Agouti-related protein (AgRP), an endogenous melanocortin 3/4 receptor antagonist, appears to play an important role in the control of food intake and energy balance because exogenous administration in rats and overexpression in mice result in hyperphagia and body mass gain. Furthermore, arcuate nucleus AgRP mRNA is increased with fasting in laboratory rats and mice and is decreased with refeeding. In Siberian hamsters, fasting also increases arcuate nucleus AgRP mRNA, but these animals increase food hoarding, rather than food intake with refeeding. Therefore, we tested whether exogenous AgRP increased food hoarding in this species. Hamsters were trained in a hoarding/foraging apparatus to run a programmed number of wheel revolutions to earn food pellets. Four doses of AgRP-(83-132) or vehicle were injected into the third ventricle at the beginning of the dark phase, and food hoarding, food intake, and foraging were measured at various time points subsequently. Overall, food hoarding was stimulated as much as 10 times more than food intake, and both responses occurred as early as 1 h after injection. Food hoarding was increased the greatest at the lowest dose (0.1 nmol), whereas food intake was increased the greatest at the second lowest dose (1 nmol). Food intake and especially food hoarding were increased up to seven days after the AgRP injections. Foraging was increased at all AgRP doses except the highest dose (100 nmol). These results suggest that AgRP triggers the search for food in this species, and once they find it, hoarding predominates over eating.
- energy balance
- body weight
animals have evolved species-specific strategies to meet the perpetually high energetic demands of the central nervous system (CNS). Small mammals attempt to meet their energetic demands either by utilizing internal energy stores (primarily lipid stored in adipose tissue), by utilizing external energy stores (food hoard), or by using both energy stores. To maintain both energy stores, animals must forage for food and then partition the foraged food energy into immediate use (eat it) or one of these two energy depots for future use. The reliance on internally or externally stored energy is highly species specific (for review, see Ref. 4).
Unlike laboratory rats and mice and most animals (for review, see Ref. 5), hamster species do not increase their food intake after a fast (e.g., 6, 30, 33), and it was hypothesized that instead they may increase their food hoarding (33) because of their reputation as prodigious food hoarders (for review, see Ref. 4). Indeed, fasted-refed Siberian hamsters (Phodopus sungorus) markedly increase their food hoarding after a fast (2, 3, 12, 13, 36).
The mechanisms underlying the fasting-induced food hoarding in this species, or the mechanisms underlying food hoarding in general, are infrequently studied, especially the role of the CNS in the control of food hoarding (for review, see Ref. 4). One likely candidate may be agouti-related protein (AgRP). AgRP is part of the melanocortin system that recently has been implicated in the control of food intake and body weight/fat regulation (for review, see Ref. 22). Briefly, pro-opiomelanocortin (POMC) is a precursor protein that produces three melanocortins, α-, β-, and γ-melanocyte-stimulating hormones (MSH), with α-MSH as the ligand for the centrally located melanocortin 3 and 4 receptor subtypes (MC3-R and MC4-R). Activation of these receptor subtypes is noted for the ability to increase food intake and body mass (e.g., Ref. 28). AgRP, the naturally occurring MC3/4-R receptor antagonist, is expressed in the arcuate nucleus of the hypothalamus (1, 8) and its central administration profoundly stimulates food intake (15, 35). Most relevant to the fasting-induced increases in food hoarding by Siberian hamsters is that fasting increases arcuate nucleus AgRP gene expression in this species (21), as it does in laboratory rats (e.g., Ref. 17). Further indirect support for the possible role of the melanocortin system in fasting-induced food hoarding is that food intake by rats and mice can be inhibited by a synthetic melanocortin system agonist, melanotan-II (MTII; Refs. 7, 9, 20), and MTII also inhibits nocturnal feeding by Siberian hamsters (31). Therefore, we asked: Does central administration of AgRP increase foraging, food hoarding, and/or food intake in Siberian hamsters? This was accomplished by testing the effects of AgRP in our recently developed hoarding/foraging system to measure these appetitive (foraging, hoarding) and consummatory (intake) ingestive behaviors (11). This system allows us to separate the effects of experimental treatments such as central peptide administration on these aspects of ingestive behavior. This was accomplished by training Siberian hamsters to earn food after completing a programmed number of wheel revolutions. Animals were implanted with a guide cannula aimed at the third ventricle, injected with several doses of AgRP, and food intake, food hoarding, and foraging were subsequently measured at various time points postinjection.
