0021-972x/92/7406-1296$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Galanin Decreases in Acromegaly”
Vol. 74, No. 6 Printed in U.S.A.
Circulating
Growth
ANDREA GIUSTINA, CORRADO BODINI, MAURO GIUSEPPE PIZZOCOLO, AND GIANNI GIUSTINA Cattedra di Clinica Medica
and Chimica
(G.P.), University
DOGA,
Hormone
MAURIZIO
Levels
SCHETTINO,
of Bresciu, Italy
ABSTRACT.
Galanin is able to elicit GH secretion in normal man. In acromegaly, circulating GH levels are elevated, and GH secretory dynamics are usually abnormal. The aim of our study was to investigate the effects of galanin on GH secretion in acromegalic subjects. Six acromegalic patients (four males and two females) and seven healthy adult subjects (five males and two females) underwent in randomized order: 1) iv infusion of 100 mL saline from O-45 min, and 2) iv infusion of synthetic porcine galanin (0.5 mg in 100 mL saline) from O-45 min. In normal subjects, peak GH levels after porcine galanin administration (8.2 f 1.9 pg/L) were significantly higher than after saline infusion (1.3 + 0.1 fig/L, P < 0.05). In acromegalic patients, GH values fell from baseline (32.5 + 12 rg/L) to a
G
mean nadir of 24.5 f 12.7 rg/L after galanin infusion. The mean change in GH values from baseline after galanin treatment in these subjects significantly differed from that observed after saline infusion from 15-90 min. Serum PRL levels were not significantly affected by galanin in either normal or acromegalic patients. Our results give the first evidence that the same dose of galanin, acting as a GH secretagogue in normal man, is, on the contrary, able to significantly inhibit GH in acromegalic patients. The cause of this paradoxical GH fall after galanin treatment in acromegaly remains to be explained. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by GH-secreting adenomatous cells. (J Clin Endocrirwl Metab 74: 1296-1300,1992)
ALANIN is a 29-amino acid, straight chain, biologically active peptide, derived from a 123-amino acid precursor protein, preprogalanin (1). Galanin was originally isolated from porcine intestine (2), but galanin-like immunoreactivity is widely distributed in central and peripheral neurons of several mammalian species, including humans (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 is able to influence the secretion of several pituitary hormones in man and rats
tients are usually abnormal (11). These abnormalities include incomplete inhibition or paradoxical elevation of GH after glucose ingestion, a fall in GH after administration of L-dopa, stimulation of GH by TRH or, less commonly, by LHRH, and exaggerated GH response to GHRH (12-15). The precise nature of the defects leading to these abnormalities is not understood. The aim of our study was to investigate the effects of galanin on GH secretion in patients with acromegaly.
(6-8).
Subjects
Galanin probably plays a significant role in the regulation of GH secretion in man. Porcine galanin is able to elicit GH secretion when given alone (6) and to increase the GH response to GH-releasing hormone (GHRH) in normal man (9). The mechanism underlying this action of galanin is unknown; evidence seems to suggest that galanin may act at the hypothalamic level (10). In acromegaly, circulating GH levels are elevated. Moreover, GH secretory dynamics in acromegalic pa-
Four men and two womenwith active acromegaly[meanage, 49.2 + 4.1 yr (range, 35-63 yr); body massindex, 27.2f 1.3 kg/ m2 (range, 24.6-33.3 kg/m2)] were studied (Table 1). Two patients (no. 1 and 2) were untreated. Four patients with GHsecreting pituitary macroadenomahad previously undergone transsphenoidalpituitary surgery, followed in one case(no. 4) by radiotherapy. All but one these patients were submitted to a l-month wash-out period without taking any pharmacological therapy before entering the study. Only patient 6 continued to receive octreotide (Sandostatin, Sandoz, Basel, Switzerland, 450 rg/day in three daily SCinjections), which was the only
Received June 19,199l. Address all correspondence and requests of reprints to: Andrea Giustina, M.D., Clinica Medica c/o 2a Medicina, Spedali Civili, 25125 Brescia, Italy. * This work was supported by the Centro Studie Ricerche di Neuroendocrinologia (Brescia, Italy).
Subjects and Methods
drug
effective
in relieving
his
headache;
in
this
last
case,
the
tests were performed 12 h after the last doseof octreotide. All of the patients had the typical clinical signsof acromegalyand elevatedinsulin-like growth factor-I (IGF-I) levelsfor their age; 1296
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GALANIN,
GH, AND
all had elevated baseline GH levels (calculated in at least four samples), i.e. more than 6 rg/L. A 100-g glucose load failed to suppress GH levels below 2.5 rg/L in all cases. A paradoxical GH response to TRH, defined as an increase of at least twice the basal GH level to a value greater than 5 pg/L (13, 14), was seen in three patients (Table 1). Seven healthy adult volunteers (five men and two women) with no family history of endocrine or metabolic diseases [mean age, 37.4 + 7.1 yr (range, 24-76 yr); body mass index, 25 f 1.5 kg/m* (range, 19.4-32.1 kg/m*)] with normal thyroid and adrenal function served as controls. No significant differences in age or body mass index between acromegalic and normal subjects were observed. None of the subjects was taking any drugs at the start of the study, and all subjects were examined on two different occasions with at least 7-day intervals. The study protocol was approved by the local ethical committee. Methods After an overnight fast, each subject was admitted to our Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. Two antecubital venous catheters (for independent infusion and blood sampling) were inserted percutaneously and kept patent by slow saline infusion. After a 30-min stabilization period, the following treatments were initiated in randomized order: 1) iv infusion of 100 mL saline from O-45 min; and 2) iv infusion of synthetic porcine galanin (0.5 mg in 100 mL saline) from O-45 min. Sterile endotoxin-free synthetic porcine galanin (Inalco, Milan, Italy) was reconstituted in 2 mL normal saline. Blood samples for GH, PRL, and glucose assay were taken at 15 min before and 0,15,30,45,60,90, and 120 min after the beginning of the infusion. Pulse rate and supine blood pressure were measured at 0, 20,45, and 60 min during each infusion. Results were expressed as the mean + SEM. GH secretory responses were expressed either as absolute values (micrograms per L), peak or nadir (micrograms per L), or a percentage of the basal level [mean of -15 and 0 min samples; maximum percent GH change]. Absolute and maximum GH change values were compared using the analysis of variance for repeated measures. Peak or nadir GH levels were compared with Student’s t test for paired data. Clinical characteristics of normal TABLE
1. Clinical Patient
characteristics no.
