- Email: [email protected]
Effects of chronic growth hormone overexpression on appetite-regulating brain gene expression in coho salmon
Accepted Manuscript Effects of chronic growth hormone overexpression on appetite-regulating brain gene expression in coho salmon Jin-Hyoung Kim, Rosalind A. Leggatt, Michelle Chan, Hélène Volkoff, Robert H. Devlin PII:
S0303-7207(15)30003-4
DOI:
10.1016/j.mce.2015.06.024
Reference:
MCE 9196
To appear in:
Molecular and Cellular Endocrinology
Received Date: 1 May 2015 Accepted Date: 22 June 2015
Please cite this article as: Kim, J.-H., Leggatt, R.A., Chan, M., Volkoff, H., Devlin, R.H., Effects of chronic growth hormone overexpression on appetite-regulating brain gene expression in coho salmon, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.06.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of chronic growth hormone overexpression on
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appetite-regulating brain gene expression in coho salmon
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Jin-Hyoung Kim1, Rosalind A. Leggatt1, Michelle Chan1, Hélène Volkoff2, and Robert H.
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Devlin1*
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Marine Drive, West Vancouver, BC V7V 1N6 Canada
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Fisheries and Oceans Canada, Centre for Aquaculture and Environmental Research, 4160
Departments of Biology and Biochemistry, Memorial University of Newfoundland, St. John's,
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NL A1B 3X9 Canada
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R.H. Devlin
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E-mail address: [email protected]
Corresponding author:
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Abstract:
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Organisms must carefully regulate energy intake and expenditure to balance growth and trade-
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offs with other physiological processes. This regulation is influenced by key pathways
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controlling appetite, feeding behaviour and energy homeostasis. Growth hormone (GH)
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transgenesis provides a model where food intake can be elevated, and is associated with dramatic
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modifications of growth, metabolism, and feeding behaviour, particularly in fish. RNA-Seq and
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qPCR analyses were used to compare the expression of multiple genes important in appetite
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regulation within brain regions and the pituitary gland (PIT) of GH transgenic (fed fully to
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satiation or restricted to a wild-type ration throughout their lifetime) and wild-type coho salmon
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(Oncorhynchus kisutch). RNA-Seq results showed that differences in both genotype and ration
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levels resulted in differentially expressed genes associated with appetite regulation in transgenic
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fish, including elevated Agrp1 in hypothalamus (HYP) and reduced Mch in PIT. Altered mRNA
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levels for Agrp1, Npy, Gh, Ghr, Igf1, Mch and Pomc were also assessed using qPCR analysis.
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Levels of mRNA for Agrp1, Gh, and Ghr were higher in transgenic than wild-type fish in HYP
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and in the preoptic area (POA), with Agrp1 more than 7-fold higher in POA and 12-fold higher
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in HYP of transgenic salmon compared to wild-type fish. These data are consistent with the
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known roles of orexigenic factors on foraging behaviour acting via GH and through MC4R
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receptor-mediated signalling. Igf1 mRNA was elevated in fully-fed transgenic fish in HYP and
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POA, but not in ration-restricted fish, yet both of these types of transgenic animals have very
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pronounced feeding behaviour relative to wild-type fish, suggesting IGF1 is not playing a direct
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role in appetite stimulation acting via paracrine or autocrine mechanisms. The present findings
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provide new insights on mechanisms ruling altered appetite regulation in response to chronically
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elevated GH, and on potential pathways by which elevated feeding response is controlled,
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independently of food availability and growth.
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Keywords: appetite; growth hormone, GH; transgenic; coho salmon; insulin-like growth factor 1,
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IGF1; agouti related neuropeptide, AGRP; nutrition; homeostasis
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1.
Introduction
59 The mechanisms controlling food intake are complex and involve many organ systems,
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endocrine pathways, and neuronal circuits that integrate environmental signals with endogenous
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physiological states. Appetite regulation is crucial to appropriate growth and energy homeostasis
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for an organism. A major pathway controlling metabolic rate, growth, and food intake is the
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growth hormone (GH)/insulin-like growth factor (IGF) axis. In fish as in mammals, GH is
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secreted into circulation by the pituitary (PIT) and acts through the growth hormone receptor
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(GHR) to stimulate IGF1 production in hepatic and other tissues, which induces somatic growth
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and exerts a negative feedback on GH secretion. Somatostatins [SST, produced by the
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hypothalamus (HYP)] inhibit both GH secretion and Igf1 gene expression whereas ghrelin
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(GHRL, produced by stomach and intestine) stimulates GH secretion (Won and Borski, 2013). In
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addition to regulating growth, GH is a pleiotropic hormone involved in many functions including
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appetite, stress response, energy homeostasis, and reproduction (Björnsson et al., 2002). Gh
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genes have been overexpressed or knocked out to examine physiological responses in species
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with determinate growth (i.e. grow to a final body size), and effects on multiple appetite-
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regulating genes have been observed (Arora and Anubhuti, 2006; Bohlool-Y et al., 2005;
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Kopchick et al., 1999). In fish, model transgenic strains overexpressing GH have also been
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developed for species that possess determinate growth (Figueiredo et al., 2007), as well as for
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those with indeterminate growth (i.e. growing throughout their entire life), including carp
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(Cyprinus carpio) (Wan et al., 2012; Zhong et al., 2013), tilapia (Oreochromis niloticus) (Lu et
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al., 2009; Rahman et al., 1998), loach (Misgurnus mizolepis) (Nam et al., 2001), and several
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salmonid species (Devlin et al., 1994; Devlin et al., 2004a; Du et al., 1992). GH transgenic (T)
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fish can show highly elevated feeding behaviour, growth, and metabolic rate, and possess
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modifications of other physiological processes at the levels of gene expression, enzyme activities,
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and the whole animal (Devlin et al., 2001; Devlin et al., 2004a; Lõhmus et al., 2008; Raven et al.,
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2008). Despite this body of literature, the mechanisms by which GH influences fish appetite
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regulation are not yet fully understood, in part because control of food intake in fish tends to
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differ among species to a greater extent than in mammals (Hoskins and Volkoff, 2012).
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Feeding and energy balance are regulated by centres in the brain, which produce and are
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affected by appetite-regulating peptides. Examples of these peptides are orexigenic factors such
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as agouti related neuropeptide 1 (AGRP1), neuropeptide Y (NPY), and orexin (HCRT), and
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anorexigenic peptides such as cocaine and amphetamine regulated transcript (CART),
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cholecystokinin (CCK), and α-melanocyte stimulating hormone (α-MSH, processed from
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proopiomelanocortin, POMC). In mammals, the paraventricular nucleus (PVN) and arcuate
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nucleus (ARC) in the hypothalamic region of the brain are recognized as command centres for
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controlling energy balance. The preoptic area (POA) was recently defined as the PVN-
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homologous region in the HYP of fish (Herget et al., 2014), and has previously been found to
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display altered mRNA levels for Npy in hungry fish (Silverstein et al., 1998). In teleost fish,
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hypothalamic AGRP and POMC neurons associated with appetite and growth project directly
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into the PIT (Zhang et al., 2012) and the HYP/POA/PIT axis is thought to play a pivotal role in
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multiple pathways including appetite regulation, feeding behaviour, and energy use. These
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actions are regulated in part by production and release of, and response to, GH (Forlano and
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Cone, 2007; Herget et al., 2014; Zhang et al., 2012).
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Due to the magnitude of phenotypic changes seen in T fish, they provide a useful model
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organism to understand the relationship between appetite regulation, growth, and behaviour. The
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pleiotropic effects of GH are believed to be largely mediated by IGF1 produced in liver and other
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tissues in response to GH stimulation (de Azevedo Figueiredo et al., 2007; Frago et al., 2002). It
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is well established that GH overexpression in fish elevates Igf1 gene expression in multiple
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tissues and increases circulating IGF1 protein levels (Beckman, 2011), and this is correlated with
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strongly elevated feeding behaviour and food intake in animals fed ad libitum. However, T fish
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reared on a wild-type (restricted) ration level have normal levels of Igf1 gene expression and
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IGF1 circulating hormone, yet possess the same heightened feeding motivation seen in fully-fed
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transgenic fish (Devlin, 2011; Raven et al., 2008). These data show that IGF1 production is
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influenced by nutritional state (Beckman, 2011), and suggest that elevated appetite in T fish is
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not directly mediated either by peripheral IGF1 levels, or by increased nutrient utilization signals
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associated with elevated somatic growth. Rather, appetite is likely elevated by direct stimulation
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of central feeding centres by GH or by other peripheral signals affected by GH independently of
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IGF1. Although the mechanisms ruling the central effects of GH on feeding behaviour and
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growth are not fully understood, recent studies in T fish suggest important roles for appetite-
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related neuropeptides. For example, T coho salmon (Oncorhynchus kisutch) have lower
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telencephalic expression of Npy and winter levels of Cck relative to wild-type fish (Lõhmus et al.,
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2008; Raven et al., 2008), whereas in T carp, both the hypothalamic and telencephalic expression
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of Agrp1 is 2-fold higher relative to wild-type fish, although transgenesis does not seem to affect
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Npy, Hcrt, Pomc, Cck, or Cart expressions (Zhong et al., 2013).
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To further understand the mechanisms controlling appetite and growth in fish, the current
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study has undertaken a comprehensive examination of mRNA levels of appetite-regulating genes
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producing orexigenic, anorexigenic, and metabolic effects, in the HYP (with POA separately)
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and the PIT of wild-type (NT), fast-growing GH transgenic (TF), and ration-restricted GH
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transgenic (TR) coho salmon.
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2.
Materials and Methods
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2.1. Experimental Animals.
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The experiment was performed September 23-27, 2013 at the Centre for Aquaculture and
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Environmental Research (CAER), Fisheries and Oceans Canada (DFO), West Vancouver,
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Canada. This facility contains specific containment measures to prevent the escape of genetically
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modified fish to the natural environment. All experimental procedures were carried out in
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compliance with the Canadian Council for Animal Care guidelines under permit from DFO’s
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Pacific Regional Animal Committee. Three size-matched groups of coho salmon (Oncorhynchus
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kisutch; 95.8 ± 15 g) were examined: (i) wild-type coho salmon (non-transgenic, NT), (ii) GH
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transgenic coho salmon fully fed to satiation throughout their lifespan (TF) and growing 2-3-fold
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faster than wild-type fish (Devlin et al., 2004b), and (iii) GH transgenic salmon that were ration-
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restricted to the NT satiety ration level throughout their lifespan (TR). All fish were of the same
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genetic background (Chehalis River hatchery coho salmon from Fisheries and Oceans Canada
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Chehalis River Enhancement Facility Agassiz, BC). Transgenic coho salmon (T) contained the
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OnMTGH1 gene construct (Devlin et al., 1994) (strain M77), and were produced at CAER
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(Devlin et al., 2004b) and maintained in a wild-type genetic background by crossing T at each
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generation to NT coho salmon collected from nature. NT salmon were produced by crossing
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wild-type males to the same females used to produce TR salmon. NT and TR fish were produced
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in January 2012, and TF fish were produced in January 2013. Thus, TF fish were of same size
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and developmental stage as TR and NT fish, but were one year younger. All groups of fish were
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reared under the same standard conditions (400 fish / 4000 L fibreglass tanks, 1 group of fish per
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tank, 10 ± 1 ºC well water, and simulated daylight set to the natural photoperiod). Fish were fed
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stage-appropriate commercial salmonid diets (Skretting Ltd., Canada) at fixed times of day (9
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AM and 2 PM) for at least 3 months prior to the experiment to standardize physiological
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responses to feeding. Foraging and schooling behaviour of each group was visually observed
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prior to and during feeding events.
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2.2. Sampling and Dissection.
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Three time points were chosen to represent different stages of feeding: pre-feeding, and
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two post-prandial stages [one hour post-feeding (1 hpf) for satiation, and four hours post-feeding
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(4 hpf) for active digestion]. Fish were sampled over a five-day period as follows: Day 1: TR
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pre-feeding; Day 2: NT pre-feeding; Day 3: TF pre-feeding, TR 1 hpf, and TR 4 hpf; Day 4: NT
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1 hpf and NT 4 hpf; Day 5: TF 1 hpf and TF 4 hpf. This sampling approach provided a two-day
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recovery period between pre-feeding and post-feeding samplings for each group. No differences
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in feeding behaviours of a population were noted between pre-experimental and sampling
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periods. Fish were fed normal feeding levels between pre-feeding and post-feeding sample days,
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and then fed to satiation at the 9 AM feeding time prior to post-feeding sample times. For the
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pre-feeding samples, 12 fish were rapidly (30 sec) selected from their population immediately
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prior to the 9 AM feeding time (i.e. at 8:45 AM), after which the population was fed to satiation.
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For 1 hpf and 4 hpf time points, 12 fish were selected for sampling at 10 AM and 1 PM,
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respectively. In order to minimize time delay between samples and effect of handling on gene
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expression, four sampling stations were simultaneously used and tissues were collected in less
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than 3 minutes per fish. The fish selected for sampling were rapidly euthanized in a bath
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containing a buffered tricaine methanesulphonate (200 mg/L; 400 mg/L sodium bicarbonate;
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Syndel Laboratories Ltd., Vancouver, BC, Canada). On cessation of ventilatory activity, fish
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were bled by caudal severance and whole brain and PIT from each fish were rapidly collected
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and placed in RNAlater (Ambion, Austin, TX, USA) for overnight storage at 4 ºC, followed by
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long-term storage at -20 ºC. This process was repeated three times to allow sampling of all 12
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selected fish per group and time point to be completed within 15 minutes. Later, POA and HYP
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were dissected from whole brain under a dissecting scope following salmon anatomical
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description (Billard and Peter, 1982; Forlano and Cone, 2007; Herget et al., 2014; see Figure 5a).
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Total RNA was extracted via RNeasy mini kits (Qiagen, Valencia, CA, USA) from
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individual samples, and concentration and purity measured using a Nanodrop (Thermo Scientific,
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Wilmington, DE, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent
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Technologies, Palo Alto, CA, USA) and all samples possessed a RNA integrity number (RIN) >
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9.0. For RNA-Seq (pre-feeding samples only), 2 µg of total RNA was pooled from five
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individuals from each group (NT, TF, and TR) to create two biological replicates (i.e. two pools
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of five fish for each genotype). For qPCR analysis, first-strand cDNA was synthesized from total
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RNA (0.5 µg) using the High Capacity cDNA synthesis kit with RNase inhibitor (Applied
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Biosystem, Foster City, CA, USA).
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RNA-Seq analysis was performed using the Illumina HiSeq2000 platform (Illumina, San
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Diego, CA, USA) using two samples (n=2) of five fish per group. BLAST-based gene
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identification was performed to annotate a coho salmon transcriptome constructed for this study
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(to be reported elsewhere) and downstream differential expression analysis was used to
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determine differentially expressed genes (DEGs) between groups of fish. For normalization of
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raw reads in RNA-Seq analysis, a scaling factor in DESeq method (Anders and Huber, 2010) and
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Ubiquitin were used. RobiNA software [http://mapman.gabipd.org/web/guest/robin (Lohse et al.,
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2012)] was used to perform DESeq, which uses a Bioconductor Software Package that assumes a
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negative binomial distribution of sequence count data.
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2.5. qPCR
209 Selection of appetite related genes for qPCR was based on previous studies of feeding
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regulation in fish (MacDonald and Volkoff, 2009; Penney and Volkoff, 2014; Tuziak and Volkoff,
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2013; Zhong et al., 2013). Primers and/or probes (Supplementary Table 1) were designed using
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Primer Express software (Applied Biosystem, version 3.0.1) with sequences from the coho
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salmon transcriptome. The specificity of all pairs of primer was validated by PCR and
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electrophoresis analyses to confirm proper size of amplicons. Ubiquitin was used as a reference
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gene to normalize mRNA levels, it displayed consistently stable mRNA expression levels among
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experimental groups and among several reference genes examined (β-actin, Ef-1a and
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Ubiquitin). qPCR reactions were performed as previously described (Kim et al., 2015). Relative
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mRNA expression levels were calculated relative to the Ct value obtained for the housekeeping
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control gene (Ubiquitin) using the 2-∆∆Ct method. (Livak and Schmittgen, 2001).
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2.6. Statistical Analysis
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One-way or two-way ANOVA followed by Duncan’s multiple range tests were used to
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evaluate differences across the time series and among genotypes, using SPSS statistical package,
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version 18.0 (SPSS Inc., Chicago, IL, USA). If normality or equal variance failed and could not
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be restored by transformation, Kruskal-Wallis tests were used. All results are expressed as the
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mean ± SEM, and statistical significance was determined at P < 0.05.
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3.
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Results
3.1. Fish Behaviour
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Before feeding, NT fish grouped tightly together at the bottom of the tank, while T fish
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dispersed much more widely in the tank and were located close to the surface of the water.
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During feeding, high levels of foraging behaviour in T fish were observed, while NT fish showed 8
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less feeding response and displayed behaviour typically associated with balancing predation risk
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avoidance with foraging.
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Differentially expressed genes (DEGs) among fish groups (NT, TF and TR) in brain tissues
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(POA, HYP and PIT) are shown in Supplementary Table 2. Twenty-three DEGs were detected
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in POA, whereas 36 and 70 DEGs were found in HYP and PIT, respectively. The greatest
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number of DEGs (60) was found between TR and NT in PIT. Among the well-known appetite
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relevant genes, mRNA levels for two genes in particular showed very large differences: Agrp1
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levels in HYP were 8- and 13-fold greater in TF and TR than in NT, respectively, whereas Mch
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levels in PIT were reduced in TF when compared to NT. Specifically, Mch levels were 9-fold and
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17-fold lower in TF in PIT, while Mch levels were 2.5-fold lower in TF than in TR in the HYP.
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For GH axis genes, only Igf1 mRNA levels were found to be significantly different in HYP,
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where TF was 2.7-fold higher than TR.
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Although only a few appetite related genes were found to be differently expressed based on
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RNA-Seq data, further analysis of the raw reads of 23 selected appetite regulating genes were
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analyzed by two normalization methods: 1) by normalizing using the scaling factor method in
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DESeq (Supplementary Table 3a), and 2) by normalizing relative to Ubiquitin as an internal
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control (Supplementary Table 3b). Similar patterns of significant differences among groups
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were found using both methods when mRNA levels were compared on an individual gene level
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for Agrp1, Cart, Gh, growth hormone receptor (Ghr), gonadotropin releasing hormone (Gnrh),
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Igf1, and melanin-concentrating hormone (Mch). However, Agrp2, growth hormone releasing
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hormone (Ghrh) and orexin (Hcrt) showed significant differences when normalized with
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Ubiquitin only, and bombesin-like peptides (Bbs) and galanin (Gal) showed significant
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differences when normalized by scaling factor only.
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3.3. qPCR Validation of Appetite-regulating Genes mRNA Relative to Reads Measured by RNASeq
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qPCR analyses were undertaken to further validate the mRNA levels for 23 appetite-
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associated genes in POA, HYP and PIT from RNA-Seq analysis. Relative levels of mRNA
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among tissues examined are shown for NT salmon (Supplementary Fig. 1). Numerical qPCR
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data are given in Supplementary Tables 4-6. qPCR analyses were performed on three DEGs
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(Agrp1, Mch and Igf1) that was detected as differentially expressed from RNA-Seq analysis (Fig.
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1a-1c). Overall, a high correlation was seen between RNA-Seq and qPCR data (R2 = 0.91,
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Supplementary Fig. 2). For genes showing greater than 2-fold differences between T and NT in
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pre-feeding samples, additional qPCR analyses were performed using 4 hpf samples. For genes
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showing significant differences at 4 hpf, mRNA levels were also measured for 1 hpf samples
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(Fig. 1a-1c).
