JY. Effects of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor I, and bone metabolism in postmenopausal women. Acta Endocrinol (Copenh). 1989;120:121-8. 22. Parker ML, Hammonds JM, Daughaday WH. The arginine provocative test: an aid in the diagnosis of hyposomatotropism. J Clin Endocrinol Metab. 1967;27:1129-36.
23. Alba-Roth J, Albrecht Muller 0, Schopohl J, Von Werder K. Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab. 1988;67:1186-9. 24. Ghigo E, Bellone J, Mazza E, et al. Arginine potentiates the GHRH- but not the pyridostigmine-induced GH secretion in normal short children. Further evidence for a somatostatin suppressing effect of arginine. Clin Endocrinol (Oxf). 1990;32:763-7. 25. Gershberg H. Growth hormone content and metabolic actions of human pituitary glands. Endocrinology. 1957;61:160-5. 26. Yu-Kai Sun YP, Fenoglio CM, Pushparaj N, et al. The effect of age on the number of pituitary cells immunoreactive to growth hormone and prolactin. Hum Pathol. 1984;15:169-80. 27. Robberecht P, Gillard M, Waelbroeck M, Camus JC, De Neef P, Christophe J. Decreased stimulation of adenylate cyclase by growth hormone releasing factor in the anterior pituitary of old rats. Neuroendocrinology. 1986;44:429-32. 28. Ceda GP, Valenti G, Butturini U, Hoffman AR. Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats. Endocrinology. 1986;118:2109-14. 29. Morimoto N, Kawakami F, Makino S, Chihara K, Hasegawa M, Ibata Y. Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus. Neuroendocrinology. 1988;47:459-64. 30. De Gennaro Colonna V, Zoli M, Cocchi D, et al. Reduced growth hormone releasing factor (GHRH)-like immunoreactivity and GHRH gene expression in hypothalamus of aged rats. Peptides. 1989;10:705-9. 31. Evain-Brion D, Donadieu M, Liapi C, et al. Plasma growth hormone releasing factor levels in children: physiologically and pharmacologically induced variations. Horm Res. 1986;24:116-21. 32. Richardson SB, Hollander CS, D'Eletto R, et al. Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology. 1980;107:1837-42. 33. Locatelli V, Torsello A, Redaelli M, et al. Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone-releasing hormone in the rat: evidence for mediation by somatostatin. J Endocrinol. 1986;lll:271-8. 34. Torsello A, Panzeri G, Cermenati P, et al. Involvement of somatostatin and cholinergic system in the mechanism of growth hormone autofeedback regulation in the rat. J Endocrinol. 1988;117:273-81. 35. Massara F, Ghigo E, Demislis K, et al. Cholinergic involvement in the growth hormone releasing factor-induced growth hormone release: studies in normal and acromegalics subjects. Neuroendocri-
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ARG ON GHRH-INDUCED GH RISE IN AGING nology. 1986;43:670-5. 36. Ghigo E, Mazza E, Imperiale E, et al. Enhancement of cholinergic tone by pyridostigmine promotes both basal and growth hormone(GH)-releasing hormone-induced GH secretion in children of short stature. J Clin Endocrinol Metab. 1987;65:452-6. 37. 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 Endocrinol (Oxf). 1987;27:727-33. 38. Page MD, Dieguez C, Valcavi R, Edwards C, Hak R, Scanlon MF. Growth hormone (GH) responses to arginine and 1-DOPA alone and after GHRH pretreatment. Clin Endocrinol (Oxf). 1988;28:551-8.
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39. Forman LJ, Sonntag WE, Hylka VW, Meites J. Pituitary growth hormone and hypothalamic somatostatin in young female rats versus old constant estrous female rats. Experientia. 1985;41:6534. 40. Takahashi S, Gottschall PE, Quigley KL, Goya RG, Meites J. Growth hormone secretory patterns in young, middle-aged and old female rats. Neuroendocrinology. 1987;46:137-42. 41. Sonntag WE, Steger RW, Forman LJ, Meites J. Decreased pulsatile release of growth hormone in old male rats. Endocrinology. 1980;107:1875-9. 42. Sonntag WE, Gottschall PE, Meites J. Increased secretion of somatostatin-28 from hypothalamic neurons of aged rats in vitro. Brain Res. 1986;380:229-34.
