Food and water deprivation: Changes in rat feeding, drinking, activity and body weight

Neuroscience & Biobehaviora! Reviews, Vol. 4, pp. 377-402. Printed in the U.S.A.

Food and Water Deprivation: Changes in Rat Feeding, Drinking,

Activity and Body Weight

S T U A R T A R M S T R O N G , G R A H A M E C O L E M A N A N D G E O R G E S I N G E R

Depar tment o f Psychology, La Trobe University, Bundoora, Australia 3083

R e c e i v e d 13 D e c e m b e r 1979

ARMSTRONG, S., G. COLEMAN AND G. SINGER. Food and water deprivation: Changes in rat feeding, drinking, activity and body weight. NEUROSCI. BIOBEHAV. REV. 4(3) 377--402, 1980.nGroups of male rats totally food deprived for periods up to 96 hr, water ad lib do not totally regain body weight lost through deprivation. Degree of body weight defence was estimated by fitting statistically a curve to the predeprivation baseline data and extrapolating the curve over the deprivation and postdeprivation period. From group data, body weight is not fully defended. However, marked individual differences in degree of body weight defence were noted. During food deprivation the daily drinking cycle persists for approximately 48 hr, then a reduction in nocturnal drinking appears. General activity increases but only in the light period. After food restitution, the main hyperphagia is confined to the first post-deprivation day. Additional, subtle long term effects of overeating in the light period are also found. In contrast to food deprived groups, water deprived groups accurately defend body weight. During water deprivation daily food intakes decrease immediately whereas nocturnal activity decreases after approximately 72 hr of deprivation have elapsed. After the termination of water deprivation the immediate response is gross hyperdipsia, but again subtle long term effects are apparent. Total daily food intake increases for many days but the increase is not confined to either light or dark periods. Water intake is similarly affected. These results are evaluated in relation to the notion of a body weight set-point regulating feeding behavior. Some limitations on the value of the findings in terms of species differences and lack of ecological validity are also discussed.

Body weight Feeding Drinking Activity Set-point Daily rhythms Light-dark cycle

ONE of the basic premises that has arisen from the concepts of homeostasis is that of body weight set-point (bwsp) theory [5, 27, 34, 35, 38, 40, 41, 46, 47, 58, 59, 60]. For any adult individual of any given mammalian species, there is an optimal body weight or set-point. Any deviation from this op- timum is strongly defended by calorie repletion and deple- tion. Feeding behavior is subservient to body weight control.

If feeding behavior is controlled by a bwsp mechanism, lowering body weight by food deprivation should instigate an increase in feeding behavior upon restitution of food, until there is a realignment of body weight to the reference value. While there is a widely held notion that rats are hyperphagic in the postdeprivation period until body weight lost during deprivation is regained (e.g., [50]) several studies show that this predicted increase in food intake does not occur. Over 30 years ago [1] it was demonstrated that food deprivation lasting up to 6 days had little effect on subsequent food intake in rats. As there was no subsequent over-consumption of food, it was concluded that food deprivation constituted little stimulus to eating. Despite the fact that rats did not compensate for the food deficit resulting from deprivation, it was observed that body weight slowly recovered over the ensuing days [1]. To account for this, it was suggested that the rat has a large margin of dispensable metabolism above that which it employs on an ad lib diet. Normal daily food intake therefore is not economical. Rats recover body weight after 24, 48, 72 and 96 hr of food deprivation but total over-

eating is limited in all groups to the equivalent of a normal total 24 hr food intake [33] unlike earlier reports [32]. On this basis it was concluded that postdeprivation eating is independent of length of deprivation and body weight loss, and that regulation of body weight is under the control of additional mechanisms to those that control food intake [33]. In contrast, it has been shown that while body weight of 96 hr deprived rats recovers by the third day of food restitution, daily food intakes are significantly greater than baseline intakes for 7 postdeprivation days [48]. In other words, postdeprivation daily hyperphagia continued well past the day of body weight recovery and certainly totalled more than the equivalent of a normal predeprivation 24 hr intake.

Findings on recovery of body weight such as those quoted above may be misleading. Close scrutiny of published graphs in the literature indicates that in rats (Fig. 3 in [25]) and the guinea pig (Fig. l in [26]) body weight is not in fact totally regained. Body weight after food restitution is maintained at a lower level but remains parallel to a line in a hypothetical curve representing body weight if the rat had not been food deprived. Whether this effect is observable or not depends upon the number of predeprivation baseline days of body weight shown in published graphs.

In the case of the guinea pig, a lack of recovery of body weight following food deprivation is not surprising. After 72 hr food deprivation there is an ingestion of an initial meal which is followed by a reduction in food ingestion for several

C o p y r i g h t © 1980 A N K H O I n t e r n a t i o n a l Inc .n0149-7634/80/030377-26503.10/0

378 ARMSTRONG, COLEMAN AND SINGER

days. Similarly, when the calorific density of food is diluted, the guinea pig, unlike the rat, does not adjust its total daily intake to compensate [26]. In both instances, therefore, there is no massive overcompensation for calorie deficit. The failure to adjust to the calorie dilution is taken as a reflection of the guinea pig being a herbivore whose natural diet does not vary greatly in calorific density. The rat, being an omnivore, would have a diet which varies greatly in calorific density and has evolved mechanisms which regulate in the face of a continually changing calorie intake [14].

The feeding behavior of the hamster is of interest because it represents a regulatory mechanism midway between the rat and guinea pig. The hamster shows no postdeprivation food compensation but does respond to a dietary dilution [48]. Fifteen days after the termination of 96 hr food deprivation, hamsters had not recovered body weight. Body weight stabilized at 93% of the predeprivation value. It was suggested that hoarding and hibernation were behaviors that had evolved in the hamster as adaptations to food restric- tions rather than compensatory mechanisms as in the rat. However , on this particular point it should be noted that several rat species carry out substantial amounts of food hoarding [6], although there is no evidence to suggest that this food is ingested under conditions of adequate food sup- ply in the environment.

It is clear that the relationship between food deprivation, postdeprivation feeding behavior and body weight defence varies amongst different rodent families, reflecting evolution of the species in relation to a particular ecological niche. While the findings from food deprivation in the hamster do not support a bwsp theory of feeding, those from the laboratory rat are more equivocal. There is a general acceptance in the literature that rats regain body weight after a period of food deprivation, although whether they increase their daily food intake in the postdeprivation period, or not, is more uncertain.

EXPERIMENT 1

As, for the reasons outlined above, it was felt that body weight was not in fact totally regained by rats in the postdeprivation period, the present experiment was carried out. It was designed to investigate the defence of body weight and to set the parameters for experimentation involving the monitoring of food and water intake and general activity. To what degree is body weight defended after long term food deprivation? If it is defended, then how long does this take? The answer to the latter question should give an indication of how many days overeating are involved if overeating should in fact occur.

METHOD

Animals

Thirty rats were used of a Wistar derived strain. Rats entering the laboratory at I20 days of age were given a further 3 weeks to adjust to laboratory conditions. This was to ensure that an adult, stable body weight increase had been achieved. Rats were assigned to 5 groups; control, 24, 48, 72 and 96 hr of food deprivation.

Apparatus

Rats were housed, 1 per cage, in a holding rack of 30 cages, 5 cages to a row. Cages measured 20x23x33 cm.

Mecon rat cubes were supplied from a hopper in the front and tap water at the back. A 12:12 LD cycle (lights on 0500 hr) was maintained.

Procedure

Body weight was measured to the nearest gramme every day for a 23 day period. Weighing took place at 1200 hr in the light period. This time was chosen to coincide with food replenishment and tray cleaning, thereby reducing distur- bance to the rats. At the end of the 23 day period rats were selected for the 5 groups. Selection was aimed at achieving approximately equivalent mean body weights for all groups. At the same time, care was taken to try and randomize position in the rack with respect to position in the 6 rows. At midday on Day 23 rats were taken off food for 0, 1, 2 .3 or 4 days. Body weight was recorded on these days and for a subsequent 40 days.

