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Adrenal modulation of the enhanced fat intake subsequent to fasting
Physiology& Behavior, Vol. 48, pp. 373-381. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0031-9384/90 $3.00 + .00
Adrenal Modulation of the Enhanced Fat Intake Subsequent to Fasting M A R Y E L L E N B L I G H , M A R I E B. D E S T E F A N O , S U S A N K. K R A M L I K , LARRY W. DOUGLASS,* PAUL DUBUCt AND THOMAS W. CASTONGUAY t
Department of Human Nutrition and Food Systems and *Department of Animal Sciences, University of Maryland, College Park, MD 20742 and ~Sansum Medical Research Foundation, Santa Barbara, CA 93102 R e c e i v e d 18 July 1990
BLIGH, M. E., M. B. DESTEFANO, S. K. KRAMLIK, L. W. DOUGLASS, P. DUBUC AND T. W. CASTONGUAY. Adrenal modulation of the enhancedfat intake subsequent tofasting. PHYSIOL BEHAV 48(3) 373-381, 1990.- Elevations in corticosterone have been linked with the enhanced fat appetite of genetically obese Zucker rats. The present study set out to describe the effects of elevations in corticosterone in adult male Sprague-Dawley rats. Previous studies have shown that food deprivation leads to a time-dependent increase in basal corticosterone concentrations. It was predicted that rats would select a high fat diet during initial refeeding subsequent to a 24-hour fast and more severe food deprivation (48 hours) would promote greater fat consumption. Dependence upon adrenal hormones for this enhanced fat intake was examined with adrenalectomized animals. It was hypothesized that adrenalectomy would prevent the increase in fat intake seen in intact animals. Two experiments were performed. In the first, rats were given access to three separate macronutrient sources and allowed to self-select a diet for 7 days. They were then divided into groups and deprived of food for 0, 24, or 48 hours. At the end of the restriction period each rat was tail bled and macronutrient access was restored. Intakes were measured and blood samples taken at 1, 3, 6, 12, and 24 hours following restored access. During the first hour of refeeding, food-deprived animals ate significantly more fat than nondeprived animals. The enhanced fat intake was positively correlated with the elevations in corticosterone observed at the start of the refeeding period (r= .72). In the second experiment, rats were allowed to self-select a diet for 9 days. On Day l0 the rats received either bilateral adrenalectomies or sham operations. They were allowed to recover for 5 days. On Day 15 they were assigned to deprivation groups and deprived of food for 0, 24, or 48 hours. After their respective restriction periods, the rats were tail bled and food access was restored. During the first hour of refeeding, sham animals deprived of food ate significantly more fat than all other groups. Enhanced fat intake was not observed in the adrenalectomized animals, suggesting that adrenal hormones mediate dietary fat intake. Corticosterone
Fasting
Fat intake
Adrenalectomy
Caloric intake
SEVERAL lines of evidence suggest that the glucocorticoids play a mediating role in the control of daily caloric intake (3, 5, 21, 23). Recent studies of the effects of adrenalectomy (ADX) and corticosterone replacement in Zucker obese (fa/fa) and lean (Fa/?) rats have demonstrated that the adrenal glucocorticoids are a necessary component for the expression of hyperphagia and increased weight gain observed in the fa/fa obese rat (6,21). Further, these studies replicate the much earlier report that body weight gain in lean rats is at least partially controlled by circulating corticosterone (17). Corticosterone is also a necessary condition for the expression of obesity promoted by highly palatable diets (12) and obesity that results from lesions of the ventromedial nucleus of the hypothalamus (10). Although the extent to which the rate of weight gain that is attenuated following A D X is diet dependent (8), it seems clear that glucocorticoid activity provides at least some of the necessary metabolic conditions that result in normal rate of weight gain in lean animals and in increased rate of weight gain in obese animals.
Dietary selection
We have noted that ADX reduces the total dally caloric intake of obese Zucker rats (3). Fat intake in particular is dramatically reduced following ADX in both lean and obese Zucker rats. Further, we have demonstrated that corticosterone replacement restores the daily fat intake of A D X rats in a dose-dependent fashion. Although these observations have not been in uniform agreement with the reports of decreased carbohydrate intake following A D X in Sprague-Dawley rats (11,13), several procedural and dietary differences between the two sets of experiments have prevented a more complete resolution of the differing observations. Another effect of ADX that is not fully understood has been the observation that ADX Sprague-Dawley rats fail to compensate for deprivation. Wurdeman, Berdanier and Tobin (22) have shown that adult ADX rats that have been fasted for 24 hours fall to increase their intake to compensate for the deprivation once food access is restored. By comparison, sham-operated fasted animals increase food intake when permitted food access. Wurdeman et al.
1Requests for reprints should he addressed to Thomas W. Castonguay, Ph.D., Department of Human Nutrition and Food Systems, University of Maryland, College Park, MD 20742.
373
374
Ingredient Cornstarch* Casein* DL-Methioninet Cellulose:~ Vitamin Mix§ Mineral Mix¶ Lard#
BLIGHETAL.
TABLE 1
TABLE 2
DIET COMPOSITION
BODY WEIGHTS AND BODY WEIGHT CHANGES IN RATS DEPRIVED OF ALL THREE MACRONUTRIENTS FOR 0, 24 OR 48 HOURS
Carbohydrate Source %
Protein Source %
Fat Source %
88.50 --4.00 1.50 6.00 --
-87.76 0.74 4.00 1.50 6.00 --
---4.00 1.50 6.00 88.50
*Bio-Serv Inc., Frenchtown, NJ. tlCN Biochemicals, Cleveland, OH. SAlphacel Non-nutritive Bulk, ICN Biochemicals, Cleveland, OH. §Vitamin Diet Fortification Mixture, ICN Biochemicals, Cleveland, OH. ¶A1N 76 Mineral Mixture, ICN Biochemicals, Cleveland, OH. #Esskay brand, Baltimore, MD.
have shown that glucocorticoid replacement restores the refeeding effect to sham-operated control levels. The purpose of the present experiment was to determine the effects of deprivation on the composition of the diet selected by Sprague-Dawley rats that had been fasted for up to 48 hours, and to determine the role of circulating corticosterone in mediating the selection of macronutrients during refeeding. Based upon our observations in obese Zucker rats, we hypothesized fasting would result in increased levels of circulating corticosterone, increased corticosterone would be correlated with an increase in the selection of dietary fat, and ADX would prevent the increased fat intake following deprivation. METHOD
Experiment 1 Eighteen male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 250-300 g were individually housed in standard stainless steel cages (Wahman Mfg., Timonium, MD) and adapted to a 12/12 light/dark cycle (lights on 0900 hr and off 2100 hr) at a room temperatur~of 22 ± 3°C. They were provided with Purina Chow (Ralston Purina Co., St. Louis, MO) and water ad lib. On the first day of the experiment Purina Chow was removed from the cages, and the rats were given access to three separate macronutrient sources (see Table 1) and allowed to self-select their diets. Macronutrient intakes and body weights were measured daily. On the 7th day macronutrient intakes were measured at 0900, 1000, 1200, 1500, 2100 hr and once again 12 hours later at 0900 hr. On the 8th day, rats were assigned to one of three food restriction periods (0, 24 or 48 hr) and restricted of food beginning at 0900 hr. At the end of their respective deprivation periods, rats were tail bled and access to the macronutrient sources was restored. At hours 1, 3, 6, and 12 after restored intake, rats were tall bled and macronutrient intake measured. Blood samples were then centrifuged, plasma removed and corticosterone levels determined by 125I radioimmunoassay (ICN Biochemicals, Costa Mesa, CA).
Experiment 2 Eighteen male Sprague-Dawley rats (Charles River, Wilming-
Group
Postdeprivation Weight (grams)
Mean Weight Change (grams)
Percent Weight Change (% change)
0hdep 24hdep 48hdep
357.5 _ 5,5 a 333.0 ± 5,5 b 317.1 - 4.3 c
9.9 -t- 5.5 a -12.8 ± 1.0b -27.6 ± 1.7c
2.9 -- 0.6 a -3.7 ± 0.3 b - 8 . 0 - 0.5 ~
There were no differences between groups in body weight prior to deprivation.Values not sharing a common superscript are significantly different from one another at the 0.05 level.
ton, MA) weighing 150-200 g were initially treated as in Experiment 1 except they were adapted to a 12/12 light/dark cycle with lights on at 1100 and off at 2300 hr and they were allowed to self-select their diets for 9 days, On the 10th day, animals were given bilateral ADX or sham operations under sodium pentobarbital (60 mg/kg) anesthesia. They were then returned to their home cages and allowed to recover for 5 days while continuing to self-select their diets. ADX animals were provided with 0.9% NaC1 drinking water ad lib. Intakes of all three macronutrient sources and body weights were monitored daily. On day 15, rats were assigned to one of three food restriction periods (0, 24 or 48 hr, as in Experiment 1). The animals were food restricted and given restored access to all three macronutrients. Intakes were measured after the first (0-1 hr), the third (1-3 hr), the sixth (3-6 hr), the twelfth (6-12 hr), and the twenty-fourth hour (12-24 hr) of access. Blood samples (150 t~1) were also taken to verify completeness of adrenalectomy. The experiment was conducted in two replications, with the second replicate using 14 animals weighing 250-360 g, and the same procedures were followed as above.
