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Am J Physiol Regul Integr Comp Physiol 279: R1025-R1034, 2000;
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Vol. 279, Issue 3, R1025-R1034, September 2000

Regulation of food intake by neuropeptide Y in goldfish

Yuwaraj K. Narnaware1, Pierre P. Peyon2, Xinwei Lin1, and Richard E. Peter1

1 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9; and 2 Laboratory de Physiologie Generale et Comparee Museum National d'Histoire Naturalle, Unité de Recherche Associée 90 Centre National de la Recherche Scientifique, 75005 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In mammals, neuropeptide Y (NPY) is a potent orexigenic factor. In the present study, third brain ventricle (intracerebroventricular) injection of goldfish NPY (gNPY) caused a dose-dependent increase in food intake in goldfish, and intracerebroventricular administration of NPY Y1-receptor antagonist BIBP-3226 decreased food intake; the actions of gNPY were blocked by simultaneous injection of BIBP-3226. Goldfish maintained on a daily scheduled feeding regimen display an increase in NPY mRNA levels in the telencephalon-preoptic area and hypothalamus shortly before feeding; however, a decrease occured in optic tectum-thalamus. In both fed and unfed fish, brain NPY mRNA levels decreased after scheduled feeding. Restriction in daily food ration intake for 1 wk or food deprivation for 72 h resulted in increased brain NPY mRNA levels. Results from these studies demonstrate that NPY is a physiological brain signal involved in feeding behavior in goldfish, mediating its effects, at least in part, through Y1-like receptors in the brain.

feeding; gene expression; growth hormone; brain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN MAMMALS, THE REGULATION of appetite and food intake have been demonstrated to be an extremely complex process involving both orexigenic and anorexigenic neuronal systems that are morphologically and functionally connected in the hypothalamus (16). Among this network, neuropeptide Y (NPY) is the most potent orexigenic signal molecule (18, 28, 29, 31) as the central injection of NPY either in the cerebroventricular system or directly into various hypothalamic nuclei stimulates feeding behavior, food intake, and body weight gain (7, 17, 29, 31). The effects of NPY in stimulating food intake are mediated by NPY Y1- and Y5-receptor subtypes (10, 28). On the other hand, hypothalamic NPY is regulated by the feeding state in rats. Food deprivation (2, 3) or diet restriction (3) induces an increase in NPY concentration in hypothalamus (9, 14, 17) and NPY mRNA gene expression (3, 16) in various brain nuclei, and refeeding normalizes both NPY concentrations and NPY mRNA levels (14).

The presence of NPY in fish was first demonstrated in goldfish by immunological and chromatographic studies (15). In goldfish, NPY immunoreactive neurons are present in the ventromedial posterior hypothalamus and hypothalamic inferior lobes (27), which have been demonstrated to be involved in the organization and control of feeding behavior in fish (for review, see Ref. 23). Localization of NPY immunoreactive neurons in the telencephalon-preoptic area (POA), hypothalamus, and optic tectum-thalamus region has also been reported in several fish species (8, 15, 27). NPY-binding sites are also localized in the "hypothalamic feeding area" (HFA; B. A. Himick, S. R. Vigna, and R. E. Peter, unpublished results). The sequence of goldfish NPY cDNA has been determined and shows strong evolutionary conservation among vertebrate species (19). The goldfish NPY (gNPY) has only five residues difference from rat NPY (19). In situ hybridization and Northern blot studies have shown that NPY mRNA is mainly expressed in forebrain regions of goldfish, particularly in the nucleus entopeduncularis of the ventral telencephalon, POA, olfactory bulbs, and various thalamic regions. In midbrain of goldfish, NPY mRNA is present in the optic tectum and locus ceruleus (22). A similar brain distribution of NPY mRNA has been described in coho salmon (30). In addition, only the POA of the hypothalamus demonstrated an increase in NPY gene expression with extended fasting in coho salmon (30)

