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Refeeding after the late increase in nitrogen excretion during prolonged fasting in the rat
Physiology& Behavior,Vol. 50, pp. 345-349. ©PergamonPress plc, 1991. Printedin the U.S.A.
0031-9384/91 $3.00 + .00
Refeeding After the Late Increase in Nitrogen Excretion During Prolonged Fasting in the Rat YVES CHEREL AND YVON LE MAHO
Laboratoire d'Etude des R(gulations Physiologiques, associ~ ~ l'Universit~ Louis Pasteur Centre National de la Recherche Scientifique, 23 rue Becquerel, 67087 Strasbourg, France Received 21 N o v e m b e r 1990 CHEREL, Y. AND Y. LE MAHO. Refeeding after the late increase in nitrogen excretion during prolonged fasting in the rat. PHYSIOL BEHAV 50(2) 345-349, 1991.--Recovery of body mass, food intake and body composition was studied in the laboratory rat after the late increase in nitrogen excretion that characterizes prolonged fasting in mammals and birds. The rats lost 43% of their body mass during 13 days of food deprivation. They all regained their prefasting body mass within a shorter period of 11 days of refeeding. These results confirm that the late increase in nitrogen excretion in rats, as in spontaneously fasting birds, is reversible and is a part of the physiological adaptations to long-term food deprivation. Water intake of the rats continuously decreased during fasting, and the animals virtually stopped drinking as protein utilization increased. On refeeding, changes in water intake paralleled those in food intake. The refed rats progressively increased their daily food intake, that was always higher than the prefasting value (8.0-10.4 vs. 6.7% of body mass). The comparison of organ weights between fed and ad lib refed rats of similar body weight indicates that muscle mass was regained earlier than body fat during refeeding. The laboratory rat therefore appears to be a good experimental model to investigate the metabolic and behavioural changes that occur during spontaneous anorexia and refeeding in wild animals. Starvation
Body mass changes
Food intake
Water intake
MAMMALS and birds adapt to food deprivation by a decrease in total energy expenditure, a decrease in nitrogen excretion and a mobilization of fat stores (4,14). This strategy of protein conservation and lipid utilization ends with the occurrence of a late increase in protein utilization that is concomitant with a progressive exhaustion of fat stores (1, 4, 7, 22). The rate of decrease in body mass may be used as a simple and noninvasive index of these different metabolic phases (1,12). In starved geese, daily body mass changes in parallel with daily nitrogen excretion; i.e., it decreases at the beginning of fasting, remains low during the phase of protein sparing and increases thereafter (13,21). Until recently [reviews in (16,18)], the late phase of increase in protein utilization was called "the premortal rise in nitrogen elimination" and was usually considered to reflect a degenerating and irreversible state. However, in long-term fasting emperor penguins (22), the increase in net proteolysis was not associated with inactivity due to weakness but, on the contrary, with increased locomotor activity. These birds leave their breeding colony at the beginning of this phase and walk on sea ice to feed in the open sea. In laboratory rats, accordingly, locomotor activity rises sharply as the rate of protein utilization increases (12). There is also a change in the nycthemeral pattern of locomotor activity, rats becoming active during both day and night (12), as well as an increase in the proportion of wakefulness at the expense of sleep (5). In the rat also, refeeding after this criticai stage suppresses the fasting-induced rise in locomotor activity (12). The increase in protein utilization during prolonged starvation therefore appears to be highly adaptive, since it is linked to behavioural changes anticipating lethal depletion of en-
Organ weights
ergy reserves and promoting food foraging. Many authors have previously investigated the effect of total starvation on subsequent food intake and mass gain in the rat. In most instances, refeeding was done after only a few days of fasting in 10(O200 g rats, that do not possess sufficient fat stores to conserve efficiently their body proteins during food deprivation (7). We previously observed that individual rats with large reserves could refeed after having reached the stage of increasing protein utilization (1, 3, 12). However, these were only scattered data. More detailed information was needed, not only to confLrm the notion that the late increase in protein utilization is still a reversible process, but also to gain insight into the patterns of body mass recovery and of food and water intakes. The changes in body composition during such large decreases and increases in body mass were also investigated by comparing the organ masses of fed, fasted and refed rats. METHOD
Animals Male Sprague-Dawley rats were obtained from M~rieux-Iffa Credo (Lyon, France) and kept in individual cages. They were fed chow pellets (mass percentage: 50% carbohydrate, 5% fat, and 24% protein) with water ad lib. The animal quarter was maintained at 25 °C with a light-dark cycle of 12 hours (8 a.m. to 8 p.m.).
Experimental Procedures The rats weighed approximately 400 g when they were ran-
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FIG. 1• Daily nitrogen excretion plotted against daily body mass loss during prolonged fasting for the 7 rats of the starved group. Prolonged starvation corresponds to the 5 last days of food deprivation for each rat, i.e., both to the end of the phase of protein sparing and to the phase of increase in nitrogen excretion.
domly divided into 3 groups. The fed group (n = 7) was immediately sacrified. The rats of the second group (n = 7) underwent a fast. They were kept in single metabolism cages• Urine was collected dally over H2SO,* 1 N and stored at - 2 0 °C until analysis. Urinary nitrogen was measured by the method of Kjeldahl using selenium as catalyser. The rats were weighed daily throughout the fast. They were killed after the rate of mass loss increased for 3 days; this corresponded to about 12 days of food deprivation (see the Results section). The rats of the third group were also fasted. They were refed (refed group, n = 9) using the same criterion as for the starved group, i.e., after 3 days of increase in daily body mass loss. They had food and water ad lib and were sacrified when they had regained their prefasting body mass. Daily records were kept of body mass changes, food and water intakes during the control period (3 days before fasting) and during refeeding, body mass loss and water intake being followed during fasting. Urine was not collected in this group because feeding rats were found to spill food through the wire mesh of the metabolic cages inducing errors in the quantification of food intake in fed and refed rats. Rats of the 3 groups were killed by decapitation. The extensor digitorum longus (EDL), soleus, heart, liver, kidneys and epididymal white adipose tissues were then quickly removed and weighed.
