The branching of insulin-like growth factor 1 and insulin: an immunohistochemical analysis during phylogeny

The branching of insulin-like growth factor 1 and insulin: an immunohistochemical analysis during phylogeny
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  Regulatory Peptides, 48 (1993) 65-76 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/ 06.00 65 REGPEP 01568 The branching of insulin-like growth factor 1 and insulin: an immunohistochemical analysis during phylogeny Manfred Reinecke a, Caroline Maake a, Sture Falkmer b and Vicki R. Sara c a nstitute of Anatomy, Division of Neuroendocrinology, University of Zi2rich, Zarich (Switzerland), b Department of Tumor Pathology, Karolinska Institute and Hospital, Stockholm (Sweden) and c School of Life Science, Q UT Gardens Point Campus, Brisbane (Australia) (Received 26 April 1993; accepted 6 May 1993) Key words: IGF-1; Insulin; Co-existence; Pancreas; Bony fish; Cartilaginous fish; Cyclostomes Summary The co-existence of insulin-like growth factor 1 (IGF-1) with the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP) in the endocrine pancreas of repre- sentative species of cyclostomes (Myxine glutinosa), cartilaginous fish (Raja clavata, Squalus acanthias) and bony fish (Cottus scorpius, Carassius auratus, Cyprinus carpio, Anguilla anguilla) was studied by the use of monoclonal and polyclonal antisera and the double immunofluorescence technique. In all species investigated, IGF-l-like- immunoreactive cells were found in the endocrine pancreas, however, in varying localization. In Myxine glutinosa, all INS-immunoreactive cells and some of the SOM-immunoreactive cells contained IGF-l-like- immunoreactivity. In Raja and Squalus, only a minority of the INS-immunoreactive cells also displayed IGF- 1-1ike-immunoreactivity. The majority of the IGF-l-like-immunoreactivity was observed in SOM- and in GLUC-immunoreactive cells. Different results were obtained in bony fish. In Cottus, in the Brockmann bod- ies and the small islets IGF-l-like- and INS-immunoreactivities co-existed to 100%. In contrast, in the other bony fish studied IGF-l-like-immunoreactivity was not observed in INS-immunoreactive cells: in Cyprinus, IGF-1-1ike-immunoreactivity was found in GLUC-, PP- and SOM-immunoreactive cells and in Carassius and Anguilla, in SOM-immunoreactive cells only. Thus, in all bony fish species with the exception of Cottus, IGF-1 and insulin display a distinct cellular distribution, similar to that of mammals. The present results, thus, may indicate that the branching of IGF-1 and insulin has occurred at the phylogenetic level of bony fish. Correspondence to: M. Reinecke, Institute of Anatomy, Division of Neuroendocrinology, University of ZOrich-Irchel, Winterthurer Str. 190, CH-8057 ZOrich, Switzerland.  66 Introduction The insulin-like growth factors (IGFs) constitute a family of polypeptides that consists of two major forms, IGF-1 and IGF-2, and some molecular vari- ants [1-3]. They exert insulin-like and growth- promoting effects in their target cells. Because con- siderable similarities exist in primary and tertiary structure [2], it has been presumed that in phylogeny the IGFs and proinsulin have branched from a com- mon ancestor [4-7]. This precursor molecule has been suggested to be coded by a gene that underwent duplication at the time of the appearence of the first vertebrates [8,9]. Recently, this hypothesis has gained support from studies on protochordates. From the cephalochor- .°. date Branchiostoma californiens a hybrid insulin/ insulin-like growth factor cDNA has been cloned [6]. In Branchiostoma lanceolatum and in the uro- chordate Ciona intestinalis, the co-existence of INS- and IGF-l-like-immunoreactivities in entero-endo- crine cells and central neurons has been shown [7] suggesting these cells as the storage sites of the hy- brid molecule found in Branchiostoma californiensis. Thus, a common insulin/IGF-1 ancestor molecule seems to occur on the phylogenetic level of proto- chordates. For mammals, the presence of IGF-l-immunore- activity has been shown in rat [10-15] as well as in human and dog endocrine pancreas [ 15]. In the rat islets, the distinct localization of IGF-l-immunore- activity is still under discussion [12,14]. While there Fig. 1. lmmunohistochemical localization of IGF-1, INS and SOM in the endocrine pancreas of Myxine glut osa. (a, b) Section incu- bated with the IGF-1 antiserum K37 (a) and with the INS-antiserum (b) in double immunofluorescence. There is 100Yo co-existence of IGF-l-like- and INS-immunoreactivities. Original magnifiaction x 140. (c, d) Double immunofluorescence of the IGF-l-antiserum 118 (c) and of the SOM-antiserum (d). Some of the SOM-immunoreactive cells (arrows) also display IGF-l-like-immunoreactivity. Original magnification x 210.  & .o, e 67 Fig. 2. Immunohistochemical localization of IGF-1 (a, c, e), INS (b), GLUC (d) and SOM (f) in the endocrine pancreas of Squalus acanthias (a, b) Section incubated with the IGF-1 antiserum 116 (a) and with INS-antiserum (b) in double immunofluorescence. A mi- nority of the INS-immunoreactive cells also contains IGF-l-like-irnmunoreactivity arrows). (c, d) Double immunofluorescence of the IGF-l-antiserum K37 (c) and of the GLUC-antiserum (d). Several of the GLUC-immunoreactive cells show also IGF-l-like- immunoreactivity. (e, f) Double immunofluorescence of the IGF-l-antiserum 118 (e) and of the SOM-antiserum (f). There is a high de- gree of co-existence of IGF-l-like-and SOM-immunoreactivities. Original magnification x 210. is consent that no IGF- 1-immunoreactivity occurs in B-cells, both the D-cells [12] and the A-cells [15] have been described to represent the major store of IGF-l-immunoreactivity. A detailed analysis using several specific antisera in double immunofluores- cence revealed the exclusive presence of IGF-1- immunoreactivity in A-cells of rat and dog, but in man singular D-cells also contained IGF-l-immu- noreactivity [ 15 ]. In contrast to protochordates [7] and mammals  68  69 [ 15], for lower vertebrates only the presence of IGF- l-like peptides in the islet parenchyma [ 14,16,17 ] has been shown, but no results have been presented yet on a possible co-existence of IGF-1 and insulin. However, information on the localization of insulin and IGF-1 may help to elucidate the phylogenetic level of the branching of these hormones from the likely precursor molecule. Thus, the present study using the double immunofluorescence technique aims: (1) to localize IGF-1 in the endocrine pancreas of representative bony fish, cartilaginous fish and cy- clostomes; (2) to compare the cellular storage sites of IGF-1 with those of insulin; and (3) in case of non- coexistence or minor partial co-existence of insulin and IGF-1 to localize IGF-1 and the classical islet hormones glucagon, somatostatin and pancreatic polypeptide. Materials and Methods Animals. The species studied were selected on the basis of the results of preceding studies on the phylogeny of IGF-1 [14,16]. However, in order to obtain more valid results other species were added to those investigated earlier. Thus, as representatives of the three lowest vertebrate classes the following spe- cies (number of individuals in parentheses) were used: Osteichthyes - Cottus scorpius (n = 8), Cyprinus carpio (n = 3), Carassius auratus (n = 5), Anguilla an- guilla (n = 5); Chondrichthyes - Raja clavata (n = 6), Squalus acanthias (n = 4); Cyclostomes - Myxine glu- tinosa (n = 8). Fixation and embedding. Adult specimens of the species investigated were anesthetized by adding M S 222 R (Sandoz, Basel, Switzerland) to the surround- ing water. Except for Carasshts and Anguilla, an iso- tonic saline solution was perfused via the heart for about 10-15 min to prevent blood clotting and to maintain colloid osmotic pressure, followed by per- fusion with acid-free Bouin's fixative for another 15 to 30 rain. After dissection, the pancreas were post- fixed by immersion in the fixative for 2 h. In Carassius and Anguilla, the pancreas were fixed by immersion in acid-free Bouin's solution for 4 h. All specimens were dehydrated in ascending series of ethanol and routinely embedded in paraplast. Sections were cut at 4/~m. Antisera and hormones. For the immunohis- tochemical localization of IGF-1, three different an- tisera (codes: 116, 118, K37) raised in rabbit were used which have been shown to be specific for IGF-1 (116, 118, see Ref. 18; K37, see Refs. 12 and 14). For further information on the antisera against IGF-1 and the classical islet hormones insulin (INS), glu- cagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP) see [15]. Recombinant human (h)IGF-1 was purchased from Bachem (Bubendorf, Switzerland), bovine (b)INS, bGLUC and SOM from Sigma (Buchs, Switzerland) and hPP from Peninsula (Heidelberg, Germany). Immunohistochemical technique. At first, unspe- cific reactions were blocked by the use of phosphate- buffered saline (PBS) containing 2~o bovine serum albumine (BSA) and 2~o normal goat serum. For double immunofluorescence, single sections were in- cubated consecutively with the INS-antiserum 72C and with one of the IGF-1-antisera. Additional single sections were incubated consecutively: (1) with the mouse GLUC-antibody and an IGF-1-antiserum; (2) with the rat SOM-antiserum and an IGF-l-antise- rum. The possible co-existence of IGF-1 and PP was Fig. 3. Double immunofluorescence of IGF-1 (a, c: antiserum 116, e: antiserum 118) and INS (b, d, O in the pyloric islet (a-d) and in a small islet (e, f) of Cottus scorpius. (a, b) Low power micrograph of the pyloric slet. IGF-l-like- and IN S-immunoreactivities display dentical distribution patterns. Original magnification x 85. (c, d) Higher magnification of a portion of the pyloric islet shown in (a, b). There is a 100~o co-localisation of IGF-l-like-and INS-immunoreactivities. e, f) All INS-immunoreactive f) cells in the small islet contain also IGF-l-like-immunoreactivity e). Original magnification × 340.
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