Physiology & Behavior,Vol. 45, pp. 249-254. Copyright©PergamonPress plc, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Reversibility of Metabolic Changes Induced by Feeding Schedule in Rats R. C U R I , R. B. B A Z O T T E , N . S. H E L L A N D C. T I M O - I A R I A
Institute o f Biomedical Sciences, University o f S6o Paulo Department of Physiology and Biophysics, 05508, S6o Paulo, SP, Brazil R e c e i v e d 4 A p r i l 1987 CUR1, R., R. B. BAZOTTE, N. S. HELL AND C. TIMO-IARIA. Reversibility of metabolic changes induced by feeding schedule in rats. PHYSIOL BEHAV 45(2) 249-254, 1989.--This study aimed to investigate the effect of free-feeding on rats kept on meal-feeding schedule for a prolonged period. Thus, rats meal-fed for 4 and 20 weeks were given free access to food for subsequent 5 weeks. The metabolic adaptation of higher hepatic glycogen content, low plasma FFA values and sustained glycemia during 22-hr fast, reported for rats subjected to meal-feeding, completely disappeared after free-eating period. The rate of body weight gain increased as a consequence of the free access to food in both groups but the control values (group with food ad lib all the time) were attained only in rats previously submitted to meal-feeding for the shorter period of time (4 weeks). The findings of this study suggest that the recovery of body weight by meal-fed rats, for the control values, seems to depend on the duration of the meal-feeding schedule and the age when it is imposed. Meal-feeding
ably associated with meal-feeding. Wistar rats maintained on 4 or 20-week meal-feeding schedules were given free access to food for the subsequent 5-week period.
RATS which are restricted to a single daily meal over a prolonged period show a number of physiological adaptive changes; among which are increased intestinal absorption of nutrients (25), higher values for the stomach fresh weights (6,7), enhanced liver and muscle glycogen (10, 12, 24), slower depletion o f fasting hepatic glycogen (7, 15, 27), raised lipid synthesis and storage in adipose tissue and liver (20-22) and maintenance of normal blood glucose concentrations even after a 22-hr fast (7). We previously reported (7) that food restricted (2-hr feeding/22-hr fast) adult male Wistar rats gradually adapted to food restriction over a four week period. Adrenal hormones appeared to play a crucial role in the adaptive process (1,7). The established metabolic and hormonal pattern was not changed even when this feeding schedule was prolonged for 20 weeks (6). The body weights of the meal-fed animals remained lower than those of the free-fed group for both the shorter (4 weeks) and prolonged (20 weeks) training periods. In contrast, Hollifield and Parson (18) reported that the body weight of SpragueDawley rats maintained on a similar feeding schedule for ten weeks increased more than that of the controls; enhanced activity of lipogenesis in the meal-fed rats was also reported (20-23). Although strain differences may be invoked to explain the above, it is an intriguing observation and one deserving further investigation. The present study aims to investigate the effect of free access to food on the quantity and pattern of food intake and increase in body weight, and also to evaluate possible reversion of the adaptive hormonal and metabolic pattern invari-
METHOD One hundred adult male Wistar rats were used. The animals were individually caged in a temperature-controlled (23_+ I°C) room, brightly illuminated from 5:00 a.m. to 7:00 p.m. A free-fed (FF) group had ad lib access to food during the experiment period. The meal-fed (MF) rats had access to food for only two hours daily, from 0730 to 0930 hours. After 4 or 20 weeks these rats were allowed free access to food, under the same condition as for the control ( F F group). All animals had free access to water. Rats weighing some 220 g (body weight increasing by 20 g per week) were kept on a restricted food schedule for either 4 or 20 weeks and then were given free access to food for the subsequent 5-week period. Body weight and food intake were evaluated daily. Animals were killed after 22 hr of food deprivation at the end of the meal-feeding and free-feeding periods. The experimental procedures and measurements were performed as described previously by Curi et al. (7) and Curi and Hell (6). Blood glucose was measured by the method of Dubowski (9) and the liver glycogen content by the method of Hassid and Abraham (14). Plasma free fatty acids ( F F A ) were evaluated according to Falholt et al. (11) and plasma insulin levels were determined by a modified radioimmunoassay procedure (28), in which polyethyl-
~This research was supported by FINEP, CNPq and FAPESP. 2A preliminary communication of this study has already been published (8).
CURl ET AL.