RESULTS
Body Mass Loss and Urinary Nitrogen During Fasting The rats of the starved group weighed 400 g at the beginning of the experiment. After about 12 days of food deprivation, their mean body mass was 231 g, a 42% total decrease in mass. Ac-
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FIG. 2. Daily change in body mass vs. time of starvation and refeeding for the 9 rats of the refed group. *p<0.05 when compared to the fed value.
cording to previous data on rats of that size (3), the daily loss in body mass decreased, from 36.9---0.9 g.24 h - I the fwst day of fasting to 11.7---0.6 g.24 h -~ the third day. It stabilized thereafter at 10-11 g-24 h - n and later increased up to 18.0± 1.1 g.24 h - ~. Daily changes in nitrogen excretion followed a similar pattern, i.e., urinary nitrogen decreased from 477 --- 19 mgN.24 h - n in the fed state, to 2 4 3 - 1 3 mgN.24 h - 1 on the third day. It remained constant at 220 mgN.24 h - ~ until the ninth day of starvation and rose thereafter up to 464-+26 mgN.24 h - 1 . As indicated in Fig. 1, daily body mass loss reflects daily nitrogen excretion during long-term fasting since there is a significant (p<0.001) positive correlation between these two parameters. The total duration of fasting, the initial and final body mass and total body mass loss during starvation were similar in both starved and refed groups (Table 1). The rats of the refed group also followed the same pattern of changes in daily body mass loss during fasting as the rats of the starved group (Fig. 2). They
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REFEEDING AFTER PROLONGED FASTING
347
TABLE 1 DURATIONOF FASTINGAND DECREASEIN BODYMASS DURINGPROLONGEDSTARVATION IN TWO GROUPSOF ADULTMALESPRAOUE-DAWLEYRATS
Group
n
Duration of Starvation, Days
Initial Body Mass, g
Final Body Mass, g
Decrease in Body Mass, %
Starved Refed
7 9
11.6 _ 0.9 12.9 -+ 0.6
400.4 +_ 2.5 400.9 _ 1.8
231.2 _+ 2.5 227.6 _+ 3.6
42.2 _ 0.8 43.2 _ 0.8
Daily Body Mass Loss, g.24 h 6th Day
Last Day
9.9 +- 0.7 10.0 _+ 0.4
18.0 -+ 1.1 15.8 -+ 0.3
Values are means -+ SE.
were refed after 3 days of increase in daily body mass loss, i.e., in the situation of large increase in daily nitrogen excretion, as indicated by the results obtained from the rats of the starved group (Fig. 1). Importantly, none of the rats died from starvation or during the refeeding period. They all appeared in good health both during prolonged fasting and refeeding.
Body Mass Gain During Refeeding The rats of the refed group starved for 13 days, tolerating a 43% decrease in mass (Table 1). They regained their initial body mass (400.5-+1.8 g) within a shorter period (10.8_+0.5 days, p<0.05). The daily body mass gain during refeeding was always significantly higher (range: 10.9-+ 1.4-21.7-+ 1.5 g.24 h -L) than the prefasting gain (6.5-+0.7 g.24 h-L), with however a tendancy to decrease at the time the experiment was stopped (Fig. 2).
Water and Food Intakes Before fasting, water intake was 46.8---1.3 ml.24 h -1. It decreased continuously during starvation and was 3.3---0.2 ml.24 h-L on the last day of food deprivation, a 93% decrease (Fig. 3). Losses due to evaporation and manipulation of water bottles were measured during three consecutive days. They were of 2.9-+0.2 ml.24 h - L , a value not significantly different from the water intake of the rats on the last day of fasting (see above). These data therefore indicate that the rats virtually stopped drinking at that time. During refeeding, water intake was significantly lower on days 2, 3 and 4 when compared to the prefasting value. It increased thereafter and stabilized at 48-53 ml.24 h -~ (Fig. 3). The first days of refeeding were marked by a hypophagia (20.8-+0.8 g.24 h -1 on day 2) when compared to the prefasting value (26.3 -+0.4 g.24 h - 1. Thereafter, hyperphagia progressively developed, daily food intake being 35.6+-1.8 g.24 h -~
the last day of refeeding; this corresponds to 35% and 71% increases over the respective values measured the day before fasting and on day 2 of refeeding (Fig. 3).
Changes in Organ Masses During Fasting and Refeeding There was a large decrease in all the organ weights during starvation, the larger loss being 97% for the epididymal white adipose tissue and the smaller (14%) for the soleus muscle (Table 2). In refed rats, the skeletal muscles and heart regained their initial mass, whereas liver became 23% heavier and epididymal fat pads 32% lighter (Table 2). DISCUSSION The present findings confirm that the late increase in protein utilization during prolonged starvation in rats, as in large birds (4, 20, 22), is reversible and appears to be a part of the physiological adaptations to long-term food deprivation. In rats, the sharp rise in locomotor activity that is observed during the fLrSt tWO tO three days of increased protein utilization (12) does not support the idea that the animals are weak and in a terminal pathological state. Indeed, none of the rats of this study died from starvation, and all animals appeared in good health during both periods of fasting and refeeding. As previously discussed (4), how long the period of increasing nitrogen excretion could be extended was not investigated, since it would have required prolongation of the fast until the rats died. Whatever the u'ue duration of the reversible state is, it presumably cannot be more than a few days. Apparently, the shift in fuel u "tflization serves as a signal for the animals to search for food. Upon refeeding, such a behavior stops and, accordingly, locomotor activity decreases (12).