Statistics Daily intake and body weight measurements were statistically evaluated using an analysis of variance (ANOVA) procedure adjusted for repeated measures. Differences between group means were subsequently tested using the Duncan's new multiple range test. Statistical significance was assumed using a p-value of -<0.05. Because ANOVA applied to the results of Experiment 2 failed to reveal differences attributable to the replicate factor (with the exception of initial body weight), data from the two replicates were pooled. All group data are presented as means ± SEM. RESULTS
Experiment 1 Body weight. Prior to deprivation, body weights increased steadily. There were no significant differences in body weight between groups prior to deprivation. On the day after the respective deprivation periods, significant differences in body weight between groups in response to deprivation were observed (refer to Table 2). Rats assigned to the 48hdep group lost significantly more weight than those assigned to the 24hdep group. Both groups lost significantly more weight than 0hdep, both in grams and in relative proportion of initial body weight (% of predeprivation weight). Food intake. Prior to deprivation, average daily carbohydrate,
ADRENAL
MODULATION
OF FAT
INTAKE
375
TABLE
3
M A C R O N U T R I E N T INTAKE (grams) A N D DIETARY C O M P O S I T I O N (%kcal) D U R I N G EACH O F THE FIVE CONSECUTIVE TESTING PERIODS B O T H W I T H O U T DEPRIVATION A N D AFTER 0, 24 OR 48 HR F O O D DEPRIVATION Group
0hdep
24hdep
48hdep
0hdep
24hdep
48hdep
Test period 0-1 hr (grams)
(%kcal)
Carbohydrate Without dep With dep
1.6 ± 0 . 3 ~ 0.1 ± 0 . 0 a
1.3 0.5
± 0.3 ~ +__ 0 . 4 ~
1.2 0.9
Without dep
0 . 3 -+ 0 . 1 a
With dep
0.1 ± 0 . 0 b
Fat Without dep
0.5 ± 0.1"
0.22 ± 0.1 b
With dep
0.1 ± 0.1 b
1.7
± 0.2 a ± 0.4 ~
53.2 ± 6.9 ~ 5 1 . 5 --_ 19.8 b
60.6 m 7.4 ±
9.0 ~ 5.1 ~
5 8 . 8 + 12.6 a 9 . 5 -+ 3 . 4 ~
0.4
± 0.2 ~
0.7
_ 0.4 a
9.3 ±
2.6"
18.8 ±
8.2 ~
22.0 ±
9.0 ~
2.4
± 0.6 ~
3.2
± 0.3 a
9.7 ±
7.4 b
37.6 ±
12.0 ~
36.7 ±
4.6 ~
0 . 1 8 ± 0.1 b
3 7 . 5 ---
6.4 ~
20.6 ±
7.7 a
19.1 ±
9.7 ~
2.4
3 8 . 8 --- 17.0 ~
55,1 -
12.5 ~
53.8 ±
3.9 ~
Protein
± 0.5 a
± 0.5 a
Test period 1-3 hr Carbohydrate Without dep
0.6 ± 0.2 a
0.5
± 0.1 ~
0.3
± 0.1 ~
69.6 ~
12.9 a
53.6 ±
12.2 a
60.7 ±
19.0 ~
With dep
0.0 ± 0.0 ~
0.1
± 0 . 0 ~b
0.2
-
0.1 a
20.0 ± 20.0 ~
4.6 ±
2.6 ~
11.3 ±
4.3 ~
Withoutdep
0,1 ± 0 . 0 ~
0.1
± 0.1 a
1.1
±
1.1 ~
13.8 ±
9.8 ~
12.1 ±
7.6 a
18.8 ±
11.7 ~
With dep
0.0 ± 0.0 a
0.7
± 0.4 ~
0.7
± 0.4 a
0.0 ±
0.0 ~
40.8 ±
14.5 ~
37.6 ±
17.1 ~
Fat Without dep
0.1 ± 0.1 ~
0.3
--+ 0 . 2 ~
0.2
± 0.2 ~
16.6 ±
10.2 ~b
With dep
0.1 ± 0.1 a
0.2
± 0.1 a
0.2
± 0.1 a
40.0 ± 24.5 a
Protein
34.4 ±
12.0 ~
3.9 ±
3.9 b
54.6 ±
15.9 ~
34.5 ±
14.5 ~
Test period 3-6 hr Carbohydrate Without dep
0 . 4 ± 0.1 a
0.2
± 0.0 ~
0.8
± 0.4 ~
46.6 ±
12.8"
34.6 ±
8.3 a
43.3 -
8.4 ~
With dep
0.0 ± 0.0 ~
0.0
± 0.0 ~
0.3
± 0.3 ~
0.0 ±
0.0 ~
1.9 ±
1.9 a
9.5 ±
3.4 ~
Without dep
0.1 ± 0 . 0 ~
0.2
± 0.1 ~
0.5
± 0.3 ~
7 . 5 --_
3.3 a
17.7 ±
8.7 a
26.4 ±
With dep
0.0 ± 0.0 ~
0.0
+ 0.0 a
0.0
--- 0 . 0 ~
0.0 ±
0.0 ~
18.6 ±
16.4 a
Fat Without dep
0.3 ± 0,2 ~
0.2
± 0.1 a
0.3
± 0.2 ~
45.9 ±
16.0 ~
47.7 ±
13.4"
30.3 ±
With dep
0.0 z 0.0 ~
0.3
± 0.2 ~
0.1
± 0.0 ~
0.0 ±
0.0 ~
29.5 ±
18.9 a
54.2 ± 20.8 ~
13.0 a
13.9 ±
Protein 0 . 0 -±
5.6 a 0.0 ~ 10.6 a
Test period 6-12 hr Carbohydrate Without dep With dep
0.4 a
0.3
± 0.1 b
0.5
± 0.2 b
37.0 ±
0 . 5 --- 0 . 3 a
1.2 -
0.1
± 0.0 a
0.7
± 0.2 a
52.8 ± 20.5 a
2 . 7 -±
5.1 a
19.8 ±
6.8 a
0.4 b
9.5 ±
3.4 b
Protein Without dep
0 . 7 --_ 0 . 5 ~
1.4
--- 0 . 3 ~
2.1
--+ 0 . 5 a
18.2 ±
12.8 b
45.4 ±
10.7 ab
58.2 ±
9.6 a
With dep
0.1 ± 0 . 0 c
1.5
± 0.4 b
3.9
± 0.5 a
25.8 ±
18.7 a
35.8 ±
11.1 a
60.0 ±
2.6 a
Without dep
0 . 6 ± 0.1 ~
0.8
± 0.4 ~
0.48 ± 0.2
44.7 ±
10.9 ~
40.6 ±
13.1 ~
22.0 ±
9.6 ~
With dep
0 . 2 ± 0.1 b
1.3
--- 0 . 3 ~
0.8
21.4 ±
14.9 b
61.5 ±
11.2 ~
30.5 ±
4 . 3 ~b
Fat ± 0.1 ~
Test period 12-24 hr Carbohydrate Without dep
5.8 ±
1.8 ~
1.6
--- 0 . 7 b
2.6
--- 0 . 9 ~b
22.6 -
7.0 ~
7.6 ±
3.1 b
12.2 ±
3 . 9 ab
With dep
4.7 ±
1.7 a
0.2
± 0.1 b
2.5
± 0 . 8 ab
21.7 ±
8.8 a
1.2 ±
1.1 b
14.6 ±
4.1 ~b
11.7 ± 2 . 2 ~
11.6
±
1.9 ~
11.9
±
1.0 ~
45.7 ±
8.3 a
54.8 ±
7.3 ~
57.7 ±
4.8 ~
9.5 ± 2.6 a
7.6
±
1.4 ~
6.8
±
1.1 a
39.5 ±
7.7 ~
44.7 ±
5.8 ~
39.7 ±
5.5 ~
Without dep
3.5 ± 0.6 ~
3.4
± 0.6 a
2.8
± 0.4 ~
31.7 ±
5.1 a
37.5 ±
7.4 ~
30.1 ±
4.7 ~
With dep
3.7 ± 0.3 ~
3.9
± 0.4 a
3.3
± 0.4 ~
38.8 ±
5.4 ~
54.1 ±
5.2 a
45.7 ±
5.5 a
Protein Without dep With dep Fat
V a l u e s as g r a m s o r % k c a l s s h a r i n g a c o m m o n s u p e r s c r i p t w i t h i n e a c h o f the t w o t e s t i n g p e r i o d s are n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one another (p>0.05).
376
BLIGHETAL.
TABLE 4 TOTAL CALORICINTAKEDURINGEACHOF THE FIVE MONITORING PERIODS EITHERWITHOUTA PRECEDINGDEPRIVATIONPERIOD OR FOLLOWINGONE OF THREE DURATIONSOF DEPRIVATION Group
0hdep
24hdep
48hdep
Test period 0-1 hr Without deprivation Following deprivation
10.8 - 1.6a 1.7 _ 0.6 b
7.9 - 1.2a 23.7 +__4.0 a
8.2 +__ 1.8a 33.4 _+ 5.2 a
Test period 1-3 hr Without deprivation Following deprivation
2.8 -+ 0.8 a 1.0 _ 0.6 ~
4.2 _ 1.6~ 4.5 - 1.4~
6.3 + 4.9 a 4.7 _ 1.4a
Test period 3-6 hr Without deprivation Following deprivation
4.5 _ 1.2~ 0.0 - 0.0 ~
3.0 -+ 0.7 a 2.8 - 1.8a
7.1 _ 3.1 a 2.1 +_ 0.5 a
Test period 6-12 hr Without deprivation Following deprivation
11.7 4- 1.1 a 3.9 -+_ 1.6~
Without deprivation Following deprivation
89.8 -+ 7.4 a 79.6 -4- 8.3 ~
12.3 +- 3.8 ~ 16.1 - 1.7b
12.9 _ 2.9 a 22.9 + 2.8 a
Test period 12-24 hr 74.1 _ 0.2 b 58.7 -+ 4.4 b
73.3 - 0.l b 59.0 +_ 4.2 b
NB: Animals were assigned to each of the three deprivation groups (Oh, 24h or 48h) randomly. Thus, differences observed in total caloric intake during the without deprivation test reflect random error, i.e., the animals had been treated identically throughout the experiment up to that time.
protein, fat and calorie intakes did not differ between treatment groups. One day prior to the start of deprivation, the intakes of all rats were monitored starting at 0900 hr. Intake of each source was measured at 0900, 1000, 1200, 1500, 2100 and 0900 hr the next day. Analysis of macronutrient intake during these 5 test periods prior to deprivation revealed only minimal group differences in the patterns of intakes of any of the three macronutrients. For example, fat intake differed only during the first hour when 24hdep and 48hdep animals ate significantly less fat than did the 0hdep control group. Intakes of carbohydrate, protein and calories did not differ significantly between groups at this time (refer to Tables 3 and 4). From hours 1-3 and 3-6, none of the groups had significantly different intakes of any of the macronutrient sources or of total calories. At hours 6-12, 24hdep and 48hdep animals ate less CHO than did the 0hdep control group. Intakes of protein, fat and calories did not differ significantly between groups. At 12-24 hours, 24hdep ate significantly less carbohydrate than 0hdep animals (5.84---1.80 vs. 1.62---0.67 g, p<0.05). The 48hdep intake did not differ significantly from either group. Calorie intakes also differed during the 12-24 hour period with 24hdep and 48hdep groups consuming significantly less calories than 0hdep controls. Finally, it should be noted that the daytime intakes of all three macronutrients were uniformly low relative to nighttime (12-24 hr) intakes. The diets that were composed during each of these testing periods (as assessed by calculating the percentage of calories consumed during each of these test periods) were also remarkably similar between groups. There were no significant differences between the diets composed by any group during test periods 0-1 hr, 3-6 hr, and 12-24 hr. Fat intake as a percentage of total
calories differed only during hours 1-3 when 24hdep ate significantly more than 48hdep. Percentage intakes of CHO and protein were not significantly different during this time. Percent CHO intakes differed only during hours 6-12 with 0hdep composing a diet that was significantly higher in CHO than the diet composed by the 24hdep group. Percent calories as protein was significantly different during hours 6-12 with the 48hdep composing a diet that was significantly higher in protein than was the diet composed by the 0hdep group. Again, it should be noted that although these differences between groups in diet composition attained statistical significance, actual intakes during the daytime hours was uniformly low when compared to nighttime (12-24 hr) intakes. Intake after deprivation. Significant increases in both protein and fat intakes were observed during the first hour of refeeding subsequent to fasting (0-1 hr) when comparing postdeprivation intakes with predeprivation levels within groups. There were no significant changes in intake of any of the macronutrient sources when compared to the predeprivation day during the second testing period (1-3 hr). The 48hdep and 0hdep groups ate less CHO during the 3-6 hr test period than they did during the predeprivation test period, Again, refer to Table 3. From 6-12 hours, the 48hdep group ate more CHO during the refeeding day while the 0hdep group ate less CHO when compared to predeprivation intakes. The 24hdep group's CHO intake did not differ from either group. During this testing period, all groups ate less protein during the refeeding day when compared to the predeprivation test period. The 24hdep group ate significantly more fat during the refeeding period compared to the predeprivation day while 0hdep ate less fat. During the first hour of refeeding (0-1 hr), carbohydrate intake (grams) was not significantly different among groups. The 24hdep and 48hdep groups ate significantly more protein and fat than did the 0hdep group. As a consequence, the 24hdep and 48hdep groups ate significantly more calories than did the 0hdep group. There were no differences between the 24hdep and 48hdep groups in either protein or fat intakes during the first hour of refeeding. Refer to Tables 3 and 4. From hours 1-3 of refeeding, the 48hdep group ate significantly more CHO than did the 0hdep group. Protein, fat and calorie intakes did not differ among treatment groups. During the 3-6 hr refeeding period, there were no significant differences in intake between groups for any of the macronutrients or total calories. From hours 6-12 of refeeding, CHO intake did not differ between groups. The 48hdep group ate significantly more protein than did the 24hdep group. Similarly, the 24hdep group ate significantly more protein than did the 0hdep group. Both the 24hdep and the 48hdep groups ate significantly more fat than did 0hdep controls. The total caloric intakes of the three groups differed significantly, with 48hdep rats eating significantly more than the 24hdep group, and both eating more than the 0hdep controls. During the 12-24 hour refeeding period, the CHO intake of the 24hdep group was significantly lower than that of the 0hdep group. Protein and fat intakes were not significantly different between groups. The 24hdep and 48hdep groups ate significantly less calories than did the 0hdep group. As percentage of calories, the 24hdep and 48hdep groups ate significantly less CHO than did the 0hdep group during the first hour of refeeding. The 24hdep and 48hdep groups composed diets that were significantly higher in percent protein than the diet composed by the 0hdep group. Percent calorie intake as fat did not differ between groups. Because intake levels during the subsequent daytime monitoring periods were so low, the dietary composition analyses (percentage of calories) were widely variable. Corticosterone concentrations. The 48hdep group had significantly higher corticosterone levels than 24hdep and 0hdep at the beginning of the refeeding period (15.13 +_3.62 vs. 5.42 - 2.74
ADRENAL MODULATION OF FAT INTAKE
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REFEEDING
i 2
I 3
I 4
I 5
0 6
I 7
I 8
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I I I I t 10 11 12 13 14
DAYS
FIG. 1. Effect of 0, 24 and 48 hr of food deprivation on circulating corticosterone. All values represent means and standard errors o f the means. Note that after the first hour o f refeeding, all three groups had comparably low concentrations.
