© 2000 by The Society for Integrative and Comparative Biology
The Effects of NPY and Insulin on Food Intake Regulation in Fish1
1 U.S. Department of Agriculture, Agricultural Research Service, Catfish Genetics Research Unit, P.O. Box 38, Thad Cochran National Warmwater Aquaculture Center, Stoneville, Mississippi 38776
2 University of Washington, School of Fisheries, 355100, Seattle, Washington 98195
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Recent abundant studies report that in rodents starvation induces increased neuropeptide Y (NPY) mRNA expression and peptide secretion in the hypothalamus which reduces autonomic nervous activity and promotes food intake, and intracerebroventricular (ICV) injection of NPY has potent orexigenic effects. Conversely, the effect of insulin in the central nervous system is to inhibit food intake and NPY biosynthesis and secretion. In mammals body fatness is regulated and insulin acts as one intake inhibitory signal related to fatness. In salmon (Oncorhynchus sp.) we have demonstrated a rise in NPY-like mRNA expression and a coincident decrease in plasma insulin levels during 2 to 3 weeks of starvation. Additionally, experimentally manipulating body fatness with high and low fat diets has demonstrated that body fatness affects food intake in teleost fishes, raising the possibility that NPY and insulin act to regulate their food intake. Therefore, we hypothesized that as in rodents, ICV treatment with NPY would stimulate food intake while ICV insulin would reduce food intake. Preliminary results suggest that ICV NPY administration does stimulate food intake in channel catfish (Ictalurus punctatus), but central injection of insulin has no effect. Results of treatments with the sulfated octapeptide of cholecystokinin and the recombinant fragment of rat leptin 22–56 are also discussed.
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INTRODUCTION |
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Neuropeptide Y (NPY), a member of the pancreatic polypeptide (PP) superfamily, was discovered in 1982 by Tatemoto et al. (Tatemoto et al., 1982
). A few years later Andrews et al. (1985) found the first piscine member of the PP family in the pancreas of anglerfish, Lophius americanus. This peptide is presently known as anglerfish peptide Y (APY). The next year, Kimmel, Plisetskaya et al. isolated and characterized a peptide from salmon pancreas with 83% homology to porcine NPY (Kimmel et al., 1986). Larhammar (1996) later classified this salmon peptide as a peptide YY (PYY) member of the PP superfamily. It was not until 1992 that NPY members of the PP superfamily were isolated and amino acid sequences elucidated from goldfish, Carassius auratus, and Torpedo ray, Torpedo marmorata (Blomqvist et al., 1992) Atlantic cod, Gadus morhua, and rainbow trout, Oncorhynchus mykiss (Jensen and Conlon, 1992), and dogfish, Scyliorhinus canicula (Conlon et al., 1992). However, because of the remarkable sequence conservation of NPY over evolutionary time (see Larhammar, 1996), immunohistochemical studies on localization of neuropeptide Y in piscine tissues were carried out using antibodies to mammalian NPY (Vallarino et al., 1988; Pontet et al., 1989; Danger et al., 1991). NPY appears to have exclusively neuronal origin whereas PYY is distributed in both gut/pancreas and neural tissues. Pancreatic polypeptide, though the namesake of this peptide family, is not found in fishes and apparently evolved from a gene duplication event in an early tetrapod (Jensen and Conlon, 1992; Larhammar, 1996).
NPY is known to influence a number of physiological parameters, one of the most avidly researched being food intake and energy balance. In mammals, NPY is a potent stimulator of food intake causing as much as 20-fold increase in food intake of adult female rats injected intracerebroventrically (ICV) at the optimal dosage rate (Clark et al., 1987). Research on rodents with genetic lesions causing tremendous obesity (e.g., ob/ob and db/db mice, Zucker fa/fa rats) has shown high levels of NPY gene expression accompanying extreme hyperphagia in obese animals compared to normal littermates (reviewed by White, 1993). In normal rodents, repeated ICV administration of NPY leads to sustained hyperphagia and weight gain (Stanley et al., 1986). In addition to potent stimulation of food intake, the involvement of NPY in energy balance regulation in mammals has been demonstrated via several lines of research. Food deprivation is one condition under which hypothalamic NPY mRNA and peptide levels rise (Marks et al., 1992). NPY also acts to reduce autonomic nervous activity and energy expenditure (Billington et al., 1991), which may be one reason for elevation of NPY peptide and mRNA levels when food is restricted (Marks et al., 1992; Schwartz and Seeley, 1997).