RESULTS

Curves were fitted to the data from the 23 day predeprivational period for each individual rat, and then were extrapolated for the next 40 days. The method of analysis entailed choosing the line of best fit from two functions: S,=c~ +/3x (linear) or y=c~ + /3y x (exponential) [51]. It was reasoned that the extrapolated curve should approximate the future body weight of the rat if it had not been food deprived. One rat died (control group) and one rat became sick (48 hr group) during experimentation and the data from these were discarded.

In all 28 cases a linear fit was found. Figure 1 shows the results for the 5 groups. The validity of the extrapolation technique for group data over these time periods is con- firmed by the regulation of the non-deprived control group seen in Fig. 1. In spite of minor fluctuations, the control group kept to the linear body weight gain and as a group lay within 2% of the predicted value at the end of the 40 days. Over long postdeprivation time periods this technique would be invalid due to the slight asymptote of body weight gain with age, and new curves would have to be extrapolated. The 24 hr deprived group also regained the predicted weight level but not until 15 days had elapsed. This is a surprisingly long time considering that some of the body weight loss during deprivation is only apparent loss due, for example, to loss of food bulk in the gut, e.g., empty stomach and intesti- nal loss through defaecation. The 48, 72 and 96 hr deprived groups never totally made up for their body weight loss. A new, stable plateau was maintained below the original predicted level.

A potential criticism of these findings is that the amount not regained by the rats is small in proportion to the rats ' total body weight. While this is true, an appropriate evaluation of the weight not regained is in terms of the total weight lost through deprivation and not in terms of total body weight.

While the extrapolation procedure is valid for group data, its reliability for individual rats leaves much to be desired. This is illustrated in Fig. 2 where the individual non-deprived control rats are shown, This raises a major problem in deal- ing with individual rats from the deprived groups. The group data of the deprived groups is misleading, for only some rats in each of the deprived groups could be said to be strictly typical of the group function, e.g., 72 hr group (Fig. 3). At the same time, because the accuracy of the curve fitting technique for the individual control rats is questionable, then

F O O D A N D W A T E R D E P R I V A T I O N 379

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FIG. I. Mean changes in body weight as a function of 0, 24, 48, 72 and 96 hr of food deprivation. Body weight was recorded for a 23 day, baseline period. A curve was fitted statistically for all individual rats in each group for the baseline period and then extrapolated for the next 40 days, thereby indicating what the future body weight for each rat would have been had it not been food deprived.


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the same criticism must apply to the individual deprived rats. One method of solving this problem would be to consider the R value as an index of goodness-of-fit of the curve over the baseline days. R is defined as 1 - Vat Ej/Var Pj where Ej is the error on occasion j and Pj the observed value on occasion j. However , a low R value can be misleading. Curves with small slopes will tend to have a smaller Var Pj which will tend to deflate the value of R. Hence, even small deviations from the fitted line may produce a poor goodness-of-fit for curves that are nearly horizontal. Thus, Rat 12 (72 hr deprived group, Fig. 3) has a very low R value because of the nearly horizontal slope of the curve, even though the deviations from the curve are small. Also, it may be argued that it is scientifically indefensible to exclude from consideration up to, in some cases, 50% of the experimental population on this basis. There is no justification for excluding certain individuals because their growth slope is small and their daily defence of body weight is less precise than in other rats.

In spite of this, it is necessary to examine individual rats. Ultimately, it can only be by consideration of why certain rats regain their body weight totally and others do not, that the scientific understanding of body weight regulation and

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FOOD AND WATER DEPRIVATION 381

defence will be achieved. When individual rats are examined and only those rats with high R values (0.7, and above) are selected, it is still clear that many rats do not make up for the body weight loss while others do. Figure 3 illustrates some individual cases among the 72 hr food deprived group and these exemplify several points. The data from rat 7 represent a clear illustration of a rat where the former body weight level is not regained. In this particular case it remains at a much lower level. The result is very similar to that of the lateral hypothalamic (LHA) lesioned rats reported in the literature [27,40]. It is therefore obvious that the L H A does not have to be destroyed in order to obtain a new body weight plateau. In contrast, rat 22 represents the phenomenon idealized in the bwsp theory. Body weight after deprivation is regained and maintained at just below the original level. Therefore, the rat does not attempt to make up for the weight it had never put on, i.e., weight it would have put on during the deprivation period if it had not been deprived. Compari- son of rats 4 and 22 demonstrates the important point that a high rate of body weight gain is not the main cause of failure to regain the original level. Over the 23 day baseline period, rat 4 displayed a very modest increase in body weight, whereas rat 22 added 20 g. Yet it was rat 22, which had a greater deficit in body weight because of its potential rate of weight gain during the food deprivation period, that precisely defended its body weight.

Rat 12 is a very interesting case, although of rather rare occurrence in this particular rat population. During the baseline measurement period in fact it slowly loses weight (and therefore has a low R value) at approximately the same rate as rat 4 gains weight. It should be emphasized that rat 12 was not sick; it ate and drank normal amounts, groomed itself and in external appearance was no different from other rats. After food deprivation, rat 12 now regained its weight, but above the extrapolated level: its rate of body weight growth is now comparable to rat 22. One rat in the 48 hr food deprived group showed a similar phenomenon.

These findings indicate that attention to individual differences is a sorely neglected aspect in studies of body weight maintenance and more data are needed. At the same time, a criterion is needed for diagnosing whether body weight has attained the extrapolated line of growth, if individual rats are to be considered. Future research will have to solve this problem before major advances can be made in this area.

DISCUSSION

Whether or not one interprets postdeprivation body weight level as evidence for bw~p theory may require a more detailed analysis of predeprivation weight trends than has hitherto been the practice. The present data suggest that as a group, body weight after food restitution remains parallel to but at a lower level than would be the case if deprivation had not taken place (Fig. II. But the picture becomes less clear when data for individual rats are considered (Figs. 2 and 3). Clearly there are marked individual differences. While the rats appear to defend body weight, there is no clearcut evidence supporting a bwsp hypothesis when defined a priori . Existing evidence in favour of such a set-point is the result of either: (l) a statistical artifact arising from averaging across rats and over trends within a given rat: or (2) the use of mean experimental body weights expressed as a percentage of mean control, non-deprived weights. An example of the inappropriateness of the latter method can be seen in previously published works (Figs. I and 2 in [33]). In

these results, body weight gain in the predeprivation period is clearly accelerating in Fig. 1 but in Fig. 2 body weight gain is decelerating [37]. The extrapolation technique used in the present experiments would not have led to the conclusion that body weight has recovered: in one case there is under- compensation and in the other over-compensation.

While it has been stated that the extrapolation technique is fallible for individual rats, this criticism, of course, applies even more so to the method of averaging across the baseline period. Averaging across the baseline assumes a zero-order body weight growth rate and this leads to completely false predictions on postdeprivation body weight defence. As the present experiment illustrates, because of gross individual differences, the technique of comparison with a control weight is also inappropriate, unless individuals from control and deprived groups can be yoked on the basis of predeprivation baseline data. In the light of the present data, rats must be matched on at least two variables: (1) day to day rate of growth; (2) final body weight before experimental manipu- lation. It is insufficient to take the last criterion alone, espe- cially when small groups of rats are used. If daily food and water intakes are also taken into account for the yoking procedure, then a formidable task, involving a large population of rats to be selected from, has to be faced. However , this may be the only presently available solution to the problem.

