Comparative aspects of the immunology and biology of insulin

Diabetologia, Dec 1967

Summary Some immunological and physiological properties of insulin preparations from a wide range of vertebrate and invertebrate species have been compared with those of crystalline ox and cod insulin, and the yields of insulin from several of these islet preparations are given. — Highly potent guinea pig and horse antisera to ox insulin were unable to provoke hyperglycaemia in the marine TeleostCottus scorpius (the sculpin) and in the CyclostomeMyxine glutinosa (the hagfish), respectively. — Crystalline ox and cod insulin provoked hypoglycaemia and death in both the sculpin and the hagfish, but only after large doses and several days' observation time. These findings contrast with the ease and fair rapidity of eliciting a hypoglycaemic response in the hagfish when the hagfish's own insulin was used. No marked quantitative differences were noted between the effects of ox, cod, and sculpin insulins in the sculpin, neither between ox and cod insulin in the hagfish. — The results indicate a species-specificity of insulin, both immunologically and hormonally.

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Comparative aspects of the immunology and biology of insulin

Comparative Aspects of the Immunology and Biology of Insulin* STUI~EFALKMER 0 STI~ATIIEARNWILSOn 0 0 I n s t i t u t e of Pathology, University of Ulnes Umes 6, the Kristineberg Zoological Station, Fiskeb~ckskil, Sweden, and the Connaught Medical Research Laboratories , Willowdale, Ont. , Canada Summary. Some immunological and physiological properties of insulin preparations from a wide range of vertebrate and invertebrate species have been compared with those of crystalline ox and cod insulin, and the yields of insulin from several of these islet preparations are given. -Highly potent guinea pig a n d horse antisera to ox insulin were unable to provoke hyperglycaemia in the marine Teleost Cottus scorpius (the sculpin) and in the Cyclostome Myxine glutinosa (the hagfish), respectively. -- Crystalline ox a n d cod insulin provoked hypoglycaemia and death in both the sculpin and the hagfish, b u t only after large doses and several days' observation time. These findings contrast with the ease and fair rapidity of elicit i n t a hypoglycaemic response in the hagfish when the hagfish's own insulin was used. No marked quantitative differences were noted between the effects of ox, cod, and sculpin insulins in the sculpin, neither between ox and cod insulin in the hagfish. -- The results indicate a species-specificity of insulin, both immuno]ogically and hormonally. - Aspects corr~paratifs de l'immunologie et de la biologic de l'insuline Rgsumd. On a eompar6 quelques propri6tgs immunologiques et physiologiques de pr@arations d'insuline obtenues ~ partir d ' u n grand hombre de vert~brgs et d'invert~br6s avee les propri6t6s de l'insuline cristalline de boeuf et de morue. La quantit6 d'insuline obtenue ~ partir de ees pr@arations insulaires est indiqu6e. -- De trgs puissants s6rums anti-insuline de boeuf obtenus chez le cobaye et le cheval se m o n t r a i e n t incapables de provoquer de l'hyperglyc6mie ehez le T61gost6en m a r i n Cottus scorplus (le sculpin) ct chez le Cyclostome Myxine glutinosa (la myxine). -- L'insuline cristalline de boeuf et de morue provoquait de l'hypoglye6mie et la mort aussi bien du sculpin que de la myxine mais seulement aprgs de fortes doses et u n temps d'observation de plusieurs jours. Au contraire, on obtenait avec faeilit6 ct avee une promptitude assez prononc6e une r6ponse hypoglyc6mique chez la myxine en utilisant sa propre insuline. On n ' a pas pu noter de diff6renees quantitatives bien prononc6es entre les effets de l'insuline de boeuf, de morue et de sculpin Introduction K n o w l e d g e a b o u t those regions of the i n s u l i n molecule t h a t g o v e r n its i m m u n o l o g i c a l properties a n d * Presented in parts at the International Diabetes Federation Vth Congress in Toronto, July 20--24, 1964 (WILSON and FALKMER, 1964) , at the 3rd Conference of European Comparative Endocrinologists in Copenhagen, August 2--5, 1965 (FA_LKMEI% and WILSON, 1965) , and at the British Insulin Manufacturers' Colloquium on the Immunology of Insulin, London, September 16, 1965 (of. COBB et al., 1966). The investigation was supported by grants from the Nordic Insulin F u n d . The authors are indebted to Dr. A. KOTAXI for gifts of bonito and t u n n y insulins, and to I)r. P. J. MOLONEu for chicken insulin. ehez le sculpin, ni entre l'insuline de boeuf et de morue chez la myxine. -- Les r6sultats indiquent qu'il existe nne sp6eifieit6 d'esp~ce de l'insuline aussi bien immunologique qu'hormonale. Vergleichende Untersuchungen zur Immunologic und Biologic verschiedener Insuline Zusammenfassung. Einige immunologische u n d physiologische Eigenschaften der bei einer l%eihe yon Wirbeltieren und Wirbellosen gewonnenen Insulinpr/~parate wurden mit entspreehenden Eigenschaften kristalliner Insuline vom R i n d u n d vom I)orsch verglichen. Gleichzeitig wird tiber die jeweiligen Ausbeuten an I n s u l i n bei den E x t r a k t i o n e n berichtet. -- Anti-Rinderinsulin-Seren vom Meerschweinehen u n d vom Pferd, die bei der R a t t e eine starke Hyperglyk~mie hervorrufen, bewirken beim Seeskorpion oder Seeteufel (Cottus scorpius) aus der Klasse der Knochenfische u n d beim gemeinen Inger oder Schleimaal (Myxine glutinosa) aus der Klasse der I~undm~uler keine Hyperglyk/hnie. Die Gabe kristallinen Rinder- bzw. Dorschinsulins bewirkte beim Seeskorpion u n d auch beim Schleimaal eine in einigen F~llen tSdliche Hypoglyk/~mie, jcdoch erst bei sehr hoher Dosis u n d nach mehrt/igiger