Interactions E. Ghigo,
of Galanin
M. Maccario,
and Arginine on Growth Hormone, Insulin Secretion in Man
E. At-vat, M.R. Valetto,
F. Valente,
M. Nicolosi,
E. Mazza,
Prolactin, V. Martina,
and
D. Cocchi,
and F. Camanni Galanin (GAL), a 29 amino acid neuropeptide, is known to increase both basal and growth hormone-releasing hormone (GHRH)-induced growth hormone (GH) secretion while not significantly increasing prolactin (PRL) secretion in man. GAL is also endowed with an inhibiting effect on glucose-stimulated insulin release in animals, but not in man. We studied the effect of GAL (80 pmol/kg/min infused over 60 minutes) on the arginine- (ARG, 30 g infused over 30 minutes) stimulated GH, PRL, insulin, and C-peptide secretion in eight healthy volunteers (age, 20 to 30 years). GAL induced an increase of GH (GAL v saline, area under curve [AUC], mean 2 SEM: 316.5 -+ 73.9 v 93.2 + 20.9 pg/L/ h, P < .05), but failed to modify both PRL and insulin secretion. GAL enhanced the ARG-induced stimulation of both GH (1.634.1 ? 293.1 v 566.9 ? 144.0 pg/L/h, P < .02) and PRL secretion (1.541.9 ? 248.8 v 1.023.8 a 158.7 kg/L/h, P < .02). On the contrary, GAL blunted the ARG-stimulated insulin (816.3 f 87.7 v 1,322.7 f 240.9 mu/L/h, P < .05), as well as C-peptide secretion (105.1 ? 9.8 v 132.8 2 17.3 pg/L/h, P < .02). ARG administration induced a transient increase of glucose levels (P < .Ol v baseline) followed by a significant decrease (P < .05 v baseline). This latter effect was prevented by the coadministration of GAL. In conclusion, these results show that in man GAL potentiates the GH response to ARG, suggesting that these drugs act at the hypothalamic level, at least in part, via different mechanisms. The coadministration of GAL and ARG induces a marked increase of PRL secretion, although the mechanisms underlying this effect are unclear. The blunting effect of GAL on the ARG-induced insulin and C-peptide secretion suggests that this neiripeptide may also modulate p-cell secretion in man. Copyright 0 1992 by W. B. Saunders Company
I
T IS WELL KNOWN that arginine
(ARG) is able to induce an increase of growth hormone (GH), prolactin (PRL), and insulin levels in man.‘” On the other hand, it has been shown in man that galanin (GAL), a recently discovered 29 amino acid neuropeptideJ” stimulates GH secretion, but does not significantly increase PRL secretion.’ GAL is also endowed with an inhibiting effect on stimulated insulin release in animals,s-‘3 but not in rnan.‘14 Although many studies have recently been performed, the mechanisms underlying the effects of both ARG and GAL on GH, PRL, and insulin secretion are still unclear. To throw further light onto these mechanisms, we studied the interactions of GAL and ARG on GH, PRL, insulin and C-peptide secretion in normal adult volunteers.
(Sorin) for determination of PRL. The sensitivity of the assay was 0.5 kg/L. The interassay and intraassay coefficients of variation were 6.5% and 2.5%, respectively. (3) INSIK-5 (Sorin) for determination of insulin. The sensitivity of the assay was 2.5 2 0.27 mu/L. The interassay and intraassay coefficients of variation were between 6.2% and 10.8% and between 5.5% and 10.6%, respectively. (4) C-Peptide kit (Ares-Serono) for determination of C-peptide. The sensitivity of the assay was 0.1 p,g/L. The interassay and intraassay coefficients of variation were between 8.7% and 11.6% and between 3.9% and 7.6%, respectively. Plasma glucose was measured by a glucose-oxidase method (Beckman II glucose analyzer, Beckman Instruments, Palo Alto, CA). All responses were expressed either as absolute values or as areas under curve (AUC), calculated by trapezoidal integration. Results were expressed as mean f SEM. Statistical analysis was performed using the Wilcoxon signed-rank test.