of acromegalic Sex/age
(yr)
ACROMEGALY and acromegalic subjects were compared with one-way analysis of variance. Assays Commercial kits were used for the estimation of GH (immunoradiometric assay; Nichols Institute, San Juan Capistrano, CA; inter- and intraassay coefficients of variation, f 5.4% and &2.3%, respectively; sensitivity limit of the assay, 0.06 fig/L), PRL (immunoradiometric assay; Ares-Serono, Milan, Italy; inter- and intraassay coefficients of variation, f 6.4% and &2.1%, respectively; sensitivity limit of the assay, 0.3 pg/L), and insulin-like growth factor-I (RIA; Nichols Institute; inter- and intraassay coefficients of variation, +5.2% and f 9.4%, respectively; sensitivity limit of the assay, 0.1 U/mL). Blood glucose levels were measured with the glucooxidase method (Beckman II glucose analyzer, Palo Alto, CA). All samples from the same subject were assayed together in duplicate.
Results The kinetics of the GH responses to porcine galanin and saline infusions in normal and acromegalic subjects are shown in Figs. 1 and 2. Normal
subjects
Galanin infusion caused a significant increase in GH levels compared to those after saline infusion. The mean peak GH level after galanin (8.2 + 0.9 pg/L) was significantly higher than the mean GH peak after saline infusion (1.3 f 0.1 pg/L; P < 0.05). Galanin infusion caused in all instances an increase in GH levels. The GH peak occurred between 25-40 min after the beginning of the infusion (Fig. 1). Acromegalic
patients
Baseline GH values were significantly elevated compared to those in normal subjects. In five of the six
patients BMI (k/m2)
Previous therapy
IGF-I W/ml)
GH bg/L) 27.3 6.7 40.0
+
+ + -
1 2 3
F M M
58 49 44
24.6 33.3 26.6
TS
3.5 4.9 6.2
4 5 6
F M M
63 46 35
24.7 25.9 28.2
TS, RT TS TS
2.8 2.3 7.5
20.8 12.5 98.0”
49.2 4.1
27.2 1.3
4.5 0.8
34.2 13.6
Mean +SEM
BMI, Body mass index; TS, transsphenoidal surgery; RT, radiotherapy; IGF-I, as the mean of four samples taken on different days at 0800 h. TRH, Paradoxical the basal GH level to a level above 5 pg/L. 0 IGF-I and GH levels under chronic administration of octreotide (450 pg/day,
TRH
-
insulin-like growth factor-I. IGF-I and GH levels were calculated GH response to TRH, defined as an increase of at least twice SC); samples
were taken
12 h after
the last dose of the drug.
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GIUSTINA
1298
ET AL.
JCE & M .1992 Vol74.No6
1L
b
hfw
ill-
I)-
3 .
6. z
3
4.
2.
O15
0
15
30
45
v///i///y///I Galanin or saline i.v. infusion
co
90
120
Time
(min)
FIG. 1. Serum GH concentrations (mean + SEM; micrograms per L) after iv infusion of saline (0) or galanin (W) in seven normal subjects. *, P < 0.05 vs. saline.
patients, GH values clearly fell during galanin infusion compared to those during saline treatment (range, 43.964.4% with respect to baseline levels), with nadirs between 30-45 min after the beginning of the infusion (Fig. 2, a and b). In patient 6, only a slight GH decrease after galanin (nadir, 90% of basal levels) was observed. The mean curve for changes in GH values (expressed as a percentage of the mean of -15 and 0 min basal levels) after galanin treatment in the whole group of acromegalic subjects differed from that observed after saline infusion in a significant manner from 15-90 min (Fig. 2~). Serum PRL levels did not show significant variations after galanin compared to baseline levels and those after saline treatment in either normal or acromegalic subjects (Table 2). Neither normal nor acromegalic subjects showed significant fluctuations in blood glucose levels after galanin or saline infusions. All of the subjects experienced a bitter taste in the mouth, which started immediately after the beginning of galanin infusion and ended at the end of the infusion. No 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 is able to cause a clear and significant increase in plasma GH levels in normal subjects can decrease GH values in
0
15
V///////f///f//A
30
45
60
90
120 TIYE(rni”)
FIG. 2. a, Individual serum GH concentrations (micrograms per L) in six acromegalic patients after iv infusion of saline. b, Individual serum GH levels (micrograms per L) in six acromegalic patients after iv infusion of galinin. c, Mean change in GH levels (A GH; +SEM; percentage of basal levels) after iv infusion of saline (0) or galanin (m) in six acromegalic patients. *, P < 0.05 vs. saline.
