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The effects of prior weight reduction on the running wheel-induced feeding suppression in rats
Behavioural Processes 82 (2009) 56–61
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The effects of prior weight reduction on the running wheel-induced feeding suppression in rats Amanda Hertel, Laura Botzang, Graham G. Parfeniuk, Roelof Eikelboom ∗ Department of Psychology, Wilfrid Laurier University, 75 University Ave West, Waterloo, ON, Canada N2L 3C5
a r t i c l e
i n f o
Article history: Received 4 December 2008 Received in revised form 25 March 2009 Accepted 23 April 2009 Keywords: Wheel running Feeding Rats Food restriction Body weight
a b s t r a c t Adult male rats given ad lib access to food and a running wheel show an initial feeding and weight suppression. Over 6–10 days feeding recovers, but body weight remains low. It is not clear which effect is primary, the wheel-induced feeding or weight change. To test this, rats were first restricted to 15 g of food a day for 8 or 16 days to reduce their weight relative to control non-restricted rats. They were then returned to ad lib feeding and half the restricted and non-restricted control rats were introduced to the wheel either immediately (Experiment 1) or 4 days later (Experiment 2). Food intake, body weight, and wheel running were monitored throughout the experiments. At the return to ad lib feeding, prior food restriction elevated feeding. Both immediate and delayed wheel access suppressed feeding in both groups of wheel access rats compared to the appropriate control rats. Feeding history did not have a significant effect on wheel running. The wheel-induced reductions in feeding from baseline were similar in the weight reduced and normal weight animals suggesting that prior weight restriction did not prevent the onset of the wheel-induced feeding suppression. It is therefore suggested that the feeding suppression is not driven by a reduced weight set point. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In some situations the relationship between energy intake and expenditure seems paradoxical. When male rats are started on an exercise program, increasing their energy expenditure, one might expect to see an increase in food intake, but in fact initially their food intake goes down. This feeding suppression is seen with forced exercise (Crews et al., 1969; Pitts et al., 1971; Rivest and Richard, 1990a,b; Stevenson et al., 1966) which may be stressful, as well as with voluntary, self-initiated exercise (Afonso and Eikelboom, 2003; Eckel and Moore, 2004; Kawaguchi et al., 2005; Lattanzio and Eikelboom, 2003; Looy and Eikelboom, 1989; Tokuyama et al., 1982). The suppression in feeding induced by wheel introduction is even evident when animals are on a restricted feeding schedule (Dwyer and Boakes, 1997; Lett et al., 2001; Routtenberg, 1968; Routtenberg and Kuznesof, 1967). If the food restriction is severe enough (1 h of food a day) it can prove fatal for most rats, a procedure that has been labeled the activity anorexia paradigm and has been suggested as an animal model of anorexia nervosa (Epling and Pierce, 1988; Epling et al., 1983). The wheel-induced feeding suppression in ad lib fed rats results in a drop in food consumption by about 25% from their baseline for several days before food intake
∗ Corresponding author. Tel.: +1 519 884 1970x3465; fax: +1 519 746 7605. E-mail address: [email protected] (R. Eikelboom). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.04.010
recovers over about 8–10 days (Afonso and Eikelboom, 2003). Eventually the feeding is restored and replaced by an increase in food intake to levels above that of animals without wheel access. A second effect evident with wheel introduction is a failure to gain weight at a rate comparable to controls, and occasionally a decrease in body weight, that is maintained for as long as the animal has wheel access (Afonso and Eikelboom, 2003; Collier, 1970; Looy and Eikelboom, 1989). It should be noted that male rats housed in a laboratory setting given ad lib food will gain weight steadily over the ages used in these experiments. The relationship between the reduced weight gain and the feeding suppression is not clear. Is the reduced weight gain a consequence of a wheel-induced feeding reduction or does the exercise induce a change in the internal weight set point which in turn reduces feeding? The feeding suppression may be secondary to the reduced weight gain as the feeding reduction is only temporary, while the reduced weight gain lasts as long as the animal has wheel access (Collier, 1970). Therefore, one might expect that if the animal returned to its control body weight the feeding suppression should be elicited again. This is what occurs. When a wheel is reintroduced after a 10 day absence period, during which animals have the opportunity to gain weight at a normal rate, there is a reoccurrence of the feeding suppression (Looy and Eikelboom, 1989; Mueller et al., 1999). Additionally, animals given alternate day wheel access show a much longer feeding suppression; perhaps because weight and food consumption was increased on the intervening no wheel days (Mueller et al., 1997).
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Wheel access may cause a feeding suppression because running induces an affective change that results in food avoidance (Lett et al., 1996, 1998). Rats are sensitive to their internal state and events which change their internal affective state (in either a positive or a negative direction) result in a food avoidance (Parker, 1995). The recovery of feeding might represent an increasing familiarity with the internal affective state induced by running. The finding that with a novel, attractive food the wheel-induced feeding suppression is stronger and longer lasting is consistent with this affective learning explanation (Satvat and Eikelboom, 2006). In this case the reduced weight gain might be either an indirect consequence of the period of suppressed feeding or an unrelated effect of the increased energy expenditure induced by the running. One way to address the food/weight relationship is to determine how feeding is affected if the rats’ weights are reduced prior to wheel introduction. If wheel running induces a change in the rats’ energy balance and weight “set point” which results in a feeding suppression then with a reduced weight there should be no feeding suppression. Alternately, if the wheel induces a change in affective state leading to food avoidance, changing the weight prior to wheel introduction should not significantly change the wheelinduced feeding suppression. Thus in these experiments the food available to animals was restricted, reducing their weight to a point well below that seen with ad lib feeding and wheel access. Rats were then put back on an ad lib feeding schedule and introduced to a wheel either immediately (Experiment 1) or 4 days later (Experiment 2) to see if wheel access still suppressed feeding. The 4 day gap between the return to ad lib food access and wheel introduction in Experiment 2 permitted the immediate effects of the running wheel on feeding to be assessed without the confound of the initial large increase and other changes in feeding evident over the first few days at the reintroduction of ad lib food. 2. Methods 2.1. Subjects Sixty-four (Experiment 1) and eighty (Experiment 2) male Sprague–Dawley rats from Charles River Canada, St. Constant Quebec, weighing between 200 and 225 g (47–50 days old) at arrival were housed individually in plastic shoebox cages (46 cm × 24 cm × 20 cm) with limited environmental enrichment (8 cm diameter ABS tubes for shelter). The animals were housed in a colony room with a 12:12 light:dark cycle (lights on at 07:00 h) and maintained at a temperature of approximately 21 ◦ C. Harlan Teklad Rodent 8640 diet pellets and tap water were available ad lib upon arrival and during the baseline period. All handling and manipulations of the rats took place during the light phase between 10:00 and 11:30 h throughout both experiments. The cages were changed twice a week when rats were in the wheel cages and once a week when they were in the shoebox cages (less bedding material was used in the wheel cages to prevent blocking the wheels). All procedures in this experiment were approved by the Wilfrid Laurier University Animal Care Committee following the guidelines and policies of the Canadian Council on Animal Care. 2.2. Apparatus 16 (Experiment 1) and 20 (Experiment 2) Nalgene (33 cm diameter × 11 cm wide) wheel cages (46 cm × 24 cm × 20 cm, the same size as the no wheel cages) were used with the Vital View IV data collection system, MiniMitter Ltd., which recorded wheel turns every second when the rats were in the wheels.
Fig. 1. (A) Mean food consumption (±SEM) for rats in the four groups over the course of Experiment 1; NW refers to rats without wheel access; W for rats with wheel access; NR for rats without food restriction and R for rats with food restriction in Phase I. In Phase II prior restriction elevated and wheel access suppressed food consumption but these effects did not interact. (B) Mean body weight (±SEM) for rats in the four groups over the experiment. In Phase II, wheel access resulted in decreased weight compared to controls a difference that became more pronounced over days. (C) Mean night time wheel turns (±SEM) for rats in the two groups with wheel access over Phase II of the experiment. Wheel running during Phase II increased over days but was not different between the previously restricted and non-restricted groups.