Fasting Period The duration of fasting and the total body mass loss agree with the previous data obtained with rats of the same strain (3).
TABLE 2 OF STARVATIONAND REFEEDINGON ORGANMASSES(ABSOLUTEVALUESAND PERCENTOF INITIALORGANMASS) IN ADULTMALESPRAGUE-DAWLEYRATS Epididymal Fat Pad
Liver
Kidney
Heart
EDL
Soleus
Group
g
%
g
%
g
%
g
%
mg
%
mg
%
Fed Starved Refed
2.38 -+ 0.27 0.18 __ 0.01" 1.61 +- 0.07*
7 68
14.94 _+ 0.34 3.71 -+- 0.18" 18.35 _+ 0.37*
25 123
1.33 -+ 0.03 0.85 - 0.01" 1.42 _ 0.02*
64 107
1.21 -- 0.03 0.72 - 0.02* 1.23 - 0.03
59 102
198 __ 4 133 __ 3* 198 _ 3
67 100
180 -+ 6 155 -- 4 188 - 3
86 104
Values are means ± SE. *p<0.05 when compared to the fed group.
348
The pattern of changes in daily body mass loss was typical of animals that possess sufficient initial fat stores to adapt to starvation by an efficient conservation of their body proteins during the major part of the fast (4, 7, 12, 22). Daily nitrogen excretion, accordingly, remained low and steady from the third to the ninth day of fasting. It increased thereafter, similarly to the daily body mass loss (Fig. 1). Such parallel changes in protein utilization and body mass loss were previously found in long-term fasting birds and mammals (1, 12, 13, 21, 22). As previously described (9, 17, 19), daily water intake drastically decreased in rats during long-term fasting. Our results moreover show that the animals virtually stopped drinking when nitrogen excretion increased. Since 3-4 ml of intracellular water are associated with each gramme of stored protein, as protein utilization rises, an increasing amount of body water may be used for the osmotic regulation and the excretion of metabolic endproducts. Most of this water is probably lost through excretion, as indicated by the parallel increase in the daily body mass loss (Fig. 2). The percent reductions in organ mass during starvation in this study were generally larger than those previously reported in the rat (6,8), indicating that fasting was stopped after a longer phase of protein utilization. The reduction in epididymal fat pads and liver mass was larger than that of the body mass, whereas the decline in skeletal muscle mass was smaller (Table 2). At the end of the fast, fat stores were essentially exhausted and about 33% of the total muscle was lost [assuming that the mass loss of skeletal muscles during the fast was similar to that of the EDL (8)]. These results indicate that animals can tolerate very large decreases in their body reserves, the muscle mass being relatively spared as compared to fat stores.
Refeeding Period To our knowledge, this study presents the first data on body mass gain, as well as water and food intakes, during refeeding after the late critical stage of starvation marked by an increase in protein utilization in the rat. Refeeding after fasting in this species is generally marked by an initial hypophagia as compared to the prefasting value (2,9). This "poststarvation anorexia" is a function of the duration of food deprivation, the longer the starvation period, the lower the food intake (9). Hyperphagia develops thereafter and is still maintained after the rats have regained their prefasting body mass (2,9). In this study, hypophagia was not observed during the first day of refeeding, but only during the 2 subsequent days. Hyperphagia occurred from day 7 of refeeding until the end of the experiment (Fig. 3). Changes in water intake during refeeding paraUelled those in food intake, and there was a slight progressive hyperdypsia [(9), this study]. Because of the large increase in body mass (from 230 to 400 g) observed during refeeding, it is informative to express daily food intake as a function of actual body mass (Fig. 4). During the first day of refeeding, food intake was very high, as it represented 10.4% of actual weight. Daily food intake was thereafter proportional (p<0.001, Fig. 4) to body mass (8.0-9.2% of body weight). These values are higher than that obtained in the fed state (6.7%), indicating that, relative to body mass, hyperphagia occurred until the experiment was stopped. A large decrease in locomotor activity has been found at that time (12). The association therefore between this decrease and hyperphagia accords with the fact that the further increase in locomotor activity during prolonged fasting reflects the food foraging behavior of the rats (l 2). In this study, the rats had regained their prefasting body mass
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BODY MASS, g FIG. 4. Daily food intake vs. body mass during refeeding for the 9 rats of the refed group. Calculation of the regression line was made with individual values (n = 88), but only mean values were in the figure. Values obtained before fasting (Fed) and during the first day of refeeding (R1) were excluded from the calculation. in 11 days of refeeding. Together with high food and water intakes, this rapid weight gain indicates that the animals still possess sufficient functional intestinal absorption and elevated metabolic capacities to replenish their energy reserves. Such resuits argue against the concept of "the premortal rise in nitrogen excretion" (16,18) and show that animals, even after a dramatic decrease in their energy stores, are still able to refeed rapidly and efficiently. The observed changes in organ mass at the same body mass (Table 2) suggest that body composition is different before fasting and at the end of refeeding. Muscle weights were identical in the two groups, but mass of the epididymal fat pad was lower in the refed rats. Similar results were previously obtained with the same tissues from rats that were starved for a shorter period ' (2). Analysis of body composition has demonstrated that during refeeding organ proteins are almost totally replaced before there is a replenishment of lipid reserves (1,11). However, several authors (15,23) have shown a transient increase in liver mass, mainly due to an early lipid accumulation in this organ during refeeding. Such a phenomenon may then explain the 25% increase in liver weight above control values that we observed in the refed rats (Table 2). This study, complementing a recent paper (12), shows that laboratory rats respond to prolonged fasting by the same metabolic and behavioral adaptations than those previously described in wild birds (4,22), and that refeeding after this critical situation is associated with a rapid recovery of body mass. These data allow a generalization of these adaptations to bird and mammal species that possess sufficient fat stores to sustain long-term food deprivation. In addition to this generalization for birds and mammals, the particular interest of the laboratory rat is related to its general use as a biomedical animal model. The comparison between the late phase of starvation and the refeeding period could provide new information about body mass regulation (regulation of body fat mass and/or of lean body mass) in relation to metabolic and behavioral control of food intake. ACKNOWLEDGEMENTS The authors thank J.-P. Robin for his suggestions in the preparation of the manuscript, and R. Stewart for his help in editing.
