Short Communications 90
Comparative Effect of Porcine and Rat Galanin on Growth Hormone Secretion in Normal Adult Men A. Giustina , Angela Girelli , C. Bodini, C. Bonfanti2 , M.Doga1, M. Schettino1 and A. Negro-Vilar3 1 Cattedra di Clinica Medica and 2Microbiologia, University of Brescia, Italy Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, U. S. A.
Sterile endotoxin-free synthetic p- and r-galanin (Peninsula Laboratories, Belmont, CA) were reconstituted in 2 ml of saline. Blood samples for GH assay were taken at — 15, 0, + 15, + 30, + 45, + 60, + 90 and + 120 min from the beginning of the infusion.
Galanin is a 29-aminoacid peptide isolated from porcine intestine and widely distributed in the central nervous system of several animal species (Rokaeus 1987). Recently, it has been demonstrated that systemic administration of synthetic porcine (p) galanin is Pulse rate and supine blood pressure were measured able to elicit GH secretion in men {Bauer, Ginsberg, Venetikou, at 0,20,40 and 60 min during each infusion. MacKay, Burrin and Bloom 1986b). GH secretory responses were expressed either as absolute values (ug/L), as GH peak (ug/L) or as area under curve (AUC, Chromatographic and immunological analysis of ug/L x 120 min). Absolute GH values were compared using the analhuman galanin-like immunoreactivity in tissue extracts shows definite differences from p-galanin (Bauer, Adrian, Christofides, Ferri, Yanai- ysis of variance for repeated measures. AUC and peak GH levels were hara, Polak and Bloom 1986a). Due to these structural differences, it compared with the Student's t-test for paired data. has been hypothesized that in man p-galanin may not have full biological potency (Bauer et al. 1986b). A commercial immunoradiometric kit was used for the estimation of GH (Nichols Institute, S. Juan Capistrano, CA, U. S. A.; inter- and intra-assay coefficients of variation + 5.4% and Recently, it has been described the aminoacid se2.3%, respectively; sensitivity limit of assay 0.02 ug/L). quence of rat (r) galanin, which differs from p-galanin by three aminoacid residue substitutions at positions located in the C-terminal area of the molecule. Synthetic r-galanin has become available for in vivo studies (Schnuerer, Rokaeus, Carlquist, Bergman, Dupre and McDonald 1990) and it has been shown to be effective in stimulating GH secretion in rats {Murakami, Kato, Koshiyama, Inoue, Yanaihara and Imura 1987). The aim of our study was to compare the relative potencies of r- and p-galanin in eliciting GH secretion in normal human subjects.
Subjects and Methods Subjects We studied five healthy adult males with no family history of endocrine or metabolic diseases, age range 25—31 years, body mass index range 19—24 Kg/m2, with normal thyroid and adrenal function. None of the subjects was taking any drugs at the start of the study and all subjects were examined on three 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 vein 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 250 ml saline from 0 to 60 min; 2) iv infusion of synthetic p-galanin (0.5 mg in 250 ml saline) from 0 to 60 min; 3) iv infusion of synthetic r-galanin (0.5 mg in 250 ml saline) from 0 to 60 min. Horm.metab.Res.24(1992)90-91 © GeorgThieme Verlag Stuttgart-New York
Fig. 1 Serum GH levels (mean±SEM; ug/L) after iv infusion of saline ( • ) , porcine galanin ( • ) and rat galanin ( • ) from 0 to 60 min in five normal young adult subjects. °p < 0.05 vs saline; *p < 0.05 vs pgalanin.
Received: 2 July 1991
Accepted: 4 Nov. 1991
Downloaded by: Universite Laval. Copyrighted material.
Introduction
Galanin and GH
The kinetics of the GH responses during p- and rgalanin and saline infusions are shown in Fig. 1. Both p- and r-galanin infusions caused a significant increase in GH levels with respect to saline infusion (Fig. 1). Peak GH levels after p-galanin (5.9 ± 1.6 u,g/L) and r-galanin infusion (10.7 ± 2.3 ug/L) were significantly higher (p < 0.05) with respect to the GH peak after saline infusion (1.3 + 0.2 ug/L). Absolute GH levels (+ 30, 45 and 60 min) (Fig. 1), GH AUC (701.9 ± 158.8 ug/L x 120 min) and GH peak were significantly higher (p < 0.05) after r-galanin with respect to p-galanin (GH AUC: 401.3 ± 111.1 ug/L x 120 min) infusion. All the five subjects experienced a bitter taste in mouth which started immediately after the beginning of both p- and rgalanin infusions and ended at the stopping of the infusion. No other side effects were observed in any of the subjects. Blood pressure and pulse rate were not significantly altered by either r- or p-galanin infusion. Discussion Our results confirm that p-galanin is able to increase GH secretion in humans. Moreover, our results show that r-galanin, 0.5 mg, is more effective than the same dose of p-galanin in eliciting GH secretion in man.