2.3. Procedure 2.3.1. Experiment 1 Due to equipment limitations both experiments were run in two equivalent replications. Upon their arrival in the lab, the rats were placed in plastic shoebox cages and habituated for 12 days to the colony and the weighing procedure. The next 3 days were the baseline period (Days −2 to 0). The experiment proper was divided into two phases; Phase I, the food restriction phase (Days 1–8) and Phase II, the wheel introduction phase (Days 9–16). Rats were randomly assigned to four treatment groups (n = 16). During the food restriction phase, two groups of rats were put on food restriction (R groups), with ad lib access to water. These rats were given 15 g of food each day at 11:00 h, for those 8 days (about 50% of their daily ad lib consumption, see Fig. 1). As a precaution, if a rat’s weight had dropped below 85% of its initial baseline weight the food provided would have been increased, but this proved unnecessary. During these days the animals in the two non-restricted groups (NR groups)
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were maintained on ad lib food and water and continued to increase in weight. At the start of Phase II (Day 9) all food restricted rats were returned to ad lib food access, so all animals received ad lib food and water for the remainder of the experiment. On this same day, two groups of rats (one food restricted and one ad lib fed group) were given ad lib access to a running wheel (W groups; NW refers to the rats without wheel access). Rats were monitored for a further 8 days (Days 9–16). For the duration of the experiment wheel running data, food and weight were collected daily. Food intake was measured by weighing the initial lab pellets and the remaining pellets and large crumbs 24 h later. Wheel turn counts were collected once every second and then analyzed as total number of wheel turns a night, as most running occurred in the night. 2.3.2. Experiment 2 The design and procedure in this experiment were the same as the first except the duration of various phases differed and food restoration and wheel access were offset by 4 days. Phase I where half the rats were food restricted (as in Experiment 1) lasted 16 days (Days 1–16) so that after Phase II at wheel introduction the groups would still differ in weight. Then ad lib food was made available to all rats from Days 17 to 20 (Phase II). In Phase III, wheel introduction to half the rats in the previously restricted and chronically ad lib fed groups occurred after food was measured on Day 20 and rats were followed for 16 days of wheel access (Days 21–36). 2.4. Analysis Wheel × Restriction × Days analyses of variance (ANOVA) were performed on feeding and weight. The 3 days of baseline (Days −2 to 0) were analyzed as a 3 day repeated measures design. For Phase I, II, and III (in Experiment 2) the number of days used in the ANOVA were the duration of the phase (8 or 16 days). When appropriate, these repeated measure ANOVAs were followed by Wheel × Restriction ANOVAs for each day. In one replication of Experiment 1, wheel running data were not collected for days 11–14 due to an equipment malfunction. Thus wheel running data were analyzed for the first and last 2 days of wheel access in a Restriction × Days ANOVA. Significance was set at p < 0.05 and for all repeated factors results are reported as significant only if also significant with the Greenhouse–Geisser correction for violations of sphericity. 3. Results
significant Wheel effects for Days 9–13 (smallest F(1, 60) = 6.72, p < 0.01), and significant Restriction effects for Days 9–16 (smallest F(1, 60) = 4.83, p < 0.05). The Wheel × Restriction interaction was never significant; suggesting that the wheel-induced feeding suppression was not changed by prior food restriction. It is evident from Fig. 1A that although both previously food restricted rats demonstrated a hyperphagia over these days of ad lib food access, the animals that received wheel access showed a feeding suppression relative to those in the parallel control group that did not. 3.1.2. Weight Fig. 1B shows the mean weight of the animals for the entire experiment. The weights during the baseline period did not differ for the four groups. For the three baseline days the mixed ANOVA revealed only a significant days effect, F(2, 120) = 366.75, p < 0.001, but no group differences or interactions. The ANOVA of the weight data for Phase I revealed significant Restriction, F(1, 60) = 88.12, p < 0.001, Days, F(7, 420) = 137.73, p < 0.001 and a Restriction × Days interaction, F(7, 420) = 358.83, p < 0.001. In the individual days ANOVA only the Restriction effect was significant (smallest F (1, 60) = 7.73, p < 0.01). By the end of Phase I the restricted rats had a much lower average weight than the ad lib fed rats (approximately 81% of ad lib fed rats). During Phase II the Wheel × Restriction × Days ANOVA of body weight revealed significant Wheel, F (1, 60) = 4.13, p < 0.05, Restriction, F (1, 60) = 16.27, p < 0.001 and Days effects, F(7, 420) = 540.57, p < 0.001. The Wheel × Days, F(7, 420) = 10.74, p < 0.001, and Restriction × Days, F(7, 420) = 29.84, p < 0.001, interactions were also both significant. The previously restricted animals were increasing their weight rapidly over these days but did not reach the weight of ad lib fed rats. Wheel access rats were gaining weight at a slower pace than rats without wheel access. ANOVAs for individual days revealed a significant Restriction (smallest F(1, 60) = 4.91, p < 0.05) effect for all these days and a significant Wheel effect for Days 11–16 (smallest F(1, 60) = 4.04, p < 0.05). By the end of Phase II the wheel access rats weighed less than rats without wheels. The effects of prior food restriction were still evident, but were decreasing over days. 3.1.3. Wheel running There were no significant differences in wheel running between the two groups with wheel access (see Fig. 1C). Only on the last 2 days of running, Days 15 and 16, did a 2 day ANOVA reveal a significant days effect, F(1, 30) = 10.38, p < 0.01, suggesting that rats were still increasing their running over these days. The similar levels of running imply that any group differences in feeding and weight could not be attributed to a difference in amount of running.
3.1. Experiment 1 3.2. Experiment 2 3.1.1. Feeding Fig. 1A shows the mean daily food consumption of each group of rats over the duration of the experiment. The repeated measures ANOVA analyzing the food consumption data during the three baseline days (Days −2 to 0) found only a significant Days effect, F(2, 120) = 23.42, p < 0.001. Before the experimental manipulations of Phase I and II there were no differences in food consumption. Phase I food data were not analyzed as the restricted rats were eating a fixed amount of food and the two ad lib feeding groups did not differ in consumption (Fig. 1A). The 8 day repeated measures ANOVA for Phase II, Days 9–16, revealed a significant effect of Wheel, F(1, 60) = 14.62, p < 0.001, Restriction, F(1, 60) = 39.80, p < 0.001, and Days, F(7, 420) = 10.94, p < 0.001 on feeding. There were also significant Wheel × Days, F(7, 420) = 6.79, p < 0.001, and Restriction × Days, F(7, 420) = 26.53, p < 0.001, interactions. Subsequent single day ANOVAs revealed
3.2.1. Feeding Fig. 2A shows the mean daily food consumption for rats in the four groups over the duration of the second experiment. The mixed ANOVA analyzing the feeding data for the 3 baseline days found a significant Days effect, F(2, 152) = 5.73, p < 0.01, a Restriction × Days, F(2, 152) = 3.10, p < 0.05, and a Restriction × Wheel × Days triple interaction, F(2, 152) = 3.25, p < 0.05. Feeding data for Phase I were not analyzed as the restricted animals were consuming a fixed amount of food. Fig. 2A suggests the two ad lib fed groups did not differ in food consumption. The data for Phase II (Days 17–20) were analyzed using a mixed ANOVA, and there was a main effect of Restriction, F(1, 76) = 23.53, p < 0.001, Days, F(3, 228) = 34.44, p < 0.001, and also a significant Restriction × Days interaction, F(3, 228) = 32.42, p < 0.001. Individual day ANOVAs revealed significant Restriction effects on feeding only for Day 17, F(1, 76) = 99.61, p < 0.001, and Day 19, F(1, 76) = 6.12,
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3.2.2. Weight Fig. 2B shows the mean daily weight for each group of rats for the duration of the experiment. For the 3 days of baseline, a mixed ANOVA revealed only a significant effect of Days, F(2, 152) = 99.77, p < 0.001. Prior to the manipulations in Phase II and III, there were no differences in the weights of the rats in the four groups. The weight data for Phase I were analyzed using a 16 day Restriction × Wheel × Days mixed ANOVAs. In this phase the Restriction, F(1, 76) = 260.60, p < 0.001, Days, F(15, 1140) = 183.35, p < 0.001, effects and the Restriction × Days interaction, F(15, 1140) = 362.15, p < 0.001. Food restriction was successful in reducing the weight of the rats, an effect that increased over days. The weight data for Phase II, Days 17–20, were also analyzed using a mixed ANOVA. There were significant effects of Restriction, F(1, 76) = 141.99, p < 0.001, Days, F(3, 228) = 147.08, p < 0.001, and a Restriction × Days interaction, F(3, 228) = 14.81, p < 0.001. A one-way Restriction × Wheel ANOVA on the final day of Phase II (Day 20) showed that while there was still a weight difference due to prior restriction, F(1, 76) = 112.46, p < 0.001, there were no weight differences between the wheel and non-wheel access groups. At wheel introduction the animals with prior food restriction weighed 83% of the weight of ad lib fed rats. For Phase III, Days 21–36, the weight data were analyzed using a Wheel × Restriction × Days ANOVAs which showed effects of Wheel F(1, 76) = 4.42, p < 0.05, Restriction, F(1, 76) = 30.45, p < 0.001) and Days, F(15, 1140) = 421.14, p < 0.001. The Wheel × Days, F(15, 1140) = 12.00, p < 0.001, and Restriction × Days, F(15, 1140) = 28.38, p < 0.001, interactions were also significant. Individual day Wheel × Restriction ANOVAs revealed significant Restriction effects for all days (smallest F(1, 76) = 10.31, p < 0.01) and significant Wheel effects from Day 27 onward (smallest F(1, 76) = 4.55, p < 0.05). Never was the Wheel × Restriction interaction significant. Rats with wheel access were gaining weight at a slower rate than those without wheels, and the weight difference became more evident over the 16 days of wheel access.
