Upsala J Med Sci 83: 141-144, 1978 Prolonged Serum Insulin Decreasing Effects of Two Synthetic Somatostatin Analogues Studied in vivo by a New Animal Method Preliminary Communication GEORG HERBAI,' SIGHILD WESTMAN-NAESER,' L h R S CARLSSON3 and KLAS NILSSON3 From the Department of 'Internal Medicine (Endocrinological Section) and Department of 2Histology and Clinical Cytology, University Hospital, Uppsala, and 3Department of Peptide Chemistry, Ferring AB, Malmo, Sweden ABSTRACT A newly developed in vivo method, using the ob-ob strain of obese-hyperglycaemic mice with permanently very high serum insulin values, makes it possible to detect more pro- longed serum insulin lowering properties than in normal animals. Two newly synthesized analogues of somatostatin, o-alanine-somatostatin and des-alanine-des glycine-des- amino-somatostatin produced a more prolonged and greater decrease in the serum insulin values of ob-ob mice than did somatostatin. Our new in vivo method makes it possible to investigate the duration of insulin suppression of new derivatives. INTRODUCTION Somatostatin research has undergone an explosive development in the last few years and an abundant literature has resulted. Several extensive and de- tailed reviews have been published ( 6 , 8 , 23). A n i m a l e x p e r i m e n t s . Insulin release inhibiting ef- fects of somatostatin have been studied in isolated rat pancreas (3, 5, 12) and in perfused canine pancreas (1 1). Glucose, triglycerides and casein administered intraduodenally in conscious dogs cause an increasing glycagon-like immunoreactivity (GLI) in serum. Somatostatin infusion inhibits such GLI increases (19). Somatostatin has been detected in the hypothalamus, posterior pituitary, pancreatic islets (A, or D-cells), stomach and intestine in rats with an irnmunofluorescence technique (10). All these experiments indicate the complex biological effects of somatostatin and several interactions with other hormone systems. H u m a n e x p e r i m e n t s . Somatostatin inhibits the arginine-dependent growth hormone release and its basal secretion in the normal male (21). Moreover, somatostatin suppresses the release of glucagon, insulin and growth hormone in normal, diabetic, acromegalic and in hypopituitary patients (14). In normal and acromegalic patients somatostatin infu- sion inhibits the sleep-related peak of growth hormone release (15). TRH-elicited TSH release is also inhibited by somatostatin (16, 2 2 , 2 5 ) . Serum glucose and insulin values are depressed by somatostatin injections (1) and somatostatin also lowers the serum insulin values stimulated by glucagon and tolbutamide injections (4). The basal and foot-stimulated secretions of gastrin and gastric acid are also decreased by somatostatin ( 2 ) . The aim of our studies was to develop a suitable in vivo method for following the time course after a single injection of somatostatin into the animal. Similar experiments with insulin lowering effects were made in humans given somatostatin infusions (1, 13, 14, 17). In all these studies somatostatin elicited a short insulin inhibition followed by a re- bound effect. The biological half-life of somatosta- tin is very short: 4-6 min (8). Our in vivo animal method, using ob-ob mice with very high serum insulin values demonstrated a dose-dependent somatostatin effect with a decrease in the serum insulin values which lasted for only a short time (9). In this preliminary communication two newly synthesized somatostatin derivatives are described which exhibit markedly prolonged serum insulin depressing effects. MATERIAL AND METHODS Male 5-7 months old obese-hyperglycaemic mice, with high serum insulin values (150-250 ngiml), were selected, Unsalo J Mrd .5( i 83 142 G. Herbai et al. Table I . The amino acid sequence of the original somatostatin (1) and of two recently synthesised ( 2 , 3 ) somatostatin analogues 1. Original GH-RIH: H-ALA-GLY-CYS-LY S-ASN-PHE-PHE-TRP-LYS-THR-PHE-THR-SER-CYS-OH 2. Somatostatin analogue synthesised by changing the first amino acid to D-alanin: H-D-ALA-GLY-CYS-LYS-ASN-PHE-PHE-TRP-LY S-THR-PHE-THR-SER-CYS-OH 3 . Somatostatin analogue produced newly by omission of the two first amino acids and the amino group of the third (des-ALA-des-GLY-desamino polypeptide): SCHZCHZCO-LY S-ASN-PHEC-PHE-TRP-LY S-THR-PHE-THR-SER-CY S-OH 6 animals per group. All animals had body weights be- tween 48 and 55 g. Somatostatin was synthesized by a step-wise frag- mentary condensation technique (9). The two ana- logues were synthesized by a classical step-wise frag- mentary condensation technique at the Department of Peptide Chemistry, Ferring AB, Malmo, Sweden. They were purified by ion-exchange chromatography on carboxymethyl cellulose (Whatman CM 2 3 ) . Analytical data D-Ala l-somatostatin: Homogeneous in three different TLC-systems. [a]i4: -41", c 0.48 in 1 % acetic acid. Amino acid analysis: Ala 0.99, Gly l.