General and Comparative Endocrinology 171 (2011) 359–366
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General and Comparative Endocrinology
journal homepage: www.elsevier.com/locate/ygcen
Postprandial effects on appetite-related neuropeptide expression in the brain
of Atlantic salmon, Salmo salar
R. Valen a,⇑, A.-E.O. Jordal a, K. Murashita a,b, I. Rønnestad a
a
b
Department of Biology, University of Bergen, NO-5020 Bergen, Norway
Aquaculture Systems Division, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan
a r t i c l e
i n f o
Article history:
Received 17 August 2010
Revised 24 February 2011
Accepted 27 February 2011
Available online 4 March 2011
Keywords:
cart
npy
pyy
cck
agrp
pomc
Isoforms
Teleost
Appetite
Neuropeptide
a b s t r a c t
Following feeding of a single meal to Atlantic salmon, the temporal changes in the brain mRNA expression of neuropeptide y (npy), cocaine-amphetamine regulated transcript (cart), peptide yy (pyy), two isoforms of agouti-related protein (agrp), two isoforms of cholecystokinin (cck), and four isoforms of
proopiomelanocortin (pomc) were assessed by q-PCR. In the course of 24 h post-feeding (hpf), several
of the brain neuropeptides displayed changes in mRNA expression compared to an unfed control group,
indicating that food intake and processing affect the regulation of expression of these genes in Atlantic
salmon. Expression of cart, cck-l, pomc-a1 and pomc-b all increased within 3 h of feeding, while most
of the feed was still in the stomach, suggesting that these neuropeptides play central anorexigenic roles
similar to those described in higher vertebrates, including determining meal intervals. On the other hand,
the npy and agrp isoforms which have been described as playing orexigenic roles in mammals, showed an
opposite response in salmon and both were elevated in the first 3 h after feeding. The different isoforms
of cck, agrp and pomc had different mRNA expression patterns, which indicate specific roles related to
feeding regulation. The minimal effect of feeding and digestion on pyy expression in the brain indicates
that PYY plays a minor role in the central control of short-term food intake in Atlantic salmon.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
The physiological process of appetite regulation involves a
complex integration of peripheral and central signals by the brain.
The nervous system, the gastrointestinal (GI) tract, adipose tissue
and the external environment mediate the afferent signals that
are involved in maintaining energy homeostasis. The efferent message may be anabolic or catabolic, depending on body energy levels
[48]. Mammalian studies have shown that certain centers in the
brain, in particular the arcuate nucleus (ARC) of the hypothalamus,
activate neuronal circuits which produce either orexigenic factors
that stimulate appetite, or anorexigenic factors that inhibit it
[48]. Studies employing electrical stimulation or lesions of specific
brain areas have shown that the hypothalamus is involved in controlling feed intake in fish as well as mammals [12,41].
Until recently, most research on appetite and feeding in teleosts
has focused on the effect of diet composition and the impact of
environmental factors such as photoperiod and temperature, with
less attention paid to the endocrine systems that control feeding
[3,44,56]. However, recent studies have shown that many of the
same appetite-regulating factors that are homologues to mamma⇑ Corresponding author.
E-mail address: [email protected] (R. Valen).
0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2011.02.027
lian neuropeptides, are also found in fish (reviewed by:
[25,30,59,60]), although their precise physiological roles remain
to be explored.
Satiety signals from the GI tract have a major impact on appetite, and studies on rainbow trout (Oncorhynchus mykiss) have
shown that appetite returned when 80–90% of the stomach content from the previous meal was transferred downstream into
the proximal gut [17,20]. This suggests that the contents of the
stomach can serve as an indicator of the return of appetite. The
goldfish (Carassius auratus) is a model frequently used in appetite
studies, and have provided much of our current knowledge of
appetite in fish [30]. Several of the neuropeptides studied in goldfish have been shown to have an appetite regulatory effect similar
to that described in mammals [60,64]. However, since teleosts are
a highly diverse group of species, the differences in habitats, feeding habits and response to feeding and fasting strongly suggest that
a species-specific approach needs to be adopted in studies of
appetite.