Fig. 2. (A) Mean food consumption (±SEM) for rats in the four groups over the course of Experiment 2; NW refers to rats without wheel access; W for rats with wheel access; NR for rats without food restriction and R for rats with food restriction in Phase I. In Phase II prior restriction elevated food consumption and in Phase III wheel introduction suppressed feeding for both previously restricted and non-restricted groups. (B) Mean body weight (±SEM) for rats in the four groups over the course of the experiment. Food restriction in Phase I decreased rats’ weight. The rats that received a wheel in Phase III showed a significantly lower weight when compared to the appropriate controls, a difference that became more pronounced over days. (C) Mean night time wheel turns (±SEM) for rats in the two groups with wheel access over Phase III of the experiment. Wheel running increased over days but did not show any differences between the previously restricted and non-restricted rats.
p < 0.05. There were no differences between the groups that would or would not receive wheel access. The data for Phase III were analyzed using a 16 Day × Wheel × Restriction ANOVA. The ANOVA revealed significant effects of Wheel, F(1, 76) = 10.91, p < 0.001, Restriction, F(1, 76) = 8.57, p < 0.01, and Days, F(15, 1140) = 5.47, p < 0.001. The Wheel × Days, F(15, 1140) = 8.51, p < 0.001, and Restriction × Days, F(15, 1140) = 6.49, p < 0.001, interactions were also significant. The Wheel × Restriction ANOVA for each day revealed significant Wheel effects from Days 21 to 29 (smallest F(1, 76) = 4.20, p < 0.05) and significant Restriction from Days 21 to 29 (except for Day 27) (smallest F(1, 76) = 6.63, p < 0.05). None of the Wheel × Restriction interactions were significant. Prior food restriction increased food consumption for about 13 days from the reintroduction of ad lib food on Day 17. Wheel access reduced food consumption relative to the appropriate controls from the first day of wheel introduction for about 9 days.
3.2.3. Wheel running For two rats (one in each wheel group) the data collection were compromised so the running data for these animals were removed from the analysis resulting in 19 animals in each wheel access group. There were no significant differences in wheel running between the two groups with wheel access (see Fig. 2C). An analysis of Days 21–36 of wheel access revealed only effects of Days F(15, 540) = 22.49, p < 0.001). Both groups increased running over days at a comparable rate. 4. Discussion The results from the present experiments demonstrate that the wheel-induced reductions in feeding were similar in the weight reduced and normal weight animals suggesting that prior weight restriction did not prevent the onset of the wheel-induced feeding suppression. It is therefore suggested that the feeding suppression is not driven by a reduced weight set point. The degree of weight suppression induced by our restricted feeding, 81% (Experiment 1) or 83% (Experiment 2; at wheel introduction), is similar to that seen in prior work in our laboratory with similar rats and food in which the wheel-induced weight ranges from 84% to 90% relative to nonwheel rats (Afonso and Eikelboom, 2003; Lattanzio and Eikelboom, 2003; Looy and Eikelboom, 1989; Mueller et al., 1997). In Experiment 1 there appeared to be a 1 day delay in the effect of the wheel on feeding, probably due to the initial hyperphagia induced by the return of ad lib food to food restricted rats. Experiment 2 involved a 4 day delay between the return to ad lib food access and the introduction of the running wheel. This design eliminated the delay in
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the effect of the wheel and the wheel-induced feeding suppression was evident on the first day of wheel access. Both the prior food restriction and wheel access had independent effects on feeding. Prior food restriction increased feeding when ad lib access was restored, an increase that became smaller over days but shows considerable variability as noted before (Harris and Martin, 1984). Wheel introduction suppressed feeding relative to no wheel control rats; in both experiments the suppression was strongest on the second and third day before gradually returning to levels similar to controls. The duration of the suppression appears longer in Experiment 2 perhaps because of the procedural difference from Experiment 1. In both experiments the previously food restricted rats given wheel access showed a feeding suppression compared to rats with an equivalent feeding history but no wheel access, even though they ate as much (in relative terms) as the no-wheel no-restriction control rats. The results of these experiments suggest that the feeding suppression is not a consequence of a wheel-induced change in body weight “set point” as suggested by Collier (1970), in that previously restricted rats were still showing a feeding suppression despite their reduced weight. In Experiment 2, on the first day of Phase III, the feeding suppression seen in the previously restricted rats with wheel access relative to the previously restricted rats without wheel access appears as more an increase in consumption in the no wheel controls than a decrease in consumption in the wheel access rats. The reason for this unusual feeding comparison maybe explained by considering the nature of the hyperphagia induced by previous restriction after a return to ad lib food access. What is unusual is that on the days following this binge, rats show a “post-gorging behavioral low” during which they consume less food, regardless of the remaining caloric deficit (Lockard, 1967; see also Armstrong et al., 1980) followed by increasing feeding above the levels of nonpreviously restricted control rats. The rats with wheel access failed to show this rebound to higher levels of consumption, assumedly as a result of the wheel access suppressing feeding. The complex ad lib feeding behavior after deprivation in control rats is something we are exploring further and may represent an affective reaction to the first days over consumption (Hertel & Eikelboom unpublished). In previous work it was suggested the degree of feeding suppression was a function of the intensity of the exercise (Epling and Pierce, 1992; Katch et al., 1979). In both experiments there were no obvious differences in wheel running in the two groups of rats (previously restricted and ad lib) given wheel access, so it is unlikely that any feeding suppression difference was being masked by differences in running. While the relationship between the wheel-induced suppression with ad lib feeding and the activity anorexia procedure is not clear, a number of activity anorexia studies have manipulated weight prior to the introduction of the wheel (Dwyer and Boakes, 1997; Lett et al., 2001; Routtenberg, 1968). Activity anorexia is complicated by the fact that with restricted feeding the animals have to adjust to the timing of the food availability and body weight may not be free to change at wheel introduction. These activity anorexia studies have focused on the effects of learning about food restriction before wheel introduction and then determining if the wheel access can still result in a suppressed intake. While not every study ran all the appropriate controls, it can generally be concluded that even with prior weight loss in activity anorexia the wheel-induced feeding suppression is still evident (Boakes and Dwyer, 1997; Lett et al., 2001; Routtenberg, 1968). Restraint stress has the ability to suppress eating for a short period and also results in a long lasting weight change (Harris et al., 1998, 2002; Rybkin et al., 1997). Three days of 3 h a day restraint reduces feeding by an amount similar to the wheel-induced feed-
