European Journal of Endocrinology (2000) 143 99±104 ISSN 0804-4643

CLINICAL STUDY Interaction between glucagon and human corticotropin- releasing hormone or vasopressin on ACTH and cortisol secretion in humans

Emanuela Arvat, Barbara Maccagno, Jose®na Ramunni, Mauro Maccario, Roberta Giordano, Fabio Broglio, Franco Camanni and Ezio Ghigo Division of Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy (Correspondence should be addressed to E Ghigo, Divisione di Endocrinologia, Ospedale Molinette, Corso Dogliotti 14, 10126 Torino, Italy; Email: [email protected])

Abstract Objective: It is known that glucagon administration elicits ACTH and cortisol responses in humans, although this effect takes place after intramuscular or subcutaneous but not after the intravenous route of administration. The mechanisms underlying this stimulatory effect on corticotroph secretion are unknown but they are unrelated to glucose variations and stress-mediated actions. Design and Methods: To throw further light on the stimulatory effect of i.m. glucagon on the pituitary± adrenal axis, using six normalyoung female volunteers (26±32 years, body mass index 19.7±22.5 kg/m2) we studied the interaction between glucagon (GLU; 0.017 mg/kg i.m.) and human corticotropin- releasing hormone (hCRH; 2.0 mg/kg i.v.) or vasopressin (AVP; 0.17 U/kg i.m.). The interactions between hCRH and AVP on the hypothalamo±pituitary±adrenal (HPA) axis and the GH response to GLU alone or combined with hCRH or AVP were also studied. Results: GLU i.m. administration elicited a clear increase in ACTH (peak vs baseline, means 6 S.E.M.: 11.6 6 3.3 vs 4.2 6 0.3 pmol/l, P < 0.05), cortisol (613.5 6 65.6 vs 436.9 6 19.3 nmol/l, P < 0.05) and GH levels (11.6 6 3.4 vs 3.3 6 0.7 mg/l, P < 0.05). The ACTH response to GLU (area under the curve: 426.4 6 80.9 pmol/l per 120 min) was higher than that to AVP (206.3 6 38.8 pmol/l per 120 min, P < 0.02) and that to hCRH (299.8 6 39.8 pmol/l per 120 min) although this latter difference did not attain statistical signi®cance. The GLU-induced cortisol response (28 336.9 6 2430.7 nmol/l per 120 min) was similar to those after hCRH (24 099.2 6 2075.2 nmol/l per 120 min) and AVP (21 808.7 6 1948.2 nmol/l per 120 min). GLU and hCRH had an additive effect on ACTH (964.9 6 106.6 pmol/l per 120 min, P < 0.02) and a less than additive effect on cortisol levels (35 542.5 6 2720.2 nmol/l per 120 min). Similarly, GLU and AVP had an additive effect on ACTH (825.6 6 139.6 pmol/l per 120 min, P < 0.02) and an effect less than additive on cortisol levels (33 059.2 6 1965.3 nmol/l per 120 min). The effects of GLU co-administered with hCRH or AVP were similar to those of the combined administration of hCRH and AVP on ACTH (906.0 6 152.7 pmol/l per 120 min) and cortisol (34 383.5 6 1669.2 nmol/l per 120 min) levels. The GH response to GLU was not modi®ed by hCRH or AVP. Conclusions: These results show that i.m. glucagon administration is a provocative stimulus of ACTH and cortisol secretion, at least as potent as hCRH and AVP.The ACTH-releasing effect of i.m. glucagon is not mediated by selective CRH or AVP stimulation but the possibility that both neurohormones play a role could be hypothesized.