All procedures were approved by the Georgia State University Institutional Animal Care and Use Committee and are in accordance with Public Health Service and United States Department of Agriculture guidelines. Adult male Siberian hamsters ∼3.5 mo old and weighing 38-46 g were obtained from our breeding colony. The outbreeding history of this colony has been described recently (11). Hamsters were group-housed and raised in a long-day photoperiod (16:8-h light-dark cycle; lights on 0200) from birth. Room temperature was maintained at 21 ± 2.0°C, and relative humidity was 50 ± 10%.
Animals and housing. Sixty-four hamsters were acclimated for 2 wk in custom-designed hoarding apparatuses as described previously (11). Briefly, two cages were connected with a convoluted polyvinlychloride tubing system (38.1 mm ID and ∼1.5 m long) with corners and straightways for both horizontal and vertical climbs. The top or food cage was 456 × 234 × 200 mm (length × width × height) and was equipped with a water bottle. The bottom or burrow cage was 290 × 180 × 130 mm, was covered to simulate the darkness of a natural burrow, and contained bedding and cotton nesting material. The test diet (75 mg pellets: Purified Rodent Diet; Research Diets, New Brunswick, NJ) and tap water were available ad libitum during this period. A running wheel (524 mm circumference) and pellet dispenser were attached to the food (top) cage. Wheel revolutions and pellet delivery were counted using a magnetic detection system and monitored by a computer-based hardware-software system (Med Associates, Lancaster, NH).
Measurement of food hoarding and food intake. Pellets earned were defined as the number of pellets delivered by the computer-based hardware-software system on completion of the requisite wheel revolutions. Food hoarding was defined as the number of pellets found in the bottom burrow cage in addition to those removed from the cheek pouches. Surplus pellets were defined as the number of pellets removed from the top food cage that were neither eaten nor hoarded. For the 10 and 50 revolutions/pellet groups, food intake was measured by using the following formula: pellets earned - surplus pellets - hoarded pellets = food intake. For the free and blocked wheel groups, food intake was measured by using the following formula: pellets given - pellets left in the top cage - hoarded pellets = food intake.
Foraging training regimen. We used a wheel-running training regimen that eases the hamsters into the higher foraging efforts without large changes in body mass or food intake (11). Specifically, hamsters were given free access to the food pellets for 2 days while they adapted to the running wheel. In addition to the free food, a 75-mg food pellet was dispensed on completion of every 10 wheel revolutions. On the third day, the free food condition was replaced by a response-contingent condition where only every 10 wheel revolutions triggered the delivery of a pellet. This condition was in effect for 4 days during which time body mass, food intake and food hoarding, wheel revolutions, and pellets earned were measured daily. On the seventh day, the foraging effort was increased to 25 revolutions/pellet, and this remained in effect 3 days. On the 10th day, the foraging effort was increased to 50 revolutions/pellet, and this remained in effect for 5 days. Foraging is defined as the number of pellets earned.
At the end of this acclimation period (14 days total), all animals were removed from the foraging apparatuses and housed in a shoebox cage where food was available ad libitum with no foraging requirements. Guide cannulas were then surgically implanted in all hamsters (see Cannula implantation for details). After a 1-wk postsurgical recovery period, all hamsters were transferred back to the hoarding/foraging apparatus and retrained to the following schedule: 5 days at 10, 3 days at 25 and 6 days at 50 revolutions/pellet.
Experimental design. At the end of this 14-day retraining period, the hamsters were separated into four foraging effort groups matched for their current body mass and average hoard size across these last 6 days of training at 50 revolutions/pellet (n = 16/group). The four groups consisted of 10 or 50 revolutions/pellet or no foraging requirement [an active running wheel (free wheel; exercise control group) or blocked wheel (blocked wheel; sedentary control group) each with food available noncontingently]. Selection of these foraging effort levels was based on our previous study in Siberian hamsters using this foraging/hoarding system (11) that, in turn, was based on the wheel-based foraging paradigm of Perrigo and Bronson (23, 25, 26) in mice. Each foraging effort group received all doses of AgRP or its vehicle, and they were given in a counterbalanced schedule to control for possible order effects of peptide administration (see below).
