Effect of pre-feeding ammonium acetate on food intake of rats fed high protein diets

Physiology & Behavior, Vol. 42, pp. 471--476.Copyright©PergamonPress plc, 1988. Printedin the U.S.A.

0031-9384/88$3.00 + .00

Effect of Pre-Feeding Ammonium Acetate on Food Intake of Rats Fed High Protein Diets B. A. SEMON, P. M. B. L E U N G A N D Q. R. R O G E R S Department o f Physiological Sciences and F o o d Intake Laboratory School o f Veterinary Medicine, University o f California, Davis, CA 95616 R e c e i v e d 8 S e p t e m b e r 1987 SEMON, B. A., P. M. B. LEUNG AND Q. R. ROGERS. Effect ofpre-feeding ammohium acetate on food intake of rats fed high protein diets. PHYSIOL BEHAV 42(5) 471-476, 1988.--The effect on intake of a 75% casein diet after prefeeding for one week a 6% casein basal diet with additional 0%, 2%, 5%, 8% or 15% ammoniumacetate was examined in rats trained to eat in three hours per day. Food intake was measured from 0-15, 15-30, 30-90, and 90-180 minutes for the first two days that the ammonium acetate diets were presented. Rats eating 5% and 8% or 15% ammonium acetate diet depressed their intake significantly for one day and for four days respectively. Rats eating 2%, 5%, 8%, or 15% ammonium acetate diets depressed their intake significantly from 0-30 minutes. When presented with the 75% casein diet, rats prefed 0% to 5% and 8% and 15% ammonium acetate diets ate 55% to 58% and 72% and 94% of their respective baseline intakes. It is suggested that prefeeding 15% ammonium acetate apparently induces sufficient metabolic adaptation to ammonia intake so that the rat is able to offset the metabolic consequences of intake of the 75% casein diet, thus preventing the usual food intake depressing effect of the high protein diet. Casein

High protein

Caloric intake

Ammonium acetate

RATS prefed a low protein diet decrease their food intake when a high protein diet is presented [1-5, 7, 9-11, 13-16, 19-23, 25-26]. The reason for this decrease in intake is not known. Plasma amino acids rise when the high protein diet is fed [1]. Ashida and Harper [2], Harper et al. [3,4] and Peters and Harper [21] have suggested that the extra protein intake exceeds the animal's capacity to catabolize amino acids, especially essential amino acids [21]. However, no mechanism has been shown for how changes in amino acid concentrations influence food intake. An increase in dietary protein may cause an increase in serum ammonia, resulting in a decrease in food intake. Leung and Rogers [12] found that carotid infusions of 0.001 millimoles of ammonium acetate/day reduced food intake by 50% whereas 4.67 millimoles of ammonium acetate per day infused into the rat jugular vein were required to reduce food intake 40%. These findings indicate that the brain is sensitive to ammonia but increases in ammonia have not been shown to occur in brain or plasma correlated with the decreased food intake that occurs when an animal is presented with a high protein diet. Lardy [8] and Rose [24] fed diammonium citrate to rats to determine if ammonia would substitute in the diet for dispensable amino acid nitrogen and they found that rats would grow on a mixture of essential amino acids and ammonia. Kiriyama [6] fed diammonium citrate from zero to eight percent of the diet to determine the effect of dietary ammonia on liver arginase activity. Specific and total liver arginase activity decreased as diammonium citrate increased in the diet

even though urea excretion increased. When ammonium acetate or citrate was force fed to rats receiving either rat chow containing 23% crude protein [5] or a 27% casein diet [28], no increase was noted in urea cycle enzyme activities. Hutchinson et al. [5] found that the specific activity of liver arginase decreased and the specific activity of liver glutamic dehydrogenase increased after force feeding ammonium acetate. Noda [17] found that addition of 2% ammonium acetate to an amino acid imbalance diet decreased food intake when compared to the imbalanced diet alone. Noda [18] also found that rats with lesions of the prepyriform cortex did not decrease their food intake over a 6 hour period when presented with a basal 5% casein diet (supplemented with 0.3% methionine and 0.2% L-threonine) containing an additional 3% ammonium chloride diet, and selected basal diet over ammonium chloride containing diet non-significantly; whereas, intact rats decreased their intake of a 3% ammonium chloride diet over a 6 hour period and selected basal diet over the ammonium chloride diet significantly. Leung and Rogers [10] found that rats with lesions of the prepyriform cortex did not decrease intake of an amino acid imbalanced diet as much as control rats but did decrease their intake of a 75% casein diet. It would appear that rats with such lesions are also not as sensitive to ammonia in the diet [18]. The reason why rats with prepyriform cortex lesions are less sensitive to amino acid imbalance and less sensitive to ammonia in the diet but are still sensitive to high protein in the diet is not known.

