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Am J Physiol Regul Integr Comp Physiol 284: R751-R759, 2003. First published November 27, 2002; doi:10.1152/ajpregu.00268.2002
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Vol. 284, Issue 3, R751-R759, March 2003

Energy restriction with protein restriction increases basal metabolism and meal-induced thermogenesis in rats

Patrick C. Even1, Eric Bertin2, Marie-Noelle Gangnerau2, Suzanne Roseau1, Daniel Tomé1, and Bernard Portha2

1 Laboratoire de Physiologie de la Nutrition et du Comportement Alimentaire, Unité Mixte de Recherche 914, Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, 75231 Paris Cedex 05; and 2 Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique-Unité Mixte de Recherches 7059, Université Paris, 75251 Paris Cedex 05, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously observed an increased sympathetic nervous system (SNS) activity that was partly responsible for a defect in the insulin secretion response to glucose after postweaning protein-energy restriction (PER) in female rats. These results, together with other data on low-protein feeding, suggested that a low protein-to-energy ratio (P/E) in the diet could stimulate energy expenditure (EE), but direct measurements of EE have never been reported under conditions of PER. The goal of the present study was thus to quantify the changes induced by PER to body composition, the various parameters of EE, and plasma triiodothyronine levels. PER induced severe growth retardation, but the subcutaneous white and interscapular brown adipose tissue masses were preserved. Basal metabolism, meal-induced thermogenesis, and triiodothyronine levels were increased, but substrate utilization by the working muscles was unaffected. Meal-induced thermogenesis was increased by spontaneous activity in PER rats only. These results suggest that rats adapt to a low P/E in the diet by burning part of their excess nonprotein energy and storing the remaining excess in subcutaneous adipose tissue.

malnutrition; brown adipose tissue; white adipose tissue; indirect calorimetry; triiodothyronine; spontaneous activity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SYMPATHETIC NERVOUS SYSTEM (SNS) activity and/or the utilization of ingested energy are unchanged or moderately decreased by calorie restriction without protein restriction (12, 14, 16, 25), but increased SNS activity has repeatedly been reported in rats fed a low-protein diet, as evidenced by elevated norepinephrine turnover in the heart and urinary norepinephrine excretion. This increased SNS activity, observed in both young and adult animals, was shown to induce an increase in interscapular brown adipose tissue (IBAT) mass and activity and sometimes in thermogenesis (26, 27, 34). Therefore, a low protein-to-energy ratio (P/E) in the diet was proposed as an explanation for the above disturbances in SNS activity and in the utilization of the ingested energy in response to a low-protein diet (26). During a recent study concerning the impact of postweaning protein-energy restriction (65% of normal ad libitum daily food intake, with a 5% protein diet) on glucose metabolism, we noted a considerable stimulation of SNS activity that was shown to be partly responsible for reduced glucose-stimulated insulin secretion and resistance of the hepatic glucose production pathway to insulin action (19, 24). These two phenomena were not observed when subjecting rats to energy restriction alone (65% of normal ad libitum daily food intake, with a 15% protein diet) (24). Thus these data also suggest that the impact of protein-energy restriction on SNS activity and metabolism may mainly be due to a low P/E in the diet.

On the other hand, the above data support the hypothesis that the stimulation of SNS activity observed under low-protein diets might be responsible for increased energy expenditure, leading to reduced food efficiency and body weight gain. However, simultaneous changes to body composition, especially in the adipose tissue-to-total body mass ratio, may instead explain the changes in energy metabolism previously attributed to protein restriction. For this reason, the significance of determining the basal metabolism without corrections for parallel changes in body composition would make any conclusions hazardous. Furthermore, to our knowledge, no data are presently available on spontaneous activity, its energy cost, and the substrates used to fuel this cost under low-protein feeding conditions. Modifications to these important components of energy expenditure may be implicated in the reduced food efficiency reported under protein or energy restriction conditions.