SUBJECTS AND METHODS Eight healthy female volunteers
(age, 25 to 32 years), were studied in the follicular phase of the menstrual cycle. All gave their informed consent to the study. All subjects underwent four tests in random order and at least 2 to 3 days apart: (1) ARG (arginine chlorydrate, Damor, Naples, Italy; 30 g in 100 mL infused over 30 minutes from 0 to +30 minutes); (2) GAL (Bachem, Bubendorf, Switzerland, 80 pmol/kg/ min infused over 60 minutes from 0 to +60 minutes); (3) ARG + GAL; (4) 0.9% saline infusion from 0 to +60 minutes. All tests were begun between 8:30 and 9:00 AM after an overnight fast and 30 minutes after cannulation of two cubital veins, kept patent by slow infusion of 0.9% saline. Blood samples were taken basally at - 15 and 0 minutes and then at 5,15,30,45,60,75,90,105, and 120 minutes. At each time, serum GH, PRL, insulin, C-peptide, and plasma glucose were assayed in duplicate. All samples from an individual subject were analyzed together. Serum GH, PRL, insulin, and plasma C-peptide were measured by immunoradiometric assay using reagents provided by Sorin, Saluggia, Italy or Ares-Serono, Milan, Italy. The following kits were used: (1) HGH-CTK-IRMA (Sorin) for determination of GH. The sensitivity of the assay was 0.03 kg/L. The interassay and intraassay coefficients of variation were between 2.3% and 5.5% and between 1.9% and 3.9%, respectively. (2) PROL-CTK-IRMA Metabolism, Vol41,
No 1
(January), 1992: pp 85-89
RESULTS
Growth Hormone
Basal GH levels did not significantly vary in different tests. When administered alone, ARG (AUC, 566.9 2 144.0 hg/L/h) and GAL (316.5 2 73.9 kg/L/h) induced a similar GH increase, which was higher than that which occurred during saline infusion (93.3 2 20.9 kg/L/h). The GH response to coadministration of ARG and GAL (1,634.l + 293.0 kg/L/h) was significantly higher (P < .02) than that after ARG or GAL alone, and even higher (P < .02) than that obtained by the sum of the GH responses to ARG and GAL, thus representing a true potentiating effect (Fig 1).
From the Division of Endocrinology, Department of Clinical Pathophysiology, University of Turin; and the Biophannacology Depatiment, University of Bari, Italy. Address reprint requests to Professor F. Camanni, Division of Emiocrinology, Ospedale Molinette, Corso Polonia 14, 10126 Torino, Italy Copyright 0 1992 by W. B. Saunders Company 0026-0495/92/4101-0015$03.00/O
85
GHIGO ET AL
86
Prolactin Basal PRL levels did not significantly vary in different tests. ARG administration induced a significant increase of PRL secretion with respect to saline (LO23.8 ? 158.7 v 566.0 2 98.5 kg/L/h, P < .02), while GAL administration failed to do so (713.9 -t 98.0 kg/L/h). Coadministration of ARG and GAL induced a marked increase of PRL secretion (1541.9 2 248.8 pg/L/h), which was significantly higher (P < .02) than that observed during infusion of ARG or GAL alone (Fig 2). Insulin Basal insulin levels did not significantly vary in different tests. ARG administration induced a marked increase of insulin secretion with respect to saline (1,322.7 + 240.9 v 472.5 + 60.9 mu/L/h, P < .02), while GAL administration failed to do so (476.6 of-85.9 mu/L/h). Insulin response to ARG was significantly blunted (P < .05) by GAL coadministration (816.3 t 87.7 mu/L/h) (Fig 3A). C-Peptide Basal C-peptide levels did not significantly vary in different tests. ARG administration induced a marked increase of C-peptide secretion with respect to saline (132.8 * 17.3 v 57.7 ? 7.4 kg/L/h, P < .02), while GAL administration failed to do so (64.8 * 6.3 pg/L/h). C-peptide response to ARG was significantly blunted by GAL coadministration (105.1 ? 9.8 kg/L/h, P < .02) (Fig 3B). Glucose Plasma glucose levels were not modified during saline or GAL infusion. Arginine induced a transient increase of plasma glucose levels at 15 and 30 minutes (P < .Ol v baseline), which was not affected by GAL coadministration. The increase in plasma glucose after ARG administration was followed by a significant decrease (P < .05 at 75
minutes v baseline), tration (Fig 4).
which was blunted
by GAL coadminis-
Side Effects GAL induced a bitter taste in all subjects. were observed after arginine administration.
