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Dissociation of somatostatin effects. Peptides inhibiting the release of growth hormone but not glucagon or insulin in rats
Life Scinaces Vol . 19, pp . 629-632, 1976 . Printed in the U .S .A .
Pergamon Press
DISSOCIATION OF SOMAIOSTATIN EFFECTS . PEPTIDES INHIBITING THE RELEASE OF GROWTH HORZANB BUT NOT GLUCAGON OR INSULIN IN RATS Norman Grant s Donald Clarks Victor Garsky~ Ivars Jaunakais, William McGregor and Dimitrios Sarantakis Research Divieion~ Wyeth Laboratories Philadelphias Pennsylvania 19101 (Received in final form May 3, 1976) Two analogs of somatostatin . were tested for their effects on release of growth hormone s glucagon, and insulin after subcutaneous injection into rats . These peptides significantly suppressed pentobarbital-stimulated growth hormone release but showed m effect on arginine-stimulated glucagon or insulin release at dosages greater than 2 mg/kg . Somatostatin acts on all three secretions at dosages below 200 u~/kg . The concept of one chemically distinct hypothalamic control factor for each of six anterior pituitary hormones (1) hâs, within a few years, disintegrated in the face of multiple functione~ multiple controls multiple target organs and multiple sites of origin. Somatostatin epitomizes the present recognition that hormones are apt to work in concert to achieve a single effect (for example, glucose homeostasis) and that hormones controlling release can modulate disparate secretions . An earlier report from this laboratory showed that somatostatin .could be structurally altered to lower plasma growth hormone and insulin levels without significantly affecting plasma glucagon (2) . The present paper shows that the linkage between suppressions of the release of growth hormone, insulin and glucagon can be broken with the retention of only the growth hormone effect . METHODS Ia vivo hormone secretion was measured in groups of 5-10 male Charles River CD rata weighing about 250 g after stimulation of GH secretion by intraperitoneal injection of Na pentobarbital (50 mg/kg ; zero time) and of glucagon and insulin secretions by intracardiac arginine (150 m$/rat; + 25 . minutes) . Somatostatia or its analog in saline, or saline alone, was injected eubcutaaeously at + 15 minutes into experimental rate or matched controls . The rate were decapitated at + 30 minutes and trunk plasma, collected in Trasylol-EDTA (3), was assayed for the three hormones by double antibody radioimmunoaseays~ using GH reagents from the NIAMDD (A . Parlow), commercial insulin reagente,~and Urger 30R aatiglucagon serum . In preparing peptides I and II, the sequences 1-10 and 1-11, respectively~ were synthesized by the solid phase method (4) . The protected peptides were subsequently cleaved from the resin by hydrazinolysis and the synthesis completed by classical methods . The peptides were purified by gel filtration and partition chromatography . The purity of the samples was established by amino acid analysis and two or more TLC systems . Details 629
630
Dissociation of Somatostatia Effects
Vol . 19, No . 5
of the synthesis will be published elsewhere. RESULTS AND DISCUSSION Somatostatia, isolated from sheep hypo thalami by Brazesu et al is a cyclic tetradecapeptide :
(5),
S H-Ala-Gly-Cys-Lye-Aan-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
In the peptides reported here the cystine bridges were replaced with nonsulfur linkages (I and II) . 1 11 rLys-Asa-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-G1~-0H
I (Wy- 19,840)
1 12 Abu-Lys-Aan-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Asp-0H
II (Wy-40,05ô)
r,~
In the eaperimeatal model employed, somatoetatin is most potent against GH release, inhibiting sign _if,~i_ cantly at 1-10 u$~8i it inhibits glucagon release as low ae 50-100 utY°`ai and it is weakest against insulin Thus, "specificity" may release, acting only as low as 100-200 ug/kg. become a function of dosage unless it is judged at levels high enough to overcome an intrinsic spread in eecretory responses . Both analogs of somatostatin displayed strong inhibition of growth Peptide II was hormone release in the range of 2-4 mg/kg (Table 1) . studied at lower dosages and retained the ability to inhibit GH secretion at 20 and 10 u$~gi its potency with respect to growth hormone is about oaefifth that of somatostatin . Neither I nor II altered the release of insulin even at levels 15-30 times higher than that needed for somatostatin, and neither altered the release of glucagoa at levels 30-f>0 times higher than that needed for somatostatin. Deletion of the inhibitory activity toward glucagon and insulin secretions raises the expectation of further selective modifications of the somatoetatia molecule, resulting in various combinations of intrinsic responses or relative enrichments . Moreover an array of peptides capable of narrowly blocking secretions from the pituitary, pancreas, stomach, and dodenum would provide valuable research tools and perhaps new approaches Peptides of the type described in this paper are of to chemotherapy . evident interest in studies of growth hormone-dependent pathologies, such as acromegaly, and, perhaps, certain mammary tumors (ô) and diabetic angiopathy (7) .