In a recent study, brain intracerbroventricular injection of porcine NPY was demonstrated to stimulate food intake in goldfish (20). However, the authors failed to demonstrate dose-dependent action of NPY, and an extraordinarily high dosage of NPY was used to find a significant effect (20). In goldfish, NPY stimulates pituitary growth hormone (GH) secretion in vitro and in vivo (24). NPY also stimulates release of gonadotropin-releasing hormone (GnRH) in goldfish; GnRH acts as GH-stimulating factor in goldfish and several other fish species (24). In the present study, we investigated the direct actions of NPY on feeding behavior in goldfish. We also examined the possible changes in NPY gene expression at the time of feeding, the effects of diet restriction and food deprivation on NPY gene expression, and the correlation between NPY gene expression and serum GH levels. Our results provide evidence for orexigenic actions of NPY in goldfish.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Male and female goldfish (Carassius auratus) of the common or comet varieties, body weight range 25-45 g, were purchased from Mount Parnell Fisheries (Mercersburg, PA). The fish were maintained in 1,800-l flow-through aquariums at 17°C under a simulated natural photoperiod of Edmonton, Alberta, Canada. Fish were fed a normal ration at 2% body weight of commercially prepared fish pellets (pellet size 5.0 mm; United Feeds, Calgary, Alberta, Canada). All fish were acclimatized to aquarium conditions for 10 days and fed at a regularly scheduled time of the day.

Northern Blot Analysis

DNA probes. To generate a DNA probe for specific detection and measurement of NPY mRNA in goldfish brain, PCR was performed using two primers corresponding to the untranslated regions of the gNPY cDNA sequence (19). The sequence of antisense primer is 5'-GCAAGAAGTTCAATCAAGACC and sense primer is 5'-ATCCATCGTGTTGTTGCTGG. PCR conditions are denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a total of 30 cycles. PCR product (365 bp) was separated on 1.5% agarose gel and purified from using GeneClean II Kit (Bio 101, La Jolla, CA). As an internal control, a goldfish 18S rRNA probe was synthesized by PCR as described in previous studies (26). The DNA probes were labeled with [alpha -32P]deoxycytidine 5'-triphosphate (dCTP) using T7QuickPrime Kit (Pharmacia Biotech, Baie d'Urfe, Quebec, Canada).

RNA extraction and Northern blot. Tissues of five discrete goldfish brain areas [olfactory bulbs and tracts, telencephalon (including optic nerve) and preoptic region, hypothalamus, optic tectum and thalamus, and posterior brain (cerebellum and medulla) and the pituitary gland] were freshly excised and homogenized for extraction of total RNA using Trizol RNA isolation reagent (GIBCO, Gaithersburg, MD) based on the acid guanidinium thiocyanate-phenol-chloroform extraction method (5). Total RNA from an individual fish or pooled tissues were fractionated by electrophoresis in a denaturing agarose gel (1.5%) with formaldehyde and blotted onto Nybond nylon membrane (Amersham Life Science, Buckinghamshire, UK) by capillary transfer.

Hybridization was performed using methods described by Church and Gilbert (6). Briefly, the membranes were prehybridized in hybridization solution (0.5 M Na2HPO4, 7% SDS, 1 mM EDTA, and 1% BSA) for at least 1 h. The hybridization solution was then changed, and the [alpha -32P]dCTP-labeled NPY probe was added. After hybridization overnight at 65°C, the membranes were washed three times with washing solution (0.04 M Na2HPO4, 1 mM EDTA, and 1% SDS) and exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) screen for 72 h. Afterward, the membranes were stripped and reprobed with a [alpha -32P]dCTP-labeled probe for 18S rRNA as internal control. This probe for 18S rRNA was synthesized by PCR as described in previous studies (26). The hybridization signals were scanned using PhosphorImager (Molecular Dynamics) and quantified by ImageQuant Software (Molecular Dynamics). The NPY mRNA levels were expressed as a ratio of NPY mRNA to 18S rRNA.

Brain Intracerebroventricular Injection Of NPY and its Antagonist on Feeding Behavior and Food Intake

Male and female goldfish were randomly assigned to 65-l observational aquariums supplied with flow-through water at 17°C (photoperiod 16:8-h light-dark cycle). All test aquariums contained gravel substrate and were covered by opaque barriers to minimize external disturbances. Fish were identified by individual markings and acclimatized under these conditions before the feeding-behavior experiment. Previous studies on goldfish in our laboratory demonstrated that cumulative food intake is similar between male and female goldfish in either gonadal recrudescent (September-April) or mature (April-May) stages of reproduction (11). In the present study, fish were in the late stages of gonadal recrudescence [gonadosomatic index (GSI): weight of gonad/total body weight × 100; female GSI = 7.42 ± 1.50; male GSI = 3.73 ± 0.38].

