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Nutritional Neurosciences
Research Communication

A Very High 70%-Protein Diet Does Not Induce Conditioned Taste Aversion in Rats

Unité INRA 914 de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, F75231 Paris Cedex 05, France

¹To whom correspondence should be addressed. E-mail: gilles.fromentin{at}inapg.inra.fr.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study was designed to assess the effects of transition and adaptation to a very high protein diet on behavioral food responses, energy intake, body weight gain, and body composition in rats. For this purpose, adult male Wistar rats were fed either a diet with 70% of energy as protein (P70 group) or a diet with 14% of energy as protein (P14 group) for 16 d. These two groups were compared with a P14 pair-fed (P14-pf) group. A behavioral satiety sequence was also examined. The P70 group ate 21% less than the P14 rats (P < 0.001) and gained less body weight (P < 0.01). The P70 group gained more carcass weight than either P14 or P14-pf rats (P < 0.05). Behavior and food intake data were affected in P70 rats on d 1 of eating the very high protein diet and then returned to baseline values as early as d 2 of consuming the P70 diet. Rats that adapted to the very high protein diet did not acquire a conditioned taste aversion but rather exhibited satiety and a normal behavioral satiety sequence.

KEY WORDS: • behavioral satiety sequence • conditioned taste aversion • satiety • palatability • high-protein diet

The benefits or adverse health consequences of a long-term or chronic high-protein diet are puzzling and not well understood, despite being increasingly employed in weight loss therapy. In rats, shifting from a normal to a high-protein diet reduces food intake on d 1 followed by a gradual but incomplete return to the initial intake over subsequent days, usually associated with a reduction in adipose tissue (1–5). The nature of both the initial and long-term depression in food intake remains unclear and has been reported to result from the poor palatability of the high-protein meal, the induction of a conditioned food aversion, the time required for metabolic adaptation, or a greater satiating effect of protein. A previous study using different paradigms, including meal pattern analysis, two-choice testing, flavor testing, a behavioral satiety sequence (BSS),² and taste reactivity, showed that the food depression produced by a high-protein diet (55% energy) was due to satiety rather than to the acquisition of a conditioned taste aversion (CTA) (4). However, the 55% protein level may be just at the limit of adaptation, and numerous studies have been conducted with diets containing 70 or 75% of energy as protein (5–7). The present study was designed to characterize the behavioral responses, body weight gain, and tissue composition in rats fed a very high (70%) protein diet.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Male Wistar rats (n = 36; Depre) weighing 200–210 g were housed in individual cylindrical cages in a room controlled for temperature (23 ± 1°C) under a 12-h light:dark cycle (lights off, 1700–0500 h). All experimental procedures complied with the guidelines of the French National Animal Care Committee. Two AIN-93M modified diets were used (4,8). Instead of casein and cystine, the control (14% protein; P14) diet contained (g/kg diet): total milk protein (Armor Proteines), 140.0; cornstarch (Cerestar), 622.4; sucrose (Eurosucre), 100.3; soy bean oil (Medias Filtrants Durieux), 40.0; vitamin mixture AIN-93M (ICN Biomedicals), 10.0; salt mixture AIN-93M (ICN Biomedicals), 35.0; cellulose (Bailly SA), 50.0; and choline chloride (ICN Biomedicals), 2.3. The diet had a metabolizable energy density of 14.6 kJ/g (% energy protein 14%, lipid 10%, carbohydrate 76%). The very high protein diet (P70) contained (g/kg diet): total milk protein, 680.0; cornstarch, 157.1; and sucrose, 25.6. The remaining ingredients were identical and in the same quantities as in the control diet. The diet had a metabolizable energy density of 14.6 kJ/g (% energy protein 70%, lipid 10%, carbohydrate 20%). Total milk protein is a mixture of casein (85%) and other milk proteins (albumins and globulins). These other milk proteins constitute a direct source of limiting sulfur amino acids. The P14 and P70 diets were moistened (powdered diet to water; 1:1 and 1:2, respectively) to minimize spillage. Food containers were refilled daily with fresh food at 1700 h. During all experiments, the rats consumed water and food ad libitum from 1700 to 0900 h and then were food deprived from 0900 to 1700 h. Food intake was determined from the difference in the weight of food cups before and after each experimental period, corrected for spillage and evaporation. Three groups, matched for body weight, were defined.

For 10 d (prefeeding period, just before d 1), 36 rats were adapted to the laboratory conditions. After 1 wk, they were divided into the 3 following groups matched for body weight: P14 (n = 12), P14-pair fed (n = 6) (P14-pf), and P70 (n = 18). During this prefeeding period, the 3 groups had free access to the standard diet, P14. Thereafter they were fed as follows for 16 d (d 1–16): the P14 group (n = 12) was fed the P14 diet; the P70 group (n = 18) was switched to the P70 diet; and the P14-pf group (n = 6) was fed the P14 diet with each rat fed the mean daily intake of the P70 group.

