Research in Veterinary Science 97 (2014) 587–591
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
An immunohistochemical study of the endocrine pancreas in raptors C. Palmieri a, H.L. Shivaprasad b,* a
School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton 4343, QLD, Australia California Animal Health and Food Safety Laboratory System, Tulare Branch, School of Veterinary Medicine, University of California – Davis, Tulare, CA 93274-9042, USA b
A R T I C L E
I N F O
Article history: Received 4 July 2014 Accepted 27 October 2014 Keywords: Avian Raptor Endocrine pancreas Immunohistochemistry
A B S T R A C T
The cytoarchitecture of the endocrine pancreas of 10 raptors (golden eagles, peregrine falcons, Saker falcon, turkey vultures, red-tailed hawk and unspecified falcon) was examined by immunohistochemistry. Three islet types were identified: type A mixed islets composed mainly by glucagon (A)-secreting cells, type B mixed islets with predominantly insulin (B)-secreting cell component and type M mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells. The latter were further characterized into Type I, II or III according to the cell distribution of the three cell types. A and D cells were also randomly scattered within the exocrine pancreas. The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, reflecting an evolutionary adaptation to different dietary habits and variation in developmental mechanisms. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction A great diversity of islet structure exists among vertebrates, suggesting multiple mechanisms of islet formation and variation in developmental processes, although different metabolic requirements and physiological conditions may play a more prominent role in determining islet structure (Steiner et al., 2010). The endocrine portion of the avian pancreas occupies more tissue mass than in mammals and the distribution of cell types differs as well. Three types of islets have been classically described; dark islets composed mostly of glucagon-secreting A cells with some D cells producing somatostatin, light islets containing a mixture of D and insulin-producing B cells, and mixed islets with all three cell types (Pilny, 2008). Clara (1924) and Nagleschmidt (1939) have indicated that the bird pancreas contains proportionately more A cells than does the mammalian pancreas, thus confirming the important role played by glucagon in maintaining the blood glucose balance in birds. However, some physiological differences have been observed among different avian species. For example, glucagon does not affect plasma insulin concentration in fowl, whereas it markedly increases this concentration in ducks (as well as in man and dogs) (Langslow and Hales, 1971). Moreover, Algauhari and Amer (1966) have noticed striking differences in the proportional
* Corresponding author. School of Veterinary Science, The University of Queensland, Gatton Qld 4343 Australia. Tel.: 0061 7 5460 1828; fax: 0061 7 5460 1922. E-mail address: [email protected] (C. Palmieri). http://dx.doi.org/10.1016/j.rvsc.2014.10.011 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
population of A and B cell types in the pancreatic islets of granivorous and carnivorous birds. Seasonal changes in the islet tissue have been observed in the European blackbird (Turdus merula) (Epple, 1961) and the starling (Sturnus vulgaris) (George and Naik, 1964). In pigeon, though A and B cell types undergo cyclic changes in the secretory activity, there is no significant alteration in the numerical population of these cells types (Guha, 1977). In some birds, such as the zebra finch (Taeniopygia guttata), B cells are rare and D cells are more common, but the effect of this difference on the islets function is unknown (Kim et al., 2010). Studies on the distribution of hormonal peptides in the endocrine pancreas have been performed on domestic fowl (Do Prado et al., 1989; Falkmer, 1985; Iwanaga et al., 1983; Mikami and Ono, 1962; Rawdon, 1998; Schwarz et al., 1983; Tomita et al., 1985; Watanabe and Nagatsu, 1991; Watanabe et al., 1988, 1990), duck (Falck and Hellman, 1963; Lucini et al., 1996), swan (Schwarz et al., 1983), pigeon (Roth, 1968), sparrowhawk (Kara et al., 2014), Japanese quail (Mikami et al., 1985), Brazilian sparrow (Nascimento et al., 2007), Houbara bustard (Mensah-Brown et al., 2000), goose (Gulmez et al., 2004), parakeet (Gupta and Kumar, 1980) and buzzard (Bayrakdar et al., 2011). To the authors’ knowledge, only two publications on the endocrine pancreas in raptors are available (Edwin and Leigh, 1993; Simsek et al., 2008). However they are mostly incomplete since the islet structure is described only in one species each (eagle the former, falcon the latter) and the immunohistochemical characterization performed by Simsek et al. (2008) does not include somatostatin and glucagon. Therefore, the aim of this study is to characterize the immunohistochemical features of the endocrine pancreas of different raptors
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C. Palmieri, H.L. Shivaprasad/Research in Veterinary Science 97 (2014) 587–591