NPY does appear to have a food intake and energy balance regulatory role in non-mammalian vertebrates, too. Morris and Crews (1990) showed increased food intake in snakes injected ICV with NPY, and some birds have shown a positive response in food intake upon NPY treatment (Kuenzel et al., 1987; Richardson et al., 1995; Furuse et al., 1997). Functional studies on NPY in teleosts had been limited to its role as a pituitary hormone secretagogue (Kah et al., 1989; Breton et al., 1989; Peng et al., 1993a, b), although it was reported that salmon PYY exhibits NPY-like activities in rats. When administered directly into the rat hypothalamus it induced a food intake response comparable to that induced by NPY (Balasubramaniam et al., 1990). Finally, during the last year, the first studies have emerged on the involvement of NPY in food intake and energy balance in fishes. Food deprivation for 2–3 weeks led to increased hypothalamic NPY-like mRNA expression in chinook and coho salmon (Oncorhynchus tshawytscha and O. kisutch) (Silverstein et al., 1998), and de Pedro et al. (1998) have shown an increase in food intake with ICV injected NPY in goldfish, Carassius aurata.
The role of insulin in food intake regulation has also been examined extensively. Insulin's ability to increase food intake when injected peripherally in mammals was recognized over 70 years ago (ref. from Morley, 1987). This acute effect of insulin is probably a result of induced hypoglycemia (Schwartz et al., 1992a; Baskin et al., 1993). At the same time, several lines of evidence suggest that on a long term basis insulin acts to reduce food intake via the central nervous system (CNS). The prevailing hypothesis, although not accepted by everyone (see Devaskar et al., 1994), is that brain insulin at least in adult mammals, comes from the pancreas and crosses the blood brain barrier via a saturable transporter mechanism (Baura et al., 1993). Insulin receptors are widely distributed in the brain, being most numerous in the cortex, olfactory bulbs and hypothalamus, particularly in the arcuate nucleus (Schwartz et al., 1992a).
Direct ICV injection of insulin suppresses food intake and body weight gain in rats (Ikeda et al., 1986), and furthermore, genetically obese Zucker rats have reduced insulin concentrations in the hypothalamus (Baskin et al., 1985). When plasma insulin is chronically elevated and glucose levels are clamped, food intake decreases in rodents (reviewed by Baskin et al., 1993). Moreover, because plasma insulin levels have been found to correlate with body fatness, insulin is thought to be one of the major signals informing the brain of body fatness and energy balance (Schwartz et al., 1992a; Kaiyala et al., 1995). Counter to NPY's suppressive effect on energy expenditure, insulin stimulates energy use (Menendez and Atrens, 1991).
In actively feeding fish, plasma insulin increases after a meal, decreases between meals and drops sharply with starvation (reviewed by Mommsen and Plisetskaya, 1991), a profile consistent with mammals. The presence of insulin receptors in membrane preparations of piscine brain (Gutiérrez and Plisetskaya, 1994; Leibush et al., 1996) and in the olfactory bulbs of juvenile salmon (D. Baskin, unpublished) suggest that fish brains are insulin sensitive organs. Nevertheless, evidence of an effect of insulin on food intake in fishes is absent.
Interaction of insulin and NPY in the regulation of food intake and energy expenditure has been reviewed for mammals. A well supported mammalian model shows that regulation of body fatness is a prime homeostatic objective and NPY and insulin are regulators in this system (Kaiyala et al., 1995). Hypoinsulinemic conditions, such as fasting and diabetes, are known to activate NPY synthesis and secretion (Schwartz et al., 1992b; reviewed by Schwartz and Seeley, 1997). Conversely, centrally administered insulin inhibited NPY mRNA expression in the arcuate nucleus and reduced immunoreactive NPY concentrations in the paraventricular nucleus (Schwartz et al., 1992b). Furthermore, it appears that not only is NPY regulated by insulin, but NPY exerts some control over insulin levels as well. NPY administration resulting in elevated pancreatic insulin secretion has been reported (Moltz and MacDonald, 1985). This may be the result of preliminary increase in hepatic glucose output, followed by a rise in plasma insulin (reviewed by White, 1993). Increases in insulin secretion could contribute to the increased fat deposition seen with NPY administration. The rise in plasma insulin associated with NPY treatment also suggests a feedback mechanism to limit NPY synthesis and secretion (McMinn et al., 1998). Outside of mammals, there has been little investigation on the interaction of NPY and insulin in tetrapods, and no published work on fish. Kuenzel and McMurtry (1988) reported that ICV administration of NPY caused elevation of plasma insulin in chickens.