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Vol. 71, No. 6 Printed in U.S.A.
Growth Hormone (GH) Responsiveness to Combined Administration of Arginine and GH-Releasing Hormone Does not Vary with Age in Man* EZIO GHIGO, STEFANIA GOFFI, MARIO NICOLOSI, EMANUELA ARVAT, FRANCESCA VALENTE, ENRICO MAZZA, MARIA CRISTINA GHIGO, and FRANCO CAMANNI Department of Clinical Physiopathology, Division of Endocrinology, University of Turin, Turin, Italy
ABSTRACT. At present, the mechanism(s) underlying the reduced spontaneous and stimulated GH secretion in aging is still unclear. To obtain new information on this mechanism(s), the GH responses to both single and combined administration of GH-releasing hormone (GHRH; 1 Mg/kg iv) and arginine (ARG; 30 g infused over 30 min), a well known GH secretagogue probably acting via inhibition of hypothalamic somatostatin release, were studied in seven elderly normal subjects and seven young healthy subjects. Basal GH levels were similar in both groups, while insulinlike growth factor-I levels were lower in elderly subjects (76.7 ± 9.2 us. 258.3 ± 29.2 ^g/L; P = 0.01). In aged subjects GHRH induced a GH increase (area under the curve, 314.9 ± 91.9 ng/ L-h) which was lower (P = 0.01) than that in young subjects (709.1 ± 114.4 Mg/Lh). On the other hand, the ARG-induced GH increase in the elderly was not significantly different from
A
N AGE-related decrease in GH secretion has been shown in both animals and man. In normal aging, basal GH levels were reported decreased (1, 2) or unchanged (3, 4), while spontaneous peak amplitude has been shown to be reduced, mainly during sleep (4-7). In agreement with the existence of impaired GH secretion, insulin-like growth factor-I (IGF-I) levels have been shown to be decreased in aged humans (1). At present, the mechanism(s) underlying the reduced GH secretion in aging is still unclear. In animals, evidence for a hypothalamic pathogenesis of this GH hyposecretory state has been presented (8-15). In man, a low somatotroph responsiveness to some stimuli (16, 17) and even to GH-releasing hormone (GHRH) (18-21) has been reported. However, the GHRH-induced GH rise seems to be partly restored by repetitive GHRH pretreatment (20, 21), suggesting an altered GHRH control of
that in young subjects (372.8 ± 81.8 us. 470.6 ± 126.5 ARG potentiated GH responsiveness to GHRH in both elderly (1787.1 ± 226.0 Mg/L-h; P = 0.0001 us. GHRH alone) and young subjects (2113.0 ± 444.3 Mg/L-h; P = 0.001 us. GHRH alone). The potentiating effect of ARG on the GHRH-induced GH response was greater in elderly than in young subjects (1013.0 ± 553.5% us. 237.9 ± 79.1%; P = 0.0001); thus, the GH increase induced by combined administration of ARG and GHRH overlapped in two groups. In conclusion, these results show that, differently from the GHRH-induced GH increase, the somatotroph response to combined administration of ARG and GHRH does not vary with age. Our finding suggests that an increased somatostatinergic activity may underlie the reduced GH secretion in normal aging. (J Clin Endocrinol Metab 7 1 : 1481-1485, 1990)
somatotrophs in aging. Recently, it has been shown that arginine, a well known GH secretagogue (22), clearly potentiates the maximal somatotroph responsiveness to GHRH in man (23, 24), suggesting that this effect is mediated by suppression of endogenous hypothalamic somatostatin release. Based on the foregoing, the aim of this study was to verify the effect of arginine on both basal and GHRHstimulated GH secretion in normal aging.
Subjects and Methods
Received March 13, 1990. Address requests for reprints to: Prof. F. Camanni, Divisione di Endocrinologia, Ospedale Molinette, Corso Polonia 14, 10126 Torino, Italy. * This work was supported by a grant from the Consiglio Nazionale delle Ricerche.