The Atlantic salmon (Salmo salar) is an important coldwater
species in both aquaculture and research. Understanding the processes that regulate appetite is a key to optimizing food intake,
feed conversion ratio and growth. Recently, the appetite-regulating
peptide hormones neuropeptide Y (NPY), cocaine-amphetamineregulated transcript (CART), two isoforms of Agouti related protein
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R. Valen et al. / General and Comparative Endocrinology 171 (2011) 359–366
(AgRP-1 and AgRP-2), three isoforms of proopiomelanocortin
(POMC-A1, -A2 and A2s), two isoforms of cholecystokinin (CCK-L
and CCK-N) and peptide YY (PYY) were cloned and their tissue distribution characterized in Atlantic salmon [34–36]. The aim of our
study was to gain a better understanding of how the concerted actions of these neuropeptides participate in the physiological control of appetite regulation.
NPY is a 36 amino acid-amidated peptide which belongs to the
pancreatic polypeptide family [57]. NPY is highly conserved among
vertebrates including fish, and it has been suggested that it is a key
peptide in the regulation of appetite in teleosts [4,27,64]. In mammals, NPY has been shown to be the most potent orexigenic peptide [54], and has also been shown to increase appetite in
teleosts including goldfish [31,39], rainbow trout [2] and channel
catfish (Ictalurus punctatus) [51]. In these species, npy expression
increases both before feeding and after 1–4 weeks of fasting, with
some species variations [21,32,38,50].
CART was initially discovered in rats as a transcript that is upregulated following administration of psychomotor stimulants
[13], and it was later found to regulate food intake in both mammals [24] and fish [23,62,63]. In mammals, CART is a potent anorexigen [24]. Although CART has been identified in several species
including goldfish [62], Atlantic cod (Gadus morhua) [21], Atlantic
salmon [35] and catfish [52], its physiological role is still unclear.
In Atlantic salmon, cart mRNA decreases after 6 days of fasting
[35], although an immediate postprandial effect has not been described. Altogether, CART is a strong candidate neuropeptide for
the regulation of short- and long-term feeding, and the central control of body weight in fish.
Mammalian AgRP is an orexigenic peptide which is coexpressed
together with NPY in the ARC [45]. The orexigenic effect of AgRP is
mainly through the antagonism of central melanocortin (MC)
receptors [40,43]. AgRP has been identified in several fish species
[5,22,26,53], including Atlantic salmon [35]. In goldfish, agrp
mRNA increase after 3 days of fasting, and it may exert its effect
through MC receptors [5,6]. In conclusion, the identification of
AgRP in several fish species and the effects of fasting on agrp
expression in goldfish, suggest that it plays a role in appetite regulation, possibly through MC receptors and hence that a melanocortin system is also found in fish.
Proopiomelanocortin (POMC) is a precursor peptide which is
post-transcriptionally processed into melanocortins including
melanocyte-stimulating hormones (a-, b- and c-MSH) and adrenocorticotropic hormone (ACTH) [47]. In mammals, melanocortins
are believed to be involved in a wide range of physiological functions, including energy homeostasis [9]. In goldfish, fasting did
not change pomc expression [7]. However, ICV injection of a melanocortin agonist reduced feed intake, which suggests the existence
of a functional melanocortin system in fish [7]. Atlantic salmon
express four isoforms of POMC (Murashita et al., unpublished;
Supplemental Fig. 1). However, only one of the four isoforms
(POMC-A1) is affected by 6 days of fasting (Murashita et al., unpublished; Supplemental Fig. 2). This paper is the first report on the
dynamic changes in pomc expression during the complete processing of a meal in any teleost.
Cholecystokinin (CCK) was originally identified as a factor that
induced gallbladder contraction, and was later shown to inhibit
appetite [19,29,33]. In goldfish, cck expression increases shortly
after feeding, and in rainbow trout appetite increases when CCK
antagonists are used [15,42]. Altogether, these studies indicate that
CCK acts as a satiety signal in fish, as it does in higher vertebrates.
In mammals, CCK has been shown to induce satiety both directly as
a neurotransmitter in the brain, and indirectly through an endocrine pathway that delays gastric emptying [29].