No side effects
DISCUSSION
The present results show that in man, GAL, a 29 amino acid neuropeptide, clearly enhances both GH and PRL responses to ARG. Moreover, our data demonstrate that GAL is able to blunt the ARG-induced insulin and C-peptide release. The mechanisms underlying the effects of GAL and ARG on hormonal secretion are still unclear. Concerning GH secretion, there is evidence favoring the hypothesis that GAL positively influences somatotrope secretion by acting at the hypothalamic level both increasing growth hormone-releasing hormone (GHRH) and reducing somatostatin (SS) release. In fact, GAL: (1) does not stimulate GH secretion by acting directly at the pituitary level,“~” although this has not been uniformly found”‘; (2) induces a clear-cut GH increase, which in rat is prevented by both anti-GHRH serum and inhibitors of hypothalamic norepinephrine and epinephrine biosynthesis,‘h-‘x thus suggesting that its GH-releasing effect is being mediated by adrenergic receptors causing GHRH stimulation”; (3) enhances the GH response to maximal doses of GHRH in man22.23and counteracts the inhibitory effect of both chohnergic antagonistsz4.” and glucose on GH secretion,‘4 thus suggesting that the GH-releasing effect of GAL is, at least partially, mediated by inhibition of endogenous SS release. In agreement with the latter hypothesis, an inhibitory effect of GAL on SS release has been shown in animals6 On the other hand, there is evidence suggesting that the GH-releasing effect of ARG is likely mediated by inhibition of SS release.‘6~z” In fact, ARG fails to influence both basal
Saline Galanin Arginine m A Arginine + Galanin M
t -15
11 0
11
11
15 30 45
60
Time
1 75
( min )
‘I
90 105120
Fig 1. Mean + SEM serum GH response ewvas and AUCr after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
ENDOCRINE INTERACTIONS
67
OF GALANIN AND ARGININE
0
Saline Galanin = Arginine : A Arginine + Galanin E
01
Fig 2. Mean k SEM serum PRL response curves and AUCs after saline, GAL, ARG, and ARG plus GAL infusion in eight normal sub-
1 -15
’
’
0
15
’
’
8
c
’
’
1
30 45 60 75 90105120 Time (min)
jects.
60. 0
Saline
i
n
Galrnin
a
0
Arginine
UIIlIrI
A Arginine + Galanin m
2 5 _ E 4o
140
I
120
-100 -80
r 3 c)
3
0
= %
-60
$
- A0
OL i -
15
I-
’
’
1
0
15
30
j
45
60
1
75
lrme (mm)
(
90
105 120
z -
Fig 3. Mean + SEM serum insulin (A) and C-peptide (8) response curves and AUCs after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
GHIGO ET AL
80
0
saline
l 9ahin 0 Arginino ~Afdnim+Wn
I -15
0
15
I
I
30
45
1 60 75 rim0 ( min )
, 90
105
120
Fig 4. Mean k SEM plasma glucose response curves after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
and GHRH-stimulated GH release from rat anterior pituitaries in vitro,26 while in man this amino acid potentiates the GH response to maximal doses of GHRHz6.” even reinstating it when inhibited by a previous GHRH administration.29 Our demonstration that combined administration of GAL and ARG has a potentiating effect on GH secretion suggests that these compounds, in man, positively influence GH secretion at least partially by acting via different mechanisms, ie, stimulation of GHRH and inhibition of SS, respectively. This hypothesis seems to disagree with our previous data3’ showing that pyridostigmine, a cholinesterase inhibitor acting via suppression of hypothalamic SS,” failed to modify both GAL- and ARG-induced GH secretion. Based on those findings, we suggested that all these drugs may act via a common mechanism, ie, by an inhibition of hypothalamic somatostatinergic tone, and thus one would expect no additive effect of pyridostigmine with GAL or ARG coadministration. This discrepancy might be due to different kinds of subjects studied, normal adults (present data) or children3’ considering an age-dependent different mode of neuroendocrine controL3* On the other hand, it has to be considered that there are data showing an
inhibitory effect of GAL on acetylcholine release in the central nervous system.“” Based on this latter evidence, one would assume that in our previous experiments pyridostigmine failed to modify GH secretion induced by GAL because acetylcholine release had been blocked by this neuropeptide. Considering PRL secretion, the mechanisms by which both GAL and ARG influence the release of this hormone are even less clear. A significant PRL-releasing effect of GAL was previously shown in animals.‘s~2” but not in man.’ In the present study, a significant increase of PRL secretion was not found after administration of GAL, despite the high dose used, while the neuropeptide greatly increased the ARG-induced PRL response. The positive influence of GAL on PRL secretion may be presumed to be exerted directly on lactotropes.“.3’.3h At this level, the presence of specific receptors has been shown in animals.3s~“” Moreover, anterior pituitary cells synthesize and secrete GAL, which may act by an autocrine mechanism amplified by estrogens.~‘,“h,‘X.‘” The effect of GAL on PRL secretion may also be related to changes in the hypothalamic balance of releasing and inhibiting factors. In fact. GAL has an inhibitory effect on dopamine release at the median eminence.” Moreover, passive immunization with specific anti-vasoactive intestinal peptide (VIP) rabbit serum suppresses the plasma PRL response to GAL in rats.j’ To our knowledge, there are no data in the literature to explain the PRL-releasing effect of ARG and, therefore. the real meaning of its interaction with GAL. Further studies are needed to evaluate this problem. Concerning insulin secretion, a striking inhibitory effect of GAL on both basal and stimulated p-cell secretion has been widely shown in animals both in vitro and in viva.‘.‘” On the other hand, GAL failed to modify serum insulin and plasma glucose responses to intravenous glucose tolerance tests in man.” thus ruling out the possibility of a major role of GAL in regulating insulin secretion in humans. Our present data showing the blunting effect of GAL on the ARG-induced insulin and C-peptide release suggest that even in man GAL may negatively modulate p-cell secretion, in agreement with the finding of specificGAL receptors in the islets.h.4’-44A direct effect on f3 cells has also been shown for ARG.“.” Interestingly, the negative modulatory effect of GAL on insulin secretion seems to be confirmed by the finding that this neuropeptide blunted the ARG-induced plasma glucose decrease.