Beobaehtungszeit. Diese Befunde standen im Gegensatz zur ausgepr~gten und raseh einsetzenden Hypoglyk/~mie beim Schleimaal wenn homologes Insulin gegeben wurde. Rinder- u n d Dorsehinsulin erwiesen sich beim Sehleimaal als etwa gleich wirksam. Beim Seeskorpion zeigten Insuline vom Rind, vom Dorsch, und das homologe Insulin des Seeskorpions ann/ihernd gleiehe blutzuekersenkende Wirkungen. -- Die Versuchsergebnisse zeigen, dal3 sich neben der immunologischen Speziesspezifit~it verschiedener Insuline in einigen F~llen auch eine Speziesspezifit/it der biologischen Wirkung nachweisen l~il~t. Key-words: Insulin, invertebrates, Cyclostomes, Elasmobranchs, Teleost fish, Amphibians, Chicken, Ox, Guinea Pig, comparative endocrinology, comparative immunology, blood glucose, anti-insulin sera, passive cutaneous anaphylaxis test. its biological a c t i o n is n o t o n l y of general i n t e r e s t i n t h a t field of h o r m o n e research dealing w i t h the relationship of s t r u c t u r e to biological a c t i v i t y of a h o r m o n e b u t is also of great i m p o r t a n c e i n m a k i n g s y n t h e t i c insulin. Here, scientific i n v e s t i g a t i o n s i n the field of c o m p a r a t i v e endocrinology m a y be valuable. R e c e n t work with isolated A- a n d B-chains i n c l u d i n g r e s y n t h e sis of cod a n d ox insulins, p r e p a r a t i o n of cod-ox " h y b r i d " i n s u l i n s (WILson et al., 1962, 1966) , studies w i t h sulfated i n s u l i n (MoLoNEu et al., 1964), a n d c o m p a r a t i v e studies of the a m i n o acid composition of several types of v e r t e b r a t e (WILSO~ a n d D i x o n , 1961 ; FALK~EI~ a n d W ~ s o ~ ~, 1965; SmTH, 1966) a n d inv e r t e b r a t e (WILSO)r a n d S. FAT,K~XEI~and S. WILSON: Comparative Immunology and Biology of Insulin have indicated t h a t the a c t i v i t y of the insulin molecule is dependent on the correct, i n t a c t three-dimensional organization of the three SS-bridges (cf. CArPEnTEr, 1966; SMIT~, 1966), and t h a t the a n t i b o d y . c o m b i n i n g site m a i n l y concerns the A-chain (WILSO~ et al., 1962), particularly the As_l~ regions, b u t also loci within the sequences B1-8ano B24_3o(WILSON et al., 1967). I n regard to the biological a c t i v i t y of various t y p e s of insulins, no significant variation was previously f o u n d when the a c t i v i t y was measured b y the s t a n d a r d mouse convulsion or blood glucose assays, n o t even with guinea pig a n d some fish insulins which differ m a r k e d l y in amino acid sequences a n d in immunological properties ( E ~ i s c ~ I , 1965; SMITH, 1966) . l~ecently, however, preliminary work with guinea pig insulin suggested t h a t it m a y be only a b o u t a quarter as active as cattle insulin when measured in mouse convulsion a n d mouse blood glucose assays, a l t h o u g h almost fully active when measured b y guinea pig blood glucose assay (SmTH, 1966). Against this b a c k g r o u n d it was considered w o r t h while to e x t e n d our previous immunological studies of insulins from lower vertebrates in order to compare the biological activities of various t y p e s of v e r t e b r a t e insulins in n o n - m a m m a l i a n test systems. A n o t h e r reason for extending our previous studies was t h a t it has been questioned whether insulin in lower vertebrates, such as marine teleosts, actually is involved in fat and carb o h y d r a t e metabolism a n d thus the regulation of the blood sugar, or whether its main role concerns protein metabolism (TAs~I~X and CXHILL, 1964). present report gives the results of some immunological studies of vertebrate and i n v e r t e b r a t e insulins, m a i n l y fish insulins, and reports the variations in blood glucose in the marine teleost Cottus scorpius a n d the cyclostome M y x i n e glutinosa, evoked b y administration of anti-ox insulin sera as well as b y ox and cod insulins. The effects of the foreign insulins on the level of blood glucose are c o m p a r e d with those evoked b y acid alcohol extracts from the isolated islet tissue of Cottus a n d Myxine, respectively. Material and methods I m m u n o l o g i c a l E x p e r i m e n t s Acid alcohol extracts for obtaining insulin preparations were made from the pancreas, the principal islets, or parts of the digestive tract from the following species: Mammals: Ox (Bos taurus) Guinea Pig (Cavia porcellus) Birds: Chicken (GaUus domesticus ) Amphibians: American Bullfrog (Rana catesbeiana) Bony fish: Sculpin (Cottus scorpius) T u n n y (Thunnus thynnus) Bonito (Katsuwonus pelamis) Pollock (Poltachius virens) Cod (Gadus eallarias) Catfish (Ictalurus punctatus) Bowfin (Amia calva) Cartilaginous fish: Spiny Dogfish (Squalus acanthias) Lemon Shark (Negaprion brevirostris) Jawless fish: Tunicates : Echinoderms: Molluscs : Hagfish (Myxine glutinosa) Sea Squirt (Ciona intestinalis ) Purple Starfish (Pisaster ochraeeus) Red Starfish (Asterias rubens) Octopus (Eledone cirrata) Oyster (Ostrea lurida) Certain of these preparations were purified further (WILso~ and F~_LKMEa, 1965). Where enough material was available, the insulin content was assayed b y the mouse convulsion test (Yov~o and LEWIS, 1947) , otherwise the mouse hemidiaphragm assay (WA~DLAW and ~r 1961) was employed. The tests for insulin-like material in extracts from the digestive tract of invertebrates were more comprehensive, including correlated biochemical, immunological, histological, and histochemical investigations as described previously (WILso:~ and FiI, KM~,~, 1965; WILSO~ et al., 1966). For the immunological studies guinea pig antisera to purified ox, chicken, and cod insulins were employed. I n pilot studies insulin preparations from the cod, catfish, and purple starfish were assayed by the doubleantibody procedure of HAL~s and RA:NDLE (1963) (tests kindly performed by Dr. K. S. I~AS~OGIand Dr. J. C A ~ B]~LL). Passive Cutaneous Anaphylaxis Test Depilated albino guinea pigs weighing 250--300 g were injected intracutaneously in sites 5 cm apart with 0.1 ml aliquots of undiluted guinea pig antisera to S-sulpho-ox-A-chain, S-sulpho-ox-B-chain, ox, chicken, and cod insulins (WILsoN et al., 1966, 1967) . The guinea pigs were challenged intravenously 20--22 hours later with 0.5 ml of a saline solution containing the insulin preparation, together with 5 mg Pontamine sky blue 6 BX. The diameters of the blue spots appearing at the sites of the serum injections were measured 10-20 rain later. 