Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas

Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas

G Model ACTHIS-50988; No. of Pages 6 ARTICLE IN PRESS Acta Histochemica xxx (2015) xxx–xxx Contents lists available at ScienceDirect Acta Histochem...

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G Model ACTHIS-50988; No. of Pages 6

ARTICLE IN PRESS Acta Histochemica xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

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Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas Izabela Janiuk a,∗ , Krzysztof Młynek b a b

Department of Dietetics and Food Assessment, Institute of Health Sciences, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland Department of Cattle Breeding and Milk Evaluation, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland

a r t i c l e

i n f o

Article history: Received 17 February 2015 Received in revised form 14 April 2015 Accepted 15 April 2015 Available online xxx Keywords: CART (cocaine and amphetamine regulated transcript) Pancreas Cattle Immunohistochemistry

a b s t r a c t This study was aimed at identifying and determining the configuration of structures which contain the cocaine- and amphetamine-regulated transcript peptide (CART) in the bovine pancreas. The study material was collected from 20 animals. The distribution of CART in the bovine pancreas was investigated, by an immunohistochemical evaluation. CART peptide in the normal pancreas has been identified in intrapancreatic ganglia, nerve fibres and in endocrine cells of Langerhans islets and exocrine pancreas. CART immunoreactive nerve fibres innervate the exocrine and endocrine regions and the intrapancreatic ganglia, where they form a moderate number of networks, encircling the cell bodies. The few CART-immunoreactive endocrine cells, that appear in the bovine pancreas, are not limited to the islet cells, where they form a subpopulation of CART-containing cells, but are also individually distributed in the exocrine region. Furthermore, CART has been visualized in nerve fibres, innervating pancreatic outlet ducts and blood vessels. CART plays a physiological role in the integrated mechanisms that regulate both endocrine and exocrine pancreatic secretion. These results are consistent with the hypothesis that CART expression in nerve fibres and intrapancreatic ganglia is a common feature of the mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to single cells of the bovine pancreas. © 2015 Elsevier GmbH. All rights reserved.

Introduction The pancreas, as a double secretion organ, is specific to vertebrates. Its stroma contains numerous dispersed and relatively loosely positioned endocrine cell clusters, called Langerhans islets, as well as cells, which are located beyond the islets in the exocrine region and in sections of the pancreatic outlet ducts. Despite constituting barely 2% of the organ, the endocrine part of the tissue contains several types of cells, participating in the secretion of hormones and biologically active substances. Pancreatic hormones influence the regulation of carbohydrate metabolism and, consequently, they maintain the systemic biochemical balance. Apart from the hormones, the pancreas produces and secretes

Abbreviations: CART, cocaine and amphetamine regulated transcript; IR, immunoreactive; NF, nerve fibres; IP, immunopositive. ∗ Corresponding author at: Department of Dietetics and Food Assessment, Institute of Health Sciences, Siedlce University of Natural Sciences and Humanities, 14 Prusa Street, 08-110 Siedlce, Poland. E-mail address: [email protected] (I. Janiuk).

active peptides that perform the role of growth, regeneration and metabolic activity regulators (Kasacka et al., 2012). Such peptides include the P (SP) substance and the Y (NPY) neuropeptide, vasoactive intestinal peptide (VIP) (De Giorgio et al., 1992; Myojin et al., 2000), pituitary adenylate cyclase-activating polypeptide (PACAP) (Fridolf et al., 1992), calcitonin gene-related peptide (CGRP) (Fujimura et al., 1988; Myojin et al., 2000; Sternini et al., 1992), galanin (Gal) (Adeghate and Ponery, 2001; Baltazar et al., 2000; Myojin et al., 2000) and somatostatin (ST) (Fujimura et al., 1988). At present, the number of factors, which regulate the exocrine and endocrine activity of the pancreas, has been expanded with another peptide: the cocaine and amphetamine regulated transcript (CART) (Kasacka et al., 2012; Wierup and Sundler, 2006). It is widely distributed in numerous normal (Ekblad et al., 2003; Hunter et al., 2004; Kasacka et al., 2012; Okumura et al., 2000) and abnormal organs and tissues (Janiuk and Kasacka, 2013; Kasacka and Piotrowska, 2012). The multifunctional neuropeptide CART is secreted from the hypothalamus, the pituitary, the adrenal gland and the pancreas. It can also be found in the circulatory system. Several lines of evidence

http://dx.doi.org/10.1016/j.acthis.2015.04.004 0065-1281/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Janiuk I, Młynek K. Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.04.004