REFERENCES 1. Merimee TJ, Lillicrap DA, Rabinowitz D: Effect of arginine on serum levels of human growth hormone. Lancet 2:668-670,1965 2. Fajans SS, Floyd JC, Knopf RF, et al: A difference in mechanism by which leucine and other amino acids induce insulin release. J Clin Endocrinol Metab 27:1600-1606,1967 3. Delitala G, Frulio T, Pacific0 A, et al: Participation of cholinergic muscarinic receptors in glucagon and arginine mediated growth hormone secretion in man. J Clin Endocrinol Metab 55:1231-1233,1982 4. Tatemoto K, Rokaeus A, Jornval H, et al: Galanin, a novel biologically active peptide from porcine intestine. FEBS Lctt 164:124-128,1983
5. 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 6. Dunning BE, Ahren B, Veith RC, et al: Galanin: A novel pancreatic neuropeptide. Am J Physiol251:E127-E133, 1986 7. Bauer FE, Ginsberg L, Venetikou M, et al: Growth hormone release in man induced by galanin, a new hypothalamic peptide. Lancet 2:192-196,1986 8. McDonald TJ, Dupre J, Greenberg GR. et al: The effect of galanin on canine plasma glucose and gastroenteropancreatic hormone responses to oral nutrients and intravenous arginine. Endocrinology 119:2340-2345, 1986
ENDOCRINE INTERACTIONS
OF GALANIN AND ARGININE
9. Lindskog S, Ahren B: Galanin: Effects on basal and stimulated insulin and glucagon secretion in the mouse. Acta Physiol Stand 129:305-309,1987 10. Silvestre RA, Miralles P, Monge L, et al: Effects of galanin on hormone secretion from the in situ perfused rat pancreas and on glucose production in rat hepatocytes in vitro. Endocrinology 121:378-383, 1987 11. Hermansen K: Effects of galanin on the release of insulin, glucagon and somatostatin from the isolated, perfused dog pancreas. Acta Endocrinol 119:91-98, 1988 12. Greenberg GR, McDonald TJ: Effect of galanin and vagal integrity on insulin release in dogs. Pancreas 3:122-127,1988 13. Tajiri Y, Sako Y, Umeda F, et al: Effect of galanin on arginine-stimulated pancreatic hormone release from isolated perfused rat islets. Horm Metab Res 22:1-6, 1990 14. Gilbey SG, Stephenson J, O’Halloran DJ, et al: High-dose porcine galanin infusion and effect on intravenous glucose tolerance in humans. Diabetes 38:1114-1116,1989 15. Ottlecz A, Snyder GD, McCann SM: Regulatory role of galanin in control of hypothalamic-anterior pituitary function. Proc Nat1 Acad Sci USA 85:9861-98651988 16. Murakami Y, Kato Y, Koshiyama H, et al: Galanin stimulates growth hormone (GH) secretion via GH-releasing factor (GRF) in conscious rats. Eur J Pharrnacol136:415-418,1987 17. Maiter DM, Hooi SG, Koenig JI, et al: Galanin is a physiological regulator of spontaneous pulsatile secretion of growth hormone in the male rat. Endocrinology 126:1216-1222,199O 18. Cella SG, Locatelli V, De Gennaro V, et al: Epinephrine mediates the growth hormone-releasing effect of galanin in infant rats. Endocrinology 122:855-859,1988 19. Sato M, Takahara J, Nijmi M, et al: Direct effect of galanin on pituitary GH release via cyclic AMP-independent mechanism. 72nd Annual Meeting of the Endocrine Society, Atlanta, GA, 1990 (abstr 54) 20. Gabriel SM, Milbury CM, Nathanson JA, et al: Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci 42:1981-1986,1988 21. Muller EE: Neural control of somatotropic function. Physiol Rev 67:962-1053,1987 22. 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 65:1248-1252, 1987 23. Loche S, Vista A, Ghigo E, et al: Evidence for involvement of endogenous somatostatin in the galanin-induced growth hormone secretion in children. Pediatr Res 27:405-407, 1990 24. Chatterjee VKK, Ball JA, Davis TME, et al: The effect of cholinergic blockade on the growth hormone response to galanin in humans. Metabolism 37:1089-1091,1988 25. Chatterjee VKK, Ball JA, Proby C, et al: Galanin abolishes the inhibitory effect of cholinergic blockade on growth hormonereleasing hormone-induced secretion of growth hormone in man. J Endocrinol 116:Rl-R2, 1988 26. Alba-Roth J, Miiller AO, Schopohl J, et al: Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J Clin Endocrinol Metab 51:781-788, 1988 27. 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 Endocrinol32:763-767, 1990 28. Page MD, Dieguez C, Valcavi R, et al: Growth hormone (GH) response to arginine and L-Dopa alone and after GHRH pretreatment. Clin Endocrinol28:551-558,1988 29. Arvat E, Valente F, Maccario M, et al: Arginine reinstates
89