European Journal of Endocrinology 143 99±104

Introduction insulin-induced hypoglycemia for the diagnosis of secondary adrenal insuf®ciency (4, 5, 7). It has been known for many years that intramuscular or Several reports have shown that glucagon stimulates subcutaneous administration of glucagon induces a somatotroph and corticotroph secretion after intramus- clear increase in growth hormone (GH) and cortisol cular or subcutaneous but not after acute intravenous levels in humans (1±8). In fact, glucagon is considered administration (9±12), suggesting that glucagon per se a classical provocative stimulus of GH secretion for is not a GH and an adrenocorticotropin (ACTH) the diagnosis of GH de®ciency (2±5, 8) and has secretagogue. It has been hypothesized that intra- been proposed by some authors as an alternative to muscular glucagon proteolysis could generate a peptidyl q 2000 Society of the European Journal of Endocrinology Online version via http://www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access 100 E Arvat and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 fragment endowed with GH- and ACTH-releasing 90 min); (d) GLU (at 0 min)+hCRH (at 90 min); (e) GLU activity (10, 11). This could represent a new factor (at 0 min†‡AVP (at 90 min); (f) hCRH (at 90 min†‡AVP involved in the control of anterior pituitary function. (at 90 min) preceded by placebo (at 0 min). The mechanisms underlying the GH-releasing effect The tests started between 0730 and 0800 h after an of glucagon could involve the inhibition of hypothala- overnight fast and 30 min after venous cannulation mic somatostatin release, while a GH-releasing hor- which was kept patent by slow infusion of isotonic mone (GHRH)-mediated action or a direct pituitary saline. Blood samples for ACTH, cortisol, GH and effect seem to be unlikely (10, 12). On the other hand, a glucose measurements were taken at 30-min intervals role of glucagon-induced free fatty acids (13), but not from 0 to 90 min and at 15-min intervals from 90 to glycemic (1, 4, 10, 11, 14) variations have also been 210 min. All samples from an individual subject were suggested. analyzed at the same time. The mechanisms underlying the stimulatory effect of Plasma ACTH levels were measured in duplicate by glucagon on the hypothalamo±pituitary±adrenal (HPA) immunoradiometric assay (Allegro HS-ACTH; Nichols axis are even less clear. Until now, it has been known Institute Diagnostic, San Juan Capistrano, CA, USA). that the stimulatory effect on cortisol secretion is The sensitivity of the assay was 0.22 pmol/l. The inter- dependent on the ACTH response which follows i.m. and intra-assay coef®cients of variation ranged from 6.9 glucagon administration (4). Moreover, the HPA to 8.9% and from 1.1 to 3.0% respectively. response to i.m. glucagon is unlikely to be related to Serum cortisol levels were measured in duplicate by glucose variations and stress-mediated actions (1, 4, 10, radioimmunoassay (CORT-CTK 125 RIA; Sorin, Salug- 11, 14). Moreover, glucagon does not act at the gia, Italy). The sensitivity of the assay was 11.0 nmol/l. pituitary level (10). The inter- and intra-assay coef®cients of variation In order to clarify the ACTH-releasing activity of ranged from 6.6 to 7.5% and from 3.8 to 6.6% i.m. glucagon in humans we studied its interaction respectively. with human corticotropin-releasing hormone (hCRH) Serum GH levels were measured in duplicate by and arginine-vasopressin (AVP), two hypophysiotropic immunoradiometric assay (hGH-CTK IRMA; Sorin, neurohormones which play a major role in the neural Seluggia, Italy). The sensitivity of the assay was 0.15 control of the HPA axis (15). These results were mg/l. The inter- and intra-assay coef®cients of variation compared with those observed after the combined were 2.9±4.5% and 2.4±4.0% respectively. administration of hCRH and AVP. As CRH has been Plasma glucose levels (nmol/l) were measured reported to play an inhibitory role on somatotroph by gluco-oxidase colorimetric method (Menarini secretion (16±20), we also veri®ed the effect of hCRH or Diagnostic, Florence, Italy). AVP on the glucagon-induced GH rise. Hormonal responses (means 6 S.E.M.) are expressed as absolute levels and areas under response curve (AUC; from 90 to 210 min). Subjects and methods The statistical analysis was carried out using a non- parametric ANOVA (Freedman test) and then with the Peptides and drugs Wilcoxon test. Vials containing 1 mg glucagon (GLU) were purchased from Novo Nordisk (Bagsvaeu, Denmark), vials contain- ing 100 mg lyophilized hCRH were purchased from Results Ferring (Copenhagen, Denmark) and vials containing Administration of GLU i.m. elicited a signi®cant increase 10 IU AVP were purchased from Parke-Davis (New in the levels of ACTH (peak vs baseline, at 165 min: York, USA). 11.6 6 3.3 vs 4.2 6 0.3 pmol/l, P < 0.05), cortisol (peak at 180 min: 613.5 6 65.6 vs 436.9 6 19.3 nmol/l, P < 0.05) and GH (peak at 150 min: 11.6 6 3.4 vs Study design 3.3 6 0.7 mg/l, P < 0.05) (Figs 1 and 2). Six normal young women (age 26±32 years, body mass The ACTH response to GLU (AUC: 426.4 6 80.9 pmol/l index 19.7±22.5 kg/m2) were studied in their early per 120 min) was higher than that to AVP follicular phase. The study had been approved by the (206.3 6 38.8 pmol/l per 120 min, P < 0.02) but not independent Ethical Committee of the University of signi®cantly different from that to hCRH (299.8 6 Turin and informed consent was obtained from all 39.8 pmol/l per 120 min). On the other hand, the GLU- subjects. induced cortisol response (28 336.9 6 2430.7 nmol/l per All subjects underwent the following treatments, at 120 min) was similar to those after hCRH (24 099.2 6 least 3 days apart. (a) i.m. GLU (0.017 mg/kg as a bolus 2075.2 nmol/l per 120 min) and AVP (21 808.7 6 at 0 min) followed by i.v. placebo (1.0 ml saline at 1948.2 nmol/l per 120 min) (Figs 1 and 3). 90 min); (b) i.m. placebo (at 0 min) followed by i.v. GLU and hCRH had additive effects on ACTH hCRH (2.0 mg/kg as a bolus at 90 min); (c) i.m. placebo (964.9 6 106.6 pmol/l per 120 min, P < 0.02) and (at 0 min) followed by i.m. AVP (0.17 IU/kg as a bolus at less than additive effects on cortisol levels (35 542.0 6 www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 Glucagon and HPA axis in humans 101