Recent studies have shown negative effects of body fatness on food intake in salmonids (Metcalfe and Thorpe, 1992; Jobling and Miglavs, 1993; Shearer et al., 1997), an effect reminiscent of insulin's action in mammals (Schwartz et al., 1992a). These results considered together with studies showing stimulation of goldfish food intake by NPY (de Pedro et al., 1998) and increased NPY-like mRNA in the hypothalamus of fasted salmon (Silverstein et al., 1998) raise the possibility that NPY and insulin act to regulate food intake in fish. Among a number of other peptides that could also be involved in such regulation one stands out and should be mentioned. This peptide, leptin, produced in mammalian fat tissue, has a central position in the regulation of energy balance and food intake in mammals (Campfield et al., 1995; reviewed by Bray and York, 1998). Leptin might be considered a counterpart of insulin in long-term regulation of food intake and energy balance in healthy animals (Schwartz and Seeley, 1997; McMinn et al., 1998). As far as we know there are no confirmed data on either leptin presence or its physiological role in fish. In this paper we present new data on the effect of body fatness on food intake, and on the effects of centrally injected NPY, insulin and leptin on food intake in the teleost, channel catfish, Ictalurus punctatus.
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MATERIALS AND METHODS |
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Effect of fatness on food consumption
Channel catfish of the USDA-103 strain (Li et al., 1998
) weighing an average of 4.2g were distributed (150 fish/tank) into six 160 liter tanks on 19 August 1998 and supplied with flow through aerated well water at 26°C. After a 1 week period of acclimation, 2 dietary treatments, A and B were initiated. Diet A was a standard commercial catfish diet (SF Services, Greenville) 32% protein, 4% fat (LF). Diet B was the same food topped with 9% fish oil (a mixture of catfish and menhaden oil) to make a diet with approximately 13% fat (HF). These diets were fed at 3% of body weight each day, 5 days each week to triplicate tanks. Samples for fat determination were collected on 25 September, 14 October, 30 October, and 19 November, in addition to initial samples collected on 19 August 1998. For the initial sample, 5 pools of 5 fish were collected, and at all other sampling times two samples of 5 fish each were collected from each tank. Sampled fish were then anesthetized and frozen at –20°C until analysis of whole body fat. All sampling was conducted on fish that were fasted for 24 hr. Fat was extracted from samples of whole ground fish. The ground tissue samples were first dried to constant weight at 105°C and moisture content determined. Fat was then extracted from dried samples using petroleum ether as solvent, refluxed for 2 hr. Whole body fat in these groups diverged, and beginning on 19 October 1998 all groups of fish were fed the same diet with an intermediate (8%) fat level to apparent satiation. For satiation feeding, food was delivered to the tank a few pellets at a time until no food was consumed for 2 min. The amount of feed consumed by fish in each tank was recorded daily. Data on feed consumed are presented as percent of body weight calculated as the weight of food consumed by all the fish in the tank divided by the weight of fish in the tank based on weight samples collected periodically.
On two occasions, at the first day of satiation feeding and 14 days later, labeled food containing x-ray opaque leaded glass "ballotini" beads mixed into the diet at 1% of the weight of the diet, was fed to the fish. The labeled diet was fed to apparent satiation. The procedure for radiographic evaluation of feed intake has been described previously (Silverstein et al., in press). This procedure allowed quantification of body fatness effects on feed intake and on variation in feed intake for individual fish as well as for replicate tanks. Approximately 50 individuals from each tank were anesthetized, then weighed to the nearest 0.1 g and radiographed. To calculate percent of feed consumed per body weight for individual fish, the weight of dry food consumed was subtracted from the weight of the fish giving the corrected fish weight. The weight of dry food was then divided by the corrected fish weight. Treatment effects were identified by two sample t-test analyses. Differences were considered significant at P 
0.05.