For the present, it is concluded that body weight is defended after food deprivation but whether this defended level coincides with the predeprivational level depends on the individual rat. The data from rat 22 (Fig. 3) strongly suggest that for this particular animal there is a mechanism which regulates an optimal weight level for the chronological age of the animal, as embodied in the bswp theory. However , as in approximately 50% of cases the defended level is clearly below that of the extrapolated level, this indicates that control of feeding behavior in the predeprivational period cannot be explained adequately by the bwsp theory.

EXPERIMENT 2

It has been reported that when rats were deprived of both food and water they initially overate by 40% after food and water restitution [ 1]. However , enigmatically, further experimentation showed that this hyperphagia was attributable more to water deprivation than to food deprivation [1]. On this basis it could be argued that body weight is defended by overeating after water deprivation alone, and after food and water deprivation, but not after food deprivation alone. If this is true, then it would provide a potential explanation of the findings [40] that the rats rapidly increase their body weight to predeprivation levels after food restitution and that they do this by overeating. The overeating may lie in the fact that the deprived rats were yoked controls for LHA lesioned rats which were adipsic as well as aphagic and therefore the controls were deprived of water as well as food. The experiments involving lesions to the L H A may interfere with fluid regulation and body weight, rather than feeding behavior.

The present experiment investigated defence of body weight after total water deprivation, food ad lib, for periods equivalent to those used in the food deprivation experiment.

METHOD

Experimental animals and apparatus were similar to those used for Experiment 1. Thirty naive rats were used and given 3 weeks to acclimatize to the laboratory. The procedure fol-


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lowed was exactly the same as that for Experiment 1 except that a longer baseline period of 30 days was used in an attempt to improve accuracy for extrapolation purposes.

R E S U L T S

One rat from the 24 hr deprived group was discarded due to sickness. From Fig. 4 it is clear that rats defended their loss in body weight after water restitution. However, the individual control curves shown in Fig. 5 again indicate that a methodological problem exists for individual rats. The 48, 72 and 96 hr water deprived groups took approximately 12 to 15 days to reach the extrapolated line for body weight, The 24 hr water deprived group overdefended their body weight in a manner parallel to, but above the extrapolated value. However, this overdefence was attributable to one rat. This rat was similar to rat 12 in the food deprivation experiment,

in that it was slowly losing weight over the baseline period. After 24 hr water deprivation, body weight growth steeply accelerated. Data on food and water intake are needed to ascertain whether short term water deprivation of this sort is a stimulus to over-compensation by overeating or overdrink- ing.

Examination of individual rats with high R values as seen in Fig. 6, showed that the majority of these rats defend body weight after water deprivation in accordance with the group data. In this respect the 72 hr water deprived group may be compared to the 72 hr food deprived group (Experiment 1). When individuals with low growth rates are looked at, e.g., rats 4 and 24, overdefence of body weight rather than under- defence is the rule, which can again be compared to the food deprived rats.


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Comparison of Food and Water Deprivation

The changes in body weight for the water deprived groups are directly compared with those of food deprivation (Exper- iment 1) in Fig. 7. The data are plotted in terms of daily percentage deviation of each mean group weight from the appropriate extrapolated lines.

It is immediately clear that a major difference between the food deprived and water deprived groups is the fact that food deprivation results in far greater body weight loss than does water deprivation for equivalent lengths of deprivation. For instance the 96 hr food deprived group loses 19.4% body weight whereas the 96 hr water deprived group only loses 12.7% body weight. This difference is partly but not wholly accounted for by body weight loss during the first

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deprivation day; 24 hr of food deprivation results in an average body weight loss of 8% whereas water deprivation results in 4% body weight loss. In this respect 48 hr of water deprivation is approximately equivalent to 24 hr of food deprivation. Therefore it would appear that a major reason for the failure of food deprived rats to regain their body weight loss when compared to water deprived rats lies in the fact that the effects of food deprivation are more severe than those of water deprivation. Ninety-six hours of water deprivation is not equivalent to 96 hr of food deprivation in terms of body weight reduction.

However, this finding alone cannot account for the fact that water deprived groups successfully defended body weight while food deprived groups (except the 24 hr group) did not reach the extrapolated line in the postdeprivation period. In terms of body weight lost during deprivation, the 96 hr water deprived group is equivalent to the 48 hr food deprived group (losing 12.5% and 12.7% body weight, respectively). The 48 hr water deprived group is similar to the 24 hr food deprived group (losing 8% and 7% body weight, respectively). Direct comparison of these two pairs should be revealing. In Fig. 8 the last day of food deprivation is aligned with the last day of water deprivation for each pair. It is apparent that on the first day of restitution, the water deprived groups compensate far better for their body weight deficit than the food deprived groups. It would appear that this is the major difference between the food and water deprived groups in the postdeprivation period and Fig. 9 confirms this. In Fig. 9 the curves are aligned on the first postdeprivation day and it can be seen that from this point onwards the difference between the food and water deprived groups is negligible. The curves faithfully follow each other and oscillate around the extrapolated line. On this basis it can be stated that if, on the first day of restitution, the 48 hr food deprived group had responded to its body weight deficit to the same degree as the 96 hr water deprived group, then body weight after food deprivation would have reached the extrapolated line.

The transposed data presented in Fig. 9 indicate that both food and water deprived groups regained body weight until the extrapolated line was reached. This suggests that both groups have mechanisms for defence of body weight which accelerate body weight gain until days l0 to 14 of the postdeprivation period, and then level off parallel to the normal body weight growth curve. Therefore. it may be hy- pothesized that after total deprivation, rats have a critical period of approximately 2 weeks in which to regain the deficit in body weight. After this time body weight will remain at a lower level, parallel to a growth curve representing body weight if rats had not been deprived. Within the 2 week period, the first day of restitution is the crucial one.

DISCUSSION

The results are clear and need little detailed discussion. The data support the finding I l l that body weight is better defended after water deprivation than food deprivation. Other investigators have also reported a more rapid weight recovery for water deprived than food deprived rats 1181. Whether this is due to increased overeating in the immediate period of water restitution needs confirmation. If this were to be found and physiological correlates ascertained of the events taking place in the immediate postdeprivational period, then important advances might be forthcoming in our

knowledge of the peripheral and central mechanisms of control of consummatory behavior.

The findings on one rat in the 24 hr water deprived group in this experiment and two rats from food deprivation experiment (e.g., rat 12, 72 hr food deprived group), suggest that for certain individuals food or water deprivation is a stimulus to body weight growth. Not only would it be important to investigate the mechanisms by which this takes place in rats, but it would also be of interest to know whether there is any counterpart in the "normal" human population.

Comparison of food and water deprived groups revealed that food deprived rats lose more weight than water deprived rats. The opposite has also been reported; water deprived rats lose more weight than food deprived rats [12]. However, this latter experiment involved restriction of food and water over a long time period rather than acute deprivation, as in the present case. Inspection of the published data reveals that the enhanced ability of water deprived rats to regain body weight on the first day of restitution is also marked (Fig. 8 in [12]), i.e., although water restricted rats have a lower body weight than food restricted rats. at the end of the first day of restitution body weight of the water restricted rats reaches that of food restricted rats.

From these two experiments it is concluded that in acute body weight reduction experiments, there are two important factors to be investigated. The first is the amount of body weight lost during deprivation. The second is the amount of body weight regained on the first day of restitution. Further. it would seem that the second factor is the most critical variable. The changes to feeding and drinking behavior and metabolic processes in the first 24 hr of restitution are the critical variables to study if insight is to be gained into mechanisms regulating body weight defence.