PYY belongs to the NPY family of peptides, which includes NPY
[27]. The effect of PYY as a satiety factor is thought to be through
Y2 receptors on vagal afferents, signaling to hypothalamic ARC
via the nucleus tractus solitarius (NTS) in the brainstem [58].
PYY has been characterized in a number of fish species, although
its role is still uncertain. In goldfish, pyy expression increased 3 h
post-feeding (hpf), while it decreased in fasted fish [16], suggesting
that it plays an anorectic role in fish similar to that in mammals.
This study investigated, the effect of feeding a single meal to
Atlantic salmon on changes in the mRNA expression of npy, cart,
pyy, cck (-l and -n), agrp (-1 and -2) and pomc (-a, a2, a2s and -b).
The temporal changes in mRNA expression of these key peptide
hormones were assessed by quantitative real-time PCR (q-PCR) in
whole brain of fed fish and unfed control fish within 24 h after a
meal. Here, we use the term ‘‘appetite’’ to cover the sensations related to food desire (hunger) and satiety. Food intake is used to describe the physical behaviour of food uptake. Since changes in
appetite and feeding behavior usually include modulation of gene
expression, our aim was to describe the role of these key
hormones/neuropeptides in regulating appetite.
2. Materials and methods
2.1. Animals and sampling strategy
Atlantic salmon (AquaGen strain), average body weight
44.7 ± 2.1 g, were reared at the Bergen High-Technology Centre
in indoor tanks supplied with a continuous flow of fresh water at
8 °C and under a 12 h:12 h light regime. The fish were kept in
two 500 L tanks at a density of ca. 4.6 kg m3 and were handfed to
satiation at 0900 every morning with a commercial pellet diet
(EWOS Innovation, Bergen; see Supplemental Table 1 for composition). The fish were acclimated to feeding and tank conditions for
2 weeks prior to sampling, in order to reduce stress. No differences
were observed between the tanks with regards to behavior and
feeding activity. The fed fish were fed to satiation prior to sampling; hence the ad lib meal size should not have been affected
by stress. Further, any stress effects caused by repeated sampling
in the same tank would be identical in the control tank and the test
tank.
On the day of sampling, three fish were sampled by dipnet at
selected times after termination of feeding [0 (prior to feeding–
unfed group only), 0.5, 1.5, 3, 6, 9, 12 and 24 h post-feeding, hpf].
The sampling of only unfed fish prior to feeding (time 0) was done
to assess gene expression prior to normal feeding time, reducing
the disturbance and potential stress effects on normal feeding
behavior and gene expression of the fed fish group. In the current
study we use short-term effects to cover effects observed immediately following a meal (within hours), while long-term effects is
used at the end of the digestive process and when food is fully digested and no longer present in the gastrointestinal (GI) tract. The
transit of food through the GI tract is supported by our observations of stomach filling/emptying and content of food and chyme
in respective segments of gut in a pilot study (see below). The sampling times were based on pilot data of gut transit rate in similarly
sized fish. For practical reasons, unfed (control) and fed fish were
sampled on two consecutive days. Control fish were sampled at
the same time of day as the fed fish, hence time 0 corresponds to
the scheduled feeding time for this group (24 h since last meal)
and time 24 corresponds to 48 hpf. The control group had thus
fasted for 48 h at the time of the final sampling.
At sampling fish were anaesthetized with 50 mg L 1 MS-222
and killed. The whole brain was dissected out, flash-frozen in liquid nitrogen, and stored at 80 °C.
In order to better understand the role of the GI tract in providing satiety (and possibly hunger) signals related to stomach and
gut filling and gut transit, the tract, including the gallbladder,
R. Valen et al. / General and Comparative Endocrinology 171 (2011) 359–366
was removed from the same fish and analysed for content. The
tract was dissected out and carefully cut into four segments without loss of content; stomach, pyloric cecae (PC), midgut (MG) and
hindgut (HG). These segments were then individually emptied of
food and chyme by gently stroking the content out onto preweighed pieces of aluminium foil. The contents of each segment
were then determined on a wet weight basis.
2.2. RNA extraction, cDNA synthesis and q-PCR
Total RNA was isolated from whole brain using TRI ReagentÒ
(Sigma, USA) for phenol–chloroform extraction according to [8].