A stock solution of gNPY and NPY Y1-receptor antagonist BIBP-3226 (Peninsula Laboratories, Belmont, CA) was made in teleost physiological saline, aliquoted, and stored at -20°C. Aliquots were then thawed and diluted in fish physiological saline just before use. Fish were given intracerebroventricular injections of 2 µl of fish physiological saline or gNPY in doses of 0.5, 1, 2, 4, 5, 7, and 8 ng/g body wt. NPY Y1-receptor antagonist BIBP-3226 was injected in doses of 5, 10, 20, 40, and 80 ng/g body wt. To determine whether BIBP-3226 could block the actions of gNPY, fish were either injected intracerebroventricularly with fish saline or the combination of gNPY (doses 0.5, 1, and 2 ng/g) and BIBP-3226 (doses 5, 10, and 20 ng/g), respectively.

Brain intracerebroventricular injections. Brain third ventricle injections of these agents were given using routine procedures outlined in the stereotaxic atlas for the goldfish forebrain (25). Briefly, fish were deeply anesthetized in 0.05% tricane methanesulphonate (TMS, Syndel Laboratories, Vancuover, British Columbia, Canada) weighed to the nearest 0.1 g. With the use of a dentist drill equipped with a circular saw, the roof of the skull was cut along three sides of a square, and the skull bone flap then folded to one side. After the brain was exposed, the injection needle (5-µl microsyringe, Hamilton, Reno, Nevada) was placed stereotaxically in the third brain ventricle (coordinates +1.0 median, down 1.2) in the region of the nucleus preopticus nucleus preopticus periventricularis (25, 32). After injection of 2 µl of test solutions, the needle was withdrawn and the space in the cranial cavity filled with teleost physiological saline. The skull flap was put back in place and secured by surgical thread.

Feeding-behavior observations. Fish were returned to test aquariums immediately after intracerebroventricular injections and normally recovered from anesthesia within 2-5 min. To accurately measure the amount of food consumed by each fish, basal feeding levels were recorded on the day before intracerebroventricular injections for 30 min. Goldfish given a 2% body weight ration normally consumed ~50-75% of the ration within 30 min. On experimental day, fish were unhandled, or intracerebroventricularly injected with saline or treatment solutions. The investigator observing feeding behavior was aware of the treatment of each fish. Fifteen minutes postintracerebroventricular injection, a 4% body weight ration of pellets was added in each tank. In preliminary tests, NPY-injected fish were observed to consume a 3-4% body weight ration in the 30-min observation period, and therefore, a 4% ration size was kept constant throughout the feeding experiments.

Observations of feeding behavior began immediately on entry of the pellets into the tank. As described by Volkoff et al. (32), feeding behavior was defined as "complete" and "incomplete feeding acts." A complete feeding act was when a fish approached a pellet and consumed it. An incomplete feeding act was when fish approached a pellet and touched or bumped it with a closed mouth or when a pellet was taken into the mouth and then spat out. Both complete and incomplete acts were recorded over 30 min after pellet administration (15 min postinjection). Each pellet consumed by an individual fish within each tank was recorded during 30 min. Over the 30-min feeding time, the size of ration was not found to be limiting. Food consumption by an individual fish then was converted to milligrams of food consumed per wet body weight per 30 min, using the average weight of individual pellets as the basis for calculation.

NPY Gene Expression Studies

Effects of daily feeding regimen. Seven experimental groups, each containing 10-12 fish were acclimatized to 65-liter aquariums. All fish were fed a 2% body weight ration daily at a scheduled time (11:00 AM). On experimental days, groups of goldfish were killed 3 or 1 h before the scheduled feeding time or at the scheduled feeding time (0 h). Of the remaining four groups, two were unfed, and two were fed with the normal ration; one fed and one unfed group were killed 1 h after the scheduled feeding time, and remaining two groups (1 fed and 1 unfed) were killed 3 h after the scheduled feeding time.

Effects of restricted diet. Three experimental groups, each containing 12 goldfish, were acclimatized to 65-l aquariums before restricted diet administration. One group was fed with normal 2% body weight ration, and the remaining two groups were fed with 1% and 0.5% body weight rations, respectively, for 7 days. At the end of these treatments, fish were killed and brain tissues collected.

Effects of food deprivation. Four experimental groups, each containing 12 fish, were acclimatized to 65-l aquariums before food deprivation. Control (fed) fish were fed a normal ration at 2% body weight (0 h), and the remaining three groups were food deprived for 24, 48, and 72 h. All four groups were killed at the end of food-deprivation periods for brain tissues.