91
(Bauer et al. 1986a). Due to these structural differences, it has been hypothesized that in man the porcine peptide may not have full biological potency (Bauer et al. 1986b). The finding that galanin 1 — 15 did not inhibit insulin secretion as much as did native galanin or galanin 2—29 suggests that the whole structure of the molecule is of some importance for the maintenance of the full biological potency of galanin (Hermansen, Yanaihara and Ahren 1989). No data concerning the structural requirements for full galanin potency in eliciting GH secretion in man are yet available. Our data show that r-galanin is more effective than p-galanin in stimulating GH secretion in normal adult subjects, when an identical dose of the two compounds is tested. R-galanin differs from p-galanin at three amino acid residues in the C-terminal heptapeptide region (residues 23,26, and 29). Therefore, it can be hypothesized that the full sequence of galanin is required for its biological effect on GH secretion. Moreover, our data suggest that the structure of human galanin may be more similar to that of r-galanin as compared to p-galanin. In conclusion, it can be hypothesized that r-galanin may be a better tool for physiological or pharmacological studies of GH secretion in man with respect to p-galanin. Acknowledgements Research was supported by the Centra Studi e Ricerche di Neuroendocrinologia (Brescia, Italy). References
Galanin is a 29 amino acid peptide isolated from porcine intestinal extracts which has been detected in the central nervous system of several animal species including humans (Rokaeus 1987). The two most prominent metabolic effects of systemic administration of synthetic p-galanin in humans are an increase in GH secretion and a decrease in insulin secretion (Bauer et al. 1986b).
Bauer, F. E., T. E. Adrian, N. D. Christofides, G. L. Ferri, N. Yanaihara, J. M. Polak, S. R. Bloom: Gastroenterology 91: 877-883 (1986a) Bauer, F. E., L. Ginsberg, M. Venetikou, D. J. MacKay, J. M. Burrin, S. R. Bloom: Lancet 2:192-195 (1986b) Davis, T. M. E., J. M. Burrin, S. R. Bloom: J. Clin. Endocrinol. Metab. 65:1248-1252(1987) GH secretion in man is controlled by the stimulating Giustina, A., S. Bossoni, C. Bodini, M. Doga, A. Girelli, M. G. Buffoli, influence of GHRH on one hand and the inhibitory action of somaM. Schettino, W. B. Wehrenberg:Metabolism40: 519-523 (1991) tostatin on the other (Giustina, Girelli, Doga, Bodini, Bossoni, Ro- Giustina, A., A. Girelli, M. Doga, C. Bodini, S. Bossoni, G. Romanelli, manelli and Wehrenberg 1990). Previous studies have shown that pW. B. Wehrenberg:). Clin. Endocrinol. Metab. 71:580-584(1990) galanin infused in doses similar to those used by us causes a significant Hermansen, K., N. Yanaihara, B. Ahren: Acta Endocrinol. (Copenh) GH response both in normal adults {Bauer et al. 1986b) and children 121:545-550(1989.) (Loche, Cella, Puggioni, Stabilini, Pintor and Mutter 1989). When p- Loche, S., S. G. Cella, R. Puggioni, L. Stabilini, C. Pintor, E. E. Mutter: galanin is co-administered with a maximal GHRH dose (Giustina, Pediatr.Res. 26:316-319(1989) Bossoni, Bodini, Doga, Girelli, Buffoli, Schettino and Wehrenberg Murakami, Y, Y. Kato, H. Koshiyama, T. Inoue, N. Yanaihara, H. 1991) the GH response to GHRH is significantly increased and it is Imura.EmJ. Pharmacol. 136:415-418 (1987) greater than the sum of the independent responses (Davis, Burrin and Rokaeus, A/Trends Neurosci. 10:158-164(1987) Bloom 1987). These findings suggest an indirect action of galanin on Schnuerer, E. M., A. Rokaeus, M. Carlquist, T. Bergman, J. Dupre, T. J. the somatotrophs, possibly through a decrease in hypothalamic somaMcDonald:Pancreas 5: 70-74 (1990) tostatin tone. Chromatographic and immunological analysis of human galanin-like-immunoreactivity in tissue extracts shows small but definite differences from p-galanin. Antibodies against the C-terminal sequence of p-galanin are not able to detect the peptide in human extracts and in rats. Therefore, it has been concluded that differences in the amino acid sequences between these species could be located at or near the C-terminal sequence of the galanin molecule
Requests of reprints should be addressed to: Andrea Giustina, MD Clinica Medica c/o 2a Medicina Spedali Civilli 1-25125 Brescia (Italy)
Downloaded by: Universite Laval. Copyrighted material.
