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Nutritional Neurosciences

Rats Rapidly Reject Diets Deficient in Essential Amino Acids

Thomas J. Koehnle^*,,2, Matthew C. Russell^* and Dorothy W. Gietzen^*,

^* School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology and Animal Behavior Graduate Group, University of California-Davis, Davis, CA 95616

²To whom correspondence should be addressed. E-mail: tjkoehnle{at}ucdavis.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS Experiment 1. Effect of... Experiment 2. Nonessential amino... Experiment 3. Using the... RESULTS DISCUSSION LITERATURE CITED

 
Omnivores must obtain diets balanced with respect to amino acids to support growth and protein synthesis. The standard paradigm used to study behavioral responses to amino acid deficiency combines deficient diets with dietary novelty. The objective of this study was to examine the effects of amino acid deficiency on the first meal of rats without the confounding effects of novelty. We report on a series of five studies of feeding behavior in rats. Rats were fed low protein diets for 5–7 d and then exposed to diets with and without essential amino acids. Rats consistently demonstrated recognition of essential amino acid deficiency within the first meal by a significant reduction in first meal duration, rejecting the deficient diets after just 12–16 min exposure. This is the first report of a rapid effect of amino acid–deficient diets without the confounding effects of dietary novelty.

KEY WORDS: • amino acid imbalance • feeding behavior • rodents • microstructure

Free amino acids cannot be stored in the body (1–3); thus, they must be readily synthesized or obtained from an animal’s diet if protein synthesis is to be sustained. Of the 20 amino acids necessary for protein synthesis, only 12–13 can be synthesized in sufficient amounts by animals, depending on age and the species (4). Omnivores can obtain great advantage from a system that allows them to recognize rapidly foodstuffs with imbalanced or deficient amino acid profiles. Such a system does exist, and has been well studied in rats, Rattus norvegicus [reviewed in (5)].

The dominant paradigm in the study of the behavioral effects of amino acid imbalanced diets has been as follows. Rats are brought into the laboratory and fed a low protein (Basal) diet limiting with respect to a single essential amino acid for 5–14 d [reviewed in (6,7)]. After this period, rats are given either a high protein diet with a balanced amino acid profile (Corrected diet, Table 1), or a test diet with high levels of all of the amino acids except the one limiting in the Basal diet (Imbalanced diet). Thus, in this model, both groups are fed a diet with an increased proportion of free amino acids (6). In an alternative paradigm, the test diet is totally devoid of the limiting amino acid (Devoid diet). Both the Imbalanced and Devoid diets ultimately lead to a complex cascade of neural signals that rapidly reduces the food intake of rats (5,8).

Below we describe a series of experiments documenting the rapid changes in feeding behavior produced by diets devoid of essential amino acids. The purpose of this study was twofold: 1) to confirm that rats rapidly recognize amino acid–deficient diets (12), and 2) to develop a paradigm that will allow the study of the behavioral effects of amino acid disproportion without the confounding effects of dietary novelty.

	MATERIALS AND METHODS

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Animals and diets.

The experiments described below were approved by the UC Davis Animal Care and Use Committee. Male Sprague-Dawley rats weighing 100–110 g were purchased from a commercial supplier (Harlan Sprague Dawley, Indianapolis, IN, except as noted). All cages were kept in a vivarium maintained at 22°C, with a 12-h light:dark cycle, following NIH guidelines for animal care and use. The lights were set to go out at 1600 h. Cage maintenance was conducted during the light cycle to avoid interference with behavioral measurements.

All rats were housed individually in hanging wire cages so that their food intake could be monitored (see below), and fed the Basal diet (one group received a 6% casein diet, see below). All diets were manufactured in our laboratory using purified ingredients and free L-amino acids (Table 1). Each rat was deprived of food for 3 h before the onset of the dark cycle throughout the study. Rats housed in this way were fed the Basal diet for 5–7 d before the feeding experiments. In all experiments, treatment groups were balanced with respect to body weight. Feeding behaviors recorded included total food intake during the 21 h of the test day, food intake and duration of the first meal, number of meals consumed during the 21 h and the delay between the first and second meal. The rate of eating during the first meal was also examined, as described below.

Apparatus.