Figure 1 Mean 6 S.E.M. ACTH and cortisol responses to i.m. glucagon, hCRH or AVP in six normal subjects.

2720.2 nmol/l per 120 min). Similarly, GLU and AVP single stimulation) and an additive effect on cortisol had additive effects on ACTH (825.6 6 139.6 pmol/l per (34 383.5 6 1669.2 nmol/l per 120 min, P < 0.05 vs 120 min, P < 0.02 vs the hormonal responses to the the hormonal responses to the single stimulation) levels single stimulation) and an effect less than additive on (Fig. 3). cortisol levels (33 059.2 6 1965.3 nmol/l per 120 min) The GH response to GLU (479.1 6 115.7 mg/l (Fig. 3). per 120 min) was not signi®cantly modi®ed by hCRH The ACTH- and cortisol-releasing effect of the co- (377.9 6 61.7 mg/l per 120 min) or AVP (425.3 6 administration of GLU and hCRH or AVP overlapped 157.6 mg/l per 120 min) (Fig. 2). that of the combined administration of hCRH and AVP GLU administration induced biphasic variations in which showed, however, a synergistic effect on ACTH blood glucose levels: a signi®cant increase was recorded (906.0 6 152.7 pmol/l per 120 min, P < 0.05 vs the at 30 min (5.9 6 0.2 vs 4.1 6 0.14 nmol/l, P < 0.05) arithmetic sum of the hormonal responses to the followed by a decrease at 180 min (3.2 6 0.2 nmol/l,

Figure 2 Mean 6 S.E.M. GH responses to i.m. GLU alone or combined with hCRH or AVP in six normal subjects.

www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access 102 E Arvat and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143

Figure 3 Mean 6 S.E.M. ACTH and cortisol AUCs after i.m. glucagon and/or hCRH and/or AVP in six normal subjects.