ICV injection
To measure the effects of ICV injected peptides on feeding behavior, groups of 6 to 12 fish were placed in a 160 liter tank containing flow through aerated well water at 26°C. A 12 hr light:12 hr dark schedule was maintained indoors. Fish weighed between 60 and 120 g and were fed 2% of body weight daily during acclimation. Ventricular injections were administered by following coordinates verified for accurate placement into the third ventricle with methylene blue dye and histological examination of brain tissues. Prior to injection, the fish was anesthetized in MS-222 (1:6,000 dilution) and placed on a plexiglas board with velcro straps adjusted to hold fish in place. The whole plexiglas board with fish attached was placed into a piece of rain gutter material with a 6 mm diameter nozzle on one end. The fish's mouth was placed around the nozzle, a padded bar was lowered over the skull to hold the fish's head stationary and anesthetic laden water was pumped across the fish's gills during the injection procedure. A 26 gauge needle on a 10 µl Hamilton syringe was aligned with the 6th preorbital bone at the rear of the eye socket, using a three dimension micro-manipulator. The needle was then raised straight up, moved horizontally and then down so that it rested against the skin of the fish in the lateral center of the head, in direct line with the 6th preorbital bone. This point was considered the zero point. From this zero point the syringe was moved posteriorly 6 mm and then down 6 mm through the space in the frontal bone into the third ventricle. The plunger on the syringe was slowly depressed to dispense the appropriate volume of solution (from 1 to 3 µl). A period of 20 sec was allowed to elapse before withdrawing the syringe to reduce any loss of material from the injection site. Prior to injection, all fish from one tank were anesthetized, and left or right barbels or caudal fins were clipped to indicate treatment. In all ICV injection trials, all treatments were housed together in a single tank and therefore had the same exposure to food when it was presented. By housing all treatments in a single tank and replicating tanks, variation between tanks and day to day variation, due perhaps to tank location, noise in the hatchery or foot traffic past certain tanks and not others, did not confound treatment effects. Fish were returned to their tank and allowed to recover from the anesthetic after injection with either peptide or vehicle. Food labeled with leaded glass beads was delivered to excess by automatic feeder. Timing and duration of food presentation varied as detailed below. One hour after feeding was ended the fish were anesthetized, weighed to the nearest 0.1 g and radiographed to determine the quantity of food consumed. Percent consumption was calculated as described above for measurement of individual food intake following radiography.
Compounds injected
Tests of the ICV injection technique were conducted with the sulfated octapeptide of cholecystokinin, CCK-8s (Peninsula Laboratories 7183). This compound had a potent and rapid inhibitory effect on food intake in goldfish (Himick and Peter, 1994a). The CCK-8s was mixed with 1 M PBS to a concentration of either 0.5 µg/µl or 1 µg/µl and a 2 µl volume was injected (
12.5 or 25 ng/g body weight [BW]) following the procedures outlined above. Control animals received PBS only. Food intake was tested at 2 intervals after injection. First, 3 hr after injection food was presented for 1 hr and then food intake quantified. Subsequently, 10 minutes after injection food was presented for 30 minutes and then intake measured. The sample size was 12 fish for all treatments in each trial.
Porcine NPY (Peninsula Laboratories 7172) dissolved in 1 M PBS was injected at doses from 2 to 6 µg per fish or 25 to 75 ng/g BW, injected in volumes between 1 and 3 µl. The effect of NPY injection on food intake was tested 1 hour after injection by presenting food for 1 hour and then quantifying intake. Several trials were conducted. In the first trial ICV injection of 50 ng/g BW NPY was compared to a vehicle control (n = 12 fish for each treatment). In trial two we compared vehicle treated control (n = 4), and fish ICV injected with 50 ng/g BW of NPY (n = 5). Trial three consisted of a vehicle treatment and 3 doses of NPY, 25 ng/g, 50 ng/g and 75 ng/g BW (n = 6 for all treatments). Another experiment with NPY, injecting fish daily for 4 days, was conducted. Each day at 9:00 AM fish were netted, anesthetized, and injected with 50 ng/g BW NPY or PBS vehicle. The fish were allowed 1 hour of recovery and then fed to excess for 1 hour. Weights of individually identified fish were monitored daily (n = 6).