Seven young healthy subjects (seven males aged 20-30 yr; mean, 26.1 ± 1.5 yr) and seven elderly normal subjects (seven males aged 68-81 yr; mean, 75.4 ± 2 . 1 yr) were studied. All subjects were within 15% of their ideal body weight and in good health, as evaluated by clinical examination and blood biochemistry. None was taking any medication known to influence GH secretion. Informed consent was obtained from all subjects. All subjects underwent three tests in random order and at least 3 days apart: 1) GHRH-29 (Kabi Vitrum, Stockholm, Sweden; 1 Mg/kg, iv, as a bolus at 0 min), 2) arginine (ARG; 30 g in 100 mL infused from 0-30 min), and 3) GHRH plus ARG.
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GHIGO ETAL.
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All tests were begun between 0800-0830 h after an overnight fast and 30 min after cannulation of a cubital vein of one side, kept patent by slow infusion of 0.9% saline. Blood samples were taken basally at —15 and 0 min and then every 15 min until 90 min. GH was measured in duplicate by a double antibody RIA, using reagents provided by Sorin (Saluggia, Italy). All samples from an individual subject were analyzed together. The sensitivity of the assay was 0.2 Mg/L- The intra- and interassay coefficients of variation were 4.5% and 7.9%, respectively. The GH secretory responses were expressed as either absolute values (micrograms per L) or areas under the curve (AUC; micrograms per L/h) calculated by trapezoidal integration. The percent potentiating effect of arginine on the GH response to GHRH was evaluated as the difference between the AUC response to GHRH with and without ARG and the AUC response to GHRH alone. IGF-I levels were measured in duplicate by RIA, using reagents provided by Nichols Institute Diagnostics (San Juan Capistrano, CA). The sensitivity of this assay was 90 pg/mL. The interassay coefficient of variation was 14%. To avoid interference by binding proteins, all plasma samples were treated with acid-ethanol. IGF-I concentrations were expressed as absolute values (micrograms per L) with reference to a pure recombinant IGF-I preparation. Results were expressed as the mean ± SEM. Statistical analysis of the data was carried out using a nonparametric analysis of variance (Kruskall-Wallis test) and the unpaired Student's t test where applicable.
Results Basal plasma GH levels were similar in elderly and young subjects (0.9 ± 0.2 us. 0.9 ± 0.3 /xg/L)- In aged subjects GHRH induced an increase (peak, 7.9 ± 2.4 ng/ L; AUC, 314.9 ± 91.9 /xg/L-h), which was significantly lower (P < 0.01) than that observed in young subjects (peak, 18.3 ± 2.2 Mg/L; AUC, 709.1 ± 114.4 Mg/L-h; Figs. 1 and 2). On the other hand, the ARG-induced GH increase in the elderly was not significantly different from that in young subjects (peak, 11.1 ± 2.2 us. 13.8 ± 4.6 Afg/L; AUC, 372.8 ± 81.8 us. 470.6 ± 126.5 /zg/L-h; Figs. 1 and 2). ARG potentiated the GH responsiveness to GHRH in both elderly (peak, 44.5 ± 5.2 Mg/L; AUC, 1787.1 ± 226.0 jug/L-h; P = 0.0001 us. GHRH alone) and young subjects (peak, 56.8 ± 12.3 fig/L; AUC, 2113.0 ± 444.3 Mg/L-h; P = 0.001 us. GHRH alone; Figs. 1 and 2). The potentiating effect of ARG on the GH response to GHRH was higher in elderly than in young subjects (1013.0 ± 553.5% us. 237.9 ± 79.1%; P = 0.0001); thus, the GH increase induced by combined administration of ARG and GHRH overlapped in the two groups. IGF-I levels were lower in elderly than in young subjects (76.7 ± 9.2 us. 258.3 ± 29.2 ^ig/L; P = 0.01). A transient facial flushing was observed in eight sub-
JCE & M • 1990 Vol 71 • No 6
jects after GHRH administration. No side-effects were observed after ARG infusion.