ing suppression. While the feeding reduction usually lasts as long as the restraint occurs or only a few days longer, the weight reduction induced by this procedure lasts for a much longer period (Harris et al., 1998). In an experiment similar to the present experiment it was found that prior weight reduction did not markedly change the restraint-induced feeding effects (Harris et al., 2002). Restricted food (50% of ad lib feeding) access rats were restrained for 3 h a day for 3 days and then some were returned to ad lib feeding. Compared to non-restrained rats with the same feeding history the restraint-induced a feeding suppression when these rats were returned to ad lib feeding. When returned to ad lib feeding these restricted and restrained rats also regained their weight at a much slower rate resulting in a significant weight difference over the post-manipulation period. Like the wheel-induced feeding suppression (Kawaguchi et al., 2005) the restraint-induced feeding effect is reduced by a corticotropin-releasing factor receptor antagonist (Smagin et al., 1999). These similarities suggest that the wheel-induced feeding suppression and weight change may be mediated by voluntary running induced stress which may be more pronounced in the activity anorexia procedure. The extended weight gain reduction compared to controls after a decrease in feeding is not an uncommon pattern. In a number of experimental manipulations weight changes of male rats are very slow to return to baseline, if they ever do. A period of food restriction can result in a long lasting change in body weight (Brownlow et al., 1993). Restraint stress (Harris et al., 1998, 2002; Rybkin et al., 1997; Shimizu et al., 1989), a short exposure to shocks (Rickards et al., 1997) and social defeat (Meerlo et al., 1997) all have short-term effects on feeding and longer term consequences on body weight. Even pair housing male rats after a period of individual housing suppresses feeding for a day or two while body weight is suppressed for an extended period (O’Conner and Eikelboom, 2000). While the effects of wheel access on feeding and weight are noticeable in normal rats, in situations where animals are likely to become obese either due to diet (Eckel and Moore, 2004; Levin and Dunn-Meynell, 2006; Levin et al., 2003; Ricci and Levin, 2003) or genetics (Bi et al., 2005) the effects of exercise are often enough to prevent the obesity completely. With normal rat chow, over a longer period, feeding in male rats with wheel access increases above that of rats without wheel access, but weight of wheel access animals remains below that of rats living without wheels (Afonso and Eikelboom, 2003). In this context it should be pointed out that the weight of male rats living in what are typical laboratory conditions with ad lib food and very little space may be “abnormal”. There may be an optimal level of weight gain that is easily achieved and the absolute weight may not be as important as the weight gain. After the initial feeding suppression the weight gain seems similar, over the longer term, in animals with and without wheel access and in other procedures that induce a long-term weight difference. The findings of the present experiment extend the relevance of the wheel-induced feeding suppression model for the human disorder of anorexia nervosa (Epling and Pierce, 1988; Epling et al., 1983; Lattanzio and Eikelboom, 2003). In anorexia nervosa exercise is being recognized as an important factor in weight regulation and the severity of the disorder (Epling and Pierce, 1988; Davis, 1997; Hebebrand et al., 2003). A past qualitative study of humans who practice consistent exercise has shown that they are more prone to developing an eating disorder (Lane et al., 2005). Animal models like the activity anorexia and the wheel-induced feeding suppression procedures have focused more on the likelihood of exercise being a factor that can result in the development of eating disorders, with the initiation of exercise resulting in a feeding suppression. This study suggests that it might not matter when in the development of the disorder an exercise program is initiated, it might suppress feeding even in individuals who are already very weight reduced.
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Acknowledgments This research was funded, in part, by the Science and Technology Endowment Program at Wilfrid Laurier University to LB and by Ontario Mental Health Foundation funding to RE. References Afonso, V.M., Eikelboom, R., 2003. Relationship between wheel running, feeding, drinking, and body weight in male rats. Physiol. Behav. 80 (19–26), 35. Armstrong, S., Coleman, G., Singer, G., 1980. Food and water deprivation: changes in rat feeding, drinking, activity and body weight. Neurosci. Biobehav. Rev. 4, 377–402. Bi, S., Scott, K.A., Hyun, J., Ladenheim, E.E., Moran, T.H., 2005. Running wheel activity prevents hyperphagia and obesity in Otsuka Long-Evans Tokushima fatty rats: role of hypothalamic signaling. Endocrinology 146, 1676–1685. Boakes, R.A., Dwyer, D.M., 1997. Weight loss in rats produced by running: effects of prior experience and individual housing. Q. J. Exp. Psychol. 50, 129–148. Brownlow, B.S., Park, C.R., Schwartz, R.S., Woods, S.C., 1993. Effects of meal pattern during food restriction on body weight loss and recovery after refeeding. Physiol. Behav. 53, 421–424. Collier, G.H., 1970. Work: a weak reinforcer. Trans. N. Y. Acad. Sci. 32, 557–576. Crews, E.L., Fuge, W.K., Oscai, L.B., Holloszy, J.O., Shank, R.E., 1969. Weight, food intake, and body composition: effects of exercise and of protein deficiency. Am. J. Physiol. 216, 359–363. Davis, C., 1997. Eating disorders and hyperactivity: a psycho-biological perspective. Can. J. Psychiatry 42, 168–175. Dwyer, D.M., Boakes, R.A., 1997. Activity-based anorexia in rats as failure to adapt to a feeding schedule. Behav. Neurosci. 111, 195–205. Eckel, L.A., Moore, S.R., 2004. Diet-induced hyperphasia in the rat is influenced by sex and exercise. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R1800–R1805. Epling, W.F., Pierce, W.D., 1988. Activity-based anorexia: a biobehavioural perspective. Int. J. Eat. Disord. 7, 475–485. Epling, W.F., Pierce, W.D., 1992. Solving the Anorexia Puzzle. Hogrefe & Huber, Toronto. Epling, W.F., Pierce, W.D., Stephan, L., 1983. Theory of activity based anorexia. Int. J. Eat. Disord. 3, 27–46. Harris, R.B.S., Martin, R.J., 1984. Recovery of body weight from below “set point” in mature female rats. J. Nutr. 114, 1143–1150. Harris, R.B., Mitchell, T.D., Simpson, J., Redmann Jr., S.M., Youngblood, B.D., Ryan, D.H., 2002. Weight loss in rats exposed to repeated acute restraint stress is independent of energy or leptin status. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R77–R88. Harris, R.B., Zhou, J., Youngblood, B.D., Rybkin, I.I., Smagin, G.N., Ryan, D.H., 1998. Effect of repeated stress on body weight and body composition of rats fed lowand high-fat diets. Am. J. Physiol. 275 (6 Pt 2), R1928–R1938. Hebebrand, J., Exner, C., Hebebrand, K., Holtkamp, C., Casper, R.C., Remschmidt, H., Herpertz-Dahlmann, B., Klingenspor, M., 2003. Hyperactivity in patients with anorexia nervosa and in semistarved rats: evidence for a pivotal role of hypoleptinemia. Physiol. Behav. 79, 25–37. Katch, V.L., Martin, R., Martin, J., 1979. Effects of exercise intensity on food consumption in the male rat. Am. J. Clin. Nutr. 32, 1401–1407. Kawaguchi, M., Scott, K.A., Moran, T.H., Bi, S., 2005. Dorsomedial hypothalamic corticotrophin-releasing factor mediation of exercise-induced anorexia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R1800–R1805. Lane, H., Whyte, G., McConell, A., MacLaren, D.P.M., Matheson, H., 2005. A qualitative study of sport and exercise participants eating attitudes and eating behaviour. J. Sports Sci. 2, 230–258.