Results
Horm. metab. Res. 24 (1992)
Comparative Effect of Porcine and Rat Galanin on Growth Hormone Secretion in Normal Adult Men A. Giustina , Angela Girelli , C. Bodini, C. Bonfanti2 , M.Doga1, M. Schettino1 and A. Negro-Vilar3 1 Cattedra di Clinica Medica and 2Microbiologia, University of Brescia, Italy Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, U. S. A.
Sterile endotoxin-free synthetic p- and r-galanin (Peninsula Laboratories, Belmont, CA) were reconstituted in 2 ml of saline. Blood samples for GH assay were taken at — 15, 0, + 15, + 30, + 45, + 60, + 90 and + 120 min from the beginning of the infusion.
Galanin is a 29-aminoacid peptide isolated from porcine intestine and widely distributed in the central nervous system of several animal species (Rokaeus 1987). Recently, it has been demonstrated that systemic administration of synthetic porcine (p) galanin is Pulse rate and supine blood pressure were measured able to elicit GH secretion in men {Bauer, Ginsberg, Venetikou, at 0,20,40 and 60 min during each infusion. MacKay, Burrin and Bloom 1986b). GH secretory responses were expressed either as absolute values (ug/L), as GH peak (ug/L) or as area under curve (AUC, Chromatographic and immunological analysis of ug/L x 120 min). Absolute GH values were compared using the analhuman galanin-like immunoreactivity in tissue extracts shows definite differences from p-galanin (Bauer, Adrian, Christofides, Ferri, Yanai- ysis of variance for repeated measures. AUC and peak GH levels were hara, Polak and Bloom 1986a). Due to these structural differences, it compared with the Student's t-test for paired data. has been hypothesized that in man p-galanin may not have full biological potency (Bauer et al. 1986b). A commercial immunoradiometric kit was used for the estimation of GH (Nichols Institute, S. Juan Capistrano, CA, U. S. A.; inter- and intra-assay coefficients of variation + 5.4% and Recently, it has been described the aminoacid se2.3%, respectively; sensitivity limit of assay 0.02 ug/L). quence of rat (r) galanin, which differs from p-galanin by three aminoacid residue substitutions at positions located in the C-terminal area of the molecule. Synthetic r-galanin has become available for in vivo studies (Schnuerer, Rokaeus, Carlquist, Bergman, Dupre and McDonald 1990) and it has been shown to be effective in stimulating GH secretion in rats {Murakami, Kato, Koshiyama, Inoue, Yanaihara and Imura 1987). The aim of our study was to compare the relative potencies of r- and p-galanin in eliciting GH secretion in normal human subjects.
Subjects and Methods Subjects We studied five healthy adult males with no family history of endocrine or metabolic diseases, age range 25—31 years, body mass index range 19—24 Kg/m2, with normal thyroid and adrenal function. None of the subjects was taking any drugs at the start of the study and all subjects were examined on three 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 vein 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 250 ml saline from 0 to 60 min; 2) iv infusion of synthetic p-galanin (0.5 mg in 250 ml saline) from 0 to 60 min; 3) iv infusion of synthetic r-galanin (0.5 mg in 250 ml saline) from 0 to 60 min. Horm.metab.Res.24(1992)90-91 © GeorgThieme Verlag Stuttgart-New York
Fig. 1 Serum GH levels (mean±SEM; ug/L) after iv infusion of saline ( • ) , porcine galanin ( • ) and rat galanin ( • ) from 0 to 60 min in five normal young adult subjects. °p < 0.05 vs saline; *p < 0.05 vs pgalanin.
Received: 2 July 1991
Accepted: 4 Nov. 1991
Downloaded by: Universite Laval. Copyrighted material.
Introduction
Galanin and GH
The kinetics of the GH responses during p- and rgalanin and saline infusions are shown in Fig. 1. Both p- and r-galanin infusions caused a significant increase in GH levels with respect to saline infusion (Fig. 1). Peak GH levels after p-galanin (5.9 ± 1.6 u,g/L) and r-galanin infusion (10.7 ± 2.3 ug/L) were significantly higher (p < 0.05) with respect to the GH peak after saline infusion (1.3 + 0.2 ug/L). Absolute GH levels (+ 30, 45 and 60 min) (Fig. 1), GH AUC (701.9 ± 158.8 ug/L x 120 min) and GH peak were significantly higher (p < 0.05) after r-galanin with respect to p-galanin (GH AUC: 401.3 ± 111.1 ug/L x 120 min) infusion. All the five subjects experienced a bitter taste in mouth which started immediately after the beginning of both p- and rgalanin infusions and ended at the stopping of the infusion. No other side effects were observed in any of the subjects. Blood pressure and pulse rate were not significantly altered by either r- or p-galanin infusion. Discussion Our results confirm that p-galanin is able to increase GH secretion in humans. Moreover, our results show that r-galanin, 0.5 mg, is more effective than the same dose of p-galanin in eliciting GH secretion in man.