Discussion The present data confirm the age-dependent reduction of GH responsiveness to GHRH (18-21), but show that the GH responsiveness to combined administration of ARG and GHRH in the elderly strictly overlaps with that in young subjects. Interestingly, in contrast to other stimuli (16, 17), the GH response to ARG alone is not different in elderly and young subjects. This evidence is in agreement with a previous report (3). Our findings show that GH secretory capacity of somatotroph cells to an acute stimulus is preserved in normal aged humans. In aged man, the pituitary content of GH was found to be unchanged by some researchers (25), but not by others (26). In animals, the pituitary GH content was generally reported to be preserved (10, 11, 14). Moreover, in uitro the somatotroph responsiveness to GHRH was reported to be unchanged in aged rats by some researchers (10, 11, 14), but not by others (27, 28). The mechanism of action by which ARG stimulates GH secretion is still unclear. In rats, ARG failed to influence both basal and GHRH-induced GH secretion from anterior pituitary in uitro (23). This finding seems to rule out a direct effect of ARG at the pituitary level. However, in old rats a decreased pituitary sensitivity to GHRH has been reported (27, 28), and the possibility that ARG influences this mechanism cannot be excluded. Actually, there is more evidence favoring the hypothesis that the GH hyposecretory state, which connotes normal aging, is due to hypothalamic impairment. A decreased hypothalamic GHRH content in old rats has been reported (29, 30). Moreover, in old dogs prolonged treatment with clonidine, an «2-adrenergic agonist known to stimulate GH secretion via stimulation of endogenous GHRH release, leads to a GH increase similar to that in young dogs (15). In further agreement with the involvement of an altered GHRH control in aging, the GH response to GHRH in humans has been shown to be fully (20) or partly (21) restored by repetitive GHRH administration. However, the possibility that ARG acts at the hypothalamic level by stimulating endogenous GHRH release seems unlikely. In fact, ARG is able to strikingly potentiate the maximal somatotroph responsiveness to GHRH (23, 24). Moreover, differently from L-dopa, ornithine, the active form of arginine, is unable to modify plasma GHRH levels in humans (31). The more plausible mechanism by which ARG is so effective on GH secretion even in normal aging consists of inhibition of hypothalamic somatostatin release. Favoring a somatostatin-mediated mechanism of action for arginine are the following findings. 1) As mentioned
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ARG ON GHRH-INDUCED GH RISE IN AGING
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YOUNG
FIG. 1. Plasma GH levels (mean ± SEM) after GHRH, ARG, and ARG plus GHRH in normal young and elderly subjects.
ELDERLY
70
70
60
60
50
50
•> 40
40
X O 30 m
30
M
20
E
JS 20 a.
10
10
0
0 15
-15
30
46
60
75
90
-15
15
30
45
60
75 90
Time(min) 2800CHI YOUNG
2400- • i
ELDERLY
2000D)
FIG. 2. Plasma GH AUCs (mean ± SEM) after GHRH, ARG, and ARG plus GHRH in normal young and elderly subjects.
O jj -^ O E iS
1600
p < 0.01
1200 800
Q.
400-
oGHRH
above, this amino acid potentiates the GH response to the maximal GHRH dose (23, 24). 2) The potentiating effect of ARG on GHRH-induced GH secretion strictly overlaps with that of pyridostigmine (24), a cholinergic drug that inhibits cholinesterases, probably acting via somatostatin inhibition (32-37). 3) The combined administration of ARG and pyridostigmine has no additive effect on GH secretion in normal short children (24), suggesting their common mechanism of action. 4) In contrast to GHRH, ARG induced a pronounced GH rise even when preceded by prior GHRH stimulation of GH secretion (38). The marked potentiating effect of ARG on the blunted GHRH-induced GH rise in normal aging may be due to
•
ARQ
ARQ*QHRH
inhibition of a somatostatin hypertone. This hypothesis agrees with evidence that in old rats both somatostatin antiserum and pilocarpine, a muscarinic cholinergic agonist, increase GH levels and antagonize the impaired GHRH-induced GH secretion (9, 12, 13). Although the hypothalamic somatostatin content has been reported to be increased (14, 39) or decreased (40, 41), other data showed an increase of fractional efflux of somatostatin in old rats (14, 42). In conclusion, our results show that, differently from the GH response to GHRH alone, the GH response to combined administration of arginine and GHRH does not vary with age, suggesting the presence of an increased somatostatinergic activity in aging.
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GHIGO ETAL.
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Acknowledgments The authors wish to thank Dr. J. Bellone, M. Procopio, M. R. Valetto, and Prof. G. Baracchi for their cooperation in performing this study.