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For both TF and TR, Agrp1 mRNA levels were significantly higher than in NT, in both
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POA and HYP (Fig. 1a) with the highest difference (12-fold) observed between NT and TR
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animals in HYP in pre-feeding samples. In contrast, in PIT, elevated Agrp1 mRNA levels above
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NT were only evident in TR fish. T fish did not differ in Agrp1 mRNA levels with time, whereas
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NT fish had higher levels at 1 hpf, but not 4 hpf, than the pre-feeding sample in HYP, and lower
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levels at 4 hpf than pre-feeding in PIT samples (Fig. 1a).
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Mch mRNA was found at lower levels in TF than NT and TR salmon in all brain regions
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examined, although these differences were not significant at all time points in POA as there was
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very high variance among the individual samples. TR fish had lower levels of Mch than NT fish
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in HYP at 1 hpf and in PIT at 4 hpf (Fig. 1b). When compared to pre-feeding samples, Mch
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mRNA levels in TR and TF fish increased in HYP and decreased in PIT at 4 hpf.
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Igf1 mRNA levels were generally elevated in TF in all tissues, although this was not
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significant at pre-feeding in PIT. Levels in TR were greater than NT in POA at 1 and 4 hpf, were
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lower than NT in PIT at 4 hpf, and were similar to NT levels at all other time points/tissues (Fig.
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1c). In HYP, levels of Igf1 increased at 1 hpf and decreased at 4 hpf in all fish groups, although
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this was not significant in TR. In POA, levels in TF increased at 1 hpf and decreased at 4 hpf,
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whereas levels in TR fish increased at 1 hpf but there was no significant difference at 4 hpf.
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3.4. Assessment of Other Appetite and Metabolism Gene mRNA Levels
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NT and T fish did not differ significantly in Npy mRNA levels at pre-feeding in any tissue.
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However, TF had greater levels of Npy mRNA than TR in POA. In PIT and HYP, a general trend
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in reduction of Npy expression was noted in NT and TR fish after feeding, whereas expression in
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HYP rose at 1 hpf and decreased at 4 hpf (Fig. 2a).
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In POA, TF and TR had higher Gh mRNA levels than NT at pre-feeding and 4 hpf. In HYP,
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TF and TR also showed significantly higher levels than NT at pre-feeding, but only TF had a
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higher level at 1 hpf and there were no significant differences at 4 hpf. In PIT, TF Gh mRNA
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levels were significantly lower at all time points than in NT, were lower than TR at pre-feeding
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and 1 hpf, and increased with feeding (Fig. 2b).
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In POA and HYP, TF and TR showed higher mRNA levels of Ghr than NT, whereas in PIT,
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TF levels were lower at pre-feeding than other groups and increased with feeding. There were no
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other differences among groups or time periods (Fig. 2c).
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Levels of Pomc mRNA in POA were significantly lower in TF fish than in NT at 4 hpf.
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There were no major differences in Pomc mRNA levels in HYP among groups, and Pomc
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decreased with feeding in TF fish only. In PIT, Pomc levels were higher in TF at 1 hpf than in all
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other genotypes (Fig. 3a).
Cart mRNA levels in POA were lower in TF than in other groups at pre-feeding only. In
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HYP and PIT, TF and TR had overall lower levels of Cart than NT fish, although this was not
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significant at all individual time points (Fig. 3b).
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Sst2 mRNA levels were higher in TF and TR than NT in PIT at pre-feeding and 4 hpf, and
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higher in TF than in NT and TR in HYP at 4 hpf only (Fig. 3c). There were significant
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differences in Sst1 in PIT only, where TF fish had the lowest levels at pre-feeding, and levels
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increased with feeding in both TF and TR so that they were greater than NT levels at 4 hpf (see
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Supplementary Table 6). There were no significant differences among fish groups and/or time
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points for any other gene examined (e.g. Agrp2, Bbs, Cck, Gal, Ghrh, Glp, Gnrh, Hcrt, Lep,
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Mc4r, Trh, Tsh, see Supplementary Tables 4-6)
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Ternary plots of appetite related mRNA levels for genes visually integrate significant
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differences observed among the three experimental groups (Fig. 4). Most orexigenic and GH-
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axis genes with significant differences tended to be over-represented in TF and TR fish, primarily
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in the POA and HYP (i.e. Agrp1 in all tissues, Gh and Ghr in POA and HYP), or in TF fish only 11
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(i.e. Npy and Igf1 in all tissues). For these genes, feeding tended to decrease the association with
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TF fish (i.e. arrows moved from high to low along TF axis in red), with the exceptions of Gh and
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Agrp1 in HYP where TF representation was increased with feeding, and for genes in PIT where
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feeding had a lesser effect. Orexigenic and GH system genes that were more associated with NT
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and TR fish included Mch (all tissues), and Gh and Ghr (PIT only). Feeding increased high
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representation of Mch in NT in all tissues (only at 1 hpf in POA and HYP), and decreased Gh
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and Ghr high representation in TR in PIT. There were fewer patterns apparent for the relative
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expression of anorexigenic genes (Fig. 4a-4c), although Cart was highly represented in NT fish
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in all tissues (indicating reduced expression in TR and TR), and this tended to increase at 4 hpf in
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HYP and PIT. Pomc was highly represented in TR fish in POA pre-feeding, but this switched to
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high NT representation post-feeding. In the HYP, Pomc expression was highly represented in TF
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pre-feeding, but was similar in all groups post-feeding, whereas in the PIT, Pomc was highly
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represented in TF fish regardless of feeding. Sst1 and Sst2 were highly represented in TR fish in
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PIT, and feeding increased Sst2 representation in TF fish and decreased NT fish representation
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post-feeding, while Sst2 was highly represented in TF fish in HYP, particularly post-feeding.
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When food is not limiting, GH transgenic (T) coho salmon grow dramatically faster than
346
non-transgenic fish (NT) due to increased appetite and food intake (Devlin et al., 1994; Devlin et
347
al., 2004a; Devlin, 2011). This behavioural transformation in T fish is likely mediated by an
348
altered balance between orexigenic and anorexigenic factors in the brain and pituitary gland
349
(PIT), as well as from peripheral signals arising from altered anabolic activity and nutritional
350
signals from processed food. It is currently not completely clear whether the stimulated appetite
351
of T salmon arises from direct effects of GH on the brain, or is due to the stimulation of growth
352
of peripheral tissues which in turn send signals to the brain to alter feeding behaviour, or both.
353
Hypothetically, following satiation, hunger signals in T fish might return more rapidly to pre-
354
feeding levels than in NT fish, thus increasing meal frequency. GH overexpression could also
355
sustain higher expression of orexigenic signals and lower protein levels and/or expression of
356
anorexigenic signals in T relative to NT to cause greater food intake at each meal. Both of these
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feeding responses (increased meal frequency and food intake) are observed in GH T salmon
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relative to NT. Elevated Agrp1 and reduced Cart expression levels found in T in the current
359
study are consistent with feeding-related peptides playing a role in mediating increased appetite.
360
However, inconsistent effects were observed for other appetite-related genes including Npy and
361
Pomc. In particular, Mch and Sst2 had expression patterns opposite to what was expected. The
362
reduced expression of genes encoding known orexigenic factors and the increased expression of
363
genes encoding appetite inhibitors in T fish might be indicative of compensatory mechanisms
364
aimed at returning feeding levels to normal.
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Elevated Agrp1 mRNA levels in the preoptic area (POA) and hypothalamus (HYP),
366
regardless of ration level, are consistent with this neuropeptide being involved in the stimulation
367
of appetite and food intake seen in T salmon. AGRP is part of the melanocortin system, which
368
acts in the endocrine regulation of growth and appetite through control of neurons affecting
369
hypothalamic release of hormones (Cone, 2005; Zhang et al., 2012). The orexigenic actions of
370
AGRP are mediated by antagonism of α-MSH (derived by processing of POMC) stimulation of
371
the melanocortin-4 receptor (MC4R), a system that has been reported in a range of vertebrates
372
from fish to mammals (Belgardt et al., 2009; Cone, 2005; Zhang et al., 2012; Zhong et al., 2013;
373
Zhu et al., 2013). In another GH transgenic fish, the common carp (Cyprinus carpio L), elevated
374
Agrp1 expression has also been demonstrated, and direct treatment of isolated NT carp HYP
375
slices with GH increases Agrp1 expression, suggesting GH acts directly on brain feeding
376
pathways. In T carp, Agrp1 expression was elevated 2-fold in HYP (Zhong et al., 2013), whereas
377
the present study found 4- to 10-fold increases in the POA of T salmon. The differences in Agrp1
378
mRNA between T and NT carp and salmon do not correlate with GH levels between the two
379
transgenic models: both peripheral GH protein and hypothalamic levels of Gh mRNA are over
380
100-fold greater in T carp, whereas T coho salmon have only a 2- to 3-fold elevation of
381
circulating GH (Raven et al., 2008), and 2- to 6-fold increases in hypothalamic and other tissue
382
Gh mRNA expression (Raven et al., 2008). This difference in response between these two studies
383
could be due to species-specific differences in GH/IGF1 axis regulation, as well as the use of
384
different promoters within the transgene (β-actin promoter in carp and metallothionein-B
385
promoter in the current study) (Devlin et al., 1994; Zhu et al., 2013) that may produce different
386
distributions and levels of Gh mRNA among brain tissues. As well, central Agrp levels are
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correlated with stimulated appetite in rats (Kamegai et al., 2001), and are decreased in fasted
388
salmon (Murashita et al., 2009), although are unaffected by temperature-derived suppressed
389
appetite in salmon (Hevrøy et al., 2012). In mammals, the melanocortin system is comprised of
390
POMC expressing neurons and AGRP/NPY co-expressing neurons in the (arcuate nucleus) ARC.
391
In POMC neurons, POMC is cleaved to form α-MSH, which acts on MC4R located in the
392
paraventricular nucleus (PVN) of the HYP to decrease food intake and increase energy
393
expenditure. It is noteworthy that although AGRP administration stimulates feeding and Agrp
394
overexpression leads to obesity in both fish and mammals (Barsh and Schwartz, 2002; Song and
395
Cone, 2007), Agrp and Npy knockout mice do not show any alteration of feeding and body
396
weight (Qian et al., 2002). Elevated Agrp1 mRNA in T fish may act to increase appetite by
397
blocking the α-MSH stimulation of MC4R anorexigenic action. However, in the current study, T
398
did not show consistent changes in either Pomc or Mc4r mRNA expression levels across tissues
399
and sampling points compared to NT fish, suggesting that neither POMC nor MC4R at the
400
mRNA level directly mediate increases in feeding observed in T fish. In mammals, although
401
AGRP and POMC neurons have been shown to be in close proximity in several brain regions
402
and act to counter-balance each other to regulate appetite, they do not appear to function simply
403
as antagonists in their responses to metabolic signals (Warne and Xu, 2013). Indeed, in an
404
optogenetic study using light-activated neuron signals, POMC suppression of feeding was
405
dependent on melanocortin receptor signalling in mice, whereas stimulation of AGRP neuron
406
activation rapidly induced food intake independent of the melanocortin pathway (Aponte et al.,
407
2011). Increased Agrp expression without changes in Pomc and Mc4r expression in T fish might
408
suggest that the actions of AGRP are independent of POMC and MC4R. It is noteworthy that
409
significantly higher Pomc expression levels were observed in the PIT of TF. POMC is cleaved
410
into several peptides other than the anorexigenic α-MSH, including opioid neuropeptides such as
411
the orexigenic factor β-endorphin (Lin et al., 2000). In both goldfish (Carassius auratus) and
412
tench (Tinca tinca), intracerebroventricular (ICV) administration of β-endorphin induces
413
increases in food intake, whereas intraperitoneal (IP) injections do not (de Pedro et al., 1995;
414
Guijarro et al., 1999), suggesting that β-endorphin stimulates food intake by activating central
415
pathways (Baile et al., 1986; Morley, 1995). It is possible that high Pomc mRNA levels of TF in
416
PIT, the main region of POMC synthesis, may induce increased β-endorphin synthesis resulting
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in increased appetite. If the appetite-stimulating actions of β-endorphin are greater than the
418
suppressive effects of α-MSH, then, coupled with highly increased AGRP, appetite would overall
419
be stimulated. The role of AGRP and the influences of the balance between α-MSH and β-
420
endorphin levels in feeding behaviour require further examination.
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MCH is a peptide synthesized in both HYP and PIT, and is associated with multiple
422
functions such as pigmentation (Naito et al., 1985) and energy homeostasis (Pissios et al., 2006).
423
Mch was expressed at lower levels in TF salmon compared to slower growing NT and TR fish in
424
POA and HYP as well as PIT, and these differences were not abolished by feeding to satiation.
425
Thus, it appears that, in salmon, Mch mRNA expression is inversely correlated with slower
426
growth and low ration level, and is not strongly influenced by short-term differences in food
427
intake (i.e. 0 to 4 hrs post-feeding). Further, MCH likely does not play a direct role in elevating
428
appetite in T fish since both TF and TR have highly stimulated feeding behaviour, yet Mch
429
mRNA levels are similar in TR and NT fish, indicating Mch levels are primarily influenced by
430
the chronic high feeding rates in TF fish. In mammals, the role of MCH in feeding regulation has
431
consistently been reported as orexigenic (Elliott et al., 2004; Pissios et al., 2006; Segal-
432
Lieberman et al., 2006), whereas, there are inconsistent reports on the role of MCH in feeding in
433
fish (Volkoff, 2014). For example, MCH acts as an orexigenic factor in several fish including
434
Atlantic cod (Gadus morhua) (Tuziak and Volkoff, 2013), zebrafish (Berman et al., 2009), winter
435
flounder (Pseudopleuronectes americanus) and barfin flounder (Verasper moseri) (Takahashi et
436
al., 2004), whereas an anorexigenic action of MCH has been reported in goldfish (Matsuda et al.,
437
2006; Matsuda et al., 2009). It is possible that MCH may act as both an orexigenic or
438
anorexigenic factor through interactions with other appetite factors, as it can stimulate some
439
orexigenic systems (i.e. HCRT and apelin) and can inhibit others (i.e. NPY, CART) (Volkoff,
440
2014). In the present study, Mch mRNA levels in the PIT were correlated with Gh expression. As
441
the PIT is the major site of GH production in NT fish, this might indicate that, as in mammals
442
(Segal-Lieberman et al., 2006) and the cichlid Cichlasoma dimerus (Pérez Sirkin et al., 2012),
443
MCH stimulates GH secretion in salmon. However, further studies are needed to determine the
444
exact roles of MCH in the regulation of feeding and GH secretion in fish. It is noteworthy that
445
the present study found large variations in Mch mRNA levels among individual fish for both
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qPCR and RNA-Seq data. Whether this variation was due to natural biological differences in
447
expression among individuals is not known. NPY is considered one of the most potent orexigenic factors in vertebrates (Volkoff et al.,
449
2005). Npy mRNA expression levels in the POA are found be increased in food-deprived wild-
450
type coho salmon (Silverstein et al., 1998), and decreased in HYP of Atlantic salmon with
451
temperature-induced suppression of feeding (Hevrøy et al., 2012), suggesting an orexigenic role
452
for NPY in salmon. It was thus expected that T fish may have elevated Npy relative to NT fish to
453
account for increased appetite. However, this was inconsistently observed during post-feeding in
454
HYP and PIT, suggesting NPY likely does not have a major role in increased appetite in T fish.
455
Similarly, changes in Npy mRNA levels between T and NT carp was not observed (Zhong et al.,
456
2013), and previous studies show lower telencephalic or hypothalamic Npy in T salmon
457
depending on ration level (Raven et al., 2008). In NT carp, in vitro studies have shown that GH
458
increases the hypothalamic mRNA expression levels of Npy only when incubated with moderate
459
doses (20 ng ml−1 of GH for 2 h), and not with high doses or long exposures to GH (Zhong et al.,
460
2013). It is possible that the effects of GH on NPY are inhibited by sustained exposure to high
461
GH levels, as observed in T fish, perhaps by receptor down-regulation. Similar receptor
462
desensitizations have been shown for other peptides in fish. For example, in goldfish, treatment
463
with high doses of either NPY (Hoskins and Volkoff, 2012; Narnaware et al., 2000) or orexin
464
(HCRT) (Volkoff et al., 1999) have no or inhibitory effects on feeding. Npy expression was
465
inconsistently affected by feeding in T and not affected by feeding in NT in POA and HYP.
466
However, PIT Npy levels decreased after feeding to satiation in both NT and TR fish, suggesting
467
that feeding has region-specific effects on Npy expression. Postprandial decreases in Npy
468
expression have been shown in several fish species, including goldfish HYP and telencephalon
469
(Narnaware et al., 2000), Brazilian flounder HYP (Campos et al., 2012) and cobia (Rachycentron
470
canadum) brain (Nguyen et al., 2013), and could be due to satiation signals inhibiting expression
471
of Npy.
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472
The anorexigenic factor Cart showed higher mRNA levels in NT than in TR and TF fish in
473
most tissues, suggesting that elevated peripheral GH levels inhibited CART expression, and
474
thereby diminished the anorexigenic actions of CART in T fish. Indeed, CART injections inhibit
475
feeding in goldfish (Volkoff and Peter, 2001) and Cart mRNA levels in the brain of carp, 16
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goldfish, and Atlantic salmon decrease after food deprivation and increase after refeeding,
477
suggesting a role as a short-term regulator of feeding (Murashita et al., 2009; Volkoff and Peter,
478
2001; Wan et al., 2012). In contrast, there was no effect of starvation on Cart mRNA levels in the
479
winter skate, Raja ocellata (MacDonald and Volkoff, 2009), and feeding did not increase mRNA
480
levels in any genotype in the present study. Thus, CART may be important in long-term
481
maintenance of feeding behaviour but not play a major role in short-term feeding and satiation.
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In both mammals and fish, SST is produced mainly in HYP and POA, inhibits the release
483
of GH from the PIT (Brazeau et al., 1973; Yada and Hirano, 1992), and is involved in feedback
484
regulatory controls of the GH/IGF1 system (Sheridan and Hagemeister, 2010). While there were
485
no significant differences in Sst mRNA levels in POA and HYP at pre-feeding in the present
486
study, in PIT, both TF and TR fish had higher mRNA expression levels than NT for Sst2 at all
487
time points and for Sst1 at 4 hpf. As stimulation of PIT Sst mRNA expression by exogenous GH
488
and IGF1 has previously been reported in rainbow trout (Melroe et al., 2004), the increase in PIT
489
Sst levels seen in T fish might thus represent a response to high GH levels, in an effort to lower
490
GH production.
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Previous studies have shown that both TF and TR fish have greater non-pituitary Gh
492
mRNA and circulating GH levels than NT fish, regardless of feeding and growth level, whereas
493
only TF fish have lower PIT Gh mRNA than NT fish (Caelers et al., 2005; Mori and Devlin,
494
1999; Raven et al., 2012). This concurs with the present findings showing an increase in POA
495
and HYP Gh levels in all T fish and a decrease in PIT Gh levels in TF fish. ICV injections of GH
496
protein do not stimulate feeding in rainbow trout (Johansson et al., 2005), whereas peripheral
497
injections of GH do (Higgs et al., 1977), suggesting GH-mediated appetite regulation may be
498
through indirect or peripheral pathways. Decreased PIT Gh in TF fish may be due to a negative
499
feedback control of IGF1 and GH acting on the PIT (Caelers et al., 2005; Mori and Devlin, 1999),
500
and/or to the actions of other appetite factors that have been shown to influence PIT GH
501
secretion [e.g. MCH, NPY, SST (Brazeau et al., 1973; Pérez Sirkin et al., 2012; Peng et al., 1993;
502
Yada and Hirano, 1992)]. However, since NT and TR salmon have similar food intake levels,
503
growth rates, and IGF1 levels, whereas TF have elevated IGF1 levels, the reduced expression
504
levels of PIT Gh observed in TF salmon may be explained by the actions of IGF1 rather than GH
505
feedback.