RNA concentration was estimated using NanoDrop ND-1000 absorbance technology (Thermo Scientific, USA). The 260/280 and 260/
230 absorbance ratios were used as indicators of sample quality
in terms of sample purity. RNA integrity was estimated using Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) in conjunction with RNA 6000 Nano and RNA 6000 Pico LabChip kits
(Agilent Technologies). All procedures followed the manufacturers’
guidelines. The RNA was found to be intact. Isolated total RNA was
DNase-treated using TURBO DNA-free Kit™ (Ambion, TX, USA) following the manufacturer’s instructions for routine DNase treatment. First-strand cDNA was synthesized using the reverse
transcription kit Superscript III First-Strand Synthesis System
(Invitrogen, CA, USA) on 5 lg total RNA template. Oligo(dT) (Sigma,
MO, USA) was chosen as primer. A negative control of pooled RNA
from all samples and no reverse transcriptase was included. No signal was observed in the negative control qPCR. The temporal
mRNA expression of NPY (GenBank Acc. No.: AB455539), CART
(GenBank Acc. No.: AB455538), AgRP-1 and AgRP-2 (GenBank
Acc. No.: AB455536 and AB455537, respectively), POMC-B (GenBank Acc. No.: DQ508935), POMC-A1/-A2/A2s (GenBank Acc. No.:
AB462418, AB462419 and AB462420, respectively), PYY (GenBank
Acc. No.: AB443435) and CCK-L and CCK-N (GenBank Acc. No.:
AB443433 and AB443434, respectively) in brain was analyzed by
q-PCR using Power SYBRÒ Green PCR Master Mix (Applied Biosystems, CA, USA) and Chromo4 Continuous Fluorescence Detector
(Bio-Rad, USA). The primer sets used in each assay, except for the
POMCs, have already been described by us [34,35], and more information about the primers is provided in Supplemental Table 2. PCR
parameters for all assays, except POMC-B were as follows; a first
degradation at 94 °C for 3 min then 40 cycles at 94 °C for 30 s,
60 °C for 30 s and 72 °C for 30 s. POMC-B had an annealing temperature of 62 °C. A melting curve was performed for each assay, in order to verify the absence of primer dimers (60–95 °C read every
0.2 °C and held for 1 s). The melting curve analysis showed a single
peak for each assay, confirming PCR specificity (data not shown).
Atlantic salmon elongation factor 1a (EF1a) was amplified as an
internal standard reference gene. Cycle threshold was set manually
to 0.010, which was within the exponential phase and above background noise for all assays. Q-PCR data was analyzed in MJ Opticom 3.2.32 (Bio-Rad) software. The PCR efficiency (%) of the
standards was for ef1a: 86, npy: 92, cart: 91, pyy: 89, cck-l: 84,
cck-n: 91, agrp-1: 97, agrp-2: 96, pomc-a1: 98, pomc-a2: 92,
pomc-a2s: 96 and pomc-b: 94, with R2 > 0.99 for all standard
curves. Copy numbers of mRNA for all samples were normalized
against Atlantic salmon eEF1a by dividing sample copy number
on ef1a copy number (target gene/reference gene).
361
mality, all data were transformed to logarithms. A main effect ANOVA was used to test significant effect of treatment (unfed, fed
fish) and time. One-way ANOVA was performed in order to identify changes caused by time alone within each treatment group,
and an independent between-variable t-test was performed to
determine significant differences between treatment groups at
each time point. The null hypothesis was rejected at significance
level a < 0.05, and a Tukey HSD post hoc test was then used to follow up.
3. Results
The compartmental gut filling data showed that 30 min after
termination of the meal, most of the ingested food (ca 87% estimated from wet weight analysis) was still in the stomach, with
only a small fraction transferred to the PC and MG segments
(Fig. 1). Subsequently, there was a rapid emptying of stomach content and by 4.5 hpf, about 50% had been transferred from the stomach. Most of the stomach content (about 83%) was gone by 9–
12 hpf, and by 24 hpf the stomach was empty. The unfed fish had
an empty stomach throughout the experiment, as confirmed by visual inspection and content weight (Fig. 1). The content of the PC
compartment in fed fish (Fig. 1) displayed a more gradual rise,
lacking a distinct peak but showed a falling trend from 9 to
24 hpf, by when it was empty. The MG and HG compartments in
fed fish displayed a weak gradual increase in chyme content until
12 hpf, followed by a gradual decline and minimal content at
24 hpf. In unfed fish, all the gut compartments had minimal content within the time of the experiment (Fig. 1).