References 1. Rudman D, Kutner MH, Rogers M, Lubin MF, Fleming GA, Bain RP. Impaired growth hormone secretion in the adult population. J Clin Invest. 1981;67:1361-9. 2. Zadik Z, Chalew SA, McCarter RJ, Meistas M, Kowarski AA. The influence of age on the 24-hour integrated concentration of growth hormone in normal individuals. J Clin Endocrinol Metab. 1985;60:513-6. 3. Dudl RJ, Ensinck JW, Palmer HE, Williams RH. Effect of age on growth hormone secretion in man. J Clin Endocrinol Metab. 1973;37:ll-6. 4. Vermeulen A. Nyctohemeral growth hormone profiles in young and aged men: correlation with somatomedin-C levels. J Clin Endocrinol Metab. 1987;64:884-8. 5. Finkelstein JW, Roffwarg HP, Boyar RM, Kream J, Hellman L. Age-related change in the twenty-four hour spontaneous secretion of growth hormone. J Clin Endocrinol Metab. 1972;35:665-70. 6. Carlson HE, Gillin JC, Gorden P, Snyder F. Absence of sleeprelated growth hormone peaks in aged normal subjects and in acromegaly. J Clin Endocrinol Metab. 1972;34:1102-5. 7. Ho KY, Evans WS, Blizzard RM, et al. Effect of sex and age on the 24-hour secretory profile of GH secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab. 1987;64:51-8. 8. Burek CL, Frohman LA. Growth hormone synthesis by rat pituitaries in vitro. Endocrinology. 1970;86:1361-7. 9. Sonntag WE, Forman LJ, Miki N, et al. Effects of CNS active drugs and somatostatin antiserum on growth hormone release in young and old male rats. Neuroendocrinology. 1981;33:73-8. 10. Sonntag WE, Hylka VW, Meites J. Impaired ability of old male rats to secrete growth hormone in vivo but not in vitro in response to hpGRF-(l-44). Endocrinology. 1983;113:2305-7. 11. Weherenberg WB, Ling N. The absence of an age-related change in the pituitary response to growth hormone-releasing factor in rats. Neuroendocrinology. 1983;37:463-6. 12. Locatelli V, Arimura A, Torsello A, Cella SG, Muller EE. Somatostatin antiserum antagonized the impaired ability of hpGRF-40 to stimulate growth hormone release in old unanesthetized male rats. Neuroendocrinol Lett. 1984;5:261-6. 13. Locatelli V, Cella SG, Cermenati P, Panzeri G, Sellan R, Muller EE. Defective growth hormone (GH) secretion in aging mammals: contribution of central and peripheral inhibitory influences and of GH-releasing hormone. In: Valenti G, ed. Psychoneuroendocrinology of aging: basic and clinical aspects. Fidia Res Ser. Padova: Liviana Press; 1988; 16:61-7. 14. Ge F, Tsagarakis S, Rees LH, Besser GM, Grossman A. Relationship between growth hormone-releasing hormone and somatostatin in the rat: effects of age and sex on content and in-vitro release from hypothalamic explants. J Endocrinol. 1989;123:53-8. 15. Cella SG, Moiraghi V, Minuto F, et al. Prolonged fasting or clonidine can restore the defective growth hormone secretion in old dogs. Acta Endocrinol (Copenh) 1989;121:177-84. 16. Muggeo M, Fedele D, Tiengo A, Molinari M, Crepaldi G. Human growth hormone and cortisol responses to insulin stimulation in aging. J Gerontol. 1975;30:546-50. 17. Gil-Ad I, Gurewitz R, Marcovici 0, Rosenfeld J, Laron Z. Effects of aging on human plasma growth hormone response to clonidine. Mech Age Dev. 1984;27:97-100.
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18. Shibasaki T, Shizume K, Nakahara M, et al. Age-related changes in plasma growth hormone response to growth hormone-releasing factor in man. J Clin Endocrinol Metab. 1984;58:212-4. 19. Lang I, Schernthaner G, Pietschmann P, Kurz R, Stephenson JM, Tempi H. Effects of sex and age on growth hormone response to growth hormone-releasing hormone in healthy individuals. J Clin Endocrinol Metab. 1987;65:535-40. 20. Iovino M, Monteleone P, Steardo L. Repetitive growth hormonereleasing hormone administration restores the attenuated growth hormone (GH) response to GH-releasing hormone testing in normal aging. J Clin Endocrinol Metab. 1989;69:910-3. 21. Franchimont P, Urbain-Choffray D, Lambelin P, Fontaine MA, Frangin G, Reginster JY. Effects of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor I, and bone metabolism in postmenopausal women. Acta Endocrinol (Copenh). 1989;120:121-8. 22. Parker ML, Hammonds JM, Daughaday WH. The arginine provocative test: an aid in the diagnosis of hyposomatotropism. J Clin Endocrinol Metab. 1967;27:1129-36.