the growth hormone responsiveness to intermittent administration of growth hormone-releasing hormone in man. J Endocrinol Invest 13:145,1990 (suppl 1) 30. Evain-Brion D, Donnadieu M, Liapi C, et al: Plasma growth hormone releasing factor levels in children: Physiologically and pharmacologically induced variations. Horm Res 24:116-121, 1986 31. Ghigo E, Bellone J, Imperiale E, et al: Pyridostigmine potentiates t-Dopa, but not arginine-and galanin-induced growth hormone secretion in children. Neuroendocrinology 52:42-45,199O 32. Ghigo E, Goffi S, Mazza E, et al: Repeated GH-releasing hormone administration unravels different GH secretory patterns in normal adults and children. Acta Endocrinol 120;598-601, 1989 33. Fisone G, Wu F, Consolo S, et al: Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: Histochemical, autoradiographic and in vitro studies. Proc Nat1 Acad Sci USA 84:7339-7343,1987 34. Koshiyama H, Kato Y, Inoue T, et al: Central galanin stimulates pituitary prolactin secretion in rats: Possible involvement of hypothalamic vasoactive intestinal polypeptide. Neurosci Lett 75:49-54, 1987 35. Vrontakis M, Friesen HG: Galanin and estrogen regulated hormone of the anterior pituitary gland, in Casanueva FF, Dieguez C (eds): Recent Advances in Basic and Clinical Neuroendocrinology. Amsterdam, The Netherlands, Elsevier, 1989, pp 25-33 36. O’Halloran DJ, Jones PM, Steel JH, et al: Effect of endocrine manipulation on anterior pituitary galanin in the rat. Endocrinology 127:467-475,199O 37. Kaplan LM, Spindel ER, Isselbacher KJ, et al: Tissuespecific expression of the rat galanin gene. Proc Nat1 Acad Sci USA 83:6287-6291,1988 38. Kaplan LM, Gabriel SM, Koenig JI, et al: Galanin is an oestrogen inducible secretory product of the rat anterior pituitary. Proc Nat1 Acad Sci USA 85:7408-7412,1988 39. Vrontakis M, Yamamoto T, Schroedter I, et al: Estrogen induction of galanin synthesis in the rat anterior-pituitary gland demonstrated by in situ hybridization and immunohistochemistry. Neurosci Lett 100:59-64, 1989 40. Vrontakis ME, Sano T, Kovacs K, et al: Presence of galanin-like immunoreactivity in non-tumorous corticotrophs and corticotroph adenoma of the human pituitary. Clin Res 37:364a, 1989 (abstr) 41. Nordstrom 0, Melander T, Hokfelt T, et al: Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett 73:21-26,1987 42. Ahren B, Arkhammar P, Berggren PO, et al: Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmatic free Car+ concentration. Biochem Biophys Res Commun 140:1059-1063,1986 43. Arniranoff B, Servin AL, Rouyer-Fessard C, et al: Galanin receptors in a hamster pancreatic beta-cell tumor: Identification and molecular characterization. Endocrinology 121:284-289,1987 44. Lagny-Pourmir I, Amiranoff B, Lorinet AM, et al: Characterization of galanin receptors in the insulin-secreting cell line Rin m SF: Evidence for coupling with a pertussis toxin-sensitive guanosine triphosphate regulatory protein. Endocrinology 124:26352641,1989 45. Charles S, Tamagawa T, Henquin JC: A single mechanism for the stimulation of insulin release and “6Rb’ efflux from rat islets by cationic aminoacids. Biochem J 208:301-308,1982 46. Blachier F, Mourtada A, Jener A, et al: Stimulus-secretion coupling of arginine-induced insulin release uptake of methabolized and nonmethabolized cationic amino acids by pancreatic islets. Endocrinology 124:134-141, 1989
of Galanin
M. Maccario,
and Arginine on Growth Hormone, Insulin Secretion in Man
E. At-vat, M.R. Valetto,
F. Valente,
M. Nicolosi,
E. Mazza,
Prolactin, V. Martina,
and
D. Cocchi,
and F. Camanni Galanin (GAL), a 29 amino acid neuropeptide, is known to increase both basal and growth hormone-releasing hormone (GHRH)-induced growth hormone (GH) secretion while not significantly increasing prolactin (PRL) secretion in man. GAL is also endowed with an inhibiting effect on glucose-stimulated insulin release in animals, but not in man. We studied the effect of GAL (80 pmol/kg/min infused over 60 minutes) on the arginine- (ARG, 30 g infused over 30 minutes) stimulated GH, PRL, insulin, and C-peptide secretion in eight healthy volunteers (age, 20 to 30 years). GAL induced an increase of GH (GAL v saline, area under curve [AUC], mean 2 SEM: 316.5 -+ 73.9 v 93.2 + 20.9 pg/L/ h, P < .05), but failed to modify both PRL and insulin secretion. GAL enhanced the ARG-induced stimulation of both GH (1.634.1 ? 293.1 v 566.9 ? 144.0 pg/L/h, P < .02) and PRL secretion (1.541.9 ? 248.8 v 1.023.8 a 158.7 kg/L/h, P < .02). On the contrary, GAL blunted the ARG-stimulated insulin (816.3 f 87.7 v 1,322.7 f 240.9 mu/L/h, P < .05), as well as C-peptide secretion (105.1 ? 