FIG. 2. Body weights of adrenalectomized and sham-operated rats prior to food deprivation. Surgery was performed on Day 10. All values represent means and standard errors of the means.
Daily carbohydrate Intake of ADX and eham-operated Sprague-Dawley rats T
hi "
m
g
2o~o I
°:t
•
~
•
II~iX
I
I
I
I
I
10
11
12
13
14
I
I
12
13
"I
Daily proteinsprague_Dawleyin ADX and sham-operatedrats
g
Io~o
-
I
I
10
11
-
14
Dally fat Intake of ADX and eham-opemtsd Sprogue-Dawley rots
~ / I
o ~ o ,Im-lm, d
I
I
I
I
I
10
11
12
13
14
DAYS FIG. 3. Effect o f adrenalectomy on carbohydrate (top), protein (nrdddle) and fat (bottom) intakes prior to food deprivation.
BLIGH ET AL.
378
vs. 0.80-+0.55 p3/dl, p<0.05). Refer to Fig. 1. At 1 hour after refeeding there were no significant differences between groups. At hour 3 of refeeding the 24hdep group had significantly higher corticosterone levels than the 0hdep and 48hdep groups (6.63 -+ 1.19 vs. 2.10 -+0.43 vs. 1.95 -+ 0.46 I~g/dl, p<0.05). This difference was also observed at the 6-hr measurement (15.08-+3.74 vs. 3.42±1.17 vs. 1.10±0.24 txg/dl, p<0.05). By hour 12 there were no significant differences between groups. During the first hour of refeeding neither CHO nor protein intake were correlated significantly with corticosterone levels. However, fat intake during this hour was significantly correlated with circulating corticosterone (r= .72, p<0.001).
Experiment 2 Although this experiment was conducted in two replications, the rats in the second replicate weighed significantly more than the rats in the first. Results from the two were pooled after statistical evaluation failed to reveal any significant interactions of the treatment effects (duration of deprivation, surgical procedure effects or their interactions) with the replicate factor. Data from any ADX rat having corticosterone values greater than 5 ~g/dl at any sampling time (n = 5) were dropped from the study due to incomplete adrenalectomy and/or the development of accessory adrenals (19). Body weight. Throughout the first 9 days of the study, body weight increased steadily. Prior to surgery, there were no significant differences in weight gain between groups assigned to different surgical treatments. Refer to Fig. 2. After surgery on Day 10, the 24hAdx group gained significantly less weight/day than did any of the sham-operated groups. Refer to Table 5. On the day prior to the institution of deprivation conditions, the 24hAdx and 0hAdx groups weighed significantly more than the 48hAdx group. The body weights of the ADX groups did not differ significantly from any of the sham-operated groups. After their respective deprivation periods, 0hSham, 24hAdx and 0hAdx were significantly heavier than the 48hAdx group. Refer to Table 6. The amount of weight lost during the deprivation period was determined by the duration of fast, F(2,21)=31.4, p<0.0001. Similarly, the percent change in body weight was determined by the duration of fast, F(2,21) = 25.16, p<0.04. Predeprivationfood intake. During the first 9 days, the 0hAdx and 24hAdx groups ate significantly more CHO than did the 24hSham, 48hAdx and 0hSham groups. Protein intakes were not significantly different between groups, while the 0hSham, 24hSham and 24hAdx groups ate more fat than did the 48hSham, 48hAdx and 0hAdx groups. The 24hAdx group consumed significantly more calories than 0hAdx, 48hSham and 48hAdx groups. The 0hSham and 24hSham ate more calories than the 48hAdx group (86.20_-_2.46 vs. 84.43___2.74 vs. 77.05---2.28 kcal, p<0.05). Although there were a number of significant differences in macronutrient intakes following surgery, ANOVA revealed no significant surgical effect on carbohydrate intake. Refer to Fig. 3. By contrast, ANOVA revealed that protein and fat intakes were significantly decreased by ADX. Adrenalectomy changed the composition of the diets that were selected. ADX rats selected diets that were higher in carbohydrate and lower in protein than the diets composed by sham-operated rats. The sham-operated rats selected diets that provided 1 9 . 3 +- 1.5% of total calories as CHO, 56.5-+ 1.5% calories as protein and 24.2-+ 1.0% calories as fat. By contrast, ADX rats composed a diet yielding 30.6 -+ 2.6% CHO, 41.5 -+ 2.4% protein and 28.0-+2.3% fat.
Day of refeeding. Deprivation promoted several shifts in the intake of all three macronutrient sources. During the first hour of refeeding (0-1 hr) the 48hSham and 24hSham groups ate significantly more fat when compared to the 0hSham controls. Refer to Fig. 4a. The protein intakes of the 24hSham and 48hSham groups were significantly higher than that of the 0hSham controls. Refer to Fig. 4b. Adrenalectomy did not affect carbohydrate intake. All groups of adrenalectomized animals consumed carbohydrate in amounts that were not different from that of their controls. Refer to Fig. 4c. By contrast, the 24hAdx and 48hAdx groups failed to eat more of any of the three sources when compared to both nondeprived controls (0hAdx or 0hSham groups). During hours 1-3 of refeeding, the intakes of all three macronutrients were uniformly low, irrespective of duration of deprivation or surgical treatment. CHO intake between groups was not significantly different. The 24hSham group consumed more protein than did 0hAdx and 0hSham controls. The 48hSham consumed significantly more fat than did 24hAdx, 0hAdx and 0hSham groups. These and other differences are noted in Table 7. During hours 3-6 of refeeding, intakes of all three macronutrients remained low. CHO intakes did not differ between groups. The 48hAdx group ate significantly more protein than all other groups. The 48hAdx consumed more fat during this time than did 24hSham, 24hAdx, 0hAdx and 0hSham. Fat intakes of 48hSham and 48hAdx were not significantly different. During hours 6-12, 24hAdx ate significantly more CHO than did the 24hSham and 0hSham groups while the 48hSham group ate significantly more than CHO than did the 0hSham group. The 48hAdx group ate significantly more protein than did the 0hAdx controls. No other differences in protein intake were found. The 24hSham group ate more fat than did the 0hAdx group. No other differences in fat intake between groups were found during the 6-12 hr test period. Caloric intakes were not different among any of the groups. During hours 12-24, CHO intakes did not differ between groups. The 0hSham group ate more protein and fat than did any other group. As a percentage of calories, during the first hour of refeeding, the 24hAdx group composed a diet higher in carbohydrate than did the 0hSham and 48hSham groups (60.09 _+23.37 vs. 12.50 - 12.50 vs. 11.72 - 4.57%, p<0.05). The percentage of calories of protein and fat did not differ between groups during this hour.
DISCUSSION
Several aspects of the present experiments are noteworthy. The most important finding was that during the initial hour of restored access subsequent to food deprivation, rats refeed primarily on fat and protein. Second, deprivation leads to elevated corticosterone levels which are correlated with the enhanced fat intake. Third, adrenalectomy attenuates the increase in fat and protein intake that is observed subsequent to food deprivation while not altering carbohydrate selection. Fourth, adrenalectomized self-selecting rats fail to increase total caloric intake after food deprivation. These observations are consistent with our laboratory's working hypothesis that the adrenal gland plays a role as a modulator of fat selection. Results from these experiments replicate the findings of Berdanier and colleagues who first reported that adrenalectomized rats fail to fully compensate for food deprivation (22). They showed that ADX rats fed a composite diet after being deprived of food for 24 or 48 hours failed to eat more calories/day than control rats that had not been fasted. Like those animals, self-selecting ADX rats also under-eat under these conditions. These results also rep-
ADRENAL MODULATION OF FAT INTAKE
2.50-
379
a
_I_
2.00-
AVERAGEDAILY WEIGHT GAIN AND TOTAL CALORIC INTAKEOF ADX AND SHAM-OPERATEDRATS PRIORTO DEPRIVATION (EXPERIMENTALDAYS 10-14)
_i_
1.50-
v
TABLE 5
Ill
Daily Weight Gain (g/day)
bJ
1.00-
Z
0.50 0.00
i
Oh Adx
2.50
Oh Sham
24h
24h
Adx
48h
48h
Sham
Adx
Sham
b
Itl
1
2.00
Sham-operated 0hSham 24hSham 48hSham Adrenalectomized 0hAdx 24hAdx 48hAdx
5.8 3.5 4.4
Daily Total Caloric Intake (kcals/day)
- 1.8= ± 1.3=b ± 0.5 ab
85.0 _+ 5.5 = 77.1 ---4.4= 79.8 ± 3.2 a
1.4 __. 1.4~ -0.85 _ 2.2 ¢ 0.9 - 1.9b~
60.2 ± 4.4 b 55.5 --- 6.7 b 52.8 ± 5.3 b
Groups not sharing common superscripts are different from one another at the p<0.05 level.
h,
1.50. z
1.00.
n¢
n
0.50. ,= Oh Adx
0.00
1.75,,,
i
Oh Sham
24h Adx
C
24h Sham
48h
48h
Adx
Sham
lit T
1.50T
1.25t~
_~
1.00-
t:a >-1o m
0.750.50-
o<
0.25.