1. Administration of Anti-Ox-Insulin Sera a) To Cottus scorpius : Details concerning the capture and maintenance of the fish, as well as the general outline of the blood glucose experiments have been given in preceding papers (FALKMER,1961; FALKMEtr and MATTY, 1966a) . The investigation was performed in December at a water temperature of 8~ Freeze-dried guinea pig antiserum to ox insulin was redissolved in glass-redistilled water to make a solution with an ox-insulin-inhibiting potency of 1 I.U. of insulin per ml, as determined by the mouse convulsion assay. The same amount of freezedried normal guinea pig serum was reconstituted with redistilled water to be used in the control experiments. Ten fasting adult sculpin of both sexes, with the same female predominance as previously (FALKMER, 1961) and with a mean body weight of 125 g (range 75-- 185 g), were injected intramuscularly with approximately 1 ml/kg body weight (range 0.6--1.3 ml/kg) of the guinea pig anti-ox-insulin serum solution. The blood glucose content was assayed b y the glucose oxidase procedure at the following observation times: 0, 6, 12, 24, 48, 72, and 96 hours after the injection. After the last sampling of blood the fish were killed, the principal islets excised and fixed in Bouin's fluid. After alcohol dehydration and clearing in xylene and methylbenzoate, the islets were embedded in paraffin. Sections, approximately 5tt thick, were stained with van Gieson's stain and Maske's modification of the aldehyde fuchsin procedure (FALK~E~, 1961). The control experiments with the solution described above of normal guinea pig serum were performed on 11 fasting adult sculpin with a similar sex and weight distribution (mean 135 g ; range 95--195 g). The blood glucose investigations and the histological examinations were identical to those of the experiments with anti-ox-insulin serum. b) To Myxine glutinosa: Details concerning the capture and maintenance of the animals, as well as the blood sampling, haematocrit, and glucose assay procedures have been given in preceding papers (FALKMEI~and WINBLAI)H, 1964a and b ; FaI~xN_EI~and lCLkT~y, t966b). The experiments were performed in 0etober and November at a water temperature of 1O~ - 14~ and at a water salinity of 33 per thousand. All animals were fasting. Freeze-dried, horse anti-ox-insulin serum (lot No. 2712) was reconstituted with redistilled water giving an estimated ox-insulin-inhibiting capacity in the mouse convulsion assay of 1.6 I.U. of insulin per ml. Approximately the same concentration (per mg dry substance) of similarly redissolved freeze-dried, normal horse serum was used for control experiments. Ten adult hagfish of both sexes, weighing between 25--40 g, were injected intramuscularly with 1 ml per fish of the solution of horse anti-oxinsulin serum. Two animals were killed at each of the following time intervals after injection: I, 4, 8, 20, and 44 hours. Exactly analogous experiments with the solution of normal horse serum were performed on I0 hagfish of the same sex and size distribution. In another experiment 15 adult hagfish of both sexes with a mean body weight of 44~ (range 30--60) g were injected intramuscularly with 2 ml of the same solution of horse anti-ox-insulin serum as above, a n d 5 animals killed a t each of the time intervals 8, 20, and 44 hours after the administration. Fifteen adult hagfish of both sexes and of the same size were used for analogous control experiments with the solution of norreal horse serum. W h e n the animals were killed, the islet tissue was excised for light microscopical examination as described above for the corresponding sculpin experiments. I n some eases pieces of islet tissue were also fixed in osmium tetroxide for electron microscopical investigations, using the same methods as previously described (FALK~EI~ and WINBSADH, 1964a and b) . The potencies of the anti-ox-insulin sera were also tested in pilot experiments in rats of both sexes weighing about 200 g. Animals were injected with a series of doses from 0 . 1 - 20 U per animal, and the blood glucose level was checked 20 rain after each injection. 