471

472

SEMON, L E U N G AND ROGERS TABLE 1 THE COMPOSITIONOF EXPERIMENTALDIETS Casein 6%

15%

Ammonium Acetate 75%

2%

5%

8c~

15%

1.0 5.0 5.0 3.0 26.8 53.6 6.0 0.3 2.0

1.0 5.0 5.0 0.3 25.8 51.6 6.0 0.3 5.0

1.0 5.0 5.0 0.3 24.8 49.6 6.0 0.3 8.0

1.0 5.0 5.0 0.3 22.5 44.9 6.0 0.3 15.0

100.0

100.0

100.0

100.0

% of Diet Vitamin premix* Salt mixturet Corn oil:~ Choline chloride Sucrose Corn starch§ Casein¶ L-methionine Ammonium acetate Total

1.0 5.0 5.0 0.3 27.5 54.9 6.0 0.3 100.0

1.0 5.0 5.0 0.1 24.55 49.05 15.0 0.3 100.0

1.0 5.0 5.0 0,1 4.62 9.28 75,0

100.0

*Total Vitamin Supp., US Biochemical Corp., Cleveland, OH. tRogers and Harper's Salt Mixture, US Biochemical Corp., Cleveland, OH. ~Mazola Corn Oil, Best Food, Englewood Cliffs, NJ. §Melojel, Food Grade Corn Starch, National Starch and Chemical Corp., Bridgewater, NJ. ¶Vita-Free Casein, US Biochemical Corp., Cleveland, OH.

When Kelly [5] prefed rats a 6% casein diet plus 20% ammonium acetate for ten days and then fed the 75% casein diet ad lib, he found that rats did not decrease their intake when presented with the 75% casein diet. In the present study, rats were fed varying levels of ammonium acetate from 0 to 15% added to a 6% casein diet for seven days and their subsequent responses to the feeding of the 75% casein diet were determined for seven more days. METHOD Thirty Sprague-Dawley male albino rats weighing 120-140 grams were obtained from Charles River, Wilmington, MA. The rats were placed in stainless steel cages with wire mesh floors. They were allowed free access to water at all times. The room was lighted from 2000 to 0800 hours. The animals were fed chow for two days and were then placed on a three hour meal feeding regimen with the 15% casein diet presented at the beginning of the dark period. The composition of all experimental diets is shown in Table 1. The food was presented in ceramic cups placed in tall cans with a circular opening in the side for access to the food. This procedure minimized spillage. Paper towels were placed under the cages to collect any spillage that occurred. After the rats were all gaining weight (nine days), their diet was changed to 6.0% casein. After four days on this regimen, the food cups were removed daily and weighed at 15 minutes, 30 minutes, 90 minutes and 180 minutes after food presentation to accustom the animals to having their food intake monitored at these intervals. The food intake measurements of the 6.0% casein diet from days 9, 10 and 11 were averaged to provide baseline data for comparison with food intake on the experimental days. The rats were separated into five groups having equal mean food intakes according to the averaged amount they ate on these three days. On day 12, each group of rats received a 6% casein diet supplemented with one level of either 0%, 2%, 5%, 8% or 15% ammonium acetate. Food intake measurements were taken at intervals as described above for two days and the

total daily food intake of the ammonium acetate containing diets was measured for five more days. At the end of the 7 days of ammonium acetate diet feeding, the rats on lower levels of ammonium acetate had gained weight and the rats eating 15% ammonium acetate had lost weight (Table 3). Therefore because of the difference in body size, the food intake from days six and seven on the ammonium acetate diet was averaged to provide a baseline for intake on the days the 75% casein diet was fed. On days I%25, the rats were all offered a 75% casein diet for the same three hour period per day. Food intake was measured daily. The following flow chart represents the entire feeding schedule mentioned. Flow Chart Purina Laboratory Chow (ad lib) 2 days