patients with acromegaly. Galanin is a 29-amino acid peptide that has been detected in the central nervous system of rats (16) and humans (17). Previous studies have shown that porcine galanin, infused in doses similar to those used by us, causes a significant increase in baseline GH levels in both adults (6) and normal children (18, 19). GH secretion in man is controlled by the stimulating influence of GHRH, on the 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 (20). Our understanding of
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GALANIN,
GH, AND
ACROMEGALY
1299
TABLE 2. Mean (+SEM) serum PRL levels (micrograms per L) in six acromegalic patients and seven normal controls after galanin (Gal) or saline (Sal) iv infusion from O-45 min Time (min) Test Patients Gal Sal P Controls Gal Sal P
-15
0
15
30
45
60
90
120
10.9 (2.3) 10.3 (2.2)
11.2 (2.3) 12.5 (2.7)
10.8 (2.6) 12.2 (1.8)
12.3 (2.6) 11.3 (1.9)
12.0 (2.6) 10.8 (2.0)
11.5 (2.7) 10.2 (2.2)
11.0 (2.3) 9.8 (2.4)
10.8 (2.2) 9.3 (2.5)
NS
NS
NS
NS
NS
NS
NS
NS
8.7 (2.7) 9.1 (2.5)
8.3 (2.5) 10.0 (2.9)
7.8 (2.1) 9.9 (2.8)
8.0 (1.9, 9.0 (“.2)
8.8 (2.2) 9.0 (2.0)
9.0 (2.4) 8.6 (1.7)
8.4 (2.5) 7.5 (1.6)
7.8 (2.2) 7.8 (2.0)
NS
NS
NS
NS
NS
NS
NS
NS
Values are the mean, with ~SEM in parentheses.
the mechanism underlying the action of galanin is still fragmentary. Neuroanatomical data provide the basis for a putative pituitary site of action of galanin in affecting GH release in both rats and man (4, 16, 17). However, contrasting in vitro results have been obtained testing the direct effect of galanin on pituitary cells. Either an absent (10) or slight (21) GH-releasing effect of galanin on dispersed rat anterior pituitary cells has been observed. On the contrary, other researchers have shown that galanin is able to inhibit GH release from dispersed anterior pituitary cells of normal adult rats (22, 23). Pretreatment with GHRH antibodies suppresses the plasma GH increase induced by iv and intracerebroventricular infusion of galanin in conscious rats (24, 25). These findings suggest that the stimulating effects of galanin on GH secretion are mediated in the rat by hypothalamic GHRH as a final common pathway. Studies in man suggest that galanin may stimulate GH secretion by inhibition of tonic endogenous somatostatin secretion from the hypothalamus. Davis et al. (9) have reported that the GH response to a maximal GHRH dose (26) is augmented by simultaneous administration of galanin. Moreover, galanin is able to prevent in man the GH inhibitory effect of cholinergic blockade (27), which is postulated to be mediated by hypothalamic somatostatin (28). Our results give the first evidence that the same dose of galanin that results in stimulation of GH secretion in normal man (6) is, on the contrary, able to induce significant GH inhibition in acromegalic patients. The magnitude of the observed GH fall after galanin in five of our acromegalic patients ranged between about 44-64s of baseline levels. However, it is not known whether the dose of galanin used by us is able to induce the maximal GH-inhibiting effect in acromegaly. In only one of the patients studied was the GH-decreasing effect of galanin not clear-cut. It can be hypothesized that in this case the adenomatous cells were partially resistant to the action
of galanin, as reported for other GH-lowering agents (29). On the other hand, we cannot exclude a significant effect of chronic octreotide therapy on the GH response to galanin in this patient. At present, the mechanism by which galanin can decrease GH levels in acromegaly remains to be explained. In keeping with recent in vitro studies (22, 23) it may be hypothesized that galanin as well as dopamine agonist drugs (30) interacting .at the pituitary level with their own receptors expressed by the adenomatous tissue are able to directly inhibit pituitary GH secretion in acromegalic patients. This hypothesis is also supported by the observation that galanin reduces GH levels in acromegalic patients with persistently elevated GH levels after pituitary surgery. Dopaminergic drugs were the only previously known agents able to cause a paradoxical GH fall in acromegaly (31, 32). Thus, the galanin-mediated decrease in GH levels in acromegaly may also be hypothesized to be indirect, via a dopaminergic pathway. However, due to the lack of effect of galanin on PRL levels (6, 20) in both normal and acromegalic patients, this latter hypothesis seems unlikely. Obesity is known to blunt the GH response to galanin in normal subjects (33), and galanin decreases GH release from pituitaries of old rats (23). Our control group was slightly younger and had slightly lower body mass indexes with respect to acromegalic patients. However, these differences did not reach a statistically significant level. In summary, we can hypothesize that in normal subjects the hypothalamic stimulating effects of galanin predominate, whereas in acromegaly, characterized by an at least partial escape of the adenoma from hypothalamic control, the pituitary GH-inhibiting effect of galanin could be evidenced. To elucidate the mechanism of the paradoxical decreasing effect of galanin on plasma GH levels in acromegaly, further in uitro and in uiuo studies on the effect of galanin on the GH response to
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GIUSTINA
1300
other neuropeptides, necessary.