    Meal pattern analysis. Food intake was recorded by means of food cups placed on a strain-gauge (Entran SA; accuracy of 0.1 g) connected to a 34970A data acquisition/switch unit (Agilent Technology) and to a personal computer programmed to record data every second via HP BenchLink Data Logger software (Agilent Technology).

The cumulative food intake was recorded every second for a period of several consecutive days (d 14–16 for 12 P14 rats, last day of P14 diet, d 1–2 and d 14–16 for 12 P70 rats). In practice, during the transition phase, an intragroup comparison was made (P14, last day; P70, d 1 and 2). After adaptation (d 16), an intergroup comparison was made between the P14 and P70 groups. The cumulative quantity of ingested dry matter (g) was calculated as the metabolizable energy ingested (kJ), using the conversion factor (14.6 kJ/g) and examined during the first 12 h of presentation of food (i.e., 1700–0900 h) and also during each quartile (3-h period).

    Video analysis (BSS). On the day before testing, 6 rats were acclimated to their test chambers between 1400 and 1800 h. The experimental chamber consisted of a circular Plexiglas tank (height, 320 mm; diameter, 300 mm), with a ring attached to the inside wall to hold the food cup, and a water bottle on the opposite side. The video cameras (JVC, Digital Still Camera GR-DVL557) were placed outside the chamber, 300 mm from the food cup ring. The video signal was recorded on conventional VHS tape (VHS-C, EC-60) at 50 frames/s using a recorder (Compact Super VHS, JVC: GR-SXM307). The behavior of 6 rats was video-recorded during h 1 of food presentation. Videotapes were analyzed by a slow-motion playback (frame by frame at one sixth of the normal playing speed) to count behavioral satiety or taste reactivity components.

Food intake was measured at 1800 h. Behavior was recorded from 1700 to 1800 h on the last day of the P14 diet and on d 1, 2, and 16 of the P70 diet. For analysis, the behavior of each rat was categorized as follows (4): Activity: this included locomotion (e.g., walking and running), rearing (defined as a rat standing up on its two hind limbs and jumping in a manner directed at escape from the experimental chamber), and sniffing. Grooming: scratching, licking, or biting of coat. Resting: inactivity, standing, sitting or lying, sleeping with occasional changes of position. Eating: biting, gnawing, swallowing food. The data for each eating bout (number, duration) were measured. A bout was defined as a period of eating as recorded by the video. Its minimum duration was therefore 1 s. The mean rate of eating was calculated for each rat by dividing the total 1-h energy intake by the total 1-h bout duration. Drinking phases were also determined.

    Body tissue weights. At the end of the experiment, the rats (P14, n = 7; P70, n = 9; P14-pf, n = 6) were deprived of food overnight and then killed by an injection of sodium pentobarbital (42 mg/kg body weight). Four white adipose tissue pads were collected and weighed (epididymal, retroperitoneal, visceral, and subcutaneous tissue). The liver, intestine, and kidneys and finally the stripped carcass (the sum of bones and muscles without the feet fingers, the head, and skin,) were also excised and weighed

    Statistical analysis. Results are presented as means ± SEM. Differences between days or groups were tested using one-way ANOVA or ANOVA for repeated measures, when appropriate (PROC GLM, SAS version 6.11). When the ANOVA was significant, a post-hoc test (Tukey’s test) was used to compare between- or within-group means. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Daily energy intake. During the last day of the P14 prefeeding period, the groups did not differ in terms of their daily P14 diet intake (P70, 431.2 ± 6.8 kJ; P14-pf, 435.7 ± 15.0 kJ; and P14, 433.3 ± 9.5 kJ) and body weight (P70, 333 ± 3 g; P14-pf, 342 ± 8 g; and P14, 330 ± 3 g). The transition from the P14 to the P70 diet induced an immediate depression in intake on d 1 and a gradual adaptation to the diet over subsequent days (data not shown). During the next 16 d, the energy intake of the P70 group remained lower than that of the P14 group, with daily energy intakes of 349.7 ± 3.1 and 442.9 ± 7.0 kJ/d, respectively (P < 0.001). The cumulative energy intake of rats fed the P70 diet is presented in Figure 1A. The depression in food intake in rats fed the P70 diet was immediate (Fig. 1B). As early as 2 min after presentation of the diet, the cumulative energy intake of rats fed the P70 diet was lower than that of rats fed the P14 diet during the transition phase (P14, last day and P70, d 1 and 2, P < 0.001) and after adaptation (P70, d 16; P14, d 16, P < 0.001). In the first 3 time periods, we observed a significant decrease in energy intake on d 1 in rats fed the P70 diet compared with their last day of consuming the P14 diet (Fig. 1C). As early as d 2 of consuming the P70 diet, only the first two quartiles (0000–0300 and 0300–0600 h of the dark period) differed. After adaptation to the P70 diet (d 16), only the first and fourth quartiles differed.

