Effect of Galanin on the Growth Hormone Response to Growth Hormone-Releasing Hormone in Acromegaly Andrea Giustina, Maurizio Schettino, Corrado Bodini, Mauro Doga, Massimo Licini, and Gianni Giustina Galanin enhances growth hormone (GH)-releasing hormone (GHRH)-stimulated GH secretion in normal man. In acromegaly, circulating GH levels are increased and the GH response to GHRH may be exaggerated. Galanin has been recently shown to decrease circulating GH levels in acromegaly. The aim of our study was to investigate the effects of galanin on the GH response to GHRH in acromegalic subjects. Five acromegalic patients (three men and two women) and seven healthy adult subjects (five men and two women) were studied. GHRH-induced GH secretion was evaluated during a 40.minute intravenous (IV) infusion of saline (100 mL) or porcine galanin (12.5 hg/min in 100 mL saline). In normal subjects, 6GH levels after GHRH + porcine galanin administration (47 -t 7.5 pg/L) were significantly higher in comparison to levels obtained with GHRH + saline (21.7 + 3.5 kg/L, P < .05). In acromegalic patients, GH responses to GHRH (6GH. 18.8 + 8.6 pg/L) were not altered by galanin infusion (ZiGH, 17.8 f 5 pg/ L). Our results give the first evidence that the same dose of galanin that induces a significant enhancement of the GH response to GHRH in normal subjects has no effect on the GH response to GHRH in acromegalic patients. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by somatotropes independent of GHRH. Failure of galanin to enhance GH response to GHRH in acromegalic patients could be due to a change in function of the galanin receptor on GH-secreting adenomatous cells. Copyright 8 1992 by W.B. Saunders Company
bioALANIN IS A 2Y-AMINO ACID straight-chain logically active peptide derived from a 123-amino acid precursor protein, preprogalanin.’ Galanin was originally isolated from porcine intestine,? but galanin-like immunoreactivity is widely distributed in central and peripheral neurons of several mammalian species, including hun~ans.3 Galanin has been hypothesized to be a neurotransmitter or a neuromodulator in the central nervous system.4,5 The physiological role of galanin has not yet been identified; however, galanin influences the secretion of several pituitary hormones in man and rat.h-H Galanin probably plays a significant role in the regulation of growth hormone (GH) secretion in man. Porcine galanin elicits GH secretion when given alone6 and when given to increase the GH response to GH-releasing hormone (GHRH) in normal man.” The mechanism underlying this action of galanin is unknown; evidence suggests that galanin may act at the hypothalamic lcvcl.“’ In acromegaly, circulating GH levels are increased; moreover, GH secretory dynamics in acromegalic patients are usually abnormal.” These abnormalities include incomplel.e inhibition or paradoxical elevation of GH after glucose ingestion, a decrease in GH level after administration of L-dopa, and stimulation of GH by thyrotropinreleasing hormone (TRH) or, less commonly, by luteinizing hormone-releasing hormone (LHRH).‘z-*4 Some acromegalic patients also show exaggerated GH responses to GHRH.” Recently, it has been shown that porcine galanin infusion decreases circulating GH levels in some patients with acromegaly.‘“.” The aim of our study was to investigate the effects of galanin on the GH response to GHRH in patients with acromegaly. SUBJECTS AND METHODS Subjects Three men and two women with active acromegaly, with a mean age of 52 ir 3.6 years (range, 44 to 63 years) and a body mass index
of 27 2 1.6 kg/m’ (range, 24.6 to 33.3 kg/m$ were studied (Table 1). Two patients (no. I and no. 2) were untreated, and three patients with GH-secreting pituitary macroadenoma had previously undergone transsphenoidal pituitary surgery, followed in one case (no. 4) by radiotherapy. All of these patients were submitted to a l-month wash-out period without taking any pharmacological therapy before entering the study. All patients had the typical clinical signs of acromegaly and increased insulin-like growth factor-l (IGF-1) levels for their age; all had increased baseline GH levels (calculated on at least four samples). ie. levels higher than 6 &L. A 100-g glucose load failed to suppress C;H levels below 2.5 Kg/L in all cases. A paradoxical GH response to TRH, defined as an increase of at least twice the basal GH level to a level higher than 5 Fg/L.‘-?.