REFEEDING AFTER PROLONGED FASTING
349
REFERENCES 1. Belkhou, R.; Cherel, Y.; Heitz, A.; Robin J. P.; Le Maho, Y. Energy contribution of proteins and lipids during prolonged fasting in the rat. Nutr. Res. 11:365-374; 1991. 2. BjOrntorp, P.; Yang, M. U. Refeeding after fasting in the rat: effects on body composition and food efficiency. Am. J. Clin. Nutr. 36:a.A.A, A.A.9;1982. 3. Cherel, Y.; Bumol, A. F.; Leturque, A.; Le Maho, Y. In vivo glucose utilization in rat tissues during the three phases of starvation. Metabolism 37:1033-1039; 1988. 4. Cherel, Y.; Robin, J. P.; Le Maho, Y. Physiology and biochemistry of long-term fasting in birds. Can. J. Zool. 66:159-166; 1988. 5. Dewasmes, G.; Duchamp, C.; Minaire, Y. Sleep changes in fasting rats. Physiol. Behav. 46:179-184; 1989. 6. Goodman, M. N.; Lowell, B.; Belur, E.; Ruderman, N. B. Sites of protein conservation and loss during starvation: influence of adiposity. Am. J. Physiol. 246(Endocrinnl. Metab. 9):E383-E390; 1984. 7. Goodman, M. N.; Larsen, P. R.; Kaplan, M. M.; Aoki, T. T.; Young, V. R.; Ruderman, N. B. Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am. J. Physiol. 239(Endocrinol. Metab. 2):E277-E286; 1980. 8. Goodman, M. N.; Ruderman, N. B. Starvation in the rat. I. Effect of age and obesity on organ weights, RNA, DNA, and protein. Am. J. Physiol. 239(Endocrinnl. Metab. 2):E269-E276; 1980. 9. Hamilton, C. L. Problems of refeeding after starvation in the rat. Ann. NY Acad. Sci. 157:10(O-1017; 1969. 10. Harper, J. F. Peritz' F test: basic program of a robust multiple comparison test for statistical analysis of all differences among group means. Comput. Biol. Med. 14:437-445; 1984. 11. Harris, R. B. S.; Kasser, T. R.; Martin, R. J. Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats. J. Nutr. 116:2536-2546; 1986. 12. Koubi, H. E.; Robin, J. P.; Dewasmes, G.; Le Maho, Y.; Frntoso, J.; Minaire, Y. Fasting-induced rise in locomotor activity in rats coincides with increased protein utilization. Physiol. Behav. 50: 337343; 1991. 13. Le Maho, Y.; Vu Van Kha, H.; Koubi, H.; Dewasmes, G.; Girard,
14. 15. 16. 17. 18.
19. 20.
21. 22.
23.
J.; Ferr6 P.; Cagnard, M. Body composition, energy expenditure, and plasma metabolites in long-term fasting geese. Am. J. Physiol. 241(Eodocrinol. Metab. 4):E342-E354; 1981. I_~ Ninan, F.; Cherel, Y.; Robin, J. P.; Lcloup, J.; Le Maho, Y. Early changes in plasma hormones and metabolites during fasting in king penguin chicks. J. Comp. Physiol. [13] 158:395-401; 1988. McDonald, B. E.; Johnson, B. C. Metabolic response to realimentation following chronic starvation in the adult male rat. J. Nutr. 87:161-167; 1965. Morgulis, S. Fasting and undernutrition. A biological and sociological study of inanition. New York: E. P. Dutton and Co.; 1923. Penicand L.; Le Magnen, J. Recovery of body weight following starvation or food restriction in rats. Neurosci. Biobehav. Rev. 4 (Suppl. 1):47-52; 1980. Peret, J.; Jaccluot, R. Nitrogen excretion on complete fasting and on a nitrogen-free diet. Endogenous nitrogen. In: Bigwood, E. J., ed. International encyclopedia of food and nutrition, vol. 11. Protein and amino acid functions. Oxford: Pergamon Press; 1972:73118. Richter, C. P.; Rice, K. K. Comparison of the effects produced by fasting on gross bodily activity of wild and domesticated norway rats. Am. J. Physiol. 179:305-308; 1954. Robin, J. P.; Cherel, Y.; Girard, H.; Chaban, C.; Le Maho, Y. Augmentation du rendement azot6 et hyperphagie associ~es a la rtalimentation apr~s un jefine prolong6 chez l'oie domestique. C. R. Acad. Sci. Paris Strie III 306:375-379; 1988. Robin, J. P.; Cherel, Y.; Girard, H.; C~loen, A.; Le Maho, Y. Uric acid and urea in relation to protein catabolism in long-term fasting geese. J. Comp. Physiol. [B] 157:491-499; 1987. Robin, J. P.; Frain, M.; Sardet, C.; Groscolas,R.; L¢ Maho, Y. Protein and lipid utilization during long-term fasting in emperor penguins. Am. J. Physiol. 254(Regulatory Integrative Comp Physiol 23):R61-R68; 1988. Sober6n, G.; S(mchez, Q. E. Changes in effective enzyme concentration in the growing rat liver. I. Effects of fasting followed by repletion. J. Biol. Chem. 236:1602-1606; 1961.