The monitoring equipment used during this study has been described previously (12). Briefly, a small Plexiglass chamber overhangs a Sartorius Type L-610 digital balance, on which the food cup is mounted. Food spillage is caught in real time by a Tupperware pan mounted beneath the food cup. Measurements are taken at a rate of 1 Hz, using software developed on site (LabView v. 5.0; National Instruments, Austin, TX). Feeding behavior was recorded over the full 21 h that food was available to the rats.

Data gathered using this monitoring system are inherently noisy due to air currents and rat activities in the vivarium. To measure microstructural elements within the first meal, a short program was written using Microsoft Quick Basic (v. 3.0, Redmond, WA). The rate of eating during the first meal was obtained by linear interpolation between periods of 2 s or longer during which the balance output fluctuated within ± 2 SD of the noise from the same balance while not being disturbed by the rat. The end of the meal was defined operationally as a pause ≥ 10 min in duration (13,14).

	Experiment 1. Effect of novel diets

TOP ABSTRACT MATERIALS AND METHODS Experiment 1. Effect of... Experiment 2. Nonessential amino... Experiment 3. Using the... RESULTS DISCUSSION LITERATURE CITED

 
To test the possibility that dietary novelty affects detection of amino acid deficiency per se, the normal paradigm was modified to compare a 6% casein prefeed diet, with its different texture and flavor, to the normal Basal diet prefeed.

Experiment 1A.

Rats (n = 16) were obtained and housed as described above. The control group received the standard high protein diet with a balanced amino acid profile (Corrected diet), whereas the experimental group received a high amino acid diet completely missing the essential amino acid threonine (Devoid diet). Their feeding behavior was then monitored for 21 h.

Experiment 1B.

In this experiment, rats [n = 16; obtained from B&K Universal (Freemont, CA), due to difficulties with vendor supplies] were housed as described above, except that they were fed a low protein diet with 6 g/100 g casein supplemented with L-methionine as the sole protein source. The control group received the low protein Basal diet, whereas the test group received the same diet minus the essential amino acid threonine (Basal-Thr diet).

	Experiment 2. Nonessential amino acid controls

TOP ABSTRACT MATERIALS AND METHODS Experiment 1. Effect of... Experiment 2. Nonessential amino... Experiment 3. Using the... RESULTS DISCUSSION LITERATURE CITED

 
Although there is no historical precedent, it is possible that rapid reactions to amino acid deficiencies are not specific to essential amino acid levels in the diet, but depend at least in part on the texture or flavor effects of removing an arbitrary amino acid from the diet, or adding a large bolus of other amino acids, as in the Corrected, Imbalanced and Devoid diets. Also, the historical emphasis on threonine deficiency may prevent generalization of results to the effects of deficits in other essential amino acids. We employed four modified Basal diet recipes to address these questions in Experiment 2.

Experiment 2A.

Rats (n = 16) were obtained and housed as described above. The control group received the low protein Basal diet minus the nonessential amino acid serine (Basal-Ser), whereas the test group received the Basal-Thr diet. Two rats were excluded from the analysis of this study. One module malfunctioned, and another rat’s feeding behavior was > 2 SD from the mean of the rest of its cohort over the basal feeding period. As a result, there were 8 rats in the Basal-Thr group and 6 in the Basal-Ser group.

Experiment 2B.

Rats (n = 16) were obtained and housed as described above. The control group received the low protein Basal diet devoid of the nonessential amino acid arginine (Basal-Arg), whereas the test group received the Basal diet devoid of the essential amino acid isoleucine (Basal-Ile). Three rats had to be excluded from the analysis of this experiment. One rat escaped, and two rats were > 2 SD away from the mean of the rest of their cohort over the basal feeding period. As a result, there were 6 rats in the Basal-Arg group and 7 in the Basal-Ile group.

	Experiment 3. Using the Basal diet as its own control

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Having controlled for the novelty of dietary texture, flavor, and composition, we sought a simplified paradigm that did not require the time, expense and potential confounds of using a variety of prefeed and test diets. Therefore, in Experiment 3, we attempted to use the basal diet as a control for the Basal-Thr diet. Rats (n = 16) were obtained from B&K Universal, and housed as described above. After 5 d of consuming the Basal diet, the control group received the Basal diet, whereas the test group received the Basal-Thr diet.

Statistical analyses.