‘4 was seen in three patients (Table I). Seven healthy adult volunteers (five men and two women) with no family history of endocrine or metabolic diseases, a mean age of 43 + 8.9 years (range, 25 to 84 years), a body mass index of 22 + 0.8 kg/m? (range, 19 to 25 kg/m’). and normal thyroid and adrenal function served as controls. No subjects were taking any drugs at the beginning of the study, and all subjects were examined on two different occasions with at least ‘I-day intervals. The study protocol was approved by the local Ethical Committee. Methods The study was performed according to a single-blind crossover design. After an overnight fast. each subject was admitted to our Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. Two antecubital vein catheters (for independent infusion and blood sampling) were inserted percutaneously and kept patent by slow saline infusion. After a 30.minute stabilization period. the following treatments were initiated in randomized order from -10 to 30 minutes: (1) intravenous (IV) infusion of saline, 100 mL; and (2) IV infusion of synthetic porcine galanin
From the Cattedra di Clinica Medica, Univer~sity ofBrescia, Italy. Supported by u grant from the “Centro Stttdi e Ricer&c di Neuroendocrinologia, *’Brescia, Italy. Address reprint requests to Andrea Giustina, MD, Clinica Medica clo 2a Medicina, Spedali Civili, 25125 Brescia, ItaJv. Copyright 0 1992 by W.B. Saunders Cornparr> 00260495192/4112-0004$03.00/0
GIUSTINA ET AL
Table 1. Clinical Characteristics
of Acromegalic Patients
52.0 k 3.6
27.0 + 1.6
3.9 + 0.7
21.5 k 5.8
and GH levels were calculated as the mean of four
samples taken on different days at 8:00 AM. Abbreviations: BMI, body mass index; TS, transsphenoidal
RT, radiotherapy; TRH. paradoxical GH response to TRH. defined as an increase of at least twice the basal GH level to a level greater than 5 PLg/L.
(Inalco, Milano, Italy), 0.5 mg in 100 mL saline. An IV bolus of human GHRH (l-29)NHz (Geref, Serono, Italy), 100 pg in 1 mL saline, was injected in both cases at 0 minutes. Blood samples for GH assay were taken at -15, -10 (at initiation of saline or galanin infusion), 0 (time of GHRH injection), +15, +30, +45, +60, +90, and +120 minutes. The two studies were divided by at least a 7-day interval. Results were expressed as the mean ? SEM. GH secretory responses to GHRH + saline and GHRH + galanin were expressed as absolute values (kg/L) or as Svalues (p,g/L calculated by subtracting the mean of - 15- and -lo-minute levels from peak GH levels). Absolute GH values were compared using ANOVA for repeated measures; 6GH levels were compared with one-way
ANOVA. Assays Commercial kits were used for the estimation of GH (IRMA, Nichols Institute, San Juan Capistrano, CA; interassay and intraassay coefficients of variation, 25.4% and *2.3%, respectively; sensitivity limit of the assay, 0.02 ug/L) and IGF-1 levels (RIA, Nichols Institute; interassay and intraassay coefficients of variation, 25.2% and ?9.4%, respectively; sensitivity limit of the assay, 0.1 U/mL). Blood glucose levels were measured with the glucoseoxidase method (Beckman II glucose analyzer, Beckman Instruments, Palo Alto, CA). All samples from the same subject were assayed together in duplicate.
Fig 1. Serum GH levels (mean f SEM; pg/L) after IV injection of GHRH + saline (A) or GHRH + galanin (0) in seven normal subjects. lP c .05 v saline.
kg/L) were similar or lower in comparison to those of normal subjects (SGH range, 11.8 to 34.6 kg/L). Only in subject no. 5 was an apparently exaggerated GH response to GHRH observed (peak GH level, 97.8 kg/L, SGH, 52.2 kg/L). In the whole group of acromegalic patients, galanin infusion did not significantly affect the mean GH response to GHRH (SGH, 17.6 2 5 kg/L) in comparison to results obtained with saline infusion (SGH, 18.8 + 8.6 ug/L; Fig 2). However, in the patient with GH hyperresponsiveness to GHRH, galanin infusion caused a clear decrease in GH release after GHRH in comparison to saline infusion @GH, 33.9 v 52.2 kg/L). Neither normal nor acromegalic subjects showed significant fluctuations in blood glucose levels after either galanin or saline infusion. All subjects experienced a bitter taste in the mouth, which began immediately after initiation of the galanin infusion and ended when the infusion was discontinued; no GH (WIL)
The kinetics of the GH responses to GHRH + saline and GHRH + porcine galanin in normal and acromegalic subjects are shown in Figs 1 and 2. Normal Subjects
Galanin infusion caused a significant increase in GH response to GHRH when compared with saline infusion. The mean 6GH level after galanin infusion (47 + 7.5 kg/L) was significantly higher in comparison to the mean 6GH level after saline infusion (21.7 + 3.5 kg/L, P < .05; Fig 1).