2. Administration of Ox Insulin K i n d l y supplied b y Mr. R. BEI~GlgAN,Vitrum AB. S. FALKMEt~and S. WILSOl~: Comparative Immunology and Biology of Insulin b) To Myxine glutinosa : Previous investigations (FALK~E~ and MALTY, 1966b) were supplemented by identically performed experiments with the dose levels 100 and 20 I.U./kg. The water temperature was 10~ (adjusted b y a cooling device). The experiments were made in July. The animals were of the same sex a n d size distribution as previously used. No morphological examination of the islet tissue was made this time. 3. Administration of Cod Insulin I n all experiments with cod insulin, a preparation (lot 10/3c/F4) of the same type as described previously (WiLso~ and DIxoN, 1961) was used. Its potency was 15 I . U . / m g (95 ~/o confidence limits: 12--20 I.U./mg). I t was dissolved (4 mg/ml) in 0.01 M HC1 and further diluted with physiological saline to a concentration suitable for the dosage desired. The stock solution was kept at + 4~ a) To Cottus seorpius: The dosages were 100 (range 69--125), 20, 5, and 1 I . U . per kg body weight. The general outline of the investigations followed t h a t of the corresponding ox insulin experiments as closely as possible. They were performed in June, July, and Decem. ber. b) To Myxine glutinosa : The general outline of the cod insulin investigations in the haggish also followed t h a t of the corresponding ox insulin experiments as closely as possible, and were performed at the same time. The dosages wore i00, 20, and 5 l.U./kg. 4. Administration of Crude Cottus and Myxine Insulin All the details concerning the preparations of hagfish insulin have been reported previously (FALx~XEIZ and MATTY, 1966b) . The general outline of the sculpin insulin experiments was almost exactly the same. Principal islets from 91 adult sculpin of both sexes were dissected, making up a total a m o u n t of tissue of 492 mg wet weight, and a crude sculpin insulin preparation was obtained b y the same procedure as t h a t used for the hagfish islets (FAr.XME~and MATT:g,1966b) . The potency of the sculpin and hagfish preparations was assayed by the mouse convulsion procedure (see Results). The experiments were performed in J u n e and December at the water temperatures 15~ and 8~ 10~ Three series of fish were used, each containing 6 adult sculpin of both sexes with a mean body weight of 100 g (range 80--150). The crude sculpin insulin preparation was injected intramuscularly at dose levels estimated to be approximately 20, 5 and 1 I . U . per kg body weight. Blood glucose determinations were made at the same time intervals as in the experiments with administration of ox and cod insulin. No morphological examination was performed at the end of the investigations, as this has been made previously (FALKI~:S:a, 1961) . I n all types of experiments crystalline ox insulin (Vitr u m AB, Stockholm K ; lot No. 6241) with a potency of 25.9 I.U. per mg air dry substance was used. I t was dissolved in either 0.01 M HCI or 0.1 31 NaC1-NaOt{ buffer p H 9.0 (P]~AIzs~, 1960) . F r o m preceding investigations it was known (FALy:~mz, 1961; F~tLKME~ and MATTY, 1966b) t h a t these solvents, at the volumes and concentrations used did not affect the blood glucose levels of the sculpin and the hagfish. Only a single dose was given. a) ~o Cottus scorpius: The dosages were: 1000, 100, 20, 5, and 1 I . U . per kg body weight. Six adult fish of Results both sexes, weighing between 80 and 250 g, were injected at each dosage. Both fed and fasting fish were used, and before the experiments the available lot was evenly dis- C o n t e n t a n d I m m u n o l o g y of I n s u l i n from Various t r i b u t e d with regard to size, sex, and feeding state into Types of Islet Tissue 6 groups. The dose level used in each group was determined by lot. The water temperature was approximately The yields of i n s u l i n a c t i v i t y in the acid alcohol either 10~ or 15~ At the dosage 1 I.U./kg the experiments were performed at both temperatures; for the extracts of tissues supposed to c o n t a i n insulin-producother dose levels the water temperatures are given in ing cells are given in Table 1. A s u m m a r y of p r e v i o u s l y Tables 3 and 4 and in the diagrams. The experiments were o b t a i n e d results of i n h i b i t i o n e x p e r i m e n t s with g u i n e a performed in J u n e and July. Blood glucose assay was pig a n t i - o x - i n s u l i n a n d a n t i - c o d - i n s u l i n sera are also paperpfloyrimnged p0r,e vi,io2u,s3ly, 4,de5s,carnibded6 dparyosceadftuerresthe (FiAnLje~cr~tai-oEnlZ,, i n c l u d e d in the Table. 1961; FALZ~MERand 2MAmTY, 1966a) . No morphological A f a i n t b u t definite i n s u l i n a c t i v i t y was f o u n d i n t h e investigations were made. Sea S q u i r t a n d the Starfish where p s e u d o i s o c y a n i n positive cells occur i n the digestive t r a c t epithelium. The insulin-like a c t i v i t y of the Sea S q u i r t was abolished S. FALKMER and S. WILSOl~: Comparative I m m u n o l o g y and Biology of Insulin ( P ~ 0.001) b y a 66-fold excess of guinea pig a n t i s e r u m to ox insulin. A more d e t a i l e d r e p o r t of these results is p r e s e n t l y being p r e p a r e d . So far, possible i n s u l i n - p r o d u c i n g cells h a v e n o t been f o u n d in t h e digestive t r a c t s of t h e o c t o p u s or p h y l o g e n e t i c o r d e r ( N o I ~ A ~ a n d GREEI~WOOD, 1963) a n d n e u t r a l i z a b i l i t y w i t h i n t h e g r o u p of Telcosts. There was r e l a t i v e l y little c r o s s - r e a c t i v i t y b e t w e e n ox a n d cod insulins, b u t insulin from a low-order t e l e o s t (Bowfin) was f o u n d to be r e a d i l y n e u t r a l i z a b l e b y b o t h 1 Potency of ox, guinea pig, frog, chicken, pollock, cod and catfish insulin preparations b y mouse convulsion assay (YouNG and LEwis, 1947) ; other preparations b y 4-point mouse hemidiaphragm assay (WARDLAW and I~OLOI~IEu 1961) . 