Measurement of Serum GH Levels

After experimental treatments, fish were anesthetized with 0.05% TMS, and blood samples were collected from the caudal vasculature using a 25-gauge <FR><NU>5</NU><DE>8</DE></FR>-in. needle attached to a 1-ml syringe. After blood was clotted for ~12 h at 4°C, samples were centrifuged and serum was collected and stored at -20°C. Concentrations of serum GH were determined by a specific RIA (21).

Statistical Analysis

Relative NPY mRNA levels, food intake, incomplete feeding acts, and serum GH levels are presented as the means ± SE. Data were subjected to one-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Intracerebroventricular Injection of gNPY and its Antagonist BIBP-3226 on Feeding Behavior and Food Intake

Basal food intake was measured in unhandled fish on day 1 to compare the amount of food consumed by these fish when handled, netted, anesthetized, and injected intracerebroventricularly with saline on day 2 (Figs. 1A, 2A, and 3A). Data presented here, as well as in earlier studies in our laboratory (11, 32), demonstrate that experimental manipulations of goldfish do not alter their subsequent food intake within the time frame used in the present study. Results presented in Fig. 1A showed that intracerebroventricular administration of gNPY caused a dose-dependent stimulation of mean food intake with a significant action occurring at 0.5 ng/g, the lowest dosage tested, and maximal stimulation occurring at 5 ng/g. Notably, at 7 ng/g, intracerebroventricular gNPY did not alter food intake compared with controls; however, at 8 ng/g, food intake was significantly decreased compared with controls (Fig. 1A).


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  		Fig. 1.   Effects of third brain ventricle [intracerebroventricular (ICV)] injection of goldfish neuropeptide Y (gNPY) on mean food intake (A) and the number of incomplete feeding acts (B) in goldfish. Mean food intake was measured in unhandled fish on day 1 (n = 19). On day 2, fish were either intracerebroventricularly injected with saline (n = 19) or gNPY in doses of 0.5, 1, 2, 4, 5, 7, and 8 ng/g (n = 8-10 fish/dose). Food intake and the number of incomplete feeding acts were recorded over 30 min after ICV injection. Data are expressed as mean food intake (mg) per body wt (g) per 30 min. Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.



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  		Fig. 2.   Effect of ICV injection of neuropeptide Y (NPY) Y1-receptor antagonist BIBP-3226 on mean food intake (A) and the number of incomplete feeding acts (B) in goldfish. Mean food intake was measured in unhandled fish on day 1 (n = 10). On day 2, fish were either intracerebroventricularly injected with saline (n = 12) or BIBP-3226 in doses of 5, 10, 20, 40, and 80 ng/g (n = 8-10 fish/dose). Food intake and the number of incomplete feeding acts were recorded over 30 min after ICV injection. Data are expressed as mean food intake (mg) per body wt (g) per 30 min. Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.



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  		Fig. 3.   Combined effects of ICV injection of gNPY and NPY Y1-receptor antagonist on mean food intake (A) and the number of incomplete feeding acts (B) in goldfish. Mean food intake was measured in unhandled fish on day 1 (n = 6). On day 2, fish were either intracerebroventricularly injected with saline (n = 6) or gNPY and BIBP-3226 in doses of 0.5, 1, and 2 ng/g (n = 6/dose) and BIBP-3226 in doses of 5, 10, and 20 ng/g, respectively (n = 6-8 fish/dose). Food intake and the number of incomplete feeding acts were recorded over 30 min after injection. Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.

Intracerebroventricular injection of the NPY Y1-receptor antagonist BIBP-3226 caused a dose-dependent decrease in food intake (Fig. 2A). BIBP-3226 caused a maximal suppression of food intake at 20 and 40 ng/g compared with 5 and 10 ng/g dosages (Fig. 2A). Three doses of gNPY (0.5, 1, and 2 ng/g) and three respective doses of BIBP-3226 at 5, 10, and 20 ng/g were selected to evaluate the combined effects of gNPY and BIBP-3226 on food intake. As shown in Fig. 3A, intracerebroventricular injection of BIBP-3226 significantly suppressed the stimulatory effects of gNPY on food intake.