View larger version (35K): [in this window] [in a new window]   FIGURE 1 Energy intake of rats fed a normal (P14) diet or that diet followed by a high-protein (P70) diet for 16 d. (A) 12-h cumulative energy intake of P70 rats during the transition period and after adaptation to the P70 diet (d 1, 2, and 16). (B) 30 min-cumulative energy intake. (C) Daily energy intake by quartile. Values are means ± SEM, n = 12. Differences from P14 are represented by letters: a (P < 0.001); b (P < 0.01), and c (P < 0.05). *P70, d 1 and 2 different from P14, last day (P < 0.001). $P70, d 16 different from P14, d 16 (P < 0.001).

View larger version (22K): [in this window] [in a new window]   FIGURE 2 Temporal profile of the BSS of rats fed a normal (P14) diet (last day) or that diet followed by a high-protein (P70) diet (d 1, 2, and 16). Each behavior (eating, activity, grooming, and resting) is expressed as a percentage of time during a 5-min period. Values are means ± SEM, n = 6. *Resting different from resting during P14, last day (P < 0.05).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The results confirmed that rats switched to a very high protein diet experienced an immediate depression in food intake followed by an incomplete return to the level of energy intake of rats fed the control diet. An examination of the BSS confirmed that the reduction in food intake was not due to a CTA to the very high protein diet, extending the results obtained with a high (50%) protein diet (4,9). In h 1 of consuming the very high-protein diet, the BSS, although altered, did not show signs of conditioned aversion. Rats spent more time eating and in activity behaviors and less time resting, with no difference in grooming behavior compared with the basal level of rats fed a normal protein diet. Even if the BSS was extremely disturbed during d 1 of consuming of the diet, the order of behaviors in the satiety sequence was not dramatically modified, contrary to what was observed in the case of a CTA induced by LiCl (9). As early as d 2 of consuming the very high protein diet, the disturbance to the BSS regressed, and after adaptation, no significant differences could be detected in any of the behaviors measured. This finding agrees with our hypothesis that rats do not develop a CTA when they are fed a 70% protein diet (7). If this had been the case, the BSS would have remained disturbed, even after adaptation, and should have been similar to the BSS after treatment with LiCl or after consuming a diet devoid of amino acids (10).

A postabsorptive increase in blood amino acid levels is probably an important reason for the decrease in high-protein diet intake; we showed previously that the subdiaphragmatic vagus nerve does not constitute an obligatory pathway for the transfer of information to the brain, resulting in a depression of high-protein diet intake (11). Several other processes, including orosensorial factors (such as palatability, nature of proteins), or vagus-mediated signals produced by higher amino acid levels in the gut and in the hepatoportal area, cannot be totally excluded. As suggested by different authors (1,2,9), disturbances to the BSS during d 1 (increase in the number of bouts, reduction in the rate of ingestion) and the reduction in food intake observed on d 1 may be due in part to the low palatability of the very high protein diet. Even after 16 d of consuming the diet, the reduction in food intake persisted immediately after food presentation, suggesting that the palatability of the very high protein diet did not improve, even after adaptation. In our opinion, it is unlikely that the nature of the protein used to formulate the food was involved in the depression of food intake during our experiments because we used a well-balanced, high-protein diet of total milk protein (casein, albumins, and globulins). Using such a protein mixture avoided studying the effect of a high-protein diet with an imbalanced pattern of essential amino acids. Moreover, the same depression in food intake was observed with high-protein diets comprising other proteins such as soy, ovalbumin, or lactalbumin (1). It is also unlikely that the different amounts of water added to the diet were involved because eating a high-protein diet induces a depression in food intake whether the food is solid (1,12) or moistened (3,4), and rats fed the P70 diet always needed to drink. More generally, further studies are warranted to determine whether a higher water intake could, for instance, be involved in the depression of high-protein diet intake through a drastic increase in the gastric volume, inducing a vagus-dependent satiating message.

The reduction in energy intake induced by the very high protein diet resulted in lower body weight gain over the 16-d experimental period. To better discriminate between the effects of the lower energy intake and the effects of the diet composition itself, we introduced a 3rd group of rats that consumed the P14 diet at the energy level of rats fed the P70 diet. Interestingly, rats in the P70 group had a higher body weight than those in the P14-pf group. This difference was due to the increased weight in lean tissues, whereas adipose tissue weights did not differ. The principal effect on body composition in the P14-pf group (because of the reduction in energy intake without any change in macronutrient diet composition) was a decrease in body weight associated with an overall decrease in tissue and organ weights, including lean body mass, adipose tissue, and liver. Rats fed the P14 diet were heavier, but this was due mainly to a higher fat mass (3). Both the stripped carcass increase and fat mass reduction in response to a high-protein diet could be related to the specific orientation of energy metabolism, with amino acids as the principal energy substrate. The precise mechanisms involved in these different processes remain to be elucidated.