n,,"
0.00
l T _]_ Oh Adx
Oh Sham
24h
24h
48h
Adx
Sham
Adx
48h
enhanced fat intake observed in the present experiment is under the exclusive control of corticosterone, preliminary results from our laboratory suggest that corticosterone replacement in ADX rats restores the enhanced fat intake that was observed subsequent to food deprivation (1). Grogan and Romsos (8) have demonstrated that the effects of ADX can be modulated by altering the fat content of a composite diet. They showed that the attenuated rate of weight gain that is observed subsequent to ADX in Sprague-Dawley rats can be blocked by increasing the fat content of the diet. ADX rats fed a high fat diet grow at rates comparable to sham-operated controls, suggesting that the metabolic effects of ADX on weight gain and intake can be modified by dietary manipulations. This suggestion has also been made by Sklar et al. (20) who demonstrated that ADX Sprague-Dawley rats given access to a sucrose supplement in addition to a semipurified diet (AIN-76 rat diet) also grow at sham-operated control rates. The importance of these observations is that dietary manipulations can play a major role in the outcome of this type of experiment. Castonguay et al. (3) reported that ADX self-selecting Zucker rats differ from sham-operated controls primarily by reducing fat intake. By contrast, Leibowitz and her colleagues reported that ADX Sprague-Dawley rats reduce both CHO and fat intake. The reason for the discrepancy between
Sham
FIG. 4. (a) Effect of adrenalectomy on fat intake during the first hour of refeeding subsequent to food deprivation. (b) Effect of adrenalectomy on protein intake during the first hour of refeeding subsequent to food deprivation. (c) Effect of adrenalectomy on carbohydrate intake during the first hour of refeeding subsequent to food deprivation. (a) and (b) *Significantlydifferentfrom 0hSham and Oh ADX (p<0.05). (c) *Significantly different from OhAdx and OhSham (p<0.05).
licate the findings of Shutz and Pilgrim (18) who reported that food-deprived rats increase dietary fat intake subsequent to restored access. The present experiments extend those of Wurdeman et al. and Shutz and Pilgrim by demonstrating that ADX selectively interferes with the refeeding response by attenuating fat intake. These results are consistent with the hypothesis that dietary fat intake is influenced by adrenal hormones. Previous work from our laboratory has demonstrated that the enhanced fat appetite of the genetically obese fa/fa Zucker rat is mediated by circulating corticosterone (3). Although it remains to be demonstrated that the
TABLE 6 WEIGHT LOSS AS A FUNCTION OF SURGICALCONDITION (ADX OR SHAM) AND DURATIONOF DEPRIVATION
Sham-operated 0hSham 24hSham 48hSham Adrenalectomized 0hAdx 24hAdx 48hAdx
Predeprivation Weight (g)
Weight Change (g)
Relative Weight Loss (% initial body weight)
305.4 ± 12.9=b 290.9 -- 28.7 ab 309.5 -- 19.2=b
4.9 ___ 1.0a -22.2 - 1.9b -34.7 ± 2.4 b
1.6 --- 0.4 a - 7 . 3 +- 0.8 b -11.4 -- 1.2b
304.5 ± 20.1ab 362.6 ± 12.6a 277.6 ± 12.0b
0.9 -- 3.5 a -21.7 ± 2.8 b - 3 4 . 6 ± 8.0 b
0.4 ± 1.0= - 6 . 0 -+ 0.6 b - 9 . 3 ± 3.1 b
380
B L I G H E T AL.
TABLE 7 MACRONUTRIENT INTAKES OF ADX AND SHAM-OPERATED RATS DURING THE REMAINING 4 TESTING PERIODS FOLLOWING DEPRIVATION
Group 0hAdx
0hSham
24hAdx
24hSharn
48hAdx
48hSham
Test period 1-3 hr CHO Pro Fat kcals
0.2 0.2 0.1 2.2
-+ -+ ± ---
0.1 ~ 0.1 b 0.1 b 1.2 b~
0.02 0.05 0.1 1.0
± 0.02 ~ ----- 0 . 0 3 b
± 0.04 b --- 0.4 c
0 + 0a 0.4 --- 0.4 ab 0.2 ± 0 . 1 b 3.0 --- 1.7 b~
1.2 1.6 0.7 15.6
4+ 4±
0.5 a 0.6 a 0.2 ab 3.7 a
1.0 __ 0.5 ~ 0.6 4- 0.2 ab 0.9 ±- 0 . 4 ab 13.1 -+ 5.0 "b
1.0 1.1 1.1 16.4
___ 0.4 a ~
0.3 ab
±
0.4 a 4.2 ~
Test period 3-6 hr CHO PrO Fat kcals
0.2 0.02 0.1 1.8
4- 0.2 a -----0.02 b - 0.04 b 4- 0.8 b
0.05 0.1 0.05 0.8
_+ 0.03 ~ 4- 0.05 b --- 0.03 b ± 0.3 b
0.3 0.3 0.2 3.6
~ 0.1 ~ --+-0.2 b +-- 0.1 b --- 0.6 b
0.5 0.2 0.3 5.1
4- 0.4" -----0.1 b -+ 0.2 b -+ 2.5 b
0.5 1.0 1.1 13.6
----.
0.2 a 4- 0.4 a --- 0.3 a ± 4.0"
0.6 0.3 0.5 7.2
1.5 2.0 1.1 21.3
2.6 3.6 1.7 35.6
4- 0.3 a ----. 0.2 b ±
0 . 2 ab
±
3.0 "b
_+ 0.4 abe 4- 0.2 a _ 0.5 "b ± 5.3"
1.6 1.9 1.3 22.8
± ± 4-_ --_
0.6 "b 0.6 ab 0.3 ab 4.1 ~
4- 0.9 a __. 0.6 b _+ 0.2 b -+ 4.5 b
5.1 5.9 1.9 54.1
+__ 2.8 a ± 1.0b __. 0.4 b ± 13.7 ab
Test period 6-12 hr CHO Pro Fat kcals
1.3 0.6 0.4 9.7
4- 0.4 a~ + 0.2 b 4- 0.3 b __. 4.0 a
0.2 1.0 0.6 9.0
± ± ± 4-
0.2 c 0.3 ab 0.1 ab 2.5 a
2.3 0.8 0.7 16.8
± ± 44-
0.2" 0.3 ab 0.3 ab 3.1 ~
0.8 - 0.2 e¢ 1.4 ± 0 . 4 ab 1.7 ~ 0.4 a 20.9 - 4.3 a
Test period 12-24 hr CHO Pro Fat kcals
3.4 5.2 2.2 48.6
___ 0.6" ___ 1.1 b ± 0.5 b ± 6.6 at'
1.1 8.7 4.2 68.1
± 0.2 a ___ 0.8 a _+ 0.7 a ___ 8.0"
3.3 4.6 1.8 42.3
± 0.5" --- 0.6 b --- 0.7 b ± 6.7 ~b
e x p e r i m e n t s w a s s u g g e s t e d to be strain and age related. H o w e v e r , the present results, like those o f Leibowitz and her colleagues (11,13), were b a s e d u p o n the selection patterns o f y o u n g SpragueD a w l e y rats. A l t h o u g h m o r e subtle differences in strain and age r e m a i n possible explanations, the present e x p e r i m e n t s u g g e s t s that differences in macronutrient source c o m p o s i t i o n m a y be a m o r e likely influencing factor. Unlike the sources u s e d in these studies, those u s e d by Leibowitz et al. were a modification of the formulation first described by K a n a r e k and H o (9). T h e primary difference b e t w e e n the Leibowitz macronutrient sources and those used in these studies is in the level o f v i t a m i n and mineral s u p p l e m e n t a t i o n o f the fat source. T h e c o m p o s i t i o n o f the Leibowitz fat source is lard s u p p l e m e n t e d with v i t a m i n s and minerals. H o w e v e r , the level o f s u p p l e m e n t a t i o n is b a s e d u p o n grams/kcal, w h e r e a s the v i t a m i n and mineral s u p p l e m e n t a t i o n o f the fat source used in these studies was b a s e d g r a m s o f s u p p l e m e n t s per g r a m o f food. T h u s , it is our s u g g e s t i o n that fat intake c a n be m o d u l a t e d in A D X rats b a s e d u p o n the mineral content o f the macronutrient sources. T h e salt-specific appetite o f the A D X rat h a s been thoroughly d o c u m e n t e d , starting with the pioneering work of Richter (16). Perhaps the drop in fat intake s u b s e q u e n t to adrenale c t o m y that is routinely o b s e r v e d in o u r laboratory m a y be c a u s e d by a shift in the hedonic value o f the protein and carbohydrate sources/kcal intake w h e n c o m p a r e d to the relatively dilute concentration o f s o d i u m in the s u p p l e m e n t e d fat source. T h e s e results are also in a g r e e m e n t with those reported by D e v e n p o r t et al. (5) w h o h a v e recently reported a selective decrease in fat intake s u b s e q u e n t to A D X in S p r a g u e - D a w l e y rats. W h e t h e r or not dietary differences determine all or only part o f this effect, it r e m a i n s a fact that w h e n the present sources are m a d e available to
2.3 3.9 2.4 40.8
± 0.7" + 0.7 b ± 0.6 b ___ 4.4 b
either Z u c k e r rats or to S p r a g u e - D a w l e y rats, fat intake is specifically attenuated by A D X . T h e s e results s u g g e s t that preference testing in A D X and s h a m - o p e r a t e d rats m a y resolve s o m e o f the c o n f u s i o n about the effects o f A D X on macronutrient selection. O n e of the m o s t intriguing aspects o f this e x p e r i m e n t is " W h y do food-restricted rats select f a t ? " S o m e insight into an a n s w e r to this question m a y be f o u n d in considering a food utilization strategy like that first proposed by F r i e d m a n and Stricker (7). T h e thesis proposed by these authors is that diabetic rats fail to maintain h y p e r p h a g i a w h e n put on a h i g h fat diet b e c a u s e fat utilization is the primary m e c h a n i s m u s e d by the diabetic rat to meet e n e r g y needs, since carbohydrate-derived glucose utilization is for the m o s t part insulin dependent. W e s u g g e s t that both the obese Z u c k e r rat and the food-restricted rat select fat in disproportionate a m o u n t s at least in part b e c a u s e they have relatively low levels o f glucose utilization. A l t h o u g h the o b e s e Z u c k e r rat h a s been reported to be mildly h y p e r g l y c e m i c , it h a s m o r e frequently been reported to be insulin insensitive (4, 15, 24). Like the Z u c k e r rat, the starved rat h a s very low levels o f circulating insulin resulting in low levels o f glucose utilization. Also note that coincident with these divergent m o d e l s is the finding that corticosterone is elevated (2, 3, 23). T h u s , both the obese rat and the starved rat c a n m o r e readily and effectively m e e t e n e r g y needs by selecting fat. O n e possible influence to account for the observation that a d r e n a l e c t o m y selectively attenuates the increase in fat intake is the w e l l - k n o w n increase in glucose utilization induced b y A D X first described H o u s s a y . A n o t h e r possible influence is an A D X induced increase in insulin sensitivity in the obese rat m o r e recently described by O h s h i m a et al. (14). This e m p h a s i s on
ADRENAL M O D U L A T I O N OF FAT INTAKE
381
selection guided by fuel utilization is similar to that fast proposed by Friedman and Stricker, but was formulated on the observations on obese or starved-refed rats instead of based around the feeding of diabetic rats. Nevertheless, the hypothesis that selection is guided by current metabolic conditions is supported by these observations. Future studies will be directed at determining the nature and location of the receptors that mediate fat intake. It remains plausible that dietary fat is selected in preference to carbohydrate
under some metabolic conditions because carbohydrate is not as readily used. In that sense, fat intake is increased out of default. It also remains possible that fat intake is actively monitored and not simply consumed as a second-best alternative to meeting energy needs. ACKNOWLEDGEMENT This work was supported by a grant from the Whitehall Foundation.