Cannula implantation. The animals were anesthetized with isoflurane, and the fur at the top of the head was removed to expose the area to be incised. Guide cannulas (26-gauge stainless steel; Plastics One, Roanoke, VA) were implanted stereotaxically targeted for placement just above the third ventricle. Specifically, the skull was trephined at the intersection of bregma and the midsagittal sinus, and the cannula was lowered using the following stereotaxic coordinates (level skull, anterposterior 0 mm, mediolateral 0 mm, and dorsoventral -5.5 mm from the top of the skull). The guide cannula was secured to the skull using cyanoacrylate ester gel, 3/16 mm jeweler's screws, and dental acrylic. A removable obturator sealed the opening in the guide cannula throughout the experiment except when it was removed for the injections.
Intracerebroventicular injection protocol. Injections consisted of either vehicle (sterile 0.15 M NaCl) or one of four doses (0.1, 1, 10, or 100 nmol) of AgRP (Phoenix Pharmaceuticals, Belmont, CA) via an inner cannula (33-gauge stainless steel, Plastics One) that penetrated 5.5 mm below the top of the skull into the third ventricle. We tested the animals one time per week with one dose of AgRP-(18-132) or vehicle. Each animal received two different doses of AgRP (0.1 and 10 nmol or 1 and 100 nmol) alternating with saline over a 4-wk period. The order of the vehicle and drug injections was counterbalanced to minimize drug order effects. These doses of AgRP were selected because they bracketed those that effectively increase food intake in laboratory rats (15). The inner cannula was connected to a microsyringe via polyethylene tubing, and the injection volume for the vehicle (sterile saline) or AgRP was 0.4 μl.
Two hours before dark onset, food was removed from the pouches of the hamsters, they were placed in clean burrow cages, and access to the tubes was blocked. Animals were restrained by hand during the 30-s injection period, and the injection needle remained in place ∼30 s before withdrawal. Hamsters were returned to their respective cages, and access to the tubes was reinstated. Food hoarding, food intake, and foraging were measured 1, 2, 4, and 24 h postinjection. Measurements also were taken every 24 h afterward for 7 days to observe any long-term effects of AgRP on ingestive behavior that have been reported previously for laboratory rats (14, 15).
Terminal measures. After the end of the last test, an injection of 0.4 μl methylene blue dye was injected to confirm placement of the cannula in the third ventricle. The animals were killed with an overdose of pentobarbital sodium (70 mg/kg Nembutal); their brains were removed and postfixed in 10% paraformaldehyde for a minimum of 2 days. Each brain was sliced, and the cannula was considered located in the third ventricle if the dye was visible in any part of the third ventricle. Only the data from these animals were analyzed.
Statistical analysis. Body mass, food intake, and food hoarding data were analyzed as a percentage of saline using a four-way mixed-model ANOVA with repeated measures (5 × 4 × 2 × 4; peptide × foraging condition × peptide order × time) using NCSS Statistical Software v 2000 (Kaysville, UT). A separate, non-repeated-measures ANOVA (5 × 4 × 2; peptide × foraging condition × peptide order) was done on the cumulative intake (0-24 h) for the initial test day. Duncan's new multiple range tests (18) were used for post hoc tests when appropriate. Differences among groups were considered statistically significant if P < 0.05. Exact probabilities and test values were omitted for simplicity and clarity of the presentation of the results.
Because the effects of AgRP were markedly different for the foraging, food intake, food hoarding, and surplus pellet measures in terms of the magnitude of the percent changes from the intracerebroventricular saline controls values, note that the scale range of percentages on the ordinates is not the same both across these dependent variables as well as within the variable across doses of the peptide.
Wheel revolutions. One potential confounding factor for the present experiment would be if AgRP increased overall wheel running such that it would appear that the peptide stimulated foraging, when instead it only stimulated locomotor activity; or, conversely, if it decreased wheel running, then this might be interpreted as inhibiting foraging rather than as a general inhibition of locomotor activity. There was only one significant increase in wheel revolutions and no significant decreases. The increase in wheel running was by a nonforaging group (the free wheel group) for the total (0-24 h) revolutions run the first day at the 10 nmol AgRP dose (∼45%, data not shown). Thus any changes in foraging do not appear be due to nonspecific alterations of locomotor activity.