All results are reported as means ± SEM. Rates of eating were calculated by determining the change in weight on each balance at 2-min intervals. Only rats still eating during the entire 2-min interval were included in the calculation of the rate during that period. Data from the test day were analyzed using a one factor (diet) between groups ANOVA. Differences were considered significant at P < 0.05. Effect sizes are reported in terms of Cohen’s d, where effects with an absolute value ≥ 0.8 are defined as large (15).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS Experiment 1. Effect of... Experiment 2. Nonessential amino... Experiment 3. Using the... RESULTS DISCUSSION LITERATURE CITED

 
Food intake during the first meal.

In Experiment 1A, rats fed the Devoid diet ate less food in the first meal than rats given the Corrected diet (P = 0.001, Fig. 1). In Experiment 1B, food intake was reduced in the Basal-Thr group relative to the Basal controls during the first meal (P = 0.003). In Experiment 2A, rats fed the Basal-Thr diet tended to eat less during the first meal than the Basal-Ser group (P = 0.052). In Experiment 2B, the Basal-Ile group tended to decrease their intake during the first meal relative to the Basal-Arg group (P = 0.089). In Experiment 3, first meal food intake of the Basal-Thr group was less than that of the Basal group (P = 0.021).

View larger version (57K): [in this window] [in a new window]   FIGURE 1 Food intake during the first meal by rats fed amino acid–deficient diets. Rats fed the deficient diets consistently ate less than their respective controls. Bars are means ± SEM, n (control/deficient) 1A: 8/8; 1B: 8/8; 2A: 6/8; 2B: 6/7; 3: 8/8. Stippled bars represent the control group, and hatched bars represent the deficient group. Values above the bars are effect sizes, given as Cohen’s d (15), where |d| > 0.8 is considered large. Negative effect sizes reflect the number of SD by which the test group mean is lower than the control mean. *P < 0.05.

In Experiment 1A, the first meals of rats given the Corrected diet were longer than the first meals of rats fed the Devoid diet (P = 0.005; Fig. 2), accounting at least in part for the reduction in food intake described above. A similar effect was observed in Experiment 1B, in which rats given the Basal-Thr diet ate for a much shorter period of time than the Basal diet controls (P = 0.041). The absence of nonessential amino acids from the test diet did not affect this relationship because the rats fed Basal-Ser diet had a longer first meal than the Basal-Thr group (P = 0.009), whereas the Basal-Arg group had a longer meal duration than the Basal-Ile group (P = 0.005). Finally, Experiment 3 revealed that feeding rats the Basal-Thr diet shortened the meal duration relative to the Basal control diet (P = 0.016).

View larger version (61K): [in this window] [in a new window]   FIGURE 2 First meal duration by rats fed amino acid–deficient diets. Rats fed the deficient diets terminated their meals significantly earlier than control rats in all experiments. Bars are means ± SEM,n (control/deficient) 1A: 8/8; 1B: 8/8; 2A: 6/8; 2B: 6/7; 3: 8/8. Stippled bars represent the control group, and hatched bars represent the deficient group. Values above the bars are effect sizes, given as Cohen’s d (15), where |d| > 0.8 is considered large. Negative effect sizes reflect the number of SD by which the test group mean is lower than the control mean. *P < 0.05.

There were no consistent differences in the rate of eating during the first meal between diet groups in any experiment, other than the large decrease in first meal duration described above. Because many rats eating diets devoid of essential amino acids ceased eating before those in their respective control groups, sample size suffered over the duration of the first meal, and statistical tests could not be performed. Those rats in the deficient groups still eating at later times did not differ when compared visually with their controls (data not shown).

Behavioral effects after the first meal.

In Experiment 1A, rats fed the Devoid diet ate less food than rats fed the Corrected diet after the first meal (P < 0.001; Table 2). Due to a power outage at 68 min into the experiment, only 3 rats in each group had commenced their second meal before loss of data. The delay between the first and second meal was determined on this basis, and was not altered by amino acid deficiency (Table 3). The total number of meals consumed over the full 21 h could not be determined in this experiment (Table 4). In Experiment 1B, food intake after meal 1 tended to be less in rats fed the Basal-Thr diet than rats fed the Basal diet (P = 0.148). The delay between the first and second meals (Table 3), and the number of meals eaten during the 21 h (Table 4) were not altered by amino acid deficiency.