2 Only performed as 3-point mouse hemidiaphragm assays. oyster, a l t h o u g h a n a c i d - a l c o h o l e x t r a c t from t h e o c t o p u s e n h a n c e d g l y c o g e n d e p o s i t i o n in t h e mouse d i a p h r a g m a s s a y of insulin a c t i v i t y (Table 1). I t is obvious from T a b l e 1 t h a t w i t h t h e n o t a b l e e x c e p t i o n of t h e g u i n e a pig, h i g h - o r d e r species a p p e a r e d to h a v e n e u t r a l i z a b l e insulins, whereas t h e m a j o r i t y of t h e fish insulins t e s t e d were i m m u n o l o g i c a l l y u n l i k e ox insulin. H o w e v e r , t h e r e was no r e l a t i o n s h i p b e t w e e n antisera, suggesting t h a t i t h a d a n t i b o d y - c o m b i n i n g sites in c o m m o n w i t h b o t h insulins. The m o s t interesting finding, however, was t h a t t h e h o r m o n a l a c t i v i t y of all t h e insulins could be n e u t r a l i z e d specifically b y b o t h a n t i s e r a , p r o v i d i n g t h a t e n o u g h of t h e l a t t e r was a d d e d . The o n l y e x c e p t i o n was g u i n e a pig insulin, which was n o t n e u t r a l i z e d b y a t least a 250 fold excess of a n t i - o x - i n s u l i n serum. 35 E 30 o o E' o tj D ..J 15 El 1o3 10 O _.1 El 5 O, .... .. S. FALK~E~ and S. WILSOX: Comparative Immunology and Biology of Insulin P r e l i m i n a r y results (Table 2) w i t h some i n s u l i n p r e p a r a t i o n s i n the P C A test (of. WILSO~ et al., 1966, 1967) are largely i n a g r e e m e n t w i t h those from the h o r m o n e - i n h i b i t i o n test a n d show t h a t insulins from widely differing species m a y h a v e a n t i g e n i c deterinsulins are a n t i g e n i c a l l y like ox i n s u l i n ; catfish a n d sea s q u i r t insulins are like chicken insulin, whereas sculpin a n d pollock insulins are like cod insulin. I n s u l i n s from the cod, catfish, a n d starfish did n o t affect the binding of ox insulin-I T M to guinea pig antii ANTI-OX-INSULIN GUINEA-PIG SERUM (3--0 NORMAL GUINEA-PI6 SERUM . . . . . . . . . . , . . . . . , . . . . . , . . . . . , . . . . . , . . . . . , . . . . . , 6 12 18 24 30 36 h2 4.8 HOURS AFTER INJECTION Fig. 2. Mean blood glucose values (A--z~) of 10 Myxine glutinosa (the hagfish) at various time intervals after a n intramuscular injection of anti-ox-insulin horse serum at a dosage found to make 200 g rat hyperglycaemic. Ten hagfish injected with the same a m o u n t of normal horse serum were used as controls (curve of mean values: 9 O). Same signs and symbols as in Fig. 1 ox-insulin serum i n the d o u b l e - a n t i b o d y procedure. The biological potencies of the three solutions supplied were 10 a n d 1 U / m l (mouse convulsion assay) for cod a n d catfish i n s u l i n a n d 0.02 U / m l (mouse hemidiap h r a g m assay) for starfish insulin. Thus, these i n s u l i n s c a n n o t be assayed b y this tracer technique. I n the pilot i n v e s t i g a t i o n s on r a t s 1 U of the guinea pig a n t i - o x - i n s u l i n 8'4 9'6 serum used was found to make a 200 g rat hyperg]ycaemic as early as 90 min after the injection. Previously published comparisons between the guinea pig and horse antisera used in this investigation (MoLoNEu and API~ILW, 1960) showed that the horse anti-ox-insulin serum was diabetogenic although less avidly than that from the guinea pig. The results of the experiments with the sculpin are given in Fig. 1, and those of the first type of the experiments with the hagfish in Fig. 2. On the basis of previous findings (FALKM]~I~, 1961; FALI{MER a n d " MATTu 1966a) one sculpin of the control group with Diabetologia, Vol. 3 S. F~d~KMEH and S. WILson: Comparative Immunology and Biology of Insulin m a r k e d hyperglycaemia, a p p a r e n t l y connected with clumsy blood samplings, skin ulcers, a n d anemia, was excluded from the diagrams. A l t h o u g h the ranges of the i n d i v i d u a l o b s e r v a t i o n t i m e values h a v e b e e n o m i t t e d from t h e diagrams, it is obvious from the given m e a n values t h a t the endogenous i n s u l i n i n the sculpin a n d the hagfish was n o t readily n e u t r a l i z e d in rive b y the two types of a n t i s e r u m . The second t y p e of the e x p e r i m e n t s w i t h the hagfish showed t h a t n o t even the double dose of antiserum i n a larger series caused a n y obvious blood glucose rise. The m e a n values a n d the range of b o t h the blood glucose a n d h a e m a t o c r i t values were a l m o s t exactly the same as i n the control group, a n d were w i t h i n the n o r m a l range a t all three o b s e r v a t i o n times. Neither the light microscopical n o r the u l t r a s t r u c 100 II 1 I 5" 1"'20 Fasting [[ Fasbing [ Fasting [ "Fasting i~ ~2 ; ~ ~ ~,: ~ ~ ~;~2 ~ ~;2~ ~ ;~L~;2 ~ ~ ~;2~ L2;2L~ ~ ~;2~;2 ~ ~;~ Insulin~ (Atstar~ of the experim.) . 49--72. 0--48 73--96 97--120 ~[21--144 (At end of the experim.) The term " H y p o " (hypoglyeaemia) signifes a blood glucose value t h a t is both significantly outside the range of 2 times the Standard Deviation of the initial blood glucose level of the animals and less t h a n 10 mg/100 ml (ef. F~A~K~EH t 961).I n the term "Normo" (normoglycaemia) isolated fish with obviously hyperglyeacmie blood glucose levels are also included. The figures in the columns give the n u m b e r of fish. - A long hyphen in a broad major column signifies t h a t no observations were made at the time interval of t h a t column. All hagfish were fasting. - The term " H y p o " (hypoglyeaemia) signifies a blood glucose value t h a t is both significantly outside the range of 2 times the Standard Deviation of the initial blood glucose level of the animals and less t h a n 10 rag/ 100 ml (of. F ~ X ~ E H and MAWTY,1966b) . - I n the term "Normo" (normoglycaemia) isolated f s h w i t h obviously hyperglycaemie blood glucose levels are also included. The figures in the columns give the n u m b e r of hagfish. - A long hyphen in a broad major column signifies t h a t no observations were made at the time interval of t h a t column. Fig. 3. I n d i v i d u a l blood glucose fluctuations of 6 Co#us scorpius injected intramuscularly with 20 I . U . / k g of crystalline ox insulin. The h e a v y black line gives the mean initial blood glucose level of ~he animals and the dashed lines limit the range of 2 times the s t a n d a r d deviation. The sign ~ indicates d e a t h of a fish OX INSULIN IN THE SCULPIN. ~Nx~_ 1000 ].U./kg. 15'C. Fed Fish A \ / k _/__.