such as TRH
or GHRH,
will be
References 1. Rokaeus A, Brownstein M. Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc Nat1 Acad Sci USA. 1986;83:6287-91. 2. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V. Galanin-a novel biologically active peptide from porcine intestine. FEBS Lett. 1983;164:124-8. 3. Rokaeus A. Galanin: a newly isolated biologically active neuropeptide. Trends Neurosci. 1987:10:158-64. 4. Palkovits M, Rokaeus A, Antony FA, Kiss A. Galanin in the hypothalamo-hypophyseal system. Neuroendocrinology. 1987; 46417-23. 5. Melander T, Hokfelt T, Rokaeus A, et al. Co-existence of galaninlike immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J Neurosci. 1986;6:364054. 6. Bauer FE, Ginsberg L, Venetikou M, MacKay DJ, Burrin JM, Bloom SR. Growth hormone release in man induced by galanin, a new hypothalamic peptide. Lancet. 1986;2:192-5. 7. Melander T, Fuxe K, Harstrand A, Enerother P, Hokfelt T. Effect of intraventricular injections of galanin on neuroendocrine functions in the male rat; possible involvement of hypothalamic catecholamine neuronal systems. Acta Physiol Stand. 1987;131: 25-32. 8. Hooi SC, Maiter DM, Martin JB, Koenig JI. Galaninergic mechanisms are involved in the regulation of corticotropin and thyrotropin secretion in the rat. Endocrinology. 1990;127:2281-9. 9. Davis TME, Burrin JM, Bloom SR. Growth hormone (GH) release in response to GH-releasing hormone in man is 3-fold enhanced by galanin. J Clin Endocrinol Metab. 1987;65:1248-52. 10. Ottlecz A, Samson WK, McCann SM. Galanin: evidence for a hypothalamic site of action to release growth hormone. Peptides. 1986;7:51-3. 11. Lawrence AM, Goldfine ID, Kirsteins L. Growth hormone dvnamits in acromegaly. J Clin Endocrinol Metab. 1970;31:239-43: 12. Chiodini PG. Liuzzi A. Botalla L. Cremascoli G. Silvestrini F. Inhibitory effects of dopaminergic ‘stimulation on’ GH release in acromegaly. J Clin Endocrinol Metab. 1974;38:200-2. 13. Faglia G, Beck-Peccoz P, Ferrari C, Travaglini P, Ambrosi B, Spada A. Plasma growth hormone response to thyrotropin-releasing hormone in patients with active acromegaly. J Clin Endocrinol Metab. 1973;36:1259-62. 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. 1974;38:906-9. 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. 1983;286:1686-91. 16. Rokaeus A, Melander T, Hokfelt T, et al. A galanin-like peptide in
ET AL.
JCE&M.1992 Vol74.No6
the central nervous system and intestine of the rat. Neurosci Lett. 1984;47:161-6. 17. Gentleman SM, Falkai P, Bogerts B, Herrero MT, Polak JM, Roberts JW. Distribution of galanin-like immunoreactivity in the human brain. Brain Res. 1989;505:311-5. 18. Loche S. Cella SG, Puggioni R, Stabilini L, Pintor C, Muller EE. The effects of galanin on growth hormone secretion in children of normal and short stature. Pediatr Res. 1989;26:316-9. 19. Loche S, Vista N, Ghigo E, et al. Evidence for involvement of endogenous somatostatin in the galanin-induced growth hormone secretion in children. Pediatr Res. 1990;27:405-7. 20. Giustina A, Girelli A, Doga M, et al. Pyridostigmine blocks the inhibitory effect of glucocorticoids on growth hormone-releasing hormone stimulated growth hormone secretion in normal man. J Clin Endocrinol Metab. 1990;71:580-4. 21. Gabriel SM, Milbury CM, Nathanson JA, Martin JB. Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci. 1988;42:1981-6. 22. Torsello A, Sellan R, Cella SWG, Locatelli V, Muller EE. Agedependent modulation by galanin of growth hormone release from rat pituitary cells in culture. Life Stir 1990;47:1861-6. 23. Meister B, Hultina AL. Influence of coexistinz hvnothalamic messengers on growth hormone secretion from rat anterior pituitary cells in uitro. Neuroendocrinology. 1987;46:387-94. 24. Murakami Y, Kato Y, Shimatsu A, et al. Possible mechanisms involved in growth hormone secretion induced by galanin in the rat. Endocrinology. 198%124:1224-g. 25. Murakami Y, Kato Y, Koshiyama H, Inoue T, Yanaihara N, Imura H. Galanin stimulates growth hormone (GH) secretion via GHreleasing factor (GRF) in conscious rats. Eur J Pharmacol. 1987;136:415-8. 26. Giustina A, Bossoni S, Bodini C, et al. The role of choline@ tone in modulating the growth hormone response to growth hormonereleasing hormone in normal man. Metabolism. 1991;40:519-23. 27. Chatterjee VKK, Ball JA, Proby C, Burrin JM, Bloom SR. Galanin abolishes the inhibitory effect of choline& blockade on growth hormone-releasing hormone-induced secretion of growth hormone in man. J Endocrinol. 1988;116:Rl-2. 28. Massara F, Ghigo E, Goffi S, Molinatti GM, Muller EE, Camanni F. Blockade of hp-GRF 40 induced GH release in normal men by a choline& muscarinic antagonist. J Clin Endocrinol Metab. 1984,59:1025-6. 29. Besser GM, Wass JAH. The medical management of acromegaly. In: Black P, ed. Secretory tumors of the pituitary gland. New York: Raven Press; 1984;155-68. 30. Peillon F, Cesselin F, Bression D, et al. In uitro effect of dopamine and L-dopa on prolactin and growth hormone release from human pituitary adenomas. J Clin Endocrinol Metab. 1979;49:737-41. 31. Liuzzi A, Chiodini PG, Botalla L, Cremascoli G, Silvestrini F. Inhibitory effect of L-dopa on GH release in acromegalic patients. J Clin Endocrinol Metab. 1972:35:941-3. 32. Liuzzi A, Chiodini PG, Botalla L, Cremascoli G, Muller EE, Silvestrini F. Decreased plasma growth hormone (GH) levels in acromegalics following CB 154 (2-Br-alpha-eraocrvntine) administration. J Clin Endocrinol Metab. 1974;38:91&2. -33. Loche S. Pintus S. Cella SG. et al. The effect of ealanin on baseline. and GHRH-induced growth hormone secretioi in obese children. Clin Endocrinol (Oxf). 1990;33:187-92.