Trials with bovine insulin (Sigma: I1882) were done under a variety of timing conditions. Insulin was first dissolved in 1/10th the final volume in 0.12% acetic acid. After solubilizing the insulin, 1M PBS was added to the appropriate final volume. In most experiments with insulin the dosage used was 2 µg/fish (25 ng/g BW), though 4 µg/fish (50 ng/g BW) was used on one occasion (when food was presented 10 minutes after injection). Presentation of food varied between 10 min and 24 hr after injection (10 min 1, 4, 12 and 24 hr). In each case food intake over 1 hr was measured. The sample size was 12 fish for each treatment.
The rat leptin fragment, leptin 22–56 inhibits food intake with potency equal to the whole peptide in rats (Samson et al., 1996). The leptin 22–56 fragment (Peninsula Laboratories 9204) was dissolved in 1 M PBS and injected at a dose of 50 ng/g BW. Food was presented for 1 hr, 60 min after injection. Three trials with leptin were conducted. In the first trial, all fish had been fed the previous day. In trial 2 fish had been starved for 2 days prior to injection, and in trial 3 fish had been fasted 4 days prior to injection. Six fish were used for each treatment.
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RESULTS |
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Effect of fatness on food consumption
Mortality during the 3 month experiment was low (
2%). Fish in all tanks consumed the entire daily ration, 3%/day, during the dietary pre-treatment phase of the experiment. Initial body fat of the fish was 6.4 ± 0.1% on a wet weight basis. Body fat diverged rapidly on the 4% (LF) and 13% (HF) fat dietary treatments (Fig. 1). Separation in body fat was greater than 2% on a wet weight basis by 25 September 1998 and thus feeding to apparent satiation with a diet intermediate in fat (8%) was begun on 14 October 1998 when there was approximately 2.2% difference in body fatness. Body fatness increased in both treatments with satiation feeding, but the increase in the LF fish was steeper such that body fatness of LF and HF groups converged. In the samples taken 19 November 1998, approximately 1 month after feeding to apparent satiation, body fatness did not differ between the LF and HF groups (Fig. 1).
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A difference in food intake was clear between the HF and LF treatments for the first 2 weeks of feeding to apparent satiation, LF fish eating more (Fig. 2). Both groups consumed more than the 3%/day that they were being fed during the dietary treatment. Over the last 3 weeks the average daily consumption did not differ significantly between the treatments, and in both groups consumption as a percent of body weight declined (Fig. 2). Investigation of food consumption by individuals gave similar results, LF treated fish consumed more than HF fish. Percent consumption was normally distributed for both treatment groups and there was no difference in sample variance (P > 0.34) for food intake between the 2 groups.
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Weights of the fish in the HF and LF treatments did not differ at any sampling point over the course of the experiment. In the last weighing 12 November 1998 the mean sizes were beginning to diverge (Fig. 3), though the difference was not significant.
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ICV injections
ICV injection of 12.5 ng/g of the sulfated CCK octapeptide caused a significant and reproducible decline of 50 to 65% in food intake when food was presented 10 min after ICV injection for 30 min (Fig. 4). The response was rapid, and the effect was not long lasting. There were no differences in food intake of fish injected with 25 ng/g CCK-8s or PBS in feeding trials conducted for 1 hr, 3 hr after CCK-8s injection.
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Porcine NPY injected ICV at a dose of 50 ng/g had a stimulatory effect on food intake in channel catfish. In three trials the dose of NPY of 50 ng/g stimulated an increase in food intake ranging from 45 to 100% (Fig. 5). A dose effect was seen when NPY was injected at 3 doses ranging from 25 to 75 ng/g body weight (Fig. 6). Catfish injected with 50 ng/g or more NPY consumed significantly more food than controls injected with vehicle only (Fig. 6), however, at the dose of 25 ng/g intake did not differ from controls. Daily ICV injection of NPY did not cause any weight difference compared to PBS injected controls (data not shown).
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The amount of feed consumed by controls in replicated feed intake trials was variable (see Figs. 4, 5). This variability between tanks and between days was normal and was most likely attributable to subtle differences in conditions. Nevertheless, the effect of bioactive peptides injected ICV was clear when compared to food intake levels of control treatments.
There was no significant effect of ICV injected insulin on food intake at any time period investigated (Fig. 7). Even the highest dosage used, 50 ng/g when examining the effect on food intake 10 minutes post-injection, did not result in a significant effect (P > 0.23, n = 12).