0031-9384/91 $3.00 + .00
Refeeding After the Late Increase in Nitrogen Excretion During Prolonged Fasting in the Rat YVES CHEREL AND YVON LE MAHO
Laboratoire d'Etude des R(gulations Physiologiques, associ~ ~ l'Universit~ Louis Pasteur Centre National de la Recherche Scientifique, 23 rue Becquerel, 67087 Strasbourg, France Received 21 N o v e m b e r 1990 CHEREL, Y. AND Y. LE MAHO. Refeeding after the late increase in nitrogen excretion during prolonged fasting in the rat. PHYSIOL BEHAV 50(2) 345-349, 1991.--Recovery of body mass, food intake and body composition was studied in the laboratory rat after the late increase in nitrogen excretion that characterizes prolonged fasting in mammals and birds. The rats lost 43% of their body mass during 13 days of food deprivation. They all regained their prefasting body mass within a shorter period of 11 days of refeeding. These results confirm that the late increase in nitrogen excretion in rats, as in spontaneously fasting birds, is reversible and is a part of the physiological adaptations to long-term food deprivation. Water intake of the rats continuously decreased during fasting, and the animals virtually stopped drinking as protein utilization increased. On refeeding, changes in water intake paralleled those in food intake. The refed rats progressively increased their daily food intake, that was always higher than the prefasting value (8.0-10.4 vs. 6.7% of body mass). The comparison of organ weights between fed and ad lib refed rats of similar body weight indicates that muscle mass was regained earlier than body fat during refeeding. The laboratory rat therefore appears to be a good experimental model to investigate the metabolic and behavioural changes that occur during spontaneous anorexia and refeeding in wild animals. Starvation
Body mass changes
Food intake
Water intake
MAMMALS and birds adapt to food deprivation by a decrease in total energy expenditure, a decrease in nitrogen excretion and a mobilization of fat stores (4,14). This strategy of protein conservation and lipid utilization ends with the occurrence of a late increase in protein utilization that is concomitant with a progressive exhaustion of fat stores (1, 4, 7, 22). The rate of decrease in body mass may be used as a simple and noninvasive index of these different metabolic phases (1,12). In starved geese, daily body mass changes in parallel with daily nitrogen excretion; i.e., it decreases at the beginning of fasting, remains low during the phase of protein sparing and increases thereafter (13,21). Until recently [reviews in (16,18)], the late phase of increase in protein utilization was called "the premortal rise in nitrogen elimination" and was usually considered to reflect a degenerating and irreversible state. However, in long-term fasting emperor penguins (22), the increase in net proteolysis was not associated with inactivity due to weakness but, on the contrary, with increased locomotor activity. These birds leave their breeding colony at the beginning of this phase and walk on sea ice to feed in the open sea. In laboratory rats, accordingly, locomotor activity rises sharply as the rate of protein utilization increases (12). There is also a change in the nycthemeral pattern of locomotor activity, rats becoming active during both day and night (12), as well as an increase in the proportion of wakefulness at the expense of sleep (5). In the rat also, refeeding after this criticai stage suppresses the fasting-induced rise in locomotor activity (12). The increase in protein utilization during prolonged starvation therefore appears to be highly adaptive, since it is linked to behavioural changes anticipating lethal depletion of en-
Organ weights
ergy reserves and promoting food foraging. Many authors have previously investigated the effect of total starvation on subsequent food intake and mass gain in the rat. In most instances, refeeding was done after only a few days of fasting in 10(O200 g rats, that do not possess sufficient fat stores to conserve efficiently their body proteins during food deprivation (7). We previously observed that individual rats with large reserves could refeed after having reached the stage of increasing protein utilization (1, 3, 12). However, these were only scattered data. More detailed information was needed, not only to confLrm the notion that the late increase in protein utilization is still a reversible process, but also to gain insight into the patterns of body mass recovery and of food and water intakes. The changes in body composition during such large decreases and increases in body mass were also investigated by comparing the organ masses of fed, fasted and refed rats. METHOD
Animals Male Sprague-Dawley rats were obtained from M~rieux-Iffa Credo (Lyon, France) and kept in individual cages. They were fed chow pellets (mass percentage: 50% carbohydrate, 5% fat, and 24% protein) with water ad lib. The animal quarter was maintained at 25 °C with a light-dark cycle of 12 hours (8 a.m. to 8 p.m.).
Experimental Procedures The rats weighed approximately 400 g when they were ran-
345
346
CHEREL AND LE MAHO 25
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.
.
.
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m
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I
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300
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:[ -10 >-
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I
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400
I
I
500
EXCRETION,
~-J
-3o
o
-4o
600
I ~
!
I
0
5
10 TIME,
m g N . 2 4 h "1
FIG. 1• Daily nitrogen excretion plotted against daily body mass loss during prolonged fasting for the 7 rats of the starved group. Prolonged starvation corresponds to the 5 last days of food deprivation for each rat, i.e., both to the end of the phase of protein sparing and to the phase of increase in nitrogen excretion.
domly divided into 3 groups. The fed group (n = 7) was immediately sacrified. The rats of the second group (n = 7) underwent a fast. They were kept in single metabolism cages• Urine was collected dally over H2SO,* 1 N and stored at - 2 0 °C until analysis. Urinary nitrogen was measured by the method of Kjeldahl using selenium as catalyser. The rats were weighed daily throughout the fast. They were killed after the rate of mass loss increased for 3 days; this corresponded to about 12 days of food deprivation (see the Results section). The rats of the third group were also fasted. They were refed (refed group, n = 9) using the same criterion as for the starved group, i.e., after 3 days of increase in daily body mass loss. They had food and water ad lib and were sacrified when they had regained their prefasting body mass. Daily records were kept of body mass changes, food and water intakes during the control period (3 days before fasting) and during refeeding, body mass loss and water intake being followed during fasting. Urine was not collected in this group because feeding rats were found to spill food through the wire mesh of the metabolic cages inducing errors in the quantification of food intake in fed and refed rats. Rats of the 3 groups were killed by decapitation. The extensor digitorum longus (EDL), soleus, heart, liver, kidneys and epididymal white adipose tissues were then quickly removed and weighed.