9.8 v 132.8 2 17.3 pg/L/h, P < .02). ARG administration induced a transient increase of glucose levels (P < .Ol v baseline) followed by a significant decrease (P < .05 v baseline). This latter effect was prevented by the coadministration of GAL. In conclusion, these results show that in man GAL potentiates the GH response to ARG, suggesting that these drugs act at the hypothalamic level, at least in part, via different mechanisms. The coadministration of GAL and ARG induces a marked increase of PRL secretion, although the mechanisms underlying this effect are unclear. The blunting effect of GAL on the ARG-induced insulin and C-peptide secretion suggests that this neiripeptide may also modulate p-cell secretion in man. Copyright 0 1992 by W. B. Saunders Company
I
T IS WELL KNOWN that arginine
(ARG) is able to induce an increase of growth hormone (GH), prolactin (PRL), and insulin levels in man.‘” On the other hand, it has been shown in man that galanin (GAL), a recently discovered 29 amino acid neuropeptideJ” stimulates GH secretion, but does not significantly increase PRL secretion.’ GAL is also endowed with an inhibiting effect on stimulated insulin release in animals,s-‘3 but not in rnan.‘14 Although many studies have recently been performed, the mechanisms underlying the effects of both ARG and GAL on GH, PRL, and insulin secretion are still unclear. To throw further light onto these mechanisms, we studied the interactions of GAL and ARG on GH, PRL, insulin and C-peptide secretion in normal adult volunteers.
(Sorin) for determination of PRL. The sensitivity of the assay was 0.5 kg/L. The interassay and intraassay coefficients of variation were 6.5% and 2.5%, respectively. (3) INSIK-5 (Sorin) for determination of insulin. The sensitivity of the assay was 2.5 2 0.27 mu/L. The interassay and intraassay coefficients of variation were between 6.2% and 10.8% and between 5.5% and 10.6%, respectively. (4) C-Peptide kit (Ares-Serono) for determination of C-peptide. The sensitivity of the assay was 0.1 p,g/L. The interassay and intraassay coefficients of variation were between 8.7% and 11.6% and between 3.9% and 7.6%, respectively. Plasma glucose was measured by a glucose-oxidase method (Beckman II glucose analyzer, Beckman Instruments, Palo Alto, CA). All responses were expressed either as absolute values or as areas under curve (AUC), calculated by trapezoidal integration. Results were expressed as mean f SEM. Statistical analysis was performed using the Wilcoxon signed-rank test.
SUBJECTS AND METHODS Eight healthy female volunteers
(age, 25 to 32 years), were studied in the follicular phase of the menstrual cycle. All gave their informed consent to the study. All subjects underwent four tests in random order and at least 2 to 3 days apart: (1) ARG (arginine chlorydrate, Damor, Naples, Italy; 30 g in 100 mL infused over 30 minutes from 0 to +30 minutes); (2) GAL (Bachem, Bubendorf, Switzerland, 80 pmol/kg/ min infused over 60 minutes from 0 to +60 minutes); (3) ARG + GAL; (4) 0.9% saline infusion from 0 to +60 minutes. All tests were begun between 8:30 and 9:00 AM after an overnight fast and 30 minutes after cannulation of two cubital veins, kept patent by slow infusion of 0.9% saline. Blood samples were taken basally at - 15 and 0 minutes and then at 5,15,30,45,60,75,90,105, and 120 minutes. At each time, serum GH, PRL, insulin, C-peptide, and plasma glucose were assayed in duplicate. All samples from an individual subject were analyzed together. Serum GH, PRL, insulin, and plasma C-peptide were measured by immunoradiometric assay using reagents provided by Sorin, Saluggia, Italy or Ares-Serono, Milan, Italy. The following kits were used: (1) HGH-CTK-IRMA (Sorin) for determination of GH. The sensitivity of the assay was 0.03 kg/L. The interassay and intraassay coefficients of variation were between 2.3% and 5.5% and between 1.9% and 3.9%, respectively. (2) PROL-CTK-IRMA Metabolism, Vol41,
No 1
(January), 1992: pp 85-89
RESULTS
Growth Hormone
Basal GH levels did not significantly vary in different tests. When administered alone, ARG (AUC, 566.9 2 144.0 hg/L/h) and GAL (316.5 2 73.9 kg/L/h) induced a similar GH increase, which was higher than that which occurred during saline infusion (93.3 2 20.9 kg/L/h). The GH response to coadministration of ARG and GAL (1,634.l + 293.0 kg/L/h) was significantly higher (P < .02) than that after ARG or GAL alone, and even higher (P < .02) than that obtained by the sum of the GH responses to ARG and GAL, thus representing a true potentiating effect (Fig 1).