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ICV injection of the rat leptin fragment 22–56 had no effect on food intake measured for one hour 60 minutes after injection. Although food intake increased somewhat after a fasting period, there were no differences between control and leptin injected fish (Fig. 8).
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DISCUSSION |
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Food intake by the LF and HF treated groups was clearly different when fish were allowed to feed to satiation. The differences in intake were not related to size of the fish which remained the same in LF and HF groups. Furthermore, intake was expressed relative to size (feed consumed/body weight) to account for individual differences in size. The information on individual food intake measured on 2 occasions showed that the differences were consistent between treatments and not due to a few fish in a treatment.
The difference in intake between LF and HF fish suggests that body fatness affected food intake. Intake remained higher in leaner fish until body fat differences were no longer detected. It is interesting that when satiation feeding began, body fatness in both treatments increased. This suggests that the fat storage capacity of the HF group was not filled, but relative to the LF groups food intake was suppressed. Evidence from other studies supports an effect of body fatness on food intake in salmonids (Metcalfe and Thorpe, 1992; Jobling and Miglavs, 1993; Shearer et al., 1997). The reduced food intake by fatter fish in all these studies may be due to an inhibitory effect of body fatness. The mechanism for such an effect has not been elucidated in fish. It is known that mammalian plasma insulin levels correlate with body fatness (Schwartz et al., 1992a) and the food intake inhibitory hormone leptin is produced in proportion to the size of the fat store. These mechanisms have not been tested for fishes in detail. In the study by Shearer et al. (1997), insulin levels measured in fat and lean fish did not correlate with body fatness, however, the sample size was small and the study could not be considered definitive. Although mean insulin levels did correlate with body weight in rainbow trout (O. mykiss) in the size range from 290 to 560 g, as well as in other salmonids (see Mommsen and Plisetskaya, 1991), the relationship with body fatness needs further testing.
A shortcoming to all these studies on body fatness and food intake was that food deprivation or restriction was used to produce fish of different fatness. Metcalfe and Thorpe (1992) and Jobling and Miglavs (1993) used acute starvation to generate differences in body fatness. In order to avoid using acute starvation Shearer et al. (1997) used a feeding procedure that led to differences in body fatness over 7 months. Nevertheless, while acute starvation was avoided, chronic food restriction of the lean fish was required. The problem with food restriction in studies comparing food intake between fat and lean fish is that fat fish may eat less because their food intake is depressed by body fatness, or lean fish may eat more because they have been food deprived and are showing compensatory growth. These 2 possibilities cannot be clearly separated. The current study, though attempting to minimize differences in satiety during the first part of the experiment, could not solve this problem completely. Both LF and HF groups were fed 3% BW/day during the feeding treatment, however, the total energy content of food provided was less for the LF groups. To address this problem, another model may be needed. Surgical removal of body fat and experimental overfeeding are methods used to study mammalian regulation of adiposity (Kaiyala et al., 1995) that might be applied to fish to manipulate energy stores without confounding compensatory growth of lean, food restricted fish and food intake depression in fat fish.
Identification of hormonal and neuropeptide regulators of food intake would allow tests of whether fish regulate body adiposity. To this end, an ICV injection technique has been developed.
Responses of the fish to this technique were unequivocal and reproducible. As shown by Himick and Peter (1994a) for goldfish, CCK-8s had a rapid and significant inhibitory effect on food intake in channel catfish. The dose used in this study was between the highest (50 ng/g) and lowest (5 ng/g) dose used in goldfish. The degree of inhibition of food intake, between 50 and 65%, was similar to that found in goldfish. This result showed the technique to be effective and reliable. ICV injected CCK has been shown to inhibit feeding in goldfish (Himick and Peter, 1994a) and now in channel catfish. Brain and gut distribution of CCK-like immunoreactivity has been documented in fishes as primitive as lamprey (Holmquist et al., 1979), and in more derived fishes (e.g., Onchorynchus mykiss, Vigna et al., 1985; Dicentrarchus labrax, Moons et al., 1992). These results taken together with in vitro work showing CCK effects on smooth muscle contraction (Aldman and Holmgren, 1987; Jonsson et al., 1987), and Himick and Peter's (1994a) inhibitory effect on feeding with peripheral injection of CCK suggest that CCK may have both central and peripheral roles in regulation of feed intake.