The results of the q-PCR assessment showed that feeding affected npy expression in the brain (Fig. 2); there was a significant
effect of fish group (fed/unfed) (p < 0.001), but no effects related
to time after feeding. Of the individual sampling points npy was
significantly increased in fed fish 0.5 and 9 hpf (p < 0.05). The cart
mRNA was higher in fed fish 1.5 hpf (p < 0.001) than in unfed control fish (Fig. 2). However, there was no effect of fish group or time
on cart expression.
There were no differences in pyy between fish groups at any
time point. However, a trend in the direction of a decrease in pyy
expression at 24 hpf was observed (p = 0.09) (Fig. 2). cck-l had an
increased expression in fed fish at all sampling times (p < 0.05), except at 24 hpf (Fig. 2). Both fish group and time (p < 0.001) had an
effect on expression. An effect of time was detected in fed fish
(p < 0.01). There was a lower cck-n expression in fed fish at
24 hpf than in unfed controls (p < 0.05) (Fig. 2). An effect of time
on cck-n was detected in fed fish, in which cck-n expression at
24 hpf was lower than at 1.5, 3, 6 and 12 hpf.
pomc-a1 showed increased expression at 3 hpf (p < 0.01), while
pomc-b expression was higher at 0.5 and 6 hpf (Fig. 2). The expression of pomc-a1 in fed fish was found to be affected by time between 3 and 24 hpf. For pomc-a2 and pomc-a2s, no effect of
feeding or time on expression was detected (Fig. 2). For agrp-1
expression in fed fish was higher at 3 hpf than in controls, while
agrp-2 was more highly expressed at 1.5 hpf in fed fish (p < 0.05)
(Fig. 2). An effect of fish group was only detected for agrp-2
(p < 0.001). No effect of time on expression of any of the agrp isoforms was detected.
2.3. Statistics
4. Discussion
All statistical analysis was performed in Statistica 8.0 (StatSoft
Inc., USA). Both unfed and fed fish groups were tested for normality within all time groups using the Shapiro–Wilk W-test. Homogeneity of variance was tested on the same groups with Levene’s
F-test. To compensate for variance heterogeneity and lack of nor-
Gastric emptying was relatively rapid and some 50% of the
stomach content had been transferred into the proximal gut at
4.5 hpf, about 85% at 9–12 hpf, and at 24 hpf the stomach was
empty (Fig. 1). Previous studies in rainbow trout showed that emptying 80–90% of stomach content produces a return in appetite
R. Valen et al. / General and Comparative Endocrinology 171 (2011) 359–366
Compartment filling (g)
362
2.5
(-1 and -2) and POMC (-A1 and -B) are involved in central pathways that regulate appetite in Atlantic salmon.
Fed fish
Unfed fish
2.0
4.1. npy
The current data show an increase in npy expression in fed fish
compared to unfed control fish, indicating that food intake has an
effect on expression levels. However, the increase in npy mRNA at
1.5 and 9 hpf, suggests the existence of a response pattern opposite to that observed in mammals and goldfish [39,49]. At the
same time, most of the stomach content had been emptied by
9 hpf, which might indicate a return of appetite. The increase in
npy expression at this time may thus be an effect of orexigenic
stimuli from the GI tract. The cause of increased npy at 1.5 hpf,
however, is not known. The expression of npy was not higher
prior to the scheduled feeding time, as might have been expected
if NPY plays an orexigenic role related to the anticipation of a
meal. In goldfish, npy expression in the brain increases 1–3 h
ahead of feeding, and falls 1–3 h after feeding [39]. These authors
showed that different brain regions display variations in patterns
of npy expression, indicating regional specificity. Furthermore, it
has been shown that an increase in npy mRNA in goldfish that
had been food-deprived for 72 h is reversed by feeding [38]. All
in all, studies in goldfish suggest that npy has an orexigenic function in this species [11,31,39]. The results of our study are in line
with those describing periprandial changes in juvenile Atlantic
cod, in which the expression of npy increases concurrently with
the onset of feeding (not prior to) and did not change 2 hpf
[21]. In a study of Atlantic salmon, brain npy expression was not
affected by 6 days of fasting [35], which suggests that most of
the effects on central npy take place within 24 hpf. In the present
study, the whole brain was used in gene expression analysis, an
approach that does not take into account potential region-specific
responses in npy expression. However, taken together, the results
show a clear effect of feeding on npy, suggesting that npy is involved in appetite regulation.
