General and Comparative Endocrinology 161 (2009) 252–261 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting Erin MacDonald, Hélène Volkoff * Department of Biology, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 article info abstract Article history: cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and Received 11 November 2008 cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution Revised 20 January 2009 was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Accepted 21 January 2009 Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general Available online 1 February 2009 the degree of identity is relatively low (50%). All three peptides are present in brain and in several periph- eral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothal- Keywords: amus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in Winter skate telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART NPY CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation CCK of feeding in winter skate. Fasting Ó 2009 Elsevier Inc. All rights reserved. Expression Cloning Distribution 1. Introduction (Lopez-Patino et al., 1999) cause an increase in food intake whereas ICV injections of NPY antagonists decrease feeding in In fish as in all vertebrates, appetite is regulated by central and goldfish (Lopez-Patino et al., 1999). In addition, increases in brain peripheral appetite-stimulating (orexigenic) or appetite-inhibiting NPY mRNA expression levels are seen in food restricted Pacific sal- (anorexigenic) factors. Neuropeptide Y (NPY) and cocaine- and mon (Oncorhyncus sp.) (Silverstein et al., 1998) and goldfish (Nar- amphetamine-regulated transcript (CART) are examples of central naware and Peter, 2002). orexigenic and anorexigenic factors, respectively, whereas chole- Cocaine- and amphetamine-regulated transcript (CART) was cystokinin (CCK), although also produced by the brain, is mostly first discovered in rats as the transcript of a brain mRNA up-regu- synthesized in the gut and acts as a peripheral satiety factor (Volk- lated following administration of cocaine and amphetamine and off et al., 2005). later shown to have a role in the regulation of feeding, as centrally Neuropeptide Y is a 36 amino acid peptide, member of a peptide injected CART dose-dependently inhibit food intake in rats (Goris- family that also includes pancreatic polypeptide and peptide YY. In sen et al., 2006). To date, CART has been cloned from mammals mammals, NPY is one of the most potent orexigenic factors known (Adams et al., 1999; Douglass and Daoud, 1996; Douglass et al., to date (Chee and Colmers, 2008). NPY has been isolated and char- 1995), amphibians (Lazar et al., 2004) and fish, including goldfish acterized in a number of fish species, including goldfish, Carassius (Volkoff and Peter, 2001), Atlantic cod (Kehoe and Volkoff, 2007) auratus (Blomqvist et al., 1992), perch, Siniperca chuatsi (Liang and catfish (Kobayashi et al., 2008). ICV injections of CART decrease et al., 2007), trout, Oncorhynchus mykiss (Doyon et al., 2003), and food intake in goldfish (Volkoff and Peter, 2000) and fasting in- cod, Gadus morhua (Kehoe and Volkoff, 2007). In fish as in other duces decreases in CART mRNA in goldfish (Volkoff and Peter, vertebrates, NPY appears to be involved in the regulation of feed- 2001), cod (Kehoe and Volkoff, 2007) and catfish (Kobayashi ing. Intracerebroventricular (ICV) injections of NPY in channel cat- et al., 2008), suggesting that CART regulates feeding in fish. fish (Ictalurus punctatus)(Silverstein et al., 2001) and goldfish Cholecystokinin (CCK) is synthesized by intestinal endocrine cells as a 115 amino acid prepro-CCK polypeptide that is cleaved * Corresponding author. Fax: +1 709 737 3018. post-translationally to generate gastrin/CCK-like peptides that E-mail address: [email protected] (H. Volkoff). share similar carboxy-terminal ends (Chandra and Liddle, 2007; 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.01.021 E. MacDonald, H. Volkoff / General and Comparative Endocrinology 161 (2009) 252–261 253 Vishnuvardhan and Beinfeld, 2002). In mammals, CCK-8, the most plings, the weights of fish were measured and the sex and sexual abundant form of CCK (Moran and Kinzig, 2004), acts via vagal maturity were noted. afferent pathways to stimulate gallbladder contractions and pan- creatic and gastric secretions and to decrease food intake (Chandra 2.2. RNA extraction and Liddle, 2007; Rehfeld et al., 2007). CCK/gastrin-like immunore- activity has been shown in the nervous system and gut of several For cloning and tissue distribution studies, four fed fish were fish species including Atlantic cod (Jonsson et al., 1987), goldfish dissected to obtain samples of brain and