TABLE 1 DAILY FOOD INTAKE(g/100g b.wt.) OF FF AND MF RATS DURING 5 WEEK FREE-FEEDINGPERIOD Groups FF
TABLE 2 DALLYFOOD INTAKE (g/100 g b.wt.) OF FREE FED (FF) AND MEAL-FED (MF) RATS AFTER 20 WEEKS IN FOOD RESTRICTION SCHEDULE DURING 5 SUBSEQUENTWEEKS HAVINGFREE ACCESS TO FOOD
Groups FF(2 hr)
After 4 Weeks of Food-Restriction
7.9 -+ 0.4
Free-eating period (24 hr for both groups) 1st day 2nd day 1st week 2nd week 3rd week 4th week 5th week
8.1 8.2 6.4 6.2 6.5 5.8 6.4
± 0.3 ± 0.2 _+ 0.3t -+ 0.2 ± 0.3 ± 0.3 ± 0.2
6.9 ± 0.1*
12.0 --- 0.3*-t 12.0 _± 0.4* 9.4 ± 0.6* 7.1 ± 0.3* 7.9 ± 0.2* 6.1 ± 0.2t 6.2 ± 0.1
*(p<0.05) different of FF rats. tip<0.05) in relation to the precendent week in each group. The animals were previously MF or FF for 4 weeks. Mean _ SEM of 10 rats in each group is presented.
eneglycol was used instead of the second antibody. In another experiment, rats weighing some 360 g (body weight increasing by 5 g per week) were kept on the above mentioned food restriction schedule for 3 weeks but were then allowed free access to food for the subsequent 5-week period. The body weight changes and daily food intake were recorded but biochemical and hormonal measurements were not made. For the statistical treatment of results, unpaired or paired Student t-test was used when appropriate taking p =0.05 as the minimum. RESULTS The absolute food intake of rats meal-fed over 4-week period was 16.0---2.4 g/day ( m e a n - s t a n d a r d deviation-SD) as compared to 23.6-+4.6 g/day (mean-+SD) for free-fed rats. When food access was not restricted, meal-fed rats showed a mean food intake of 26.5-+5.2 g/day on the first day, 23.9-+3.5 g/day on the first week and 21.0+2.9 g/day for the following 4 weeks. Rats meal-fed for 20 weeks consumed 16.5--_2.3 g/day as compared to 26.6-+4.3 g/day for free-fed rats. During the 5-week of free access to food, rats meal-fed for 20 weeks consumed a mean of 31.3---5.6 g/day on the first day, 28.4-+4.1 g/day for the first week and 25.0-+3.8 g/day over the subsequent 4 weeks. These values for absolute food intake become completely modified when food intake is expressed as g of food consumed/100 g body weight (Tables 1 and 2). As presented in Table 1, the rats restricted to mealfeeding for 4 weeks showed lower values than did the freefed animals, however, when this feeding schedule was extended to 20 weeks, there was no difference between both groups (Table 2). These results suggest that the gastric capacity of meal fed rats increases with long term training which certainly contributed to their reaching the relative
After 20 Weeks of Meal-Feeding
6,1 ± 0.2
5.6 ± 0.2
Free-eating period (24 hr for both groups) 1st day 2nd day 4th day 1st week 2nd week 3rd week 4tb week 5th week
6.4 6.2 6.0 6.9 6.1 6.0 6.3 6.6
± ± ± ± ± ± ±
0.3 0.2 0.2 0.2 0.2 0.3 0.4 0.2
9.4 7.4 5.6 8.0 6.6 6.4 6.9 6.9
± 0.2*? ± 0.2*t -+ 0.4t ± 0.2*? ± 0.2~ ± 0.2 ± 0.2 ± 0.3
*(p<0.05) for the comparison between FF and MF rats. t(p<0.05) in relation to the precendent period of each group. The values are expressed as mean _+ standard error mean (S.E.M.) of 10 rats.