REFERENCES 1. Bligh, M. E.; Castonguay, T. W. Glucocorticoid control of macronutrient choice subsequent to fasting; in preparation. 2. Bray, G. A.; York, D. A. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev. 59:719-809; 1979. 3. Castonguay, T. W.; Dallman, M. F.; Stern, J. S. Some metabolic and behavioral effects of adrenalectomy on obese Zucker rats. Am. J. Physiol. 251:R923-R933; 1986. 4. Chan, C. P.; Stern, J. S. Adipose lipoprotein lipase in insulin-treated diabetic lean and obese rats. Am. J. Physiol. 242:F_,445-E450; 1982. 5. Devenport, L.; Knehans, A.; Thomas, T.; Sundstrom, A. Macronutrient intake and utilization by rats: interactions with type I adrenocorticoid receptor stimulation. Am. J. Physiol.; in press. 6. Freedman, M. R.; Horwitz, B. A.; Stern, J. S. Effect of adrenalectomy and glucocorticoid replacement on development of obesity. Am. J. Physiol. 250:R595-R607; 1986. 7. Friedman, M. I.; Stricker, F. M. The physiological psychology of hunger: a physiological perspective. Psychol. Bull. 83:409-431; 1976. 8. Grogan, C. K.; Kim, H.; Romsos, D. R. Effects of adrenalectomy on energy balance in obese (ob/ob) mice fed high carbohydrate or high fat diets. J. Nutr. 117:1115-1120; 1987. 9. Kanarek, R. B.; Ho, L. Patterns of nutrient selection in rats with streptozotocin-induced diabetes. Physiol. Behav. 32:639--645; 1983. 10. King, B. M.; Banta, A. R.; Tharel, G. N.; Bruce, B. K.; Frohman, L. A. Hypothalamic hyperinsulinemia and obesity: role of adrenal glucocorticoids. Am. J. Physiol. 245:EI94-E199; 1983. 11. Kumar, B. A.; Leibowitz, S. F. Impact of acute corticosterone administration on feeding and macronutrient self-selection patterns. Am. J. Physiol. 254:R222-R228; 1988. 12. Langely, S. C.; York, D. A. The effects of the antiglucocorticoid RU-486 on the development of obesity in the obese 'fafa' Zucker rat. Am. J. Physiol.; in press.
13. Lucas, D. J.; Leibowitz, K. L.; Jhanwar, Y. S.; Leibowitz, S. F. Developmental patterns of macronutrient selection and body weight gain in male and female rats. Proc. Abstr. Annu. Meet. Eastern Psychological Assoc. 61:47; 1990. 14. Ohshima, K.; Shargill, N. S.; Chan, T. M.; Bray, G. A. Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice. Am. J. Physiol. 246:E193-E197; 1984. 15. Reaven, G. M.; Bernstein, R.; Davis, B.; Olefsky, J. M. Nonketotic diabetes mellitus: insulin deficiency or resistance. Am. J. Med. 60:80-84; 1976. 16. Richter, C. P. Decreased carbohydrate appetite of adrenalectomized rats. Proc. Soc. Exp. Biol. Med. 48:577-579; 1941. 17. Schiffer, F.; Wertheimer, E. Leanness in adrenalectomized rats. J. Endocrinol. 5:147-151; 1947. 18. Schutz, H. G.; Pilgrim, F. J. Changes in the self-selection pattern for purified dietary components by rats after starvation. J. Comp. Physiol. Psychol. 47:44A. a.~9; 1954. 19. Schwabedal, P. E.; Partenheimer, U. Uber vorkommen und strnktur accessorischer nebennieren bei der Wistar-Ratte. Leipzig 97:753-768; 1983. 20. Sklar, T. N.; Castonguay, T. W.; Horwitz, B. A.; Stem, J. S. Effect of adrenalectomy on rats fed a 32% sucrose solution. Am. J. Physiol.; submitted. 21. Solomon, J.; Mayer, J. The effect of adrenalectomy on the development of the obese-hyperglycemic syndrome in ob/ob mice. Endocrinology 93:510-513; 1973. 22. Wurdeman, R.; Berdanier, C. D.; Tobin, R. B. Enzyme overshoot in starved-refed rats: Role of the adrenal glucocorticoid. J. Nutr. 108:1457-1461; 1978. 23. Yukimura, Y.; Bray, G. A.; Wolfsen, A. R. Some effects of adrenalectomy in the fatty rat. Endocrinology 103:1924-1928; 1978. 24. Zucker, L. M.; Zucker, T. F. Fatty, a new mutation in the rat. J. Hered. 52:275-278; 1961.
0031-9384/90 $3.00 + .00
Adrenal Modulation of the Enhanced Fat Intake Subsequent to Fasting M A R Y E L L E N B L I G H , M A R I E B. D E S T E F A N O , S U S A N K. K R A M L I K , LARRY W. DOUGLASS,* PAUL DUBUCt AND THOMAS W. CASTONGUAY t
Department of Human Nutrition and Food Systems and *Department of Animal Sciences, University of Maryland, College Park, MD 20742 and ~Sansum Medical Research Foundation, Santa Barbara, CA 93102 R e c e i v e d 18 July 1990
BLIGH, M. E., M. B. DESTEFANO, S. K. KRAMLIK, L. W. DOUGLASS, P. DUBUC AND T. W. CASTONGUAY. Adrenal modulation of the enhancedfat intake subsequent tofasting. PHYSIOL BEHAV 48(3) 373-381, 1990.- Elevations in corticosterone have been linked with the enhanced fat appetite of genetically obese Zucker rats. The present study set out to describe the effects of elevations in corticosterone in adult male Sprague-Dawley rats. Previous studies have shown that food deprivation leads to a time-dependent increase in basal corticosterone concentrations. It was predicted that rats would select a high fat diet during initial refeeding subsequent to a 24-hour fast and more severe food deprivation (48 hours) would promote greater fat consumption. Dependence upon adrenal hormones for this enhanced fat intake was examined with adrenalectomized animals. It was hypothesized that adrenalectomy would prevent the increase in fat intake seen in intact animals. Two experiments were performed. In the first, rats were given access to three separate macronutrient sources and allowed to self-select a diet for 7 days. They were then divided into groups and deprived of food for 0, 24, or 48 hours. At the end of the restriction period each rat was tail bled and macronutrient access was restored. Intakes were measured and blood samples taken at 1, 3, 6, 12, and 24 hours following restored access. During the first hour of refeeding, food-deprived animals ate significantly more fat than nondeprived animals. The enhanced fat intake was positively correlated with the elevations in corticosterone observed at the start of the refeeding period (r= .72). In the second experiment, rats were allowed to self-select a diet for 9 days. On Day l0 the rats received either bilateral adrenalectomies or sham operations. They were allowed to recover for 5 days. On Day 15 they were assigned to deprivation groups and deprived of food for 0, 24, or 48 hours. After their respective restriction periods, the rats were tail bled and food access was restored. During the first hour of refeeding, sham animals deprived of food ate significantly more fat than all other groups. Enhanced fat intake was not observed in the adrenalectomized animals, suggesting that adrenal hormones mediate dietary fat intake. Corticosterone
Fasting
Fat intake
Adrenalectomy
Caloric intake
SEVERAL lines of evidence suggest that the glucocorticoids play a mediating role in the control of daily caloric intake (3, 5, 21, 23). Recent studies of the effects of adrenalectomy (ADX) and corticosterone replacement in Zucker obese (fa/fa) and lean (Fa/?) rats have demonstrated that the adrenal glucocorticoids are a necessary component for the expression of hyperphagia and increased weight gain observed in the fa/fa obese rat (6,21). Further, these studies replicate the much earlier report that body weight gain in lean rats is at least partially controlled by circulating corticosterone (17). Corticosterone is also a necessary condition for the expression of obesity promoted by highly palatable diets (12) and obesity that results from lesions of the ventromedial nucleus of the hypothalamus (10). Although the extent to which the rate of weight gain that is attenuated following A D X is diet dependent (8), it seems clear that glucocorticoid activity provides at least some of the necessary metabolic conditions that result in normal rate of weight gain in lean animals and in increased rate of weight gain in obese animals.
Dietary selection
We have noted that ADX reduces the total dally caloric intake of obese Zucker rats (3). Fat intake in particular is dramatically reduced following ADX in both lean and obese Zucker rats. Further, we have demonstrated that corticosterone replacement restores the daily fat intake of A D X rats in a dose-dependent fashion. Although these observations have not been in uniform agreement with the reports of decreased carbohydrate intake following A D X in Sprague-Dawley rats (11,13), several procedural and dietary differences between the two sets of experiments have prevented a more complete resolution of the differing observations. Another effect of ADX that is not fully understood has been the observation that ADX Sprague-Dawley rats fail to compensate for deprivation. Wurdeman, Berdanier and Tobin (22) have shown that adult ADX rats that have been fasted for 24 hours fall to increase their intake to compensate for the deprivation once food access is restored. By comparison, sham-operated fasted animals increase food intake when permitted food access. Wurdeman et al.
1Requests for reprints should he addressed to Thomas W. Castonguay, Ph.D., Department of Human Nutrition and Food Systems, University of Maryland, College Park, MD 20742.
373
374
Ingredient Cornstarch* Casein* DL-Methioninet Cellulose:~ Vitamin Mix§ Mineral Mix¶ Lard#
BLIGHETAL.
TABLE 1
TABLE 2
DIET COMPOSITION
BODY WEIGHTS AND BODY WEIGHT CHANGES IN RATS DEPRIVED OF ALL THREE MACRONUTRIENTS FOR 0, 24 OR 48 HOURS
Carbohydrate Source %
Protein Source %
Fat Source %
88.50 --4.00 1.50 6.00 --
-87.76 0.74 4.00 1.50 6.00 --
---4.00 1.50 6.00 88.50
*Bio-Serv Inc., Frenchtown, NJ. tlCN Biochemicals, Cleveland, OH. SAlphacel Non-nutritive Bulk, ICN Biochemicals, Cleveland, OH. §Vitamin Diet Fortification Mixture, ICN Biochemicals, Cleveland, OH. ¶A1N 76 Mineral Mixture, ICN Biochemicals, Cleveland, OH. #Esskay brand, Baltimore, MD.
have shown that glucocorticoid replacement restores the refeeding effect to sham-operated control levels. The purpose of the present experiment was to determine the effects of deprivation on the composition of the diet selected by Sprague-Dawley rats that had been fasted for up to 48 hours, and to determine the role of circulating corticosterone in mediating the selection of macronutrients during refeeding. Based upon our observations in obese Zucker rats, we hypothesized fasting would result in increased levels of circulating corticosterone, increased corticosterone would be correlated with an increase in the selection of dietary fat, and ADX would prevent the increased fat intake following deprivation. METHOD
Experiment 1 Eighteen male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 250-300 g were individually housed in standard stainless steel cages (Wahman Mfg., Timonium, MD) and adapted to a 12/12 light/dark cycle (lights on 0900 hr and off 2100 hr) at a room temperatur~of 22 ± 3°C. They were provided with Purina Chow (Ralston Purina Co., St. Louis, MO) and water ad lib. On the first day of the experiment Purina Chow was removed from the cages, and the rats were given access to three separate macronutrient sources (see Table 1) and allowed to self-select their diets. Macronutrient intakes and body weights were measured daily. On the 7th day macronutrient intakes were measured at 0900, 1000, 1200, 1500, 2100 hr and once again 12 hours later at 0900 hr. On the 8th day, rats were assigned to one of three food restriction periods (0, 24 or 48 hr) and restricted of food beginning at 0900 hr. At the end of their respective deprivation periods, rats were tail bled and access to the macronutrient sources was restored. At hours 1, 3, 6, and 12 after restored intake, rats were tall bled and macronutrient intake measured. Blood samples were then centrifuged, plasma removed and corticosterone levels determined by 125I radioimmunoassay (ICN Biochemicals, Costa Mesa, CA).