The aim of the present study was therefore to analyze the impact of a double protein-energy restriction on energy metabolism. For that purpose, we measured the various components of energy expenditure (basal and associated with feeding and activity) (7, 9) in free-moving female rats subjected to the above model of early double protein-energy restriction. To ensure greater accuracy in interpreting these data, they were adjusted to the body composition data using a newly developed model based on the weight of the rat organs and tissues (10). Our results are discussed with reference to data in the literature so as to identify the principal determinant of SNS activity and of its metabolic coupling between protein and energy supply.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

All experiments complied strictly with the European Convention on the Protection of Laboratory Animals (6). Female Wistar rats were studied because the previous experiments by Picarel-Blanchot et al. (24) and Léon-Quinto Adnot and Portha (19) were performed on females. The rats, bred in our colony, were housed in a temperature-controlled room (24°C ± 1) with a 12:12-h light-dark cycle (lights on at 0600). They were weaned 28 days after birth and from this age were grouped in pairs and fed either the standard [control (C) rats] or protein- and energy-restricted diet (PER rats) for 4 further weeks. The C member of each pair of littermates was fed ad libitum with the standard diet, and its food intake was measured daily. The PER member was fed the low-protein diet, restricted to 65% of the caloric intake measured in the C rat. The food cups were refilled every day, 1 h before the onset of darkness.

We verified that PER rats never consumed their daily food allowance in one short meal and had an excess of food available most of the time (i.e., for at least 11 h) during the nocturnal feeding period and checked that no major alterations to the feeding pattern occurred. It could therefore be considered that the duration of fasting was comparable in the two groups when the energy metabolism was analyzed and blood samples were collected during this study. This hypothesis was further reinforced by the observation, after completion of the calorimetric studies, that the energy expended through activity during the 2 h preceding the presentation of the test meal was similar in both the C and PER rats, i.e., 3.94 ± 0.56 and 4.33 ± 0.64 kJ/kg, respectively (P = 0.65). Therefore, PER rats did not exhibit any increased anticipatory activity indicative of an increased feeling of hunger.

Diets

The powdered, semisynthetic standard diet contained by weight (g/100 g) 68% starch, 4% cellulose, 5% lipid (corn oil), and 15% protein (casein) and by calories 72% carbohydrate, 12% lipid, and 15% protein. The powdered, semisynthetic low-protein diet contained by weight (g/100 g) 78% starch, 4% cellulose, 5% lipid (corn oil), and 5% protein (casein) and by calories 83% carbohydrate, 12% lipid, and 5% protein. Assuming 16.7 kJ/g for carbohydrates and proteins and 37.7 kJ/g for lipids, the energy content per 1 g diet was the same in both diets (15.78 kJ). Both diets contained yeast (2 g/100 g), a salt mixture (3.5 g/100 g), and a vitamin mixture (2.2 g/100 g), as described in the study by Picarel-Blanchot et al. (24). Assuming oxidation quotients of 1.0 for carbohydrates, 0.7 for lipids, and 0.825 for proteins, the oxidation quotients of the C and PER diets were, respectively, 0.928 and 0.955.

Measurement of Components of Energy Expenditure

Methodology. This was performed by indirect calorimetry after the rats were housed in turn in a 7-liter, cylindrical, air-proof cage (floor area 408 cm2, height 17.5 cm) linked to an open-circuit, flow-through calorimetric device connected to a computer-controlled system of data acquisition, the description and operating procedures of which have been described extensively in previous publications (7, 9). A brief description of the method is, however, given in Fig. 1. To limit energy expenditure related to thermoregulation, the temperature in the metabolic cage was set at 26°C ± 1 (7, 8, 10).