__~_ 25 oE 20 r u r a l i n v e s t i g a t i o n s of t h e sculpin a n d hagfish islet I n t h e sculpin this h y p o g l y c a e m i a was often a s s o c i a t e d tissue disclosed a n y significant d e v i a t i o n s f r o m t h e w i t h i m b a l a n c e a n d u n c o o r d i n a t e d s w i m m i n g m o v e n o r m a l in a n y of t h e g r o u p s a t a n y of t h e o b s e r v a t i o n m e n t s a l t e r n a t i n g w i t h a e o m a t o u s s t a t e (el. F ~ K ~ , times. i J 1961). N o such effects were n o t e d in t h e hagfish (cf. FALK~tER a n d MATTY, 1966 b) . C O D INSULIN IN THE SCULPIN O 1;;d CFihs O / ....~.i...-./.~. ~ "~ 1 3 4 5 6 DAYS AFTER INSULIN INJECTION Fig. 4. I n d i v i d u a l blood glucose variations of 6 Cottus scorpius injected intramuscularly with 1000 I . U . / k g of crystalline ox insulin. The h e a v y black line gives the m e a n for u n t r e a t e d fish and the dashed line limits the upper range of 2.58 times the s t a n d a r d deviation. Otherwise same signs and symbols as in Fig. 3 Administration of Various Types of Insulin T a b l e s 3 a n d 4 a n d Figs. 3 - - 8 c l e a r l y show t h a t h y p o g l y c a e m i a a n d d e a t h could be e v o k e d in b o t h t y p e s of fish w i t h all t h e t h r e e t y p e s of insulin t e s t e d . DAYS AFTER INSULIN INJECTION Fig. 5. I n d i v i d u a l blood glucose variations of 6 Cottus scorpius injected intramuscularly with 20 I . U . / k g of crystalline cod insulin. Same signs and symbols as in Fig. 3 Fig. 6. I n d i v i d u a l blood glucose variations of 6 Cottus scorpius injected intramuscularly with 100 I . U . / k g of crystalline cod insulin. S a m e signs and symbols as in Fig. 3 In the experiments with the sculpin the results do not, however, allow a n y detailed quantitative comparison between the effects of the various types of insulin used, as it is obvious that the feeding state of t h e fish a n d t h e t e m p e r a t u r e of t h e w a t e r influenced t h e b l o o d glucose response t o a t l e a s t t h e same e x t e n t as d i d t h e insulin itself (el. Figs. 3 a n d 4). As f o u n d p r e v i o u s l y (FALKMER, 1961; F A L K ~ a n d M~TmY, S. FAT~KME~and S. WILSON: Comparative Immunology and Biology of Insulin 1966a and b), hypoglycaemia and death were b o t h fairly easily elicited in fasting fish at higher temperatures, whereas fed fish at lower temperatures were markedly resistant to even tremendous doses of ox and cod insulin (Tables 3 and 4). I n general, however, 25 , 1 2 3 4 5 6 DAYS AFTER INJECTION OF ISLET EXTRACT Fig. 7. Individual blood glucose fluctuations of 6 Go#us scorpius injected intramuscularly with an acid-alcohol extract of sculpin islet tissue, constituting a crude insulin preparation corresponding to a dosage of 1 I.U./kg of sculpin insulin. Signs and symbols are analogous to those of Fig. 3 ' 2 4 ' 4'8 ' 7'2 ' 9'6 ' 1890 HOURS AFTER INJECTION OF ISLET EXTRACT Fig. 8. Similar type of experiment as in Fig. 7 but using a dose of about 20 I.U./kg of crude sculpin insulin. Signs and symbols are analogous to those of Fig. 3 increasing the insulin dosage increased the frequency of hypoglycaemia and death in both fish, and in the sculpin no m a r k e d differences were noted between ox, cod, and sculpin insulins. 25 ( : ~ -~ 20 i ~ o s UA CO (D D.J ld f~ O O .J m 5 EXTRACT OF SCULPIN ISLETS. APPROX. DOSE:0.04g/FISH (Aboui 20 I.U./kg.of sculpininsulin) ~0"c Fasting Fish I n the experiments with the hagfish, where only fasting fish at more strictly controlled temperatures were used, the hagfish's own insulin was m a r k e d l y more effective t h a n either ox or cod insulin in evoking hypoglycaemia. No m a r k e d difference between ox and cod insulin was noted in the hagfish either. The transient hypoglycaemia produced in the hagfish b y 5 I . U . hagfish insulin per kg occurred earlier and was more pronounced t h a n t h a t caused b y either 20 I . U . cod insulin or 100 I. U. ox insulin per kg. A rough comparison of cod and ox insulins in the mouse hemidiaphragm assay at 37~ and 17~ showed t h a t the relative potency of purified cod insulin versus crystalline ox insulin standard was 0.9 (0.7--1.4) at 37~ and 0.6 (0.2--2.2) at 17~ Thus, the two hormones had apparently similar t e m p e r a t u r e coefficients with respect to their effects on glycogen synthesis in the m a m m a l i a n system. Discussion The observation t h a t also the sea squirt seems to contain insulin-producing cells, whereas so far no convincing evidence for insulin production has been obtained in the Molluscs, conforms with our present working hypothesis t h a t insulin mainly occurs in the Deuterostomes (Fig. 9). Here, however, further in MAMMALIA I AVES REPTILIA AMPHIBIA OSTEICHTHYEB CHONDRIDHTHYES AGNATHA ARTHROPODA I | I TUNICATA MOLLUSCA1 PROTO- ~ STOMIA y [ ECHINODERMATA ~ DEUTERO S T O N I A (PROTOZOA) l~ig. 9. Much simplified phylogenetie tree showing some groups of animals where tests have been made on the occurrence of insulin vestigations are in progress, as this question m a y be of great phylogenetical interest. The high yield of insulin from sculpin islets is in good conformity with the old reports of McCorMICK and NOBLE (1924 ) and VInCEnT et al. (1925 ), and with the quantitative morphological studies of the islet tissue of this species (FALKME~, 1961), which showed " g i a n t " pure islets with about 