From the Division of Endocrinology, Department of Clinical Pathophysiology, University of Turin; and the Biophannacology Depatiment, University of Bari, Italy. Address reprint requests to Professor F. Camanni, Division of Emiocrinology, Ospedale Molinette, Corso Polonia 14, 10126 Torino, Italy Copyright 0 1992 by W. B. Saunders Company 0026-0495/92/4101-0015$03.00/O
85
GHIGO ET AL
86
Prolactin Basal PRL levels did not significantly vary in different tests. ARG administration induced a significant increase of PRL secretion with respect to saline (LO23.8 ? 158.7 v 566.0 2 98.5 kg/L/h, P < .02), while GAL administration failed to do so (713.9 -t 98.0 kg/L/h). Coadministration of ARG and GAL induced a marked increase of PRL secretion (1541.9 2 248.8 pg/L/h), which was significantly higher (P < .02) than that observed during infusion of ARG or GAL alone (Fig 2). Insulin Basal insulin levels did not significantly vary in different tests. ARG administration induced a marked increase of insulin secretion with respect to saline (1,322.7 + 240.9 v 472.5 + 60.9 mu/L/h, P < .02), while GAL administration failed to do so (476.6 of-85.9 mu/L/h). Insulin response to ARG was significantly blunted (P < .05) by GAL coadministration (816.3 t 87.7 mu/L/h) (Fig 3A). C-Peptide Basal C-peptide levels did not significantly vary in different tests. ARG administration induced a marked increase of C-peptide secretion with respect to saline (132.8 * 17.3 v 57.7 ? 7.4 kg/L/h, P < .02), while GAL administration failed to do so (64.8 * 6.3 pg/L/h). C-peptide response to ARG was significantly blunted by GAL coadministration (105.1 ? 9.8 kg/L/h, P < .02) (Fig 3B). Glucose Plasma glucose levels were not modified during saline or GAL infusion. Arginine induced a transient increase of plasma glucose levels at 15 and 30 minutes (P < .Ol v baseline), which was not affected by GAL coadministration. The increase in plasma glucose after ARG administration was followed by a significant decrease (P < .05 at 75
minutes v baseline), tration (Fig 4).
which was blunted
by GAL coadminis-
Side Effects GAL induced a bitter taste in all subjects. were observed after arginine administration.
No side effects
DISCUSSION
The present results show that in man, GAL, a 29 amino acid neuropeptide, clearly enhances both GH and PRL responses to ARG. Moreover, our data demonstrate that GAL is able to blunt the ARG-induced insulin and C-peptide release. The mechanisms underlying the effects of GAL and ARG on hormonal secretion are still unclear. Concerning GH secretion, there is evidence favoring the hypothesis that GAL positively influences somatotrope secretion by acting at the hypothalamic level both increasing growth hormone-releasing hormone (GHRH) and reducing somatostatin (SS) release. In fact, GAL: (1) does not stimulate GH secretion by acting directly at the pituitary level,“~” although this has not been uniformly found”‘; (2) induces a clear-cut GH increase, which in rat is prevented by both anti-GHRH serum and inhibitors of hypothalamic norepinephrine and epinephrine biosynthesis,‘h-‘x thus suggesting that its GH-releasing effect is being mediated by adrenergic receptors causing GHRH stimulation”; (3) enhances the GH response to maximal doses of GHRH in man22.23and counteracts the inhibitory effect of both chohnergic antagonistsz4.” and glucose on GH secretion,‘4 thus suggesting that the GH-releasing effect of GAL is, at least partially, mediated by inhibition of endogenous SS release. In agreement with the latter hypothesis, an inhibitory effect of GAL on SS release has been shown in animals6 On the other hand, there is evidence suggesting that the GH-releasing effect of ARG is likely mediated by inhibition of SS release.‘6~z” In fact, ARG fails to influence both basal
Saline Galanin Arginine m A Arginine + Galanin M
t -15
11 0
11
11
15 30 45
60
Time
1 75
( min )
‘I
90 105120
Fig 1. Mean + SEM serum GH response ewvas and AUCr after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
ENDOCRINE INTERACTIONS
67
OF GALANIN AND ARGININE
0
Saline Galanin = Arginine : A Arginine + Galanin E
01
Fig 2. Mean k SEM serum PRL response curves and AUCs after saline, GAL, ARG, and ARG plus GAL infusion in eight normal sub-
1 -15
’
’
0
15
’
’
8
c
’
’
1
30 45 60 75 90105120 Time (min)
jects.