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Galanin Decreases in Acromegaly”
Vol. 74, No. 6 Printed in U.S.A.
Circulating
Growth
ANDREA GIUSTINA, CORRADO BODINI, MAURO GIUSEPPE PIZZOCOLO, AND GIANNI GIUSTINA Cattedra di Clinica Medica
and Chimica
(G.P.), University
DOGA,
Hormone
MAURIZIO
Levels
SCHETTINO,
of Bresciu, Italy
ABSTRACT.
Galanin is able to elicit GH secretion in normal man. In acromegaly, circulating GH levels are elevated, and GH secretory dynamics are usually abnormal. The aim of our study was to investigate the effects of galanin on GH secretion in acromegalic subjects. Six acromegalic patients (four males and two females) and seven healthy adult subjects (five males and two females) underwent in randomized order: 1) iv infusion of 100 mL saline from O-45 min, and 2) iv infusion of synthetic porcine galanin (0.5 mg in 100 mL saline) from O-45 min. In normal subjects, peak GH levels after porcine galanin administration (8.2 f 1.9 pg/L) were significantly higher than after saline infusion (1.3 + 0.1 fig/L, P < 0.05). In acromegalic patients, GH values fell from baseline (32.5 + 12 rg/L) to a
G
mean nadir of 24.5 f 12.7 rg/L after galanin infusion. The mean change in GH values from baseline after galanin treatment in these subjects significantly differed from that observed after saline infusion from 15-90 min. Serum PRL levels were not significantly affected by galanin in either normal or acromegalic patients. Our results give the first evidence that the same dose of galanin, acting as a GH secretagogue in normal man, is, on the contrary, able to significantly inhibit GH in acromegalic patients. The cause of this paradoxical GH fall after galanin treatment in acromegaly remains to be explained. It can be hypothesized that galanin may interact at the pituitary level with its own receptors expressed by GH-secreting adenomatous cells. (J Clin Endocrirwl Metab 74: 1296-1300,1992)
ALANIN is a 29-amino acid, straight chain, biologically active peptide, derived from a 123-amino acid precursor protein, preprogalanin (1). Galanin was originally isolated from porcine intestine (2), but galanin-like immunoreactivity is widely distributed in central and peripheral neurons of several mammalian species, including humans (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 is able to influence the secretion of several pituitary hormones in man and rats
tients are usually abnormal (11). These abnormalities include incomplete inhibition or paradoxical elevation of GH after glucose ingestion, a fall in GH after administration of L-dopa, stimulation of GH by TRH or, less commonly, by LHRH, and exaggerated GH response to GHRH (12-15). The precise nature of the defects leading to these abnormalities is not understood. The aim of our study was to investigate the effects of galanin on GH secretion in patients with acromegaly.
(6-8).
Subjects
Galanin probably plays a significant role in the regulation of GH secretion in man. Porcine galanin is able to elicit GH secretion when given alone (6) and to increase the GH response to GH-releasing hormone (GHRH) in normal man (9). The mechanism underlying this action of galanin is unknown; evidence seems to suggest that galanin may act at the hypothalamic level (10). In acromegaly, circulating GH levels are elevated. Moreover, GH secretory dynamics in acromegalic pa-
Four men and two womenwith active acromegaly[meanage, 49.2 + 4.1 yr (range, 35-63 yr); body massindex, 27.2f 1.3 kg/ m2 (range, 24.6-33.3 kg/m2)] were studied (Table 1). Two patients (no. 1 and 2) were untreated. Four patients with GHsecreting pituitary macroadenomahad previously undergone transsphenoidalpituitary surgery, followed in one case(no. 4) by radiotherapy. All but one these patients were submitted to a l-month wash-out period without taking any pharmacological therapy before entering the study. Only patient 6 continued to receive octreotide (Sandostatin, Sandoz, Basel, Switzerland, 450 rg/day in three daily SCinjections), which was the only
Received June 19,199l. Address all correspondence and requests of reprints to: Andrea Giustina, M.D., Clinica Medica c/o 2a Medicina, Spedali Civili, 25125 Brescia, Italy. * This work was supported by the Centro Studie Ricerche di Neuroendocrinologia (Brescia, Italy).