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  		Fig. 1.   Example of computation of resting metabolic rate (top) and resting rates of glucose (Gox) and lipid oxidation (Lox) (bottom) in 1 rat studied. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were determined using the Kalman filtering method. In summary, a measurement was taken every 10 s, a diffusion model of the respiratory exchanges in the metabolic cage predicting the changes in VO2 and VCO2 that ought to be induced by activity; the Kalman filtering process adjusted the model's prediction and the actual measurements by estimating the source of the discrepancy (the resting metabolism and/or the cost of activity) and then by gradually adjusting these values to keep the predictions in line with the measurements. Because we acquired data at 10-s intervals, this process was repeated 360 times/h (i.e., 1700 times in this example). This procedure allowed us to compute the changes in resting VO2 and VCO2, including during activity, and therefore to compute the total and resting rates of energy expenditure (EE) and Gox and Lox according to the classic formulas EE = 16.3VO2 + 4.57VCO2, Gox = 4.57VCO2 - 3.23VO2, and Lox = 1.69VO2 - 1.69VCO2, with total VCO2 and VO2 used to compute total EE, Gox, and Lox and resting VCO2 and VO2 used to compute resting EE, Gox, and Lox. With VO2 and VCO2 in l/min, the rates of oxidation were obtained in kcal/min for EE and g/min for Gox and Lox. They were further converted into W assuming 1 cal = 4.18 J, 15.65 J/g for glucose, and 39.6 J/g for lipids. Protein oxidation (Pox) was discarded from calculations because collection of urine during study in metabolic chamber indicated that average Pox values during the study were 0.191 mg/min (0.0128 W) in control (C) rats and 0.04 mg/min (0.00240 W) in protein-energy restriction (PER) rats, or 1.33 and 0.27% of basal metabolism, respectively. Rat described in this example exhibited a resting respiratory quotient (RQ) close to 0.7 and bursts of activity inducing sharp increases in the total metabolic rate and RQ. Examination of Gox and Lox rates showed that activity increased the rate of Gox 2 to 3 times more than Lox rate. Application of this procedure to all rats in the study during various time periods (see MATERIAL AND METHODS) was used to compare their basal metabolism, their thermogenic response to feeding and activity, and the relative utilization of glucose and lipid by working muscles. EE-act, EE in relation to spontaneous activity.

Basal metabolism and meal-induced thermogenesis. Basal metabolism and meal-induced thermogenesis were measured in 10 C rats and 9 PER rats according to the following procedure. The rats were housed in the metabolic chamber at 1000 with water but no food available. At 1800, a 3.0-g test meal of the rat's usual maintenance diet was placed in the food cup. Respiratory exchanges and spontaneous activity were continuously recorded until 0900 the next day.

Metabolic response to spontaneous activity. The precise onset of all periods of spontaneous activity was recorded, and changes to glucose and lipid oxidation induced by periods of spontaneous activity were computed from 30 min before to 1 h after the onset of each period of activity. The data were then pooled as a function of the time elapsing between the onset of activity and the test meal: I) early postingestive period between 1 and 3 h after the test meal, II) mid postingestive period between 3 and 6 h after the test meal, and III) late postingestive period between 6 and 12 h after the test meal.

Measurement of Body Composition

Body composition was analyzed immediately after the end of calorimetric studies in 7 of the 10 C rats and 7 of the 9 PER rats. The live body weight at exit from the metabolic chamber was measured, and then the rat was anesthetized (pentobarbital sodium, 20 mg/kg, Sanofi Santé, France) and exsanguinated by removal of blood via the abdominal aorta. The heart, liver, kidneys, brain, skin, white adipose pads (mesenteric, retroperitoneal, epididymal, abdominal subcutaneous), and tail were removed, blotted dry, and immediately weighed to the nearest 0.01 g. The other internal organs (lungs, genital apparatus, intestines, etc.) were also removed but not weighed. Once dissection was completed, the empty body, i.e., muscle mass and skeleton (excluding tail), was weighed and classified as carcass (Carc). The white fat mass (Fat) was defined as the sum of all white adipose pads dissected out. For the purpose of analysis, three other components were derived: organs and tissues with high metabolic activity (brain, liver, kidney, heart) were grouped under the "High" component; tissues with a low metabolic activity (white adipose depots, skin, tail) were grouped under the "Low" component, and lean body mass (LBM) was taken as body weight minus the Low component (10). The weight of brown adipose tissue (BAT) in the interscapular space and around the kidneys was also determined after minute dissection.