In Experiment 3, food intake after the first meal was significantly suppressed in the Basal-Thr group compared with the Basal diet–fed controls (P < 0.001; Table 2). The delay between the first and second meals tended to be longer in the Basal-Thr group (P = 0.073; Table 3). The Basal-Thr diet did not affect the number of meals eaten during the 21-h feeding period (Table 4).

	DISCUSSION

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The role of novelty in recognizing amino acid deficient diets.

This is the first report on behavioral responses to diets deficient in an essential amino acid when the test diet is not completely novel. This shift in empirical paradigms is necessary because the Basal to Corrected diet transition activates many of the same signaling pathways in the rat brain as those involved in the Basal to Imbalanced transition (9–11).

If novel tastes, textures and odors affect the behavioral detection of essential amino acid deficiency, then the results of Experiments 1A and 1B should have been dramatically different from the results of Experiments 2 and 3. Similarly, if the deficiency of individual amino acids was detectable by taste, the removal of serine and arginine in Experiment 2 would be expected to mimic the absence of an essential amino acid. Indeed, past research on the detection of amino acid deficiencies many hours after presentation of the test diets has shown that detection is not dependent on an intact olfactory system (16) or any other peripheral tissue (5). Amino acid deficiencies are detected in the central nervous system by the anterior piriform cortex (APC) (5,17). Signals from the APC are relayed to the amygdala, where aversions and dietary rejections are controlled, and the lateral hypothalamus, an important brain nucleus in the control of food intake (8).

First meal duration as a robust indicator of recognition.

Gietzen and co-workers (18) found that food intake was significantly reduced within 15–30 min after the first exposure of rats to a threonine-imbalanced diet. They did not report whether this reduction in intake was the result of a decrease in feeding bout frequency or duration, or due to a change in the rate of eating. In the present study, the reduction in first meal duration was specific to dietary deficiencies in essential amino acids only, and this response was independent of the particular prefeeding regimen and exact composition of the test diets. Most importantly, our results show the striking quantitative reliability of this behavioral phenomenon (Fig. 2), suggesting that the duration of the first meal is a robust indicator of dietary amino acid quality.

On the other hand, measures other than total food intake after the first meal are less robust in response to amino acid deficiency. The delay to the second meal was consistently, but not always significantly increased by deficient diets. This agrees well with our earlier observations (18). Although food intake was markedly suppressed after the first meal (Table 2), the number of meals eaten during the full 21 h was both increased and decreased, with no consistent significant differences. This observation deviates somewhat from the accepted view that amino acid deficiency reduces the frequency of meals (18,19). It may be that the smaller rats used in these studies have a higher demand for food during their early life than those studied previously. Alternatively, the absence of novelty in most of the test diets may have abolished the change in meal frequency. Without a clear choice between an amino acid–deficient diet and another, adequate diet, laboratory rats normally continue to eat the deficient diet (5).

Modulation of the rate of eating in response to amino acid deficiency.

Finally, some comment must be made about the lack of evidence for a decrease in the rate of eating in response to amino acid–deficient diets (Fig. 2). In contrast to earlier studies (5,12), the present results did not support any change in the rate of eating in response to amino acid deficiency during the first meal. Two alternatives might account for this discrepancy. First, any change in the rate of eating might be relatively small, making it difficult to detect empirically. Indeed, Koehnle et al. (12) reported that the change in the rate of eating was not significant. Alternatively, it is possible that the rate of eating is not modulated at all in response to amino acid deficiency or imbalance, and the difference reported in earlier studies was in error. More research aimed at understanding the behavioral mechanisms that support recognition of amino acid deficiencies must be conducted to separate these alternatives.

	FOOTNOTES

 
¹ Funded by National Institutes of Health grant NS-33347 and U.S. Department of Agriculture grant 2000–01049.

³ Abbreviations used: APC, anterior piriform cortex; Basal-Thr, Basal diet minus threonine; Basal-Ser, Basal diet minus serine; Basal-Ile, Basal diet minus isoleucine; Basal-Arg, Basal diet minus arginine.