These results may challenge the consensus concerning the amount of protein required in the diet. Rats fed a control diet (P14) consumed a mean quantity of 3.7 g protein/d. By comparison, rats fed a very high protein diet consumed 10 g protein/d. Rats fed high-protein diets ingested a level of protein that exceeded the requirements defined by the NRC (8). During our study, the difference in protein intake could not be explained by the significant but small between-group difference in weight (23 g). The question of a possible regulation of protein intake at a higher level than that recommended by the NRC committee remains to be answered. It would mean that protein needs may have to cover unknown requirements other than the replacement of essential amino acids and nitrogen. This supports the idea that consumption of high-protein diets should not have deleterious consequences in the long term, which agrees with the concept that omnivores are capable not only of selecting food to meet their needs but also of avoiding toxic foods. Interestingly, the principal differences between the energy intakes of rats fed the P14 or P70 diets occurred during the first quartile of the night. This finding agrees with those of other authors (13) who observed that when rats were given a choice among 3 macronutrients, protein was avoided most during the 1st quartile and then eaten most during the 4th quartile. One explanation could be the influence of circadian cycle hormones on the metabolic state of rats, which in turn influences their protein intake.

	FOOTNOTES

 
² Abbreviations used: BSS, behavioral sequence satiety; CTA, conditioned taste aversion; P14 diet, normal protein (14% energy) diet; P14-pf, pair-fed P14 diet; P50, high protein (50% energy) diet; P70 diet, very high protein (70% energy) diet.

Manuscript received 24 October 2003. Initial review completed 23 November 2003. Revision accepted 17 February 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Semon, B. A., Leung, P.M.B., Rogers, Q. R. & Gietzen, D. W. (1987) Effect of type of protein on food intake of rats fed high protein diets. Physiol. Behav. 41:451-458.[Medline]

2. McArthur, L. H., Kelly, W. F., Gietzen, D. W. & Rogers, Q. R. (1993) The role of palatability in the food intake response of rats fed high-protein intake response. Appetite 20:191-196.

3. Jean, C., Rome, S., Mathé, V., Huneau, J. F., Aattouri, N., Fromentin, G., Larue-Achagiotis, C. & Tomé, D. (2001) Metabolic evidence for adaptation to a high protein diet. J. Nutr. 131:91-98.[Abstract/Free Full Text]

4. Bensaïd, A., Tomé, D., L’Heureux-Bouron, D., Even, P., Gietzen, D., Morens, C., Gaudichon, C., Larue-Achagiotis, C. & Fromentin, G. (2003) A high-protein diet enhances satiety without conditioned taste aversion in the rat. Physiol. Behav. 78:311-320.[Medline]

5. Harper, A. E. & Peters, J. C. (1989) Protein intake, brain amino acid and serotonin concentrations and protein self-selection. J. Nutr. 119:677-689.

6. Leung, P.M.B. & Rogers, Q. R. (1987) The effect of amino acid and protein on dietary choice. Kawamura, Y. Kare, M. R. eds. Umami: A Basic Taste 1987:565-610 Marcel Dekker New York, NY. .

7. Fromentin, G., Feurté, S., Nicolaidis, S. & Norgren, R. (2000) Parabrachial lesions disrupt responses of rats to amino acid devoid diets, to protein-free diets, but not to high-protein diets. Physiol. Behav. 70:381-389.[Medline]

8. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76 Rodent diet. J. Nutr. 123:1939-1951.

9. Blundell, J. E., Rogers, P. J. & Hill, A. J. (1985) Behavioural structure and mechanisms of anorexia: calibration of natural and abnormal inhibition of eating. Brain Res. Bull. 15:371-376.[Medline]

10. Feurté, S., Tomé, D., Gietzen, D. W., Even, P. C., Nicolaïdis, S. & Fromentin, G. (2001) Feeding patterns and meal microstructure during development of a taste aversion to a threonine devoid diet. Nutr. Neurosci. 5:269-278.

11. L’Heureux-Bouron, D., Tome, D., Rampin, O., Even, P. C., Larue-Achagiotis, C. & Fromentin, G. (2003) Total subdiaphragmatic vagotomy does not suppress high protein diet-induced food intake depression in rats. J. Nutr. 133:2639-2642.[Abstract/Free Full Text]

12. Harper, A. E., Benevenga, N. J. & Wohlhueter, R. M. (1970) Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50:428-558.[Free Full Text]

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