Subjects and Methods
drug
effective
in relieving
his
headache;
in
this
last
case,
the
tests were performed 12 h after the last doseof octreotide. All of the patients had the typical clinical signsof acromegalyand elevatedinsulin-like growth factor-I (IGF-I) levelsfor their age; 1296
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 November 2015. at 15:15 For personal use only. No other uses without permission. . All rights reserved.
GALANIN,
GH, AND
all had elevated baseline GH levels (calculated in at least four samples), i.e. more than 6 rg/L. A 100-g glucose load failed to suppress GH levels below 2.5 rg/L in all cases. A paradoxical GH response to TRH, defined as an increase of at least twice the basal GH level to a value greater than 5 pg/L (13, 14), was seen in three patients (Table 1). Seven healthy adult volunteers (five men and two women) with no family history of endocrine or metabolic diseases [mean age, 37.4 + 7.1 yr (range, 24-76 yr); body mass index, 25 f 1.5 kg/m* (range, 19.4-32.1 kg/m*)] with normal thyroid and adrenal function served as controls. No significant differences in age or body mass index between acromegalic and normal subjects were observed. None of the subjects was taking any drugs at the start of the study, and all subjects were examined on two different occasions with at least 7-day intervals. The study protocol was approved by the local ethical committee. Methods After an overnight fast, each subject was admitted to our Clinical Research Unit. Patients rested in a recumbent position throughout the experiment. Two antecubital venous catheters (for independent infusion and blood sampling) were inserted percutaneously and kept patent by slow saline infusion. After a 30-min stabilization period, the following treatments were initiated in randomized order: 1) iv infusion of 100 mL saline from O-45 min; and 2) iv infusion of synthetic porcine galanin (0.5 mg in 100 mL saline) from O-45 min. Sterile endotoxin-free synthetic porcine galanin (Inalco, Milan, Italy) was reconstituted in 2 mL normal saline. Blood samples for GH, PRL, and glucose assay were taken at 15 min before and 0,15,30,45,60,90, and 120 min after the beginning of the infusion. Pulse rate and supine blood pressure were measured at 0, 20,45, and 60 min during each infusion. Results were expressed as the mean + SEM. GH secretory responses were expressed either as absolute values (micrograms per L), peak or nadir (micrograms per L), or a percentage of the basal level [mean of -15 and 0 min samples; maximum percent GH change]. Absolute and maximum GH change values were compared using the analysis of variance for repeated measures. Peak or nadir GH levels were compared with Student’s t test for paired data. Clinical characteristics of normal TABLE
1. Clinical Patient
characteristics no.
of acromegalic Sex/age
(yr)
ACROMEGALY and acromegalic subjects were compared with one-way analysis of variance. Assays Commercial kits were used for the estimation of GH (immunoradiometric assay; Nichols Institute, San Juan Capistrano, CA; inter- and intraassay coefficients of variation, f 5.4% and &2.3%, respectively; sensitivity limit of the assay, 0.06 fig/L), PRL (immunoradiometric assay; Ares-Serono, Milan, Italy; inter- and intraassay coefficients of variation, f 6.4% and &2.1%, respectively; sensitivity limit of the assay, 0.3 pg/L), and insulin-like growth factor-I (RIA; Nichols Institute; inter- and intraassay coefficients of variation, +5.2% and f 9.4%, respectively; sensitivity limit of the assay, 0.1 U/mL). Blood glucose levels were measured with the glucooxidase method (Beckman II glucose analyzer, Palo Alto, CA). All samples from the same subject were assayed together in duplicate.
Results The kinetics of the GH responses to porcine galanin and saline infusions in normal and acromegalic subjects are shown in Figs. 1 and 2. Normal
subjects
Galanin infusion caused a significant increase in GH levels compared to those after saline infusion. The mean peak GH level after galanin (8.2 + 0.9 pg/L) was significantly higher than the mean GH peak after saline infusion (1.3 f 0.1 pg/L; P < 0.05). Galanin infusion caused in all instances an increase in GH levels. The GH peak occurred between 25-40 min after the beginning of the infusion (Fig. 1). Acromegalic
patients
Baseline GH values were significantly elevated compared to those in normal subjects. In five of the six
patients BMI (k/m2)
Previous therapy
IGF-I W/ml)
GH bg/L) 27.3 6.7 40.0
+
+ + -
1 2 3
F M M
58 49 44
24.6 33.3 26.6
TS
3.5 4.9 6.2
4 5 6
F M M
63 46 35
24.7 25.9 28.2
TS, RT TS TS
2.8 2.3 7.5
20.8 12.5 98.0”
49.2 4.1
27.2 1.3
4.5 0.8
34.2 13.6
Mean +SEM
BMI, Body mass index; TS, transsphenoidal surgery; RT, radiotherapy; IGF-I, as the mean of four samples taken on different days at 0800 h. TRH, Paradoxical the basal GH level to a level above 5 pg/L. 0 IGF-I and GH levels under chronic administration of octreotide (450 pg/day,
TRH
-
insulin-like growth factor-I. IGF-I and GH levels were calculated GH response to TRH, defined as an increase of at least twice SC); samples
were taken
12 h after
the last dose of the drug.
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GIUSTINA
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ET AL.
JCE & M .1992 Vol74.No6
1L
b
hfw
ill-
I)-
3 .
6. z
3
4.
2.