and to ascertain whether the distribution of endocrine cells is similar to those of other avian species and mammals. 2. Materials and methods 2.1. Samples Ten formalin-fixed paraffin-embedded samples from the database of the California Animal Health and Food Safety Laboratory System (CAHFS, Fresno, CA, USA) were included in this study. The samples consisted of pancreatic tissue collected from raptors and specifically 3 golden eagles (Aquila chrysaetos), 2 peregrine falcons (Falco peregrinus), 1 unspecified falcon, 1 Saker falcon (Falco cherrug), 2 turkey vultures (Cathartes aura) and 1 red-tailed hawk (Buteo jamaicensis). None of the birds had any evidence of pancreatic damage such as vacuolations, degeneration, necrosis or inflammation. Three birds were euthanized because of multiple fractures, three other birds had respiratory aspergillosis, one bird was affected by respiratory cryptosporidiosis and rest of the birds had miscellaneous conditions of unknown significance. The distribution of endocrine cells was compared with that reported in other species (Heller, 2010; Steiner et al., 2010). Specimens were representative of the three lobes of the pancreas: the dorsal, ventral, and splenic lobes. An overall histological evaluation of the exocrine and endocrine pancreas was performed on hematoxylin & eosin-stained slides. 2.2. Immunohistochemical staining For immunohistochemistry (IHC), tissue sections were dewaxed in xylene and rehydrated in an alcohol series. After inhibition of the endogenous peroxidase activity and blocking with 10% normal goat serum, the slides were incubated for 60 minutes with the following primary antibodies: polyclonal guinea pig anti-insulin (1:600; Dako), polyclonal guinea pig anti-glucagon (1:100; LINCO Research) and polyclonal rabbit anti-somatostatin (1:1000; ImmunoStar). The incubation with the secondary biotinylated goat anti-guinea pig IgG (1:250) was followed by the streptavidin–biotin– peroxidase complex. The presence of antibody binding was ‘visualized’ with aminoethylcarbazole and sections were counterstained with Mayer’s hematoxylin. Positive control slides were used for each antibody using pancreas of dogs and different avian species (turkey, chicken, duck, psittacines). A negative control was
performed in all instances by omitting the primary antibody and incubating tissue sections with Tris buffered saline (TBS).
3. Results All three types of endocrine cells were detected using antisera against insulin, glucagon and somatostatin in the pancreatic islets. Lack of cross-reactivity of the anti-somatostatin antibody was observed in the three golden eagles. Most of the positive cells were organized into islets, although they were also seen throughout the exocrine parenchyma. No segregation into pure A and B islets was observed but all the endocrine cell types were represented in each islet, although with variable number and distribution. Therefore three islet types were identified in the pancreas of raptors: type A mixed islets composed mainly by glucagon (A)-secreting cells (Fig. 1), type B mixed islets with predominantly insulin (B)-secreting cell component (Fig. 2) and pure mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells (Fig. 3). The latter were the most prevalent in the endocrine pancreas of 1/3 golden eagle and 2/2 turkey vultures. The three types of islets were equally represented in the other raptors. Overall, insulin-immunoreactive cells (B cells) in type M islets were more numerous compared with A and D cells. The islets were distributed throughout the lobes without any predilection in 1/3 golden eagle, 1/1 unspecified falcon and 2/2 turkey vultures, while in 2/3 golden eagles, in 2/2 peregrine falcons and 1/1 Saker falcon the splenic lobes contained the highest number of types A and B islets. A cells (glucagon-positive) were mainly distributed in the type A islets and in the M islets. Few scattered A cells were also observed at the periphery of the type B islets and within the exocrine pancreas. Occasionally, A cells showed cytoplasmic processes. B cells (insulin-positive) were mainly observed in type B islets with occasional circular arrangement around a central small capillary (1/3 golden eagles; 2/2 peregrine falcons; 2/2 turkey vultures; 1/1 red-tail hawk) or as a round central aggregate (1/1 Saker falcon; 1/2 unspecified falcons; 2/2 turkey vultures; 1/1 red-tail hawk), as well as in M islets. Few isolated B cells were distributed at the periphery of the type A islets or scattered within the exocrine tissue. Somatostatin-positive D cells were observed in A, B and M islets, although more numerous in the former (2/2 peregrine falcons) and latter ones (1/1 Saker falcon; 1/1 unspecified falcon; 2/2 turkey
Fig. 1. Type A mixed islets in the endocrine pancreas of a turkey vulture. Numerous glucagon-positive A cells (A) in the outer layer encircling a small aggregate of insulinpositive B cells (B). Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
C. Palmieri, H.L. Shivaprasad/Research in Veterinary Science 97 (2014) 587–591
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Fig. 2. Type B mixed islet in the endocrine pancreas of a golden eagle. Few peripheral glucagon-positive A cells (A) and high number of insulin-positive B cells (B) in the center. Note A and B cells (arrows) randomly scattered in the exocrine pancreas. Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
