Effect of Galanin on Growth Hormone-Releasing Hormone-Stimulated Growth Hormone Secretion in Adult Patients With Nonendocrine Diseases on Long-Term Daily Glucocorticoid Treatment Andrea Giustina, Angela Girelli, Simonetta Bossoni, Fabio Legati, Maurizio Schettino, and William B. Wehrenberg Glucocorticoids are thought to inhibit growth hormone (GH) secretion through an enhancement of endogenous somatostatin tone. The aim of our study was to evaluate the effect of galanin, a neuropeptide that stimulates GH secretion, on GH-releasing hormone (GHRHj-induced GH secretion in adult patients with nonendocrine diseases who were under daily immunosuppressive glucocorticoid therapy. Six normal subjects (four men, two women) and seven steroid-treated subjects (three men, four women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (IV) infusion of saline or porcine galanin (12.5 pg/min). During saline infusion, steroid-treated patients showed a blunted GH response to GHRH (GH peak, 8.1 2 2.8 pg/L), as compared with normal subjects (GH peak, 23.8 f 3.9 ug/L). During galanin infusion, the GH response to GHRH was significantly enhanced (GH peak, 46.8 + 9.4 pg/L, P < .05), as compared with saline infusion in normal subjects. In contrast, galanin infusion did not enhance the GH response to GHRH (GH peak, 16.6 f 6.5 pg/L), as compared with saline infusion in steroid-treated patients. The area under the GH-response curves was also significantly (P < .05) lower in steroid-treated subjects, as compared with normal subjects. Thus, galanin failed to normalize or enhance the GH response to GHRH in patients treated long-term with glucocorticoids. It can be hypothesized that galanin does not elicit GH secretion by decreasing hypothalamic somatostatin tone. Copyright 0 1992 by W.B. Saunders Company

G fere with normal somatic growth in laboratory animals’ and in man. Children receiving high-dose glucocortiLUCOCORTICOIDS

have long been known to inter-

coid treatment* or who have Gushing’s disease3 exhibit impaired growth. It has been hypothesized that glucocorticoids may reduce insulin-like growth factor 1 (IGF-1) levels and activity.4,5 On the other hand, normal subjects, after administration of supraphysiological doses of glucocorticoids, and patients with Gushing’s disease, have blunted growth hormone (GH) responses to the stimuli.6.9 Finally, glucocorticoids enhance GH release by cultured, human pituitary somatotropes. lo Moreover, GH deficiency in patients with idiopathic corticotropin deficiency resolves during glucocorticoid replacement therapy.” GH synthesis and secretion are regulated by the hypothalamic peptides, GH-releasing hormone (GHRH), which has an excitatory role, and somatostatin, which has an inhibitory role.12 The in vivo inhibitory effects of glucocorticoids on GH secretion are hypothesized to be due to the enhancement of hypothalamic somatostatin release, both in the rat and in man.r3.14 Galanin is a 29-amino acid peptide originally isolated from porcine intestine. Is Galanin is thought to be a neurotransmitter in the central nervous system of several mammalian species, including humans.i6J7 Synthetic porcine galanin elicits GH secretion when given alone,18 and enhances

From the Clinica Medica, University of Brescia, Italy; and the Department of Health Sciences, University of Wisconsin, Milwaukee, WI. Supported by the “Centro Studi e Ricerche di Neuroendocrinologia, ” Brescia, Italy, and by National Institutes of Health Grants No. 5 ROl DK38324 and 5 K04 DK01874. Address reprint requests to Andrea Giustina, MD, Cattedra di Clinica Medica, Universita’di Brescia clo 2a Medicina-Spedali Civili, 25125 Brescia, Italy. Copyright 0 1992 by W.B. Saunders Company 0026-0495/92/4105-0019$03.OOlO