EXPERIMENT 3

The results of the two experiments qualify the bwsp theory of rat feeding behavior and may support arguments against the theory. Food consumption on the first postdeprivation day after 72 hr of food deprivation has been reported as being no larger than for that ingested after 24 hr of deprivation [32]. Similarly, it has been found that even after a restricted diet for approximately 35 days, rats only overcompensated by overeating for a day or two [ 12l. Such findings contrast with reports that the overeating after food deprivation does not take place until many days later 1251. The amounts ingested in the first few postdeprivation days are a function of the length of deprivation; the longer the deprivation period, the less the initial intake immediately following food restitution. More recently, clear evidence was presented that the postdeprivation consumption excess was in all deprived groups (24 to 96 hr) only equivalent to 24 hr total daily intake [33]. The 24 hr deprived group ingested in excess of the normal daily intake for 3 to 4 days, the 48 hr group for 2 days but the 72 and 96 hr groups for approximately a week. Therefore, the consensus of opinion is that gross postdeprivation feeding is limited to the days immediately following food restitution but more subtle effects of deprivation are still apparent several days later for longer term deprivation periods. In this respect there is the possibility of a sex difference. After 5 days of food deprivation, the published graphs in the literature I11] show that for female rats, daily food consumption is still above the control value six postdeprivation days later, while the daily intake of male rats fluctuates but still appears to be mainly above the

FOOD A N D WATER DEPRIVATION 385

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control values. Other investigators have also found a long period of increased postdeprivation daily eating for female rats [48].

Investigation of longer periods of food deprivation affords the opportunity of examining activity and drinking patterns during the deprivation period. Previous unpublished experiments in this laboratory have demonstrated that with 24 hr food deprivation, the normal daily drinking and activity cy-

cles persisted; but it is not known for how long they would persist. By the late 1950"s it was well established that conditions of severe food deprivation resulted in increased running wheel activity by laboratory rats [49]. Laboratory rats increase their running wheel activity by 32% during 5 days of food deprivation, while young, wild Norway rats increased their activity by 142% [43]. This suggests that the increased running phenomenon is not an artifact of domesticity but may have some survival advantage in the wild. Explanations put forward to account for the phenomenon encompass psy- chological, ecological and physiological paradigms [13]. A basic problem in evaluating the findings in the literature is the non-equivalence of the differing devices for measuring activity. For instance, both food and water deprivation increase activity in running wheels but only food deprivation increases activity in stabilimeter cages [9]. In addition, different aged rats show different responses at different environmental temperatures. In contrast, it has been reported that 7 days of total food deprivation had no effect on the daily cycle of wheel running [42]. In a blinded rat, nocturnal predominance remained striking, and further, the activity pattern continued to free-run [42]. The implication from this observation is that the large increases in activity wheel running reported above are solely due to increased nocturnality without accompanying change in diurnal running. Attention to photoperiodic changes may therefore be useful in explain- ing the increased running, e.g., if the daily temporal organ- ization of rest and waking is not changed, such findings are unlikely to support any theories encompassing notions of increased "arousal".

The same may be true of the daily drinking cycle. As already stated, initially, during food deprivation, the daily drinking pattern continues with a nocturnal predominance being evident [37]. At the same time, it is also established that severe food deprivation leads to hypodipsia [11]. During 5 days of food deprivation daily water intakes were reduced to 40, 32, 34, 25 and 25% of controls. The water intake of wild Norway rats is also reduced during 5 days of food deprivation [43]. Reduction in total daily water intake presumably implies a loss of nocturnal drinking. It is not clear from the literature at what stage during food deprivation regulation of the daily drinking cycle breaks down. In the postdeprivation period, there is a major increase in water consumption on the first day of food restitution, which rapidly decreases over the next four days. This is apparent for both sexes [11].

The present experiment was designed to evaluate the effects of 48, 72 and 96 hr food deprivation on subsequent food intake and changes to drinking and activity during food deprivation. As the results in activity, feeding and drinking from all 3 groups were similar, and are available elsewhere I2] only the 96 hr group data are presented as this represents the most extreme case. Results from the 48 and 72 hr groups are alluded to when necessary.

METHOD

Animals and Apparatus

Six adult male Wistar rats were housed in a specialized 12 cage rack for continuous monitoring of food, water and activity. Details of the feeding and drinking devices are described elsewhere [4]. Activity was measured by miniature Doppler radar units sold for commercial purposes (Philips) and modified to fit on the front of the rat cages. The "horn" was removed and sensitivity reduced to allow measurement


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FIG. 1 I. Mean total daily food intakes, mean dark intakes and mean light intakes for 96 hr food deprived group over the entire experimental period. Vertical arrows indicate removal and restitution of food. Horizontal extended lines indicate predeprivation mean intake.

in close proximity to the unit. Insulation by Eccosorb (Type H- l ) boards was provided around cages to eliminate micro- wave interference between cages. A 12:12 LD cycle (lights on 0600 hr) was maintained. Food and water were replenished daily at 1200 hr and body weights were recorded at this time.

Procedure

Acclimatization to the laboratory conditions and the testing equipment preceded experimentation. The experimental design consisted of three parts: a two week baseline period, a 96 hr food deprivation period, and a two week postdeprivation period. Food was removed and returned at 1800 hr, the start of the dark period. Each individual acted as its own control in this repeated measures design. Food and water intake and activity were measured at half-hourly intervals, which were then collapsed in 4 hr or 12 hr blocks for analysis purposes.

RESULTS

Data for the first 2 days of the baseline period were lost for some rats due to an electricity blackout stopping the computer monitoring food, water and activity. Therefore, only a 12 day baseline is available for these variables.

Body Weight

Group body weight data are shown in Fig. t0A and while the postdeprivation period consists of only 14 days, the data clearly support the conclusions drawn from Experiment 1. Rats do not reach the extrapolated body weight curve, and therefore, while body weight is defended, the defended level lies below the extrapolated level.

Food Intake

In Fig. 11 food intake data is graphed in terms of mean total daily intake, dark intake and light intake. The curve fit technique already used for body weight data was not applied here. The extrapolation technique was found to be unreliable for this form of data for such a short baseline of 12 days. Minor perturbations in daily feeding had pronounced effects on the curve fitting and extrapolation, and led to invalid trend fitting. As an alternative, food intakes were averaged across the baseline period and extrapolated over the deprivation and postdeprivation period.

From Fig. 11 it is clear that relative to the extrapolated line on the total 24 hr food intakes, rats continue to overeat for many days after food restitution. This overeating continues for approximately 12 days. However, the graphs reveal a previously unreported finding: the overeating is mainly attributable to food intake during the postdeprivation light period. Any overeating during the dark appears to be confined mainly to the first night of the postdeprivation period.

All the experimental days were divided into light and dark periods and these data were analyzed by means of a one-way analysis of variance [28]. A significant change occurred, F(51,255)=20.85, p<0.05. A posteriori contrasts [44] were used to compare predeprivation and postdeprivation food intakes in the light and dark periods. This method of analysis was chosen on the basis of a published comparison evaluating the various post-hoe testing methods [44]. The comparison between all predeprivation dark period intakes and all postdeprivation dark intakes revealed no significant differenc, es, R(51,255)=0.02, p>0.05. The increase in the first postdeprivation dark period alone also was not signifi-


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cant when compared to the predeprivation dark periods, R(51,255)=0.29, p>0.05. However, comparison of food intakes of all baseline light periods with all postdeprivation light periods showed significant differences, R(51,255) =2.14, p<0.05. Therefore, 96 hr of food deprivation leads to long term increased food consumption and this increase is restricted to the light half of the 12:12 LD regimen. Figure 12 is a graph of the more detailed changes in the cyclic feeding pattern for 4 days before and after food deprivation. The data are plotted in 4 hr blocks and it can be seen that the major postdeprivation change in the light period occurs in the last 4 hr. In essence, during the predeprivation baseline period food intake drops from 0600 to 1000 hr, rises again to a small peak in the middle of the light between 1000 and 1400 hr, and drops again between 1400 and 1800 hr. The peak in the middle of the light presumably reflects a major feeding episode stimulated by the experimenter changing the food, etc. In the postdeprivation period, although all light values may be higher, the most dramatic change occurs in the period from 1400 to 1800 hr.