P < 0.05). The GLU-induced blood glucose variations It is well known that i.m. glucagon administration is were not modi®ed by hCRH or AVP co-administration. followed by a clear increase in ACTH and cortisol secretion (4, 5). In fact, testing with glucagon is considered a reliable alternative to insulin-induced Side-effects hypoglycemia for the diagnosis of secondary adrenal Mild and transient nausea was recorded in four subjects insuf®ciency (4, 5, 7). Our present data showing that after the intramuscular administration of GLU. Facial i.m. glucagon alone represents a stimulus at least as ¯ushing was induced by hCRH in all subjects. Nausea potent as hCRH and stronger than AVP further and vasoconstriction was observed in all subjects while indicates its potency as a provocative stimulus of contraction of diuresis was recorded in two subjects ACTH and cortisol reserve. after AVP administration. Co-administration of the The mechanisms underlying the stimulatory effect of various drugs slightly increased the side-effects observed glucagon on the HPAaxis are still unknown. It is worthy after single drug administration but neither stopping of note that this stimulatory effect follows the intra- the testing nor medication were required. muscular or subcutaneous but not the acute intra- venous administration of glucagon, suggesting that this hormone per se is not a true ACTH secretagogue (9±12). Discussion It has been hypothesized that intramuscular glucagon The results of the present study demonstrated that: (a) proteolysis could generate a peptidyl fragment endowed the intramuscular administration of glucagon releases with ACTH-releasing activity (11). ACTH and cortisol to the same extent as hCRH and The stimulatory effect of i.m. glucagon on the HPA more than AVP; (b) the co-administration of i.m. axis is not likely to be explained by stress-related glucagon and hCRH or AVP has an additive effect on mechanisms due to unpleasant side-effects, such as ACTH release so that these corticotroph responses nausea and vomiting. In fact, side-effects are generally became similar to that recorded after the synergistical negligible after intramuscular administration in com- effect of hCRH and AVP; and (c) the GH response which parison with those recorded after intravenous injection, follows the intramuscular administration of glucagon is which had no effect on the HPA axis (11). Moreover, not modi®ed by hCRH or AVP. though catecholamines play a well-known stimulatory www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 Glucagon and HPA axis in humans 103 role on the HPA axis (21), glucagon administration In conclusion, the results of the present study, though induces catecholamine release after both intravenous indirect, emphasize the stimulatory effect of intra- and intramuscular administration (22, 23), but only muscular glucagon administration on the HPA axis the latter is followed by hormonal release (11). and indicate that it is not mediated by selective CRH or The positive effect of i.m. glucagon on the HPA axis AVP stimulation. does not seem to be due to metabolic mechanisms. The stimulatory effect of absolute and even relative hypo- glycemia on ACTH and cortisol secretion is well known Acknowledgements (21, 24). Indeed, glucagon administration is followed by This study was supported by grants from the University a prompt increase in blood glucose levels and later of Turin and the SMEM Foundation. The authors wish relative hypoglycemia but these effects overlap after to thank Dr A Bertagna, Mrs A Barberis and M Taliano both intravenous and intramuscular administration for their skilful technical assistance. (10,11); this evidence makes it unlikely that the ACTH and cortisol increase which follow i.m. glucagon administration re¯ects the response of the HPA axis to References relative hypoglycemia. 