548

the GH response to GHRH in normal human subjects.19 The mechanism of this stimulatory action of galanin is unclear. Experimental studies suggest that the action of galanin may be mediated either by a decrease in hypothalamic somatostatin secretion,19-21 an increase in endogenous GHRH secretion,22 or by a direct stimulatory effect at the pituitary level.r3,24 The aim of our study was to analyze the GH response to GHRH alone or combined with a galanin infusion in adult patients receiving daily immunosuppressive glucocorticoid treatment for nonendocrine diseases. SUBJECTS AND METHODS Subjects We studied seven adult patients undergoing long-term immunosuppressive glucocorticoid treatment for nonendocrine diseases. The group was composed of two men and five women. The mean age was 38.7 ? 4.5 (mean rt SEM) years. The clinical characteristics of the patients are reported in Table 1. At the time of the study, all patients were medically stable with normal liver and kidney function. Their body mass index was less than 28 kg/m,2 and their blood glucose levels were in the normal range. Prednisone (orally. twice daily) was the only drug the patients received. Doses and durations are reported in Table 1. Six normal, adult, non-obese volunteers (four men and two women) with no history of endocrine or metabolic diseases served as controls. They were matched for age (mean 35 + 3.5 years) with the steroid-treated subjects. Methods The study was performed according to a single-blind, cross-over design. Galanin and saline were infused in random order. After fasting overnight, each subject spent two mornings in the Clinical Research Unit. Subjects rested in a recumbent position, with an antecubital vein catheter inserted percutaneously. After a 30.minute stabilization period, the steroid-treated patients received their oral morning dose of prednisone. Infusions were then initiated from -10 minutes to 30 minutes in random order: (1) infusion of synthetic porcine galanin (Inalco, Milano, Italy), IV,

Mefabolism, Vol41, No 5 (May). 1992: pp 548-551

GLUCOCORTICOIDS,

Table 1. Clinical Characteristics

of Seven Adult Subjects Receiving Prednisone Treatment (orally, twice daily) for Nonendocrine BMI

Subject NO.

549

GALANIN, AND GH SECRETION

AgelSex

(kg/m’)

Prednisone Diagncws

1

55/F

28

Systemic

2

49iM

27

Polymialgia

3

33/F

26

Systemic

4

44lF

28

Scleroderma

5

23/F

19

Mixed connective

6

41/M

24

Systemic

7

29/F

23.5

Churg-Strauss

Mean t SEM

38.7

+

4.51-

lmglkgld)

lupus erythematosus

of Iv4

0.10

lupus erythematosus

0.58 0.16

tissue disease

lupus erythematosus

0.45 0.17 0.29

disease

0.26 2 0.07

3.1 t 0.9

RESULTS

of the GH responses to GHRH + saline and GHRH + galanin are illustrated in Fig 1. Initial GH levels were not significantly different between the groups. Galanin did not significantly increase basal GH levels either in glucocorticoid-treated or normal subjects, as determined by no change in GH concentration between -10 and 0 minutes. Glucocorticoid-treated patients showed blunted GH responses to GHRH (GH peak, 8.1 ? 2.8 t~,g/L) (Fig lB) during saline infusion, as compared with normal controls (GH peak, 23.8 t 3.9 kg/L; P < .05) (Fig 1A). During galanin infusion, a significantly enhanced GH response to GHRH, as compared with saline infusion, was observed in control subjects (GH peak, 46.6 + 9.4 up/L; P < .05) (Fig lA), but not in glucocorticoid-treated patients (GH peak, 16.6 t 6.5 Kg/L) (Fig 1B). Mean absolute GH levels at times 15, 30. 45, and 60 minutes (Fig 1) and GH,,,, (Fig 2) were significantly lower both after saline and after galanin in glucocorticoid-treated patients, as compared with normal controls. All subjects experienced facial flushing after the GHRH injection and a bitter taste in their mouths during the galanin infusion. The kinetics

/l SSllYS A>commercial immunoradiometric kit was used for estimation GH (Allegro hGH, Nichols Institute. San Juan Capistrano, interassay and intraassay coefficients of variation were *5.40/o 2,3%, respectively; sensitivity limit of the assay, 0.02 ug/L). All samples for an individual subject were measured in one assay, all samples were measured in duplicate.

Duration Therapy

0.10

rheumatica

25.1 + 1.2

500 ug in 100 mL saline, or (2) infusion of saline, IV, 100 mL. A bolus of human GHRH (l-29)NHz (Geref. Serono, Italy), 100 ug in 1 mL saline. was injected IV at 0 minutes. Blood samples for GH assay were taken at -10 (time of initiation of saline or galanin infusion), 0 (time of GHRH injection), 15, 30.45. 60, 90, and 120 minutes. The two study mornings were divided by intervals of at least 7 days. Prednisone treatment aas unchanged during the study. The GH secretory responses to GHRH + saline and GHRH + galanin were expressed either as absolute values (ug/L), as peak values (ug/L), or as area under the cutve (AUC; kg/L x 120 minutes) calculated by trapezoidal integration. The results are expressed as the mean 2 SEM. GH absolute values after GHRH + saline and GHRH + galanin were compared within each group and between groups using the ANOVA for repeated measures. Statistical comparison of GH,,, and GH peaks after GHRH + saline and GHRH + galanin was performed with the one-way ANOVA.