&, Cys 2.0, Lys 2.11, Asp 1.00,Phe2.85, Thr 1.92, Ser0.94. Des-Ala1-des-Gly2-desarnino-somatostatin: Homogene- ous in three different TLC-systems. [a]$: -47", c 0.42 in 1 % acetic acid. Amino acid analysis: Lys 2 . 0 4 , Asp I .OO, Phe 2.88, Thr 1.94, Ser 0.93. Immuno-reactive serum insulin was determined by double-antibody radinimmuno-assay (7), using a kit ob- tained from the Radiochemical Centre, Amersham, Eng- land. Crystalline mouse insulin (24 IU/mg) was used as standard. The animals were conscious and unanesthetised during the experiments. Blood samples were obtained by puncture of the orbital venous plexus with a thin-walled Pasteur pipette before and after the injection of control saline, somatostatin, or synthesized analogues. In this preliminary report the lowest effective dosage of the drugs, found in an earlier study (9), was used: 100 pg/kg. The solutions (0.1 ml) were injected rapidly into a tail vein. Blood samples were taken from all animals at zero, 8, 16, 32, 64 and 128 min after the start. The small blood samples were collected and stored as described previ- ously (9). RESULTS The chemical structure of somatostatin and the synthetic analogues are given in Table I. The first analogue ( 2 ) has D-alanine in the first position of the CJpJulo J M P ~ S c i 83 chain in contrast to L-alanine as in somatostatin itself. The second analogue (3) lacks the first two amino acids, on the N-terminal end of the chain alanine and glycine, together with the amino group of the third, with an unchanged Cys-Cys bridge. The serum insulin values, initially and at several times up to 128 min after drug administration, are given in ng/ml in Fig. 1 . Control saline injections did not alter the insulin values. Somatostatin injection resulted in rapidly decreasing serum insulin value, which returned to the normal initial value of about 300 ng/ml after 30 min. Both synthetic analogues also caused and markedly and rapidly reduced the 300 - - E E \ 5 2 0 0 - 0 0 z _I 3 v, z r 3 - - 100- 0 0 8 16 32 6L 128 T I M E minutes Fig. I . Serum insulin values at different times after injec- tion of the drugs in vivo. Six animals per group. The controls received physiological saline. All drugs were in- jected at zero time i.v. in single doses of 100 pg/kg. Effects of somatostatin analogues on serum insulin 143 insulin values. In contrast to the fairly rapid recov- ery after somatostatin, both the synthetic deriva- tives produced a prolonged depression of the insulin value, which lasted throughout the period of obser- vation, i.e. 128 minutes. DISCUSSION The great difficulty in the therapeutic use of somatostatin in endocrine disorders has been the lack of a long-acting derivative. Several research groups have synthesized and tested a large number of chemically different derivatives (18, 20, 23). Some attempts were made to shorten the tetra- decapeptide chain by excluding one or several amino acids. Another structure-activity relation in- vestigated, was whether cleavage of the disulphide link in the polypeptide resulted in alterations of somatostatin activities. Another possibility was to analyse the separation of the activities of so- matostatin derivatives inhibiting growth hormone and influencing insulin and glucagon. The evalua- tion of the different activities was made by studying the hormone effects in vitro and in some experi- ments in vivo. In these studies it was found that many shorter amino acid analogues had lower activities than somatostatin in several types of tests. As far as we know, no new method has been described, which permits tests of somatostatin and different structural analogues, by single injections of the agent in vivo with subsequent monitoring of the serum hormone values at different times after injection. Our previous study (9) showed a statisti- cally significant dose-response effect in vivo for serum insulin-inhibiting effects with different doses of somatostatin injected into obese-hyperglycaemic mice. In our system, somatostatin had a very short- lived serum insulin diminishing effect in the dosage used. In the present investigation of the two somatostatin analogues, both were injected in rela- tively low doses into each group of animals. It was of great interest to note that both D-ala-somatostatin and des-ala-des-gly-desamino-somatostatin had markedly prolonged serum insulin depressant effects. These prolonged insulin inhibiting effects in vivo were obtained in a complex hormonal environ- ment. 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