The number of incomplete feeding acts remained the same in both unhandled and intracerebroventricular saline-injected fish, indicating that experimental manipulation did not alter feeding behavior in control fish (Figs. 1B, 2B, and 3B). Intracerebroventricular gNPY injection, however, significantly lowered the number of incomplete feeding acts at all doses (Fig. 1B). Intracerebroventricular injection of the Y1 antagonist BIBP-3226 at doses of 10, 40, and 80 ng/g also suppressed the number of incomplete feeding acts. At 5 and 20 ng/g BIBP-3226, these acts were similar to unhandled and intracerebroventricular saline-injected fish (Fig. 2B). In fish treated with the combination of gNPY and BIBP-3226, the number of incomplete feeding acts were not significantly different from unhandled and saline-injected groups, except at the combined dose of NPY and BIBP-3226 at 0.5 and 5 ng/g, respectively (Fig. 3B).

NPY Gene Expression in Goldfish Brain

NPY mRNA was detected and quantified in discrete brain regions and pituitary gland of goldfish by Northern blot analysis using specific DNA probes for gNPY mRNA (Fig. 4). NPY mRNA was detected in the telencephalon-preoptic region, hypothalamus, and optic-tectum thalamus, whereas NPY mRNA was undetectable in other brain regions examined and the pituitary. The telencephalon-preoptic region exhibited the highest levels of NPY mRNA, approximately fivefold higher than those in hypothalamus and optic-tectum thalamus (Fig. 4). In subsequent NPY gene expression experiments, NPY mRNA levels were quantified in the telencephalon-preoptic region, hypothalamus, and optic-tectum thalamus.


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  		Fig. 4.   Northern blot analysis of NPY gene expression in discrete brain regions of goldfish brain (A), NPY mRNA levels (B), compared with internal control (18s RNA) (C), and expressed as a normalized NPY gene expression (D). Dissection of the brain regions used for RNA extraction: telencephalon-preoptic area (TEL-POA), optic tectum-thalamus (OT-THAL), hypothalamus (HYP), posterior brain (POST. BRAIN), pituitary (P), and olfactory bulbs (OB).

Effects of Daily Feeding Regimen on NPY Gene Expression and Serum GH Levels

In both telencephalon-preoptic and hypothalamic regions, NPY mRNA levels were significantly lower at 3 h before the scheduled feeding time (-3 h) compared with 0 h (scheduled feeding time), with no significant differences between -1 and 0 h (Fig. 5, A and B). At 1 and 3 h after the scheduled feeding time, NPY mRNA levels in both telencephalon-preoptic and hypothalamic regions were markedly decreased in both fed and unfed fish compared with 0 h. There were no significant differences in NPY mRNA levels between fed and unfed fish at 1 and 3 h after the scheduled feeding time (Fig. 5, A and B). Optic tectum thalamus exhibited a different pattern of mRNA levels. In this region, NPY mRNA levels were significantly higher at -3 compared with 0 h, showing no significant differences between -1 and 0 h. At 1 h after feeding, NPY mRNA levels were not significantly different from 0 h; however, fed fish at 3 h had significantly lower mRNA levels than those fed at 0 h. In unfed fish, there was a significant increase in NPY mRNA levels at 1 compared with 0 h, and then levels were decreased significantly at 3 h compared with 1 and 0 h (Fig. 5C).


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  		Fig. 5.   Effect of a daily scheduled feeding regimen on NPY mRNA levels in TEL-POA (A), HYP (B), OT-THAL (C), and serum GH levels (D) of goldfish. Fish were fed daily at a fixed time of the day (0 h) for 10 days. On the experimental day, groups of fish were killed each at -3, -1, and 0 h. One fed (open bars) and one unfed (hatched bars) group were killed 1 and 3 h after 0 h. Data are expressed as means + SE (n = 12 fish/group). Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.

Serum GH levels increased significantly between -3 (3 h before scheduled feeding time) and -1 h (1 h before scheduled feeding time) and remained high through the scheduled feeding time. At 1 and 3 h after the scheduled feeding time, serum GH levels significantly decreased in both fed and unfed fish to levels similar to those found in fish at -3 h (Fig. 5D).

Effects of Diet Restriction and Food Deprivation on NPY Gene Expression and Serum GH Levels

Telencephalon-preoptic region and optic-tectum thalamus exhibited a significant increase in NPY mRNA levels when ration size was restricted from 2% body weight (control) to 1% and 0.5% body weight/day for 7 days (Fig. 6, A and C). In hypothalamus, NPY mRNA levels were significantly increased in fish fed 1% body weight ration, but levels remained the same as fed controls in fish fed the 0.5% body weight ration (Fig. 6B). Restriction in ration size also influenced the serum GH levels. Compared with fed fish, GH levels were significantly increased by 1.5- to 2-fold when ration size was reduced (Fig. 6D).