vultures; 1/1 red-tail hawk). They showed a different distribution according to the islet type: scattered throughout the A islets, at the periphery and extending toward the center of B islets and in the outermost layer of the M islets. Individual or small groups of D cells were also observed randomly scattered within the exocrine pancreas (2/2 peregrine falcons; 1/1/ Saker falcon; 2/2 turkey vultures; 1/1 red-tail hawk). D cells possessed prominent cytoplasmic extensions. The distribution of the different endocrine cells within the M islets was variable. A and B cells formed two distinct components (type I) (3/3 golden eagles; 1/1 red-tail hawk) (Fig. 4), were haphazardly intermixed (type II) (1/3 golden eagle; 2/2 peregrine falcons; 1/1 unspecified falcon; 1/1 Saker falcon) or A cells were organized in an outer layer encircling B cell aggregates (type III) (2/3 golden eagles; 2/2 peregrine falcons; 1/1 unspecified falcon; 1/1 Saker falcon; 2/2 turkey vultures) (Fig. 4). In the latter type (type III), D cells were admixed with A cells in the outermost layer, while they represented the only type of cells forming the outer layer when A and B cells were separated (type I) or intermixed (type II). 4. Discussion and conclusions The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, as already demonstrated in five wedgetailed eagles (Edwin and Leigh, 1993). Studies on islets from raptors may be confounded by the paucity of islet and pancreatic tissue for research, so that it may be difficult to normalize data generated from specimens from various donors of different species, different ages and health states. However, our results were consistent across different species and suggest that the islets of the raptors have very unique and distinct cytoarchitecture. Therefore, a prototypical avian islet type may not exist. The variability in islet cell composition and distribution might reflect evolutionary adaptations to different dietary habits (carnivorous vs. granivorous birds) and other environmental constraints. Considering the three types of M islets observed in this study, B cells represent the main cell population. This is in contrast with granivorous birds, such as chicken and duck, whose islets contain more A cells than B cells, so that glucagon levels are 10-fold higher than in the pancreas of mammals (Pollock, 2002). For example, in chickens 80% of the islets in the splenic lobe of the pancreas are composed
of type A islets (Tomita et al., 1985). Whilst glucagon is recognized as a major pancreatic hormone involved in glucoregulation in granivorous birds (Epple et al., 1980; Falkmer and Van Noorden, 1983; Hazelwood, 1984), insulin is considered to be the dominant pancreatic hormone in carnivorous birds (Stevens, 2004), as occurs in mammals (Pollock, 2002; Steiner et al., 2010). This finding is further confirmed by the predominance of B cells observed in our study. Avian insulin is a powerful anabolic hormone and it is more potent than mammalian insulin in stimulating glycogenesis and hypoglycemia (Hazelwood, 2000). Glucagon is a powerful catabolic hormone in birds and circulates at level up to eight times higher than humans (Hazelwood, 2000). Somatostatin is secreted by D-cells in pancreatic islets and inhibits both glucagon and insulin secretion (Hazelwood, 2000). D cells most likely regulate the insulin/ glucagon (I/G) ratio, a dominant factor in homeostasis, to prepare the bird for either a catabolic or anabolic state as the metabolic need dictates (Pilny, 2008). Distribution and frequency of D-cells in the islets were reported by previous immunohistochemical studies performed on goose (Gulmez et al., 2004), quail (Mikami et al., 1985) and chicken (Iwanaga et al., 1983). According to these studies, D-cells are distributed in moderate number throughout the A islets, while peripherally in B islets. In our study, marginal D cells are predominantly observed, except in prevalent A mixed islets where few D cells are randomly scattered throughout the islets. Although the organization of endocrine cells within M islets was variable among different raptors, the three types of M islets shared a common finding, i.e. the distinct segregation of non-B cells, mainly D cells but also A cells in type II and III, at the periphery of the islets. It has been hypothesized that this segregation reflects different microcirculatory pattern for the most efficient endocrine signaling within the islet (Kim et al., 2010). Two signaling models have been proposed: (1) mantle to core: A and D cells sensing changes in the external glucose levels before the signals reaches the central B cells to secrete insulin (Ohtani, 1984); (2) core to mantle: central B cells sensing the changes and secreting insulin, then A and D cells counteracting this response (Samols et al., 1988). No microcirculatory studies of islets blood flow are available in raptors; therefore both models are potentially valid, although mutually exclusive. However, the authors hypothesize that the core to mantle model most likely reflects the major role played by insulin in carnivorous glucoregulation. Considering the dominant model of mixed islets observed in our study and lack of pure A and B islet, the distribution of the
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stimulate both B- and D-cell activity, thereby closing a short-loop negative feedback (Pilny, 2008). Finally, the dissemination of A and B cells within the exocrine pancreas may suggest a local effect on the surrounding exocrine cells. The different endocrine pancreas composition may have important implications in different physiological and experimental conditions, such as normal glucoregulation, fasting and pancreatectomy. Experimentally, total pancreatectomy in granivorous birds results in fatal hypoglycemia, whereas total pancreatectomy in carnivorous birds results in hyperglycemia, glycosuria and diabetes mellitus (Cramb and Langslow, 1984) and this outcome reflects the different distribution of A and B cells in the endocrine pancreas.