Experiment 2 Eighteen male Sprague-Dawley rats (Charles River, Wilming-
Group
Postdeprivation Weight (grams)
Mean Weight Change (grams)
Percent Weight Change (% change)
0hdep 24hdep 48hdep
357.5 _ 5,5 a 333.0 ± 5,5 b 317.1 - 4.3 c
9.9 -t- 5.5 a -12.8 ± 1.0b -27.6 ± 1.7c
2.9 -- 0.6 a -3.7 ± 0.3 b - 8 . 0 - 0.5 ~
There were no differences between groups in body weight prior to deprivation.Values not sharing a common superscript are significantly different from one another at the 0.05 level.
ton, MA) weighing 150-200 g were initially treated as in Experiment 1 except they were adapted to a 12/12 light/dark cycle with lights on at 1100 and off at 2300 hr and they were allowed to self-select their diets for 9 days, On the 10th day, animals were given bilateral ADX or sham operations under sodium pentobarbital (60 mg/kg) anesthesia. They were then returned to their home cages and allowed to recover for 5 days while continuing to self-select their diets. ADX animals were provided with 0.9% NaC1 drinking water ad lib. Intakes of all three macronutrient sources and body weights were monitored daily. On day 15, rats were assigned to one of three food restriction periods (0, 24 or 48 hr, as in Experiment 1). The animals were food restricted and given restored access to all three macronutrients. Intakes were measured after the first (0-1 hr), the third (1-3 hr), the sixth (3-6 hr), the twelfth (6-12 hr), and the twenty-fourth hour (12-24 hr) of access. Blood samples (150 t~1) were also taken to verify completeness of adrenalectomy. The experiment was conducted in two replications, with the second replicate using 14 animals weighing 250-360 g, and the same procedures were followed as above.
Statistics Daily intake and body weight measurements were statistically evaluated using an analysis of variance (ANOVA) procedure adjusted for repeated measures. Differences between group means were subsequently tested using the Duncan's new multiple range test. Statistical significance was assumed using a p-value of -<0.05. Because ANOVA applied to the results of Experiment 2 failed to reveal differences attributable to the replicate factor (with the exception of initial body weight), data from the two replicates were pooled. All group data are presented as means ± SEM. RESULTS
Experiment 1 Body weight. Prior to deprivation, body weights increased steadily. There were no significant differences in body weight between groups prior to deprivation. On the day after the respective deprivation periods, significant differences in body weight between groups in response to deprivation were observed (refer to Table 2). Rats assigned to the 48hdep group lost significantly more weight than those assigned to the 24hdep group. Both groups lost significantly more weight than 0hdep, both in grams and in relative proportion of initial body weight (% of predeprivation weight). Food intake. Prior to deprivation, average daily carbohydrate,
ADRENAL
MODULATION
OF FAT
INTAKE
375
TABLE
3
M A C R O N U T R I E N T INTAKE (grams) A N D DIETARY C O M P O S I T I O N (%kcal) D U R I N G EACH O F THE FIVE CONSECUTIVE TESTING PERIODS B O T H W I T H O U T DEPRIVATION A N D AFTER 0, 24 OR 48 HR F O O D DEPRIVATION Group
0hdep
24hdep
48hdep
0hdep
24hdep
48hdep
Test period 0-1 hr (grams)
(%kcal)
Carbohydrate Without dep With dep
1.6 ± 0 . 3 ~ 0.1 ± 0 . 0 a
1.3 0.5
± 0.3 ~ +__ 0 . 4 ~
1.2 0.9
Without dep
0 . 3 -+ 0 . 1 a
With dep
0.1 ± 0 . 0 b
Fat Without dep
0.5 ± 0.1"
0.22 ± 0.1 b
With dep
0.1 ± 0.1 b
1.7
± 0.2 a ± 0.4 ~
53.2 ± 6.9 ~ 5 1 . 5 --_ 19.8 b
60.6 m 7.4 ±
9.0 ~ 5.1 ~
5 8 . 8 + 12.6 a 9 . 5 -+ 3 . 4 ~
0.4
± 0.2 ~
0.7
_ 0.4 a
9.3 ±
2.6"
18.8 ±
8.2 ~
22.0 ±
9.0 ~
2.4
± 0.6 ~
3.2
± 0.3 a
9.7 ±
7.4 b
37.6 ±
12.0 ~
36.7 ±
4.6 ~
0 . 1 8 ± 0.1 b
3 7 . 5 ---
6.4 ~
20.6 ±
7.7 a
19.1 ±
9.7 ~
2.4
3 8 . 8 --- 17.0 ~
55,1 -
12.5 ~
53.8 ±
3.9 ~
Protein
± 0.5 a
± 0.5 a
Test period 1-3 hr Carbohydrate Without dep
0.6 ± 0.2 a
0.5
± 0.1 ~
0.3
± 0.1 ~
69.6 ~
12.9 a
53.6 ±
12.2 a
60.7 ±
19.0 ~
With dep
0.0 ± 0.0 ~
0.1
± 0 . 0 ~b
0.2
-
0.1 a
20.0 ± 20.0 ~
4.6 ±
2.6 ~
11.3 ±
4.3 ~
Withoutdep
0,1 ± 0 . 0 ~
0.1
± 0.1 a
1.1
±
1.1 ~
13.8 ±
9.8 ~
12.1 ±
7.6 a
18.8 ±
11.7 ~
With dep
0.0 ± 0.0 a
0.7
± 0.4 ~
0.7
± 0.4 a
0.0 ±
0.0 ~
40.8 ±
14.5 ~
37.6 ±
17.1 ~
Fat Without dep
0.1 ± 0.1 ~
0.3
--+ 0 . 2 ~
0.2
± 0.2 ~
16.6 ±
10.2 ~b
With dep
0.1 ± 0.1 a
0.2
± 0.1 a
0.2
± 0.1 a
40.0 ± 24.5 a
Protein
34.4 ±
12.0 ~
3.9 ±
3.9 b
54.6 ±
15.9 ~
34.5 ±
14.5 ~
Test period 3-6 hr Carbohydrate Without dep
0 . 4 ± 0.1 a
0.2
± 0.0 ~
0.8
± 0.4 ~
46.6 ±
12.8"
34.6 ±
8.3 a
43.3 -
8.4 ~
With dep
0.0 ± 0.0 ~
0.0
± 0.0 ~
0.3
± 0.3 ~
0.0 ±
0.0 ~
1.9 ±
1.9 a
9.5 ±
3.4 ~
Without dep
0.1 ± 0 . 0 ~
0.2
± 0.1 ~
0.5
± 0.3 ~
7 . 5 --_
3.3 a
17.7 ±
8.7 a
26.4 ±
With dep
0.0 ± 0.0 ~
0.0
+ 0.0 a
0.0
--- 0 . 0 ~
0.0 ±
0.0 ~
18.6 ±
16.4 a
Fat Without dep
0.3 ± 0,2 ~
0.2
± 0.1 a
0.3
± 0.2 ~
45.9 ±
16.0 ~
47.7 ±
13.4"
30.3 ±
With dep
0.0 z 0.0 ~
0.3
± 0.2 ~
0.1
± 0.0 ~
0.0 ±
0.0 ~
29.5 ±
18.9 a
54.2 ± 20.8 ~
13.0 a
13.9 ±
Protein 0 . 0 -±
5.6 a 0.0 ~ 10.6 a
Test period 6-12 hr Carbohydrate Without dep With dep
0.4 a
0.3
± 0.1 b
0.5
± 0.2 b
37.0 ±
0 . 5 --- 0 . 3 a
1.2 -
0.1
± 0.0 a
0.7
± 0.2 a
52.8 ± 20.5 a
2 . 7 -±
5.1 a
19.8 ±
6.8 a
0.4 b
9.5 ±
3.4 b
Protein Without dep
0 . 7 --_ 0 . 5 ~
1.4
--- 0 . 3 ~
2.1
--+ 0 . 5 a
18.2 ±
12.8 b
45.4 ±
10.7 ab
58.2 ±
9.6 a
With dep
0.1 ± 0 . 0 c
1.5
± 0.4 b
3.9
± 0.5 a
25.8 ±
18.7 a
35.8 ±
11.1 a
60.0 ±
2.6 a
Without dep
0 . 6 ± 0.1 ~
0.8
± 0.4 ~
0.48 ± 0.2
44.7 ±
10.9 ~
40.6 ±
13.1 ~
22.0 ±
9.6 ~
With dep
0 . 2 ± 0.1 b
1.3
--- 0 . 3 ~
0.8
21.4 ±
14.9 b
61.5 ±
11.2 ~
30.5 ±
4 . 3 ~b
Fat ± 0.1 ~
Test period 12-24 hr Carbohydrate Without dep
5.8 ±
1.8 ~
1.6
--- 0 . 7 b
2.6
--- 0 . 9 ~b
22.6 -
7.0 ~
7.6 ±
3.1 b
12.2 ±
3 . 9 ab
With dep
4.7 ±
1.7 a
0.2
± 0.1 b
2.5
± 0 . 8 ab
21.7 ±
8.8 a
1.2 ±
1.1 b
14.6 ±
4.1 ~b
11.7 ± 2 . 2 ~
11.6
±
1.9 ~
11.9
±
1.0 ~
45.7 ±
8.3 a
54.8 ±
7.3 ~
57.7 ±
4.8 ~
9.5 ± 2.6 a
7.6
±
1.4 ~
6.8
±
1.1 a
39.5 ±
7.7 ~
44.7 ±
5.8 ~
39.7 ±
5.5 ~
Without dep
3.5 ± 0.6 ~
3.4
± 0.6 a
2.8
± 0.4 ~
31.7 ±
5.1 a
37.5 ±
7.4 ~
30.1 ±
4.7 ~
With dep
3.7 ± 0.3 ~
3.9
± 0.4 a
3.3
± 0.4 ~
38.8 ±
5.4 ~
54.1 ±
5.2 a
45.7 ±
5.5 a
Protein Without dep With dep Fat
V a l u e s as g r a m s o r % k c a l s s h a r i n g a c o m m o n s u p e r s c r i p t w i t h i n e a c h o f the t w o t e s t i n g p e r i o d s are n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one another (p>0.05).
376
BLIGHETAL.