food intake values of the control group. Free access to food led to a 68% increase in food intake on the first day in the 20-week meal-fed rats (Table 2), which represents a value 47% above that of the control group. The same comparison for the second day gave a 19% increase, and at the end of the first week 16% above the values described for F F rats; thereafter there was no significant difference. Table 1 shows that the 4-week meal-fed rats increased their daily food intake by 74%, representing an intake 5(1% greater than that for the free-fed group, on the first and second days of the ad lib feeding period. By the end of the first week, the daily food intake of meal-fed rats was 47% higher than that of the control group, and 15% and 22% higher, respectively, after 2 and 3 weeks. Subsequent to this period, food intake did not differ between groups. Concomitant with the elevation of food ingestion by the 4-week meal-fed group, a rapid gain in body weight was also observed resulting in a mean body weight not different from that of the free-fed group (Fig. 1). Similar results have been reported by Cleary (4) for Zucker rats weighing 180 to 300 g. However, this phenomenon was not observed in the 20-week meal-fed group (Fig. 2) which consistently showed a lower body weight in relation to the control rats, although increases in food intake were detected until the end of the first week (Table 2). The rate of body weight gain by meal-fed rats was significantly reduced after 8 weeks, when rats weighed approximately 330 g, subsequent values remaining unchanged; see Fig. 2. The effect of meal-feeding on this age class was thus investigated. Rats weighing 360 g when subjected to mealfeeding showed a 11% decrease in body weight within 1 week with no recovery during the subsequent 2 weeks (Fig. 3). During the following 5-week free-feeding period, the mealfed rats showed a steady increase in body weight until the second week, attaining the values shown by the free-fed group by the end o f the third week. Daily food intake (g/100 g b.wt.) by meal-fed rats was lower than that by free-fed rats
REVERSIBILITY OF METABOLIC ADAPTATION
FIG. 1. Body weight (mean-+S.E.M.) as a function of time (in weeks) in free-fed (open circle) and meal-fed (closed circle) rats, during 4 weeks of food-restriction and 5 weeks of free access to food in both groups. §(p~<0.05) in relation to the precedent period. *(p~<0.05) for the comparison between free-fed and meal-fed groups.
FIG. 2. Body weight (mean_+S.E.M.) as a function of time (in weeks) in free-fed (open circle) and meal-fed (closed circle) rats, during 20 weeks of food-restriction and 5 weeks of free access to food in the both groups. §(p~<0.05) in relation to the precedent period.
FIG. 3. Body weight (mean_+S.E.M.) as function of time (in weeks) in free-fed (open circle) and meal-fed (closed circle) rats, during 3 weeks of food-restriction and 5 weeks of free access to food in both groups. *(p~0.05) for the comparison between free-fed and meal-fed groups.
over the 3-week period of meal-feeding (Table 3). Under ad lib conditions, the meal-fed group increased daily food intake by 52% on the first and second days, and by 36.5% and 25% at the end of the first and second week respectively. In the subsequent weeks no changes were detected, food intake values remaining similar to those of the free-fed group. Therefore, 3-week meal-fed rats, when fed ad lib for 5 weeks
showed a clear correlation between food intake and body weight gain. The biochemical and hormonal parameters were determined after a 22-hr fast for both the free-fed and meal-fed rats (Tables 4 and 5). Four and 20-week meal-fed rats had a higher stomach fresh weight (g/100 g b.wt.) and liver glycogen content (mg/100 nag) and a lower plasma F F A level than
CUR1 E T A L .
TABLE 3 DALLYFOOD INTAKE (g/100g b.wt.) OF FF AND MF RATS WEIGHING 360 g AT THE BEGINNING OF THE EXPERIMENT, DURING 3 WEEKS IN THE RESPECTIVE FEEDING SCHEDULE AND ALSO DURING 5 SUBSEQUENT WEEKS OF FREE-FEEDING Groups Days
The ad lib condition abolished the metabolic adaptations described for either 4- and 20-week meal-fed rats (Tables 4 and 5). In fact, after this 5-week period, glycemia was l o w e r and insulinemia higher in 4-week meal-fed as c o m p a r e d with the free-fed rats. T w e n t y - w e e k meal-fed rats showed a significant reduction in liver glycogen content w h e n food was freely given (Table 5).