Foraging (pellets earned). Foraging was significantly increased at all time points and for both foraging efforts at the 0.1 nmol dose of AgRP compared with saline (P < 0.05; Fig. 1A). At the intermediate AgRP doses, foraging was significantly increased at the 1- to 2-h period for the 10 revolutions/pellet group (1 nmol; P < 0.05; Fig. 2A), and at the 0- to 1-h and 1- to 2-h periods for the 50 revolutions/pellet group (10 nmol dose; Fig. 3A). Foraging was increased at 0- to 1-h, 1- to 2-h and 0- to 24-h period for the highest AgRP dose (100 nmol; Fig. 4A).
Food intake. As with laboratory rats (15), AgRP increased food intake substantially in Siberian hamsters with significant increases as large as ∼100-150% occurring across the doses, times, and foraging efforts. Generally the most pronounced increases in food intake occurred at the lower two AgRP doses (0.1 and 1 nmol) with food intake somewhat suppressed at the higher two doses (10 and especially at 100 nmol) (P < 0.05; Figs. 1B, 2B, 3B, and 4B). More specifically, only hamsters foraging for their food (10 and 50 revolutions/pellet) had increased food intake at the lowest AgRP dose (0.1 nmol), and this occurred at 0-1, 1-2, and 2-4 h postinjection compared with saline injections (P < 0.05; Fig. 1B). When the AgRP dose was increased further (1 nmol), hamsters at the lowest foraging effort continued to increase their food intake significantly at all time points, but hamsters at the highest foraging effort (50 revolutions/pellet) did not significantly increase their food intake (Fig. 2B). At the second lowest dose of AgRP (1 nmol), there was increased food intake in the nonforaging groups (blocked wheel at 2-4 and 4-24 h, and free wheel at 1-2, 4-24, and 0-24 h; P < 0.05; Fig. 2B). At the lowest dose of AgRP (0.1 nmol), there were occasional significant increases in food intake seen by the nonforaging groups (blocked wheel at 2-4 h, free wheel at 1-2, 4-24, and 0-24 h; P < 0.05; Fig. 2). When the dose of AgRP was increased further (10 nmol), food intake was more reliably significantly increased for the nonforaging groups (free wheel across all time points, blocked wheel at 1-2 and 2-4 h), with occasional significant increased food intake for the foraging groups (10 revolutions/pellet at 1-2 h, 50 revolutions/pellet at 4-24 h; P < 0.05; Fig. 3B). Food intake, however, also was more frequently inhibited for the foraging groups at this dose (Fig. 3). Finally, at the highest dose of AgRP (100 nmol) food intake also was inhibited, especially at the highest foraging effort (50 revolutions/pellet; Fig. 4B). Food intake was sporadically significantly increased for the other groups (blocked wheel group, 1-2 and 2-4 h; free wheel group, 0-1, 1-2 and 2-4 h; 10 revolutions/pellet group, 4-24 and 0-24 h; 50 revolutions/pellet group, 1-2 h; Fig. 4B).
Sustained elevation of food intake by AgRP beyond the first 24 h was seen most prominently at the second lowest dose (1 nmol), primarily by hamsters in the blocked wheel group from days 2 to 5 with increase ranging from ∼10 to 45% (Fig. 5). Smaller persisting increased food intake was seen at the two highest doses of AgRP by several of the groups, but they were seldom significantly greater than occurred with the vehicle injections (Fig. 5).