RESULTS
Body Mass Loss and Urinary Nitrogen During Fasting The rats of the starved group weighed 400 g at the beginning of the experiment. After about 12 days of food deprivation, their mean body mass was 231 g, a 42% total decrease in mass. Ac-
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25
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FIG. 2. Daily change in body mass vs. time of starvation and refeeding for the 9 rats of the refed group. *p<0.05 when compared to the fed value.
cording to previous data on rats of that size (3), the daily loss in body mass decreased, from 36.9---0.9 g.24 h - I the fwst day of fasting to 11.7---0.6 g.24 h -~ the third day. It stabilized thereafter at 10-11 g-24 h - n and later increased up to 18.0± 1.1 g.24 h - ~. Daily changes in nitrogen excretion followed a similar pattern, i.e., urinary nitrogen decreased from 477 --- 19 mgN.24 h - n in the fed state, to 2 4 3 - 1 3 mgN.24 h - 1 on the third day. It remained constant at 220 mgN.24 h - ~ until the ninth day of starvation and rose thereafter up to 464-+26 mgN.24 h - 1 . As indicated in Fig. 1, daily body mass loss reflects daily nitrogen excretion during long-term fasting since there is a significant (p<0.001) positive correlation between these two parameters. The total duration of fasting, the initial and final body mass and total body mass loss during starvation were similar in both starved and refed groups (Table 1). The rats of the refed group also followed the same pattern of changes in daily body mass loss during fasting as the rats of the starved group (Fig. 2). They
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FIG. 3. Daily water and food intakes vs. time of starvation and refeeding for the 9 rats of the refed group. *p<0.05 when compared to the fed values.
REFEEDING AFTER PROLONGED FASTING
347
TABLE 1 DURATIONOF FASTINGAND DECREASEIN BODYMASS DURINGPROLONGEDSTARVATION IN TWO GROUPSOF ADULTMALESPRAOUE-DAWLEYRATS
Group
n
Duration of Starvation, Days
Initial Body Mass, g
Final Body Mass, g
Decrease in Body Mass, %
Starved Refed
7 9
11.6 _ 0.9 12.9 -+ 0.6
400.4 +_ 2.5 400.9 _ 1.8
231.2 _+ 2.5 227.6 _+ 3.6
42.2 _ 0.8 43.2 _ 0.8
Daily Body Mass Loss, g.24 h 6th Day
Last Day
9.9 +- 0.7 10.0 _+ 0.4
18.0 -+ 1.1 15.8 -+ 0.3
Values are means -+ SE.
were refed after 3 days of increase in daily body mass loss, i.e., in the situation of large increase in daily nitrogen excretion, as indicated by the results obtained from the rats of the starved group (Fig. 1). Importantly, none of the rats died from starvation or during the refeeding period. They all appeared in good health both during prolonged fasting and refeeding.
Body Mass Gain During Refeeding The rats of the refed group starved for 13 days, tolerating a 43% decrease in mass (Table 1). They regained their initial body mass (400.5-+1.8 g) within a shorter period (10.8_+0.5 days, p<0.05). The daily body mass gain during refeeding was always significantly higher (range: 10.9-+ 1.4-21.7-+ 1.5 g.24 h -L) than the prefasting gain (6.5-+0.7 g.24 h-L), with however a tendancy to decrease at the time the experiment was stopped (Fig. 2).
Water and Food Intakes Before fasting, water intake was 46.8---1.3 ml.24 h -1. It decreased continuously during starvation and was 3.3---0.2 ml.24 h-L on the last day of food deprivation, a 93% decrease (Fig. 3). Losses due to evaporation and manipulation of water bottles were measured during three consecutive days. They were of 2.9-+0.2 ml.24 h - L , a value not significantly different from the water intake of the rats on the last day of fasting (see above). These data therefore indicate that the rats virtually stopped drinking at that time. During refeeding, water intake was significantly lower on days 2, 3 and 4 when compared to the prefasting value. It increased thereafter and stabilized at 48-53 ml.24 h -~ (Fig. 3). The first days of refeeding were marked by a hypophagia (20.8-+0.8 g.24 h -1 on day 2) when compared to the prefasting value (26.3 -+0.4 g.24 h - 1. Thereafter, hyperphagia progressively developed, daily food intake being 35.6+-1.8 g.24 h -~
the last day of refeeding; this corresponds to 35% and 71% increases over the respective values measured the day before fasting and on day 2 of refeeding (Fig. 3).
Changes in Organ Masses During Fasting and Refeeding There was a large decrease in all the organ weights during starvation, the larger loss being 97% for the epididymal white adipose tissue and the smaller (14%) for the soleus muscle (Table 2). In refed rats, the skeletal muscles and heart regained their initial mass, whereas liver became 23% heavier and epididymal fat pads 32% lighter (Table 2). DISCUSSION The present findings confirm that the late increase in protein utilization during prolonged starvation in rats, as in large birds (4, 20, 22), is reversible and appears to be a part of the physiological adaptations to long-term food deprivation. In rats, the sharp rise in locomotor activity that is observed during the fLrSt tWO tO three days of increased protein utilization (12) does not support the idea that the animals are weak and in a terminal pathological state. Indeed, none of the rats of this study died from starvation, and all animals appeared in good health during both periods of fasting and refeeding. As previously discussed (4), how long the period of increasing nitrogen excretion could be extended was not investigated, since it would have required prolongation of the fast until the rats died. Whatever the u'ue duration of the reversible state is, it presumably cannot be more than a few days. Apparently, the shift in fuel u "tflization serves as a signal for the animals to search for food. Upon refeeding, such a behavior stops and, accordingly, locomotor activity decreases (12).