60. 0
Saline
i
n
Galrnin
a
0
Arginine
UIIlIrI
A Arginine + Galanin m
2 5 _ E 4o
140
I
120
-100 -80
r 3 c)
3
0
= %
-60
$
- A0
OL i -
15
I-
’
’
1
0
15
30
j
45
60
1
75
lrme (mm)
(
90
105 120
z -
Fig 3. Mean + SEM serum insulin (A) and C-peptide (8) response curves and AUCs after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
GHIGO ET AL
80
0
saline
l 9ahin 0 Arginino ~Afdnim+Wn
I -15
0
15
I
I
30
45
1 60 75 rim0 ( min )
, 90
105
120
Fig 4. Mean k SEM plasma glucose response curves after saline, GAL, ARG, and ARG plus GAL infusion in eight normal subjects.
and GHRH-stimulated GH release from rat anterior pituitaries in vitro,26 while in man this amino acid potentiates the GH response to maximal doses of GHRHz6.” even reinstating it when inhibited by a previous GHRH administration.29 Our demonstration that combined administration of GAL and ARG has a potentiating effect on GH secretion suggests that these compounds, in man, positively influence GH secretion at least partially by acting via different mechanisms, ie, stimulation of GHRH and inhibition of SS, respectively. This hypothesis seems to disagree with our previous data3’ showing that pyridostigmine, a cholinesterase inhibitor acting via suppression of hypothalamic SS,” failed to modify both GAL- and ARG-induced GH secretion. Based on those findings, we suggested that all these drugs may act via a common mechanism, ie, by an inhibition of hypothalamic somatostatinergic tone, and thus one would expect no additive effect of pyridostigmine with GAL or ARG coadministration. This discrepancy might be due to different kinds of subjects studied, normal adults (present data) or children3’ considering an age-dependent different mode of neuroendocrine controL3* On the other hand, it has to be considered that there are data showing an
inhibitory effect of GAL on acetylcholine release in the central nervous system.“” Based on this latter evidence, one would assume that in our previous experiments pyridostigmine failed to modify GH secretion induced by GAL because acetylcholine release had been blocked by this neuropeptide. Considering PRL secretion, the mechanisms by which both GAL and ARG influence the release of this hormone are even less clear. A significant PRL-releasing effect of GAL was previously shown in animals.‘s~2” but not in man.’ In the present study, a significant increase of PRL secretion was not found after administration of GAL, despite the high dose used, while the neuropeptide greatly increased the ARG-induced PRL response. The positive influence of GAL on PRL secretion may be presumed to be exerted directly on lactotropes.“.3’.3h At this level, the presence of specific receptors has been shown in animals.3s~“” Moreover, anterior pituitary cells synthesize and secrete GAL, which may act by an autocrine mechanism amplified by estrogens.~‘,“h,‘X.‘” The effect of GAL on PRL secretion may also be related to changes in the hypothalamic balance of releasing and inhibiting factors. In fact. GAL has an inhibitory effect on dopamine release at the median eminence.” Moreover, passive immunization with specific anti-vasoactive intestinal peptide (VIP) rabbit serum suppresses the plasma PRL response to GAL in rats.j’ To our knowledge, there are no data in the literature to explain the PRL-releasing effect of ARG and, therefore. the real meaning of its interaction with GAL. Further studies are needed to evaluate this problem. Concerning insulin secretion, a striking inhibitory effect of GAL on both basal and stimulated p-cell secretion has been widely shown in animals both in vitro and in viva.‘.‘” On the other hand, GAL failed to modify serum insulin and plasma glucose responses to intravenous glucose tolerance tests in man.” thus ruling out the possibility of a major role of GAL in regulating insulin secretion in humans. Our present data showing the blunting effect of GAL on the ARG-induced insulin and C-peptide release suggest that even in man GAL may negatively modulate p-cell secretion, in agreement with the finding of specificGAL receptors in the islets.h.4’-44A direct effect on f3 cells has also been shown for ARG.“.” Interestingly, the negative modulatory effect of GAL on insulin secretion seems to be confirmed by the finding that this neuropeptide blunted the ARG-induced plasma glucose decrease.
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