Fig. 3. A pure mixed islet (type M) in the endocrine pancreas of a turkey vulture. Note the distribution of glucagon-positive A cells (A), insulin-positive B cells (B) and somatostatin-positive D cells (C). Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
pancreatic islet cells in carnivorous birds represents a combination of that of avian and mammals (Supplementary Fig. S1), although the human pancreatic islets differ from those of raptors regarding the distribution of D cells (peripheral in raptors and randomly scattered within human mixed islets) and in few mammals (e.g. horse) a central mass of A cells is observed. The existence of D cells’ cytoplasmic processes close to A and B cells confirm the morphological evidence of the inhibitory effects of somatostatin on insulin and glucagon secretion and the hypothesis of a paracrine function of type D cells (Koerker et al., 1974). Moreover, the observation of cytoplasmic extension in rare A cells suggests that a paracrine modulation of endocrine secretion is played by these cells as well. It has been hypothesized that A cells
Fig. 4. Pure mixed islets (type M) in the endocrine pancreas of a red-tailed hawk. In type I islet (on the right), A and B cells form two distinct components, while in type III islet (on the left) A cells are organized in an outer layer encircling B cell aggregates. Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
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Understanding the physiology of the avian pancreas will allow for better treatment of sporadic conditions, such as diabetes mellitus, whose pathophysiologic mechanisms are still unknown in raptors and birds in general. Diabetes mellitus has been only described in a red-tailed hawk (Wallner-Pendleton et al., 1993), with histological lesion of balloonization of B cells, a phenomenon observed in mammals during hormonally-induced diabetes mellitus and therefore suggesting a most likely excessive stimulation. Taken together, our results have provided a picture of the islets in raptors that differs from that of prototypical islet described in other avian species. Our anatomical data point to the uniqueness of raptors’ islets but also emphasize that islet biology in this species is still poorly understood. Neither the mechanism for the production of three types of islets in birds, nor the developmental phenomena involved in their segregation into different lobes in the raptors has yet been elucidated by embryological studies so far. The diversity in islet structure (Supplementary Fig. S2) may suggest different mechanism of islet formation between avian species and their respective phylogenetic position, although the authors believe that different metabolic requirements play a more important role in determining islets cell composition in granivorous and carnivorous birds. Acknowledgments The authors would like to thank the staff of CAHFS, Fresno for their technical assistance. Special thanks to Ms. Diane Naydan of the histology laboratory of Veterinary Medical Teaching Hospital, Davis for performing immunohistochemistry. Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.rvsc.2014.10.011. References Algauhari, A.E.I., Amer, F.I., 1966. Comparative studies on the islets of Langherans in granivorous and carnivorous birds. Zoologischer Anzeiger 176, 254–258. Bayrakdar, A., Yaman, M., Atalar, P., Gencer Tarakci, B., Ceribasi, S., 2011. Distribution of neuropeptides in endocrine and exocrine pancreas of long-legged buzzard (Buteo rufinus): and immunohistochemical study. Regulatory Peptides 166 (1–3), 121–127. Clara, M., 1924. Das pankreas der vogel. Anatomische Anzeiger 57, 257–265. Cramb, G., Langslow, D.R., 1984. The endocrine pancreas: Control of secretions and actions of the hormones. In: Freeman, B.M. (Ed.), Physiology and Biochemistry of the Domestic Fowl, vol. 5. Academic Press, London, pp. 93–124. Do Prado, M.L., Campos, M.N., Ricciardi Cruz, A.R., 1989. Distribution of A, B and mixed pancreatic islets in two bird species (Anas plathyrhincus Gallus gallus) – a morphometric study. Gegenbaurs Morphologisches Jahrbuch 153, 379–384. Edwin, N., Leigh, C.M., 1993. The endocrine pancreas in the Australian wedge-tailed eagle (Aquila audax) – an immunocytochemical study. European Journal of Histochemistry 37, 219–224. Epple, A., 1961. Uber beziehungen zwischen feinbau and jahresperiodik des inselorgans von vogeln. Zeitschrift für Zellforschung und mikroskopische Anatomie 53, 731–758. Epple, A., Brinn, J.E., Young, J.B., 1980. Evolution of pancreatic islet functions. In: Pang, P.K.T., Epple, A. (Eds.), Evolution of Vertebrate Endocrine System. Texas Tech. Press, Lubbock, TX, pp. 270–321. Falck, B., Hellman, B., 1963. Evidence for the presence of biogenic amines in pancreatic islets. Experientia 19, 139–140. Falkmer, S., 1985. Comparative morphology of pancreatic islets in animals. In: Volk, B.W., Arquilla, E.R. (Eds.), The Diabetic Pancreas. Medical Book Company, New York/London, pp. 17–25. Falkmer, S., Van Noorden, S., 1983. Ontogeny and phylogeny of the glucagon cell. In: Febre, P.J. (Ed.), Handbook of Experimental Pharmacology. Springer, Berlin/ Heidelberg/New York, pp. 81–119. George, J.C., Naik, D.V., 1964. Cyclic histological and histochemical changes in the pancreas in relation to blood glucose levels in the migratory starling, Sturnus roseus (Linnaeus). Pavo 2 (2), 88–95. Guha, B., 1977. Seasonal changes in the endocrine pancreas and blood glucose level of Columba livia. Folia Biologica, Cracow 25, 81–86. Gulmez, N., Kocamis, H., Aslan, S., Nazli, M., 2004. Immunohistochemical distribution of cells containing insulin, glucagon and somatostatin in the goose