Fasting Period The duration of fasting and the total body mass loss agree with the previous data obtained with rats of the same strain (3).
TABLE 2 OF STARVATIONAND REFEEDINGON ORGANMASSES(ABSOLUTEVALUESAND PERCENTOF INITIALORGANMASS) IN ADULTMALESPRAGUE-DAWLEYRATS Epididymal Fat Pad
Liver
Kidney
Heart
EDL
Soleus
Group
g
%
g
%
g
%
g
%
mg
%
mg
%
Fed Starved Refed
2.38 -+ 0.27 0.18 __ 0.01" 1.61 +- 0.07*
7 68
14.94 _+ 0.34 3.71 -+- 0.18" 18.35 _+ 0.37*
25 123
1.33 -+ 0.03 0.85 - 0.01" 1.42 _ 0.02*
64 107
1.21 -- 0.03 0.72 - 0.02* 1.23 - 0.03
59 102
198 __ 4 133 __ 3* 198 _ 3
67 100
180 -+ 6 155 -- 4 188 - 3
86 104
Values are means ± SE. *p<0.05 when compared to the fed group.
348
The pattern of changes in daily body mass loss was typical of animals that possess sufficient initial fat stores to adapt to starvation by an efficient conservation of their body proteins during the major part of the fast (4, 7, 12, 22). Daily nitrogen excretion, accordingly, remained low and steady from the third to the ninth day of fasting. It increased thereafter, similarly to the daily body mass loss (Fig. 1). Such parallel changes in protein utilization and body mass loss were previously found in long-term fasting birds and mammals (1, 12, 13, 21, 22). As previously described (9, 17, 19), daily water intake drastically decreased in rats during long-term fasting. Our results moreover show that the animals virtually stopped drinking when nitrogen excretion increased. Since 3-4 ml of intracellular water are associated with each gramme of stored protein, as protein utilization rises, an increasing amount of body water may be used for the osmotic regulation and the excretion of metabolic endproducts. Most of this water is probably lost through excretion, as indicated by the parallel increase in the daily body mass loss (Fig. 2). The percent reductions in organ mass during starvation in this study were generally larger than those previously reported in the rat (6,8), indicating that fasting was stopped after a longer phase of protein utilization. The reduction in epididymal fat pads and liver mass was larger than that of the body mass, whereas the decline in skeletal muscle mass was smaller (Table 2). At the end of the fast, fat stores were essentially exhausted and about 33% of the total muscle was lost [assuming that the mass loss of skeletal muscles during the fast was similar to that of the EDL (8)]. These results indicate that animals can tolerate very large decreases in their body reserves, the muscle mass being relatively spared as compared to fat stores.
Refeeding Period To our knowledge, this study presents the first data on body mass gain, as well as water and food intakes, during refeeding after the late critical stage of starvation marked by an increase in protein utilization in the rat. Refeeding after fasting in this species is generally marked by an initial hypophagia as compared to the prefasting value (2,9). This "poststarvation anorexia" is a function of the duration of food deprivation, the longer the starvation period, the lower the food intake (9). Hyperphagia develops thereafter and is still maintained after the rats have regained their prefasting body mass (2,9). In this study, hypophagia was not observed during the first day of refeeding, but only during the 2 subsequent days. Hyperphagia occurred from day 7 of refeeding until the end of the experiment (Fig. 3). Changes in water intake during refeeding paraUelled those in food intake, and there was a slight progressive hyperdypsia [(9), this study]. Because of the large increase in body mass (from 230 to 400 g) observed during refeeding, it is informative to express daily food intake as a function of actual body mass (Fig. 4). During the first day of refeeding, food intake was very high, as it represented 10.4% of actual weight. Daily food intake was thereafter proportional (p<0.001, Fig. 4) to body mass (8.0-9.2% of body weight). These values are higher than that obtained in the fed state (6.7%), indicating that, relative to body mass, hyperphagia occurred until the experiment was stopped. A large decrease in locomotor activity has been found at that time (12). The association therefore between this decrease and hyperphagia accords with the fact that the further increase in locomotor activity during prolonged fasting reflects the food foraging behavior of the rats (l 2). In this study, the rats had regained their prefasting body mass
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BODY MASS, g FIG. 4. Daily food intake vs. body mass during refeeding for the 9 rats of the refed group. Calculation of the regression line was made with individual values (n = 88), but only mean values were in the figure. Values obtained before fasting (Fed) and during the first day of refeeding (R1) were excluded from the calculation. in 11 days of refeeding. Together with high food and water intakes, this rapid weight gain indicates that the animals still possess sufficient functional intestinal absorption and elevated metabolic capacities to replenish their energy reserves. Such resuits argue against the concept of "the premortal rise in nitrogen excretion" (16,18) and show that animals, even after a dramatic decrease in their energy stores, are still able to refeed rapidly and efficiently. The observed changes in organ mass at the same body mass (Table 2) suggest that body composition is different before fasting and at the end of refeeding. Muscle weights were identical in the two groups, but mass of the epididymal fat pad was lower in the refed rats. Similar results were previously obtained with the same tissues from rats that were starved for a shorter period ' (2). Analysis of body composition has demonstrated that during refeeding organ proteins are almost totally replaced before there is a replenishment of lipid reserves (1,11). However, several authors (15,23) have shown a transient increase in liver mass, mainly due to an early lipid accumulation in this organ during refeeding. Such a phenomenon may then explain the 25% increase in liver weight above control values that we observed in the refed rats (Table 2). This study, complementing a recent paper (12), shows that laboratory rats respond to prolonged fasting by the same metabolic and behavioral adaptations than those previously described in wild birds (4,22), and that refeeding after this critical situation is associated with a rapid recovery of body mass. These data allow a generalization of these adaptations to bird and mammal species that possess sufficient fat stores to sustain long-term food deprivation. In addition to this generalization for birds and mammals, the particular interest of the laboratory rat is related to its general use as a biomedical animal model. The comparison between the late phase of starvation and the refeeding period could provide new information about body mass regulation (regulation of body fat mass and/or of lean body mass) in relation to metabolic and behavioral control of food intake. ACKNOWLEDGEMENTS The authors thank J.-P. Robin for his suggestions in the preparation of the manuscript, and R. Stewart for his help in editing.