Food hoarding. In contrast to the more modest percent increased food intake by AgRP, food hoarding was strikingly increased. Food hoard size was generally inversely related to the dose of AgRP. Thus the lowest AgRP dose (0.1 nmol) had the greatest increases in food hoarding (up to ∼1,200% for the blocked wheel group at 2-4 h; P < 0.05; Fig. 1C) and was significantly increased at all time points for all foraging efforts at this dose except, inexplicably, for the 10 revolutions/pellet group at 1-2 h. Food hoard size also was significantly increased at all time points for all foraging efforts at the 1 nmol AgRP dose (∼21-473%) with the nonforaging groups (blocked and free wheel) and 10 revolutions/pellet groups having the greatest increases (P < 0.05; Fig. 2C). Food hoard size was significantly increased at the next highest dose of AgRP (10 nmol AgRP) across all time points (P < 0.05; Fig. 3C), with one exception (10 revolutions/pellet group at 0-1 h). There were fewer and smaller increases in food hoarding at the highest AgRP dose (100 nmol; Fig. 4C). By the standards of the lower doses of AgRP, these increases were modest (∼<200%) and none of the groups were consistently associated with the significant increases in food hoard size, nor in the lack of such significant increases.
As for the increases in food hoarding during the first 24 h compared with food intake, the prolonged stimulation by AgRP of food hoarding across the 7-day measurement period was much greater than for food intake (Fig. 6 vs. Fig. 5). Overall, AgRP triggered a sustained elevation of food hoarding beyond the first 24 h at each dose for all groups at one or more times across the 7-day measurement period (P < 0.05, Fig. 6). Similar to that for the acute effects of AgRP on food hoarding, the prolonged stimulation of food hoarding by the peptide tended to be inversely related to the AgRP dose (Fig. 6). Specifically, food hoard size was greatest and elevated the longest for hamsters given 0.1 nmol AgRP with increases up to ∼2,000% 3 days after injection for the 10 revolutions/pellet group and remaining elevated, but with progressive decreases to ∼500% through day 7 (P < 0.05; Fig. 6). There were smaller increases and shorter durations of elevation for the other groups at this dose [but still substantial with increases ranging from ∼1,400 to ∼250% through day 7 for the blocked wheel group (P < 0.05; Fig. 6)]. This inverse relation between peptide dose and food hoard size was evident at the two highest AgRP doses (10 and 100 nmol), despite receiving more of the peptide. Even so, increases across the groups were as high as ∼900% (day 2, 10 revolutions/pellet group) and persisted across the test period leveling off at ∼105-200% on days 4-7 for several of the groups (10 nmol blocked wheel and 10 revolutions/pellet groups; 100 nmol all groups except the blocked wheel group; P values < 0.05; Fig. 6).
Surplus pellets. Surplus pellets (food earned not eaten or hoarded) suggests a mismatch between efforts to earn food and efficient use of the food (24). Surplus pellets were significantly increased for the 10 revolutions/pellet group at the 0.1 nmol dose of AgRP at all time points (P values < 0.05; Fig. 1D) and for 0-1 and 1-2 h for the 10 nmol dose of AgRP (P values < 0.05; Fig. 3D) compared with saline. At the higher foraging effort (50 revolutions/pellet), surplus pellets were generally significantly increased across all AgRP doses compared with saline (except at 0.1 nmol at 4- to 24-h period and at 10 nmol for the 2- to 4-h, 4- to 24-h, and 0- to 24-h measures) (P < 0.05; Fig. 4D).
The results of the present study suggest a novel role for AgRP-(83-132) for ingestive behavior in that foraging and especially food hoarding were stimulated after central administration (3rd ventricular) of the neuropeptide to Siberian hamsters. In addition, AgRP stimulated food intake, as has been previously shown in laboratory rats (e.g., Refs. 15, 29, 35). Overall, food hoarding was stimulated as much as 10 times more than food intake, both responses occurred as early as 1 h after injection, and both food hoarding and intake were increased 4-7 days postinjection. Finally, within the dose range tested here (0.1-100 nmol), the stimulation of food intake and food hoarding both tended to be inversely related to the dose of AgRP, suggesting even lower doses of the peptide might be effective in stimulating these behaviors in this species.