MF(2 hr) DISCUSSION
1st 2nd 7th 14th 21st
5.8 6.1 6.1 5.8 6.4
± 0.2 _+ 0.3 ± 0.2 _+ 0.4 --- 0.4
1.9 2.1 4.2 5.1 5.2
± ± ± ±
0.1" 0.1" 0.2*t 0.5 0.2*
The clear relationship b e t w e e n food intake and weight gain (16,29) permits speculation as to w h e t h e r body weight (and thus the age of the rat) might be important for body weight profile and the adaptive response to meal-feeding. To investigate this relationship, rats weighing 360 g (body weight gain rate of 5 g per week) were submitted to mealfeeding for 3 w e e k s and then were given free access to food for 5 weeks. In this protocol (see the Method section for details), the animals restricted to meal-feeding showed l o w e r food intake values than did younger rats (7). Similarly, to the previously reported data (6), food intake increased o v e r the 3 w e e k test period although results were consistently l o w e r in the meal-fed than in the free-fed group (Table 3). The body weight of these latter rats decreased markedly (Fig. 3) in contrast with previous studies involving younger animals (5-7). H o w e v e r , the body weight of meal-fed rats was rec o v e r e d when food was given freely (Fig. 3), attaining values similar to those of the free-fed animals within two weeks. This body weight r e c o v e r y coincided with the beginning of the free-feeding period and was c o n c o m i t a n t with the period o f increased food intake (Table 3, 1st, 2nd, 7th and 14th days o f free-feeding period). In fact, w h e n the increased food intake of meal-fed rats b e c a m e attenuated, the food intake of both groups being similar, (Table 3; 3rd, 4th and 5th w e e k s of free eating period), body weight gain rate stabilized (Fig. 3). These results suggest that the more efficient utilization o f food by meal-fed rats (6, 7, 16) does not cause overweight-
Free-eating period (24 hr for both groups) 1st 2nd 7th 14th 21st 28th 35th
6.2 6.8 5.6 5.9 5.4 5.5 6.2
--- 0.1 ± 0.2 ± 0.1 ± 0.1 ± 0.2 ± 0.2 ± 0.2
7.9 8.0 7.1 6.5 5.6 5.4 6.0
-4- 0.3*t _+ 0.4* ± 0.2* ± 0.1*t ± 0.2t ± 0.1 ± 0.4
*(o<0.05) between FF and MF rats. t(p<0.05) in relation to the precendent period in each group. The values are expressed as mean +_ S.E.M. of 9 rats.
the free-fed group. Insulinemia (tzU/ml) did not differ among the groups for each experimental protocol. The blood glucose concentration (mg/100 ml) o f the 4 or 20-week meal-fed group was higher than that o f the control although the difference b e c a m e significant only after 20 weeks of meal-feeding.
TABLE 4 PARAMETERS EVALUATED IN FF AND MF RATS AFTER 4 WEEKS SUBMI'ITED TO RESPECTIVE FEEDING SCHEDULE AND AT THE END OF THE 5 WEEK PERIOD IN WHICH BOTH GROUPS HAD FREE ACCESS TO FOOD After 5 Weeks Under Free Feeding
4th Week FF
Stomach weight (g/1000 g b.wt.)
0.59 -+ 0.02 (12)
0.80 +- 0.02* (8)
0.62 ± 0.05 (9)
0.62 ± 0.03 (16)
Glycemia (mg/100 ml)
79.4 _+ 4.8 (11)
95.0 - 6.1 (11)
81.1 ± 1.1 (8)
74.3 _+ 1.2" (17)
Hepatic glycogen (mg/100 mg)
0.43 _+ 0.05 (12)
1.48 + 0.23* (6)
0.39 _+ 0.05 (8)
0.30 _+ 0.05 (16)
641 -4- 20.0 (6)
485 ± 26.7* (6)
639 ± 81.1 (8)
657 + 58.7 (16)
Plasma insulin (/~U/ml)
19.9 ± 2.6 (6)
16.1 ± 1.1 (9)
19.4 ± 0.9* (16)
22.9 _+ 1.5 (8)
*(o<0.05) for the comparison between FF and MF rats. The rats were killed after 22 hours fasting. The results are presented as mean ___SEM. ( ) number of animals.
REVERSIBILITY OF METABOLIC ADAPTATION
TABLE 5 PARAMETERS EVALUATEDIN FF AND MF RATS AFTER 20 WEEKS SUBMITTED TO RESPECTIVE FEEDING SCHEDULE AND AT THE END OF THE 5 WEEK PERIOD IN WHICH BOTH GROUPS HAD FREE ACCESS TO FOOD After 5 Weeks Under Free Feeding
20th Week FF
Stomach weight (g/100 g b.wt.)