The experimental design used in this study for evaluating the effects of ICV injection on food intake represents an improvement over previous methods. Work by Himick and Peter (1994a, b) focused on accurate placement of the injection needle, but animals were housed individually and intake of each individual observed. de Pedro et al. (1993) have used a different technique depending on free hand injection and separate confinement of treatment groups. Our technique combined the speed and accuracy of the two techniques, and allows treatment groups to be kept together, ensuring equal feeding opportunity for all fish. The radiographic method of food intake measurement permitted larger sample sizes, and accurate estimation of food consumed.
NPY was as orexigenic in channel catfish as in a number of other vertebrates. A dose of approximately 50 ng/g BW was required. This dosage is similar to that used in ICV injections for rats (Marks et al., 1996; Parikh and Marks, 1997) chickens (Furuse et al., 1997) and goldfish (de Pedro et al., 1998). A dose this large does not permit identification of the site of action because enough NPY was present to diffuse widely throughout the hypothalamus and brain. Although information on distribution of NPY immunoreactivity (Vallarino et al., 1988; Pontet et al., 1989; Danger et al., 1991 and many others) and mRNA (Peng et al., 1994; Vecino et al., 1994; Silverstein et al., 1998) is available for fish, hypothalamic binding sites for NPY have not yet been reported. Repeated ICV injections of NPY, once daily for 4 days, did not result in greater weight gain than in PBS treated controls (data not shown). This may be due to the stress of repeated handling, and therefore lack of a sustained response to NPY should not be assumed. Further trials, perhaps employing osmotic pumps (Levy and Baker, 1997) are needed and may yield different results. Our results on catfish imply a role for NPY in energy balance and food intake regulation in teleosts. These data are supported by increased levels of NPY-like mRNA in food deprived salmon (Silverstein et al., 1998) and by stimulation of food intake with NPY ICV injection in goldfish (de Pedro et al., 1998).
The lack of effect of ICV injected insulin on food intake was unexpected. Based on a mammalian model, the low insulin levels seen in fasted salmon with elevated hypothalamic NPY-like mRNA suggested a possible link between NPY synthesis and plasma insulin levels. In fish as in other vertebrates, plasma insulin levels typically decline with fasting, however, lamprey and salmon may endure long voluntary fasting periods during spawning migrations and yet maintain surprisingly stable plasma insulin levels (Plisetskaya, 1985; Mommsen and Plisetskaya, 1991). Again based on a mammalian model, we speculated that sustained insulin levels may have acted to prevent food intake during these migrations (Plisetskaya and Silverstein, unpublished). One should keep in mind that whenever non-homologous peptides are used, some reservations about the reliability of results are held.
Although ICV injected insulin clearly did not have an effect on food consumption under the current experimental conditions, it would be premature to rule out a regulatory role for insulin in the central nervous system of fish. The presence of insulin receptors in brain preparations from teleosts (Gutiérrez and Plisetskaya, 1994; Leibush et al., 1996) leaves the question of what insulin does in the brain. The fish used in these injection experiments were raised under long day (12 hr light) conditions, and the duration of food presentation was not varied. Effects of insulin may be seasonal (Plisetskaya, 1998) or in a different time frame than the one being tested, therefore the experimental conditions for insulin testing should be expanded.
Similarly for leptin, the lack of effect under the current experimental conditions do not eliminate the possibility that leptin or a related compound is active in fish. With leptin the problem of non-homologous peptides is compounded by the current paucity of evidence of any protein that resembles leptin in fish (however, see Johnson et al., 2000). This is despite the efforts of several laboratories to clone leptin using reverse transcription-PCR.
In summary, body fatness appears to have an inhibitory effect on food intake in fish, as it does in mammals. Although central injection of NPY stimulates and CCK inhibits food intake in fish like in other vertebrates, regulatory roles for insulin and leptin have not yet been supported. Nevertheless, given the inhibition of food intake by body fatness, roles for insulin and leptin cannot be dismissed.
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ACKNOWLEDGMENTS |
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The authors wish to thank W. Staub, M. Dennis, and C. Dennis for expert technical assistance, and Dr. M. Li for body fat analysis. This work was supported in part by National Science Foundation Award number IBN-9722830 to EMP and JTS.
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FOOTNOTES |
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1 From the symposium A Tribute to Erika M. Plisetskaya: New Insights on the Function and Evolution of Gastroenteropancreatic Hormones presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 6–10 January 1999, at Denver, Colorado.
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References |
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