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Lattanzio, S.B., Eikelboom, R., 2003. Wheel access duration in rats. I. Effects on feeding and running. Behav. Neurosci. 117, 496–504. Lett, B.T., Grant, V.L., Gaborko, L.L., 1996. A small amount of wheel running facilitates eating in nondeprived rats. Behav. Neurosci. 110, 1492–1495. Lett, B.T., Grant, V.L., Koh, M.T., Parsons, J.F., 1998. Chlordiazepoxide attenuates activity-induced anorexia and weight loss in rats. Exp. Clin. Psychopharmacol. 6, 360–366. Lett, B.T., Grant, V.L., Smith, J.F., Koh, M.T., 2001. Preadaptation to the feeding schedule does not eliminate activity-based anorexia in rats. Q. J. Exp. Psychol. B 54, 193–199. Levin, B.E., Dunn-Meynell, A.A., 2006. Differential effects of exercise on body weigh gain and adiposity in obesity-prone and—resistant rats. Int. J. Obes. 30, 722–727. Levin, B.E., Dunn-Meynell, A.A., McMinn, J.E., Alperovich, M., Cunningham-Bussel, A., Chua, S.C., 2003. A new obesity-prone, glucose-intolerant rat strain (F. DIO). Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R1184–R1191. Lockard, R.B., 1967. Replacement behavior following deprivation of food, water, running, or light. Psychol. Rep. 21, 753–769. Looy, H., Eikelboom, R., 1989. Wheel running, food intake, and body weight in male rats. Physiol. Behav. 45, 403–405. Meerlo, P., Overkamp, G.J., Koolhaas, J.M., 1997. Behavioural and physiological consequences of a single social defeat in Roman high- and low-avoidance rats. Psychoneuroendocrinology 22, 155–168. Mueller, D.T., Herman, G., Eikelboom, R., 1999. Effects of short- and long-term wheel deprivation on running. Physiol. Behav. 66, 101–107. Mueller, D.T., Loft, A., Eikelboom, R., 1997. Alternative-day wheel access: effects on feeding, body weight, and running. Physiol. Behav. 62, 905–908. O’Conner, R., Eikelboom, R., 2000. The effects of changes in housing on feeding and wheel running. Physiol. Behav. 68, 361–371. Parker, L.A., 1995. Rewarding drugs produce taste avoidance, but not taste aversion. Neurosci. Biobehav. Rev. 19, 143–157. Pitts, G.C., Bull, L.S., Hollifield, H., 1971. Physiologic changes in composition and mass of total body adipose tissue. Am. J. Physiol. 221, 961–966. Ricci, M.R., Levin, B.E., 2003. Ontogeny of diet-induced obesity in selectively bred Sprague–Dawley rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R610–R618. Rickards, S.L., Job, R.F.S., Boakes, R.A., 1997. Effects of acute shock on body weight are mediated by changes in food intake. Anim. Learn. Behav. 25, 437–445. Rivest, S., Richard, D., 1990a. Hypothalamic paraventricular nucleus lesions do not prevent anorectic effect of exercise in male rats. Am. J. Physiol. 259, R579–R584. Rivest, S., Richard, D., 1990b. Involvement of corticotrophin-releasing factor in the anorexia induced by exercise. Brain Res. Bull. 25, 169–172. Routtenberg, A., 1968. Self-starvation of rats living in activity wheels: adaptation effects. J. Comp. Physiol. Psychol. 66, 234–238. Routtenberg, A., Kuznesof, A.W., 1967. Self-starvation of rats living in activity wheels on a restricted feeding schedule. J. Comp. Physiol. Psychol. 64, 414–421. Rybkin, I.I., Zhou, Y., Volaufova, J., Smagin, G.N., Ryan, D.H., Harris, R.B., 1997. Effects of restraint stress on food intake and body weight is determined by time of day. Am. J. Physiol. 273 (5 Pt 2), R1612–R1622. Satvat, E., Eikelboom, R., 2006. Dissociation of conditioned and unconditioned factors in the running-induced feeding suppression. Physiol. Behav. 89, 428–437. Shimizu, N., Oomura, Y., Kai, Y., 1989. Stress-induced anorexia in rats mediated by serotonergic mechanisms in the hypothalamus. Physiol. Behav. 46, 835–841. Smagin, G.N., Howell, L.A., Redmann Jr., S., Ryan, D.H., Harris, R., 1999. Prevention of stress-induced weight loss by third ventricle CRF receptor antagonist. Am. J. Physiol. 276 (5 Pt 2), R1461–R1468. Stevenson, J.A., Box, B.M., Feleki, V., Beaton, J.R., 1966. Bouts of exercise and food intake in the rat. J. Appl. Physiol. 21, 118–122. Tokuyama, K., Saito, M., Okuda, H., 1982. Effects of wheel running on food intake and weight gain of male and female rats. Physiol. Behav. 28, 899–903.
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Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
The effects of prior weight reduction on the running wheel-induced feeding suppression in rats Amanda Hertel, Laura Botzang, Graham G. Parfeniuk, Roelof Eikelboom ∗ Department of Psychology, Wilfrid Laurier University, 75 University Ave West, Waterloo, ON, Canada N2L 3C5
a r t i c l e
i n f o
Article history: Received 4 December 2008 Received in revised form 25 March 2009 Accepted 23 April 2009 Keywords: Wheel running Feeding Rats Food restriction Body weight
a b s t r a c t Adult male rats given ad lib access to food and a running wheel show an initial feeding and weight suppression. Over 6–10 days feeding recovers, but body weight remains low. It is not clear which effect is primary, the wheel-induced feeding or weight change. To test this, rats were first restricted to 15 g of food a day for 8 or 16 days to reduce their weight relative to control non-restricted rats. They were then returned to ad lib feeding and half the restricted and non-restricted control rats were introduced to the wheel either immediately (Experiment 1) or 4 days later (Experiment 2). Food intake, body weight, and wheel running were monitored throughout the experiments. At the return to ad lib feeding, prior food restriction elevated feeding. Both immediate and delayed wheel access suppressed feeding in both groups of wheel access rats compared to the appropriate control rats. Feeding history did not have a significant effect on wheel running. The wheel-induced reductions in feeding from baseline were similar in the weight reduced and normal weight animals suggesting that prior weight restriction did not prevent the onset of the wheel-induced feeding suppression. It is therefore suggested that the feeding suppression is not driven by a reduced weight set point. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In some situations the relationship between energy intake and expenditure seems paradoxical. When male rats are started on an exercise program, increasing their energy expenditure, one might expect to see an increase in food intake, but in fact initially their food intake goes down. This feeding suppression is seen with forced exercise (Crews et al., 1969; Pitts et al., 1971; Rivest and Richard, 1990a,b; Stevenson et al., 1966) which may be stressful, as well as with voluntary, self-initiated exercise (Afonso and Eikelboom, 2003; Eckel and Moore, 2004; Kawaguchi et al., 2005; Lattanzio and Eikelboom, 2003; Looy and Eikelboom, 1989; Tokuyama et al., 1982). The suppression in feeding induced by wheel introduction is even evident when animals are on a restricted feeding schedule (Dwyer and Boakes, 1997; Lett et al., 2001; Routtenberg, 1968; Routtenberg and Kuznesof, 1967). If the food restriction is severe enough (1 h of food a day) it can prove fatal for most rats, a procedure that has been labeled the activity anorexia paradigm and has been suggested as an animal model of anorexia nervosa (Epling and Pierce, 1988; Epling et al., 1983). The wheel-induced feeding suppression in ad lib fed rats results in a drop in food consumption by about 25% from their baseline for several days before food intake
∗ Corresponding author. Tel.: +1 519 884 1970x3465; fax: +1 519 746 7605. E-mail address: [email protected] (R. Eikelboom). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.04.010
recovers over about 8–10 days (Afonso and Eikelboom, 2003). Eventually the feeding is restored and replaced by an increase in food intake to levels above that of animals without wheel access. A second effect evident with wheel introduction is a failure to gain weight at a rate comparable to controls, and occasionally a decrease in body weight, that is maintained for as long as the animal has wheel access (Afonso and Eikelboom, 2003; Collier, 1970; Looy and Eikelboom, 1989). It should be noted that male rats housed in a laboratory setting given ad lib food will gain weight steadily over the ages used in these experiments. The relationship between the reduced weight gain and the feeding suppression is not clear. Is the reduced weight gain a consequence of a wheel-induced feeding reduction or does the exercise induce a change in the internal weight set point which in turn reduces feeding? The feeding suppression may be secondary to the reduced weight gain as the feeding reduction is only temporary, while the reduced weight gain lasts as long as the animal has wheel access (Collier, 1970). Therefore, one might expect that if the animal returned to its control body weight the feeding suppression should be elicited again. This is what occurs. When a wheel is reintroduced after a 10 day absence period, during which animals have the opportunity to gain weight at a normal rate, there is a reoccurrence of the feeding suppression (Looy and Eikelboom, 1989; Mueller et al., 1999). Additionally, animals given alternate day wheel access show a much longer feeding suppression; perhaps because weight and food consumption was increased on the intervening no wheel days (Mueller et al., 1997).