15% casein (3-hour meal feeding) 9 days

6% casein (3-hour meal feeding) 11 days Either 0%, 2%, 5%, 8%, or 15% ammonium acetate + 6% casein diet (meal feeding) 7 days

75% casein (meal feeding) 7 days The differences in intake between the test days when the ammonium acetate containing diets were fed and the three day baseline average were analyzed by paired t-test both for

A M M O N I U M A C E T A T E A N D I N T A K E O F H I G H PROTEIN DIET

473

FOOD INTAKE (g per day) 22

t 20

• o

18 _

x

DIET 0% ammonium acetate 2% ammonium acetate 5% ammonium acetate 8% ammonium acetate 15% ammonium acetate

16 14

10

\

d

4 4

0

:~e

I

0

i

i

i

i

i

i

i

1

2

3

4

5

6

7

DAY FIG. 1. The average daily food intake with standard errors of the mean for each group of rats (n=6) during the feeding of the 6% casein diet plus the various levels of ammonium acetate as indicated. The mean 3 day pre-test intake on the 6% casein diet is plotted as day 0. The ammonium acetate diets were presented for three hours on day 1. Significance levels are: A, p<0.05; B, p<0.025; C, p<0.01; D, p
intervals within the three hour period and for the total intake each day the ammonium acetate containing diets were offered. The food intake during the time the 75% casein diet was fed was converted to percent of baseline from the last two days of the ammonium acetate feeding and results for the seven days of 75% casein feeding were analyzed by one way analysis of variance for each day. The Tukey test was used for post-hoc testing of differences among means. RESULTS The average daily food intake for each group of rats ingesting the 6% casein diet in the three days prior to introducing the ammonium acetate diets (day 0) and for the seven days during which the ammonium acetate diets were fed are shown in Fig. 1. Rats eating 0% ammonium acetate and 2% ammonium acetate containing diets did not depress their intake during the seven day period whereas rats ingesting 5% ammonium acetate depressed their intake significantly only on day 1 and rats eating 8% and 15% ammonium acetate depressed their intakes from day 1 through day 4. On day 1, rats eating diets containing 5%, 8% and 15% ammonium acetate depressed their intakes 16% (p<0.025), 52% (p<0.001), and 70% (/9< 0.001), as compared to their re spective baseline

intakes. On day 2, rats ingesting 5%, 8% and 15% ammonium acetate, depressed their intakes 9%, 38% (p<0.005), and 59.4% (p<0.001), respectively. On day 3, only rats eating diets containing 8% and 15% ammonium acetate depressed their intake significantly, 25% (p<0.025) and 47% (p<0.005), respectively. On day 4, food intake of rats eating 8% and 15% ammonium acetate was still depressed at 25% (p<0.005) and 37% (p<0.005) respectively. From day 5 to day 7 rats eating 8% and 15% ammonium acetate containing diet continued to eat less than they had in the baseline period but the decreases were not significant. The interval intake of each group on the first and second days the high protein diet was fed was compared to that of the baseline period are shown in Table 2. During the first day from 0-15 minutes rats consuming diets containing 2%, 5%, 8%, and 15% ammonium acetate depressed their intake by 45% (p<0.001), 67% (p<0.001), 79% (p<0.001), and 70% (p<0.025) from their respective baseline interval intakes. From 15-30 minutes, the changes in food intake of rats ingesting the (F~, 2%, 5%, 8% and 15% ammonium acetate diets were: +6%, - 2 8 % (o<0.05), - 6 0 % (p<0.01), - 7 0 % (p<0.01), and - 9 0 % (p<0.005) respectively. F r o m 30 to 90 minutes rats fed the 2% ammonium acetate containing diet increased their intake 35% (p<0.025) while all other groups showed no significant depression in food intake. From 90 to

474

SEMON, L E U N G A N D R O G E R S TABLE 2 FOOD INTAKE BY INTERVALFOR THE FIRST TWO DAYSOF AMMONIUMACETATE DIET FEEDING Diet 6% Casein