O15
0
15
30
45
v///i///y///I Galanin or saline i.v. infusion
co
90
120
Time
(min)
FIG. 1. Serum GH concentrations (mean + SEM; micrograms per L) after iv infusion of saline (0) or galanin (W) in seven normal subjects. *, P < 0.05 vs. saline.
patients, GH values clearly fell during galanin infusion compared to those during saline treatment (range, 43.964.4% with respect to baseline levels), with nadirs between 30-45 min after the beginning of the infusion (Fig. 2, a and b). In patient 6, only a slight GH decrease after galanin (nadir, 90% of basal levels) was observed. The mean curve for changes in GH values (expressed as a percentage of the mean of -15 and 0 min basal levels) after galanin treatment in the whole group of acromegalic subjects differed from that observed after saline infusion in a significant manner from 15-90 min (Fig. 2~). Serum PRL levels did not show significant variations after galanin compared to baseline levels and those after saline treatment in either normal or acromegalic subjects (Table 2). Neither normal nor acromegalic subjects showed significant fluctuations in blood glucose levels after galanin or saline infusions. All of the subjects experienced a bitter taste in the mouth, which started immediately after the beginning of galanin infusion and ended at the end of the infusion. No 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 is able to cause a clear and significant increase in plasma GH levels in normal subjects can decrease GH values in
0
15
V///////f///f//A
30
45
60
90
120 TIYE(rni”)
FIG. 2. a, Individual serum GH concentrations (micrograms per L) in six acromegalic patients after iv infusion of saline. b, Individual serum GH levels (micrograms per L) in six acromegalic patients after iv infusion of galinin. c, Mean change in GH levels (A GH; +SEM; percentage of basal levels) after iv infusion of saline (0) or galanin (m) in six acromegalic patients. *, P < 0.05 vs. saline.
patients with acromegaly. Galanin is a 29-amino acid peptide that has been detected in the central nervous system of rats (16) and humans (17). Previous studies have shown that porcine galanin, infused in doses similar to those used by us, causes a significant increase in baseline GH levels in both adults (6) and normal children (18, 19). GH secretion in man is controlled by the stimulating influence of GHRH, on the 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 (20). Our understanding of
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GALANIN,
GH, AND
ACROMEGALY
1299
TABLE 2. Mean (+SEM) serum PRL levels (micrograms per L) in six acromegalic patients and seven normal controls after galanin (Gal) or saline (Sal) iv infusion from O-45 min Time (min) Test Patients Gal Sal P Controls Gal Sal P
-15
0
15
30
45
60
90
120
10.9 (2.3) 10.3 (2.2)
11.2 (2.3) 12.5 (2.7)
10.8 (2.6) 12.2 (1.8)
12.3 (2.6) 11.3 (1.9)
12.0 (2.6) 10.8 (2.0)
11.5 (2.7) 10.2 (2.2)
11.0 (2.3) 9.8 (2.4)
10.8 (2.2) 9.3 (2.5)
NS
NS
NS
NS
NS
NS
NS
NS
8.7 (2.7) 9.1 (2.5)
8.3 (2.5) 10.0 (2.9)
7.8 (2.1) 9.9 (2.8)
8.0 (1.9, 9.0 (“.2)
8.8 (2.2) 9.0 (2.0)
9.0 (2.4) 8.6 (1.7)
8.4 (2.5) 7.5 (1.6)
7.8 (2.2) 7.8 (2.0)
NS
NS
NS
NS
NS
NS
NS
NS
Values are the mean, with ~SEM in parentheses.
the mechanism underlying the action of galanin is still fragmentary. Neuroanatomical data provide the basis for a putative pituitary site of action of galanin in affecting GH release in both rats and man (4, 16, 17). However, contrasting in vitro results have been obtained testing the direct effect of galanin on pituitary cells. Either an absent (10) or slight (21) GH-releasing effect of galanin on dispersed rat anterior pituitary cells has been observed. On the contrary, other researchers have shown that galanin is able to inhibit GH release from dispersed anterior pituitary cells of normal adult rats (22, 23). Pretreatment with GHRH antibodies suppresses the plasma GH increase induced by iv and intracerebroventricular infusion of galanin in conscious rats (24, 25). These findings suggest that the stimulating effects of galanin on GH secretion are mediated in the rat by hypothalamic GHRH as a final common pathway. Studies in man suggest that galanin may stimulate GH secretion by inhibition of tonic endogenous somatostatin secretion from the hypothalamus. Davis et al. (9) have reported that the GH response to a maximal GHRH dose (26) is augmented by simultaneous administration of galanin. Moreover, galanin is able to prevent in man the GH inhibitory effect of cholinergic blockade (27), which is postulated to be mediated by hypothalamic somatostatin (28). Our results give the first evidence that the same dose of galanin that results in stimulation of GH secretion in normal man (6) is, on the contrary, able to induce significant GH inhibition in acromegalic patients. The magnitude of the observed GH fall after galanin in five of our acromegalic patients ranged between about 44-64s of baseline levels. However, it is not known whether the dose of galanin used by us is able to induce the maximal GH-inhibiting effect in acromegaly. In only one of the patients studied was the GH-decreasing effect of galanin not clear-cut. It can be hypothesized that in this case the adenomatous cells were partially resistant to the action
of galanin, as reported for other GH-lowering agents (29). On the other hand, we cannot exclude a significant effect of chronic octreotide therapy on the GH response to galanin in this patient. At present, the mechanism by which galanin can decrease GH levels in acromegaly remains to be explained. In keeping with recent in vitro studies (22, 23) it may be hypothesized that galanin as well as dopamine agonist drugs (30) interacting .at the pituitary level with their own receptors expressed by the adenomatous tissue are able to directly inhibit pituitary GH secretion in acromegalic patients. This hypothesis is also supported by the observation that galanin reduces GH levels in acromegalic patients with persistently elevated GH levels after pituitary surgery. Dopaminergic drugs were the only previously known agents able to cause a paradoxical GH fall in acromegaly (31, 32). Thus, the galanin-mediated decrease in GH levels in acromegaly may also be hypothesized to be indirect, via a dopaminergic pathway. However, due to the lack of effect of galanin on PRL levels (6, 20) in both normal and acromegalic patients, this latter hypothesis seems unlikely. Obesity is known to blunt the GH response to galanin in normal subjects (33), and galanin decreases GH release from pituitaries of old rats (23). Our control group was slightly younger and had slightly lower body mass indexes with respect to acromegalic patients. However, these differences did not reach a statistically significant level. In summary, we can hypothesize that in normal subjects the hypothalamic stimulating effects of galanin predominate, whereas in acromegaly, characterized by an at least partial escape of the adenoma from hypothalamic control, the pituitary GH-inhibiting effect of galanin could be evidenced. To elucidate the mechanism of the paradoxical decreasing effect of galanin on plasma GH levels in acromegaly, further in uitro and in uiuo studies on the effect of galanin on the GH response to
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GIUSTINA
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other neuropeptides, necessary.