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Compared to NT fish, Ghr mRNA levels were elevated in both TR and TF in HYP and
507
POA, whereas Igf1 mRNA levels were elevated in TF only. Similar trends have been reported in
508
TF and TR salmon for serum IGF1 levels and for Igf1 mRNA levels in muscle and liver (Devlin
509
et al., 2009; Overturf et al., 2010; Raven et al., 2008). IGF1 may be involved in stimulating
510
pathways necessary for enhanced growth in TF fish and appears to be related to the long-term
511
nutritional condition of T fish. High Igf1 levels in TF but not TR fish, despite high Gh levels in
512
both types, suggest that long-term ration restriction (to NT levels) in T fish has an inhibitory
513
effect on Igf1 mRNA levels. This concurs with previous studies showing that plasma IGF1 and
514
muscle, liver, and brain Igf1 mRNA levels are influenced by both feeding levels and growth
515
(Beckman, 2011), and that nutritional status can affect the response of Igf1 to GH (Beckman,
516
2011; Moriyama et al., 2000). Some biological actions of GH are mediated through interactions
517
with GHR to produce IGF1 (de Azevedo Figueiredo et al., 2007), and these actions can
518
consequently be influenced by GHR abundance on cell surfaces (Flores-Morales et al., 2006).
519
The discordance between Igf1 levels and Gh and Ghr levels in TR fish could be due to sustained
520
food restriction affecting GHR sensitivity by decreasing GHR production in other unexamined
521
tissues [see (Won and Borski, 2013)]. In the current study, T fish had higher Ghr mRNA levels in
522
the POA and HYP, but not in the PIT, corresponding to respective Gh expression levels seen for
523
these tissues. Overall, normal levels of IGF1 and Igf1 mRNA in TR indicate that the heightened
524
appetite and feeding behaviour of T fish (seen in both TF and TR) may not be mediated by either
525
elevated circulating IGF1 levels or by paracrine/autocrine actions of IGF1 in the brain regions
526
examined or in PIT. However, more specific tests of this hypothesis, involving blockage of IGF1
527
signalling in the brain, are required.
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The present data show significant responses of several genes to both long-term food intake
529
levels and to short-term effects of a meal. For genes that differed in expression between TF and
530
TR fish, intake of food and digestion decreased differences in gene expression levels for many
531
but not all orexigenic and GH system genes examined. There were some consistent trends in the
532
effects of food intake and digestion (relative to the pre-feeding state) on mRNA levels in
533
different tissues and genotypes. For some genes and tissues where expression differed between
534
TF and TR fish, food intake resulted in mRNA levels becoming more similar between TF and TR
535
fish, illustrated by vertical arrows moving to the centre in the ternary plots (i.e. for Agrp1 and
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Pomc in HYP, Gh and Ghr in PIT, Igf1 in POA, Cart in HYP and POA; see Figures 4a-4f).
537
These data are consistent with differences between TR and TF being due to long-term feeding
538
levels and show that these differences can be reduced by a single satiation/feeding event. In
539
contrast, feeding increased differences between TF and TR fish for other genes and tissues as
540
shown by arrows moving away from the centre towards the TF or TR vertex in the ternary plots
541
(i.e. for Npy in HYP and PIT, Sst2 in HYP, Pomc in PIT). Together, these responses reveal a
542
complex relationship among feeding levels, appetite, timing of nutrient processing, and gene
543
expression in T and NT fish.
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Multiple and complex responses in expression of appetite-regulating genes (including
545
Agrp1, Gh, Igf1, Ghr, Mch and Cart) have been observed in T fish compared to wild-type fish
546
(Fig. 5). Appetite regulation is likely maintained by a balance between orexigenic and
547
anorexigenic factors including those influencing the melanocortin system in neurons located in
548
the POA/HYP/PIT axis. Overexpression of GH stimulates Agrp1 expression, which in turn
549
stimulates feeding behaviour, possibly by antagonizing the anorexigenic actions of α-MSH at
550
MC4R. In addition, the decreased expression of the anorexigenic factor CART observed in T fish
551
may also further increase feeding motivation. These actions persist even when growth rate is
552
restricted in T fish by ration limitation. Interestingly, many genes known to be involved in
553
appetite regulation (i.e. Agrp2, Bbs, Cck, Gal, Ghrh, Glp, Gnrh, Hcrt, Lep, Mc4r, Trh, Tsh) did
554
not differ in brain expression between T and NT fish, indicating increased appetite and foraging
555
behaviour in T fish may be controlled by a limited number of appetite-related genes and
556
pathways. The regulation of appetite by brain signals and its integration with peripheral energy
557
and appetite-regulating pathways in chronically growth-stimulated animals deserves further
558
investigations.
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Acknowledgement
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The authors thank Carlo Biagi, Hamid Seshadri, Marcus Johansson, Janice Oakes, Christine Ng,
563
Hu Jie, Breanna Watson, Dionne Sakhrani, and Krista Woodward for assistance during team
564
sampling, and Bill Gibson for informative discussions. Funding was provided from the Canadian
565
Biotechnology Strategy grant to RHD. 19
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Figure legend Fig. 1. Quantitative PCR measurements of appetite-related mRNA levels in brain (POA,
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HYP) and pituitary (PIT) tissues for genes found to be differentially expressed by RNA-Seq. a)
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Agrp1, b) Mch c) Igf1. PF: pre-feeding; 1H and 4H: 1 hour and 4 hour post-feeding. All values
801
are means ± SEM and were normalized to the value of NT at pre-feeding. Letters indicate
802
significant differences (P < 0.05) among genotypes and time within tissues.
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Fig. 2. Quantitative PCR results of appetite-related mRNA levels in brain (POA, HYP)
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and pituitary (PIT) tissues for orexigenic and GH-axis genes. a) Npy, b) Gh, c) Ghr. PF: pre-
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feeding; 1H and 4H: 1 hour and 4 hour post-feeding. All values are means ± SEM and were
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normalized to the value of NT at pre-feeding. Letters indicate significant differences (P < 0.05)
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among genotypes and time within tissues.
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Fig. 3. Quantitative PCR results of appetite-related mRNA levels in brain (POA, HYP)
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and pituitary (PIT) tissues for anorexigenic genes. a) Pomc, b) Cart and c) Sst2. PF: pre-feeding;
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1H and 4H: 1 hour and 4 hour post-feeding. All values are means ± SEM and were normalized
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to the value of NT at pre-feeding. Letters indicate significant differences (P < 0.05) among
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genotypes and time within tissues.
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Fig. 4. Ternary plots of appetite-related mRNAs for genes with > 2-fold change relative
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to NT pre-feeding levels in the three experimental groups (NT, TF, TR) influenced by genotype
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and nutritional status. Arrows indicate the direction of change with time/feeding. Genes are
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grouped based on their role in appetite. Orexigenic and GH-axis genes: a) POA, b) HYP, c) PIT;
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Anorexigenic genes: d) POA, e) HYP, f) PIT. The ternary plots graphically depict the ratios of
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the three variables as positions in an equilateral triangle with the positions on each triangle side
822
defining the relative value for each of the groups. The three variables are the relative mRNA
823
expression level of each fish group summed to the value of 1.0, and each plot point indicates
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the proportion of the variables represented by each fish group at each time point. The relative
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ratio of gene expression in a fish group is determined by the intercept of a line through the plot
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point and axis for the fish group and parallel with the apex for the fish group. Background
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colours indicate where a relative gene expression is highest in NT (blue), TF (red), or TR
828
(yellow) fish. To give an example, Gh mRNA levels in PIT were similarly represented in TR
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and NT fish, and minimally represented in TF fish (i.e. pre-feeding plot is placed between NT
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and TR vertices on the low end of TF axis, with the rank order of TR : NT : TF = 0.49 : 0.45 :
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0.05, respectively). After a single satiation feeding event, the three fish groups were more
832
equally represented (i.e. arrow shows how plot moved towards centre of triangle, and the rank
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order of NT : TR : TF = 0.43 : 0.33 : 0.24, respectively).
SC
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Fig. 5. Endocrine regulation of appetite-related genes mediated by overexpressed growth
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hormone. a) Study regions in fish brain; POA, preoptic area of the hypothalamus; HYP,
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remainder of the hypothalamus; PIT, pituitary; OB, olfactory bulb; T, telencephalon; ON, optic
838
nerve; OT, optic tectum; MB, midbrain; C, cerebellum; HB, hindbrain; SC, spinal cord. Shaded
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areas indicate tissues examined in the current study. b) The influence of GH transgenesis and
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nutrient levels on endocrine and genetic regulation of appetite and growth in salmon. Genes and
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proteins/peptides that are increased in GH transgenic fish are shown in red, those that are
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decreased are shown in blue, and those that are unaffected by transgenesis are shown in grey. A
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yellow background within tissues indicates effects of transgenesis regardless of ration level (i.e.
844
TF and TR differ from NT fish), while green background indicates effects of transgenesis are
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present only when ration is unrestricted (i.e. only TF fish differ from NT fish). Red arrows
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indicate stimulation, blue blunt-end lines indicate suppression, and green arrows indicate effect
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of increased nutrients. ? indicates pathways which effects are not fully defined in fish. Data for
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peripheral tissues is from (Raven et al., 2008).
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Supplementary Fig. 1. Basal mRNA expression levels of appetite-related genes in brain
regions and pituitary of wild-type (NT) coho salmon.
Supplementary Fig. 2. Relationships between RNA-Seq and qPCR expression levels of
appetite-related genes.
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Fig. 1
a) Agrp1
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Mc4r
Lep
Igf1r
Igf1
Gnrh
Glp
Ghrh
Ghr
Gh
Gal
Cck
Cart
Bbs
Agrp2
Agrp1
AC C
Normalized mRNA level
0.47
2.70
S.2
ACCEPTED MANUSCRIPT
RI PT M AN U
SC
4
0
EP
TE D
-4
R2 = 91
-8
-12
AC C
Normalized mRNA level from qPCR
8
-4
0
4
8
12
Normalized reads from RNASeq
16
ACCEPTED MANUSCRIPT
Highlights GH overexpression alters expression of appetite-regulating genes in salmon brain.
•
Agrp1 is upregulated up to 12 fold in the preoptic area and hypothalamus.
•
Mch was downregulated in most brain tissues and Pomc was elevated in pituitary.
•
Igf1 affected by nutritional status and does not correlate with heightened feeding.
•
Other orexigenic and anorexigenic genes inconsistently affected by GH transgenesis.
AC C
EP
TE D
M AN U
SC
RI PT
•
ACCEPTED MANUSCRIPT Supplementary Table 1. Primers and probes used in this study
Cck Gal Gh
Ghr
Ghrh Glp
Gnrh Igf1
AC C
Igf1r
Igfbp
Lep
Mc4r
Mch
RI PT
Cart
SC
Bbs
M AN U
Agrp2
Agrp1-RT-F Agrp1-RT-R Agrp1-RT-probe Agrp2-RT-F Agrp2-RT-R Bbs-RT-F Bbs-RT-R Bbs-RT-probe Cart-RT-F Cart-RT-R Cck-RT-F Cck-RT-R Gal-RT-F Gal-RT-R Gh-RT-F Gh-RT-R Gh-RT-probe Ghr-RT-F Ghr-RT-R GHR-RT-probe Ghrh-RT-F Ghrh-RT-R Glp-RT-F Glp-RT-R Glp-RT-probe Gnrh-RT-F Gnrh-RT-R I gf1-RT-F I gf1-RT-R I gf1-RT-probe Igf1r-RT-F Igf1r-RT-R Igf1r-RT-probe Igfbp-RT-F Igfbp-RT-R Igfpb-RT-probe Lep-RT-F Lep-RT-R Lep-RT-probe Mc4r-RT-F Mc4r -RT-R Mc4r -RT-probe Mch-RT-F Mch-RT-R
Sequences (5´´ 3´´) ACCAGCAGTCCTGTCTGGGTAA AGTAGCAGATGGAGCCGAACA CTGCCCTGCTGCGACCCCTG TGTTTGCCAGGAGACGGATT AGGCTCGTGTTTCTGAAATGC CAGAACGGGATGGGAAATCTC TTTTAGAGCGGTTCTCTGTGTCAT CGCGTTGCAAGCCCAACTCAGA AGCATCAGGGTTCGCTCACT TGGCAAACAACACTGAAGACAGA TCCTCTGAAGCACGTCTTGAAG TGGCGGAGCGTGTCTGT ACAGTGCTGGCTACCTTTTGG AGGCCATGCTTGTCACTGAGT CAAGATATTCCTGCTGGACTTCTGT GGGTACTCCCAGGATTCAATCA CAGTCCTGAAGCTGC CACTGTGGAAGACATCGTGGAA CAAAGTGGCTCCCGGTTAGA AACTGGACCCTGCTGAA CAAATCTCAGCCAGGAAA TCCGGCCTTCTTCTTG AGTGGTGCTCCATCCAAACG CGCCTGGTCCTGTAGGTAGGT CGATGGGACCTACACCAGCGACGT AGCTGTCTTCCTCCCAGCAC GCATTCTCCTGCCTCACAGA GGCATTTATGTGATGTCTTCAAGAGT CCTGTTGCCGCCGAAGT TCTCACTGCTGCTGTGC CATGGAGCTGATGACTAAAGGAGAT GGAGGGAGGTTGAGACTGTAAGACT TGAAGAGCCACCTGAGG AACACCATCCGCAAGAAACTG TTGTCCAGAGCTGCATGCA TGGAACAGGGTCCCTGCCACATTG TGCTGGAGAACTGGATGATATCA GCCCTCCCTCTCCTGTCTGT CTGCCCAGGCCGCCAACAGA CTCGCTCTACGTCCACATGTTC GCAGCACGGCAATCCTCTT TGCTGGCCCGCCTGCACA GACTCTGGCCTGTGGATGAAC GCTGCAGCTCTCAGCTTGTAGA
TE D
Agrp1
Oligo name
EP
Genes
1
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
TGAACAGAGGACTTCCT Mch-RT-probe CAAGGCAGAGGTATGGGAAGAG Npy Npy-RT-F TCTCCTTTAGCAGCAGTTCTGAGA Npy-RT-R CCAGCCCTGACACACTGGATTCACTG Npy-RT-probe ATCCCACTCCTGCCGTCTT Hcrt Hcrt-RT-F CCTTGTCCCCGTCCCATT Hcrt-RT-R ACCCATTGGGCACAAACG Pomc Pomc-RT-F GGAGTCCCCCCCTTCCA Pomc-RT-R CCCTTCCAGACTGGAGGCA Pomc-RT-probe TCCGTCCGATGCCAAACT Sst1 Sst1-RT-F CTGGCAAGCTCCTGTTTGC Sst1-RT-R CGCCAGCTGCTCCAACGGTCA Sst1-RT-probe AGCCGCCGACTCCTTCA Sst2 Sst2-RT-F TGTCCTCCATCACTCGCTTACTC Sst2-RT-R CCGGCAGACTATTGACGATATCA Trh Trh-RT-F CGCCATCCTCATCCTCTATATTTT Trh-RT-R CCGAGAGCCTCCTGCTCCGCTC Trh-RT-probe ACAAGGCCAGCAGTGGTGAT Tsh Tsh-RT-F AGGCCAGGGTATGGGTAGATG Tsh-RT-R CCCCAGGTGTACCAAGCCACTCAGAA Tsh-RT-probe ACAGCTGGCCCAGAAGTACAA Ubiquitin Ubiquitin-RT-F GGCGAGCGTAGCATTTGC Ubiquitin-RT-R TGTGACAAAATGATCTGC Ubiquitin-RT-probe Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulin-like growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroidstimulating hormone
2
ACCEPTED MANUSCRIPT Supplementary Table 2. Differentially expressed genes in the coho salmon brain (DEGs shown as ratios between group pairs, fold change >2, FDR<0.001). The appetite regulation-related genes are bolded.