0.52 -+ 0.02 (8)
0.70 _+ 0.02* (7)
0.52 -+ 0.01 (8)
0.56 --- 0.01 (9)
Glycemia (mg/100 ml)
77.0 -- 3.0 (8)
98.2 +- 4.6* (6)
84.1 +_ 2.3 (8)
82.9 -+ 1.6 (10)
Hepatic glycogen (mg/100 mg)
0.82 _+ 0.07 (8)
1.30 _+ 0.09* (6)
0.88 -- 0.12 (9)
0.42 _+ 0.06* (10)
Free fatty acids (FFA) (fteq/l)
932 _+ 68.6 (8)
692 -+ 64.1" (8)
747 _+ 42.4 (6)
786 -- 43.6 (5)
Plasma insulin (/zU/ml)
35.1 _+ 2.0 (7)
32.9 --- 2.8 (8)
49.9 _+ 2.5 (10)
45.0 --- 3.4 (10)
*(0<0.05) for the comparison between FF and MF rats. The rats were killed after 22 hours fasting. The results are presented as mean _+ SEM. ( ) number of animals. ness in the Wistar rat unless accompanied by an increase in food intake. Moreover, these data suggest that the increased liver and adipose tissue lipogenic activity reported for mealfeeding schedule (10, 20--23) may only play a minor role in obesity which is contrary to what has been claimed (18). The meal-feeding imposed on young rats (body weight around 220 g) did not promote body weight loss as observed for rats weighing 360 g. The former presented a lower rate of body weight gain in relation to the free-fed group (Fig. 1), confirming the data previously reported (6-8). When this group of rats weighing 220 g was allowed free access to food, after 4 weeks under meal-feeding, an increased food intake occurred from the first to the third week (Table 1) leading to a marked increment of the rate of body weight gain (Fig. 1). After 5 weeks, the quantity of food ingested and the rate of body weight gain did not differ between the meal-fed and free-fed groups (Table 2 and Fig. 2). Twenty-week meal-fed rats, however, disclosed the increase in food intake during the first week of the free access to food period (Table 2) and body weight did not attain the values of the control group (Fig. 2). As described above, the recovery of body weight seems to depend on the age when the meal-feeding schedule is imposed and on the duration of this feeding period. However, the mechanisms involved in the regulation of this phenomenon still have to be investigated. It is possible that changes in the total number and size of adipocytes are involved in this process (2, 3, 17, 19, 29). Food-restriction imposed at an early stage (roughly before 3 months of age) and chronically maintained for a prolonged period (20 weeks) may reduce the total number of adipocytes in some fat depots (2,3). As a consequence when free access to food was again allowed an increase in the size o f the cells (hypertrophy) would be more important than an enhancement of the cell number. This would be insufficient to restore the normal body weight values (Fig. 2). Rats maintained under meal-feeding schedule for a prolonged period, until reach the condition of low rate
of body weight gain--20 weeks, would be unable to show great changes in the number of adipocytes when subjected to the condition of free-feeding (Fig. 3). Free-feeding schedule imposed on 4-week meal-fed rats (short period of food restriction) may have assured that the total number of adipocytes was reestablished (Fig. 1). Under this condition, cellular and metabolic differences among adipose depots may occur (30). The differences in blood glucose, hepatic glycogen and plasma F F A between free-fed and meal-fed rats (Table 4 and Table 5, left side) could be due to a group difference in food intake (about 40% higher in MF) immediately before the 22-hr fast period. The general metabolic adaptations of the 4-week meal-fed rats were not altered after 20 weeks (Table 4 and 5). However, sustained glycemia during the 22-hr fast by meal-fed rats was more pronounced in the prolonged group. Insulinemia was higher in the older animals for both groups. This finding might be associated with the condition o f insulin resistance reported for the ageing subjects (13). Under a free-feeding period of 5 weeks the adaptive metabolic and hormonal pattern reported for meal-fed rats (15, 26, 27) was abolished. The imposed alterations by the feeding schedule involving higher stomach mass weight, sustained glycemia, elevated liver glycogen content and lower free fatty acids mobilization during the 22-hr fast (6,7) were completely recovered to basal values in both the 4 and 20-week meal-fed groups (Tables 4 and 5). It was notable that the glycemia levels of the 4-week meal-fed rats were lower after the freefeeding period. The same was found for the liver glycogen content of the 20-week meal-fed group in comparison with those in the free-fed group. These findings demonstrate that the metabolic adaptations described for the meal-feeding schedule are entirely reversible. It seems clear that these metabolic adaptations do exist to assure the maintenance of a basic energy supply for the long term intermeal period, so that such fine metabolic adjustments disappeared when food was freely available.
CURI ET AL.
254 ACKNOWLEDGEMENTS The authors are indebted to Professor John Williams (Department of Biochemistry--The Australian National University) and Dr. John McNamara for the revision of the manuscript and to Manuel Jos6 de Oliveira, Marlene Santos Rocha, Valdecir Abflio de Oliveira and Roberto Nascimento for the technical assistance.
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