91
(Bauer et al. 1986a). Due to these structural differences, it has been hypothesized that in man the porcine peptide may not have full biological potency (Bauer et al. 1986b). The finding that galanin 1 — 15 did not inhibit insulin secretion as much as did native galanin or galanin 2—29 suggests that the whole structure of the molecule is of some importance for the maintenance of the full biological potency of galanin (Hermansen, Yanaihara and Ahren 1989). No data concerning the structural requirements for full galanin potency in eliciting GH secretion in man are yet available. Our data show that r-galanin is more effective than p-galanin in stimulating GH secretion in normal adult subjects, when an identical dose of the two compounds is tested. R-galanin differs from p-galanin at three amino acid residues in the C-terminal heptapeptide region (residues 23,26, and 29). Therefore, it can be hypothesized that the full sequence of galanin is required for its biological effect on GH secretion. Moreover, our data suggest that the structure of human galanin may be more similar to that of r-galanin as compared to p-galanin. In conclusion, it can be hypothesized that r-galanin may be a better tool for physiological or pharmacological studies of GH secretion in man with respect to p-galanin. Acknowledgements Research was supported by the Centra Studi e Ricerche di Neuroendocrinologia (Brescia, Italy). References
Galanin is a 29 amino acid peptide isolated from porcine intestinal extracts which has been detected in the central nervous system of several animal species including humans (Rokaeus 1987). The two most prominent metabolic effects of systemic administration of synthetic p-galanin in humans are an increase in GH secretion and a decrease in insulin secretion (Bauer et al. 1986b).
Bauer, F. E., T. E. Adrian, N. D. Christofides, G. L. Ferri, N. Yanaihara, J. M. Polak, S. R. Bloom: Gastroenterology 91: 877-883 (1986a) Bauer, F. E., L. Ginsberg, M. Venetikou, D. J. MacKay, J. M. Burrin, S. R. Bloom: Lancet 2:192-195 (1986b) Davis, T. M. E., J. M. Burrin, S. R. Bloom: J. Clin. Endocrinol. Metab. 65:1248-1252(1987) GH secretion in man is controlled by the stimulating Giustina, A., S. Bossoni, C. Bodini, M. Doga, A. Girelli, M. G. Buffoli, influence of GHRH on one hand and the inhibitory action of somaM. Schettino, W. B. Wehrenberg:Metabolism40: 519-523 (1991) tostatin on the other (Giustina, Girelli, Doga, Bodini, Bossoni, Ro- Giustina, A., A. Girelli, M. Doga, C. Bodini, S. Bossoni, G. Romanelli, manelli and Wehrenberg 1990). Previous studies have shown that pW. B. Wehrenberg:). Clin. Endocrinol. Metab. 71:580-584(1990) galanin infused in doses similar to those used by us causes a significant Hermansen, K., N. Yanaihara, B. Ahren: Acta Endocrinol. (Copenh) GH response both in normal adults {Bauer et al. 1986b) and children 121:545-550(1989.) (Loche, Cella, Puggioni, Stabilini, Pintor and Mutter 1989). When p- Loche, S., S. G. Cella, R. Puggioni, L. Stabilini, C. Pintor, E. E. Mutter: galanin is co-administered with a maximal GHRH dose (Giustina, Pediatr.Res. 26:316-319(1989) Bossoni, Bodini, Doga, Girelli, Buffoli, Schettino and Wehrenberg Murakami, Y, Y. Kato, H. Koshiyama, T. Inoue, N. Yanaihara, H. 1991) the GH response to GHRH is significantly increased and it is Imura.EmJ. Pharmacol. 136:415-418 (1987) greater than the sum of the independent responses (Davis, Burrin and Rokaeus, A/Trends Neurosci. 10:158-164(1987) Bloom 1987). These findings suggest an indirect action of galanin on Schnuerer, E. M., A. Rokaeus, M. Carlquist, T. Bergman, J. Dupre, T. J. the somatotrophs, possibly through a decrease in hypothalamic somaMcDonald:Pancreas 5: 70-74 (1990) tostatin tone. Chromatographic and immunological analysis of human galanin-like-immunoreactivity in tissue extracts shows small but definite differences from p-galanin. Antibodies against the C-terminal sequence of p-galanin are not able to detect the peptide in human extracts and in rats. Therefore, it has been concluded that differences in the amino acid sequences between these species could be located at or near the C-terminal sequence of the galanin molecule
Requests of reprints should be addressed to: Andrea Giustina, MD Clinica Medica c/o 2a Medicina Spedali Civilli 1-25125 Brescia (Italy)
Downloaded by: Universite Laval. Copyrighted material.
Results
Horm. metab. Res. 24 (1992)