1.5
1.0
Stomach
0.5
0.0
Pyloric cecae
Compartment filling (g)
0.5
0.0
Midgut
0.5
0.0
Hindgut
0.5
0.0
0.2
4.2. cart
0.1
Gallbladder
0.0
00
6
6
12
18
24
Time (hpf)
Fig. 1. GI tract compartment contents during processing of a single meal. Content of
feed and chyme was removed from dissected compartments. Data are represented
as mean weight (g) (n = 6) ± SEM at selected time points after feeding (fed fish), and
after scheduled feeding time (unfed fish). Data points for different fish groups are
shifted relative to each other.
[17,20]. If this also holds true for Atlantic salmon this could mean
that potential orexigenic signals may arise in the GI tract by 9 hpf
or even earlier, when most of the stomach content is evacuated,
while anorexigenic (satiety) signals are generated during the initial
phase after feeding, when the stomach is still more or less full. The
gene expression data show that the brain neuropeptides npy, cart,
(pyy), cck (-l and -n), agrp (-1 and -2) and pomc (-a1 and -b) change
during the postprandial phases after feeding a single meal, which
indicates that the respective genes/products are regulated according to the presence of food/chyme within the GI-tract (Fig. 2). Our
findings thus suggest that NPY, CART, (PYY), CCK (-L and -N), AgRP
There was a significant increase in brain cart mRNA 1.5 hpf,
indicating that expression of this neuropeptide is modulated after
a single meal, and that it thus may be a regulator of appetite. The
mammalian model describes cart as an appetite-inhibitory/anorexigenic factor [24]. However, the role of cart in fish has only recently
started to be explored and our current knowledge is limited to a
few species. In goldfish, cart mRNA have been shown to increase
2 hpf [62], which is a response pattern similar to that found in
our study. In goldfish, ICV injections of human CART have been
shown to decrease food intake, and injections of leptin lead to an
increase in cart mRNA [62], suggesting that the satiety effect of cart
is regulated through leptin. In contrast, cart expression in Atlantic
cod decreases 2 hpf, and remains low after 7 days of food deprivation [21], showing a somewhat different short-term effect. A study
using Atlantic salmon showed that cart expression fell in fish fasted
for 6 days, which would agree with an anorexigenic role repressed
by longer periods without food [35]. It has recently been shown
that CCK mediates the effect of CART through vagal afferents in rats
[10]. Our (preliminary) data show that cck-n expression in stomach
of salmon in the present study increases by 1.5 hpf, which corresponds to an increase in cart mRNA (Valen et al., unpublished),
which in turn suggests a functional relationship similar to that
found in rats. However, these data are not conclusive. It has been
shown that CART suppresses NPY-induced feeding in goldfish
[63], suggesting that CART has an inhibitory effect on NPY.