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  		Fig. 6.   Effects of restriction in diet on TEL-POA (A), HYP (B), OT-THAL (C) NPY gene expression, and serum GH levels (D) in goldfish. One group of fish was daily fed a normal 2% body weight ration, and the other 2 groups were provided with 1% and 0.5% rations for 7 days. All groups were killed at the end of these treatments. Data are expressed as means + SE (n = 12 fish/group). Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.

Compared with the fish that were fed regularly (0 h), food deprivation for 24 and 48 h did not significantly alter NPY mRNA levels either in telencephalon-preoptic region or optic tectum thalamus. However, 72 h food deprivation resulted in a significant and approximately twofold increase in NPY mRNA levels in these brain regions (Fig. 7, A and C). In hypothalamus, however, 24 and 48 h food deprivation caused a significant increase in NPY mRNA levels, which was further increased in the 72-h food-deprived group (Fig. 7B). Serum GH levels were similar in both fed and 24-h food-deprived fish but were significantly increased after 48 and 72 h food deprivation (Fig. 7D).


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  		Fig. 7.   Effects of food deprivation on NPY gene expression in TEL-POA (A), HYP (B), OT-THAL (C), and serum GH levels (D) in goldfish. Fish were either fed regularly at 2% body weight ration or food deprived for 24, 48, and 72 h and killed at the end of food-deprivation periods. Data are expressed as means + SE (n = 12 fish/group). Significant differences (P < 0.05) between different groups are compared by using 1-way ANOVA followed by Student-Newman-Keuls multiple-comparisons test. Letters not joined by underlining indicate groups that differ significantly.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, NPY gene expression in goldfish brain was examined by Northern blot analysis. The results showed that the highest level of NPY mRNA is in telencephalon-preoptic region, with comparatively less expression in hypothalamus and optic tectum thalamus. Other brain regions and the pituitary were under detection levels. Our previous studies using in situ hybridization demonstrated NPY mRNA in hypothalamic areas as well as in the telencephalon and optic tectum thalamus (22). Similarly, strong NPY signals have been detected in telencephalon in two salmonid species (30). Earlier electrical stimulation and brain-lesioning studies demonstrated that the hypothalamic inferior lobes and ventromedial area are involved in feeding behavior in fishes (23). The demonstration of the presence of NPY by immunohistochemistry (see introduction) and NPY mRNA expression in telencephalon-preoptic region and hypothalamus (22, 30, and present study) provides an anatomical basis for involvement of NPY in the regulation of feeding behavior.

Brain intracerebroventricular injection of the native gNPY stimulates food intake in a dose-dependent fashion, demonstrating that NPY is a potent orexigenic factor in the goldfish. Intracerebroventricular injection of gNPY at 0.5 ng/g body wt caused a significant increase in food intake and was increased approximately twofold higher at 5 ng/g. However, higher doses were ineffective in stimulating food intake, with a dosage of 8 ng/g suppressing food intake compared with controls. The decrease in food intake at high doses of gNPY suggests that desensitization of NPY receptor(s) may have occurred so that NPY was no longer effective in stimulating food consumption. A rapid desensitization of brain neuropeptide receptors has been reported previously in both fish and mammals. Administration of intracerebroventricular injection of orexin A in low doses of 1 and 10 ng/g increased feeding in goldfish; however, no significant effects on food intake were observed at the high dose of 100 ng/g (32). In rats, intrahypothalamic injection of NPY at 250 ng stimulated feeding; however, a dosage of 500 ng decreased feeding (4).

In mammals, central action of NPY on food intake is mediated through both Y1 (10, 13, 28) and Y5 receptors (10). NPY Y1-receptor antagonists BIBP-3226 and 1229U91 blocked the NPY-induced food intake in the rat (10, 28). Similarly, the Y5-receptor antagonists CGP 71683 or Y5 antisense oligodeoxynucleotide (10) suppressed both NPY- and fast-induced feeding, indicating that NPY acts centrally through both Y1 and Y5 receptors. In addition, Y1-receptor mRNA has been shown to be upregulated in conjunction with increased appetite in fasted rats and experimentally produced hypherphagic rats (18), indicating the involvement of both Y1- and Y5-receptor subtypes in NPY-induced feeding. These findings are consistent with some (10, 13, 28) but not others. For example, recent evidence indicates that the Y1 antagonist GR 231118 (12291U91) is ineffective in blocking NPY-induced feeding, and BIBP-3226 and its opposite enantiomer BIBP-3435, which is ineffective on Y1 receptors, were both effective in blocking NPY-induced feeding in mice, questioning the specificity and the role of Y1 receptors in feeding behavior. In mammals, therefore, it is possible that NPY-induced feeding may be mediated either by a combination of more than one receptor or by a novel receptor, as suggested by Gehlert (10). New selective NPY receptor antagonistics may be needed to help clarify the role of Y1 receptors in feeding behavior in goldfish.