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Contents lists available at ScienceDirect
Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
An immunohistochemical study of the endocrine pancreas in raptors C. Palmieri a, H.L. Shivaprasad b,* a
School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton 4343, QLD, Australia California Animal Health and Food Safety Laboratory System, Tulare Branch, School of Veterinary Medicine, University of California – Davis, Tulare, CA 93274-9042, USA b
A R T I C L E
I N F O
Article history: Received 4 July 2014 Accepted 27 October 2014 Keywords: Avian Raptor Endocrine pancreas Immunohistochemistry
A B S T R A C T
The cytoarchitecture of the endocrine pancreas of 10 raptors (golden eagles, peregrine falcons, Saker falcon, turkey vultures, red-tailed hawk and unspecified falcon) was examined by immunohistochemistry. Three islet types were identified: type A mixed islets composed mainly by glucagon (A)-secreting cells, type B mixed islets with predominantly insulin (B)-secreting cell component and type M mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells. The latter were further characterized into Type I, II or III according to the cell distribution of the three cell types. A and D cells were also randomly scattered within the exocrine pancreas. The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, reflecting an evolutionary adaptation to different dietary habits and variation in developmental mechanisms. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction A great diversity of islet structure exists among vertebrates, suggesting multiple mechanisms of islet formation and variation in developmental processes, although different metabolic requirements and physiological conditions may play a more prominent role in determining islet structure (Steiner et al., 2010). The endocrine portion of the avian pancreas occupies more tissue mass than in mammals and the distribution of cell types differs as well. Three types of islets have been classically described; dark islets composed mostly of glucagon-secreting A cells with some D cells producing somatostatin, light islets containing a mixture of D and insulin-producing B cells, and mixed islets with all three cell types (Pilny, 2008). Clara (1924) and Nagleschmidt (1939) have indicated that the bird pancreas contains proportionately more A cells than does the mammalian pancreas, thus confirming the important role played by glucagon in maintaining the blood glucose balance in birds. However, some physiological differences have been observed among different avian species. For example, glucagon does not affect plasma insulin concentration in fowl, whereas it markedly increases this concentration in ducks (as well as in man and dogs) (Langslow and Hales, 1971). Moreover, Algauhari and Amer (1966) have noticed striking differences in the proportional
* Corresponding author. School of Veterinary Science, The University of Queensland, Gatton Qld 4343 Australia. Tel.: 0061 7 5460 1828; fax: 0061 7 5460 1922. E-mail address: [email protected] (C. Palmieri). http://dx.doi.org/10.1016/j.rvsc.2014.10.011 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
population of A and B cell types in the pancreatic islets of granivorous and carnivorous birds. Seasonal changes in the islet tissue have been observed in the European blackbird (Turdus merula) (Epple, 1961) and the starling (Sturnus vulgaris) (George and Naik, 1964). In pigeon, though A and B cell types undergo cyclic changes in the secretory activity, there is no significant alteration in the numerical population of these cells types (Guha, 1977). In some birds, such as the zebra finch (Taeniopygia guttata), B cells are rare and D cells are more common, but the effect of this difference on the islets function is unknown (Kim et al., 2010). Studies on the distribution of hormonal peptides in the endocrine pancreas have been performed on domestic fowl (Do Prado et al., 1989; Falkmer, 1985; Iwanaga et al., 1983; Mikami and Ono, 1962; Rawdon, 1998; Schwarz et al., 1983; Tomita et al., 1985; Watanabe and Nagatsu, 1991; Watanabe et al., 1988, 1990), duck (Falck and Hellman, 1963; Lucini et al., 1996), swan (Schwarz et al., 1983), pigeon (Roth, 1968), sparrowhawk (Kara et al., 2014), Japanese quail (Mikami et al., 1985), Brazilian sparrow (Nascimento et al., 2007), Houbara bustard (Mensah-Brown et al., 2000), goose (Gulmez et al., 2004), parakeet (Gupta and Kumar, 1980) and buzzard (Bayrakdar et al., 2011). To the authors’ knowledge, only two publications on the endocrine pancreas in raptors are available (Edwin and Leigh, 1993; Simsek et al., 2008). However they are mostly incomplete since the islet structure is described only in one species each (eagle the former, falcon the latter) and the immunohistochemical characterization performed by Simsek et al. (2008) does not include somatostatin and glucagon. Therefore, the aim of this study is to characterize the immunohistochemical features of the endocrine pancreas of different raptors