26.8 7.9 4.8 4.1 4.0 2.8 2.7 2.6 2.5 2.3 2.3 2.3 2.1 2.1 2.1 -2.1 -2.9 -2.9 -3.1 -3.3 -10.7
21.7
17.7 17.5 10.1 8.1 5.8 5.0 3.5 3.4 3.0 3.0 2.9 2.9 2.6 2.5 -8.7 -9.3 -20.6 -68.7
AC C PIT
TR/TF
-3.5 -2.9
-2.9 -2.4 -2.9
3.5 26.4 31.4 10.0 13.0 6.4
-3.4 -3.3 -3.0 -2.3 -2.5
-11.2
-14.9 -5.4 8.5 9.0 11.0 16.0 17.3
7.5 -3.2 -2.7 -2.6 -2.3 -2.2 -2.1 2.1 2.2 2.4 2.5 2.7
70.7 62.0 16.2 5.3
Homeobox protein Hypothetical protein Prepro-urotensin II-related peptide Neurofilament light polypeptide Physical map contig Plasticin Neurofilament light polypeptide b Plasticin Neurofilament light polypeptide-like Secretagogin Neurofilament medium polypeptide isoform 2 Plasticin Neurofilament medium polypeptide Seurofilament medium polypeptide Neurofilament heavy polypeptide-like Isotocin-I precursor Vasotocin Vasotocin C-C motif chemokine precursor Vasotocin Cell division control protein precursor Uncharacterized protein 12-RF amide peptide 12-RF amide peptide Immunoglobulin heavy chain locus B Barrier-to-autointegration factor Agouti related protein-1 precursor Suppressor of cytokine signaling 2 Solute carrier family 22 member 5-like Collagen precursor Collagen precursor Collagen precursor Perforin-1 precursor Ollagen type I Collagen precursor Collagen Fatty acid-binding protein Programmed cell death 1 ligand 1 precursor Uncharacterized protein Interleukin-5 receptor Conserved noncoding element Glucose-6-phosphatase Clone BAC Homeobox protein Neurogranin High choriolytic enzyme 1 precursor Follicle-stimulating hormone Lectin precursor Neutral amino acid transporter Insulin-like growth factor I precursor Collagen precursor Arbonic anhydrase 4-like Carbonic anhydrase 4-like Thymosin beta-like Melanin-concentrating hormone Metallothionein A Melanin-concentrating hormone-like protein Melanin-concentrating hormone MHC class I heavy chain precursor Isotocin Isotocin Relaxin precursor Heat shock 70 kda protein
SC
-2.3 -2.2
Identification
RI PT
TR/NT
EP
HYP
CL16070.Contig2_All Unigene109213_All CL19203.Contig2_All Unigene88010_All Unigene48309_All Unigene27974_All CL26051.Contig1_All CL9024.Contig1_All CL15264.Contig3_All Unigene1226_All Unigene28401_All Unigene38973_All CL20386.Contig2_All Unigene55135_All Unigene69936_All Unigene41692_All CL21933.Contig1_All Unigene59744_All Unigene71783_All Unigene40388_All CL4761.Contig3_All Unigene24774_All CL10291.Contig1_All CL10291.Contig1_All Unigene27055_All Unigene57306_All Unigene76640_All CL8110.Contig4_All Unigene57540_All CL25868.Contig1_All Unigene30416_All CL3630.Contig3_All CL8644.Contig2_All Unigene36971_All Unigene30420_All Unigene36970_All CL18280.Contig2_All Unigene30893_All Unigene69802_All Unigene63064_All CL25077.Contig3_All CL26454.Contig1_All CL6927.Contig1_All CL16070.Contig2_All CL10025.Contig1_All Unigene63080_All Unigene45955_All Unigene39007_All CL8969.Contig1_All CL5146.Contig3_All CL3630.Contig2_All CL7671.Contig2_All Unigene62679_All CL26397.Contig1_All CL24390.Contig1_All Unigene56962_All CL1086.Contig7_All CL24390.Contig2_All Unigene16306_All CL12454.Contig2_All CL12454.Contig1_All Unigene108551_All Unigene77422_All
TF/NT
M AN U
POA
Contig
TE D
Tissue
-15.6 3.2
1
ACCEPTED MANUSCRIPT 7.3 -4.0 -3.8 28.6
SC
RI PT
-362.1 -293.8 -244.0 -203.1 -194.4 -165.2 -155.7 -124.1 -110.1 -88.3 -80.0 -52.2 -41.8 -38.0 -32.3 -30.9 -27.2 -26.0 -25.9 -25.0 -24.6 -24.0 -20.2 -19.8 -19.2 -18.8 -18.6 -15.7 -12.4 -12.1 -10.5 -9.7 -9.3 -8.5 -7.9 -4.6 -3.1 -2.8 -2.8 -2.7 -2.3 -2.2 -2.2 -2.1 2.0 2.1 2.2 2.2 2.4 2.6 2.8 2.9 3.1 3.3 5.1 5.6 5.8
Calreticulin precursor Neurogranin Regulator of G-protein signaling 5 Melanin-concentrating hormone Immunoglobulin heavy chain locus B Programmed cell death 1 ligand 1 precursor Melanin concentrating hormone precursor Sterile alpha motif domain-containing protein Apolipoprotein A-II precursor Serum albumin 2 precursor Alpha-1-antiproteinase-like protein precursor C1 inhibitor Antihemorragic factor Hypothetical protein Complement factor Apolipoprotein B-100-like Uncharacterized protein Apolipoprotein B Complement component C3 Complement factor H-like Orphan nuclear receptor Dax-1 Complement C3-S Alpha-2-macroglobulin Saxitoxin and tetrodotoxin-binding protein 2 Apolipoprotein Bb precursor Beta-2-glycoprotein 1 precursor Liver-type fatty acid-binding protein 4-hydroxyphenylpyruvate dioxygenase Apolipoprotein A-IV precursor AMBP protein precursor Fibrinogen beta chain-like Coagulation factor II precursor Secreted phosphoprotein 24 precursor Plasminogen precursor Fructose-bisphosphate aldolase B Fibrinogen gamma chain precursor Apolipoprotein A-I-1 precursor Complement component 3-like precursor Alpha-1-antitrypsin-like Uridine phosphorylase 2-like Hemopexin-like protein Hyaluronan-binding protein 2-like Retinol-binding protein 1 Succinyl-coa ligase subunit beta Sushi domain-containing protein 2-like Apolipoprotein C-I precursor T-complex protein 1 subunit eta-like Ultra conserved element locus Transferrin Islet amyloid polypeptide precursor Somatolactin beta Somatolactin beta precursor Phosducin-like 3-2 Nuclear prelamin A recognition factor Nose resistant to fluoxetine protein 6-like Unnamed protein product Gastrin-releasing peptide-like precursor Hypothetical protein Fibronectin type-III domain-containing protein Galectin-3-binding protein precursor Immunoglobulin heavy chain locus A Unnamed protein product Lysozyme variant LRR and PYD domains-containing protein Uncharacterized protein Collagen alpha-1(XVII) chain
TE D
M AN U
5.0 4.2 3.0 -8.8 -9.2 -12.4 -16.7 -21.4
AC C
EP
CL23238.Contig1_All CL10025.Contig1_All Unigene21424_All CL24390.Contig1_All Unigene20684_All Unigene30893_All CL24390.Contig2_All CL15919.Contig4_All Unigene44665_All CL13198.Contig1_All Unigene25835_All Unigene26038_All Unigene44347_All CL8258.Contig1_All CL5254.Contig13_All Unigene37404_All Unigene39230_All CL21575.Contig1_All CL74.Contig2_All Unigene44027_All CL23759.Contig1_All CL25254.Contig2_All Unigene639_All Unigene64340_All CL4139.Contig1_All CL13321.Contig1_All Unigene70666_All Unigene72625_All CL13442.Contig1_All CL3260.Contig2_All CL21940.Contig2_All Unigene50138_All Unigene33139_All CL18560.Contig2_All CL25565.Contig1_All CL880.Contig2_All Unigene38325_All Unigene37879_All CL10379.Contig1_All Unigene19690_All Unigene16218_All Unigene52612_All CL11335.Contig1_All Unigene43291_All Unigene31055_All Unigene21161_All CL16339.Contig1_All Unigene50081_All CL5054.Contig6_All CL12798.Contig1_All Unigene3312_All Unigene77121_All CL5646.Contig1_All CL22274.Contig1_All Unigene64369_All Unigene75867_All Unigene52686_All CL17915.Contig2_All Unigene16697_All Unigene15925_All Unigene6951_All CL15564.Contig2_All CL26463.Contig7_All CL26282.Contig1_All Unigene24774_All CL22016.Contig1_All
9.9 -6.0
NT, non-transgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary; FDR, false discovery rate. 2
ACCEPTED MANUSCRIPT Supplementary Table 3a. Normalized reads of RNA-Seq for appetite-related genes in coho salmon.
NT a
TF
PIT
TR b
8.1 12.1 72.0 51.3 63.0 10.1a 443 66.3 1.5 0.0
23.3 21.5 99.5 100 99.2 49.4b 240 62.2 4.5 0.0
9.9 20.3 127ab 16.1 767 49.9 276 94.6a 3.9 0.0
81.6 21.5 110a 16.2 747 50.5 490 213b 5.9 0.0
13.0 30.0 3.9 2.5 0.5 2.4 1.5 4.5 41.0 415 479 1.0 29.1 5.0
17.0 48.5 0.5 3.5 0.0 2.0 0.0 496 61.6 181 748 2.5 59.4 2.0
7.8 34.6 1.0a 0.0 94.8 2.5 0.5 5145b 18.8 252b 112 6.4 7.9 2.4
6.3 33.3 6.3 0.0 158 1.5 1.0 1800a 19.1 287b 164 11.8 13.2 2.9
NT
b
129 12.9 156b 18.3 1006 55.0 342 235b 5.5 0.0
ab
7.4 39.8 2.5 0.0 106 0.5 2.0 4410b 24.8 13.9a 130 31.3 14.4 0.5
TF
TR
11.9 2.9 0.0 212 0.0 0.5 482638a 86.6 0.5 0.0
53.6b 0.5 1.0 181 4.6 0.0 528853a 87.8 0.5 0.0
13.6b 0.0
1.0a 0.0
12.7b 0.0
1.9a 3.4 63.1 0.0 0.0
4.6b 5.1 112 0.0 0.0
3.0a 2.0 57.2 0.0 0.0
216c 0.5 381456 3.9 3.4 0.0 2214
12.9a 4.1 954872 15.0 2.5 0.0 4124
82.2b 1.0 652225 21.2 2.0 1.5 2578
18.5 2.9 0.0 253 1.9 1.5 681994b 86.9 0.0 0.0
a
RI PT
TR
M AN U
0.5 15.7 148 96.4 53.1 40.8ab 182 40.3 2.0 0.0 16.2 28.5 2.0 4.4 0.0 2.5 1.0 16.7 88.1 207 558 0.0 44.3 0.0
HYP
TF
TE D
NT
SC
POA Gene Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Igfbp Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
AC C
EP
All data were normalized by a scaling factor from DESeq method. The letters indicate significant difference of reads between genotypes (P < 0.05). Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulin-like growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, nontransgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary.
ACCEPTED MANUSCRIPT Supplementary Table 3b. Normalized reads of RNA-Seq for appetite-related genes in coho salmon.
NT 0.0478a 0.0979b 0.6186 0.0788ab 3.7094 0.1206 1.3591 0.0764a 0.0191 0.0382 0.1672 0.024a 0.0311 0.4610 0.0119 0.0024 25.068ab 0.0908 1.2444 0.5470 0.0311 0.3535 0.0119
HYP TF 0.3590b 0.0952ab 0.4867 0.0714a 3.3071 0.1114 2.1846 0.1568b 0.0260 0.0281 0.1471 0.0141b 0.0216 0.6965 0.0065 0.0043 7.9077a 0.0844 1.2978 0.7224 0.0519 0.5883 0.0130
TR 0.5954b 0.0595a 0.7214 0.0847b 4.6511 0.1271 1.5802 0.1805 0.0252 0.0344 0.1832 0.0057a 0.0229 0.4901 0.0023 0.0092 20.361b 0.1145 0.0641 0.5977 0.1443 0.6847 0.0023
NT 0.0334ab 0.0053 0.4580 0.0035 0.0026 1234.16b 0.0315 0.0246b 0.0018a 0.0062 0.1143 0.3912c 0.0009 690.251 0.0070 0.0062 0.0009 4.0056
PIT TF 0.0190a 0.0047 0.3267 0.0008 745.17a 0.0274 0.0008 0.0016a 0.0036b 0.0079 0.1741 0.0206a 0.0063 1482.18 0.0237 0.0040 6.4019
TR 0.0977b 0.0009 0.0018 0.3300 0.0083 0.0 966.02ab 0.0321 0.0009 0.0230ab 0.0055ab 0.0037 0.1041 0.1502b 0.0018 1191.27 0.0387 0.0037 0.0028 4.7106
RI PT
TR 0.1339 0.1225 0.5670 0.5726 0.5698 0.1410 1.3561 0.0594 0.0256b 0.0969 0.2764b 0.0028 0.0100 0.4672 0.0114 2.6724 0.3504 1.0171 4.2564 0.0142 0.9288 0.0114
SC
POA TF 0.0448 0.0672 0.4034 0.2885 0.3501 0.0280 2.4762 0.0739 0.0084a 0.0728 0.1681a 0.0112 0.0070 0.4258 0.0140 0.0084 0.0252 0.2297 2.3249 2.6807 0.0056 0.7087 0.0280
M AN U
NT 0.0028 0.0892 0.8420 0.5465 0.3011 0.1157 1.0260 0.0381 0.0112ab 0.0920 0.1617ab 0.0056 0.0125 0.4517 0.0139 0.0056 0.0948 0.4991 1.1654 3.1673 0.8587 -
TE D
Gene Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Igfbp Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
AC C
EP
All data was normalized to the read of Ubiquitin. The letters indicate significant difference of reads between genotypes (P < 0.05). Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulinlike growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary; -, not detected.
ACCEPTED MANUSCRIPT Supplementary Table 4. Relative mRNA expression levels of appetite-related genes in preoptic area (POA) of coho salmon. Mean values only provided. Average standard error among the groups was 0.023. N ranged between 11 and 12.
0.0015a 0.0133 0.4697 0.0415b 0.0159 0.0423 0.0505a 0.0799a 0.0010 0.0027 0.0664 0.0159 0.0087a 0.0367 0.0007 0.0088 0.2512ab 0.1811ab 0.0024ab 0.5824 0.1140 0.0908 0.0041
4 hpf
0.0006a
0.0018a
0.0389b 0.0100 0.0575 0.0684a 0.0798a 0.0010 0.0047 0.0525 0.0146 0.0074a 0.0384
0.0127ab
0.2369
b
0.0088c
0.0103 0.6574b 0.2938b 0.0080bc 0.4874 0.1014 0.0498 0.0067
Prefeeding 0.0074b 0.0137 0.3285 0.0207a 0.0155 0.0141 0.3173b 0.1075bc 0.0012 0.0019 0.1163 0.0131 0.0186bc 0.0365 0.0005 0.0059 0.0320a 0.3134b 0.0057ab 0.5229 0.1031 0.0879 0.0051
1 hpf
4 hpf
0.0128b
0.0092b
0.0355ab 0.0102 0.0296 0.1963b 0.1234c 0.0011 0.0038 0.0507 0.0110 0.0143bc 0.0324
Prefeeding 0.0055b 0.0190 0.3628 0.0387b 0.0155 0.0533 0.2266b 0.1180c 0.0015 0.0029 0.1172 0.0301 0.0082a 0.0366 0.0006 0.0082 0.2366a 0.1073a 0.0116bc 0.6251 0.1323 0.1202 0.0041
1 hpf
4 hpf
0.0148b
0.0108b
RI PT
Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
1 hpf
TR
M AN U
Prefeeding
TE D
Gene
TF
SC
NT
0.0553d
0.0078
d
0.0104c
0.0062 0.0710bc 0.1949ab 0.0012a 0.8868 0.1922 0.0495 0.0053
0.0218c
0.1324
c
0.0097c
0.0444b 0.0123 0.0588 0.2973b 0.1285c 0.0013 0.0040 0.0934 0.0329 0.0146bc 0.0328 0.0085 0.4948bc 0.2314b 0.0024ab 0.7824 0.1824 0.1483 0.0081
AC C
EP
All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample time (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured.
ACCEPTED MANUSCRIPT Supplementary Table 5. Relative mRNA expression levels of appetite-related genes in hypothalamus (HYP) of coho salmon. Mean values only provided. Average standard error among the groups was 0.027. N ranged between 11 and 12. NT TF TR 0.0111b
0.0072ab
0.0239cd 0.0034
0.0214bcd 0.0064 0.0565 0.1460abc 0.0157a 0.0054 0.0103 0.0001 0.0227 0.0094a 0.0467
0.1060ab
0.0274c
cd
3.2125 0.0099ab
0.0222 3.8073bcd 0.0081ab 0.0031ab 0.6825 0.0458a 0.0419 0.0080
Prefeeding 0.0204c 0.0174 0.1327 0.0172c 0.0053 0.0451 0.5008d 0.0587e 0.0045 0.0032 0.0002 0.0175 0.0411d 0.0385 0.0195 0.7218a 0.0155ab 0.0070b 0.6023 0.0659a 0.0407 0.0061
1 hpf
4 hpf
0.0450cd
0.0305cd
0.0182c 0.0036
0.0073a 0.0066 0.0463 0.1556c 0.0531de 0.0022 0.0074 0.0001 0.0213 0.0185b 0.0537
0.2674c
Prefeeding 0.0575d 0.0158 0.1308 0.0127b 0.0069 0.0581 0.6035d 0.0423bc 0.0127 0.0042 0.0002 0.0165 0.0182b 0.0339 0.0198 2.4139b 0.0114ab 0.0033ab 0.4646 0.0630a 0.0236 0.0063
1 hpf
4 hpf
0.0379cd
0.0374cd
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4 hpf
0.0121b 0.0038
0.1885bc
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1 hpf
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Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
Prefeeding 0.0048a 0.0216 0.1271 0.0287d 0.0063 0.0607 0.2430a 0.0164a 0.0054 0.0058 0.0001 0.0192 0.0184b 0.0354 0.0214 2.6986bc 0.0125ab 0.0024ab 0.5018 0.0450a 0.0419 0.0117
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0.0609e
a
0.8406 0.0138c
0.0177 2.1954bc 0.0045a 0.0030a 1.1702 0.0954b 0.0460 0.0068
0.0249bc
bc
1.7896 0.0099ab
0.0090ab 0.0124 0.0574 0.1482bc 0.0395bc 0.0090 0.0108 0.0002 0.0140 0.0127a 0.0292 0.0170 4.0790a 0.0074a 0.0027ab 0.5673 0.0506a 0.0455 0.0129
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All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample time (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured; -, not detected.
ACCEPTED MANUSCRIPT Supplementary Table 6. Relative mRNA expression levels of appetite-related genes in pituitary (PIT) of coho salmon. Mean values only provided. Average standard error among the groups was 1.413. N ranged between 9 and 12.
0.0072a
0.0083bc 0.0007 114.99cde 0.0282b
154.27e 0.0282b 0.0005
0.0035 0.0135b 0.0096
259.54ab
0.0006 0.0870c 0.0021a 187.34ab 0.0118a 0.0010b 0.0009 2.4733
1 hpf
4 hpf 0.0094ab
0.0027a 0.0003 57.542b 0.0246b
87.622c 0.0381b 0.0010
0.0045 0.0201c 0.0119
721.58c
Prefeeding 0.0195c 0.0012 0.0003 0.0064abc 0.0007 0.0004 114.49d 0.0344b 0.0007 0.0031 0.0118ab 0.0085 0.0007 0.0469c 0.0066b 120.25ab 0.0149a 0.0027c 0.0007 4.1755
0.0005 0.0025a 0.0036b 389.22bc 0.0305b 0.0022c 0.0016 3.7866
1 hpf
4 hpf 0.0181bc
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4 hpf
TR
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Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
1 hpf
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TF Prefeeding 0.0100a 0.0018 0.0003 0.0067abc 0.0004 0.0007 13.362a 0.0129a 0.0009 0.0033 0.0195bc 0.0119 0.0007 0.0085b 0.0078b 183.42ab 0.0053c 0.0021bc 0.0014 6.1057
SC
NT Prefeeding 0.0116b 0.0012 0.0003 0.0098c 0.0005 0.0003 104.53cd 0.0282b 0.0005 0.0028 0.0121b 0.0083 0.0009 0.0737c 0.0056b 129.74a 0.0104a 0.0007a 0.0009 2.5773
104.96cd 0.0268b
0.0044ab 0.0006 119.04cd 0.0296b 0.0007
0.0024 0.0086a 0.0088
215.97a
0.0007 0.0106b 0.0023a 228.44ab 0.0340b 0.0033c 0.0009 1.8243
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All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample times (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured; -, not detected.
S0303-7207(15)30003-4
DOI:
10.1016/j.mce.2015.06.024
Reference:
MCE 9196
To appear in:
Molecular and Cellular Endocrinology
Received Date: 1 May 2015 Accepted Date: 22 June 2015
Please cite this article as: Kim, J.-H., Leggatt, R.A., Chan, M., Volkoff, H., Devlin, R.H., Effects of chronic growth hormone overexpression on appetite-regulating brain gene expression in coho salmon, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.06.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of chronic growth hormone overexpression on
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appetite-regulating brain gene expression in coho salmon
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Jin-Hyoung Kim1, Rosalind A. Leggatt1, Michelle Chan1, Hélène Volkoff2, and Robert H.
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Devlin1*
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Marine Drive, West Vancouver, BC V7V 1N6 Canada
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Fisheries and Oceans Canada, Centre for Aquaculture and Environmental Research, 4160
Departments of Biology and Biochemistry, Memorial University of Newfoundland, St. John's,
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R.H. Devlin
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E-mail address: [email protected]
Corresponding author:
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Abstract:
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Organisms must carefully regulate energy intake and expenditure to balance growth and trade-
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offs with other physiological processes. This regulation is influenced by key pathways
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controlling appetite, feeding behaviour and energy homeostasis. Growth hormone (GH)
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transgenesis provides a model where food intake can be elevated, and is associated with dramatic
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modifications of growth, metabolism, and feeding behaviour, particularly in fish. RNA-Seq and
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qPCR analyses were used to compare the expression of multiple genes important in appetite
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regulation within brain regions and the pituitary gland (PIT) of GH transgenic (fed fully to
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satiation or restricted to a wild-type ration throughout their lifetime) and wild-type coho salmon
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(Oncorhynchus kisutch). RNA-Seq results showed that differences in both genotype and ration
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levels resulted in differentially expressed genes associated with appetite regulation in transgenic
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fish, including elevated Agrp1 in hypothalamus (HYP) and reduced Mch in PIT. Altered mRNA
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levels for Agrp1, Npy, Gh, Ghr, Igf1, Mch and Pomc were also assessed using qPCR analysis.