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Wheel access may cause a feeding suppression because running induces an affective change that results in food avoidance (Lett et al., 1996, 1998). Rats are sensitive to their internal state and events which change their internal affective state (in either a positive or a negative direction) result in a food avoidance (Parker, 1995). The recovery of feeding might represent an increasing familiarity with the internal affective state induced by running. The finding that with a novel, attractive food the wheel-induced feeding suppression is stronger and longer lasting is consistent with this affective learning explanation (Satvat and Eikelboom, 2006). In this case the reduced weight gain might be either an indirect consequence of the period of suppressed feeding or an unrelated effect of the increased energy expenditure induced by the running. One way to address the food/weight relationship is to determine how feeding is affected if the rats’ weights are reduced prior to wheel introduction. If wheel running induces a change in the rats’ energy balance and weight “set point” which results in a feeding suppression then with a reduced weight there should be no feeding suppression. Alternately, if the wheel induces a change in affective state leading to food avoidance, changing the weight prior to wheel introduction should not significantly change the wheelinduced feeding suppression. Thus in these experiments the food available to animals was restricted, reducing their weight to a point well below that seen with ad lib feeding and wheel access. Rats were then put back on an ad lib feeding schedule and introduced to a wheel either immediately (Experiment 1) or 4 days later (Experiment 2) to see if wheel access still suppressed feeding. The 4 day gap between the return to ad lib food access and wheel introduction in Experiment 2 permitted the immediate effects of the running wheel on feeding to be assessed without the confound of the initial large increase and other changes in feeding evident over the first few days at the reintroduction of ad lib food. 2. Methods 2.1. Subjects Sixty-four (Experiment 1) and eighty (Experiment 2) male Sprague–Dawley rats from Charles River Canada, St. Constant Quebec, weighing between 200 and 225 g (47–50 days old) at arrival were housed individually in plastic shoebox cages (46 cm × 24 cm × 20 cm) with limited environmental enrichment (8 cm diameter ABS tubes for shelter). The animals were housed in a colony room with a 12:12 light:dark cycle (lights on at 07:00 h) and maintained at a temperature of approximately 21 ◦ C. Harlan Teklad Rodent 8640 diet pellets and tap water were available ad lib upon arrival and during the baseline period. All handling and manipulations of the rats took place during the light phase between 10:00 and 11:30 h throughout both experiments. The cages were changed twice a week when rats were in the wheel cages and once a week when they were in the shoebox cages (less bedding material was used in the wheel cages to prevent blocking the wheels). All procedures in this experiment were approved by the Wilfrid Laurier University Animal Care Committee following the guidelines and policies of the Canadian Council on Animal Care. 2.2. Apparatus 16 (Experiment 1) and 20 (Experiment 2) Nalgene (33 cm diameter × 11 cm wide) wheel cages (46 cm × 24 cm × 20 cm, the same size as the no wheel cages) were used with the Vital View IV data collection system, MiniMitter Ltd., which recorded wheel turns every second when the rats were in the wheels.
Fig. 1. (A) Mean food consumption (±SEM) for rats in the four groups over the course of Experiment 1; NW refers to rats without wheel access; W for rats with wheel access; NR for rats without food restriction and R for rats with food restriction in Phase I. In Phase II prior restriction elevated and wheel access suppressed food consumption but these effects did not interact. (B) Mean body weight (±SEM) for rats in the four groups over the experiment. In Phase II, wheel access resulted in decreased weight compared to controls a difference that became more pronounced over days. (C) Mean night time wheel turns (±SEM) for rats in the two groups with wheel access over Phase II of the experiment. Wheel running during Phase II increased over days but was not different between the previously restricted and non-restricted groups.
2.3. Procedure 2.3.1. Experiment 1 Due to equipment limitations both experiments were run in two equivalent replications. Upon their arrival in the lab, the rats were placed in plastic shoebox cages and habituated for 12 days to the colony and the weighing procedure. The next 3 days were the baseline period (Days −2 to 0). The experiment proper was divided into two phases; Phase I, the food restriction phase (Days 1–8) and Phase II, the wheel introduction phase (Days 9–16). Rats were randomly assigned to four treatment groups (n = 16). During the food restriction phase, two groups of rats were put on food restriction (R groups), with ad lib access to water. These rats were given 15 g of food each day at 11:00 h, for those 8 days (about 50% of their daily ad lib consumption, see Fig. 1). As a precaution, if a rat’s weight had dropped below 85% of its initial baseline weight the food provided would have been increased, but this proved unnecessary. During these days the animals in the two non-restricted groups (NR groups)
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were maintained on ad lib food and water and continued to increase in weight. At the start of Phase II (Day 9) all food restricted rats were returned to ad lib food access, so all animals received ad lib food and water for the remainder of the experiment. On this same day, two groups of rats (one food restricted and one ad lib fed group) were given ad lib access to a running wheel (W groups; NW refers to the rats without wheel access). Rats were monitored for a further 8 days (Days 9–16). For the duration of the experiment wheel running data, food and weight were collected daily. Food intake was measured by weighing the initial lab pellets and the remaining pellets and large crumbs 24 h later. Wheel turn counts were collected once every second and then analyzed as total number of wheel turns a night, as most running occurred in the night. 2.3.2. Experiment 2 The design and procedure in this experiment were the same as the first except the duration of various phases differed and food restoration and wheel access were offset by 4 days. Phase I where half the rats were food restricted (as in Experiment 1) lasted 16 days (Days 1–16) so that after Phase II at wheel introduction the groups would still differ in weight. Then ad lib food was made available to all rats from Days 17 to 20 (Phase II). In Phase III, wheel introduction to half the rats in the previously restricted and chronically ad lib fed groups occurred after food was measured on Day 20 and rats were followed for 16 days of wheel access (Days 21–36). 2.4. Analysis Wheel × Restriction × Days analyses of variance (ANOVA) were performed on feeding and weight. The 3 days of baseline (Days −2 to 0) were analyzed as a 3 day repeated measures design. For Phase I, II, and III (in Experiment 2) the number of days used in the ANOVA were the duration of the phase (8 or 16 days). When appropriate, these repeated measure ANOVAs were followed by Wheel × Restriction ANOVAs for each day. In one replication of Experiment 1, wheel running data were not collected for days 11–14 due to an equipment malfunction. Thus wheel running data were analyzed for the first and last 2 days of wheel access in a Restriction × Days ANOVA. Significance was set at p < 0.05 and for all repeated factors results are reported as significant only if also significant with the Greenhouse–Geisser correction for violations of sphericity. 3. Results
significant Wheel effects for Days 9–13 (smallest F(1, 60) = 6.72, p < 0.01), and significant Restriction effects for Days 9–16 (smallest F(1, 60) = 4.83, p < 0.05). The Wheel × Restriction interaction was never significant; suggesting that the wheel-induced feeding suppression was not changed by prior food restriction. It is evident from Fig. 1A that although both previously food restricted rats demonstrated a hyperphagia over these days of ad lib food access, the animals that received wheel access showed a feeding suppression relative to those in the parallel control group that did not. 3.1.2. Weight Fig. 1B shows the mean weight of the animals for the entire experiment. The weights during the baseline period did not differ for the four groups. For the three baseline days the mixed ANOVA revealed only a significant days effect, F(2, 120) = 366.75, p < 0.001, but no group differences or interactions. The ANOVA of the weight data for Phase I revealed significant Restriction, F(1, 60) = 88.12, p < 0.001, Days, F(7, 420) = 137.73, p < 0.001 and a Restriction × Days interaction, F(7, 420) = 358.83, p < 0.001. In the individual days ANOVA only the Restriction effect was significant (smallest F (1, 60) = 7.73, p < 0.01). By the end of Phase I the restricted rats had a much lower average weight than the ad lib fed rats (approximately 81% of ad lib fed rats). During Phase II the Wheel × Restriction × Days ANOVA of body weight revealed significant Wheel, F (1, 60) = 4.13, p < 0.05, Restriction, F (1, 60) = 16.27, p < 0.001 and Days effects, F(7, 420) = 540.57, p < 0.001. The Wheel × Days, F(7, 420) = 10.74, p < 0.001, and Restriction × Days, F(7, 420) = 29.84, p < 0.001, interactions were also both significant. The previously restricted animals were increasing their weight rapidly over these days but did not reach the weight of ad lib fed rats. Wheel access rats were gaining weight at a slower pace than rats without wheel access. ANOVAs for individual days revealed a significant Restriction (smallest F(1, 60) = 4.91, p < 0.05) effect for all these days and a significant Wheel effect for Days 11–16 (smallest F(1, 60) = 4.04, p < 0.05). By the end of Phase II the wheel access rats weighed less than rats without wheels. The effects of prior food restriction were still evident, but were decreasing over days. 3.1.3. Wheel running There were no significant differences in wheel running between the two groups with wheel access (see Fig. 1C). Only on the last 2 days of running, Days 15 and 16, did a 2 day ANOVA reveal a significant days effect, F(1, 30) = 10.38, p < 0.01, suggesting that rats were still increasing their running over these days. The similar levels of running imply that any group differences in feeding and weight could not be attributed to a difference in amount of running.