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and to ascertain whether the distribution of endocrine cells is similar to those of other avian species and mammals. 2. Materials and methods 2.1. Samples Ten formalin-fixed paraffin-embedded samples from the database of the California Animal Health and Food Safety Laboratory System (CAHFS, Fresno, CA, USA) were included in this study. The samples consisted of pancreatic tissue collected from raptors and specifically 3 golden eagles (Aquila chrysaetos), 2 peregrine falcons (Falco peregrinus), 1 unspecified falcon, 1 Saker falcon (Falco cherrug), 2 turkey vultures (Cathartes aura) and 1 red-tailed hawk (Buteo jamaicensis). None of the birds had any evidence of pancreatic damage such as vacuolations, degeneration, necrosis or inflammation. Three birds were euthanized because of multiple fractures, three other birds had respiratory aspergillosis, one bird was affected by respiratory cryptosporidiosis and rest of the birds had miscellaneous conditions of unknown significance. The distribution of endocrine cells was compared with that reported in other species (Heller, 2010; Steiner et al., 2010). Specimens were representative of the three lobes of the pancreas: the dorsal, ventral, and splenic lobes. An overall histological evaluation of the exocrine and endocrine pancreas was performed on hematoxylin & eosin-stained slides. 2.2. Immunohistochemical staining For immunohistochemistry (IHC), tissue sections were dewaxed in xylene and rehydrated in an alcohol series. After inhibition of the endogenous peroxidase activity and blocking with 10% normal goat serum, the slides were incubated for 60 minutes with the following primary antibodies: polyclonal guinea pig anti-insulin (1:600; Dako), polyclonal guinea pig anti-glucagon (1:100; LINCO Research) and polyclonal rabbit anti-somatostatin (1:1000; ImmunoStar). The incubation with the secondary biotinylated goat anti-guinea pig IgG (1:250) was followed by the streptavidin–biotin– peroxidase complex. The presence of antibody binding was ‘visualized’ with aminoethylcarbazole and sections were counterstained with Mayer’s hematoxylin. Positive control slides were used for each antibody using pancreas of dogs and different avian species (turkey, chicken, duck, psittacines). A negative control was
performed in all instances by omitting the primary antibody and incubating tissue sections with Tris buffered saline (TBS).
3. Results All three types of endocrine cells were detected using antisera against insulin, glucagon and somatostatin in the pancreatic islets. Lack of cross-reactivity of the anti-somatostatin antibody was observed in the three golden eagles. Most of the positive cells were organized into islets, although they were also seen throughout the exocrine parenchyma. No segregation into pure A and B islets was observed but all the endocrine cell types were represented in each islet, although with variable number and distribution. Therefore three islet types were identified in the pancreas of raptors: type A mixed islets composed mainly by glucagon (A)-secreting cells (Fig. 1), type B mixed islets with predominantly insulin (B)-secreting cell component (Fig. 2) and pure mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells (Fig. 3). The latter were the most prevalent in the endocrine pancreas of 1/3 golden eagle and 2/2 turkey vultures. The three types of islets were equally represented in the other raptors. Overall, insulin-immunoreactive cells (B cells) in type M islets were more numerous compared with A and D cells. The islets were distributed throughout the lobes without any predilection in 1/3 golden eagle, 1/1 unspecified falcon and 2/2 turkey vultures, while in 2/3 golden eagles, in 2/2 peregrine falcons and 1/1 Saker falcon the splenic lobes contained the highest number of types A and B islets. A cells (glucagon-positive) were mainly distributed in the type A islets and in the M islets. Few scattered A cells were also observed at the periphery of the type B islets and within the exocrine pancreas. Occasionally, A cells showed cytoplasmic processes. B cells (insulin-positive) were mainly observed in type B islets with occasional circular arrangement around a central small capillary (1/3 golden eagles; 2/2 peregrine falcons; 2/2 turkey vultures; 1/1 red-tail hawk) or as a round central aggregate (1/1 Saker falcon; 1/2 unspecified falcons; 2/2 turkey vultures; 1/1 red-tail hawk), as well as in M islets. Few isolated B cells were distributed at the periphery of the type A islets or scattered within the exocrine tissue. Somatostatin-positive D cells were observed in A, B and M islets, although more numerous in the former (2/2 peregrine falcons) and latter ones (1/1 Saker falcon; 1/1 unspecified falcon; 2/2 turkey
Fig. 1. Type A mixed islets in the endocrine pancreas of a turkey vulture. Numerous glucagon-positive A cells (A) in the outer layer encircling a small aggregate of insulinpositive B cells (B). Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
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Fig. 2. Type B mixed islet in the endocrine pancreas of a golden eagle. Few peripheral glucagon-positive A cells (A) and high number of insulin-positive B cells (B) in the center. Note A and B cells (arrows) randomly scattered in the exocrine pancreas. Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