As far as NPY-elicited feeding through NPY Y1-receptor subtypes in fish is concerned, recently, three distinct NPY-like receptors, closely resembling mammalian Y1 receptors, have been identified by molecular cloning and shown to be distributed in the brain of Atlantic cod, zebrafish, and dogfish (1) indicating the presence of NPY-like receptors in fish. In the present study, brain intracerebroventricular injection of the Y1 antagonist BIBP-3226 caused a dose-dependent suppression of food intake in goldfish. Moreover, with co-intracerebroventricular injection of BIBP-3226 and gNPY, the stimulatory actions of gNPY on food intake were abolished. Similarly, a recent study in goldfish (20) showed that intraventricular injection of the Y1-receptor antagonist NPY-(27-36) also suppressed NPY-elicited feeding (20). These results suggest that the actions of NPY in stimulation of food intake in goldfish are mediated, at least in part, through NPY Y1-like receptors in goldfish brain.

In the present study, brain intracerebroventricular injection of gNPY also influenced feeding behavior in addition to its stimulatory actions on food intake. Interestingly, the number of incomplete feeding acts were decreased by gNPY as well as by the Y1-receptor antagonist. However, combined treatment with NPY and BIBP-3226 at the two sets of high dosages used did not significantly change the number of incomplete feeding acts compared with unhandled and intracerebroventricular saline-injected fish. An interpretation of these seemingly conflicting results is that less time is spent performing incomplete feeding acts in the situation in which gNPY stimulates food intake. On the other hand, under the influence of BIBP-3226, both food intake and incomplete feeding acts are suppressed. In mammals, studies on the actions of NPY on feeding behavior are mainly focused on food intake, and limited information is available on feeding behavior (7). Available evidence shows that NPY-injected rats spend more time eating and drinking (12). In a recent study, Ida et al. (12) demonstrated that intracerebroventricular injection of NPY caused an increase in searching acts but decreased face washing, grooming, burrowing, and smelling. Importantly, these authors reported that within 40 min of brain injection of NPY, rats performed the same number of searching acts as saline-injected controls, whereas food consumption in these rats was significantly higher within 10 min after injection, implying that the rats spent more time feeding and less time searching. In our present study, it was noticed that immediately after presentation of the food pellets, gNPY-treated goldfish approached the pellets and spent most of the time in pellet consumption, and, as result, the number of incomplete feeding acts were decreased. Nevertheless, if the number of complete and incomplete feeding acts is totaled, the total amount of feeding behavior is significantly increased over controls at all dosages except for the highest dosage of gNPY, which caused a suppression of food intake. On this basis, we conclude that NPY also stimulates feeding behavior as well as actual food intake.

The involvement of NPY in the regulation of food intake in goldfish was also supported by the correlation between daily feeding pattern and NPY gene expression in brain. Goldfish maintained on a daily scheduled feeding regimen display rapid and sequential changes in NPY mRNA levels in the telencephalon-preoptic region, hypothalamus, and optic tectum thalamus 1-3 h before the scheduled feeding time, and then levels decrease by 40-50% at 1 and 3 h after the scheduled feeding regimen. The increase in NPY mRNA levels in the telencephalon-preoptic region and hypothalamus before the scheduled feeding regimen suggests that the sensation of hunger or appetite of fish was increased at some time before their regular feeding time and then gradually subsides with the availability of food. Similarly, Kalra et al. (17) reported that rats trained to eat daily exhibited a significant increase in NPY levels in the paraventricular nucleus (PVN) before mealtime and then gradually declined to fed levels during the course of feeding, strongly supporting that NPY is involved in the regulation of daily feeding behavior in mammals (9, 16, 17). Taken together, the present results suggest that NPY plays a key role in regulation of food intake in goldfish on a multi-site-specific brain mechanism, similar to the observations reported in mammals (9, 16, 17).