3.1. Experiment 1 3.2. Experiment 2 3.1.1. Feeding Fig. 1A shows the mean daily food consumption of each group of rats over the duration of the experiment. The repeated measures ANOVA analyzing the food consumption data during the three baseline days (Days −2 to 0) found only a significant Days effect, F(2, 120) = 23.42, p < 0.001. Before the experimental manipulations of Phase I and II there were no differences in food consumption. Phase I food data were not analyzed as the restricted rats were eating a fixed amount of food and the two ad lib feeding groups did not differ in consumption (Fig. 1A). The 8 day repeated measures ANOVA for Phase II, Days 9–16, revealed a significant effect of Wheel, F(1, 60) = 14.62, p < 0.001, Restriction, F(1, 60) = 39.80, p < 0.001, and Days, F(7, 420) = 10.94, p < 0.001 on feeding. There were also significant Wheel × Days, F(7, 420) = 6.79, p < 0.001, and Restriction × Days, F(7, 420) = 26.53, p < 0.001, interactions. Subsequent single day ANOVAs revealed
3.2.1. Feeding Fig. 2A shows the mean daily food consumption for rats in the four groups over the duration of the second experiment. The mixed ANOVA analyzing the feeding data for the 3 baseline days found a significant Days effect, F(2, 152) = 5.73, p < 0.01, a Restriction × Days, F(2, 152) = 3.10, p < 0.05, and a Restriction × Wheel × Days triple interaction, F(2, 152) = 3.25, p < 0.05. Feeding data for Phase I were not analyzed as the restricted animals were consuming a fixed amount of food. Fig. 2A suggests the two ad lib fed groups did not differ in food consumption. The data for Phase II (Days 17–20) were analyzed using a mixed ANOVA, and there was a main effect of Restriction, F(1, 76) = 23.53, p < 0.001, Days, F(3, 228) = 34.44, p < 0.001, and also a significant Restriction × Days interaction, F(3, 228) = 32.42, p < 0.001. Individual day ANOVAs revealed significant Restriction effects on feeding only for Day 17, F(1, 76) = 99.61, p < 0.001, and Day 19, F(1, 76) = 6.12,
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3.2.2. Weight Fig. 2B shows the mean daily weight for each group of rats for the duration of the experiment. For the 3 days of baseline, a mixed ANOVA revealed only a significant effect of Days, F(2, 152) = 99.77, p < 0.001. Prior to the manipulations in Phase II and III, there were no differences in the weights of the rats in the four groups. The weight data for Phase I were analyzed using a 16 day Restriction × Wheel × Days mixed ANOVAs. In this phase the Restriction, F(1, 76) = 260.60, p < 0.001, Days, F(15, 1140) = 183.35, p < 0.001, effects and the Restriction × Days interaction, F(15, 1140) = 362.15, p < 0.001. Food restriction was successful in reducing the weight of the rats, an effect that increased over days. The weight data for Phase II, Days 17–20, were also analyzed using a mixed ANOVA. There were significant effects of Restriction, F(1, 76) = 141.99, p < 0.001, Days, F(3, 228) = 147.08, p < 0.001, and a Restriction × Days interaction, F(3, 228) = 14.81, p < 0.001. A one-way Restriction × Wheel ANOVA on the final day of Phase II (Day 20) showed that while there was still a weight difference due to prior restriction, F(1, 76) = 112.46, p < 0.001, there were no weight differences between the wheel and non-wheel access groups. At wheel introduction the animals with prior food restriction weighed 83% of the weight of ad lib fed rats. For Phase III, Days 21–36, the weight data were analyzed using a Wheel × Restriction × Days ANOVAs which showed effects of Wheel F(1, 76) = 4.42, p < 0.05, Restriction, F(1, 76) = 30.45, p < 0.001) and Days, F(15, 1140) = 421.14, p < 0.001. The Wheel × Days, F(15, 1140) = 12.00, p < 0.001, and Restriction × Days, F(15, 1140) = 28.38, p < 0.001, interactions were also significant. Individual day Wheel × Restriction ANOVAs revealed significant Restriction effects for all days (smallest F(1, 76) = 10.31, p < 0.01) and significant Wheel effects from Day 27 onward (smallest F(1, 76) = 4.55, p < 0.05). Never was the Wheel × Restriction interaction significant. Rats with wheel access were gaining weight at a slower rate than those without wheels, and the weight difference became more evident over the 16 days of wheel access.
Fig. 2. (A) Mean food consumption (±SEM) for rats in the four groups over the course of Experiment 2; NW refers to rats without wheel access; W for rats with wheel access; NR for rats without food restriction and R for rats with food restriction in Phase I. In Phase II prior restriction elevated food consumption and in Phase III wheel introduction suppressed feeding for both previously restricted and non-restricted groups. (B) Mean body weight (±SEM) for rats in the four groups over the course of the experiment. Food restriction in Phase I decreased rats’ weight. The rats that received a wheel in Phase III showed a significantly lower weight when compared to the appropriate controls, a difference that became more pronounced over days. (C) Mean night time wheel turns (±SEM) for rats in the two groups with wheel access over Phase III of the experiment. Wheel running increased over days but did not show any differences between the previously restricted and non-restricted rats.
p < 0.05. There were no differences between the groups that would or would not receive wheel access. The data for Phase III were analyzed using a 16 Day × Wheel × Restriction ANOVA. The ANOVA revealed significant effects of Wheel, F(1, 76) = 10.91, p < 0.001, Restriction, F(1, 76) = 8.57, p < 0.01, and Days, F(15, 1140) = 5.47, p < 0.001. The Wheel × Days, F(15, 1140) = 8.51, p < 0.001, and Restriction × Days, F(15, 1140) = 6.49, p < 0.001, interactions were also significant. The Wheel × Restriction ANOVA for each day revealed significant Wheel effects from Days 21 to 29 (smallest F(1, 76) = 4.20, p < 0.05) and significant Restriction from Days 21 to 29 (except for Day 27) (smallest F(1, 76) = 6.63, p < 0.05). None of the Wheel × Restriction interactions were significant. Prior food restriction increased food consumption for about 13 days from the reintroduction of ad lib food on Day 17. Wheel access reduced food consumption relative to the appropriate controls from the first day of wheel introduction for about 9 days.