Day Pretest

0--15 min

15-30 min

30-90 min

90-180min

1.6 + 0.2 1.7 ± 0.2 1.9 _ 0.1

4.1 ± 0.3 3.3 ± 0.8 3.6 ± 0.6

5.0 ± 0.6 5.6 ___0.8 6.0 ± 0.5*

2

3.2 ± 0.3 3.2 -- 0.2 3.2 ± 0.3

2% Ammonium acetate

Pretest 1 2

3.1 ± 0.1 1.7 ± 0.1¶ 2.4 -4- 0.2~

1.8 _+ 0.1 1.3 ± 0.1" 1.7 ± 0.1

3.4 _+ 0.2 4.6 ± 0.4t 3.8 ± 0.4

5.7 ± 0.7 6.0 ± 0.6 6.4 ± 0.9

5% Ammonium acetate

Pretest 1 2

3.0 ± 0.1 1.0 ± 0.2¶ 1.7 ± 0.1§

1.5 ± 0.1 0,6 ± 0.15 0,8 ± 0.3t

4.1 ± 0.3 3.6 ± 0.3 4.1 ± 0.6

5.5 ± 0.7 6.6 ± 0.716.2 ± 0.8

8% Ammonium acetate

Pretest 1 2

3.4 ± 0.3 0.7 ± 0.1¶ 1.6 ± 0.3t

2.0 ± 0.2 0.6 ± 0.2:~ 0,6 ± 0.2¶

3.7 ± 0.6 2.1 ± 0.6 2.6 ± 0.3

4.9 ± 0.6 3.3 ± 0.7 4.0 ± 0.5

15% Ammonium acetate

Pretest 1 2

3.0 ± 0.3 0.9 ± 0.5t 1.0 ± 0.05¶

2.0 ± 0.3 0,2 ± 0.1§ 0.6 ± 0.1:~

3.6 ± 0.5 1.8 ± 0.6 1.6 ± 0.3§

5.0 ± 0.7 1.3 ± 0.2§ 2.6 ± 0.4*

1

Intakes are expressed in grams ±SEM (n=6 per group). Significance levels are: *p<0.05; Ip<0.025; ~:p<0.01; §p<0.005; ~<0.001. 180 minutes, rats ingesting diets containing 0%, 2%, and 5% ammonium acetate increased their intake by + 12%, +5%, and + 20% (p <0.025) respectively whereas rats receiving the 8% and 15% ammonium acetate diets depressed their intake by 33% and 74% (p<0.005) respectively. On day 2, all rats except those eating 0% ammonium acetate depressed their intake in the first interval from 0 to 15 minutes (p<0.025). From 15 to 30 minutes, rats receiving 5%, 8% and 15% ammonium acetate depressed their intake significantly (p<0.025). From 30 to 90 minutes and from 90 to 180 minutes, only rats eating diets containing 15% ammonium acetate showed significant reduction in intake (p<0.05). The intakes of each group of rats as percent of baseline values during the seven days the 75% casein diet was presented are shown in Fig. 2. The intake of the 15% ammonium acetate fed rats decreased to 94% of baseline on the first day of 75% casein feeding, which was significantly different from all other groups (F=9.7, p<0.0001 and by Tukey test). On day 2, the intake of the 8% and 15% ammonium acetate fed rats rose to 91.1% and 105% of baseline respectively and these two groups were significantly different from the other groups (F=18.13, p<0.0001 and by Tukey test). On day 3, intakes of rats consuming the 5%, 8% and 15% ammonium acetate fed rats were elevated to 82%, 99%, and 119% of baseline respectively ( F = 13.84, p<0.0001). By Tukey test, the intake of 15% ammonium acetate prefed rats was significantly different from intakes of those prefed 0%, 2% and 5% ammonium acetate prefed rats while the intake of 8% ammonium acetate prefed rats was significantly different from those prefed 0% and 2% ammonium acetate. The intake of all groups rose from day 3 to day 4 while the intake of 15% ammonium acetate prefed rats leveled off at approximately 116% (F=3.71, p<0.02). The intake of the 15% ammonium acetate prefed rats was significantly different from those prefed 0% ammonium acetate on day 5 (F=3.85, p<0.02 and by Tukey test) and from those prefed 0% and 2% ammonium