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indicate CART peptides to have a diverse spectrum of biological activity (Hunter et al., 2004; Dampney et al., 2005; Koylu et al., 2006; Kuhar et al., 2002, 2005; Wierup et al., 2004; Vicentic and Jones, 2007). CART has been reported as an endogenous satiety factor, acting on specific centres in the brain which regulates the sensation of satiety. Mutations of this hormone and its receptors can lead to obesity (Cheung and Mao, 2012). Moreover, the results of studies in recent years suggest a general role for CART in different cells. Given the unique molecular structure and biological features of this peptide, it may be concluded that CART is an antioxidant peptide (or antioxidant hormone). Furthermore, some authors suggest that it may have strong therapeutic properties for diseases in which oxidative stress is strongly involved (Mao et al., 2012). A new function of CART is its antioxidant activity by which CART protects cellular lipids, protein and mitochondrial DNA against oxidant stress, thus suggesting a general cytoprotective role for CART. Apart from non-human primates, cows are one of the most important and, in many aspects, little explored research animals. Despite our updated knowledge, regarding the location and configuration of CART in the pancreas in a number of vertebrates (Arciszewski et al., 2008; Colombo et al., 2003; Kasacka et al., 2012; Wierup et al., 2007), very little is still known about the distribution and function of CART in cattle. Nerves with CART structures have been found in the bovine abomasum (Janiuk et al., 2013). In polygastric animals, studies have been carried out to investigate the localization and distribution of such peptides as Gal, CGRP or NPY (Baltazar et al., 2000; Myojin et al., 2000). In turn, Arciszewski and his team analyzed the expression of neuronal nitric oxide synthase (NOS) and co-localization of selected peptides in nerve structures of the ovine pancreas (Arciszewski and Zacharko-Siembida, 2007; Arciszewski, 2007). CART expression has been investigated in the ovine pancreas (Arciszewski et al., 2008) but it remains unknown whether CART is expressed in the bovine pancreas. The aim of the present study was to perform a detailed mapping of CART in the bovine pancreas, using a broad immunohistochemical apparatus.

Material and methods Adult cattle were used, including crossbreeds of Polish Lowland black-and-white cows (BW) and bulls, representing the Limousin breed (LIM) (aged: 543 ± 32 days, n = 20, weight ca 441.0–491.4 kg). The animals were kept in similar conditions but originated from different farms. The animals were killed in a standard slaughter procedure and sections were collected always from the same part of the pancreas, i.e. the body). The collected tissue specimens were immediately fixed in 4% buffered formaldehyde and embedded in paraffin for 4 days at room temperature, based on standard histological procedures. The paraffin blocks were cut into 4 ␮m sections with LEICA RM 2135 microtome (3 sections from each item), and attached to FLEX IHC microscope slides (K8020, Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark) and dried overnight at 37 ◦ C, with a subsequent 1-h incubation at 60 ◦ C. The specimens were stained with haematoxylin and eosin (H+E) for general histological examination and immunohistochemically processed for CART detection.