Samples and Analytical Techniques

Blood samples for determination of the free triiodothyronine (T3) level in plasma were obtained in the postabsorptive state from the tail vein of seven rats in each group. They were then centrifuged, and the plasma was separated and stored at -20°C until determination. Free T3 concentrations were obtained using an RIA kit (Amerlase-MAB, Ortho-Clinical Diagnostics, Amersham, UK).

Statistical Analyses

All values are expressed as means ± SE. ANOVA and repeated-measures ANOVA were used to assess differences between groups and were followed by post hoc Scheffé's tests when appropriate. A P value of <0.05 was considered significant. The SAS software (version 6.1; SAS, Cary, NC) was used for the analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body Weight and Body Composition

Body weights were similar in C and PER rats at weaning (78 ± 1.1 vs. 80 ± 0.8 g). The PER diet profoundly affected the development of the rats, which presented with growth arrest. At 8 wk of age, the mean body weight in PER rats was reduced to nearly half that seen in control rats (Table 1). The weight of most organs and tissues from the body, including the brain, was significantly decreased in PER rats (Table 1). In contrast, the weights of subcutaneous white adipose tissue and interscapular and perirenal brown pads were maintained, a result in line with other measurements performed in a larger sample (n = 12) of PER and C rats at 8 wk of age (data not shown).

                              
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  		Table 1.   Body composition (in g) of rats at 8-9 wk

The calculation of tissue weight relative to total body weight (Table 2) revealed that some tissues were more preserved than others. The stomach, intestine, lungs, heart, and particularly the brain were less severely affected than the carcass, skin, and urogenital apparatus. The weight of subcutaneous adipose tissue in PER rats was nearly twice that seen in C rats (2.20 vs. 1.34% of total body wt). However, the fat mass in the other depots tended to be reduced, so that the overall fat mass in PER rats was only marginally increased (5.62 vs. 4.72% of total body wt). BAT mass relative to body weight was significantly increased in PER rats (Table 2).

                              
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  		Table 2.   Body composition (% total body wt) of rats at 8-9 wk

Parameters of Energy Metabolism

The parameters of energy metabolism were determined from analyses as that illustrated and discussed in Fig. 1

Basal metabolism and basal respiratory quotient. The lowest rate of resting metabolism (defined here as the basal metabolism) was observed 15-17 h after the test meal. The basal metabolism measured in C rats was very close to the basal metabolism predicted by our newly developed model (10) based on the weight of rat organs (104%). This result suggests that, when body composition is carefully taken into account, a prediction of metabolism from organ size is valid for a broad range of body weights and fits for both male and female rats. The basal metabolism was lower in PER than in C rats. However, when adjusted to body weight or body weight(0.75), or to the weight and metabolic activity of the various tissues and organs in the body, it was higher (Table 3). The basal respiratory quotient (RQ) did not differ significantly between C and PER rats (0.79 ± 0.01 vs. 0.78 ± 0.01), indicating that in the postabsorptive state, the differences in basal metabolism were not due to differences in the relative oxidation rates of the various substrates.

                              
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  		Table 3.   Basal metabolism in C and PER rats

Postprandial energy expenditure. Meal-induced thermogenesis was 70% higher in PER rats (5.26 ± 0.47 kJ) than in C rats (3.08 ± 0.41 kJ) (Fig. 2). During the first 7 h of the postprandial period, i.e., for as long as meal-induced thermogenesis was not completed, the time course of activity was the same in C and PER rats (Fig. 3A). It then fell sharply and became significantly lower than in the C group. However, because PER rats weighed less than C rats, the energy expended in relation to spontaneous activity, i.e., the difference between total and background energy expenditure (EE-act; see Fig. 1), was smaller in PER rats during most of the postprandial period (Fig. 3B). When the energies expended in relation to the thermic effect of the test meal and to spontaneous activity were added, it was observed that the energy expended over basal metabolism was the same in the two groups and very reproducible within each group until 6-7 h after the meal (Fig. 3C). Thereafter, as meal-induced thermogenesis disappeared and activity decreased, energy expenditure fell in PER rats.