Vol . 19, No . 5
Dissociation of Somatostatin Effects
631
TABLE 1 Inhibition of Releaee of Growth Hormone, Glucagon, and Insulin by Somatostatin (SRIH) and Analogs Plasma Hormone Levels (M ± SEM) GH Glucagon Insulin ngiml p~ml utj/ml
Exp .
Peptide
Dose R$~$
375B
none SRIH I
217 2700
162 + 18 64 ± 6** 73 ± 14**
395B
none SRIH I
200 3400
177 ± 29 48 + 7** 67 ± 9**
108 + 34 26 + L2* 125 ± 43
218 ± 15 169 + 11* 191 ± 12
68C
none SRIH II
200 4000
261 ± 43 73 ± 13** 95 + 17**
21 ± 5 .7 + 16 +
184 + 14 112 ± 11** 164 + 9
63C a
none SRIH II II
20 100 20
271 94 109 184
70Ca
mne SRIH II
10 100
350 ± 46 193 ± 29** 158 ± 23**
± ± ± +
83 ± 18 23 ± 7** 80 ± 12
5 2** 4
351 ± 52 234 ± 17* 342 ± 61
25 32** 33** 29*
** p < 0 . O1 compared to saline controls by analysis of variance . * p < 0 .05 a arginiae injection omitted. REFERENCES 1. 2. 3. 4. 5. 6. 7.
G,W. HARRIS, is Hypophyeiotropic Hormones of the Hypothalamus (Ed . J . Meites), p . 9, Williams and Wilkine Co,, Baltimore (1970) . D, SARANTARIS, W, A, MCRINLEY, I . JAUNARAIS, D . CLARK, and N. H, GRANT, Proceedings of the Fifth International Conference on Endocrinology, 1975 . Loadon~ Clinical Endocrinolottv . Suppl ., 275a-278s (1976) . G . R, FALOONA and R . H. UNGSR, in Methods in Hormone Radioimmuaoassav (Ed, B . M. Jaffe and H . R, Behrman), p, 317, Academic Press, New York (1974) . R .B . MERRLFIELD, J . Am . Chem. Soc . . 85 . 2149-2154 (1963) . P . BRAZEAU, W . VALE, R . BURGUS, N . LING, M . BUTCHER, J . RIMER, and R . GUILLEMIN, Science, 179 . 77-79 (1973) . I . DE SOUZA, L . MORGAN, U .J . LEWIS, P .R, RAGGATT, H . SALIR, and J. R . HOBBS, Lancet . 182-184 (1974) . K. LUI~BAER, M,J . CHRISTIANSEN, V . A . JENSEN, T. S . OLSEN, Aa . P . HANSEN, H. ~RS~V, and R . ldSTERBY, Lancet, 2, 131-133 (1970),
5
Pergamon Press
DISSOCIATION OF SOMAIOSTATIN EFFECTS . PEPTIDES INHIBITING THE RELEASE OF GROWTH HORZANB BUT NOT GLUCAGON OR INSULIN IN RATS Norman Grant s Donald Clarks Victor Garsky~ Ivars Jaunakais, William McGregor and Dimitrios Sarantakis Research Divieion~ Wyeth Laboratories Philadelphias Pennsylvania 19101 (Received in final form May 3, 1976) Two analogs of somatostatin . were tested for their effects on release of growth hormone s glucagon, and insulin after subcutaneous injection into rats . These peptides significantly suppressed pentobarbital-stimulated growth hormone release but showed m effect on arginine-stimulated glucagon or insulin release at dosages greater than 2 mg/kg . Somatostatin acts on all three secretions at dosages below 200 u~/kg . The concept of one chemically distinct hypothalamic control factor for each of six anterior pituitary hormones (1) hâs, within a few years, disintegrated in the face of multiple functione~ multiple controls multiple target organs and multiple sites of origin. Somatostatin epitomizes the present recognition that hormones are apt to work in concert to achieve a single effect (for example, glucose homeostasis) and that hormones controlling release can modulate disparate secretions . An earlier report from this laboratory showed that somatostatin .could be structurally altered to lower plasma growth hormone and insulin levels without significantly affecting plasma glucagon (2) . The present paper shows that the linkage between suppressions of the release of growth hormone, insulin and glucagon can be broken with the retention of only the growth hormone effect . METHODS Ia vivo hormone secretion was measured in groups of 5-10 male Charles River CD rata weighing about 250 g after stimulation of GH secretion by intraperitoneal injection of Na pentobarbital (50 mg/kg ; zero time) and of glucagon and insulin secretions by intracardiac arginine (150 m$/rat; + 25 . minutes) . Somatostatia or its analog in saline, or saline alone, was injected eubcutaaeously at + 15 minutes into experimental rate or matched controls . The rate were decapitated at + 30 minutes and trunk plasma, collected in Trasylol-EDTA (3), was assayed for the three hormones by double antibody radioimmunoaseays~ using GH reagents from the NIAMDD (A . Parlow), commercial insulin reagente,~and Urger 30R aatiglucagon serum . In preparing peptides I and II, the sequences 1-10 and 1-11, respectively~ were synthesized by the solid phase method (4) . The protected peptides were subsequently cleaved from the resin by hydrazinolysis and the synthesis completed by classical methods . The peptides were purified by gel filtration and partition chromatography . The purity of the samples was established by amino acid analysis and two or more TLC systems . Details 629