Baseline GH levels were significantly increased in comparison to those of normal subjects. In four of five patients, GHRH-induced GH responses (6GH range, 4.2 to 18.8
Fig 2. Serum GH levels (mean f SEM; gg/L) after IV injection of GHRH + saline (W) or GHRH + galanin (0) in five acromegalic patients.
GHRH, AND ACROMEGALY
other side effects were observed in any of the subjects. Blood pressure and pulse rate were not significantly altered by galanin infusion. DISCUSSION
Our results show that the same dose of galanin that increases the GH response to GHRH in normal subjects does not alter the GH response to GHRH in patients with acromegaly. GH secretion in man is controlled by the stimulating influence of GHRH on one hand, and the inhibitory action of somatostatin on the other. Several substances can affect GH secretion, usually by influencing GHRH and/or somatostatin release.lx Galanin is a 29-amino acid peptide that has been detected in the central nervous system of ratsI and humanszO Porcine galanin, infused in doses similar to those used in this study, causes a significant increase in the GH response to a maximal”’ GHRH dose in normal adults.9~2’ The mechanism of this stimulating action of galanin remains to be explained. In man, galanin prevents the GH-inhibitory effect of cholinergic blockade,X3 which is postulated to be mediated by hypothalamic somatostatin.24 Therefore, it has been suggested that galanin may act via somatostatin. On the other hand, we have recently shown that galanin does not enhance the GH response to GHRH in subjects with increased somatostatin tone due to chronic glucocorticoid therapy.‘2 Moreover, galanin and GHRH have been found to be colocalized at the hypothalamic level in several animal species.?” Thus, it has been hypothesized that galanin acting at the pituitary level on its own receptors could enhance GHRH action.26 This hypothesis has been confirmed by in-vitro studies that have shown a direct effect of galanin on cultured rat somatotropes.27.2x Our results give the first evidence that the same dose of galanin that enhances the GH response to GHRH in normal man9,22 does not significantly alter the GH response to GHRH in acromegalic patients. It can be hypothesized that changes in the function of galanin receptors on adenomatous cells may be responsible for the lack of effect of galanin on the GH response to GHRH in acromegaly. 11 has previously been shown that the dopaminergic
agonist bromocriptine does not influence the GH response to either GHRH*” or TRH30 in acromegalic patients. On the other hand, bromocriptine has been demonstrated to enhance the GH response to GHRH in normal subjects.“’ The lack of interaction of bromocriptine with GHRH on GH-secreting adenomatous cells has been explained by suggesting that the effects of dopamine and GHRH may result from different intracellular actions.2Y Recently, it has been shown that while GHRH is coupled with the adenylate cyclase-cyclic adenosine monophosphate system, the transduction of the dopamine signal involves the reduction of cytosolic free-Ca2+ concentration.3Z In-vitro studies have shown that galanin may inhibit insulin release, decreasing cytoplasmic free-Ca?+ concentrations in mouse p-cell cultures.33 On the basis of these data, it seems likely that galanin also activates different intracellular messengers with respect to GHRH. Therefore, it can be hypothesized that in human tumoral somatotropes, the transduction of both the galanin- and dopamine-inhibitory signal may involve the inhibition of Ca2+ influx. On the other hand, it can be argued that the administration of GHRH alone could cause a maximal activation of GH-releasable pools in acromegaly. Therefore, on the basis of our data, it cannot be excluded that the inability of galanin to augment the GH response to GHRH may reflect an exhaustion of pituitary GH stores in our acromegalic patients. In four of five patients with acromegaly, the GH response to GHRH was similar or lower in comparison to that of normal subjects. These data confirm the existence of two subgroups of acromegalics: one with norma13j and the other with exaggeratedI GH response to GHRH. Interestingly, in one patient the GH hyperresponsiveness to GHRH was clearly inhibited by galanin, suggesting the possible existence of different sensitivities to the neuropeptide in the two subgroups of acromegalic patients. In summary, in normal subjects galanin has stimulatory effects on the GH response to GHRH, whereas in acromegaly, galanin may have either no effects or inhibitory efl’ects on the GH response to GHRH. This discrepancy can be hypothesized to be due to a change in the function of galanin receptors on GH-secreting adenomatous cells.