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  		Fig. 2.   Increases in metabolism and RQ above the premeal levels induced by the 3-g (47.3 kJ) test meal in C and PER rats. * P < 0.05. ** P < 0.01.



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  		Fig. 3.   Hourly changes in postprandial activity (A), energy expended with activity (B), energy expended with activity and the thermogenic response to feeding (C), and correlation between energy expended with activity and the thermogenic response to feeding (D). LR, linear regression. * P < 0.05.

Figure 3D demonstrates a significant positive correlation between meal-induced thermogenesis and EE-act in PER rats; the higher that EE-act was, the higher was meal-induced thermogenesis. The R2 value of 0.385 indicates that close to 40% of the variance in meal-induced thermogenesis was related to the intensity of activity occurring in parallel. Such a correlation was not found in C rats and has never been observed by our team to date.

Postprandial changes in RQ. As well as a higher level of meal-induced thermogenesis, a larger postprandial RQ increase was observed in PER rats (Fig. 2). This difference could not be attributed to minor differences in the food quotient (0.027) because postprandial RQ differences sometimes exceeded 0.10. This suggests that during the postprandial period, glucose oxidation relative to lipid oxidation was higher in PER rats.

Energy metabolism in working muscles. The results of this study are summarized in Table 4 and suggest the following general observations. The energy expended per unit of activity decreased with time after the test meal, as did glucose oxidation. In contrast, lipid oxidation increased so that the proportion of glucose used to fuel working muscles gradually declined from 90 to 60-70%. No differences were revealed between C and PER rats with respect to any of the measured parameters, showing that the control of energy metabolism in working muscles was not affected by the PER diet.

                              
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  		Table 4.   Components of energy metabolism measured in C and PER rats during the bursts of spontaneous activity

Free T3 in Plasma

Free T3 levels in plasma were significantly higher in PER rats than in C rats (19.8 ± 0.7 vs. 13.6 ± 1.1 pmol/l, respectively; P < 0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present results show that the well-recognized stunted growth resulting from early protein-energy restriction also involves a redistribution of the relative weight of various tissues and organs and an increase in the predicted basal metabolism and thermogenic and RQ responses to feeding but does not affect the energy metabolism of working muscles. Spontaneous activity was reduced during the postabsorptive period but preserved during the postprandial period.

Body Composition and Hypothesis Regarding SNS Activity

The data on body composition showed that the various tissues and organs responded differently to restriction. Some increased in size relative to total body weight [subcutaneous adipose tissue (+64%), IBAT (+58%), brain (+49%), and intestine (+22%)], others were preserved (muscles, stomach, kidneys, lungs, heart), and others decreased [skin (-13%), genital apparatus (-26%), spleen (-25%)]. These results confirm the marked protection of the brain previously observed during isocaloric low-protein feeding (11), a phenomenon that is probably sustained by the fact that the brain deserves a high priority and seems fully capable of maintaining a normal rate of protein synthesis in the context of a restricted amino acid supply (11). As for IBAT, previous studies had reported a simultaneous increase in its activity and mass under low-protein restriction (29, 34) and a strong relationship between its thermogenic capacity and the SNS activity (30). The robustness of this relationship encouraged us to use hypertrophy of the IBAT as an indication of an effective stimulation of SNS activity. The increased thermogenic capacity of PER rats observed here, based on the increased basal metabolism and thermogenic response to feeding, further confirmed that such SNS activation had indeed occurred. The increased relative weight of white adipose tissue, mainly of the subcutaneous pad, also confirms previous observations in low-protein-fed rats (20, 33), but such data have not yet been reported when combining protein and energy restriction. This hypertrophy could be considered as a positive adaptation allowing rats to store as lipids part of the excess carbohydrate ingested relative to protein that could not be oxidized through the increased basal metabolism and meal-induced thermogenesis. To attain this goal, it can be hypothesized that protein- and energy-restricted feeding did not stimulate SNS activity in the white adipose tissue (particularly subcutaneous) as in other organs but rather decreased it or reduced the sensitivity of tissue(s) to SNS stimulation, so as to diminish lipolysis. One explanation for such regional differences in the sensitivity to an increase in SNS activity could be linked to sensitivity to growth and/or steroid hormones (particularly estrogens as we were studying female rats), but this point merits further investigation. However, because it appears that male and female rats react similarly to low-protein feeding (15), it can be suggested that sex steroids were not the principal factor responsible for the difference in body composition and therefore that the response observed here was not gender specific.