acetate on day 6 (F=4.20, p<0.01 and by Tukey test). On day 7, there were no significant differences among means by Tukey test. DISCUSSION Rats prefed 15% ammonium acetate are almost completely adapted to eat 75% casein diet the first day it was presented and were completely adapted the second day whereas rats prefed 0% ammonium acetate do not show complete adaptation to the 75% casein diet even at the end of 7 days. Fifteen percent ammonium acetate in the 6% casein diet appears to induce the necessary metabolic changes in rats for the eating of a 75% casein diet and this ammonium acetate diet induces these changes as quickly as the 75% casein diet itself. Rats prefed 15% ammonium acetate for one week depressed their intake by only 6% when presented with the 75% casein diet whereas rats not prefed ammonium acetate depressed their intake to 55% of baseline. Rats prefed 15% ammonium acetate ate 105% of baseline on day 2 and 115% of baseline on day 7 whereas rats not prefed ammonium acetate ate less on day 2 (51%) than on day 1 and ate 96% of baseline on day 7. Even after 7 days of 75% casein feeding, rats are not completely adapted to 75% casein whereas rats prefed 15% ammonium acetate for 7 days are fully adapted to 75% casein after one day of 75% casein feeding. Rats prefed 8% ammonium acetate adapted to eating the 75% casein diet by day 3 while rats prefed 5% ammonium acetate consumed 94% of baseline intake on day 4. The results of interval intake show that rats respond to ammonium acetate at 2% of the diet by decreasing initial intake, even though this level in the diet does not enhance their intake of the 75% casein diet when it is presented later. Rats presented with 2% ammonium acetate in the diet depress their intake quickly, in the first 15 minutes. Ammonium acetate in the 6% casein diet would appear to make the diet less acceptable than a 6% casein diet alone either because of sensory characteristics of the ammonium acetate

AMMONIUM ACETATE AND INTAKE OF HIGH PROTEIN DIET

475

F O O D INTAKE (% of b a s e l i n e ) 200 DIET P R E F E D 180

160

• o •

0% ammonium acetate 2% ammonium acetate 5% ammonium acetate

® x

8% ammonium acetate 15% ammonium acetate

140

120

100

4

80

60

40

_

20

0

I

I

I

I

I

I

I

1

2

3

4

5

6

7

DAY

FIG. 2. The average daily food intake as percent of baseline with standard errors of the mean for each group of rats (n=6) during the 75% casein diet feeding. Rats were prefed 0% ammonium acetate (n), 2% ammonium acetate (O), 5% ammonium acetate (O), 8% ammonium acetate (®), and 15% ammonium acetate (×). The average of day 6 and day 7 of ammonium acetate feeding is plotted as 100% on day 0. Rats eating 6% casein plus 0%, 2%, 5%, 8% and 15% ammonium acetate ate 15.6-+0.8 g, 15.3-+0.7 g, 16.1___0.6 g, 12.4-+0.8 g and 11.4_+0.6 g respectively on days 6 and 7 of ammonium acetate feeding. Results of anova testing are given in the text. TABLE 3 BODY WEIGHTS OF RATS FED IN SEQUENCE 6% CASEIN, VARYING LEVELS OF AMMONIUMACETATE(7 DAYS)AND 75% CASEIN (7 DAYS) Day 0 Day 7 of of Ammonium Ammonium Acetate Acetate Feeding Feeding 0% Ammonium acetate 2% Ammonium acetate 5% Ammonium acetate 8% Ammonium acetate 15% Ammonium acetate

153.2+6.5 155.7+8.1 149.9_+8.1 153.4+6.4 154.2_+7.1

172.3_+7.1 173.8_+8.2 167.3-+8.6 154.0_+6.7 143.0-+9.3

Day 7 of 75% Casein Feeding 194.2+ 7.7 199.5-+10.9 196.7+ 8.4 184.6_+ 6.9 177.2+ 8.3

Body weights are expressed in grams -+SEM (n=6).