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Levels of mRNA for Agrp1, Gh, and Ghr were higher in transgenic than wild-type fish in HYP
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and in the preoptic area (POA), with Agrp1 more than 7-fold higher in POA and 12-fold higher
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in HYP of transgenic salmon compared to wild-type fish. These data are consistent with the
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known roles of orexigenic factors on foraging behaviour acting via GH and through MC4R
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receptor-mediated signalling. Igf1 mRNA was elevated in fully-fed transgenic fish in HYP and
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POA, but not in ration-restricted fish, yet both of these types of transgenic animals have very
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pronounced feeding behaviour relative to wild-type fish, suggesting IGF1 is not playing a direct
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role in appetite stimulation acting via paracrine or autocrine mechanisms. The present findings
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provide new insights on mechanisms ruling altered appetite regulation in response to chronically
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elevated GH, and on potential pathways by which elevated feeding response is controlled,
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independently of food availability and growth.
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Keywords: appetite; growth hormone, GH; transgenic; coho salmon; insulin-like growth factor 1,
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IGF1; agouti related neuropeptide, AGRP; nutrition; homeostasis
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1.
Introduction
59 The mechanisms controlling food intake are complex and involve many organ systems,
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endocrine pathways, and neuronal circuits that integrate environmental signals with endogenous
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physiological states. Appetite regulation is crucial to appropriate growth and energy homeostasis
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for an organism. A major pathway controlling metabolic rate, growth, and food intake is the
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growth hormone (GH)/insulin-like growth factor (IGF) axis. In fish as in mammals, GH is
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secreted into circulation by the pituitary (PIT) and acts through the growth hormone receptor
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(GHR) to stimulate IGF1 production in hepatic and other tissues, which induces somatic growth
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and exerts a negative feedback on GH secretion. Somatostatins [SST, produced by the
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hypothalamus (HYP)] inhibit both GH secretion and Igf1 gene expression whereas ghrelin
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(GHRL, produced by stomach and intestine) stimulates GH secretion (Won and Borski, 2013). In
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addition to regulating growth, GH is a pleiotropic hormone involved in many functions including
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appetite, stress response, energy homeostasis, and reproduction (Björnsson et al., 2002). Gh
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genes have been overexpressed or knocked out to examine physiological responses in species
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with determinate growth (i.e. grow to a final body size), and effects on multiple appetite-
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regulating genes have been observed (Arora and Anubhuti, 2006; Bohlool-Y et al., 2005;
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Kopchick et al., 1999). In fish, model transgenic strains overexpressing GH have also been
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developed for species that possess determinate growth (Figueiredo et al., 2007), as well as for
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those with indeterminate growth (i.e. growing throughout their entire life), including carp
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(Cyprinus carpio) (Wan et al., 2012; Zhong et al., 2013), tilapia (Oreochromis niloticus) (Lu et
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al., 2009; Rahman et al., 1998), loach (Misgurnus mizolepis) (Nam et al., 2001), and several
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salmonid species (Devlin et al., 1994; Devlin et al., 2004a; Du et al., 1992). GH transgenic (T)
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fish can show highly elevated feeding behaviour, growth, and metabolic rate, and possess
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modifications of other physiological processes at the levels of gene expression, enzyme activities,
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and the whole animal (Devlin et al., 2001; Devlin et al., 2004a; Lõhmus et al., 2008; Raven et al.,
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2008). Despite this body of literature, the mechanisms by which GH influences fish appetite
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regulation are not yet fully understood, in part because control of food intake in fish tends to
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differ among species to a greater extent than in mammals (Hoskins and Volkoff, 2012).
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Feeding and energy balance are regulated by centres in the brain, which produce and are
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affected by appetite-regulating peptides. Examples of these peptides are orexigenic factors such
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as agouti related neuropeptide 1 (AGRP1), neuropeptide Y (NPY), and orexin (HCRT), and
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anorexigenic peptides such as cocaine and amphetamine regulated transcript (CART),
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cholecystokinin (CCK), and α-melanocyte stimulating hormone (α-MSH, processed from
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proopiomelanocortin, POMC). In mammals, the paraventricular nucleus (PVN) and arcuate
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nucleus (ARC) in the hypothalamic region of the brain are recognized as command centres for
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controlling energy balance. The preoptic area (POA) was recently defined as the PVN-
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homologous region in the HYP of fish (Herget et al., 2014), and has previously been found to
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display altered mRNA levels for Npy in hungry fish (Silverstein et al., 1998). In teleost fish,
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hypothalamic AGRP and POMC neurons associated with appetite and growth project directly
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into the PIT (Zhang et al., 2012) and the HYP/POA/PIT axis is thought to play a pivotal role in
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multiple pathways including appetite regulation, feeding behaviour, and energy use. These
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actions are regulated in part by production and release of, and response to, GH (Forlano and
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Cone, 2007; Herget et al., 2014; Zhang et al., 2012).
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Due to the magnitude of phenotypic changes seen in T fish, they provide a useful model
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organism to understand the relationship between appetite regulation, growth, and behaviour. The
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pleiotropic effects of GH are believed to be largely mediated by IGF1 produced in liver and other
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tissues in response to GH stimulation (de Azevedo Figueiredo et al., 2007; Frago et al., 2002). It
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is well established that GH overexpression in fish elevates Igf1 gene expression in multiple
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tissues and increases circulating IGF1 protein levels (Beckman, 2011), and this is correlated with
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strongly elevated feeding behaviour and food intake in animals fed ad libitum. However, T fish
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reared on a wild-type (restricted) ration level have normal levels of Igf1 gene expression and
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IGF1 circulating hormone, yet possess the same heightened feeding motivation seen in fully-fed
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transgenic fish (Devlin, 2011; Raven et al., 2008). These data show that IGF1 production is
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influenced by nutritional state (Beckman, 2011), and suggest that elevated appetite in T fish is
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not directly mediated either by peripheral IGF1 levels, or by increased nutrient utilization signals
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associated with elevated somatic growth. Rather, appetite is likely elevated by direct stimulation
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of central feeding centres by GH or by other peripheral signals affected by GH independently of
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IGF1. Although the mechanisms ruling the central effects of GH on feeding behaviour and
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growth are not fully understood, recent studies in T fish suggest important roles for appetite-
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related neuropeptides. For example, T coho salmon (Oncorhynchus kisutch) have lower
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telencephalic expression of Npy and winter levels of Cck relative to wild-type fish (Lõhmus et al.,
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2008; Raven et al., 2008), whereas in T carp, both the hypothalamic and telencephalic expression
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of Agrp1 is 2-fold higher relative to wild-type fish, although transgenesis does not seem to affect
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Npy, Hcrt, Pomc, Cck, or Cart expressions (Zhong et al., 2013).
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To further understand the mechanisms controlling appetite and growth in fish, the current
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study has undertaken a comprehensive examination of mRNA levels of appetite-regulating genes
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producing orexigenic, anorexigenic, and metabolic effects, in the HYP (with POA separately)
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and the PIT of wild-type (NT), fast-growing GH transgenic (TF), and ration-restricted GH
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transgenic (TR) coho salmon.
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2.
Materials and Methods
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2.1. Experimental Animals.
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The experiment was performed September 23-27, 2013 at the Centre for Aquaculture and
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Environmental Research (CAER), Fisheries and Oceans Canada (DFO), West Vancouver,
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Canada. This facility contains specific containment measures to prevent the escape of genetically
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modified fish to the natural environment. All experimental procedures were carried out in
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compliance with the Canadian Council for Animal Care guidelines under permit from DFO’s
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Pacific Regional Animal Committee. Three size-matched groups of coho salmon (Oncorhynchus
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kisutch; 95.8 ± 15 g) were examined: (i) wild-type coho salmon (non-transgenic, NT), (ii) GH
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transgenic coho salmon fully fed to satiation throughout their lifespan (TF) and growing 2-3-fold
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faster than wild-type fish (Devlin et al., 2004b), and (iii) GH transgenic salmon that were ration-
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restricted to the NT satiety ration level throughout their lifespan (TR). All fish were of the same
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genetic background (Chehalis River hatchery coho salmon from Fisheries and Oceans Canada
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Chehalis River Enhancement Facility Agassiz, BC). Transgenic coho salmon (T) contained the
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OnMTGH1 gene construct (Devlin et al., 1994) (strain M77), and were produced at CAER
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(Devlin et al., 2004b) and maintained in a wild-type genetic background by crossing T at each
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generation to NT coho salmon collected from nature. NT salmon were produced by crossing
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wild-type males to the same females used to produce TR salmon. NT and TR fish were produced
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in January 2012, and TF fish were produced in January 2013. Thus, TF fish were of same size
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and developmental stage as TR and NT fish, but were one year younger. All groups of fish were
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reared under the same standard conditions (400 fish / 4000 L fibreglass tanks, 1 group of fish per
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tank, 10 ± 1 ºC well water, and simulated daylight set to the natural photoperiod). Fish were fed
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stage-appropriate commercial salmonid diets (Skretting Ltd., Canada) at fixed times of day (9
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AM and 2 PM) for at least 3 months prior to the experiment to standardize physiological
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responses to feeding. Foraging and schooling behaviour of each group was visually observed
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prior to and during feeding events.
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2.2. Sampling and Dissection.
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Three time points were chosen to represent different stages of feeding: pre-feeding, and
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two post-prandial stages [one hour post-feeding (1 hpf) for satiation, and four hours post-feeding
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(4 hpf) for active digestion]. Fish were sampled over a five-day period as follows: Day 1: TR
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pre-feeding; Day 2: NT pre-feeding; Day 3: TF pre-feeding, TR 1 hpf, and TR 4 hpf; Day 4: NT
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1 hpf and NT 4 hpf; Day 5: TF 1 hpf and TF 4 hpf. This sampling approach provided a two-day
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recovery period between pre-feeding and post-feeding samplings for each group. No differences
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in feeding behaviours of a population were noted between pre-experimental and sampling
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periods. Fish were fed normal feeding levels between pre-feeding and post-feeding sample days,
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and then fed to satiation at the 9 AM feeding time prior to post-feeding sample times. For the
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pre-feeding samples, 12 fish were rapidly (30 sec) selected from their population immediately
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prior to the 9 AM feeding time (i.e. at 8:45 AM), after which the population was fed to satiation.
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For 1 hpf and 4 hpf time points, 12 fish were selected for sampling at 10 AM and 1 PM,
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respectively. In order to minimize time delay between samples and effect of handling on gene
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expression, four sampling stations were simultaneously used and tissues were collected in less
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than 3 minutes per fish. The fish selected for sampling were rapidly euthanized in a bath
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containing a buffered tricaine methanesulphonate (200 mg/L; 400 mg/L sodium bicarbonate;
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Syndel Laboratories Ltd., Vancouver, BC, Canada). On cessation of ventilatory activity, fish
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were bled by caudal severance and whole brain and PIT from each fish were rapidly collected
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and placed in RNAlater (Ambion, Austin, TX, USA) for overnight storage at 4 ºC, followed by
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long-term storage at -20 ºC. This process was repeated three times to allow sampling of all 12
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selected fish per group and time point to be completed within 15 minutes. Later, POA and HYP
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were dissected from whole brain under a dissecting scope following salmon anatomical
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description (Billard and Peter, 1982; Forlano and Cone, 2007; Herget et al., 2014; see Figure 5a).
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Total RNA was extracted via RNeasy mini kits (Qiagen, Valencia, CA, USA) from
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individual samples, and concentration and purity measured using a Nanodrop (Thermo Scientific,
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Wilmington, DE, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent
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Technologies, Palo Alto, CA, USA) and all samples possessed a RNA integrity number (RIN) >
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9.0. For RNA-Seq (pre-feeding samples only), 2 µg of total RNA was pooled from five
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individuals from each group (NT, TF, and TR) to create two biological replicates (i.e. two pools
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of five fish for each genotype). For qPCR analysis, first-strand cDNA was synthesized from total
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RNA (0.5 µg) using the High Capacity cDNA synthesis kit with RNase inhibitor (Applied
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Biosystem, Foster City, CA, USA).
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RNA-Seq analysis was performed using the Illumina HiSeq2000 platform (Illumina, San
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Diego, CA, USA) using two samples (n=2) of five fish per group. BLAST-based gene
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identification was performed to annotate a coho salmon transcriptome constructed for this study
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(to be reported elsewhere) and downstream differential expression analysis was used to
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determine differentially expressed genes (DEGs) between groups of fish. For normalization of
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raw reads in RNA-Seq analysis, a scaling factor in DESeq method (Anders and Huber, 2010) and
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Ubiquitin were used. RobiNA software [http://mapman.gabipd.org/web/guest/robin (Lohse et al.,
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2012)] was used to perform DESeq, which uses a Bioconductor Software Package that assumes a
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negative binomial distribution of sequence count data.
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2.5. qPCR
209 Selection of appetite related genes for qPCR was based on previous studies of feeding
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regulation in fish (MacDonald and Volkoff, 2009; Penney and Volkoff, 2014; Tuziak and Volkoff,
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2013; Zhong et al., 2013). Primers and/or probes (Supplementary Table 1) were designed using
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Primer Express software (Applied Biosystem, version 3.0.1) with sequences from the coho
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salmon transcriptome. The specificity of all pairs of primer was validated by PCR and
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electrophoresis analyses to confirm proper size of amplicons. Ubiquitin was used as a reference
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gene to normalize mRNA levels, it displayed consistently stable mRNA expression levels among
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experimental groups and among several reference genes examined (β-actin, Ef-1a and
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Ubiquitin). qPCR reactions were performed as previously described (Kim et al., 2015). Relative
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mRNA expression levels were calculated relative to the Ct value obtained for the housekeeping
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control gene (Ubiquitin) using the 2-∆∆Ct method. (Livak and Schmittgen, 2001).
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2.6. Statistical Analysis
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One-way or two-way ANOVA followed by Duncan’s multiple range tests were used to
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evaluate differences across the time series and among genotypes, using SPSS statistical package,
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version 18.0 (SPSS Inc., Chicago, IL, USA). If normality or equal variance failed and could not
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be restored by transformation, Kruskal-Wallis tests were used. All results are expressed as the
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mean ± SEM, and statistical significance was determined at P < 0.05.
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3.
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Results
3.1. Fish Behaviour
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Before feeding, NT fish grouped tightly together at the bottom of the tank, while T fish
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dispersed much more widely in the tank and were located close to the surface of the water.
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During feeding, high levels of foraging behaviour in T fish were observed, while NT fish showed 8
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less feeding response and displayed behaviour typically associated with balancing predation risk
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avoidance with foraging.
239 3.2. RNA-Seq Analysis.
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Differentially expressed genes (DEGs) among fish groups (NT, TF and TR) in brain tissues
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(POA, HYP and PIT) are shown in Supplementary Table 2. Twenty-three DEGs were detected
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in POA, whereas 36 and 70 DEGs were found in HYP and PIT, respectively. The greatest
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number of DEGs (60) was found between TR and NT in PIT. Among the well-known appetite
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relevant genes, mRNA levels for two genes in particular showed very large differences: Agrp1
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levels in HYP were 8- and 13-fold greater in TF and TR than in NT, respectively, whereas Mch
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levels in PIT were reduced in TF when compared to NT. Specifically, Mch levels were 9-fold and
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17-fold lower in TF in PIT, while Mch levels were 2.5-fold lower in TF than in TR in the HYP.
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For GH axis genes, only Igf1 mRNA levels were found to be significantly different in HYP,
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where TF was 2.7-fold higher than TR.
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Although only a few appetite related genes were found to be differently expressed based on
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RNA-Seq data, further analysis of the raw reads of 23 selected appetite regulating genes were
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analyzed by two normalization methods: 1) by normalizing using the scaling factor method in
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DESeq (Supplementary Table 3a), and 2) by normalizing relative to Ubiquitin as an internal
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control (Supplementary Table 3b). Similar patterns of significant differences among groups
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were found using both methods when mRNA levels were compared on an individual gene level
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for Agrp1, Cart, Gh, growth hormone receptor (Ghr), gonadotropin releasing hormone (Gnrh),
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Igf1, and melanin-concentrating hormone (Mch). However, Agrp2, growth hormone releasing
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hormone (Ghrh) and orexin (Hcrt) showed significant differences when normalized with
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Ubiquitin only, and bombesin-like peptides (Bbs) and galanin (Gal) showed significant
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differences when normalized by scaling factor only.
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qPCR analyses were undertaken to further validate the mRNA levels for 23 appetite-
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associated genes in POA, HYP and PIT from RNA-Seq analysis. Relative levels of mRNA
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among tissues examined are shown for NT salmon (Supplementary Fig. 1). Numerical qPCR
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data are given in Supplementary Tables 4-6. qPCR analyses were performed on three DEGs
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(Agrp1, Mch and Igf1) that was detected as differentially expressed from RNA-Seq analysis (Fig.
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1a-1c). Overall, a high correlation was seen between RNA-Seq and qPCR data (R2 = 0.91,
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Supplementary Fig. 2). For genes showing greater than 2-fold differences between T and NT in
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pre-feeding samples, additional qPCR analyses were performed using 4 hpf samples. For genes
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showing significant differences at 4 hpf, mRNA levels were also measured for 1 hpf samples
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(Fig. 1a-1c).
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For both TF and TR, Agrp1 mRNA levels were significantly higher than in NT, in both
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POA and HYP (Fig. 1a) with the highest difference (12-fold) observed between NT and TR
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animals in HYP in pre-feeding samples. In contrast, in PIT, elevated Agrp1 mRNA levels above
280
NT were only evident in TR fish. T fish did not differ in Agrp1 mRNA levels with time, whereas
281
NT fish had higher levels at 1 hpf, but not 4 hpf, than the pre-feeding sample in HYP, and lower
282
levels at 4 hpf than pre-feeding in PIT samples (Fig. 1a).
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Mch mRNA was found at lower levels in TF than NT and TR salmon in all brain regions
284
examined, although these differences were not significant at all time points in POA as there was
285
very high variance among the individual samples. TR fish had lower levels of Mch than NT fish
286
in HYP at 1 hpf and in PIT at 4 hpf (Fig. 1b). When compared to pre-feeding samples, Mch
287
mRNA levels in TR and TF fish increased in HYP and decreased in PIT at 4 hpf.
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Igf1 mRNA levels were generally elevated in TF in all tissues, although this was not
289
significant at pre-feeding in PIT. Levels in TR were greater than NT in POA at 1 and 4 hpf, were
290
lower than NT in PIT at 4 hpf, and were similar to NT levels at all other time points/tissues (Fig.
291
1c). In HYP, levels of Igf1 increased at 1 hpf and decreased at 4 hpf in all fish groups, although
292
this was not significant in TR. In POA, levels in TF increased at 1 hpf and decreased at 4 hpf,
293
whereas levels in TR fish increased at 1 hpf but there was no significant difference at 4 hpf.
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3.4. Assessment of Other Appetite and Metabolism Gene mRNA Levels
296 10
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NT and T fish did not differ significantly in Npy mRNA levels at pre-feeding in any tissue.
298
However, TF had greater levels of Npy mRNA than TR in POA. In PIT and HYP, a general trend
299
in reduction of Npy expression was noted in NT and TR fish after feeding, whereas expression in
300
HYP rose at 1 hpf and decreased at 4 hpf (Fig. 2a).