23. Alba-Roth J, Albrecht Muller 0, Schopohl J, Von Werder K. Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab. 1988;67:1186-9. 24. Ghigo E, Bellone J, Mazza E, et al. Arginine potentiates the GHRH- but not the pyridostigmine-induced GH secretion in normal short children. Further evidence for a somatostatin suppressing effect of arginine. Clin Endocrinol (Oxf). 1990;32:763-7. 25. Gershberg H. Growth hormone content and metabolic actions of human pituitary glands. Endocrinology. 1957;61:160-5. 26. Yu-Kai Sun YP, Fenoglio CM, Pushparaj N, et al. The effect of age on the number of pituitary cells immunoreactive to growth hormone and prolactin. Hum Pathol. 1984;15:169-80. 27. Robberecht P, Gillard M, Waelbroeck M, Camus JC, De Neef P, Christophe J. Decreased stimulation of adenylate cyclase by growth hormone releasing factor in the anterior pituitary of old rats. Neuroendocrinology. 1986;44:429-32. 28. Ceda GP, Valenti G, Butturini U, Hoffman AR. Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats. Endocrinology. 1986;118:2109-14. 29. Morimoto N, Kawakami F, Makino S, Chihara K, Hasegawa M, Ibata Y. Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus. Neuroendocrinology. 1988;47:459-64. 30. De Gennaro Colonna V, Zoli M, Cocchi D, et al. Reduced growth hormone releasing factor (GHRH)-like immunoreactivity and GHRH gene expression in hypothalamus of aged rats. Peptides. 1989;10:705-9. 31. Evain-Brion D, Donadieu M, Liapi C, et al. Plasma growth hormone releasing factor levels in children: physiologically and pharmacologically induced variations. Horm Res. 1986;24:116-21. 32. Richardson SB, Hollander CS, D'Eletto R, et al. Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology. 1980;107:1837-42. 33. Locatelli V, Torsello A, Redaelli M, et al. Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone-releasing hormone in the rat: evidence for mediation by somatostatin. J Endocrinol. 1986;lll:271-8. 34. Torsello A, Panzeri G, Cermenati P, et al. Involvement of somatostatin and cholinergic system in the mechanism of growth hormone autofeedback regulation in the rat. J Endocrinol. 1988;117:273-81. 35. Massara F, Ghigo E, Demislis K, et al. Cholinergic involvement in the growth hormone releasing factor-induced growth hormone release: studies in normal and acromegalics subjects. Neuroendocri-
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ARG ON GHRH-INDUCED GH RISE IN AGING nology. 1986;43:670-5. 36. Ghigo E, Mazza E, Imperiale E, et al. Enhancement of cholinergic tone by pyridostigmine promotes both basal and growth hormone(GH)-releasing hormone-induced GH secretion in children of short stature. J Clin Endocrinol Metab. 1987;65:452-6. 37. 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 Endocrinol (Oxf). 1987;27:727-33. 38. Page MD, Dieguez C, Valcavi R, Edwards C, Hak R, Scanlon MF. Growth hormone (GH) responses to arginine and 1-DOPA alone and after GHRH pretreatment. Clin Endocrinol (Oxf). 1988;28:551-8.
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39. Forman LJ, Sonntag WE, Hylka VW, Meites J. Pituitary growth hormone and hypothalamic somatostatin in young female rats versus old constant estrous female rats. Experientia. 1985;41:6534. 40. Takahashi S, Gottschall PE, Quigley KL, Goya RG, Meites J. Growth hormone secretory patterns in young, middle-aged and old female rats. Neuroendocrinology. 1987;46:137-42. 41. Sonntag WE, Steger RW, Forman LJ, Meites J. Decreased pulsatile release of growth hormone in old male rats. Endocrinology. 1980;107:1875-9. 42. Sonntag WE, Gottschall PE, Meites J. Increased secretion of somatostatin-28 from hypothalamic neurons of aged rats in vitro. Brain Res. 1986;380:229-34.
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