vultures; 1/1 red-tail hawk). They showed a different distribution according to the islet type: scattered throughout the A islets, at the periphery and extending toward the center of B islets and in the outermost layer of the M islets. Individual or small groups of D cells were also observed randomly scattered within the exocrine pancreas (2/2 peregrine falcons; 1/1/ Saker falcon; 2/2 turkey vultures; 1/1 red-tail hawk). D cells possessed prominent cytoplasmic extensions. The distribution of the different endocrine cells within the M islets was variable. A and B cells formed two distinct components (type I) (3/3 golden eagles; 1/1 red-tail hawk) (Fig. 4), were haphazardly intermixed (type II) (1/3 golden eagle; 2/2 peregrine falcons; 1/1 unspecified falcon; 1/1 Saker falcon) or A cells were organized in an outer layer encircling B cell aggregates (type III) (2/3 golden eagles; 2/2 peregrine falcons; 1/1 unspecified falcon; 1/1 Saker falcon; 2/2 turkey vultures) (Fig. 4). In the latter type (type III), D cells were admixed with A cells in the outermost layer, while they represented the only type of cells forming the outer layer when A and B cells were separated (type I) or intermixed (type II). 4. Discussion and conclusions The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, as already demonstrated in five wedgetailed eagles (Edwin and Leigh, 1993). Studies on islets from raptors may be confounded by the paucity of islet and pancreatic tissue for research, so that it may be difficult to normalize data generated from specimens from various donors of different species, different ages and health states. However, our results were consistent across different species and suggest that the islets of the raptors have very unique and distinct cytoarchitecture. Therefore, a prototypical avian islet type may not exist. The variability in islet cell composition and distribution might reflect evolutionary adaptations to different dietary habits (carnivorous vs. granivorous birds) and other environmental constraints. Considering the three types of M islets observed in this study, B cells represent the main cell population. This is in contrast with granivorous birds, such as chicken and duck, whose islets contain more A cells than B cells, so that glucagon levels are 10-fold higher than in the pancreas of mammals (Pollock, 2002). For example, in chickens 80% of the islets in the splenic lobe of the pancreas are composed
of type A islets (Tomita et al., 1985). Whilst glucagon is recognized as a major pancreatic hormone involved in glucoregulation in granivorous birds (Epple et al., 1980; Falkmer and Van Noorden, 1983; Hazelwood, 1984), insulin is considered to be the dominant pancreatic hormone in carnivorous birds (Stevens, 2004), as occurs in mammals (Pollock, 2002; Steiner et al., 2010). This finding is further confirmed by the predominance of B cells observed in our study. Avian insulin is a powerful anabolic hormone and it is more potent than mammalian insulin in stimulating glycogenesis and hypoglycemia (Hazelwood, 2000). Glucagon is a powerful catabolic hormone in birds and circulates at level up to eight times higher than humans (Hazelwood, 2000). Somatostatin is secreted by D-cells in pancreatic islets and inhibits both glucagon and insulin secretion (Hazelwood, 2000). D cells most likely regulate the insulin/ glucagon (I/G) ratio, a dominant factor in homeostasis, to prepare the bird for either a catabolic or anabolic state as the metabolic need dictates (Pilny, 2008). Distribution and frequency of D-cells in the islets were reported by previous immunohistochemical studies performed on goose (Gulmez et al., 2004), quail (Mikami et al., 1985) and chicken (Iwanaga et al., 1983). According to these studies, D-cells are distributed in moderate number throughout the A islets, while peripherally in B islets. In our study, marginal D cells are predominantly observed, except in prevalent A mixed islets where few D cells are randomly scattered throughout the islets. Although the organization of endocrine cells within M islets was variable among different raptors, the three types of M islets shared a common finding, i.e. the distinct segregation of non-B cells, mainly D cells but also A cells in type II and III, at the periphery of the islets. It has been hypothesized that this segregation reflects different microcirculatory pattern for the most efficient endocrine signaling within the islet (Kim et al., 2010). Two signaling models have been proposed: (1) mantle to core: A and D cells sensing changes in the external glucose levels before the signals reaches the central B cells to secrete insulin (Ohtani, 1984); (2) core to mantle: central B cells sensing the changes and secreting insulin, then A and D cells counteracting this response (Samols et al., 1988). No microcirculatory studies of islets blood flow are available in raptors; therefore both models are potentially valid, although mutually exclusive. However, the authors hypothesize that the core to mantle model most likely reflects the major role played by insulin in carnivorous glucoregulation. Considering the dominant model of mixed islets observed in our study and lack of pure A and B islet, the distribution of the
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stimulate both B- and D-cell activity, thereby closing a short-loop negative feedback (Pilny, 2008). Finally, the dissemination of A and B cells within the exocrine pancreas may suggest a local effect on the surrounding exocrine cells. The different endocrine pancreas composition may have important implications in different physiological and experimental conditions, such as normal glucoregulation, fasting and pancreatectomy. Experimentally, total pancreatectomy in granivorous birds results in fatal hypoglycemia, whereas total pancreatectomy in carnivorous birds results in hyperglycemia, glycosuria and diabetes mellitus (Cramb and Langslow, 1984) and this outcome reflects the different distribution of A and B cells in the endocrine pancreas.