Basal Metabolism

Few previous studies reported on the direct measurement of basal metabolism in low-protein-fed rats (none in low-energy/low-protein-fed rats). Rothwell et al. (27) reported increased oxygen consumption in 22-day-old rats fed a 8% casein diet, while Young et al. (34) observed increased oxygen consumption in lean Zucker rats fed low-protein diets. However, in most cases, the increased energy expenditure was inferred from reduced energy efficiency (22, 30), supporting the hypothesis of increased diet-induced thermogenesis. Classical approaches to basal metabolism adjustments to body size (body weight and body weight raised to the power 0.75) take little account of changes in the relative weight of different tissues and organs. As we anticipated major changes in body composition of PER rats, we also dissected out and weighed most of the individual organs and tissues and predicted the basal metabolism by adding up their metabolic rate. Using this approach, we observed that the basal metabolism measured in C rats fitted well to the values predicted by the weights of their various organs and tissues, demonstrating that the model is robust and can adequately predict the basal metabolism of adult male and female rats within a broad range of body weights. In contrast, the basal metabolism value seen in PER rats was 25% higher that predicted from the weight of their organs and tissues. Because these rats were also food restricted, a manipulation that generally induces a 5-10% decrease in basal metabolism (10, 12, 14), it could be expected that a low-protein diet without energy restriction would result in an even higher increase in the predicted basal metabolism. These data demonstrate for the first time that protein restriction has a greater impact on basal metabolism than energy restriction. Among the potential factors that may have increased energy expenditure per unit of organ size under the PER regimen were the rates of protein synthesis and the nervous/hormonal control of energy metabolism in different major tissues such as the muscles, liver, and gut. It is also possible that the internal composition of these organs, and in particular their lipid content, may have been affected and biased the calculations. However, because the PER rats exhibited higher fat levels, an increased rather than a decreased fat content in these tissues is more likely to have occurred, which would have reduced, rather than elevated, the basal metabolism corrected for organ size.

The increased circulating levels of free T3 observed here agreed with the findings of previous reports on protein restriction (28, 29) and were certainly partly responsible for the increased metabolic rate seen in several important organs. The well-known T3 activation of the futile energy-wasting cycle (5) would indeed allow excess nonprotein energy to be dissipated through excess heat production, as suggested by Young et al. (34). On the other hand, because food restriction generally decreases T3 levels, it appears that, as already suggested by the increased basal metabolic rate and meal-induced thermogenesis, the T3 response to protein restriction prevailed over the T3 response to food restriction. This suggests that priority is given to protein rather than to energy balance, a mechanism that can be understood if one considers the crucial importance of amino acids to renew essential proteins, enzymes, and nucleotides. To our knowledge, however, despite several reports previously reporting a T3 increase in isolated protein restriction, no data are available to further explain the determinants of this increase.