Water Intake

The water intake data are plotted in Fig. 13 in terms of total daily intakes, light intakes and dark intakes for the 3 deprivation groups. It can be seen that in terms of total daily intakes, in addition to the dramatic increase in water consumption on the first postdeprivation day, there are also indications of hyperdipsia on the subsequent postdeprivation days. The effect of food deprivation on water intake during the deprivation period is better illustrated by Fig. 14, which presents the data plotted in 4 hr blocks over the last 4 days of baseline, the 4 days during food deprivation and the first 4 days of the postdeprivation period.

The data for the entire 30 days of the experiment were analyzed in a one-way analysis of variance with the days divided into light and dark periods. A significant change was found, F(59,295)=23.94, p<0.05. Post-hoc contrasts were carried out to evaluate statistically the changes in drinking at the various stages of experimentation. In the first contrast, the amount consumed during the baseline dark periods was compared to the 4 dark periods during food deprivation and a significant reduction in drinking was found, R(59,295)=0.47, p<0.05. It can be seen from Figs. 13 and 14 that hypodipsia becomes apparent in the third dark period of deprivation and most pronounced in the fourth dark period. There is in fact a tendency for hyperdipsia on the first dark period of food deprivation (Fig. 13) but this was not significant, R(59,295)=0.05, p>0.05. There was no significant difference between water intake during the baseline light period and the 4 deprivation light periods, R(59,295)=0.19, p>0.05. From Figs. 13 and 14 it can be seen that during the first two light periods of food deprivation there is a tendency for hyperdipsia, but this was not found to be significant, R(59,295)=0.25, p>0.05. Therefore, these results indicate that during 96 hr of food deprivation hypodipsia becomes apparent after the first 48 hr and that this hypodipsia is due to a reduction in nocturnal drinking. Between 72 and 96 hr of food deprivation, total daily water intake is reduced to 52% of the predeprivation value and nocturnal drinking only ac- counts for 59% of this amount, as against 74 to 77% during the baseline. Thus, rats still continue to drink more in the dark but the light-dark differences are rapidly extinguished.

In the postdeprivation period a state of hyperdipsia is apparent (Figs. 13 and 14). Comparison of baseline dark in-

390 A R M S T R O N G , C O L E M A N A N D S I N G E R

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takes with postdeprivation dark intakes yielded no overall significant difference, R(59,295)=0.17, p>0.05. However, in the first dark postdeprivation period when rats were restored to food availability, water intake increased by 72c~ above baseline values and this change was significant, R(59,295)= 1.34, p <0.05. When postdeprivation light periods were compared to predeprivation light periods, an overall significant increase in water consumption was found, R(59,295)=0.52, p<0.05. Therefore, drinking increased in the postdeprivation period and, except for the first night of food restitution, the increase is attributable to light period consumption.

Activity

Figure 15 shows the total daily activity counts as well as activity in the light and dark periods. A total daily increase in activity over the food deprived days is seen, but activity only increases during the light period while there is a slight decrease in the dark period. In the postdeprivation period dark values remain below the extrapolated line and light values remain on or above this line. A one-way analysis of variance was carried out on the light and dark activity for the whole 28 days of the experiment. A significant change in activity took place during the period analyzed, F(59,295)=20.82, p <0.05. Post-hoe contrasts showed that there was a significant increase in light period activity during the 4 days of food deprivation when compared to all the predeprivation light periods, R(59,295)=0.95, p<0.05. As can be seen from Figs. 15 and 16, diurnal activity increased from just over 20% of total daily activity during baseline to 45.5% of total daily activity on day 4 of deprivation. This figure is slightly in- flated because there is a consistent decrease in dark period activity of about 5% during the deprivation period. There- fore, total daily activity during deprivation only increases by 33% on day 4. However, this decrease in the dark was not statistically significant, R(59,295)=0.06, p>0.05. Figure 15 also indicates that there was a further consistent decrease in dark period activity in the postdeprivation period, reaching approximately 22% in the immediate postdeprivation period. This decrease was statistically significant, R(59,295)= 1.62, p<0.05. There was no significant difference between the postdeprivation light periods and the predeprivation light periods, R(59,295)=0.04, p >0.05. Therefore, diurnal activity increased during food deprivation, nocturnal activity was defended and there was a significant, small, but consistent decrease in nocturnal activity in the postdeprivation period.

DISCUSSION

The data are interesting because not only do they reveal, once again, that body weight after food deprivation does not appear to regain the predeprivation levels, but they also reveal that postdeprivation food intakes are increased for a much longer period of time than was suspected from previous findings reviewed in the introduction to this paper. The daily increases in postdeprivation feeding are more similar to those reported by Silverman and Zucker [48] than others [33]. At first sight the findings on body weight and food intake appear contradictory. Furthermore, they may seem paradoxical because the increase in food consumption is limited to the light period. In terms of food utilization, if increased food intake was a compensation for body weight loss, the increased feeding should take place in the dark period [3]. Therefore, increased feeding in the light would not appear to be a response to loss in total body weight in the


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sense espoused by the bwsp model. If extra calories are needed to regain body weight, they should be taken during the dark. However, the observation from Fig. 12 that the major observable change in the light period feeding pattern occurs at the end of the light (1400 to 1800 hr), is extremely suggestive. This is the period when maximum lipolysis normally occurs [30,31]. The rat at this time of day is normally extremely dependent upon the mobilization of plasma free fatty acids (PFFA's ) and gluconeogenesis as an internal source of energy [3]. Therefore, the present data strongly suggest that this internal source of energy is not available to the rat in the postdeprivation period and that recourse to external calories in the form of food is undertaken. If this were shown to be true, it would indicate that in the postdeprivation period food ingested in the early dark was either: (I) being utilized for purposes other than lipogenesis, or that (2) lipogenesis was proceeding at the same rate as during the predeprivation period. If the latter were also shown to be true, it might also suggest (3) that there is a critical level of content in the adipocyte which must be met before lipolysis can occur. The second suggestion is in keep- ing with that put forward to account for data obtained from hamsters [48]. These authors suggest that there is a rate-

limiting step in lipogenesis which could account for the hamsters ' lack ofpostdeprivat ion food consumption and lack of body weight defence. In the present experiments, the lack of postdeprivation hyperphagia in the dark period suggests that a similar but more subtle limitation may also exist in the rat when on ad lib feeding. It is not clear what such limitations on iipogenesis represent. A strong endogenous rhythm of insulin secretion [39] would certainly place temporal re- strictions on lipogenesis, but it is more likely that other, more critical rate-limiting steps exist within the adipocyte.

The persistence of the nocturnal predominance of drinking lasted for 48 hr of food deprivation, before dramatically dropping on the third night of deprivation. This suggests that what has been termed as "emergency si tuation" starts at this time [37]. Emergency homeostatic mechanisms override the generation of the circadian drinking rhythm. The type of signal system involved and how the brain mechanisms direct the switching from a state of diuresis accompanying relative hyperdipsia to a hypodipsic state is not known. What is more surprising about the data from the deprivation period is the continuation of diurnal drinking. On the basis of the known aversive properties of light [21, 23, 63] and the fact that drinking in laboratory rats is more nocturnal than is food


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intake, a decrease in diurnal drinking would be predicted. Further, this decrease should have occurred before nocturnal drinking was affected. The continuation of diurnal drinking is not easily explicable. Longer periods of food deprivation combined with measurement of urine and serum osmolality might prove more revealing. The increase in water intake on the first night of food restitution is to be expected as it probably reflects food associated drinking [29]. The postdeprivation continuation of increased drinking into the light period is also explicable as increased food associated drinking. It is notable that on postdeprivation days 1, 2 and 3, the increased drinking occurs during the second half of the light period. Thus, increased diurnal feeding is accompanied by increased diurnal drinking.