xylene and hydrated in a series of pure alcohols with decreasing concentrations. For antigen retrieval, the sections were subjected to pretreatment with pressure chamber heating for 1 min at 21 psi and 125 ◦ C, using a Target Retrieval Solution with pH of 9.0 (Product No: S 2367 Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark). After cooling down to RT, the sections were incubated with a Peroxidase Blocking Reagent (Product No: S 2001, Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark) for 10 min to block endogenous peroxidase activity. Then, the tissues were washed with distilled water and a Wash Buffer (Product No: S 3003, Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark), 3 times for 5 min. The sections with the primary antibody to the CART peptide fragment (55–102) were incubated with the antibody overnight at 4 ◦ C in a humidified chamber. A specific polyclonal rabbit antibody against CART (dilution 1:12,000; Phoenix Pharmaceuticals, CA, Burlingame, USA, Product No: H 00362) was used in the analyses. The antiserum was diluted in an Antibody Diluent (Product No: S 0809, Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark). That procedure was followed up with incubation with a secondary rabbit antibody (conjugated to horseradish peroxidase-labelled polymer) (EnVision+ Kit HRP Rabbit, Product No: K 4011 Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark). The bound antibodies were visualized after 1-min incubation with liquid 3,3 -diaminobenzidine substrate chromogen. The sections were finally counterstained in haematoxylin QS (Product No: H-3404, Vector Laboratories; Burlingame, CA). A three-fold, 5-min washing was performed at RT between each step with the Wash Buffer (Product No: S 3006 Dako Denmark A/S, Produktionsvej 42, DK-2600, Glostrup, Denmark). Simultaneously, a control reaction was performed for the CART antibody. In the negative control, the specific antibody was omitted in the staining procedure and a positive control was prepared with specific tissue (as recommended by the manufacturer; rat hypothalamus). CART-positive structures were searched for and their topography was observed under a light microscope. Light microscopy and quantitative analysis The analysis of the specimens and image documentation were carried out with an Olympus BX41 light microscope, equipped with a video channel and connected to a PC with installed Cell-B image analysis software (Olympus 114 Corp., Tokyo, Japan). When recording the microscopic images, particular attention was paid to the distribution of the structures with immunoreactivity to the analyzed antigen. Three sections per animal were studied. Structures with CART expression were searched for and their topography was observed in 10 randomly selected fields of vision (0.785 mm2 ) of the analyzed pancreas section area at a 200× zoom (20× objective and 10× eyepiece). We performed a subjective semi-quantitative evaluation of the density of the CART-IR structures. An arbitrary scale was used, where: (−) = CART-IR structures absent; (+) = single; (++) = few; (+++) = a moderate number.

Immunohistochemical protocol

Results

In an immunohistochemical analysis, the EnVision technique was used, according to Herman and Elfont (1991) and Escribano et al. (1987). Immunostaining was performed, according to the following protocol: the paraffin-embedded sections were deparaffinized in

A routine histopathological examination did not reveal any significant microscopic pathological changes in the bovine pancreas, while positive results of the immunohistochemical reaction for CART were observed in the pancreas of each studied animal. The present study demonstrated CART-like immunoreactivity in the

Please cite this article in press as: Janiuk I, Młynek K. Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.04.004

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neural elements of the pancreas of cattle. CART is mainly present in nerve cell bodies in pancreatic ganglia and in nerve fibres (NF). NFs immunoreactive (IR) to CART were detected in all the histological regions of the bovine pancreas (data summarized in Table 1). They were distributed predominantly in the exocrine and endocrine compartments and the intrapancreatic ganglia, where they form a moderate number of networks, encircling the cell bodies. It was also detected in the endocrine cells of the islets and in the exocrine parts of this organ. A few CART-positive fibres were found projecting to the walls of small and medium-sized blood vessels (BV). Often were those fibres penetrating the wall BV along its course (Fig. 1 A). Whereas in the large blood vessels, no CART-positive fibres were found (not shown). The ductal system was also innervated by a few numbers

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Table 1 Numbers of CART-IR structures in bovine pancreas. A subjective semi-quantitative evaluation of CART-IR structure density. Pancreas parts

Number of CART-IR structures

Islets of Langerhans Nerve fibres Endocrine cells

+

Exocrine pancreas Pancreatic ganglia Nerve fibres Nerve fibres around blood vessels Nerve fibres innervating pancreatic ducts Nerve fibres innervating Langerhans islets Endocrine cells

+/++ ++ ++/+++ ++ ++ +/++ ++

Fig. 1. (A)–(B) Micrographs, showing CART expression in neuronal elements of the bovine pancreas. (TIF). (A) Nerve fibres (NF), penetrating the walls of blood vessels (BV). Visible NF running along BV walls. Scale bar 400 ␮m. (B) NF are distributed around the duct (arrows). Scale bar 200 ␮m. Tissue expression of CART peptide, determined by immunohistochemical (IHC) analysis.

Fig. 2. (A)–(B) IHC staining of the bovine pancreas. The pictures present: (A) short nerve fibres are located between the exocrine parts of the pancreas. Scale bar 200 ␮m, and (B) The micrographs present CART-positive nerve endings reaching the islet (arrowhead). Scale bar 200 ␮m.

Fig. 3. (A)–(B). In transverse section (A) of intrapancreatic ganglia. Visible immunohistochemical reaction in perikarya. Scale bar 400 ␮m. (B) The micrograph shows a positive staining for CART in long nerve terminals, forming fascicles (arrowhead). NFs are located in-between in the exocrine pancreas. Scale bar 100 ␮m.