Whether this is the case in salmon is still unclear, as npy and cart
do not show opposite patterns of expression. In conclusion, our re-
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R. Valen et al. / General and Comparative Endocrinology 171 (2011) 359–366
np y
Normalized mRNA expression
0.17
0.90
0.15
0.64
6
0.75
Normalized mRNA expression
Normalized mRNA expression
0.11
0.60
0
3
0.13
*
*
6
9
12
2
24
0
3
6
9
12
0
24
3
*
*
6
9
12
2
24
12
2
24
12
2
24
*
0.12
0.13
*
0.10
0.12
0.12
*
*
0.08
0.11
0.11
cck-l
cck-n
0
3
6
9
12
2
24
0.00
0
3
6
9
12
24
0.12
0.12
0
3
6
9
0.12
pomc-a2s
0.10
pomc-a1
0.06
0.10
0.10
No malized mRNA expression
0.13
0.60
0.56
5
pomc-b
0.10
0.08
0.08
*
*
0.08
0.04
pomc-a2
0.06
0.06
0.00
0.00
0
3
6
9
12
2
24
0.00
0
3
Time (h)
6
9
12
24
Time (h)
0.14
Normalized mRNA expression
pyy
0.19
*
*
0.68
6
cart
*
1.05
0.72
7
0.14
0
3
6
9
Time (h)
*
*
0.12
0.12
Unfed fish
Fed fish
0.10
0.10
agrp-2
agrp
r -1
0.08
0.08
0
3
6
9
12
2
24
0
3
Time (h)
6
9
12
24
Time (h)
Fig. 2. Postprandial mRNA expression of selected neuropeptides in Atlantic salmon brain. Data are presented as mean (n = 3) calculated mRNA copy number ± SEM
normalized against ef1a copy numbers. Mean values with an asterisk above show significant differences between fish groups at a given time point (p < 0.05). In order to
prevent sampling-related stress effects on feed ingestion, pre-feeding (time 0) was only analyzed in the unfed (control) group.
sults, in combination with those of an earlier study in Atlantic salmon [35], indicate that cart is involved in both short-term (postprandial) and long-term (days) regulation of feed intake,
probably as an anorexigenic factor.
4.3. agrp-1 and -2
Our data indicate a postprandial effect on the expression of both
isoforms of agrp. While agrp-1 levels increase at 3 hpf, those of
agrp-2 rise by 1.5 hpf, suggesting different expression patterns
which may indicate that the two isoforms related to feeding have
different functions. Murashita et al. [35] have recently shown that
Atlantic salmon fasted for 6 days displayed a lower expression of
agrp-1, while no change in agrp-2 was observed. These data may
support the notion of different roles for agrp-1 and agrp-2 in the
regulation of both short-term and long-term appetite. In mammals,
the orexigenic effect of AgRP is mediated by antagonizing the effect
of a-MSH at the MC-4 receptor [40,49]. In goldfish, the expression
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R. Valen et al. / General and Comparative Endocrinology 171 (2011) 359–366
of agrp is increased in fish fasted for 3–7 days [5], and MC-4 receptors was shown to control feed intake in this species [6]. A recent
study in seabass showed that AgRP antagonizes the effect of the
MC-1 receptor in vitro [46]. All in all, the previously reported effects of AgRP in fish suggest that this neuropeptide is involved in
controlling feed intake, possibly as an endogenous antagonist of
MC receptors. The opposite expression patterns of agrp isoforms
in Atlantic salmon, compared to the orexigenic role described in
mammals, highlight the complexity of appetite regulation in fish,
and may be an indication of one of many strategies used by fish
to control energy homeostasis.
4.4. pomc-a1/a2/a2s/b
The expression of two of four isoforms of pomc was affected by
feeding. The pomc-a1 expression increased 3 hpf, while pomc-b increased 0.5 and 6 hpf, indicating that these isoforms play a role in
the short-term control of feed intake. A pilot study on Atlantic salmon showed that pomc-a1 mRNA fell after 6 days of fasting, while
no significant changes were detected in the other pomc isoforms
(Murashita et al., unpublished; Supplemental Fig. 2). In barfin
flounder and rainbow trout, the expression of pomc-b-like transcripts was found to be unaffected by feeding, which collectively
led to the assumption that pomc-b had lost its function in energy
homeostasis [28,37,55]. However, the current expression data together with data that show that pomc-b is the quantitatively most
important variant in the brain and pituitary of Atlantic salmon
(Murashita et al., unpublished; Supplemental Fig. 1), suggest that
pomc-b is the main POMC variant involved in feeding regulation.
As one of the main products of the pomc gene is a-MSH, it is possible that the ratio of agonist (a-MSH) to antagonist (AgRP) at the
MC-receptors determines the effect on feeding in fish, as is suggested at the MC-4 receptor in mammals [1,14]. In our study, both
pomc-a1 and agrp-1 increase in the brain at 3 hpf, which indicates
an effect within the same time period. Whether this effect is functionally connected through the MC receptor is not yet known.