The increase in total daily activity during food deprivation supports many previous findings. However, the fact that the total daily increase was only 33% while diurnal activity increased by 45.5%, appears not to have been reported before. It has been reported that the percentage of waking was rela- tively high during the light phase of the third day of food deprivation, although this increase was not statistically different from controls [8]. While it is hard to evaluate the various theories put forward to account for the overall increase in activity on the basis of the present data, the fact that nocturnal activity did not increase but was reduced, (although not significantly), cannot be seen as being particularly good evidence in support of an arousal hypothesis. One would assume that arousal would increase in the dark as well as in the light. However, a better measure of arousal may be obtained by monitoring vigilance states in conjunction with the activity rhythm [8]. The present data are more suggestive of an energy regulation hypothesis. If hormones such as growth hormone (GH) normally act as iipolytic agents during the light, increased "exercise" might produce increased GH levels, thereby increasing fat mobilization [13]. Fat mobilization in turn would decrease subcutaneous fat insulation, thereby requiring an increase in muscular activity as a means to defend homeothermy. However, it has been considered that there is little, if any, direct relationship between level of activity and level of basal metabolic rate during starvation [49]. Further, 7 days of food deprivation leads to a decrease and not increase in plasma GH concentrations [16]. The episodic release of GH in the light period is extremely de- pressed after 3 days of food deprivation. The depression appears to be due to inhibitory influences of circulating somatostatin [52]. Therefore mobilization of fat reserves in the rat during food deprivation must be stimulated by some other iipolytic agent than GH.

The reduction in activity in the postdeprivation period was significant and has been described by previous authors [17,49]. It is known that the activity pattern after food restitution is made up of long feeding periods separated by long intervening rest periods [8], but whether the long rest periods could be reflected as an overall reduction in activity is not clear. Certainly, the reduction in activity appears to be confined to postdeprivation dark phase.

EXPERIMENT 4

This experiment was similar to Experiment 3 and was designed to evaluate the effects of 96 hr of total water deprivation on body weight, food intake and general activity during deprivation, and on body weight, food and water intakes, and on general activity in the postdeprivation period.

Over 5 days of water deprivation, daily food intake has

394 ARMSTRONG,COLEMAN AND SINGER

been found to reduce progressively to 52, 22, 15, 9 and 4%, of the control, baseline period [11]. This is consistent with other findings [12]. The drop in food intake is noted even on the first day of water deprivation. One interpretation is that a water deprived rat loses body weight by restricting food intake to ensure that the ratio of body water to body weight is preserved and that in water deprived rats, animal body size rather than concentration basically reflects the degree of thirst [12]. Alternatively, digestion of dry laboratory chow may be severely retarded when water supplies are restricted. However, although total food intakes are markedly reduced during water deprivation, evidence of the feeding cycle is still apparent [20], strongly suggesting that the mechanisms generating daily rhythms are still operating in the face of an emergency situation.

According to the published graphs in the literature [11], after the restitution of water availability, both daily food and water intakes are well above control levels for at least six postdeprivation days. While a sex difference is again apparent, females ingesting greater amounts than the males, there are no data in the literature indicating how these increases are achieved in respect to alterations to the daily feeding and drinking cycles. The daily variations of food intake during short-term water deprivation influence postdeprivation water intake [36].

Experiments employing running wheels show that total water deprivation increases running wheel activity in a similar manner to total food deprivation [49]. However , when stabilimeter cages are used, activity decreases with length of water deprivation [ 10]. The decrease in activity was apparent for rats of 25, 50 and 100 days of age but a gradual decrement was seen only in the 100 day old group. The 25 day old group did not decrease activity until day 4 of deprivation and the 50 day old group until day 6. This could suggest that the daily home cage activity rhythm persists for many days under conditions of water deprivation. Total water deprivation of 67 hr has no effect on the daily free-running rhythm of running wheel activity of a blinded rat 142]. This could imply that there is no increase in activity or that the temporal dis- tribution of activity is such that any increase occurs at the usual time of the activity rhythm of non-blinded rats, i.e. nocturnal predominance.

M H t t { } I )

Details of animals, apparilitl ~, and procedure are the same as for Experiment 3. Six naivc male rats were housed and studied under identical conditions but water deprived for 96 hr instead of food deprived.

RF.St I . I S

Body Weight

Group body weight data are shown in Fig. 10B. It is clear that in comparison to Fig. 10A, water deprived rats defend body weight better than food deprived rats and therefore these results support those reported in Experiment 2.

Food Intake

The mean food intakes for the entire experimental period are graphed in Fig. 17 in terms of total daily intakes, total light intakes and total dark intakes. Observation of this figure indicates that food intake was markedly reduced during the water deprivation period and that this was attributable to

reduction in feeding in both the dark and light periods. After the termination of water deprivation, overeating is apparent in both light and dark periods. The food intakes were sub- jected to a one-way analysis of variance in terms of light and dark periods for 14 days of predeprivation baseline, the 4 days of water deprivation and 14 days of postdeprivation. A significant difference was found, F(63,315)-- 28.9, p <0.05, and post-hoe contrasts 144] were used to localize the significant changes.

The decrease in food intake during the 4 days of water deprivation was significant both during the dark, R(63,315)=5.59, p<0.05, and the light, R{63,315}=0.48, p<0.05. The total daily intakes for the 14 postdeprivation days were not significantly greater than during the predeprivation days, R(63,315)=0.22, p>0.05. Even when light and dark intakes were analyzed separately for these times, no significant changes were found for the light, R(63,315) =0.20. p>0.05, or the dark periods, R(63,315)=0.05, p>0.05. How- ever, Fig. 17 indicates that any potential increase in food consumption is mainly limited to the first 8 to t0 postdeprivation days. Accordingly, when predeprivation total daily food intake was compared to total daily food intake for the first 8 postdeprivation days, a significant increase in daily intake was found, R(63,315)=0.34, p<0.05. The increase in dark intakes alone for these 8 days was not significant, R(63,315)=0.075, p>0.05, and the light intakes alone for the 8 days also failed to reach significance in comparison to predeprivation values, R(63,315)=0.22, p>0.05. Therefore 96 hr of water deprivation results in a significant change in postdeprivation food intake. This increase is limited to approximately the first 8 postdeprivation days and is found as a total daily increase rather than being restricted to light or dark periods alone. Figure 18 shows mean 4 hourly food intakes for the first 4 postdeprivation days. There is little indication of increased food intake for any specific time of day although the light values initially appear higher than on predeprivation days.

Water Intake

Figure 19 shows water intake presented in a similar manner as for food intake in the previous section. It is quite clear that there is an extremely large increase in water consumption on the first night of water restitution. It would also appear that there is an increase in daily postdeprivation water intakes in both light and dark periods. The data were analyzed by means of a one-way analysis of variance on light and dark values over the 14 days of baseline versus the 14 postdeprivation days, and a significant change was found, F(55,275)=37.36, p<0.05. Post-hoe tests showed that water consumption on the first night (first dark period) of water restitution was significantly greater than during the predeprivation dark periods, R(55,275)=6.21, p<0.05. Water consumption in the following light period was not significantly greater than during the predeprivation light periods, R(55,275)=0.08, p>0.05. The total daily increases in water intake in the 13 day postdeprivation period subsequent to the first day after water restitution were significant, R(55,275)= 0.64, p<0.05, when compared to the 14 predeprivation days. However, the increases in the 13 clark periods alone were not significant, R(55,275)=0.10, p>0.05, and the increases in the 13 light periods were also not significant, R(55,275)=0.12, p>0.05. Therefore, water deprivation exerts long term effects on daily water intakes, but this change is not limited to light or dark periods per se. Inspection of 4 hr block data


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showed that the daily drinking cycle returned immediately in the postdeprivation period.