25% fl-cells without a n y admixture of traversing strands of acinar tissue. There is also a fairly good correlation between the insulin content and the morphology of the islet tissue in b o t h S. FALKMERand S. WILSON: Comparative Immunology and Biology of Insulin the cod and the hagfish (cf. FALK?aEI~and WINBLADtI, which differs m a r k e d l y from ox insulin b o t h in 1964a) . The relatively low yield of insulin in m o s t other immunological behaviour and in amino-acid sequence species investigated can also be easily understood from (WILSO?r and D i x o n , 1961) . The sculpin's own insulin the histological appearance of the islets. This is was also surprisingly inefficient at short observation especially true of the shark pancreas, which is of the times at the lowest dose (Fig. 7). I t is thus still possible "mammalian" t y p e (cf. FEt~NE~ and KERN, 1964), and t h a t insulin is involved only to a small extent in the of the digestive epithelium of the pyloric coeca of the blood sugar regulation of Teleosts. starfish and the digestive t r a c t of the sea squirt where The finding t h a t in the hagfish the crude native the a p p a r e n t l y insulin-producing cells only constitute insulin preparation was much more effective in proa small fraction (WILSON and FALKMER, 1965) . ducing hypoglyeaemia t h a n either of the two foreign crystalline insulins, m a y be due to a real speciesBlood Glucose Experiments specificity of the hormone or to inadequate assessment of the potencies of the crude insulin preparation in Administration of A nti-Ox-Insulin Serum the m a m m a l i a n test systems used. However, if the latter explanation has some bearing, it m a y also be an P o t e n t anti-ox-insulin sera were unable to provoke expression of a species-specificity of the hormone, like hyperglycaemia in the sculpin and the hagfish, that for guinea pig insulin referred to in the Introducindicating t h a t the endogenous insulin of these two tion. Moreover, it is seen from Table 1 that fairly large species was got readily neutralized b y the antisera in fluctuations could occur in the insulin yields of vivo. The absence of a n y light microscopical or ultra- different preparations. structural changes in the islets was in conformity with Thus the main results of the present investigations this stability of the level of blood glucose. These are t h a t insulin a p p a r e n t l y induced hypoglycaemia in results contrasted with the ease of producing hyper- both Teleosts and Cyclostomes, as it does in higher glycaemia with the same antisera in the mouse and rat, species ; and t h a t there is a species-specificity of insulins but were in agreement with the immunological differ- from a wide range of animals, both immunologically ences between the fish insulins and m a m m a l i a n and with regard to its hormonal action as measured b y insulins. its effects on the blood glucose level. This speciesspecificity not only manifests itself between different Administration of Various Types of Insulin groups of animals but also within the large animal The present results, together with those previously groups. Similar reports have previously been given for obtained (FAL:K.2c[EI~,1961; F ~ : ~ n ~ and MATTY, other protein hormones, e.g. thyrotropin (ef. B~RmNG1966b) , clearly show t h a t b o t h ox and cod insulin TON, 1964). Moreover, MAcA~THV~ and FISHER affect the blood glucose level in b o t h Teleosts and (personal communication) recently found in a study on Cyclostomes. The fact t h a t TASmMA and CA~IILL(1964) the action of frog, fish, and ox insulins on frog muscle failed to obtain a n y hypoglycaemia in the marine tissue, t h a t bullfrog insulin evoked a significantly teleost Opsanus tau (the toadfish) with ox insulin a,t a faster increase in respiration of leopard frog muscle dosage of 100 I. U./kg i.v. m a y be explained b y several tissue t h a n did cod or ox insulins. interfering factors. Firstly, it is well known t h a t the The present results do not permit a n y speculations intravenous p a t h of injection is ineffective in fish, concerning the influence of t e m p e r a t u r e upon the p r o b a b l y due to sensitive vasoregulatory reflexes and activity of sculpin and hagfish insulins (el. BArRIO-Gpoor mixing conditions in the single circuit circulation TON, 1964). However, we have found t h a t cod insulin system of Teleosts (ef. lVAL~:MEg, 1961; LAZAROW, has the same potency relative to ox insulin at 17~ and 1964). Secondly, the present investigation has shown at 37~ in the mouse hemidiaphragm assay. The fish t h a t the blood glucose level of fed fish (the toadfish and m a m m a l i a n hormones, therefore, appear to have were k e p t in cribs in the open sea) is fairly resistant to similar t e m p e r a t u r e coefficients in the m a m m a l i a n test ox insulin, even when tremendously high doses were system. applied (Fig. 4). Thirdly, it is essential to follow the blood glucose level of each individual fish separately References b y repeated blood samplings in vivo in order to detect transient hypoglycaemias occurring in isolated fish. BAT~hgelNEGnTgOliNsh,E U.Jn.iWve.rs:iHtioesrmPorensess, apn.d1e0v4o-l-u1t1i8o,n.1L96o4n.don, However, even when all these precautions are t a k e n CA~:e~NTEm F . H . : Relationship of structure to biological into consideration (intramuscular p a t h of admini- activity of insulin as revealed by degradative studies. stration without lengthy manipulations, fasting fish- Amer. J. Med. 40, 750--758 (1966). and long observation times with regular blood samp- CORBeBp,oRr.t,s Tf.roHmANMLeEeYti,nI.gLs:. SA. nMtiIbToCdHieEsL LtoanidnsGu.liIn-I.. SDMiaITbeH-: ling), it is, to judge from the present