such as TRH
or GHRH,
will be
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the central nervous system and intestine of the rat. Neurosci Lett. 1984;47:161-6. 17. Gentleman SM, Falkai P, Bogerts B, Herrero MT, Polak JM, Roberts JW. Distribution of galanin-like immunoreactivity in the human brain. Brain Res. 1989;505:311-5. 18. Loche S. Cella SG, Puggioni R, Stabilini L, Pintor C, Muller EE. The effects of galanin on growth hormone secretion in children of normal and short stature. Pediatr Res. 1989;26:316-9. 19. Loche S, Vista N, Ghigo E, et al. Evidence for involvement of endogenous somatostatin in the galanin-induced growth hormone secretion in children. Pediatr Res. 1990;27:405-7. 20. Giustina A, Girelli A, Doga M, et al. Pyridostigmine blocks the inhibitory effect of glucocorticoids on growth hormone-releasing hormone stimulated growth hormone secretion in normal man. J Clin Endocrinol Metab. 1990;71:580-4. 21. Gabriel SM, Milbury CM, Nathanson JA, Martin JB. Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci. 1988;42:1981-6. 22. Torsello A, Sellan R, Cella SWG, Locatelli V, Muller EE. Agedependent modulation by galanin of growth hormone release from rat pituitary cells in culture. Life Stir 1990;47:1861-6. 23. Meister B, Hultina AL. Influence of coexistinz hvnothalamic messengers on growth hormone secretion from rat anterior pituitary cells in uitro. Neuroendocrinology. 1987;46:387-94. 24. Murakami Y, Kato Y, Shimatsu A, et al. Possible mechanisms involved in growth hormone secretion induced by galanin in the rat. Endocrinology. 198%124:1224-g. 25. Murakami Y, Kato Y, Koshiyama H, Inoue T, Yanaihara N, Imura H. Galanin stimulates growth hormone (GH) secretion via GHreleasing factor (GRF) in conscious rats. Eur J Pharmacol. 1987;136:415-8. 26. Giustina A, Bossoni S, Bodini C, et al. The role of choline@ tone in modulating the growth hormone response to growth hormonereleasing hormone in normal man. Metabolism. 1991;40:519-23. 27. Chatterjee VKK, Ball JA, Proby C, Burrin JM, Bloom SR. Galanin abolishes the inhibitory effect of choline& blockade on growth hormone-releasing hormone-induced secretion of growth hormone in man. J Endocrinol. 1988;116:Rl-2. 28. Massara F, Ghigo E, Goffi S, Molinatti GM, Muller EE, Camanni F. Blockade of hp-GRF 40 induced GH release in normal men by a choline& muscarinic antagonist. J Clin Endocrinol Metab. 1984,59:1025-6. 29. Besser GM, Wass JAH. The medical management of acromegaly. In: Black P, ed. Secretory tumors of the pituitary gland. New York: Raven Press; 1984;155-68. 30. Peillon F, Cesselin F, Bression D, et al. In uitro effect of dopamine and L-dopa on prolactin and growth hormone release from human pituitary adenomas. J Clin Endocrinol Metab. 1979;49:737-41. 31. Liuzzi A, Chiodini PG, Botalla L, Cremascoli G, Silvestrini F. Inhibitory effect of L-dopa on GH release in acromegalic patients. J Clin Endocrinol Metab. 1972:35:941-3. 32. Liuzzi A, Chiodini PG, Botalla L, Cremascoli G, Muller EE, Silvestrini F. Decreased plasma growth hormone (GH) levels in acromegalics following CB 154 (2-Br-alpha-eraocrvntine) administration. J Clin Endocrinol Metab. 1974;38:91&2. -33. Loche S. Pintus S. Cella SG. et al. The effect of ealanin on baseline. and GHRH-induced growth hormone secretioi in obese children. Clin Endocrinol (Oxf). 1990;33:187-92.
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