REFEEDING AFTER PROLONGED FASTING
349
REFERENCES 1. Belkhou, R.; Cherel, Y.; Heitz, A.; Robin J. P.; Le Maho, Y. Energy contribution of proteins and lipids during prolonged fasting in the rat. Nutr. Res. 11:365-374; 1991. 2. BjOrntorp, P.; Yang, M. U. Refeeding after fasting in the rat: effects on body composition and food efficiency. Am. J. Clin. Nutr. 36:a.A.A, A.A.9;1982. 3. Cherel, Y.; Bumol, A. F.; Leturque, A.; Le Maho, Y. In vivo glucose utilization in rat tissues during the three phases of starvation. Metabolism 37:1033-1039; 1988. 4. Cherel, Y.; Robin, J. P.; Le Maho, Y. Physiology and biochemistry of long-term fasting in birds. Can. J. Zool. 66:159-166; 1988. 5. Dewasmes, G.; Duchamp, C.; Minaire, Y. Sleep changes in fasting rats. Physiol. Behav. 46:179-184; 1989. 6. Goodman, M. N.; Lowell, B.; Belur, E.; Ruderman, N. B. Sites of protein conservation and loss during starvation: influence of adiposity. Am. J. Physiol. 246(Endocrinnl. Metab. 9):E383-E390; 1984. 7. Goodman, M. N.; Larsen, P. R.; Kaplan, M. M.; Aoki, T. T.; Young, V. R.; Ruderman, N. B. Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am. J. Physiol. 239(Endocrinol. Metab. 2):E277-E286; 1980. 8. Goodman, M. N.; Ruderman, N. B. Starvation in the rat. I. Effect of age and obesity on organ weights, RNA, DNA, and protein. Am. J. Physiol. 239(Endocrinnl. Metab. 2):E269-E276; 1980. 9. Hamilton, C. L. Problems of refeeding after starvation in the rat. Ann. NY Acad. Sci. 157:10(O-1017; 1969. 10. Harper, J. F. Peritz' F test: basic program of a robust multiple comparison test for statistical analysis of all differences among group means. Comput. Biol. Med. 14:437-445; 1984. 11. Harris, R. B. S.; Kasser, T. R.; Martin, R. J. Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats. J. Nutr. 116:2536-2546; 1986. 12. Koubi, H. E.; Robin, J. P.; Dewasmes, G.; Le Maho, Y.; Frntoso, J.; Minaire, Y. Fasting-induced rise in locomotor activity in rats coincides with increased protein utilization. Physiol. Behav. 50: 337343; 1991. 13. Le Maho, Y.; Vu Van Kha, H.; Koubi, H.; Dewasmes, G.; Girard,
14. 15. 16. 17. 18.
19. 20.
21. 22.
23.
J.; Ferr6 P.; Cagnard, M. Body composition, energy expenditure, and plasma metabolites in long-term fasting geese. Am. J. Physiol. 241(Eodocrinol. Metab. 4):E342-E354; 1981. I_~ Ninan, F.; Cherel, Y.; Robin, J. P.; Lcloup, J.; Le Maho, Y. Early changes in plasma hormones and metabolites during fasting in king penguin chicks. J. Comp. Physiol. [13] 158:395-401; 1988. McDonald, B. E.; Johnson, B. C. Metabolic response to realimentation following chronic starvation in the adult male rat. J. Nutr. 87:161-167; 1965. Morgulis, S. Fasting and undernutrition. A biological and sociological study of inanition. New York: E. P. Dutton and Co.; 1923. Penicand L.; Le Magnen, J. Recovery of body weight following starvation or food restriction in rats. Neurosci. Biobehav. Rev. 4 (Suppl. 1):47-52; 1980. Peret, J.; Jaccluot, R. Nitrogen excretion on complete fasting and on a nitrogen-free diet. Endogenous nitrogen. In: Bigwood, E. J., ed. International encyclopedia of food and nutrition, vol. 11. Protein and amino acid functions. Oxford: Pergamon Press; 1972:73118. Richter, C. P.; Rice, K. K. Comparison of the effects produced by fasting on gross bodily activity of wild and domesticated norway rats. Am. J. Physiol. 179:305-308; 1954. Robin, J. P.; Cherel, Y.; Girard, H.; Chaban, C.; Le Maho, Y. Augmentation du rendement azot6 et hyperphagie associ~es a la rtalimentation apr~s un jefine prolong6 chez l'oie domestique. C. R. Acad. Sci. Paris Strie III 306:375-379; 1988. Robin, J. P.; Cherel, Y.; Girard, H.; C~loen, A.; Le Maho, Y. Uric acid and urea in relation to protein catabolism in long-term fasting geese. J. Comp. Physiol. [B] 157:491-499; 1987. Robin, J. P.; Frain, M.; Sardet, C.; Groscolas,R.; L¢ Maho, Y. Protein and lipid utilization during long-term fasting in emperor penguins. Am. J. Physiol. 254(Regulatory Integrative Comp Physiol 23):R61-R68; 1988. Sober6n, G.; S(mchez, Q. E. Changes in effective enzyme concentration in the growing rat liver. I. Effects of fasting followed by repletion. J. Biol. Chem. 236:1602-1606; 1961.