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11. Lawrence AM, Goldfine ID, Kirsteins L: Growth hormone dynamics in acromegaly. J Clin Endocrinol Metab 31:239-243,197O 12. Chiodini PG, Liuzzi A, Botalla L, et al: Inhibitory effects of dopaminergic stimulation on GH release in acromegaly. J Clin Endocrinol Metab 38:200-202,1974 13. Faglia G, Beck-Peccoz P, Ferrari C, et al: Plasma growth hormone response to thyrotropin-releasing hormone in patients with active acromegaly. J Clin Endocrinol Metab 36:1259-1262, 1973 14. Giustina G, Reschini E, Peracchi M, et al: Failure of somatostatin to suppress thyrotropin releasing factor and luteinizing hormone releasing factor-induced growth hormone release in acromegaly. J Clin Endocrinol Metab 38:906-909,1974 15. Wood SM, Ch’ng JLC, Adams EF. et al: Abnormalities of growth hormone release in response to human pancreatic growth hormone releasing factor (GRF (l-44)) in acromegaly and hypopituitarism. Br Med J 286:1686-1691, 1983 16. Giustina A, Bodini C, Bossoni S, et al: Galanin decreases circulating growth hormone levels in acromegaly. Eleventh Congress of the European Association of Internal Medicine, Lisbon, Portugal, November 6-9,1991, p 82 (abstr) 17. Giustina A, Bodini C, Doga M, et al: Galanin decreases circulating growth hormone levels in acromegaly. J Clin Endocrinol Metab 74:1296-1300,1992 18. Giustina A, Girelli A, Doga M, et al: Pyridostigmine blocks the inhibitory effect of glucocorticoids on growth hormonereleasing hormone stimulated growth hormone secretion in normal man. J Clin Endocrinol Metab 71:580-584, 1990 19. Rokaeus A, Melander T, Hokfelt T, et al: A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 47:161-166, 1984 20. Gentleman SM, Falkai P, Bogerts B. et al: Distribution of galanin-like immunoreactivity in the human brain. Brain Res 505:311-315,1989 21. Giustina A, Bossoni S, Bodini C, et al: The role of cholinergic tone in modulating the growth hormone response to growth hormone-releasing hormone in normal man. Metabolism 40:519523,199l 22. Giustina A, Girelli A, Bossoni S, et al: Effect of galanin on growth hormone releasing hormone-stimulated growth hormone secretion in adult patients with nonendocrine diseases on chronic daily glucocorticoid treatment. Metabolism 41:548-551,1992 23. Chatterjee VKK, Ball JA, Proby C, et al: Galanin abolishes the inhibitory effect of cholinergic blockade on growth hormone-
releasing hormone-induced secretion of growth hormone in man. J Endocrinol 116:Rl-R2, 1988 24. 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 25. Meister B, Scanlon MF, Hokfelt T: Occurrence of galaninlike immunoreactivity in growth hormone releasing factor (GRF)containing neurons of the monkey (Macacafuscicularis) infundibular nucleus and median eminence. Neurosci Lett 119:136-139,199O 26. Kitajima N, Chihara K, Abe H, et al: Galanin stimulates immunoreactive growth hormone releasing factor secretion from rat hypothalamic slices perifused in vitro. Life Sci 47:2371-2376, 1990 27. Torsello A, Sellan R, Cella SWG, et al: Age-dependent modulation by galanin of growth hormone release from rat pituitary cells in culture. Life Sci 47:1861-1866, 1990 28. Meister B, Hulting AL: Influence of coexisting hypothalamic messengers on growth hormone secretion from rat anterior pituitary cells in vitro. Neuroendocrinology 46:387-394. 1987 29. Cozzi R, Dallabonzana D, Oppizzi G, et al: Bromocriptine does not alter growth hormone (GH) responsiveness to GHreleasing hormone in acromegaly. J Clin Endocrinol Metab 62:601604,1986 30. Belforte L, Camanni F, Chiodini PG, et al: Long-term treatment with 2Br-alpha ergocryptine in acromegaly. Acta Endocrinol (Copenh) 85:235-241,1977 31. Vance ML, Kaiser DL, Frohman LA, et al: Role of dopamine in the regulation of growth hormone secretion: Dopamine and bromocriptine augment growth hormone (GH)-releasing hormonestimulated GH secretion in normal man. J Clin Endocrinol Metab 64:1136-1141,1987 32. Spada A, Bassetti M, Arosio M, et al: The hypersecreting somatotroph: Functional and morphological aspects, in Muller EE, Cocchi D, Locatelli V (eds): Advances in Growth Hormone and Growth Factor Research. Roma, Italy, Pythagora, 1989, ~~411-421 33. Ahren B, Arkhammar P, Berggren P-O, et al: Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmic free Ca++ concentration. Biochem Biophys Res Commun 140:1059-1063, 1986 34. Gelato MC, Merriam GR, Vance ML, et al: Effects of growth hormone-releasing factor on growth hormone secretion in acromegaly. J Clin Endocrinol Metab 60:251-257, 1985