Activity

Figure 20 demonstrates that there is little change in activity until the fourth day of water deprivation. Nocturnal but not diurnal activity decreases. There is also a decrease in dark activity on the first postdeprivation day while the light activity lies within the normal range. The main conclusion to be drawn is that, unlike food deprivation, water deprivation does not appear to exert any change in activity in any one systematic direction for at least 3 days of total deprivation.

The daily light and dark values were analyzed as de-

scribed previously. A significant change was found. F(63,315)=22.17, p<0.05. Post-hoe contrasts showed that no sigificant change in act ivi ty occurred during the 4 light periods of deprivation, R(63,315)=0.005, p >0.05. No significant change occurred in the dark periods during deprivation, R(63,315)=0.13, p>0.05, until the fourth dark period when there was a significant reduction in activity, R(63,315)= 1.74, p<0.05. There also appeared to be a reduction in activity on the first night of water restitution but this was not significant, R(63,315) = 0.07, p >0.05. The activity on all subsequent postdeprivation nights was also not significant from baseline values, R(63,315) =0.001, p >0.05. The activity during all postdeprivation light periods was not significant, R(63,315)=0.005, p>0.05. Figure 21 shows the activity over 4 predeprivation days, 4 days of deprivation and the first 4 postdeprivation days plotted in 4 hr blocks. It is concluded that severe water deprivation does not systematically interrupt the daily activity cycle until the fourth day of deprivation and that this interruption is manifest as a reduction in nocturnality.

Comparison of Food and Water Deprivation

In Fig. 10C the body weight graphs from Figs. 10A and 10B have been directly compared by aligning the extrapolated curves of the food deprived group with those of the water deprived group. Two important observations can be made. First, unlike the data presented in Experiment 1, water deprived rats do not lose less body weight than food deprived rats. The body weight losses are similar under both conditions. There is no obvious explanation for this discrepancy. The only major differences between the experimental conditions were: (i) the rats used were of different ages, older rats being used in Experiments 1 and 2. (ii) In Experi- ments 1 and 2 the diet consisted of cubes whereas for rats investigated in Experiments 3 and 4 the diet was a mash prepared from crushed cubes. Neither of these differences represents an adequate explanation of the discrepancy between the results.

The second characteristic observable in Fig. 10C is, once again, that the crucial difference between food and water deprived groups is the ability of the latter to regain a substantial amount of the body weight lost through deprivation, on the first day of water restitution. This finding is quite marked and confirms the findings and conclusions already reached.

In Table 1 the amounts of food and water ingested during the critical first day of restitution are shown. The 48 and 72 hr groups as well as the 96 hr group are included. Both food and water deprived groups are hyperphagic and a substantial portion of this hyperphagia occurs during the dark period. This is presumably due more to time of day at which food was replaced than to any photoperiodic effect. The amount of food ingested lies in a similar range for both water and food deprived groups. While the actual percentage increase over the average baseline values ranges from 15 to 29%, the important point is that the water deprived groups collectively ingest slightly less food than the food deprived groups.

Hyperdipsia was greater than hyperphagia for both water and food deprived groups but the hyperdipsia of the water deprived group is extreme. It appears that this is the variable which distinguishes the food and water deprived group and must represent the degree of defence of body weight, It is not known how much of the increased water load was retained for extracellular and intracellular re-hydration but diuresis


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FIG. 19. Mean total daily water intakes, mean dark water intakes and mean light water intakes for 96 hr water deprived group. (Details the same as for Fig. 11.)

must have been considerable. Studies performed in a metabolic cage would be more revealing. Increased water intake may also have improved the efficiency of gastrointes- tinal food absorption and assimilation thereby ensuring more efficient calorie utilization. A monitoring of metabolic correlates such as serum and urine osmolality would be beneficial for future investigations.

It is concluded that water ingestion is the critical variable dictating body weight gain in the immediate postdeprivation period.

DISCUSSION

During water deprivation food intake is reduced, thus confirming previous findings [11,12]. By the fourth day of water deprivation total intake is markedly reduced to 16% of the control baseline values but nocturnal intake still pre- dominates over diurnal intake, thereby supporting previous findings [20]. Seventy percent of the reduced intake was ingested in the dark period, which may suggest that the daily rhythm underlying feeding was still operating even under


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these severe emergency conditions. After the restitution of water, there is an increase in daily food intake which continues for approximately the first 8 days of the postdeprivation period. This increase in food consumption is attributable both to nocturnal and diurnal changes. Therefore, long term water deprivation, unlike long term food deprivation, results in increased nocturnal food consumption but this increase by itself is not statistically significant. Only the total daily increases were statistically significant. Whether this increased daily food intake can account solely for the ability of water deprived rats to defend and regain their body weights is de- batable. Certainly, the increase in nocturnal feeding was ab- sent from the postdeprivation period of food deprived rats which do not totally regain their body weights (Experiment 3). It would also be expected that the increased calories taken in at night should be more efficiently utilized. A study of respiratory quotients and the lipogenic-lipolytic cycle would be needed to substantiate such a hypothesis. It is clear, however, that by the second postdeprivation dark

period the normal feeding cycle has been re-established (Fig. 18), which would suggest that any changes in metabolic rhythms must be subtle. It is also clear from the comparisons made on body weight defence (Experiment 2, Fig. 9 and Fig. 10, Experiment 3) that the tong term differences between food and water deprived groups are minimal after the first day of restitution. Therefore, whatever the causal factors for the long term daily overeating of the water deprived rats and diurnal hyperphagia of the food deprived rats, they do not seem to be directly related to body weight defence.

On restitution of water, extremely large increases in drinking were observed during the first dark period. Water intake of the first postdeprivation light period was not increased significantly. These increases are reflected in the large body weight increases of the first postdeprivation day and confirm earlier findings [18]. Whether such large water loads were retained by the rats is not known from the present data. While daily nocturnal and diurnal water intakes were increased in approximately the first 8 days of the


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postdeprivation period, these increases alone were not statistically significant. Only total daily intake was significant. It may be concluded therefore that long term water deprivation results in prolonged increases in daily food intake. These increases are accompanied by increases in daily water consumption. However, the latter appear to persist until the termination of the experimental period. As with the long term changes in feeding, the long term hyperdipsia seems unrelated directly to body weight defence. The comparison of food and water intakes and body weight (Table 1) demonstrates that it is the large increase in water load on the day of water restitution that is the critical factor in the water deprived rats' ability to defend body weight. As already suggested, a series of physiological measures are needed to ascertain the mechanisms by which this defence is made.

The daily activity cycle is maintained during water deprivation for up to 72 hr. This is in agreement with findings of a persistence of the rhythm in running wheel activity [42]. However, examination of individual rats in the present experiment indicated both increases and decreases in activity within this time period, suggesting that severe dehydration interferes with the expression of the rhythm. When, on the fourth day of water deprivation, the rhythm is changed, the change is due to a reduction in nocturnal activity. Noctur- nality is reduced by 44%. These results are seen as confirming and extending previous findings [10]. Although not statistically significant, activity on the first night of water restitution is also reduced below predeprivation levels. This has previously been reported for running wheel activity [18,49]. What this reduction represents cannot be ascertained from the present data. Again, measurement of vigilance states as carried out for feeding behavior [8] might yield a functional explanation.

The contrast in home cage activity between long term food and water deprivation conditions is not supportive of arousal hypotheses but is more indicative of changes in energy regulation. One possibility is that during water deprivation the decrease in activity is due to the instigation of regulatory mechanisms designed to conserve bodily water, whereas during food deprivation increased activity would mobilize calories stored in adipose tissue. Under long term water deprivation there may be decrease in arousal, whereas under long term food deprivation there may be increased state of arousal. The measurement of EEG changes under the two respective deprivation states would allow further evaluation of this arousal hypothesis.