Meal-Induced Thermogenesis

In addition to basal metabolism, we observed that meal-induced thermogenesis was also markedly increased (+70%) in PER rats, a phenomenon accompanied by a tendency for RQ to be higher during the postprandial period, thus indicating that more carbohydrates were oxidized than lipids. The difference in total energy expenditure between PER and C rats may therefore have been increased by up to 30% (25% from basal metabolism, 5-7% from meal-induced thermogenesis). Previous measurements of the acute thermogenic response to a test meal in protein-restricted rats had reported increases of 15-16% (26), 100% (31), or 0 (33). In the latter study, the absence of significant change could be explained by the fact that the meals were tube fed, a procedure that shunts orosensory responses to a meal (18, 21) and thus attenuates the sympathetic response to feeding. The elevated meal-induced thermogenesis seen in our study may have resulted from a higher insulin activity in peripheral tissues (13, 24), leading to increased rates of glucose oxidation during the postprandial period and then to increased BAT activation.

Spontaneous Activity

The absence of increased anticipatory activity before the test meal in PER rats suggests that despite energy restriction, PER rats did not exhibit a food-anticipatory activity that would have suggested some urge to eat. However, it is difficult to derive firm conclusions about the metabolic or hunger status of these rats before the test meal. Indeed, food-anticipatory activity can be sometimes observed in laboratory animals given only one meal a day, either ad libitum or in restricted amounts, but this phenomenon is not systematically observed. It may depend on the palatability of the food, the kind of activity measured (wheel running vs. spontaneous activity), the period (light vs. dark), and the level of food restriction (1, 17, 23, 32). One possible interpretation of these discrepancies is that activity depends on a light-entrainable pacemaker and a food-entrainable pacemaker that compete and may conflict (1, 17, 32). In addition, not only food-anticipatory activity, but overall activity was not increased in PER rats. In our hands, measurements of the components of energy expenditure in food-restricted rats, in the same conditions and with the same apparatus as the one used here (that is well fitted to measure precisely activity), did not show any change in daily activity when food restriction was mild and prolonged (like presently) and in contrast revealed a decreased activity after 5 days where food restriction was more severe (8). Thus food restriction per se could not be expected to induce an increase in activity. Theoretically, because activity has usually an overall favorable effect on protein balance (3, 4) and may also balance a low-protein diet in favor of growth (2), an increased spontaneous activity might have constituted a strategy for low-protein-fed rats to promote anabolism in vital organs and sustain oxidation of nonprotein energy. Why such a strategy was not used in PER rats remains to be elucidated. It seems possible to rule out alterations in the control of energy metabolism in the working muscles because we did not observe any changes, either during the postprandial period (period I) or in the postabsorptive state (period III). However, specific alterations in the processes of protein anabolism-catabolism in relation to activity may have occurred and should be investigated to definitely answer this point.

In conclusion, rats fed restricted amounts of a diet with a low P/E exhibited a range of metabolic changes that seemed to converge in favor of the oxidation of nonprotein substrates and storage of the remaining excess in the white adipose tissue(s). In contrast, no clear adaptations seem to develop to favor the retention of dietary amino acids, except perhaps the specific sensitivity of the metabolic fate of ingested nutrients to activity, although the mechanisms are still to be elucidated. One question that may guide future studies is the threshold P/E below which metabolic adaptations are required because insufficient protein is provided relative to carbohydrates and lipids. It may be difficult to answer this because metabolic adaptations are less easy to demonstrate at higher protein levels (e.g., 10-20%), but the results may be of considerable value to studies on human nutrition.


    FOOTNOTES

Address for reprint requests and other correspondence: P. C. Even, Laboratoire de UMR INRA/INA P-G 914, Physiologie de la Nutrition et du Comportement Alimentaire, 16 rue Claude Bernard, 75231 Paris Cedex 05, France (E-mail : even{at}inapg.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 27, 2002;10.1152/ajpregu.00268.2002

Received 13 May 2002; accepted in final form 25 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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3.   Biolo, G, Maggi SP, Williams BD, Tipton KD, and Wolfe RR. Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. Am J Physiol Endocrinol Metab 268: E514-E520, 1995[Abstract/Free Full Text].

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