As mentioned above, the lower doses of AgRP (0.1 and 1 nmol) tested here had a greater affect on food intake than the higher doses (10 and 100 nmol), a relation that is similar to the stimulation of feeding by the peptide in laboratory rats (15). The magnitude of the AgRP-induced increases in food intake (∼100-150%) in the present study were similar to those seen by laboratory rats (e.g., Refs. 15, 29, 35) and mice (20). It appears that AgRP induces Siberian hamsters, and perhaps other animals, to seek food (foraging was significantly stimulated by AgRP in the present study), and once found, at least for Siberian hamsters, food hoarding, rather than food intake, predominates. This apparent hierarchy of ingestive behaviors seems to fit nicely with the predominate response of hamsters to refeeding after a fast (for review, see Ref. 4). That is, hamster species, including Siberian hamsters, do not exhibit a postfast hyperphagia; rather they increase their food hoarding postfast (2, 3, 12, 13, 36). Taken together with the fasting-induced increase in arcuate nucleus AgRP gene expression by Siberian hamsters (21), it seems likely that AgRP may play a significant role in the postfast-induced increase in food hoarding and foraging shown by this, and perhaps other, hamster species. It may be that AgRP also would stimulate food hoarding by laboratory rats, but because food hoarding is not a naturally occurring ingestive behavior of even their wild counterparts (10, 19, 27), it may not do so.
AgRP triggered a prolonged stimulation of food intake, and especially food hoarding for 5-7 days, similar to its ability to elicit sustained increases in food in laboratory rats (15). As with the more acute effects of AgRP on food hoard size, the persisting increases in food hoarding tended to be inversely related to the peptide dose. Thus food hoarding was elevated to the greatest degree and for the longest period after the 0.1-nmol AgRP dose, to much lesser degree for the 1-nmol dose, and least for the two highest AgRP doses (10 and 100 nmol), the latter occurring despite the hamsters receiving more of the peptide than at the low doses. As is the case for the lingering effects on food intake by AgRP in the laboratory rats, and in the present study in Siberian hamsters, the mechanism underlying these prolonged effects is unknown.
Because we had some hamsters earning their food by wheel running, as well as others with food freely available but that had access to a running wheel, assessment of AgRP-induced nonspecific increases or decreases in locomotor activity, or indirect assessment of general malaise, as might be indicated by decreases in wheel running, was possible. AgRP did not significantly decrease wheel running and only increased wheel running in terms of the total revolutions for the first day after the 10-nmol dose in hamsters not foraging for their food (free wheel group). Thus these potential nonspecific effects of the peptide do not seem important for the interpretation of our finding that AgRP increased foraging (i.e., the increased foraging was not due to AgRP-induced increased locomotor activity). Foraging (pellets earned) was significantly increased for both foraging efforts at all time points for the lowest dose of AgRP compared with saline and was increased more sporadically as the peptide dose was increased until it was not different from saline at the highest dose of AgRP (100 nmol). One interpretation of the increased surplus pellets (earned pellets not eaten or not hoarded), seen especially by the 10 revolutions/pellet group at the 0.1- and 10-nmol doses of AgRP, is that this represents a relatively pure measure of foraging. That is, in these instances there was increased earned food that was not eaten or hoarded, and this increase was not due to nonspecific increased locomotor activity (see above), suggesting that AgRP may be a trigger for foraging, as has been suggested, but not explicitly tested for neuropeptide Y (16, 32, 34).
Collectively, the present studies demonstrated marked and prolonged increased food hoarding primarily, and food intake secondarily, by Siberian hamsters given third ventricular injections of AgRP-(83-132), as well as increased foraging for food. The increased food hoarding by AgRP may at least partially underlie the fasting-induced increases in food hoarding seen by this species and is consistent with the fasting-induced increases in arcuate nucleus AgRP gene expression by Siberian hamsters. Clearly, our understanding of the physiological mechanisms underlying this appetitive phase of the ingestive behavior sequence lags far behind advances made in the consummatory phase and seems needed for a deeper understanding of ingestive behavior. Perhaps studies such as the present one where animals are given the opportunity to display appetitive behaviors (foraging and hoarding) in addition to consummatory behavior (food intake) may help define the neurochemical underpinnings for each of these phases of ingestive behavior.
This work was supported, in part by National Science Foundation Grant IBN-9876495 and a Georgia State University Research Program Enhancement grant to T. J. Bartness.
We thank M. Acharya and C. Brothers and the Georgia State Animal Care personnel: D. Blake, D. Marshall, A. Gibson, and A. Po for assistance in keeping the equipment clean and in operating condition and Dr. R. Seeley for helpful discussions.
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. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society