Fig. 3. A pure mixed islet (type M) in the endocrine pancreas of a turkey vulture. Note the distribution of glucagon-positive A cells (A), insulin-positive B cells (B) and somatostatin-positive D cells (C). Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
pancreatic islet cells in carnivorous birds represents a combination of that of avian and mammals (Supplementary Fig. S1), although the human pancreatic islets differ from those of raptors regarding the distribution of D cells (peripheral in raptors and randomly scattered within human mixed islets) and in few mammals (e.g. horse) a central mass of A cells is observed. The existence of D cells’ cytoplasmic processes close to A and B cells confirm the morphological evidence of the inhibitory effects of somatostatin on insulin and glucagon secretion and the hypothesis of a paracrine function of type D cells (Koerker et al., 1974). Moreover, the observation of cytoplasmic extension in rare A cells suggests that a paracrine modulation of endocrine secretion is played by these cells as well. It has been hypothesized that A cells
Fig. 4. Pure mixed islets (type M) in the endocrine pancreas of a red-tailed hawk. In type I islet (on the right), A and B cells form two distinct components, while in type III islet (on the left) A cells are organized in an outer layer encircling B cell aggregates. Immunohistochemistry (IHC). Aminoethylcarbazole and Mayer’s hematoxylin counterstain. Bar = 50 μm.
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Understanding the physiology of the avian pancreas will allow for better treatment of sporadic conditions, such as diabetes mellitus, whose pathophysiologic mechanisms are still unknown in raptors and birds in general. Diabetes mellitus has been only described in a red-tailed hawk (Wallner-Pendleton et al., 1993), with histological lesion of balloonization of B cells, a phenomenon observed in mammals during hormonally-induced diabetes mellitus and therefore suggesting a most likely excessive stimulation. Taken together, our results have provided a picture of the islets in raptors that differs from that of prototypical islet described in other avian species. Our anatomical data point to the uniqueness of raptors’ islets but also emphasize that islet biology in this species is still poorly understood. Neither the mechanism for the production of three types of islets in birds, nor the developmental phenomena involved in their segregation into different lobes in the raptors has yet been elucidated by embryological studies so far. The diversity in islet structure (Supplementary Fig. S2) may suggest different mechanism of islet formation between avian species and their respective phylogenetic position, although the authors believe that different metabolic requirements play a more important role in determining islets cell composition in granivorous and carnivorous birds. Acknowledgments The authors would like to thank the staff of CAHFS, Fresno for their technical assistance. Special thanks to Ms. Diane Naydan of the histology laboratory of Veterinary Medical Teaching Hospital, Davis for performing immunohistochemistry. Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.rvsc.2014.10.011. References Algauhari, A.E.I., Amer, F.I., 1966. Comparative studies on the islets of Langherans in granivorous and carnivorous birds. Zoologischer Anzeiger 176, 254–258. Bayrakdar, A., Yaman, M., Atalar, P., Gencer Tarakci, B., Ceribasi, S., 2011. Distribution of neuropeptides in endocrine and exocrine pancreas of long-legged buzzard (Buteo rufinus): and immunohistochemical study. Regulatory Peptides 166 (1–3), 121–127. Clara, M., 1924. Das pankreas der vogel. Anatomische Anzeiger 57, 257–265. Cramb, G., Langslow, D.R., 1984. The endocrine pancreas: Control of secretions and actions of the hormones. In: Freeman, B.M. (Ed.), Physiology and Biochemistry of the Domestic Fowl, vol. 5. Academic Press, London, pp. 93–124. Do Prado, M.L., Campos, M.N., Ricciardi Cruz, A.R., 1989. Distribution of A, B and mixed pancreatic islets in two bird species (Anas plathyrhincus Gallus gallus) – a morphometric study. Gegenbaurs Morphologisches Jahrbuch 153, 379–384. Edwin, N., Leigh, C.M., 1993. The endocrine pancreas in the Australian wedge-tailed eagle (Aquila audax) – an immunocytochemical study. European Journal of Histochemistry 37, 219–224. Epple, A., 1961. Uber beziehungen zwischen feinbau and jahresperiodik des inselorgans von vogeln. Zeitschrift für Zellforschung und mikroskopische Anatomie 53, 731–758. Epple, A., Brinn, J.E., Young, J.B., 1980. Evolution of pancreatic islet functions. In: Pang, P.K.T., Epple, A. (Eds.), Evolution of Vertebrate Endocrine System. Texas Tech. Press, Lubbock, TX, pp. 270–321. Falck, B., Hellman, B., 1963. Evidence for the presence of biogenic amines in pancreatic islets. Experientia 19, 139–140. Falkmer, S., 1985. Comparative morphology of pancreatic islets in animals. In: Volk, B.W., Arquilla, E.R. (Eds.), The Diabetic Pancreas. Medical Book Company, New York/London, pp. 17–25. Falkmer, S., Van Noorden, S., 1983. Ontogeny and phylogeny of the glucagon cell. In: Febre, P.J. (Ed.), Handbook of Experimental Pharmacology. Springer, Berlin/ Heidelberg/New York, pp. 81–119. George, J.C., Naik, D.V., 1964. Cyclic histological and histochemical changes in the pancreas in relation to blood glucose levels in the migratory starling, Sturnus roseus (Linnaeus). Pavo 2 (2), 88–95. Guha, B., 1977. Seasonal changes in the endocrine pancreas and blood glucose level of Columba livia. Folia Biologica, Cracow 25, 81–86. Gulmez, N., Kocamis, H., Aslan, S., Nazli, M., 2004. Immunohistochemical distribution of cells containing insulin, glucagon and somatostatin in the goose
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