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In POA, TF and TR had higher Gh mRNA levels than NT at pre-feeding and 4 hpf. In HYP,
302
TF and TR also showed significantly higher levels than NT at pre-feeding, but only TF had a
303
higher level at 1 hpf and there were no significant differences at 4 hpf. In PIT, TF Gh mRNA
304
levels were significantly lower at all time points than in NT, were lower than TR at pre-feeding
305
and 1 hpf, and increased with feeding (Fig. 2b).
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In POA and HYP, TF and TR showed higher mRNA levels of Ghr than NT, whereas in PIT,
307
TF levels were lower at pre-feeding than other groups and increased with feeding. There were no
308
other differences among groups or time periods (Fig. 2c).
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Levels of Pomc mRNA in POA were significantly lower in TF fish than in NT at 4 hpf.
310
There were no major differences in Pomc mRNA levels in HYP among groups, and Pomc
311
decreased with feeding in TF fish only. In PIT, Pomc levels were higher in TF at 1 hpf than in all
312
other genotypes (Fig. 3a).
Cart mRNA levels in POA were lower in TF than in other groups at pre-feeding only. In
314
HYP and PIT, TF and TR had overall lower levels of Cart than NT fish, although this was not
315
significant at all individual time points (Fig. 3b).
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Sst2 mRNA levels were higher in TF and TR than NT in PIT at pre-feeding and 4 hpf, and
317
higher in TF than in NT and TR in HYP at 4 hpf only (Fig. 3c). There were significant
318
differences in Sst1 in PIT only, where TF fish had the lowest levels at pre-feeding, and levels
319
increased with feeding in both TF and TR so that they were greater than NT levels at 4 hpf (see
320
Supplementary Table 6). There were no significant differences among fish groups and/or time
321
points for any other gene examined (e.g. Agrp2, Bbs, Cck, Gal, Ghrh, Glp, Gnrh, Hcrt, Lep,
322
Mc4r, Trh, Tsh, see Supplementary Tables 4-6)
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323
Ternary plots of appetite related mRNA levels for genes visually integrate significant
324
differences observed among the three experimental groups (Fig. 4). Most orexigenic and GH-
325
axis genes with significant differences tended to be over-represented in TF and TR fish, primarily
326
in the POA and HYP (i.e. Agrp1 in all tissues, Gh and Ghr in POA and HYP), or in TF fish only 11
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(i.e. Npy and Igf1 in all tissues). For these genes, feeding tended to decrease the association with
328
TF fish (i.e. arrows moved from high to low along TF axis in red), with the exceptions of Gh and
329
Agrp1 in HYP where TF representation was increased with feeding, and for genes in PIT where
330
feeding had a lesser effect. Orexigenic and GH system genes that were more associated with NT
331
and TR fish included Mch (all tissues), and Gh and Ghr (PIT only). Feeding increased high
332
representation of Mch in NT in all tissues (only at 1 hpf in POA and HYP), and decreased Gh
333
and Ghr high representation in TR in PIT. There were fewer patterns apparent for the relative
334
expression of anorexigenic genes (Fig. 4a-4c), although Cart was highly represented in NT fish
335
in all tissues (indicating reduced expression in TR and TR), and this tended to increase at 4 hpf in
336
HYP and PIT. Pomc was highly represented in TR fish in POA pre-feeding, but this switched to
337
high NT representation post-feeding. In the HYP, Pomc expression was highly represented in TF
338
pre-feeding, but was similar in all groups post-feeding, whereas in the PIT, Pomc was highly
339
represented in TF fish regardless of feeding. Sst1 and Sst2 were highly represented in TR fish in
340
PIT, and feeding increased Sst2 representation in TF fish and decreased NT fish representation
341
post-feeding, while Sst2 was highly represented in TF fish in HYP, particularly post-feeding.
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4.
Discussion
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When food is not limiting, GH transgenic (T) coho salmon grow dramatically faster than
346
non-transgenic fish (NT) due to increased appetite and food intake (Devlin et al., 1994; Devlin et
347
al., 2004a; Devlin, 2011). This behavioural transformation in T fish is likely mediated by an
348
altered balance between orexigenic and anorexigenic factors in the brain and pituitary gland
349
(PIT), as well as from peripheral signals arising from altered anabolic activity and nutritional
350
signals from processed food. It is currently not completely clear whether the stimulated appetite
351
of T salmon arises from direct effects of GH on the brain, or is due to the stimulation of growth
352
of peripheral tissues which in turn send signals to the brain to alter feeding behaviour, or both.
353
Hypothetically, following satiation, hunger signals in T fish might return more rapidly to pre-
354
feeding levels than in NT fish, thus increasing meal frequency. GH overexpression could also
355
sustain higher expression of orexigenic signals and lower protein levels and/or expression of
356
anorexigenic signals in T relative to NT to cause greater food intake at each meal. Both of these
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feeding responses (increased meal frequency and food intake) are observed in GH T salmon
358
relative to NT. Elevated Agrp1 and reduced Cart expression levels found in T in the current
359
study are consistent with feeding-related peptides playing a role in mediating increased appetite.
360
However, inconsistent effects were observed for other appetite-related genes including Npy and
361
Pomc. In particular, Mch and Sst2 had expression patterns opposite to what was expected. The
362
reduced expression of genes encoding known orexigenic factors and the increased expression of
363
genes encoding appetite inhibitors in T fish might be indicative of compensatory mechanisms
364
aimed at returning feeding levels to normal.
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Elevated Agrp1 mRNA levels in the preoptic area (POA) and hypothalamus (HYP),
366
regardless of ration level, are consistent with this neuropeptide being involved in the stimulation
367
of appetite and food intake seen in T salmon. AGRP is part of the melanocortin system, which
368
acts in the endocrine regulation of growth and appetite through control of neurons affecting
369
hypothalamic release of hormones (Cone, 2005; Zhang et al., 2012). The orexigenic actions of
370
AGRP are mediated by antagonism of α-MSH (derived by processing of POMC) stimulation of
371
the melanocortin-4 receptor (MC4R), a system that has been reported in a range of vertebrates
372
from fish to mammals (Belgardt et al., 2009; Cone, 2005; Zhang et al., 2012; Zhong et al., 2013;
373
Zhu et al., 2013). In another GH transgenic fish, the common carp (Cyprinus carpio L), elevated
374
Agrp1 expression has also been demonstrated, and direct treatment of isolated NT carp HYP
375
slices with GH increases Agrp1 expression, suggesting GH acts directly on brain feeding
376
pathways. In T carp, Agrp1 expression was elevated 2-fold in HYP (Zhong et al., 2013), whereas
377
the present study found 4- to 10-fold increases in the POA of T salmon. The differences in Agrp1
378
mRNA between T and NT carp and salmon do not correlate with GH levels between the two
379
transgenic models: both peripheral GH protein and hypothalamic levels of Gh mRNA are over
380
100-fold greater in T carp, whereas T coho salmon have only a 2- to 3-fold elevation of
381
circulating GH (Raven et al., 2008), and 2- to 6-fold increases in hypothalamic and other tissue
382
Gh mRNA expression (Raven et al., 2008). This difference in response between these two studies
383
could be due to species-specific differences in GH/IGF1 axis regulation, as well as the use of
384
different promoters within the transgene (β-actin promoter in carp and metallothionein-B
385
promoter in the current study) (Devlin et al., 1994; Zhu et al., 2013) that may produce different
386
distributions and levels of Gh mRNA among brain tissues. As well, central Agrp levels are
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correlated with stimulated appetite in rats (Kamegai et al., 2001), and are decreased in fasted
388
salmon (Murashita et al., 2009), although are unaffected by temperature-derived suppressed
389
appetite in salmon (Hevrøy et al., 2012). In mammals, the melanocortin system is comprised of
390
POMC expressing neurons and AGRP/NPY co-expressing neurons in the (arcuate nucleus) ARC.
391
In POMC neurons, POMC is cleaved to form α-MSH, which acts on MC4R located in the
392
paraventricular nucleus (PVN) of the HYP to decrease food intake and increase energy
393
expenditure. It is noteworthy that although AGRP administration stimulates feeding and Agrp
394
overexpression leads to obesity in both fish and mammals (Barsh and Schwartz, 2002; Song and
395
Cone, 2007), Agrp and Npy knockout mice do not show any alteration of feeding and body
396
weight (Qian et al., 2002). Elevated Agrp1 mRNA in T fish may act to increase appetite by
397
blocking the α-MSH stimulation of MC4R anorexigenic action. However, in the current study, T
398
did not show consistent changes in either Pomc or Mc4r mRNA expression levels across tissues
399
and sampling points compared to NT fish, suggesting that neither POMC nor MC4R at the
400
mRNA level directly mediate increases in feeding observed in T fish. In mammals, although
401
AGRP and POMC neurons have been shown to be in close proximity in several brain regions
402
and act to counter-balance each other to regulate appetite, they do not appear to function simply
403
as antagonists in their responses to metabolic signals (Warne and Xu, 2013). Indeed, in an
404
optogenetic study using light-activated neuron signals, POMC suppression of feeding was
405
dependent on melanocortin receptor signalling in mice, whereas stimulation of AGRP neuron
406
activation rapidly induced food intake independent of the melanocortin pathway (Aponte et al.,
407
2011). Increased Agrp expression without changes in Pomc and Mc4r expression in T fish might
408
suggest that the actions of AGRP are independent of POMC and MC4R. It is noteworthy that
409
significantly higher Pomc expression levels were observed in the PIT of TF. POMC is cleaved
410
into several peptides other than the anorexigenic α-MSH, including opioid neuropeptides such as
411
the orexigenic factor β-endorphin (Lin et al., 2000). In both goldfish (Carassius auratus) and
412
tench (Tinca tinca), intracerebroventricular (ICV) administration of β-endorphin induces
413
increases in food intake, whereas intraperitoneal (IP) injections do not (de Pedro et al., 1995;
414
Guijarro et al., 1999), suggesting that β-endorphin stimulates food intake by activating central
415
pathways (Baile et al., 1986; Morley, 1995). It is possible that high Pomc mRNA levels of TF in
416
PIT, the main region of POMC synthesis, may induce increased β-endorphin synthesis resulting
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in increased appetite. If the appetite-stimulating actions of β-endorphin are greater than the
418
suppressive effects of α-MSH, then, coupled with highly increased AGRP, appetite would overall
419
be stimulated. The role of AGRP and the influences of the balance between α-MSH and β-
420
endorphin levels in feeding behaviour require further examination.
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MCH is a peptide synthesized in both HYP and PIT, and is associated with multiple
422
functions such as pigmentation (Naito et al., 1985) and energy homeostasis (Pissios et al., 2006).
423
Mch was expressed at lower levels in TF salmon compared to slower growing NT and TR fish in
424
POA and HYP as well as PIT, and these differences were not abolished by feeding to satiation.
425
Thus, it appears that, in salmon, Mch mRNA expression is inversely correlated with slower
426
growth and low ration level, and is not strongly influenced by short-term differences in food
427
intake (i.e. 0 to 4 hrs post-feeding). Further, MCH likely does not play a direct role in elevating
428
appetite in T fish since both TF and TR have highly stimulated feeding behaviour, yet Mch
429
mRNA levels are similar in TR and NT fish, indicating Mch levels are primarily influenced by
430
the chronic high feeding rates in TF fish. In mammals, the role of MCH in feeding regulation has
431
consistently been reported as orexigenic (Elliott et al., 2004; Pissios et al., 2006; Segal-
432
Lieberman et al., 2006), whereas, there are inconsistent reports on the role of MCH in feeding in
433
fish (Volkoff, 2014). For example, MCH acts as an orexigenic factor in several fish including
434
Atlantic cod (Gadus morhua) (Tuziak and Volkoff, 2013), zebrafish (Berman et al., 2009), winter
435
flounder (Pseudopleuronectes americanus) and barfin flounder (Verasper moseri) (Takahashi et
436
al., 2004), whereas an anorexigenic action of MCH has been reported in goldfish (Matsuda et al.,
437
2006; Matsuda et al., 2009). It is possible that MCH may act as both an orexigenic or
438
anorexigenic factor through interactions with other appetite factors, as it can stimulate some
439
orexigenic systems (i.e. HCRT and apelin) and can inhibit others (i.e. NPY, CART) (Volkoff,
440
2014). In the present study, Mch mRNA levels in the PIT were correlated with Gh expression. As
441
the PIT is the major site of GH production in NT fish, this might indicate that, as in mammals
442
(Segal-Lieberman et al., 2006) and the cichlid Cichlasoma dimerus (Pérez Sirkin et al., 2012),
443
MCH stimulates GH secretion in salmon. However, further studies are needed to determine the
444
exact roles of MCH in the regulation of feeding and GH secretion in fish. It is noteworthy that
445
the present study found large variations in Mch mRNA levels among individual fish for both
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446
qPCR and RNA-Seq data. Whether this variation was due to natural biological differences in
447
expression among individuals is not known. NPY is considered one of the most potent orexigenic factors in vertebrates (Volkoff et al.,
449
2005). Npy mRNA expression levels in the POA are found be increased in food-deprived wild-
450
type coho salmon (Silverstein et al., 1998), and decreased in HYP of Atlantic salmon with
451
temperature-induced suppression of feeding (Hevrøy et al., 2012), suggesting an orexigenic role
452
for NPY in salmon. It was thus expected that T fish may have elevated Npy relative to NT fish to
453
account for increased appetite. However, this was inconsistently observed during post-feeding in
454
HYP and PIT, suggesting NPY likely does not have a major role in increased appetite in T fish.
455
Similarly, changes in Npy mRNA levels between T and NT carp was not observed (Zhong et al.,
456
2013), and previous studies show lower telencephalic or hypothalamic Npy in T salmon
457
depending on ration level (Raven et al., 2008). In NT carp, in vitro studies have shown that GH
458
increases the hypothalamic mRNA expression levels of Npy only when incubated with moderate
459
doses (20 ng ml−1 of GH for 2 h), and not with high doses or long exposures to GH (Zhong et al.,
460
2013). It is possible that the effects of GH on NPY are inhibited by sustained exposure to high
461
GH levels, as observed in T fish, perhaps by receptor down-regulation. Similar receptor
462
desensitizations have been shown for other peptides in fish. For example, in goldfish, treatment
463
with high doses of either NPY (Hoskins and Volkoff, 2012; Narnaware et al., 2000) or orexin
464
(HCRT) (Volkoff et al., 1999) have no or inhibitory effects on feeding. Npy expression was
465
inconsistently affected by feeding in T and not affected by feeding in NT in POA and HYP.
466
However, PIT Npy levels decreased after feeding to satiation in both NT and TR fish, suggesting
467
that feeding has region-specific effects on Npy expression. Postprandial decreases in Npy
468
expression have been shown in several fish species, including goldfish HYP and telencephalon
469
(Narnaware et al., 2000), Brazilian flounder HYP (Campos et al., 2012) and cobia (Rachycentron
470
canadum) brain (Nguyen et al., 2013), and could be due to satiation signals inhibiting expression
471
of Npy.
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472
The anorexigenic factor Cart showed higher mRNA levels in NT than in TR and TF fish in
473
most tissues, suggesting that elevated peripheral GH levels inhibited CART expression, and
474
thereby diminished the anorexigenic actions of CART in T fish. Indeed, CART injections inhibit
475
feeding in goldfish (Volkoff and Peter, 2001) and Cart mRNA levels in the brain of carp, 16
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goldfish, and Atlantic salmon decrease after food deprivation and increase after refeeding,
477
suggesting a role as a short-term regulator of feeding (Murashita et al., 2009; Volkoff and Peter,
478
2001; Wan et al., 2012). In contrast, there was no effect of starvation on Cart mRNA levels in the
479
winter skate, Raja ocellata (MacDonald and Volkoff, 2009), and feeding did not increase mRNA
480
levels in any genotype in the present study. Thus, CART may be important in long-term
481
maintenance of feeding behaviour but not play a major role in short-term feeding and satiation.
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In both mammals and fish, SST is produced mainly in HYP and POA, inhibits the release
483
of GH from the PIT (Brazeau et al., 1973; Yada and Hirano, 1992), and is involved in feedback
484
regulatory controls of the GH/IGF1 system (Sheridan and Hagemeister, 2010). While there were
485
no significant differences in Sst mRNA levels in POA and HYP at pre-feeding in the present
486
study, in PIT, both TF and TR fish had higher mRNA expression levels than NT for Sst2 at all
487
time points and for Sst1 at 4 hpf. As stimulation of PIT Sst mRNA expression by exogenous GH
488
and IGF1 has previously been reported in rainbow trout (Melroe et al., 2004), the increase in PIT
489
Sst levels seen in T fish might thus represent a response to high GH levels, in an effort to lower
490
GH production.
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Previous studies have shown that both TF and TR fish have greater non-pituitary Gh
492
mRNA and circulating GH levels than NT fish, regardless of feeding and growth level, whereas
493
only TF fish have lower PIT Gh mRNA than NT fish (Caelers et al., 2005; Mori and Devlin,
494
1999; Raven et al., 2012). This concurs with the present findings showing an increase in POA
495
and HYP Gh levels in all T fish and a decrease in PIT Gh levels in TF fish. ICV injections of GH
496
protein do not stimulate feeding in rainbow trout (Johansson et al., 2005), whereas peripheral
497
injections of GH do (Higgs et al., 1977), suggesting GH-mediated appetite regulation may be
498
through indirect or peripheral pathways. Decreased PIT Gh in TF fish may be due to a negative
499
feedback control of IGF1 and GH acting on the PIT (Caelers et al., 2005; Mori and Devlin, 1999),
500
and/or to the actions of other appetite factors that have been shown to influence PIT GH
501
secretion [e.g. MCH, NPY, SST (Brazeau et al., 1973; Pérez Sirkin et al., 2012; Peng et al., 1993;
502
Yada and Hirano, 1992)]. However, since NT and TR salmon have similar food intake levels,
503
growth rates, and IGF1 levels, whereas TF have elevated IGF1 levels, the reduced expression
504
levels of PIT Gh observed in TF salmon may be explained by the actions of IGF1 rather than GH
505
feedback.
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Compared to NT fish, Ghr mRNA levels were elevated in both TR and TF in HYP and
507
POA, whereas Igf1 mRNA levels were elevated in TF only. Similar trends have been reported in
508
TF and TR salmon for serum IGF1 levels and for Igf1 mRNA levels in muscle and liver (Devlin
509
et al., 2009; Overturf et al., 2010; Raven et al., 2008). IGF1 may be involved in stimulating
510
pathways necessary for enhanced growth in TF fish and appears to be related to the long-term
511
nutritional condition of T fish. High Igf1 levels in TF but not TR fish, despite high Gh levels in
512
both types, suggest that long-term ration restriction (to NT levels) in T fish has an inhibitory
513
effect on Igf1 mRNA levels. This concurs with previous studies showing that plasma IGF1 and
514
muscle, liver, and brain Igf1 mRNA levels are influenced by both feeding levels and growth
515
(Beckman, 2011), and that nutritional status can affect the response of Igf1 to GH (Beckman,
516
2011; Moriyama et al., 2000). Some biological actions of GH are mediated through interactions
517
with GHR to produce IGF1 (de Azevedo Figueiredo et al., 2007), and these actions can
518
consequently be influenced by GHR abundance on cell surfaces (Flores-Morales et al., 2006).
519
The discordance between Igf1 levels and Gh and Ghr levels in TR fish could be due to sustained
520
food restriction affecting GHR sensitivity by decreasing GHR production in other unexamined
521
tissues [see (Won and Borski, 2013)]. In the current study, T fish had higher Ghr mRNA levels in
522
the POA and HYP, but not in the PIT, corresponding to respective Gh expression levels seen for
523
these tissues. Overall, normal levels of IGF1 and Igf1 mRNA in TR indicate that the heightened
524
appetite and feeding behaviour of T fish (seen in both TF and TR) may not be mediated by either
525
elevated circulating IGF1 levels or by paracrine/autocrine actions of IGF1 in the brain regions
526
examined or in PIT. However, more specific tests of this hypothesis, involving blockage of IGF1
527
signalling in the brain, are required.