3.2.3. Wheel running For two rats (one in each wheel group) the data collection were compromised so the running data for these animals were removed from the analysis resulting in 19 animals in each wheel access group. There were no significant differences in wheel running between the two groups with wheel access (see Fig. 2C). An analysis of Days 21–36 of wheel access revealed only effects of Days F(15, 540) = 22.49, p < 0.001). Both groups increased running over days at a comparable rate. 4. Discussion The results from the present experiments demonstrate that the wheel-induced reductions in feeding were similar in the weight reduced and normal weight animals suggesting that prior weight restriction did not prevent the onset of the wheel-induced feeding suppression. It is therefore suggested that the feeding suppression is not driven by a reduced weight set point. The degree of weight suppression induced by our restricted feeding, 81% (Experiment 1) or 83% (Experiment 2; at wheel introduction), is similar to that seen in prior work in our laboratory with similar rats and food in which the wheel-induced weight ranges from 84% to 90% relative to nonwheel rats (Afonso and Eikelboom, 2003; Lattanzio and Eikelboom, 2003; Looy and Eikelboom, 1989; Mueller et al., 1997). In Experiment 1 there appeared to be a 1 day delay in the effect of the wheel on feeding, probably due to the initial hyperphagia induced by the return of ad lib food to food restricted rats. Experiment 2 involved a 4 day delay between the return to ad lib food access and the introduction of the running wheel. This design eliminated the delay in
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the effect of the wheel and the wheel-induced feeding suppression was evident on the first day of wheel access. Both the prior food restriction and wheel access had independent effects on feeding. Prior food restriction increased feeding when ad lib access was restored, an increase that became smaller over days but shows considerable variability as noted before (Harris and Martin, 1984). Wheel introduction suppressed feeding relative to no wheel control rats; in both experiments the suppression was strongest on the second and third day before gradually returning to levels similar to controls. The duration of the suppression appears longer in Experiment 2 perhaps because of the procedural difference from Experiment 1. In both experiments the previously food restricted rats given wheel access showed a feeding suppression compared to rats with an equivalent feeding history but no wheel access, even though they ate as much (in relative terms) as the no-wheel no-restriction control rats. The results of these experiments suggest that the feeding suppression is not a consequence of a wheel-induced change in body weight “set point” as suggested by Collier (1970), in that previously restricted rats were still showing a feeding suppression despite their reduced weight. In Experiment 2, on the first day of Phase III, the feeding suppression seen in the previously restricted rats with wheel access relative to the previously restricted rats without wheel access appears as more an increase in consumption in the no wheel controls than a decrease in consumption in the wheel access rats. The reason for this unusual feeding comparison maybe explained by considering the nature of the hyperphagia induced by previous restriction after a return to ad lib food access. What is unusual is that on the days following this binge, rats show a “post-gorging behavioral low” during which they consume less food, regardless of the remaining caloric deficit (Lockard, 1967; see also Armstrong et al., 1980) followed by increasing feeding above the levels of nonpreviously restricted control rats. The rats with wheel access failed to show this rebound to higher levels of consumption, assumedly as a result of the wheel access suppressing feeding. The complex ad lib feeding behavior after deprivation in control rats is something we are exploring further and may represent an affective reaction to the first days over consumption (Hertel & Eikelboom unpublished). In previous work it was suggested the degree of feeding suppression was a function of the intensity of the exercise (Epling and Pierce, 1992; Katch et al., 1979). In both experiments there were no obvious differences in wheel running in the two groups of rats (previously restricted and ad lib) given wheel access, so it is unlikely that any feeding suppression difference was being masked by differences in running. While the relationship between the wheel-induced suppression with ad lib feeding and the activity anorexia procedure is not clear, a number of activity anorexia studies have manipulated weight prior to the introduction of the wheel (Dwyer and Boakes, 1997; Lett et al., 2001; Routtenberg, 1968). Activity anorexia is complicated by the fact that with restricted feeding the animals have to adjust to the timing of the food availability and body weight may not be free to change at wheel introduction. These activity anorexia studies have focused on the effects of learning about food restriction before wheel introduction and then determining if the wheel access can still result in a suppressed intake. While not every study ran all the appropriate controls, it can generally be concluded that even with prior weight loss in activity anorexia the wheel-induced feeding suppression is still evident (Boakes and Dwyer, 1997; Lett et al., 2001; Routtenberg, 1968). Restraint stress has the ability to suppress eating for a short period and also results in a long lasting weight change (Harris et al., 1998, 2002; Rybkin et al., 1997). Three days of 3 h a day restraint reduces feeding by an amount similar to the wheel-induced feed-
ing suppression. While the feeding reduction usually lasts as long as the restraint occurs or only a few days longer, the weight reduction induced by this procedure lasts for a much longer period (Harris et al., 1998). In an experiment similar to the present experiment it was found that prior weight reduction did not markedly change the restraint-induced feeding effects (Harris et al., 2002). Restricted food (50% of ad lib feeding) access rats were restrained for 3 h a day for 3 days and then some were returned to ad lib feeding. Compared to non-restrained rats with the same feeding history the restraint-induced a feeding suppression when these rats were returned to ad lib feeding. When returned to ad lib feeding these restricted and restrained rats also regained their weight at a much slower rate resulting in a significant weight difference over the post-manipulation period. Like the wheel-induced feeding suppression (Kawaguchi et al., 2005) the restraint-induced feeding effect is reduced by a corticotropin-releasing factor receptor antagonist (Smagin et al., 1999). These similarities suggest that the wheel-induced feeding suppression and weight change may be mediated by voluntary running induced stress which may be more pronounced in the activity anorexia procedure. The extended weight gain reduction compared to controls after a decrease in feeding is not an uncommon pattern. In a number of experimental manipulations weight changes of male rats are very slow to return to baseline, if they ever do. A period of food restriction can result in a long lasting change in body weight (Brownlow et al., 1993). Restraint stress (Harris et al., 1998, 2002; Rybkin et al., 1997; Shimizu et al., 1989), a short exposure to shocks (Rickards et al., 1997) and social defeat (Meerlo et al., 1997) all have short-term effects on feeding and longer term consequences on body weight. Even pair housing male rats after a period of individual housing suppresses feeding for a day or two while body weight is suppressed for an extended period (O’Conner and Eikelboom, 2000). While the effects of wheel access on feeding and weight are noticeable in normal rats, in situations where animals are likely to become obese either due to diet (Eckel and Moore, 2004; Levin and Dunn-Meynell, 2006; Levin et al., 2003; Ricci and Levin, 2003) or genetics (Bi et al., 2005) the effects of exercise are often enough to prevent the obesity completely. With normal rat chow, over a longer period, feeding in male rats with wheel access increases above that of rats without wheel access, but weight of wheel access animals remains below that of rats living without wheels (Afonso and Eikelboom, 2003). In this context it should be pointed out that the weight of male rats living in what are typical laboratory conditions with ad lib food and very little space may be “abnormal”. There may be an optimal level of weight gain that is easily achieved and the absolute weight may not be as important as the weight gain. After the initial feeding suppression the weight gain seems similar, over the longer term, in animals with and without wheel access and in other procedures that induce a long-term weight difference. The findings of the present experiment extend the relevance of the wheel-induced feeding suppression model for the human disorder of anorexia nervosa (Epling and Pierce, 1988; Epling et al., 1983; Lattanzio and Eikelboom, 2003). In anorexia nervosa exercise is being recognized as an important factor in weight regulation and the severity of the disorder (Epling and Pierce, 1988; Davis, 1997; Hebebrand et al., 2003). A past qualitative study of humans who practice consistent exercise has shown that they are more prone to developing an eating disorder (Lane et al., 2005). Animal models like the activity anorexia and the wheel-induced feeding suppression procedures have focused more on the likelihood of exercise being a factor that can result in the development of eating disorders, with the initiation of exercise resulting in a feeding suppression. This study suggests that it might not matter when in the development of the disorder an exercise program is initiated, it might suppress feeding even in individuals who are already very weight reduced.
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