TABLE 4 TOTAL CALORICINTAKEDURINGEACHOF THE FIVE MONITORING PERIODS EITHERWITHOUTA PRECEDINGDEPRIVATIONPERIOD OR FOLLOWINGONE OF THREE DURATIONSOF DEPRIVATION Group
0hdep
24hdep
48hdep
Test period 0-1 hr Without deprivation Following deprivation
10.8 - 1.6a 1.7 _ 0.6 b
7.9 - 1.2a 23.7 +__4.0 a
8.2 +__ 1.8a 33.4 _+ 5.2 a
Test period 1-3 hr Without deprivation Following deprivation
2.8 -+ 0.8 a 1.0 _ 0.6 ~
4.2 _ 1.6~ 4.5 - 1.4~
6.3 + 4.9 a 4.7 _ 1.4a
Test period 3-6 hr Without deprivation Following deprivation
4.5 _ 1.2~ 0.0 - 0.0 ~
3.0 -+ 0.7 a 2.8 - 1.8a
7.1 _ 3.1 a 2.1 +_ 0.5 a
Test period 6-12 hr Without deprivation Following deprivation
11.7 4- 1.1 a 3.9 -+_ 1.6~
Without deprivation Following deprivation
89.8 -+ 7.4 a 79.6 -4- 8.3 ~
12.3 +- 3.8 ~ 16.1 - 1.7b
12.9 _ 2.9 a 22.9 + 2.8 a
Test period 12-24 hr 74.1 _ 0.2 b 58.7 -+ 4.4 b
73.3 - 0.l b 59.0 +_ 4.2 b
NB: Animals were assigned to each of the three deprivation groups (Oh, 24h or 48h) randomly. Thus, differences observed in total caloric intake during the without deprivation test reflect random error, i.e., the animals had been treated identically throughout the experiment up to that time.
protein, fat and calorie intakes did not differ between treatment groups. One day prior to the start of deprivation, the intakes of all rats were monitored starting at 0900 hr. Intake of each source was measured at 0900, 1000, 1200, 1500, 2100 and 0900 hr the next day. Analysis of macronutrient intake during these 5 test periods prior to deprivation revealed only minimal group differences in the patterns of intakes of any of the three macronutrients. For example, fat intake differed only during the first hour when 24hdep and 48hdep animals ate significantly less fat than did the 0hdep control group. Intakes of carbohydrate, protein and calories did not differ significantly between groups at this time (refer to Tables 3 and 4). From hours 1-3 and 3-6, none of the groups had significantly different intakes of any of the macronutrient sources or of total calories. At hours 6-12, 24hdep and 48hdep animals ate less CHO than did the 0hdep control group. Intakes of protein, fat and calories did not differ significantly between groups. At 12-24 hours, 24hdep ate significantly less carbohydrate than 0hdep animals (5.84---1.80 vs. 1.62---0.67 g, p<0.05). The 48hdep intake did not differ significantly from either group. Calorie intakes also differed during the 12-24 hour period with 24hdep and 48hdep groups consuming significantly less calories than 0hdep controls. Finally, it should be noted that the daytime intakes of all three macronutrients were uniformly low relative to nighttime (12-24 hr) intakes. The diets that were composed during each of these testing periods (as assessed by calculating the percentage of calories consumed during each of these test periods) were also remarkably similar between groups. There were no significant differences between the diets composed by any group during test periods 0-1 hr, 3-6 hr, and 12-24 hr. Fat intake as a percentage of total
calories differed only during hours 1-3 when 24hdep ate significantly more than 48hdep. Percentage intakes of CHO and protein were not significantly different during this time. Percent CHO intakes differed only during hours 6-12 with 0hdep composing a diet that was significantly higher in CHO than the diet composed by the 24hdep group. Percent calories as protein was significantly different during hours 6-12 with the 48hdep composing a diet that was significantly higher in protein than was the diet composed by the 0hdep group. Again, it should be noted that although these differences between groups in diet composition attained statistical significance, actual intakes during the daytime hours was uniformly low when compared to nighttime (12-24 hr) intakes. Intake after deprivation. Significant increases in both protein and fat intakes were observed during the first hour of refeeding subsequent to fasting (0-1 hr) when comparing postdeprivation intakes with predeprivation levels within groups. There were no significant changes in intake of any of the macronutrient sources when compared to the predeprivation day during the second testing period (1-3 hr). The 48hdep and 0hdep groups ate less CHO during the 3-6 hr test period than they did during the predeprivation test period, Again, refer to Table 3. From 6-12 hours, the 48hdep group ate more CHO during the refeeding day while the 0hdep group ate less CHO when compared to predeprivation intakes. The 24hdep group's CHO intake did not differ from either group. During this testing period, all groups ate less protein during the refeeding day when compared to the predeprivation test period. The 24hdep group ate significantly more fat during the refeeding period compared to the predeprivation day while 0hdep ate less fat. During the first hour of refeeding (0-1 hr), carbohydrate intake (grams) was not significantly different among groups. The 24hdep and 48hdep groups ate significantly more protein and fat than did the 0hdep group. As a consequence, the 24hdep and 48hdep groups ate significantly more calories than did the 0hdep group. There were no differences between the 24hdep and 48hdep groups in either protein or fat intakes during the first hour of refeeding. Refer to Tables 3 and 4. From hours 1-3 of refeeding, the 48hdep group ate significantly more CHO than did the 0hdep group. Protein, fat and calorie intakes did not differ among treatment groups. During the 3-6 hr refeeding period, there were no significant differences in intake between groups for any of the macronutrients or total calories. From hours 6-12 of refeeding, CHO intake did not differ between groups. The 48hdep group ate significantly more protein than did the 24hdep group. Similarly, the 24hdep group ate significantly more protein than did the 0hdep group. Both the 24hdep and the 48hdep groups ate significantly more fat than did 0hdep controls. The total caloric intakes of the three groups differed significantly, with 48hdep rats eating significantly more than the 24hdep group, and both eating more than the 0hdep controls. During the 12-24 hour refeeding period, the CHO intake of the 24hdep group was significantly lower than that of the 0hdep group. Protein and fat intakes were not significantly different between groups. The 24hdep and 48hdep groups ate significantly less calories than did the 0hdep group. As percentage of calories, the 24hdep and 48hdep groups ate significantly less CHO than did the 0hdep group during the first hour of refeeding. The 24hdep and 48hdep groups composed diets that were significantly higher in percent protein than the diet composed by the 0hdep group. Percent calorie intake as fat did not differ between groups. Because intake levels during the subsequent daytime monitoring periods were so low, the dietary composition analyses (percentage of calories) were widely variable. Corticosterone concentrations. The 48hdep group had significantly higher corticosterone levels than 24hdep and 0hdep at the beginning of the refeeding period (15.13 +_3.62 vs. 5.42 - 2.74
ADRENAL MODULATION OF FAT INTAKE
•.-, 4-0.0-~ ~'o, 3 5 . 0 :3
vIM Z
o
0~0
Ohdep 24hdep 48hdep
e~e
z~--Zl
377
320.00-
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1
oooo
e
30.0-
280.00
25.0-
_ T
T
T
~
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20.0o
15.0-
240.00-
10.0-
m
220.00-
5.00.0 --'
0
', 1
', 2
, 3 TIME
', 4
',--, 5 6
(hours) OF
'. 7
', 8
'. 9
'
'
'
200.00
10 11 12
I 1
0
REFEEDING
i 2
I 3
I 4
I 5
0 6
I 7
I 8
I 9
I I I I t 10 11 12 13 14
DAYS
FIG. 1. Effect of 0, 24 and 48 hr of food deprivation on circulating corticosterone. All values represent means and standard errors o f the means. Note that after the first hour o f refeeding, all three groups had comparably low concentrations.
FIG. 2. Body weights of adrenalectomized and sham-operated rats prior to food deprivation. Surgery was performed on Day 10. All values represent means and standard errors of the means.
Daily carbohydrate Intake of ADX and eham-operated Sprague-Dawley rats T
hi "
m
g
2o~o I
°:t
•
~
•
II~iX
I
I
I
I
I
10
11
12
13
14
I
I
12
13
"I
Daily proteinsprague_Dawleyin ADX and sham-operatedrats
g
Io~o
-
I
I
10
11
-
14
Dally fat Intake of ADX and eham-opemtsd Sprogue-Dawley rots
~ / I
o ~ o ,Im-lm, d
I
I
I
I
I
10
11
12
13
14
DAYS FIG. 3. Effect o f adrenalectomy on carbohydrate (top), protein (nrdddle) and fat (bottom) intakes prior to food deprivation.
BLIGH ET AL.
378
vs. 0.80-+0.55 p3/dl, p<0.05). Refer to Fig. 1. At 1 hour after refeeding there were no significant differences between groups. At hour 3 of refeeding the 24hdep group had significantly higher corticosterone levels than the 0hdep and 48hdep groups (6.63 -+ 1.19 vs. 2.10 -+0.43 vs. 1.95 -+ 0.46 I~g/dl, p<0.05). This difference was also observed at the 6-hr measurement (15.08-+3.74 vs. 3.42±1.17 vs. 1.10±0.24 txg/dl, p<0.05). By hour 12 there were no significant differences between groups. During the first hour of refeeding neither CHO nor protein intake were correlated significantly with corticosterone levels. However, fat intake during this hour was significantly correlated with circulating corticosterone (r= .72, p<0.001).
Experiment 2 Although this experiment was conducted in two replications, the rats in the second replicate weighed significantly more than the rats in the first. Results from the two were pooled after statistical evaluation failed to reveal any significant interactions of the treatment effects (duration of deprivation, surgical procedure effects or their interactions) with the replicate factor. Data from any ADX rat having corticosterone values greater than 5 ~g/dl at any sampling time (n = 5) were dropped from the study due to incomplete adrenalectomy and/or the development of accessory adrenals (19). Body weight. Throughout the first 9 days of the study, body weight increased steadily. Prior to surgery, there were no significant differences in weight gain between groups assigned to different surgical treatments. Refer to Fig. 2. After surgery on Day 10, the 24hAdx group gained significantly less weight/day than did any of the sham-operated groups. Refer to Table 5. On the day prior to the institution of deprivation conditions, the 24hAdx and 0hAdx groups weighed significantly more than the 48hAdx group. The body weights of the ADX groups did not differ significantly from any of the sham-operated groups. After their respective deprivation periods, 0hSham, 24hAdx and 0hAdx were significantly heavier than the 48hAdx group. Refer to Table 6. The amount of weight lost during the deprivation period was determined by the duration of fast, F(2,21)=31.4, p<0.0001. Similarly, the percent change in body weight was determined by the duration of fast, F(2,21) = 25.16, p<0.04. Predeprivationfood intake. During the first 9 days, the 0hAdx and 24hAdx groups ate significantly more CHO than did the 24hSham, 48hAdx and 0hSham groups. Protein intakes were not significantly different between groups, while the 0hSham, 24hSham and 24hAdx groups ate more fat than did the 48hSham, 48hAdx and 0hAdx groups. The 24hAdx group consumed significantly more calories than 0hAdx, 48hSham and 48hAdx groups. The 0hSham and 24hSham ate more calories than the 48hAdx group (86.20_-_2.46 vs. 84.43___2.74 vs. 77.05---2.28 kcal, p<0.05). Although there were a number of significant differences in macronutrient intakes following surgery, ANOVA revealed no significant surgical effect on carbohydrate intake. Refer to Fig. 3. By contrast, ANOVA revealed that protein and fat intakes were significantly decreased by ADX. Adrenalectomy changed the composition of the diets that were selected. ADX rats selected diets that were higher in carbohydrate and lower in protein than the diets composed by sham-operated rats. The sham-operated rats selected diets that provided 1 9 . 3 +- 1.5% of total calories as CHO, 56.5-+ 1.5% calories as protein and 24.2-+ 1.0% calories as fat. By contrast, ADX rats composed a diet yielding 30.6 -+ 2.6% CHO, 41.5 -+ 2.4% protein and 28.0-+2.3% fat.