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The present data show significant responses of several genes to both long-term food intake
529
levels and to short-term effects of a meal. For genes that differed in expression between TF and
530
TR fish, intake of food and digestion decreased differences in gene expression levels for many
531
but not all orexigenic and GH system genes examined. There were some consistent trends in the
532
effects of food intake and digestion (relative to the pre-feeding state) on mRNA levels in
533
different tissues and genotypes. For some genes and tissues where expression differed between
534
TF and TR fish, food intake resulted in mRNA levels becoming more similar between TF and TR
535
fish, illustrated by vertical arrows moving to the centre in the ternary plots (i.e. for Agrp1 and
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Pomc in HYP, Gh and Ghr in PIT, Igf1 in POA, Cart in HYP and POA; see Figures 4a-4f).
537
These data are consistent with differences between TR and TF being due to long-term feeding
538
levels and show that these differences can be reduced by a single satiation/feeding event. In
539
contrast, feeding increased differences between TF and TR fish for other genes and tissues as
540
shown by arrows moving away from the centre towards the TF or TR vertex in the ternary plots
541
(i.e. for Npy in HYP and PIT, Sst2 in HYP, Pomc in PIT). Together, these responses reveal a
542
complex relationship among feeding levels, appetite, timing of nutrient processing, and gene
543
expression in T and NT fish.
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Multiple and complex responses in expression of appetite-regulating genes (including
545
Agrp1, Gh, Igf1, Ghr, Mch and Cart) have been observed in T fish compared to wild-type fish
546
(Fig. 5). Appetite regulation is likely maintained by a balance between orexigenic and
547
anorexigenic factors including those influencing the melanocortin system in neurons located in
548
the POA/HYP/PIT axis. Overexpression of GH stimulates Agrp1 expression, which in turn
549
stimulates feeding behaviour, possibly by antagonizing the anorexigenic actions of α-MSH at
550
MC4R. In addition, the decreased expression of the anorexigenic factor CART observed in T fish
551
may also further increase feeding motivation. These actions persist even when growth rate is
552
restricted in T fish by ration limitation. Interestingly, many genes known to be involved in
553
appetite regulation (i.e. Agrp2, Bbs, Cck, Gal, Ghrh, Glp, Gnrh, Hcrt, Lep, Mc4r, Trh, Tsh) did
554
not differ in brain expression between T and NT fish, indicating increased appetite and foraging
555
behaviour in T fish may be controlled by a limited number of appetite-related genes and
556
pathways. The regulation of appetite by brain signals and its integration with peripheral energy
557
and appetite-regulating pathways in chronically growth-stimulated animals deserves further
558
investigations.
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Acknowledgement
562
The authors thank Carlo Biagi, Hamid Seshadri, Marcus Johansson, Janice Oakes, Christine Ng,
563
Hu Jie, Breanna Watson, Dionne Sakhrani, and Krista Woodward for assistance during team
564
sampling, and Bill Gibson for informative discussions. Funding was provided from the Canadian
565
Biotechnology Strategy grant to RHD. 19
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References
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Figure legend Fig. 1. Quantitative PCR measurements of appetite-related mRNA levels in brain (POA,
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HYP) and pituitary (PIT) tissues for genes found to be differentially expressed by RNA-Seq. a)
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Agrp1, b) Mch c) Igf1. PF: pre-feeding; 1H and 4H: 1 hour and 4 hour post-feeding. All values
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are means ± SEM and were normalized to the value of NT at pre-feeding. Letters indicate
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significant differences (P < 0.05) among genotypes and time within tissues.
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Fig. 2. Quantitative PCR results of appetite-related mRNA levels in brain (POA, HYP)
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and pituitary (PIT) tissues for orexigenic and GH-axis genes. a) Npy, b) Gh, c) Ghr. PF: pre-
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feeding; 1H and 4H: 1 hour and 4 hour post-feeding. All values are means ± SEM and were
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normalized to the value of NT at pre-feeding. Letters indicate significant differences (P < 0.05)
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among genotypes and time within tissues.
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Fig. 3. Quantitative PCR results of appetite-related mRNA levels in brain (POA, HYP)
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and pituitary (PIT) tissues for anorexigenic genes. a) Pomc, b) Cart and c) Sst2. PF: pre-feeding;
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1H and 4H: 1 hour and 4 hour post-feeding. All values are means ± SEM and were normalized
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to the value of NT at pre-feeding. Letters indicate significant differences (P < 0.05) among
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genotypes and time within tissues.
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Fig. 4. Ternary plots of appetite-related mRNAs for genes with > 2-fold change relative
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to NT pre-feeding levels in the three experimental groups (NT, TF, TR) influenced by genotype
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and nutritional status. Arrows indicate the direction of change with time/feeding. Genes are
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grouped based on their role in appetite. Orexigenic and GH-axis genes: a) POA, b) HYP, c) PIT;
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Anorexigenic genes: d) POA, e) HYP, f) PIT. The ternary plots graphically depict the ratios of
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the three variables as positions in an equilateral triangle with the positions on each triangle side
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defining the relative value for each of the groups. The three variables are the relative mRNA
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expression level of each fish group summed to the value of 1.0, and each plot point indicates
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the proportion of the variables represented by each fish group at each time point. The relative
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ratio of gene expression in a fish group is determined by the intercept of a line through the plot
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point and axis for the fish group and parallel with the apex for the fish group. Background
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colours indicate where a relative gene expression is highest in NT (blue), TF (red), or TR
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(yellow) fish. To give an example, Gh mRNA levels in PIT were similarly represented in TR
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and NT fish, and minimally represented in TF fish (i.e. pre-feeding plot is placed between NT
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and TR vertices on the low end of TF axis, with the rank order of TR : NT : TF = 0.49 : 0.45 :
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0.05, respectively). After a single satiation feeding event, the three fish groups were more
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equally represented (i.e. arrow shows how plot moved towards centre of triangle, and the rank
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order of NT : TR : TF = 0.43 : 0.33 : 0.24, respectively).
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Fig. 5. Endocrine regulation of appetite-related genes mediated by overexpressed growth
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hormone. a) Study regions in fish brain; POA, preoptic area of the hypothalamus; HYP,
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remainder of the hypothalamus; PIT, pituitary; OB, olfactory bulb; T, telencephalon; ON, optic
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nerve; OT, optic tectum; MB, midbrain; C, cerebellum; HB, hindbrain; SC, spinal cord. Shaded
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areas indicate tissues examined in the current study. b) The influence of GH transgenesis and
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nutrient levels on endocrine and genetic regulation of appetite and growth in salmon. Genes and
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proteins/peptides that are increased in GH transgenic fish are shown in red, those that are
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decreased are shown in blue, and those that are unaffected by transgenesis are shown in grey. A
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yellow background within tissues indicates effects of transgenesis regardless of ration level (i.e.
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TF and TR differ from NT fish), while green background indicates effects of transgenesis are
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present only when ration is unrestricted (i.e. only TF fish differ from NT fish). Red arrows
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indicate stimulation, blue blunt-end lines indicate suppression, and green arrows indicate effect
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of increased nutrients. ? indicates pathways which effects are not fully defined in fish. Data for
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peripheral tissues is from (Raven et al., 2008).
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Supplementary Fig. 1. Basal mRNA expression levels of appetite-related genes in brain
regions and pituitary of wild-type (NT) coho salmon.
Supplementary Fig. 2. Relationships between RNA-Seq and qPCR expression levels of
appetite-related genes.
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Fig. 1
a) Agrp1
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2
gh
gh ab
gh
ab
x
xy x
xy
0 PF
4H
PF
HYP
4H
PIT
b) Cart
NT TF TR
M AN U
1.5
1H
SC
4H
1H POA
xy
xy
a
PF
RI PT
Relative mRNA level
Fig. 3
a) Pomc
8
j
b
b
1.0
ab
z
ij
hij
xyz
i
yz
xyz
i
a
h
0.5
xy
h
TE D
Relative mRNA level
b b
gh
x
g
EP
0.0
PF
4H
PF
1H
POA
4H
PF
HYP
c) Sst2
AC C
6
z
NT TF TR
5
Relative mRNA level
4H PIT
z
4
yz
z
3 h a
2
a g g
y
a
1
a a
g
g
g
a
x
0 PF
4H POA
PF
4H HYP
PF
4H PIT
Fig. 4
ACCEPTED MANUSCRIPT d) anorexigenic
a) orexigenic genes in POA
Pre-feeding
genes in POA
Pre-feeding
1 hpf
1 hpf
4 hpf
4 hpf
Igf1
Pomc
Npy
Ghr
Agrp1
RI PT
Gh
Cart
SC
Mch
Pre-feeding 1 hpf 4 hpf
Igf1 Ghr Agrp1 Npy
Pre-feeding 1 hpf 4 hpf
Pomc
Sst2
TE D
Gh
e) anorexigenic genes in HYP
M AN U
b) orexigenic genes in HYP
Cart
c) orexigenic genes in PIT
AC C
EP
Mch
Pre-feeding
f) anorexigenic genes in PIT
Pre-feeding
1 hpf
1 hpf
4 hpf
4 hpf Pomc
Igf1 Npy Sst2 Ghr
Sst1 Cart
Agrp1 Gh Mch
Fig. 5
ACCEPTED MANUSCRIPT
a)
C
OT T
MB POA
HB HYP
ON
b) ?
Appetite ?
αMSH (POMC)
AGRP
transgene
GH
peripheral tissues)
CART
IGF1
TE D
SST
MC4R
Growth (brain and
M AN U
POA / HYP
SC
PIT
SC
RI PT
OB
?
MCH
GHR
Nutrients
EP
circulating
AC C
GH
GHR
GHR
PIT
transgene
GH
SST
transgene
host GH ?
SST
GH
IGF1
IGF1
IGF1
Peripheral tissues
S.1
ACCEPTED MANUSCRIPT
RI PT
200
130
102 100 0.13
0.25 POA HYP PIT
0.18
2.58
M AN U
0.1
0.58 0.50 0.11
SC
0.24
0.08
TE D
0.06
EP
0.04
0.02
0
Tsh
Trh
Sst2
Sst1
Pomc
Hcrt
Npy
Mch
Mc4r
Lep
Igf1r
Igf1
Gnrh
Glp
Ghrh
Ghr
Gh
Gal
Cck
Cart
Bbs
Agrp2
Agrp1
AC C
Normalized mRNA level
0.47
2.70
S.2
ACCEPTED MANUSCRIPT
RI PT M AN U
SC
4
0
EP
TE D
-4
R2 = 91
-8
-12
AC C
Normalized mRNA level from qPCR
8
-4
0
4
8
12
Normalized reads from RNASeq
16
ACCEPTED MANUSCRIPT
Highlights GH overexpression alters expression of appetite-regulating genes in salmon brain.
•
Agrp1 is upregulated up to 12 fold in the preoptic area and hypothalamus.
•
Mch was downregulated in most brain tissues and Pomc was elevated in pituitary.
•
Igf1 affected by nutritional status and does not correlate with heightened feeding.
•
Other orexigenic and anorexigenic genes inconsistently affected by GH transgenesis.
AC C
EP
TE D
M AN U
SC
RI PT
•
ACCEPTED MANUSCRIPT Supplementary Table 1. Primers and probes used in this study
Cck Gal Gh
Ghr
Ghrh Glp
Gnrh Igf1
AC C
Igf1r
Igfbp
Lep
Mc4r
Mch
RI PT
Cart
SC
Bbs
M AN U
Agrp2
Agrp1-RT-F Agrp1-RT-R Agrp1-RT-probe Agrp2-RT-F Agrp2-RT-R Bbs-RT-F Bbs-RT-R Bbs-RT-probe Cart-RT-F Cart-RT-R Cck-RT-F Cck-RT-R Gal-RT-F Gal-RT-R Gh-RT-F Gh-RT-R Gh-RT-probe Ghr-RT-F Ghr-RT-R GHR-RT-probe Ghrh-RT-F Ghrh-RT-R Glp-RT-F Glp-RT-R Glp-RT-probe Gnrh-RT-F Gnrh-RT-R I gf1-RT-F I gf1-RT-R I gf1-RT-probe Igf1r-RT-F Igf1r-RT-R Igf1r-RT-probe Igfbp-RT-F Igfbp-RT-R Igfpb-RT-probe Lep-RT-F Lep-RT-R Lep-RT-probe Mc4r-RT-F Mc4r -RT-R Mc4r -RT-probe Mch-RT-F Mch-RT-R
Sequences (5´´ 3´´) ACCAGCAGTCCTGTCTGGGTAA AGTAGCAGATGGAGCCGAACA CTGCCCTGCTGCGACCCCTG TGTTTGCCAGGAGACGGATT AGGCTCGTGTTTCTGAAATGC CAGAACGGGATGGGAAATCTC TTTTAGAGCGGTTCTCTGTGTCAT CGCGTTGCAAGCCCAACTCAGA AGCATCAGGGTTCGCTCACT TGGCAAACAACACTGAAGACAGA TCCTCTGAAGCACGTCTTGAAG TGGCGGAGCGTGTCTGT ACAGTGCTGGCTACCTTTTGG AGGCCATGCTTGTCACTGAGT CAAGATATTCCTGCTGGACTTCTGT GGGTACTCCCAGGATTCAATCA CAGTCCTGAAGCTGC CACTGTGGAAGACATCGTGGAA CAAAGTGGCTCCCGGTTAGA AACTGGACCCTGCTGAA CAAATCTCAGCCAGGAAA TCCGGCCTTCTTCTTG AGTGGTGCTCCATCCAAACG CGCCTGGTCCTGTAGGTAGGT CGATGGGACCTACACCAGCGACGT AGCTGTCTTCCTCCCAGCAC GCATTCTCCTGCCTCACAGA GGCATTTATGTGATGTCTTCAAGAGT CCTGTTGCCGCCGAAGT TCTCACTGCTGCTGTGC CATGGAGCTGATGACTAAAGGAGAT GGAGGGAGGTTGAGACTGTAAGACT TGAAGAGCCACCTGAGG AACACCATCCGCAAGAAACTG TTGTCCAGAGCTGCATGCA TGGAACAGGGTCCCTGCCACATTG TGCTGGAGAACTGGATGATATCA GCCCTCCCTCTCCTGTCTGT CTGCCCAGGCCGCCAACAGA CTCGCTCTACGTCCACATGTTC GCAGCACGGCAATCCTCTT TGCTGGCCCGCCTGCACA GACTCTGGCCTGTGGATGAAC GCTGCAGCTCTCAGCTTGTAGA
TE D
Agrp1
Oligo name
EP
Genes
1
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
TGAACAGAGGACTTCCT Mch-RT-probe CAAGGCAGAGGTATGGGAAGAG Npy Npy-RT-F TCTCCTTTAGCAGCAGTTCTGAGA Npy-RT-R CCAGCCCTGACACACTGGATTCACTG Npy-RT-probe ATCCCACTCCTGCCGTCTT Hcrt Hcrt-RT-F CCTTGTCCCCGTCCCATT Hcrt-RT-R ACCCATTGGGCACAAACG Pomc Pomc-RT-F GGAGTCCCCCCCTTCCA Pomc-RT-R CCCTTCCAGACTGGAGGCA Pomc-RT-probe TCCGTCCGATGCCAAACT Sst1 Sst1-RT-F CTGGCAAGCTCCTGTTTGC Sst1-RT-R CGCCAGCTGCTCCAACGGTCA Sst1-RT-probe AGCCGCCGACTCCTTCA Sst2 Sst2-RT-F TGTCCTCCATCACTCGCTTACTC Sst2-RT-R CCGGCAGACTATTGACGATATCA Trh Trh-RT-F CGCCATCCTCATCCTCTATATTTT Trh-RT-R CCGAGAGCCTCCTGCTCCGCTC Trh-RT-probe ACAAGGCCAGCAGTGGTGAT Tsh Tsh-RT-F AGGCCAGGGTATGGGTAGATG Tsh-RT-R CCCCAGGTGTACCAAGCCACTCAGAA Tsh-RT-probe ACAGCTGGCCCAGAAGTACAA Ubiquitin Ubiquitin-RT-F GGCGAGCGTAGCATTTGC Ubiquitin-RT-R TGTGACAAAATGATCTGC Ubiquitin-RT-probe Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulin-like growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroidstimulating hormone
2
ACCEPTED MANUSCRIPT Supplementary Table 2. Differentially expressed genes in the coho salmon brain (DEGs shown as ratios between group pairs, fold change >2, FDR<0.001). The appetite regulation-related genes are bolded.