The involvement of NPY in the regulation of food intake in goldfish was further investigated by examining the effects of diet restriction and food deprivation on NPY gene expression in goldfish brain. In the present study, reduction in ration size by 50-75% for 7 days resulted in a significant increase in NPY mRNA levels in all three brain regions examined. This suggests that nutritional imbalance and/or peripheral metabolic state of goldfish can activate NPY synthesis in brain similar to observations in mammals (2, 3). In rats, food restriction not only increased NPY content in the PVN and other hypothalamic nuclei (2, 9, 14) but also enhanced NPY mRNA levels in the arcuate nucleus (3). However, other physiological factors associated with altered food conditions may also contribute to the increased NPY mRNA levels both in fish as well as mammals. Severe disturbances in daily food intake in salmonids have been shown to suppress plasma insulin and glucose, which are associated with an increase in NPY gene expression (30). In the present study, food deprivation resulted in a significant, time-related, and brain site-specific increase in NPY mRNA levels in brain. An earlier study in salmon also demonstrated that a long period of food deprivation (3 wk) increased NPY gene expression in the brain POA (30). Taken together, these results indicate that food deprivation can activate NPY gene expression and possible protein synthesis in goldfish brain regions, a phenomenon that has been well observed in mammalian models (2, 3, 9, 16). In rats, varying periods of food deprivation cause an increase in NPY levels in PVN, a main site in food regulation, and mRNA levels in other hypothalamic nuclei (2, 3, 9, 14). In these studies, the significant increase in NPY levels in PVN under food deprivation has been shown to be associated with an increased drive to eat. Another study also showed that rats consumed more food after food deprivation for as long as 3 h in light phase and increased further as the period of starvation increases (14). Similarly, goldfish consumed 2-3% body weight ration within 30 min after 48-72 h food deprivation compared with 50-75% of a 2% body weight ration consumed by normal-fed fish (Y. K. Narnaware and R. E. Peter, unpublished results), suggesting that increased endogenous NPY after food deprivation may serve as a neural signal to increase appetite in goldfish.

In addition to stimulation of food intake, NPY also caused an increase in serum GH levels, consistent with the previous findings that NPY is a potent stimulator of GH release in vivo and in vitro in goldfish (24). GH has been shown to play an important role in regulating food intake in fish (for review, see Ref. 24). In goldfish, a short-term relationship between circulating serum GH levels and feeding has been demonstrated (11). When fed a 2% wet body weight ration, goldfish exhibit an acute elevation in serum GH at 30 min after feeding. This initial rise in serum GH is followed by a sharp decrease and then, over the next 3 h, a more gradual decrease to serum GH levels significantly lower than in unfed control fish (11). In the present study, serum GH levels were also increased just before feeding, in association with the increase in NPY gene expression in the forebrain regions. In addition, diet restriction and food deprivation resulted in significant increases in serum GH levels, correlated with the increase in NPY gene expression. These results suggest that the increase in endogenous NPY may stimulate the release of GH from the pituitary.

Perspectives

The present study demonstrates that gNPY stimulates food intake and feeding behavior in goldfish, which are mediated, at least in part, through Y1-like receptors in the brain. The NPY actions on food intake in mammals are thought to be mediated through Y1 and Y5 receptors. Our results suggest the evolutionary origin of this system from the bony fishes or earlier in vertebrate phylogeny. Our gene-expression studies show that the brain NPYergic system is broadly activated as part of the normal appetite response before scheduled feeding. In addition, the brain NPYergic system is broadly activated in situations of mild food restriction and withdrawal. In mammals, NPY plays a critical role in daily management of food intake and energy balance. Our results indicates that, similar to mammals, the brain NPYergic system is dynamically involved in the regulation of daily food intake in goldfish and supports the notion that NPY may be a key orexigenic neuropeptide in goldfish.


    ACKNOWLEDGEMENTS

We thank Jim Johnson for assistance in GH RIA. gNPY was a gift from Dr. J. Rivier, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA.


    FOOTNOTES

This research was supported by Grant A6371 to R. E. Peter from the Natural Sciences and Engineering Research Council of Canada.

Address for reprint requests and other correspondence: R. E. Peter, Dept. of Biological Sciences, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2E9 (E-mail: dick.peter{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 8 November 1999; accepted in final form 24 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 279(3):R1025-R1034
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