Diseases

of CA: and GH and

GH

GHRH t

Fig 1. (A) Mean + SEM GH levels (gg/L): (Cl) after GHRH + saline; (A) after GHRH + galanin in six normal adult subjects. (B) Mean + SEM GH levels (pg/L): (Wj after GHRH + saline; (A) after GHRH + galanin in seven adult subjects receiving glucocorticoid therapy (orally, twice daily). lP < .05 Y GHRH + saline; “P < .05 v steroid-treated patients.

550

GIUSTINA ET AL

GH AUC (pg/LxlXtmin) 4000

3000

2000

1000

0

NORMALS

STEROID-n?.

RMAIS

-D-lu.

GHRH

GHRH

+SAL

+GAL

Fig 2. Mean f SEM GH,uc AUCs, pg/L x 120 minutes, after GHRH + saline (SAL) and after GHRH + galanin (GAL) in six normal adult subjects (open symbols) and seven adult subjects receiving glucocorticoid therapy orally twice daily (filled symbols). lP < 35 v GHRH + saline; “P c .05 v steroid-treated patients.

DISCUSSION

Our data demonstrate that the GH response to GHRH is inhibited in glucocorticoid-treated adult subjects, compared with normal subjects. Our data also confirm that galanin significantly enhances the GH response to GHRH in normal subjects. However, galanin infusion does not normalize or increase the GH response to GHRH in glucocorticoid-treated patients. In the normal rat, dexamethasone inhibits the GH response to GHRH.13 Normal human subjects, after shortterm and long-term exogenous glucocorticoid administration, and patients with Cushing’s disease show impaired GH responses to arginine, insulin, and GHRH.6-9 On the other hand, glucocorticoids have a direct stimulatory effect on human somatotropes in vitro.lO Moreover, hypoadrenalism causes, in vivo, a decrease in the GH response to the stimuli.11,25,26Recently, it has been shown in normal subjects that glucocorticoids do not acutely inhibit the GH response to GHRH after pretreatment with pyridostigmine,14 an acetylcholinesterase inhibitor that increases cholinergic tone and is thought to decrease hypothalamic somatostatin release.27,28 Moreover, administration of somatostatin antibodies to rats treated with dexamethasone improves growthz9 and normalizes the GH response to GHRH.i3 Therefore, it is hypothesized that pharmacological doses of glucocorticoids increase hypothalamic somatostatin secretion, while at the same time, they enhance the somatotrope response to GHRH. This dual action is obscured, since it appears that enhanced somatostatin secretion predominates enhanced somatotrope function.13,l‘W One of the most prominent metabolic effects in man of systemic administration of the neuropeptide galanin, in doses similar to those used in the present study, is an increase of GH secretion.18 Our understanding of the mechanism behind this action of galanin is still developing. Porcine galanin enhances the GH response to a maximal

GHRH dose in normal subjects.19v2iTherefore, it has been suggested that galanin may act via a decrease in hypothalamic somatostatin.19 On the other hand, pretreatment with rat GHRH antiserum markedly attenuates the GH response to galanin in the rat. 31Moreover, galanin stimulates immunoreactive GHRH secretion from perifused rat hypothalamic slices.** Finally, galanin is reported either to have no effects on GH secretion32 or to stimulate GH and augment GHRH-induced GH secretion in rat somatotrope cultures.23,24 Thus, it can be hypothesized that galanin is released together with GHRH and, acting on the GHRH receptor at the pituitary level or through a positive feedback mechanism on GHRH neurons, is able to enhance both GHRH action and secretion.22 Our data show that the GH response to GHRH is impaired in adult subjects with nonendocrine diseases who are receiving daily glucocorticoid treatment.2+6J3,34Our data also confirm that galanin enhances the GH response to GHRH in normal subjects. 19,21On the other hand, galanin is ineffective both in normalizing or increasing the GH response to GHRH in glucocorticoid-treated patients. Long-term and short-term administration of pharmacological doses of glucocorticoids are thought to decrease GH secretion through an increase in hypothalamic somatostatin tone.*3J4q29,30 That galanin cannot reverse this effect further suggests that the mechanism of action of galanin is not via a decrease in somatostatin release by the hypothalamus. The GHRH-stimulating activity of galanin may be made ineffective in the presence of a glucocorticoid-mediated increase in hypothalamic somatostatin tone.35 However, our data cannot rule out that glucocorticoids counteract the direct stimulatory action of galanin at the pituitary leve1.23.24 In the present study, both men and women were examined. In rats, females have higher galanin-like immunoreactivity concentrations in the brain and pituitary, compared with males.36 Moreover, in man, sex-based differences in the GH response to GHRH have been described.37 Due to the limited number of subjects examined, no significant differences in the GH response to galanin or in glucocorticoid-galanin interactions between males and females could have been detected in our study. However, in the group of glucocorticoid-treated patients, there was a prevalence of women, while the majority of normal subjects were men. Therefore, we cannot exclude that galanin may be less effective in eliciting GH secretion and in removing the glucocorticoid-mediated GH inhibition in females as compared with males. Finally, patients receiving long-term glucocorticoid treatment were studied in the present work; therefore, the effects of short-term glucocorticoid administrationsJ4x3*on the galanin/GHRH interaction remain to be clarified. In summary, long-term administration of pharmacological amounts of glucocorticoids inhibits the GH response to GHRH. Galanin administration significantly enhances GHRH-stimulated GH secretion in normal controls, but not in glucocorticoid-treated subjects. Thus, we hypothesize that the stimulatory action of galanin on GH secretion in man may not be mediated by a decrease of somatostatin release by the hypothalamus.