peripheral tissues (gill, (Himick and Peter, 1994) and halibut (Hippoglossus hippoglossus) heart, stomach, gut, spleen, liver, kidney, muscle and gonad). (Kamisaka et al., 2001). mRNA sequences have also been deter- For brain tissue distribution, individual brains were further dis- mined for a number of fish species including goldfish (Peyon sected into hypothalamus, telencephalon, optic tectum, and cer- et al., 1998), dogfish (Squalus acanthias)(Johnsen et al., 1997), puf- ebellum according to a previously established brain morphology ferfish (Kurokawa et al., 2003) and Japanese flounder (Paralichthys for elasmobranchs (Northcutt, 2002). For gene expression stud- olivaceus)(Kurokawa et al., 2003). Various forms of CCK, including ies experimental fish were dissected to obtain hypothalamus, CCK-8, are present in fish and have been shown to influence diges- telencephalon and gut (adjacent to the pyloric caeca) tissue. tion and appetite. In teleosts, CCK induces contractions of the gall Fish were anesthetized by immersion in 0.05% tricaine meth- bladder (Aldman and Holmgren, 1995), a decrease in gastric emp- anesulfonate (Syndel Laboratories, Vancouver, BC, Canada) and tying (Olsson et al., 1999) and an increase in gut motility (Forgan killed by spinal section. Tissues were dissected and immediately and Forster, 2007). Both central and peripheral injections of CCK placed on ice in RNAlater (Qiagen Inc., Mississauga, Ont., Can- cause a decrease in food intake in goldfish (Himick and Peter, ada) and stored at À20 °C until RNA extractions were 1994; Volkoff et al., 2003), oral administration of CCK decreases performed. food intake in sea bass (Rubio et al., 2008) and oral administration Total RNA was isolated using a trizol/chloroform extraction of a CCK antagonists causes an increase in food consumption in with Tri-reagent (BioShop, Mississauga, Ont., Canada) following both trout and sea bass (Gelineau and Boujard, 2001; Rubio the manufacturers’ protocol. Final RNA concentrations were deter- et al., 2008), suggesting that CCK also influences appetite regula- mined by optical density reading at 260 nm using a NanoDrop ND- tion in fish. In addition, CCK mRNA levels increase following a meal 1000 spectrophotometer (NanoDrop Technologies Inc., Wilming- in goldfish brain (Peyon et al., 1999) and in the pyloric caeca of yel- ton, USA). The quality of RNA samples was assessed by measuring lowtail (Murashita et al., 2007). the ratio of sample absorbance at 260 and 280 nm. Only RNA sam- The winter skate is an oviparous elasmobranch benthic species ples with a ratio between 1.8 and 2.1 were used. of the family Rajidae, which range extends from the Gulf of St. Law- rence to the south coast of Newfoundland (Scott et al., 1988). 2.3. Cloning of cDNA Although winter skates have recently become the object of specific fisheries (Frisk and Miller, 2006), little is currently known about Two micrograms of total RNA was subjected to reverse tran- their life cycle and physiology, in particular their feeding physiol- scription into cDNA with a dT-adapter primer (Table 1) using M- ogy. To date, most research on winter skates has focused on osmo- MLV Reverse Transcriptase (New England Biolabs, Pickering, Ont., regulation, as they are well-adapted to exposure to different Canada). cDNA (0.5 lg) were then submitted to PCR amplifications salinities (Sulikowski et al., 2004; Treberg and Driedzic, 2006). using degenerate primers designed in regions of high identity In order to provide new information on the mechanisms regu- among fish and various vertebrate sequences. The annealing tem- lating appetite in winter skate, we cloned cDNAs encoding three perature was optimized for each primer set. All PCR reactions were appetite-regulating hormones (NPY, CART and CCK), examined carried out in a volume of 25 ll using JumpStart Taq DNA polymer- their mRNA tissue distribution and assessed the effects of fasting ase (Sigma, St. Louis, MO, USA). PCR products were electrophoresed on their gene expression. in a 1% agarose gel, and visualized using an Epichemi Darkroom BioImaging System (UVP, Upland, CA, USA) equipped with a 12- bit cooled camera. Image processing and analysis were performed 2. Material and methods using LabWorks 4.0 software (UVP). Bands of predicted size were isolated and purified with the GenElute Gel Extraction Kit (Sigma, 2.1. Animals Oakville, Ont., Canada), cloned using a pGEM-T easy vector system (Promega, Madison, WI, USA) and sequenced by the MOBIX Lab Twenty winter skates (average weight of 1.86 ± 0.32 kg) were (McMaster University, Ont., Canada). collected by scubadivers off the shore of St. John’s (NL, Canada) In order to isolate winter skate NPY, an initial fragment was ob- in September.