Manuscript received 21 January 2003. Initial review completed 19 February 2003. Revision accepted 17 April 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS Experiment 1. Effect of... Experiment 2. Nonessential amino... Experiment 3. Using the... RESULTS DISCUSSION LITERATURE CITED

 

1. Berg, C. P. & Rose, W. P. (1929) Tryptophane and growth. J. Biol. Chem. 82:479-484.[Free Full Text]

2. Munro, H. N. (1976) Regulation of body protein and metabolism in relation to diet. Proc. Nutr. Soc. 35:297-307.[Medline]

3. Heger, J. & Frydrych, Z. (1989) Efficiency of utilization of amino acids. Friedman, M. eds. Absorption and Utilization of Amino Acids 1989:31-56 CRC Press Boca Raton, FL. .

4. Bender, D. A. (1985) Amino Acid Metabolism 1985 John Wiley & Sons Chichester, UK.

5. Gietzen, D. W. (2000) Amino acid recognition in the central nervous system. Berthoud, H. Seeley, R. J. eds. Neural and Metabolic Control of Macronutrient Intake 2000:339-357 CRC Press London, UK. .

6. 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]

7. Harper, A. E. (1976) Protein and amino acids in the regulation of food intake. Novin, D. Wyrwicka, W. Bray, G. eds. Hunger: Basic Mechanisms and Clinical Implications 1976:103-113 Raven Press New York, NY. .

8. Gietzen, D. W., Erecius, L. F. & Rogers, Q. R. (1998) Neurochemical changes after imbalanced diets suggest a brain circuit mediating anorectic responses to amino acid deficiency in rats. J. Nutr. 128:771-781.[Abstract/Free Full Text]

9. Wang, Y., Cummings, S. L. & Gietzen, D. W. (1996) Temporal-spatial pattern of c-fos expression in the rat brain in response to indispensable amino acid deficiency I. The initial recognition phase. Mol. Brain Res. 40:27-34.[Medline]

10. Sharp, J. W., Magrum, L. J. & Gietzen, D. W. (2002) Role of MAP kinase in signaling indispensable amino acid deficiency in the brain. Mol. Brain Res. 105:11-18.[Medline]

11. Sharp, J. W., Magrum, L. J., Ross, C. M. & Gietzen, D. W. (2002) Molecular signaling in the anterior piriform cortex in response to corrected and threonine devoid diets. Appetite. 39:98(abs.).

12. Koehnle, T. J., Stephens, A. & Gietzen, D. W. (2002) Alteration of the microstructure of feeding behavior during the first meal of the threonine imbalanced diet. Appetite 39:85(abs.).

13. Castonguay, T. W., Kaiser, L. L. & Stern, J. S. (1986) Meal pattern analysis: Artifacts, assumptions and implications. Brain Res. Bull. 17:439-443.[Medline]

14. Booth, D. A. (1972) Some characteristics of feeding during streptozotocin-induced diabetes in the rat. J. Comp. Physiol. Psychol. 80:238-249.[Medline]

15. Cohen, J. (1988) Statistical Power Analysis for the Behavioral Sciences 2nd ed. 1988 Erlbaum Hilldale, NJ.

16. Leung, P.M.B, Larson, D. M. & Rogers, Q. R. (1972) Food intake and preference of olfactory bulbectomized rats fed amino acid imbalanced or deficient diets. Physiol. Behav. 9:553-557.[Medline]

17. Gietzen, D. W. (1993) Neural mechanisms in the responses to amino acid deficiency. J. Nutr. 123:610-625.

18. Gietzen, D. W., Leung, P.M.B., Castonguay, T. W., Hartman, W. J. & Rogers, Q. R. (1986) Time course of food intake and plasma and brain amino acid concentrations in rats fed amino acid-imbalanced or -deficient diets. Kare, M. R. Brand, J. G. eds. Interaction of the Chemical Senses with Nutrition 1986:415-456 Academic Press Orlando, FL. .

19. Feurte, S., Tome, D., Gietzen, D. W., Even, P. C., Nicolaidis, S. & Fromentin, G. (2002) Feeding patterns and meal microstructure during development of taste aversion to a threonine devoid diet. Nutr. Neurosci. 5:269-278.[Medline]

20. Hammer, V. A., Gietzen, D. W., Sworts, V. D., Beverly, J. L. & Rogers, Q. R. (1990) Adrenal hormones and the anorectic response and adaptation of rats to amino acid imbalance. J. Nutr. 120:1617-1623.

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