1 Mitchell ML, Byrne M J & Silver J. Growth-hormone release by It is widely accepted that CRH and AVP play the glucagon. Lancet 1969 i 289±290. major neurohormonal role in the control of the HPA 2 Cain JP, Williams GH & Dluhy RG. Glucagon stimulation of axis (15, 21). Our aim was to clarify the possibility that human growth hormone. Journal of Clinical Endocrinology and Metabolism 1970 31 222±224. the stimulatory effect of i.m. glucagon on corticotroph 3 Wieland RG, Hallberg MC & Zorn EM. Growth hormone response secretion could be due to a selective mediation by CRH to intramuscular glucagon. Journal of Clinical Endocrinology and or AVP, which showed the well-known synergistical Metabolism 1973 37 329±330. effect on corticotroph release (15). We found that the 4 Spathis GS, Bloom SR, Jeffcoate WJ, Millar JG, Kurtz A, Pyasena MR et al. Subcutaneous glucagon as a test of the ability of the administration of either hCRH or AVP with i.m. pituitary to secrete GH and ACTH. Clinical Endocrinology 1974 3 glucagon had only additive effects on ACTH secretion 175±186. and this evidence makes the hypothesis that the 5 Rao RH & Spathis GS. Intramuscular glucagon as a provocative stimulatory effect of i.m. glucagon is selectively stimulus for the assessment of pituitary function: growth mediated by endogenous CRH or AVP unlikely. However, hormone and cortisol responses. Metabolism 1987 36 658±663. 6 Mukherjee JJ, De Castro JJ, Kaltsas G, Afshar F, Grossman AB, it is noteworthy that the ACTH response to the Wass JA et al. A comparison of the insulin tolerance/glucagon test combined administration of i.m. glucagon with hCRH with the short ACTH stimulation test in the assessment of the or AVP became similar to that recorded as a result of the hypothalamo±pituitary±adrenal axis in the early post-operative synergistical effect of hCRH and AVP. Thus, it could be period after hypophysectomy. Clinical Endocrinology 1997 47 51±60. 7 Orme SM, Price A, Weetman AP & Ross RJ. Comparison of the hypothesized that both neurohormones play a role in diagnostic utility of the simpli®ed and standard i.m. glucagon mediating the corticotroph rise which follows the stimulation test. Clinical Endocrinology 1998 49 773±778. intramuscular administration of glucagon. On the 8 Frasier SD. A preview of growth hormone stimulation tests in other hand, the possibility that the HPA response to children. Pediatrics 1974 53 929±937. i.m. glucagon is mediated by unknown mechanisms 9 Roth J, Glick S M, Yalow RS & Berson SA. Hypoglygemia: a potent stimulus to secretion of growth hormone. Science 1963 140 other than CRH and AVP must be taken into account. It 987±988. is clear that our study provided only indirect evidence 10 Ghigo E, Bartolotta E, Imperiale E, Bellone J, Cardinale G, and that animal studies such as those evaluating Aimaretti G et al. Glucagon stimulates GH secretion after neurohormonal levels in the portal blood could give intramuscular but not intravenous administration. Evidence against the assumption that glucagon per se has a GH-releasing more direct information about this hypothesis. activity. Journal of Endocrinological Investigation 1994 17 849±854. Our present data also con®rm the well-known 11 Arvat E, Maccagno B, Di Vito L, Gottero C, Talliano M & Ghigo E. stimulatory effect of i.m. glucagon on GH secretion in ACTH-releasing effect of glucagon in humans. Interaction with humans (1±7) and show that this effect is not modi®ed hCRH, AVP and hexarelin. Journal of Endocrinological Investigation by co-administration with both CRH and AVP. An 1999 22 (Suppl 4) 24. 12 Ghigo E, Bartolotta E, Imperiale E, Bellone J, Cardinale G, inhibition of hypothalamic somatostatin release has Aimaretti G et al. About the effects of glucagon on growth been proposed to explain the stimulatory effect of i.m. hormone release. Letter to the editor. Journal of Endocrinological glucagon on somatotroph secretion (10±13). On the Investigation 1995 18 397. other hand, it has been reported by some (17, 18, 20), 13 Pontiroli AE, Perfetti MG, Andreotti AC, Fattor B, Monti LD & Pozza G. Metabolic effects of graded glucagon infusions in man: but not by other authors (25±27), that hCRH inhibits inhibition of glucagon, insulin and somatostatin response to the GHRH-induced GH response probably via enhance- arginine. Metabolism 1993 42 1242±1246. ment of the hypothalamic somatostatin release (28). Our 14 Weber B, Helge H & Quabbe H. Glucagon-induced growth present results do not allow an increase in the under- hormone release in children. Acta Endocrinologica 1970 65 323±341. standing of the mechanisms underlying the GH-releasing 15 Orth DN. Corticotropin-releasing hormone in humans. Endocrine Reviews 1992 17 164±191. effect of intramuscular glucagon administration while 16 Rivier C & Vale W. Corticotropin-releasing factor (CRF) acts they make it more unlikely that CRH has a major centrally to inhibit growth hormone secretion in the rat. inhibitory role on somatotroph secretion in humans. Endocrinology 1984 114 2409±2411.