630
Dissociation of Somatostatia Effects
Vol . 19, No . 5
of the synthesis will be published elsewhere. RESULTS AND DISCUSSION Somatostatia, isolated from sheep hypo thalami by Brazesu et al is a cyclic tetradecapeptide :
(5),
S H-Ala-Gly-Cys-Lye-Aan-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
In the peptides reported here the cystine bridges were replaced with nonsulfur linkages (I and II) . 1 11 rLys-Asa-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-G1~-0H
I (Wy- 19,840)
1 12 Abu-Lys-Aan-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Asp-0H
II (Wy-40,05ô)
r,~
In the eaperimeatal model employed, somatoetatin is most potent against GH release, inhibiting sign _if,~i_ cantly at 1-10 u$~8i it inhibits glucagon release as low ae 50-100 utY°`ai and it is weakest against insulin Thus, "specificity" may release, acting only as low as 100-200 ug/kg. become a function of dosage unless it is judged at levels high enough to overcome an intrinsic spread in eecretory responses . Both analogs of somatostatin displayed strong inhibition of growth Peptide II was hormone release in the range of 2-4 mg/kg (Table 1) . studied at lower dosages and retained the ability to inhibit GH secretion at 20 and 10 u$~gi its potency with respect to growth hormone is about oaefifth that of somatostatin . Neither I nor II altered the release of insulin even at levels 15-30 times higher than that needed for somatostatin, and neither altered the release of glucagoa at levels 30-f>0 times higher than that needed for somatostatin. Deletion of the inhibitory activity toward glucagon and insulin secretions raises the expectation of further selective modifications of the somatoetatia molecule, resulting in various combinations of intrinsic responses or relative enrichments . Moreover an array of peptides capable of narrowly blocking secretions from the pituitary, pancreas, stomach, and dodenum would provide valuable research tools and perhaps new approaches Peptides of the type described in this paper are of to chemotherapy . evident interest in studies of growth hormone-dependent pathologies, such as acromegaly, and, perhaps, certain mammary tumors (ô) and diabetic angiopathy (7) .
Vol . 19, No . 5
Dissociation of Somatostatin Effects
631
TABLE 1 Inhibition of Releaee of Growth Hormone, Glucagon, and Insulin by Somatostatin (SRIH) and Analogs Plasma Hormone Levels (M ± SEM) GH Glucagon Insulin ngiml p~ml utj/ml
Exp .
Peptide
Dose R$~$
375B
none SRIH I
217 2700
162 + 18 64 ± 6** 73 ± 14**
395B
none SRIH I
200 3400
177 ± 29 48 + 7** 67 ± 9**
108 + 34 26 + L2* 125 ± 43
218 ± 15 169 + 11* 191 ± 12
68C
none SRIH II
200 4000
261 ± 43 73 ± 13** 95 + 17**
21 ± 5 .7 + 16 +
184 + 14 112 ± 11** 164 + 9
63C a
none SRIH II II
20 100 20
271 94 109 184
70Ca
mne SRIH II
10 100
350 ± 46 193 ± 29** 158 ± 23**
± ± ± +
83 ± 18 23 ± 7** 80 ± 12
5 2** 4
351 ± 52 234 ± 17* 342 ± 61
25 32** 33** 29*
** p < 0 . O1 compared to saline controls by analysis of variance . * p < 0 .05 a arginiae injection omitted. REFERENCES 1. 2. 3. 4. 5. 6. 7.
G,W. HARRIS, is Hypophyeiotropic Hormones of the Hypothalamus (Ed . J . Meites), p . 9, Williams and Wilkine Co,, Baltimore (1970) . D, SARANTARIS, W, A, MCRINLEY, I . JAUNARAIS, D . CLARK, and N. H, GRANT, Proceedings of the Fifth International Conference on Endocrinology, 1975 . Loadon~ Clinical Endocrinolottv . Suppl ., 275a-278s (1976) . G . R, FALOONA and R . H. UNGSR, in Methods in Hormone Radioimmuaoassav (Ed, B . M. Jaffe and H . R, Behrman), p, 317, Academic Press, New York (1974) . R .B . MERRLFIELD, J . Am . Chem. Soc . . 85 . 2149-2154 (1963) . P . BRAZEAU, W . VALE, R . BURGUS, N . LING, M . BUTCHER, J . RIMER, and R . GUILLEMIN, Science, 179 . 77-79 (1973) . I . DE SOUZA, L . MORGAN, U .J . LEWIS, P .R, RAGGATT, H . SALIR, and J. R . HOBBS, Lancet . 182-184 (1974) . K. LUI~BAER, M,J . CHRISTIANSEN, V . A . JENSEN, T. S . OLSEN, Aa . P . HANSEN, H. ~RS~V, and R . ldSTERBY, Lancet, 2, 131-133 (1970),
5