4.5. pyy
These data do not show a postprandial effect on pyy mRNA
expression during the first 12 hpf, suggesting a minimal short term
effect of feeding on pyy expression. However, there is a trend in the
direction of a difference between the fish groups in pyy expression
at 24 hpf, which might indicate an effect after the meal is processed by the GI tract. Analysis of pyy expression in the GI tract
in the same fish showed a specific decrease in hindgut at 24 hpf
Valen et al., unpublished), which supports the notion of a similar
effect of feeding on pyy in the brain. However, in contrast to data
derived from analyses of brain tissue, short-term (within 3 hpf)
changes in pyy expression were detected in the GI tract (Valen
et al., unpublished). Collectively, these data suggest that pyy responds differently to feed intake, depending on peripheral or central expression. A previous study of pyy in Atlantic salmon did not
show any changes in mRNA expression in either the GI tract or the
brain after 6 days of fasting [36], which suggests that pyy, is mainly
affected in the short term after feeding. The current results thus
contrast with a previous study in goldfish, where pyy expression
in the brain increased at 3 hpf [16]. The same study showed that
both IP and ICV injections of goldfish PYY reduced food intake,
demonstrating an anorectic function of PYY. The differences between Atlantic salmon and goldfish pyy expression are probably
due in part to the differences in these species’ ability to withstand
food deprivation, as Atlantic salmon can tolerate longer periods
without food than goldfish. Anatomical differences, including the
lack of a stomach in goldfish and the consequent physiological differences in meal transport and processing, may also underlie the
differences in the data obtained. However, in spite of the species
differences, the trend to a fall in pyy at 24 hpf suggests that PYY
has an anorectic function in salmon similar to goldfish.
4.6. cck-l and -n
The differences in brain mRNA expression between the two cck
isoforms in response to feeding in Atlantic salmon indicate that
each plays a specific role in feeding regulation. CCK has previously
been shown to decrease appetite in goldfish, and cck expression in
the goldfish brain increased at 2 hpf [18,42,61]. In our study, cck-l
expression in the brain was higher than in the unfed controls at
0.5, 1.5, 3, 6, 9 and 12 hpf, while cck-n expression was reduced,
but only at 24 hpf, when the meal had been completely processed
and the stomach was empty. This suggests that cck-l, in contrast to
cck-n, plays its role during the initial phase of feed ingestion, possibly as an anorexigenic factor in the brain. The data further indicate that cck-l is the main isoform involved in feeding regulation,
while the role of cck-n is less central. An earlier study did not find
any effect of 6 days of fasting on any of the cck isoforms in salmon
GI tract or brain [36], suggesting that cck acts within a short period
of time. All in all, current data indicate that the brain expression of
both isoforms of cck is affected by short-term nutrient status,
which suggest anorexigenic roles related to when food is present
in the GI tract.
In conclusion, the brain neuropeptides npy, cart, cck (-l and -n),
agrp (-1 and -2), (pyy) and pomc (-a1 and -b) have been shown to
undergo changes in mRNA expression during processing of a meal,
suggesting that they play central roles in the regulation of food intake and appetite in Atlantic salmon. While the expression of npy
and agrp (-1 and -2) did not show a clear pattern typical of the
orexigenic factors described in higher vertebrates, the patterns of
expression of cart, cck-l (-n), pomc-a1/b (and pyy) indicate that
these neuropeptides mediate satiety signals.
Acknowledgments
We thank Dr. Pedro Gómes-Requini, Dr. Anne-Grethe Gamst
Moen and Tharmini Kalananthan (BIO, UiB) for their assistance
during the experiments. This study was supported by Research
Council of Norway (RCN) Grant #172548/S40 (IR), UiB and HelseVest grant (IR) and Research Fellowships from RCN and JSPS for
Young Scientists to KM. The study also received funding from the
European Community’s Seventh Framework Programme (FP7/
2007-2013) under Grant Agreement No. 222719 – LIFECYCLE (IR).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ygcen.2011.02.027.
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