GENERAL DISCUSSION

The use of the term set-point has been criticized by re- searchers expert in the application of control theory analysis to models of feeding behavior [7, 15, 62]. Precise day to day regulation of body weight does not make the existence of a set-point obligatory. Simple feedback models without setpoints are able to account for the data. The findings of Ex- periment l support these criticisms of bwsp theory. Body weight is not as precisely defended as popularly believed. Group data from rats totally deprived of food for 48, 72 and 96 hr showed that body weight in the postdeprivation period was only partially regained. This finding was contrary to all previous reports (e.g. [1, 33, 48]) and is due to the different methods employed to determine postdeprivation body weight. It remains true that the amount of body weight not regained is small in comparison to other rodents, e.g. hamster [48]. Nevertheless, that the phenomenon is worth inves-


TABLE 1 CHANGES IN FOOD INTAKE. WATER INTAKE AND BODY WEIGHT ON THE

FIRST DAY OF RESTITUTION

Food deprivation Water deprivation (Experiment 3) (Experiment 4)

48 hr 72 hr 96 hr 48 hr 72 hr 96 hr

% BW lost 10.2 15.1 17.2 11.2 15.7 19.1 % BW gained 4.2 6.1 4.5 8.9 11.6 12.0

Dark food (g) 24.68 2 1 . 8 8 2 1 . 7 6 2 0 . 3 6 21.10 18.84 Light food (g) 6.92 8.21 8.46 10.13 10.23 9.55 Total food (g) 31.60 3 0 . 0 9 3 0 . 2 2 30.49 31.33 28.43 % total increase food 28.7 19.8 23.8 23.2 19.4 15.0

Dark water (g) 29.95 3 2 . 3 6 32.07 50.8 54.01 63.46 Light water (g) 10.00 12.52 10.83 9.95 13.50 10.68 Total water (g) 39.95 44.88 42.90 6 0 . 7 5 67.50 74.14 % total increase water 65.2 67.2 72.9 96.0 122.7 125.5

Percent body weight lost refers to amount lost by the end of the deprivation period. Percent body weight gained refers to the amount regained in the first 24 hr postdeprivation period. The amount lost and regained is calculated relative to the extrapolated values, i. e. the value that the rat would have reached if it had not been deprived. Percent increase in food and water intake is relative to mean daily predeprivation intakes.

Body weight on the first day of restitution does not exactly correspond with amounts ingested because body weight was monitored daily at 1200 hours whereas food and water were removed and replaced at 1800 hours.

tigating becomes clear when water deprivation is investigated. Unlike food deprived rats, water deprived rats defend their body weight as predicted by bwsp theory. The main factor determining the differential ability to defend body weight between food and water deprived rats occurs in the first 24 hr of the postdeprivation period. At a behavioral level this factor represents the gross hyperdipsia and not the hyperphagia of water deprived rats. On this basis it could be argued that a comparison of food and water deprived rats is of little value. The latter could be responding to a different physiological deficit, i.e. dehydration. Such a criticism would be misleading because the water deprived rats, particularly those deprived for 96 hr, are also food deprived. Food intake is drastically reduced during water deprivation. Yet, body weight of water deprived rats is regained, demon- strating that the recovery of body weight is more than simply re-hydration.

While the curve-fit and extrapolation technique used in these experiments is superior to other existing techniques for predicting body weight growth in the postdeprivation period, attention to individual rats shows that there is a need to develop other statistical procedures. For instance, the technique used in the present studies is biased against finding a satisfactory goodness-of-fit for rats with a low body weight growth rate. A technique is needed which will allow the precise prediction of postdeprivation body weight for non- deprived control rats. At the same time, it is clear that for some individuals it will remain impossible to predict postdeprivation body weight recovery because it bears no relationship to the predeprivation trends.

When the body weights of food and water deprived groups were compared directly, it was apparent that besides

the dramatic changes to body weight on the first day of restitution, there was a second, more subtle process involved, of accelerated body weight gain which lasts approximately two weeks. It is therefore possible that the long term hyperphagia in the light period of the food deprived group (Exper- iment 3) and the long term hyperphagia in the light and dark periods of the water deprived group (Experiment 4) contrib- ute to this regrowth. This would indicate a change in daily rhythms of anabolic systems. In the case of the food deprived groups, this would constitute a phase shift in the rhythm of lipogenesis into the light period from the dark period [30,39] to accompany the increased feeding. The testing of physiological variables is needed to clarify this issue.

After 96 hr of food deprivation, rats are markedly hyper- dipsic on the first night of food restitution. As with food intake, long term increases in water consumption are found. These are due to increased drinking in the light period. Dark intakes are only significantly increased on the first night of food restitution. After water deprivation hyperdipsia is even more apparent. As with food intake, subtle daily increases in drinking were noted during the postdeprivation period. These increases were not confined to light or dark periods.

During 96 hr of food deprivation a consistent change in the daily activity pattern emerged. Diurnal activity increased over the 4 days of deprivation for the 96 hr food deprived group. Nocturnal activity showed no statistically significant change although it was reduced. The increase in activity during deprivation is well documented although the restriction of this increase to light hours appears not to have been reported. A variety of functions encompassing physiological, ecological and behavioral explanations have been attributed to the increased activity. A parsimonious explanation lies in


the release of PFFA's as a source of energy. Exercise stimu- lates lipolysis [22]. The lipolytic agent is unknown, but after 48 hr of deprivation it is unlikely to be GH in the rat [16,52]. The energy liberation hypothesis was favoured due to the discovery that long term water deprivation, while disturbing the activity pattern in individual rats, produced no consistent increase in activity. In fact, after 3 days of water deprivation nocturnal activity dramatically decreased. It may be argued that ecological explanations of emigration and behavioral explanations of increased CNS arousal should apply equally to water deprived rats as they do to food deprived rats. The measurement of changes to hormones in the blood and metabolites in the urine during the two states of deprivation would be needed to thoroughly test a physiological explanation.

With regard to the status of GH in the bwsp model, it has been postulated that body fat stores are regulated by the ratio of plasma insulin and GH [59]. As pointed out elsewhere [3] this relationship in the rat is unlikely to exist. Release of GH in ad lib feeding rats is episodic, occurring predictably every three to four hours [53, 54, 55]. Episodic surges appear to be independent of short term metabolic changes [55], peripheral hormonal feedback [57], light dark cycles, or states of sleep and waking [54,56]. It remains possible that the relationship between GH and insulin may be important for body fat store regulation in other mammals.

Attention to individual differences and the correlation be-

tween degree of defence of body weight and amount of food eaten after deprivation is important for future research aimed at elucidating the mechanisms of body weight regulation. This is particularly so in respect to a carcass analysis of the ratio of adipose tissue to lean body tissue in various individual rats exhibiting different growth functions. Measurement of fat depot content may be more revealing than body weight per se. Recent findings relating blood glycerol concentrations to adipocyte size, food intake and body weight regulation indicate that future research should concentrate in this direction [61]. However, it is unlikely that measurement of physiological variables alone will unravel the interrelation- ship between feeding, drinking, activity and body weight defence. It is now clear that comparative physiology is needed in conjunction with comparative behavioral studies. Differ- ences between rat, hamster and guinea pig in feeding and body weight regulation have already been alluded to, Similarities and differences in these variables occur between hamster and rat during stages of reproduction [19]. Even more striking species differences are found in drinking behavior in response to food deprivation (see ref 45 for further references). It is therefore clear that comparative physiology and behavior needs an ecological framework on which to base models of feeding behavior [24]. At the present time, the scientific requirement for such information is more ur- gent than the generation and proliferation of such concepts as set-point.

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Food and water deprivation: Changes in rat feeding, drinking, activity and body weight

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