26.8 7.9 4.8 4.1 4.0 2.8 2.7 2.6 2.5 2.3 2.3 2.3 2.1 2.1 2.1 -2.1 -2.9 -2.9 -3.1 -3.3 -10.7
21.7
17.7 17.5 10.1 8.1 5.8 5.0 3.5 3.4 3.0 3.0 2.9 2.9 2.6 2.5 -8.7 -9.3 -20.6 -68.7
AC C PIT
TR/TF
-3.5 -2.9
-2.9 -2.4 -2.9
3.5 26.4 31.4 10.0 13.0 6.4
-3.4 -3.3 -3.0 -2.3 -2.5
-11.2
-14.9 -5.4 8.5 9.0 11.0 16.0 17.3
7.5 -3.2 -2.7 -2.6 -2.3 -2.2 -2.1 2.1 2.2 2.4 2.5 2.7
70.7 62.0 16.2 5.3
Homeobox protein Hypothetical protein Prepro-urotensin II-related peptide Neurofilament light polypeptide Physical map contig Plasticin Neurofilament light polypeptide b Plasticin Neurofilament light polypeptide-like Secretagogin Neurofilament medium polypeptide isoform 2 Plasticin Neurofilament medium polypeptide Seurofilament medium polypeptide Neurofilament heavy polypeptide-like Isotocin-I precursor Vasotocin Vasotocin C-C motif chemokine precursor Vasotocin Cell division control protein precursor Uncharacterized protein 12-RF amide peptide 12-RF amide peptide Immunoglobulin heavy chain locus B Barrier-to-autointegration factor Agouti related protein-1 precursor Suppressor of cytokine signaling 2 Solute carrier family 22 member 5-like Collagen precursor Collagen precursor Collagen precursor Perforin-1 precursor Ollagen type I Collagen precursor Collagen Fatty acid-binding protein Programmed cell death 1 ligand 1 precursor Uncharacterized protein Interleukin-5 receptor Conserved noncoding element Glucose-6-phosphatase Clone BAC Homeobox protein Neurogranin High choriolytic enzyme 1 precursor Follicle-stimulating hormone Lectin precursor Neutral amino acid transporter Insulin-like growth factor I precursor Collagen precursor Arbonic anhydrase 4-like Carbonic anhydrase 4-like Thymosin beta-like Melanin-concentrating hormone Metallothionein A Melanin-concentrating hormone-like protein Melanin-concentrating hormone MHC class I heavy chain precursor Isotocin Isotocin Relaxin precursor Heat shock 70 kda protein
SC
-2.3 -2.2
Identification
RI PT
TR/NT
EP
HYP
CL16070.Contig2_All Unigene109213_All CL19203.Contig2_All Unigene88010_All Unigene48309_All Unigene27974_All CL26051.Contig1_All CL9024.Contig1_All CL15264.Contig3_All Unigene1226_All Unigene28401_All Unigene38973_All CL20386.Contig2_All Unigene55135_All Unigene69936_All Unigene41692_All CL21933.Contig1_All Unigene59744_All Unigene71783_All Unigene40388_All CL4761.Contig3_All Unigene24774_All CL10291.Contig1_All CL10291.Contig1_All Unigene27055_All Unigene57306_All Unigene76640_All CL8110.Contig4_All Unigene57540_All CL25868.Contig1_All Unigene30416_All CL3630.Contig3_All CL8644.Contig2_All Unigene36971_All Unigene30420_All Unigene36970_All CL18280.Contig2_All Unigene30893_All Unigene69802_All Unigene63064_All CL25077.Contig3_All CL26454.Contig1_All CL6927.Contig1_All CL16070.Contig2_All CL10025.Contig1_All Unigene63080_All Unigene45955_All Unigene39007_All CL8969.Contig1_All CL5146.Contig3_All CL3630.Contig2_All CL7671.Contig2_All Unigene62679_All CL26397.Contig1_All CL24390.Contig1_All Unigene56962_All CL1086.Contig7_All CL24390.Contig2_All Unigene16306_All CL12454.Contig2_All CL12454.Contig1_All Unigene108551_All Unigene77422_All
TF/NT
M AN U
POA
Contig
TE D
Tissue
-15.6 3.2
1
ACCEPTED MANUSCRIPT 7.3 -4.0 -3.8 28.6
SC
RI PT
-362.1 -293.8 -244.0 -203.1 -194.4 -165.2 -155.7 -124.1 -110.1 -88.3 -80.0 -52.2 -41.8 -38.0 -32.3 -30.9 -27.2 -26.0 -25.9 -25.0 -24.6 -24.0 -20.2 -19.8 -19.2 -18.8 -18.6 -15.7 -12.4 -12.1 -10.5 -9.7 -9.3 -8.5 -7.9 -4.6 -3.1 -2.8 -2.8 -2.7 -2.3 -2.2 -2.2 -2.1 2.0 2.1 2.2 2.2 2.4 2.6 2.8 2.9 3.1 3.3 5.1 5.6 5.8
Calreticulin precursor Neurogranin Regulator of G-protein signaling 5 Melanin-concentrating hormone Immunoglobulin heavy chain locus B Programmed cell death 1 ligand 1 precursor Melanin concentrating hormone precursor Sterile alpha motif domain-containing protein Apolipoprotein A-II precursor Serum albumin 2 precursor Alpha-1-antiproteinase-like protein precursor C1 inhibitor Antihemorragic factor Hypothetical protein Complement factor Apolipoprotein B-100-like Uncharacterized protein Apolipoprotein B Complement component C3 Complement factor H-like Orphan nuclear receptor Dax-1 Complement C3-S Alpha-2-macroglobulin Saxitoxin and tetrodotoxin-binding protein 2 Apolipoprotein Bb precursor Beta-2-glycoprotein 1 precursor Liver-type fatty acid-binding protein 4-hydroxyphenylpyruvate dioxygenase Apolipoprotein A-IV precursor AMBP protein precursor Fibrinogen beta chain-like Coagulation factor II precursor Secreted phosphoprotein 24 precursor Plasminogen precursor Fructose-bisphosphate aldolase B Fibrinogen gamma chain precursor Apolipoprotein A-I-1 precursor Complement component 3-like precursor Alpha-1-antitrypsin-like Uridine phosphorylase 2-like Hemopexin-like protein Hyaluronan-binding protein 2-like Retinol-binding protein 1 Succinyl-coa ligase subunit beta Sushi domain-containing protein 2-like Apolipoprotein C-I precursor T-complex protein 1 subunit eta-like Ultra conserved element locus Transferrin Islet amyloid polypeptide precursor Somatolactin beta Somatolactin beta precursor Phosducin-like 3-2 Nuclear prelamin A recognition factor Nose resistant to fluoxetine protein 6-like Unnamed protein product Gastrin-releasing peptide-like precursor Hypothetical protein Fibronectin type-III domain-containing protein Galectin-3-binding protein precursor Immunoglobulin heavy chain locus A Unnamed protein product Lysozyme variant LRR and PYD domains-containing protein Uncharacterized protein Collagen alpha-1(XVII) chain
TE D
M AN U
5.0 4.2 3.0 -8.8 -9.2 -12.4 -16.7 -21.4
AC C
EP
CL23238.Contig1_All CL10025.Contig1_All Unigene21424_All CL24390.Contig1_All Unigene20684_All Unigene30893_All CL24390.Contig2_All CL15919.Contig4_All Unigene44665_All CL13198.Contig1_All Unigene25835_All Unigene26038_All Unigene44347_All CL8258.Contig1_All CL5254.Contig13_All Unigene37404_All Unigene39230_All CL21575.Contig1_All CL74.Contig2_All Unigene44027_All CL23759.Contig1_All CL25254.Contig2_All Unigene639_All Unigene64340_All CL4139.Contig1_All CL13321.Contig1_All Unigene70666_All Unigene72625_All CL13442.Contig1_All CL3260.Contig2_All CL21940.Contig2_All Unigene50138_All Unigene33139_All CL18560.Contig2_All CL25565.Contig1_All CL880.Contig2_All Unigene38325_All Unigene37879_All CL10379.Contig1_All Unigene19690_All Unigene16218_All Unigene52612_All CL11335.Contig1_All Unigene43291_All Unigene31055_All Unigene21161_All CL16339.Contig1_All Unigene50081_All CL5054.Contig6_All CL12798.Contig1_All Unigene3312_All Unigene77121_All CL5646.Contig1_All CL22274.Contig1_All Unigene64369_All Unigene75867_All Unigene52686_All CL17915.Contig2_All Unigene16697_All Unigene15925_All Unigene6951_All CL15564.Contig2_All CL26463.Contig7_All CL26282.Contig1_All Unigene24774_All CL22016.Contig1_All
9.9 -6.0
NT, non-transgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary; FDR, false discovery rate. 2
ACCEPTED MANUSCRIPT Supplementary Table 3a. Normalized reads of RNA-Seq for appetite-related genes in coho salmon.
NT a
TF
PIT
TR b
8.1 12.1 72.0 51.3 63.0 10.1a 443 66.3 1.5 0.0
23.3 21.5 99.5 100 99.2 49.4b 240 62.2 4.5 0.0
9.9 20.3 127ab 16.1 767 49.9 276 94.6a 3.9 0.0
81.6 21.5 110a 16.2 747 50.5 490 213b 5.9 0.0
13.0 30.0 3.9 2.5 0.5 2.4 1.5 4.5 41.0 415 479 1.0 29.1 5.0
17.0 48.5 0.5 3.5 0.0 2.0 0.0 496 61.6 181 748 2.5 59.4 2.0
7.8 34.6 1.0a 0.0 94.8 2.5 0.5 5145b 18.8 252b 112 6.4 7.9 2.4
6.3 33.3 6.3 0.0 158 1.5 1.0 1800a 19.1 287b 164 11.8 13.2 2.9
NT
b
129 12.9 156b 18.3 1006 55.0 342 235b 5.5 0.0
ab
7.4 39.8 2.5 0.0 106 0.5 2.0 4410b 24.8 13.9a 130 31.3 14.4 0.5
TF
TR
11.9 2.9 0.0 212 0.0 0.5 482638a 86.6 0.5 0.0
53.6b 0.5 1.0 181 4.6 0.0 528853a 87.8 0.5 0.0
13.6b 0.0
1.0a 0.0
12.7b 0.0
1.9a 3.4 63.1 0.0 0.0
4.6b 5.1 112 0.0 0.0
3.0a 2.0 57.2 0.0 0.0
216c 0.5 381456 3.9 3.4 0.0 2214
12.9a 4.1 954872 15.0 2.5 0.0 4124
82.2b 1.0 652225 21.2 2.0 1.5 2578
18.5 2.9 0.0 253 1.9 1.5 681994b 86.9 0.0 0.0
a
RI PT
TR
M AN U
0.5 15.7 148 96.4 53.1 40.8ab 182 40.3 2.0 0.0 16.2 28.5 2.0 4.4 0.0 2.5 1.0 16.7 88.1 207 558 0.0 44.3 0.0
HYP
TF
TE D
NT
SC
POA Gene Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Igfbp Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
AC C
EP
All data were normalized by a scaling factor from DESeq method. The letters indicate significant difference of reads between genotypes (P < 0.05). Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulin-like growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, nontransgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary.
ACCEPTED MANUSCRIPT Supplementary Table 3b. Normalized reads of RNA-Seq for appetite-related genes in coho salmon.
NT 0.0478a 0.0979b 0.6186 0.0788ab 3.7094 0.1206 1.3591 0.0764a 0.0191 0.0382 0.1672 0.024a 0.0311 0.4610 0.0119 0.0024 25.068ab 0.0908 1.2444 0.5470 0.0311 0.3535 0.0119
HYP TF 0.3590b 0.0952ab 0.4867 0.0714a 3.3071 0.1114 2.1846 0.1568b 0.0260 0.0281 0.1471 0.0141b 0.0216 0.6965 0.0065 0.0043 7.9077a 0.0844 1.2978 0.7224 0.0519 0.5883 0.0130
TR 0.5954b 0.0595a 0.7214 0.0847b 4.6511 0.1271 1.5802 0.1805 0.0252 0.0344 0.1832 0.0057a 0.0229 0.4901 0.0023 0.0092 20.361b 0.1145 0.0641 0.5977 0.1443 0.6847 0.0023
NT 0.0334ab 0.0053 0.4580 0.0035 0.0026 1234.16b 0.0315 0.0246b 0.0018a 0.0062 0.1143 0.3912c 0.0009 690.251 0.0070 0.0062 0.0009 4.0056
PIT TF 0.0190a 0.0047 0.3267 0.0008 745.17a 0.0274 0.0008 0.0016a 0.0036b 0.0079 0.1741 0.0206a 0.0063 1482.18 0.0237 0.0040 6.4019
TR 0.0977b 0.0009 0.0018 0.3300 0.0083 0.0 966.02ab 0.0321 0.0009 0.0230ab 0.0055ab 0.0037 0.1041 0.1502b 0.0018 1191.27 0.0387 0.0037 0.0028 4.7106
RI PT
TR 0.1339 0.1225 0.5670 0.5726 0.5698 0.1410 1.3561 0.0594 0.0256b 0.0969 0.2764b 0.0028 0.0100 0.4672 0.0114 2.6724 0.3504 1.0171 4.2564 0.0142 0.9288 0.0114
SC
POA TF 0.0448 0.0672 0.4034 0.2885 0.3501 0.0280 2.4762 0.0739 0.0084a 0.0728 0.1681a 0.0112 0.0070 0.4258 0.0140 0.0084 0.0252 0.2297 2.3249 2.6807 0.0056 0.7087 0.0280
M AN U
NT 0.0028 0.0892 0.8420 0.5465 0.3011 0.1157 1.0260 0.0381 0.0112ab 0.0920 0.1617ab 0.0056 0.0125 0.4517 0.0139 0.0056 0.0948 0.4991 1.1654 3.1673 0.8587 -
TE D
Gene Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Igfbp Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
AC C
EP
All data was normalized to the read of Ubiquitin. The letters indicate significant difference of reads between genotypes (P < 0.05). Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Igfbp, insulinlike growth factor bind protein; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic fully fed fish; TR, transgenic ration restricted fish. POA, preoptic area; HYP, hypothalamus; PIT, pituitary; -, not detected.
ACCEPTED MANUSCRIPT Supplementary Table 4. Relative mRNA expression levels of appetite-related genes in preoptic area (POA) of coho salmon. Mean values only provided. Average standard error among the groups was 0.023. N ranged between 11 and 12.
0.0015a 0.0133 0.4697 0.0415b 0.0159 0.0423 0.0505a 0.0799a 0.0010 0.0027 0.0664 0.0159 0.0087a 0.0367 0.0007 0.0088 0.2512ab 0.1811ab 0.0024ab 0.5824 0.1140 0.0908 0.0041
4 hpf
0.0006a
0.0018a
0.0389b 0.0100 0.0575 0.0684a 0.0798a 0.0010 0.0047 0.0525 0.0146 0.0074a 0.0384
0.0127ab
0.2369
b
0.0088c
0.0103 0.6574b 0.2938b 0.0080bc 0.4874 0.1014 0.0498 0.0067
Prefeeding 0.0074b 0.0137 0.3285 0.0207a 0.0155 0.0141 0.3173b 0.1075bc 0.0012 0.0019 0.1163 0.0131 0.0186bc 0.0365 0.0005 0.0059 0.0320a 0.3134b 0.0057ab 0.5229 0.1031 0.0879 0.0051
1 hpf
4 hpf
0.0128b
0.0092b
0.0355ab 0.0102 0.0296 0.1963b 0.1234c 0.0011 0.0038 0.0507 0.0110 0.0143bc 0.0324
Prefeeding 0.0055b 0.0190 0.3628 0.0387b 0.0155 0.0533 0.2266b 0.1180c 0.0015 0.0029 0.1172 0.0301 0.0082a 0.0366 0.0006 0.0082 0.2366a 0.1073a 0.0116bc 0.6251 0.1323 0.1202 0.0041
1 hpf
4 hpf
0.0148b
0.0108b
RI PT
Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
1 hpf
TR
M AN U
Prefeeding
TE D
Gene
TF
SC
NT
0.0553d
0.0078
d
0.0104c
0.0062 0.0710bc 0.1949ab 0.0012a 0.8868 0.1922 0.0495 0.0053
0.0218c
0.1324
c
0.0097c
0.0444b 0.0123 0.0588 0.2973b 0.1285c 0.0013 0.0040 0.0934 0.0329 0.0146bc 0.0328 0.0085 0.4948bc 0.2314b 0.0024ab 0.7824 0.1824 0.1483 0.0081
AC C
EP
All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample time (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured.
ACCEPTED MANUSCRIPT Supplementary Table 5. Relative mRNA expression levels of appetite-related genes in hypothalamus (HYP) of coho salmon. Mean values only provided. Average standard error among the groups was 0.027. N ranged between 11 and 12. NT TF TR 0.0111b
0.0072ab
0.0239cd 0.0034
0.0214bcd 0.0064 0.0565 0.1460abc 0.0157a 0.0054 0.0103 0.0001 0.0227 0.0094a 0.0467
0.1060ab
0.0274c
cd
3.2125 0.0099ab
0.0222 3.8073bcd 0.0081ab 0.0031ab 0.6825 0.0458a 0.0419 0.0080
Prefeeding 0.0204c 0.0174 0.1327 0.0172c 0.0053 0.0451 0.5008d 0.0587e 0.0045 0.0032 0.0002 0.0175 0.0411d 0.0385 0.0195 0.7218a 0.0155ab 0.0070b 0.6023 0.0659a 0.0407 0.0061
1 hpf
4 hpf
0.0450cd
0.0305cd
0.0182c 0.0036
0.0073a 0.0066 0.0463 0.1556c 0.0531de 0.0022 0.0074 0.0001 0.0213 0.0185b 0.0537
0.2674c
Prefeeding 0.0575d 0.0158 0.1308 0.0127b 0.0069 0.0581 0.6035d 0.0423bc 0.0127 0.0042 0.0002 0.0165 0.0182b 0.0339 0.0198 2.4139b 0.0114ab 0.0033ab 0.4646 0.0630a 0.0236 0.0063
1 hpf
4 hpf
0.0379cd
0.0374cd
RI PT
4 hpf
0.0121b 0.0038
0.1885bc
SC
1 hpf
M AN U
Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
Prefeeding 0.0048a 0.0216 0.1271 0.0287d 0.0063 0.0607 0.2430a 0.0164a 0.0054 0.0058 0.0001 0.0192 0.0184b 0.0354 0.0214 2.6986bc 0.0125ab 0.0024ab 0.5018 0.0450a 0.0419 0.0117
TE D
Gene
0.0609e
a
0.8406 0.0138c
0.0177 2.1954bc 0.0045a 0.0030a 1.1702 0.0954b 0.0460 0.0068
0.0249bc
bc
1.7896 0.0099ab
0.0090ab 0.0124 0.0574 0.1482bc 0.0395bc 0.0090 0.0108 0.0002 0.0140 0.0127a 0.0292 0.0170 4.0790a 0.0074a 0.0027ab 0.5673 0.0506a 0.0455 0.0129
AC C
EP
All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample time (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured; -, not detected.
ACCEPTED MANUSCRIPT Supplementary Table 6. Relative mRNA expression levels of appetite-related genes in pituitary (PIT) of coho salmon. Mean values only provided. Average standard error among the groups was 1.413. N ranged between 9 and 12.
0.0072a
0.0083bc 0.0007 114.99cde 0.0282b
154.27e 0.0282b 0.0005
0.0035 0.0135b 0.0096
259.54ab
0.0006 0.0870c 0.0021a 187.34ab 0.0118a 0.0010b 0.0009 2.4733
1 hpf
4 hpf 0.0094ab
0.0027a 0.0003 57.542b 0.0246b
87.622c 0.0381b 0.0010
0.0045 0.0201c 0.0119
721.58c
Prefeeding 0.0195c 0.0012 0.0003 0.0064abc 0.0007 0.0004 114.49d 0.0344b 0.0007 0.0031 0.0118ab 0.0085 0.0007 0.0469c 0.0066b 120.25ab 0.0149a 0.0027c 0.0007 4.1755
0.0005 0.0025a 0.0036b 389.22bc 0.0305b 0.0022c 0.0016 3.7866
1 hpf
4 hpf 0.0181bc
RI PT
4 hpf
TR
M AN U
Agrp1 Agrp2 Bbs Cart Cck Gal Gh Ghr Ghrh Glp Gnrh Hcrt Igf1 Igf1r Lep Mc4r Mch Npy Pomc Sst1 Sst2 Trh Tsh
1 hpf
TE D
Gene
TF Prefeeding 0.0100a 0.0018 0.0003 0.0067abc 0.0004 0.0007 13.362a 0.0129a 0.0009 0.0033 0.0195bc 0.0119 0.0007 0.0085b 0.0078b 183.42ab 0.0053c 0.0021bc 0.0014 6.1057
SC
NT Prefeeding 0.0116b 0.0012 0.0003 0.0098c 0.0005 0.0003 104.53cd 0.0282b 0.0005 0.0028 0.0121b 0.0083 0.0009 0.0737c 0.0056b 129.74a 0.0104a 0.0007a 0.0009 2.5773
104.96cd 0.0268b
0.0044ab 0.0006 119.04cd 0.0296b 0.0007
0.0024 0.0086a 0.0088
215.97a
0.0007 0.0106b 0.0023a 228.44ab 0.0340b 0.0033c 0.0009 1.8243
AC C
EP
All values are normalized to Ubiquitin. The letters indicate significant difference among genotypes and sample times (P < 0.05). hpf, hour post feeding; Agrp, agouti-related protein; Bbs, bombesin; Cart, cocaine and amphetamine regulated transcript; Cck, cholesytokinin; Gal, galanin; Gh, growth hormone; Ghr, growth hormone receptor; Ghrh, growth hormone release hormone; Glp, glucagon-like peptide; Gnrh, gonadotropin release hormone; Hcrt, hypocretin (orexin); Igf1, insulin-like growth factor 1; Igf1r, insulin-like growth factor 1 receptor; Lep, leptin; Mc4r, melanocortin 4 receptor; Mch, melanin-concentrating hormone; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Sst, somatostatin; Trh, Thyrotropin release hormone; Tsh, thyroid-stimulating hormone thyroid-stimulating hormone; NT, non-transgenic fish; TF, transgenic full fed fish; TR, transgenic ration restricted fish; blank, unmeasured; -, not detected.