Please cite this article in press as: Janiuk I, Młynek K. Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.04.004

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Fig. 4. (A)–(B) The pictures show either CART presence in neuroendocrine cells (NE). (A) Population NE in islet. Scale bar 100 ␮m and (B) in the exocrine pancreas. Scale bar 400 ␮m.

of CART-positive nerves (see Fig. 1 B). The neuronal elements in the form of NFs were frequently identified within the pancreatic parenchyma. In that part of the organ, the most often observed NFs were thick (see Fig. 2A), while less numerous and more delicate, supplying the hormonal part of the organ (Fig. 2B). CART-positive nerve endings reached the Langerhans islets. Within the intrapancreatic ganglia, a considerable number of CART-IR nerve cell bodies were also observed. Those nerve cells were round to oval in shape. The CART-like immunoreactivity in the pericarya appeared in the cytoplasm but was not localized in the nucleus (Fig. 3A). In the sections, long CART-IR nerve terminals were occasionally observed, forming fascicles (see Fig. 3B). The distribution of CART-like demonstrated in the immunoreactive endocrine cells. Two types of CART-IR cell location were observed: in the islets (Fig. 4A) and as single randomly distributed cells in the remaining part of the organ (Fig. 4B). The CARTsynthetising cells in the bovine pancreas were most often present on the edges of the islets, constituting a small percentage of pancreatic cells. However, they were also observed within some of the islets. Discussion Pancreatic secretions play an extremely important role in digestion and glucose homeostasis. These secretions are controlled by a host of neuronal and hormonal signalling pathways which modulate not only the secretory function but also the cellular integrity of the gland. The complex physiology and biology of the pancreas is also influenced by CART. The present work is exploratory since, to the best of our knowledge, there have been no reports in the literature describing the identification and topography of CART in the bovine pancreas. To-date’s analyses of CART incidence in the pancreas have been conducted only in rodents, pigs, sheep and man (Arciszewski et al., 2008; Kasacka et al., 2012; Wierup et al., 2004, 2007; Wierup and Sundler, 2006). What deserves attention is the presence of CART in nerve structures, namely in intrapancreatic ganglia and NFs penetrating the hormonal and exocrine pancreas, blood vessels and the ductus pancreaticus. CART location may significantly affect the secretory function of the hormonal and exocrine pancreatic regions. Our observations and literature review have led us to a conclusion that the configuration of the analyzed peptide in the hormonal and exocrine pancreas of cattle is not unique. A similar (as in cattle) expression pattern was previously described in the rat (Wierup et al., 2004, 2005), sheep (Arciszewski et al., 2008) and human pancreas (Kasacka et al., 2012). Recent studies of adult mice and pigs have shown a different expression pattern. Despite an ample presence of CART-IR structures in the mouse and porcine pancreas

(Wierup et al., 2007), this peptide was not found in the hormonal part of the organ. The analyses have revealed that there are few CART-IP cells in a fully developed bovine pancreas and they constitute a small percentage of the other cells that make up the organ, in a similar way as with rats and sheep (Arciszewski et al., 2008; Wierup et al., 2004). Nevertheless, Kasacka et al. (2012) observed definitely more numerous CART-IR islet cells in certain islets of the human pancreas. In those studies, as well as in some earlier ones (Arciszewski et al., 2008; Kasacka et al., 2012), CART-IR islet cells were found in the periphery of the Langerhans islets. In cattle, they were occasionally detected inside the islets. A different CART islet cell profile is typical of developing pancreata of rats (Wierup et al., 2004) and mice (Wierup et al., 2005). Significant quantitative and locational differences were revealed during pancreatic development in comparison with the mature organ. Wierup et al. (2004) showed CART presence already in the foetal period and revealed a strong CART expression in all the endocrine cell types, whereas it was found to be present mostly in the alfa cells in mice (Wierup et al., 2005; Wierup and Sundler, 2006). In barely two-week-old rats, postnatal CART expression was restricted to somatostatin-secreting cell populations (Wierup et al., 2004), whereas no CART IR islet cells were detected in adult mice (Wierup and Sundler, 2006). In our study, CART presence was observed in pancreatic nerve fibres and in nerve cell bodies of the intrapancreatic ganglia. The topographic distribution of CART peptide-containing NFs and of the CART-IR nerve cell bodies in the bovine pancreas was found to be in line with the data obtained in other vertebrates (Arciszewski et al., 2008; Kasacka et al., 2012; Wierup et al., 2004). It must be added that a rich CART innervation was observed in adult mice, pigs and humans. In contrast to the observations by Arciszewski et al. (2008), who extensively described CART-IR innervation of the blood vessels in the sheep pancreas, small and medium-sized blood vessels of the bovine pancreas were penetrated by few NFs. It should also be noted that no CART-IR NFs supporting blood vessels were detected in the human pancreas (Kasacka et al., 2012). It is evident that differences in the innervation of pancreatic vessels and regions are species-related. Such a anatomical location of CART clearly suggests that CART is involved in the control of islet function since these neurons are known to exert stimulatory effects on the insulin secretion rate (Wierup and Sundler, 2006). CART belongs to a large family of regulatory peptides,located and distributed in the central and peripheral nerves, as well as in neuroendocrine cells (Asnicar et al., 2001; Ekblad et al., 2003; Koylu et al., 1997; Okumura et al., 2000). Undoubtedly, its pancreatic function is essential, and the documented lowerpercentage share of CART cells in rodent islets indicates a potential role of CART