or because the ammonium acetate causes possible metabolic changes in the rat. Because rats eating 2% ammonium acetate containing diet do not depress their intake on a daily

basis and overate from 30 to 90 minutes and from from 90 to 180 minutes compared to their respective baseline intake on the first day, 2% ammonium acetate does not appear to induce any metabolic changes which affect their food intake except possibly from 0-30 minutes. Five percent ammonium acetate was initially not acceptable in the first and second 15 minute intervals but the rats apparently overate to compensate during the 90 to 180 minute period, indicating little, if any metabolic consequences caused by the consumption of 5% ammonium acetate which would influence food intake after the period from 0-30 minutes. Rats curtailed their intake of the 8% and 15% ammonium acetate containing diets in the first two 15 minute intervals indicating that these diets were not as acceptable as the 6% casein diet. In addition, the rats depressed theft intakes for at least four days when presented with the 8% and 15% ammonium acetate diets, which may reflect the time necessary for metabolic adaptation to occur. The ability of rats prefed the 15% ammonium acetate to consume more of the 75% casein diet is apparently the result of metabolic adaptation caused by the ingestion of the 15% ammonium acetate diet for one week. The nature of this metabolic adaptation cannot be ascribed with certainty be-

476

SEMON, LEUNG AND ROGERS

c a u s e no e n z y m e o r m e t a b o l i t e c o n c e n t r a t i o n m e a s u r e m e n t s w e r e m a d e in this study. It is possible t h a t t h e 75% c a s e i n diet m a y h a v e b e e n m o r e a c c e p t a b l e t h a n t h e 15% amm o n i u m a c e t a t e a n d t h a t the 15% a m m o n i u m a c e t a t e p r e f e d rats ate the 75% c a s e i n the first d a y b e c a u s e t h e 75% c a s e i n diet w a s m o r e a c c e p t a b l e t h a n the 15% a m m o n i u m a c e t a t e diet. If the 15% a m m o n i u m a c e t a t e p r e f e d rats ate the 75% c a s e i n diet t h e first day b e c a u s e it w a s m o r e a c c e p t a b l e t h a n the p r e v i o u s diet, t h e n t h e y s h o u l d h a v e e x p e r i e n c e d a m e t a b o l i c effect f r o m eating the high p r o t e i n diet, resulting in a d e c r e a s e in intake t h e s e c o n d day o f 75% c a s e i n feeding. N o s u c h d e c r e a s e w a s o b s e r v e d . R a t s p r e f e d 2% a n d 5% a m m o n i u m a c e t a t e s h o w e d no a p p a r e n t m e t a b o l i c adaptation to the a m m o n i u m a c e t a t e as i n d i c a t e d b y a c o n s i s t e n t d e c r e a s e in food intake o n the first day a n d t h e s e rats dep r e s s e d t h e i r i n t a k e o f the 75% casein diet as did the 0% a m m o n i u m a c e t a t e p r e f e d rats. T h e 4.2% a m m o n i a n i t r o g e n c o n t e n t o f t h e 15% am-

m o n i u m a c e t a t e diet is a p p a r e n t l y sufficient to i n d u c e m e t a b o l i c a d a p t a t i o n in t h e rat so t h a t rats c a n m a i n t a i n t h e i r i n t a k e o f the 75% c a s e i n diet, c o n t a i n i n g 11% a m i n o nitrogen. F u r t h e r e x p e r i m e n t s are n e e d e d to d e t e r m i n e w h e t h e r t h e r e are u r e a cycle a n d a m i n o acid c a t a b o l i c e n z y m e c h a n g e s or o t h e r e n z y m e c h a n g e s or w h e t h e r t h e r e are c h a n g e s in n e u r o n a l sensitivity to a m m o n i a a n d / o r a m i n o acids in rats fed 15% a m m o n i u m a c e t a t e in o r d e r to p r o v i d e the m e t a b o l i c e x p l a n a t i o n for the b e h a v i o r c h a n g e s observed.

ACKNOWLEDGEMENTS This paper was supported in part by NIH grants DK 13252 and AM 07355. The authors wish to acknowledge Tracy Schuster and Pam Bains for their help in manuscript preparation and Dr. A. A. Heusner and Pierre Loranger for their help in preparation of computerized graphs.

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