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AND GH SECRETION

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21. 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 27:405-407. 1990 22. 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 23. Gabriel SM, Milbury CM, Nathanson JA, et al: Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sci 42:1981-1986, 1988 24. Sato M, Takahara J, Niimi M, et al: Characterization of the stimulatory effect of galanin on growth hormone release from the rat anterior pituitary. Life Sci 48:1639-1644,199l 25. Wehrenberg WB, Baird A, Ling N: Potent interaction between glucocorticoids and growth hormone-releasing Factor in vivo. Science 221:556-558, 1983 26. Giustina A, Romanelli G. Bossoni S, et al: Effects of short-term glucocorticoid deprivation on growth hormone (GH) response to GH-releasing hormone in man. Horm Metab Res 21:693-700, 1989 27. Giustina A, Bodini C. Bossoni S, et al: Effects of calcitonin on GH response to pyridostigmine in combination with hGHRH (l-29)NHz in normal adult subjects. Clin Endocrinol (Oxf) 33:375380,199O 28. Locatelli V, Torsello A, Redaelli M, et al: Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone-releasing hormone in the rat. Evidence for mediation by somatostatin. J Endocrinol 111:271-278. 1986 29. Wehrenberg WB, Bergman PJ, Stagg L, et al: Glucocorticoid inhibition of growth in rats: Partial reversal with somatostatin antibodies. Endocrinology 127:2705-2708.1990 30. Nagakawa K, Ishizuka T, Obara T, et al: Dichotomic action of glucocorticoids on growth hormone secretion. Acta Endocrinol (Copenh) 116:165-171, 1987 31. Murakami Y, Kato Y, Shimatsu A, et al: Possible mechanisms involved in growth hormone secretion induced by galanin in the rat. Endocrinology 124:1224-1229. 1989 32. Ottlecz A, Samson WK, McCann SM: Galanin: Evidence for a hypothalamic site of action to release growth hormone. Peptides 7:51-53, 1986 33. Daly JR, Fletcher MR, Glass D, et al: Comparison of the effects of long-term corticotropin and corticosteroid treatment on responses of plasma growth hormone, ACTH, and corticosteroids to hypoglycaemia. Br Med J 2:521-524, 1974 34. Giustina A, Alberti D, Girelli A, et al: Effects of pyridostigmine on nocturnal and growth hormone (GH)-releasing hormone stimulated GH secretion in children on daily glucocorticoid therapy after liver transplantation. Program of 73rd Annual Meeting of the Endocrine Society, Washington, DC. June 19-22. 1991, p 87 35. 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. 1991 36. Gabriel SM. Kaplan LM, Martin JB, et al: Tissue-specific sex differences in galanin-like immunoreactivity and galanin mRNA during development in the rat. Peptides 10:369-374. 1989 37. Lang I, Schernthaner G, Pietschmann P, et al: Effects of sex and age on growth hormone response to growth hormone-releasing hormone in healthy individuals. J Clin Endocrinol Metab 65:535540. 1987 38. Lal S, Nair NPV, Isaac I, et al: Effect of hydrocortisone on basal and apomorphine-induced growth hormone secretion in normal subjects. Clin Invest Med 11:218-223. 1988