investigations in tologia 2, 133 (1966). the sculpin, correct to state t h a t the blood glucose Ea~iscH, A. : Zum physiologisehen und immunologisehen level of Teleosts seems m a r k e d l y resistant to admini- Insulin-Naehweis bei Neunaugen. Aeta biol. reed. gerstration of ox insulin (ef. FALKMER, 1961). I t is inter- FAmLKan~.E11%5,S1.:93E-x-p1e9r6im(1e9n6t5a)l. diabetes research in fish. On esting to note t h a t this also holds true for cod insulin, the morphology and physiology of the endocrine pan Prof. STURE FAL~MER Institute of Pathology University of Ume~ Ume~ 6, Sweden c r e a t i e tissue of t h e m a r i n e t e l e o s t Cottus scorpius w i t h special reference to t h e role of g l u t a t h i o n e in t h e mec h a n i s m of a l l o x a n d i a b e t e s using a modified n i t r o - prusside m e t h o d . A c t a endocr . (Kbh.) , Suppl . 59 ( 1961 ). - - , a n d A . J . MATTY: T h e p i t u i t a r y g l a n d a n d its role in t h e blood sugar r e g u l a t i o n in a m a r i n e teleost, Cottus scorpius. A c t a See. reed. U p s a l . 71 , 1 5 6 - - 1 7 2 ( 1966a ). -- -- B l o o d sugar r e g u l a t i o n in t h e hagfish, Myxine glutinosa . Gen. Comp. E n d o c r . 6 , 3 3 4 - - 3 4 6 ( 1966b ). - - , a n d L. WINBLAD~ : A n i n v e s t i g a t i o n of t h e p a n c r e a t i c islet tissue of t h e hagfish (Myxine glutinosa) b y light a n d electron microscopy. I n " T h e S t r u c t u r e a n d Metab o l i s m of t h e P a n c r e a t i c I s l e t s " (S. E . 13ROLIN , 13. H E L L - MAX, a n d H . KNUTSON, eds.). Oxford, P e r g a m o n Press, p. 1 7 - - 3 2 , 1964a. - - -- S o m e a s p e c t s of t h e b l o o d sugar r e g u l a t i o n of t h e hagfish, Myxine glutinosa. I n " T h e S t r u c t u r e a n d Metab o l i s m of t h e P a n c r e a t i c I s l e t s " (S.E. 13ROLIN, 13. I'IELLMAN a n d H . KNUTSON, eds.). Oxford, P e r g a m o n Press, p. 3 3 - - 4 3 , 1964b. - - , a n d S. WILSON: C o m p a r a t i v e i m m u n o l o g y of insulin . Gen. Comp. E n d o c r . 5 , 676 ( 1965 ). FERNER , H., a n d H . KERN: T h e islet o r g a n of Selachians. I n " T h e S t r u c t u r e a n d M e t a b o l i s m of t h e P a n c r e a t i c I s l e t s " (S.E . 13ROLIN , 13 . HELLMAN, a n d I-I . KNUTSON, eds.). Oxford, P e r g a m o n Press, p. 3 - - 1 0 , 1964 . HALES , C . H . , a n d P . J . RANDLE : I m m u n o a s s a y of insulin w i t h a n t i b o d y p r e c i p i t a t e . L a n c e t 1963 I, 200 . L A z ~ o w , A. : Discussion. I n " T h e S t r u c t u r e a n d Metab o l i s m of t h e P a n c r e a t i c I s l e t s " (S.E . 13ROLIN , 13 . HELLMAN a n d H . KNUTSON, eds.). Oxford, P e r g a m o n Press, p. 43 , 1964 . McCoRMICK , N . A . , a n d E . C . NOBLE: T h e yield of insulin f r o m fish . Contrib. Canad. 13iol. 2 , 115 -- 128 ( 1924 ). MOLONEY , P . J . , a n d M . A . A]~RILE : N e u t r a l i z a t i o n of cod insulin w i t h a n t i s e r u m : P r e c i p i t a t i o n of insulin-antiinsulin c o m p l e x w i t h e t h a n o l - w a t e r . Canad. J . 13iochem. 38 , 1216 -- 1218 ( 1960 ). -- - - a n d S. WILSON: S u l f a t e d insulin for t r e a t m e n t of i n s u l i n - r e s i s t a n t diabetics . J . N e w D r u g s 4, 2 5 8 - - 2 6 3 ( 1964 ). MOLONEY , P . J . , a n d L. GOLDSMITH: On t h e a n t i g e n i c i t y of insulin . Canad. J . B i o e h e m . 35 , 7 9 - - 9 2 ( 1957 ). NORMAN , J . l ~ . , a n d P . H . GREENWOOD: A H i s t o r y of Fishes. E d . 2. L o n d o n , E . Benn, 1963 . PEARSE , A . G . E . : I-Iistochemistry , T h e o r e t i c a l a n d A p p - lied. L o n d o n , Churchill , 1960 . SO~IEBLER , T.H. , and S. SCHIESSLER : ~ber den Nachweis yon Insulin mit den metachromatisch reagierenden Pseudoisocyaninen . Histoehemie I, 445 -- 465 (i 959). SMIS?H , L . F . : Species v a r i a t i o n in t h e a m i n o a c i d sequence of insulin. A m e r . J . Med . 40 , 6 6 2 - - 6 6 6 ( 1966 ). TASHIMA , L. , a n d G . F . CAmr , L J r . : l~ole of g l u c a g o n a n d insulin in t h e e a r b o h y d r a t e m e t a b o l i s m of t h e toadfish . E x t . Med. I n t . Congr. Ser . 74 , 140 ( 1964 ). WARDLAW , A.C. , a n d P . J . MOLONEu T h e assay of insulin w i t h a n t i - i n s u l i n a n d m o u s e d i a p h r a g m . Canad. J . B i o c h e m . 39 , 6 9 5 - - 7 1 2 ( 1961 ). WILSON , S., M . A . AFRILE a n d L. SAs~KI: P a s s i v e cutaneous a n a p h y l a x i s i n d u c e d in guinea-pigs b y insulins a n d t h e i r c o m p o n e n t chains . Canad. J . 13ioehem. 44 , 9 8 9 - - 9 9 6 ( 1966 ). - - -- -- T h e a n t i g e n i c loci of insulin . Canad. J . 13iochem . (in press) 1967 . - - , a n d G.I-I. DIXON: A c o m p a r i s o n of cod a n d b o v i n e insulins . N a t u r e 191, 8 7 6 - - 8 7 9 ( 1961 ). -- -- a n d A . C . W~ a~D~AW: R e s y n t h e s i s of cod insulin f r o m its p o l y p e p t i d e chains a n d t h e p r e p a r a t i o n of e o d - o x " h y b r i d " insulins. 13ioehim. b i o p h y s . A c t a 62, 4 8 3 - - 4 8 9 ( 1962 ). - - , a n d S. FALKMEa: C o m p a r a t i v e i m m u n o l o g y of insulin . E x t . Med. I n t . Congr. Ser . 74 , 174 ( 1964 ). -- -- Starfish insulin. Canad. J. Biochem . 48 , 1615 -- 1624 ( 1965 ). V I N C E N T , S., E.C. I)ODDS and F. DICKENS : The pancreas of teleostean fishes and the source of insulin . Quart. J. exp. Physiol . 15 , 313 -- 317 ( 1925 ). YOUNG , D.M. , and A.H. LEWIS : Detection of hypoglycemie reactions in the mouse assay for insulin . Science I05 , 368 -- 369 ( 1947 ).


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Sture Falkmer, Strathearn Wilson. Comparative aspects of the immunology and biology of insulin, Diabetologia, 1967, 519-528, DOI: 10.1007/BF01213571