www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access 104 E Arvat and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143

17 Barbarino A, Corsello SM, Della-Casa S, Tofani A, Scuitor R, Rota 24 Eddy RL, Jones L & Hirsh RM. Effect of exogenous glucagon CA et al. Corticotropin-releasing hormone inhibition of growth on pituitary polypeptide hormone release. Metabolism 1970 19 hormone releasing hormone induced growth hormone release in 904±912. man. Journal of Clinical Endocrinology and Metabolism 1990 71 25 Rolla M, Andreoni A, Bellitti D, Ferdeghini M, Ghigo E & Muller EE. 1368±1374. Corticotrophin-releasing hormone does not inhibit growth 18 Ghizzoni L, Vottero A, Street ME & Bernasconi S. Dose dependent hormone-releasing hormone-induced release of growth hormone inhibition of growth hormone (GH)-releasing hormone induced in control subjects but is effective in patients with eating GH release by corticotropin-releasing hormone in prepubertal disorders. Journal of Endocrinology 1994 140 327±332. children. Journal of Clinical Endocrinology and Metabolism 1996 81 26 Arvat E, Maccagno B, Ramunni J, Di Vito L, Broglio F, Deghenghi R 1397±1400. et al. Hexarelin, a synthetic growth-hormone releasing peptide, 19 Holsboer F, Von Bardeleben U & Steiger A. Effects of intravenous shows no interaction with corticotropin-releasing hormone and corticotropin-releasing hormone upon sleep-related growth hormone vasopressin on adrenocorticotropin and cortisol secretion in surge and sleep EEG in man. Neuroendocrinology 1988 48 32±38. humans. Neuroendocrinology 1997 66 432±438. 20 Raza J, Massoud AF, Hindmarsh PC, Robinson ICAF & Brook CGD. 27 Arvat E, Ramunni J, Giordano R, Maccagno B, Broglio F, Benso A Direct effects of corticotrophin-releasing hormone on stimulated et al. Effects of the combined administration of hexarelin, a growth hormone secretion. Clinical Endocrinology 1998 48 synthetic peptidyl GH secretagogue, and hCRH on ACTH, cortisol 217±222. and GH secretion in patients with Cushing's disease. Journal of 21 Muller EE & NisticoÁ G. Neurotransmitter regulation of the anterior Endocrinological Investigation 1999 22 23±28. pituitary. In Brain Messengers and the Pituitary, pp 404±537. Eds 28 Rivier C & Vale W. Involvement of corticotropin-releasing factor EE Muller & G NisticoÁ. San Diego: Academic Press Inc., 1989. and somatostatin in stress-induced inhibition of growth hormone 22 Lefebre P & Luychx A. Glucagon and energetic substrates. Bulletin secretion in the rat. Endocrinology 1985 117 2478±2482. de l'Academie Royale de Medicine de Belgique 1974 129 143±145. 23 Kuschke HJ, Klusmann H & Scholkens B. Studies on the effect of glucagon on the human sympatho-adrenal system. Klinische Received 16 November 1999 Wochenschrift 1966 44 1297±1300. Accepted 10 March 2000

www.eje.org

Downloaded from Bioscientifica.com at 09/30/2021 05:25:11PM via free access