Please cite this article in press as: Janiuk I, Młynek K. Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.04.004

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cells in the development and formation of fully functional pancreatic Langerhans cells (Wierup et al., 2004). Moreover, it has been proved that the CART peptide stimulates endocrine cell differentiation and promotes islet cell survival, as well as it is an important islet regulator (Wierup et al., 2004; Wierup and Sundler, 2006). CART participation in the control of islet function is confirmed with its coincidence with VIP in neurons, and these neurons are known to exert stimulatory effects on insulin secretion (Ahren, 2000). Intrapancreatic neurons and ganglia, blood vessel innervating nerve fibres, also constitute a substantial source of the CART peptide, which is, in turn, associated with regulation of the exocrine parts, while VIP presence at nerve endings has a stimulating effect on local blood flow (Jansson, 1994). In conclusion, based on previous studies and considering the location of the CART peptide in the cattle pancreas, it may be assumed that CART plays an important role in the control of secretion of pancreatic enzymes and hormones. Conflict of interest The authors have no conflict of interest. Acknowledgments The reported study has been supported by statutory funds of the University of Natural Sciences and Humanities of Siedlce, Grant No. S/341/13. References Adeghate E, Ponery AS. Large reduction in the number of galaninimmunoreactive cells in pancreatic islets of diabetic rats. J Neuroendocrinol 2001;13:706–10. Ahren B. Autonomic regulation of islet hormone secretionimplications for health and disease. Diabetologia 2000;43: 393–410. Arciszewski MB, Całka J, Majewski M. Cocaine- and amphetamineregulated transcript (CART) is expressed in the ovine pancreas. Ann Anat 2008;190:292–9. Arciszewski MB, Zacharko-Siembida A. A co-localization study on the ovine pancreas innervation. Ann Anat 2007;189:157–67. Arciszewski MB. Expression of neuronal nitric oxide synthase in the pancreas of the sheep. Anat Histol Embryol 2007;36:375–81. Asnicar MA, Smith DP, Yang DD, Heiman ML, Fox N, Chen YF, et al. Absence of cocaine- and amphetamine-regulated transcript results in obesity in mice fed a high caloric diet. Endocrinology 2001;142:4394–400. Baltazar ET, Kitamura N, Hondo E, Narreto EC, Yamada J. Galaninlike immunoreactive endocrine cells in bovine pancreas. J Anat 2000;196:285–91. Colombo M, Gregersen S, Xiao J, Hermansen K. Effects of ghrelin and other neuropeptides (CART, MCH, orexin A and B, and GLP1) on the release of insulin from isolated rat islets. Pancreas 2003;27:161–6. Cheung WW, Mao P. Recent advances in obesity: genetand beyond. ISRN Endocrinol 2012;53690:5, ics http://dx.doi.org/10.5402/2012/536905 [Epub 2012 March 5]. Dampney RA, Horiuchi J, Killinger S, Sheriff MJ, Tan PS, McDowall LM. Long-term regulation of arterial blood pressure by hypothalamic nuclei: some critical questions. Clin Exp Pharmacol Physiol 2005;32:419–25. De Giorgio R, Sternini C, Brecha NC, Widdison AL, Karanjia ND, Reber HA, et al. Patterns of innervation of vasoactive intestinal polypeptide, neuropeptide Y, and gastrin releasing

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Please cite this article in press as: Janiuk I, Młynek K. Immunodetection of cocaine- and amphetamine-regulated transcript in bovine pancreas. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.04.004