Day of refeeding. Deprivation promoted several shifts in the intake of all three macronutrient sources. During the first hour of refeeding (0-1 hr) the 48hSham and 24hSham groups ate significantly more fat when compared to the 0hSham controls. Refer to Fig. 4a. The protein intakes of the 24hSham and 48hSham groups were significantly higher than that of the 0hSham controls. Refer to Fig. 4b. Adrenalectomy did not affect carbohydrate intake. All groups of adrenalectomized animals consumed carbohydrate in amounts that were not different from that of their controls. Refer to Fig. 4c. By contrast, the 24hAdx and 48hAdx groups failed to eat more of any of the three sources when compared to both nondeprived controls (0hAdx or 0hSham groups). During hours 1-3 of refeeding, the intakes of all three macronutrients were uniformly low, irrespective of duration of deprivation or surgical treatment. CHO intake between groups was not significantly different. The 24hSham group consumed more protein than did 0hAdx and 0hSham controls. The 48hSham consumed significantly more fat than did 24hAdx, 0hAdx and 0hSham groups. These and other differences are noted in Table 7. During hours 3-6 of refeeding, intakes of all three macronutrients remained low. CHO intakes did not differ between groups. The 48hAdx group ate significantly more protein than all other groups. The 48hAdx consumed more fat during this time than did 24hSham, 24hAdx, 0hAdx and 0hSham. Fat intakes of 48hSham and 48hAdx were not significantly different. During hours 6-12, 24hAdx ate significantly more CHO than did the 24hSham and 0hSham groups while the 48hSham group ate significantly more than CHO than did the 0hSham group. The 48hAdx group ate significantly more protein than did the 0hAdx controls. No other differences in protein intake were found. The 24hSham group ate more fat than did the 0hAdx group. No other differences in fat intake between groups were found during the 6-12 hr test period. Caloric intakes were not different among any of the groups. During hours 12-24, CHO intakes did not differ between groups. The 0hSham group ate more protein and fat than did any other group. As a percentage of calories, during the first hour of refeeding, the 24hAdx group composed a diet higher in carbohydrate than did the 0hSham and 48hSham groups (60.09 _+23.37 vs. 12.50 - 12.50 vs. 11.72 - 4.57%, p<0.05). The percentage of calories of protein and fat did not differ between groups during this hour.
DISCUSSION
Several aspects of the present experiments are noteworthy. The most important finding was that during the initial hour of restored access subsequent to food deprivation, rats refeed primarily on fat and protein. Second, deprivation leads to elevated corticosterone levels which are correlated with the enhanced fat intake. Third, adrenalectomy attenuates the increase in fat and protein intake that is observed subsequent to food deprivation while not altering carbohydrate selection. Fourth, adrenalectomized self-selecting rats fail to increase total caloric intake after food deprivation. These observations are consistent with our laboratory's working hypothesis that the adrenal gland plays a role as a modulator of fat selection. Results from these experiments replicate the findings of Berdanier and colleagues who first reported that adrenalectomized rats fail to fully compensate for food deprivation (22). They showed that ADX rats fed a composite diet after being deprived of food for 24 or 48 hours failed to eat more calories/day than control rats that had not been fasted. Like those animals, self-selecting ADX rats also under-eat under these conditions. These results also rep-
ADRENAL MODULATION OF FAT INTAKE
2.50-
379
a
_I_
2.00-
AVERAGEDAILY WEIGHT GAIN AND TOTAL CALORIC INTAKEOF ADX AND SHAM-OPERATEDRATS PRIORTO DEPRIVATION (EXPERIMENTALDAYS 10-14)
_i_
1.50-
v
TABLE 5
Ill
Daily Weight Gain (g/day)
bJ
1.00-
Z
0.50 0.00
i
Oh Adx
2.50
Oh Sham
24h
24h
Adx
48h
48h
Sham
Adx
Sham
b
Itl
1
2.00
Sham-operated 0hSham 24hSham 48hSham Adrenalectomized 0hAdx 24hAdx 48hAdx
5.8 3.5 4.4
Daily Total Caloric Intake (kcals/day)
- 1.8= ± 1.3=b ± 0.5 ab
85.0 _+ 5.5 = 77.1 ---4.4= 79.8 ± 3.2 a
1.4 __. 1.4~ -0.85 _ 2.2 ¢ 0.9 - 1.9b~
60.2 ± 4.4 b 55.5 --- 6.7 b 52.8 ± 5.3 b
Groups not sharing common superscripts are different from one another at the p<0.05 level.
h,
1.50. z
1.00.
n¢
n
0.50. ,= Oh Adx
0.00
1.75,,,
i
Oh Sham
24h Adx
C
24h Sham
48h
48h
Adx
Sham
lit T
1.50T
1.25t~
_~
1.00-
t:a >-1o m
0.750.50-
o<
0.25.
n,,"
0.00
l T _]_ Oh Adx
Oh Sham
24h
24h
48h
Adx
Sham
Adx
48h
enhanced fat intake observed in the present experiment is under the exclusive control of corticosterone, preliminary results from our laboratory suggest that corticosterone replacement in ADX rats restores the enhanced fat intake that was observed subsequent to food deprivation (1). Grogan and Romsos (8) have demonstrated that the effects of ADX can be modulated by altering the fat content of a composite diet. They showed that the attenuated rate of weight gain that is observed subsequent to ADX in Sprague-Dawley rats can be blocked by increasing the fat content of the diet. ADX rats fed a high fat diet grow at rates comparable to sham-operated controls, suggesting that the metabolic effects of ADX on weight gain and intake can be modified by dietary manipulations. This suggestion has also been made by Sklar et al. (20) who demonstrated that ADX Sprague-Dawley rats given access to a sucrose supplement in addition to a semipurified diet (AIN-76 rat diet) also grow at sham-operated control rates. The importance of these observations is that dietary manipulations can play a major role in the outcome of this type of experiment. Castonguay et al. (3) reported that ADX self-selecting Zucker rats differ from sham-operated controls primarily by reducing fat intake. By contrast, Leibowitz and her colleagues reported that ADX Sprague-Dawley rats reduce both CHO and fat intake. The reason for the discrepancy between
Sham
FIG. 4. (a) Effect of adrenalectomy on fat intake during the first hour of refeeding subsequent to food deprivation. (b) Effect of adrenalectomy on protein intake during the first hour of refeeding subsequent to food deprivation. (c) Effect of adrenalectomy on carbohydrate intake during the first hour of refeeding subsequent to food deprivation. (a) and (b) *Significantlydifferentfrom 0hSham and Oh ADX (p<0.05). (c) *Significantly different from OhAdx and OhSham (p<0.05).
licate the findings of Shutz and Pilgrim (18) who reported that food-deprived rats increase dietary fat intake subsequent to restored access. The present experiments extend those of Wurdeman et al. and Shutz and Pilgrim by demonstrating that ADX selectively interferes with the refeeding response by attenuating fat intake. These results are consistent with the hypothesis that dietary fat intake is influenced by adrenal hormones. Previous work from our laboratory has demonstrated that the enhanced fat appetite of the genetically obese fa/fa Zucker rat is mediated by circulating corticosterone (3). Although it remains to be demonstrated that the
TABLE 6 WEIGHT LOSS AS A FUNCTION OF SURGICALCONDITION (ADX OR SHAM) AND DURATIONOF DEPRIVATION
Sham-operated 0hSham 24hSham 48hSham Adrenalectomized 0hAdx 24hAdx 48hAdx
Predeprivation Weight (g)
Weight Change (g)
Relative Weight Loss (% initial body weight)
305.4 ± 12.9=b 290.9 -- 28.7 ab 309.5 -- 19.2=b
4.9 ___ 1.0a -22.2 - 1.9b -34.7 ± 2.4 b
1.6 --- 0.4 a - 7 . 3 +- 0.8 b -11.4 -- 1.2b
304.5 ± 20.1ab 362.6 ± 12.6a 277.6 ± 12.0b
0.9 -- 3.5 a -21.7 ± 2.8 b - 3 4 . 6 ± 8.0 b
0.4 ± 1.0= - 6 . 0 -+ 0.6 b - 9 . 3 ± 3.1 b
380
B L I G H E T AL.
TABLE 7 MACRONUTRIENT INTAKES OF ADX AND SHAM-OPERATED RATS DURING THE REMAINING 4 TESTING PERIODS FOLLOWING DEPRIVATION
Group 0hAdx
0hSham
24hAdx
24hSharn
48hAdx
48hSham
Test period 1-3 hr CHO Pro Fat kcals
0.2 0.2 0.1 2.2
-+ -+ ± ---
0.1 ~ 0.1 b 0.1 b 1.2 b~
0.02 0.05 0.1 1.0
± 0.02 ~ ----- 0 . 0 3 b
± 0.04 b --- 0.4 c
0 + 0a 0.4 --- 0.4 ab 0.2 ± 0 . 1 b 3.0 --- 1.7 b~
1.2 1.6 0.7 15.6
4+ 4±
0.5 a 0.6 a 0.2 ab 3.7 a
1.0 __ 0.5 ~ 0.6 4- 0.2 ab 0.9 ±- 0 . 4 ab 13.1 -+ 5.0 "b
1.0 1.1 1.1 16.4
___ 0.4 a ~
0.3 ab
±
0.4 a 4.2 ~
Test period 3-6 hr CHO PrO Fat kcals
0.2 0.02 0.1 1.8
4- 0.2 a -----0.02 b - 0.04 b 4- 0.8 b
0.05 0.1 0.05 0.8
_+ 0.03 ~ 4- 0.05 b --- 0.03 b ± 0.3 b
0.3 0.3 0.2 3.6
~ 0.1 ~ --+-0.2 b +-- 0.1 b --- 0.6 b
0.5 0.2 0.3 5.1
4- 0.4" -----0.1 b -+ 0.2 b -+ 2.5 b
0.5 1.0 1.1 13.6
----.
0.2 a 4- 0.4 a --- 0.3 a ± 4.0"
0.6 0.3 0.5 7.2
1.5 2.0 1.1 21.3
2.6 3.6 1.7 35.6
4- 0.3 a ----. 0.2 b ±
0 . 2 ab
±
3.0 "b
_+ 0.4 abe 4- 0.2 a _ 0.5 "b ± 5.3"
1.6 1.9 1.3 22.8
± ± 4-_ --_
0.6 "b 0.6 ab 0.3 ab 4.1 ~
4- 0.9 a __. 0.6 b _+ 0.2 b -+ 4.5 b
5.1 5.9 1.9 54.1
+__ 2.8 a ± 1.0b __. 0.4 b ± 13.7 ab
Test period 6-12 hr CHO Pro Fat kcals
1.3 0.6 0.4 9.7
4- 0.4 a~ + 0.2 b 4- 0.3 b __. 4.0 a
0.2 1.0 0.6 9.0
± ± ± 4-
0.2 c 0.3 ab 0.1 ab 2.5 a
2.3 0.8 0.7 16.8
± ± 44-
0.2" 0.3 ab 0.3 ab 3.1 ~
0.8 - 0.2 e¢ 1.4 ± 0 . 4 ab 1.7 ~ 0.4 a 20.9 - 4.3 a
Test period 12-24 hr CHO Pro Fat kcals
3.4 5.2 2.2 48.6
___ 0.6" ___ 1.1 b ± 0.5 b ± 6.6 at'
1.1 8.7 4.2 68.1
± 0.2 a ___ 0.8 a _+ 0.7 a ___ 8.0"
3.3 4.6 1.8 42.3
± 0.5" --- 0.6 b --- 0.7 b ± 6.7 ~b
e x p e r i m e n t s w a s s u g g e s t e d to be strain and age related. H o w e v e r , the present results, like those o f Leibowitz and her colleagues (11,13), were b a s e d u p o n the selection patterns o f y o u n g SpragueD a w l e y rats. A l t h o u g h m o r e subtle differences in strain and age r e m a i n possible explanations, the present e x p e r i m e n t s u g g e s t s that differences in macronutrient source c o m p o s i t i o n m a y be a m o r e likely influencing factor. Unlike the sources u s e d in these studies, those u s e d by Leibowitz et al. were a modification of the formulation first described by K a n a r e k and H o (9). T h e primary difference b e t w e e n the Leibowitz macronutrient sources and those used in these studies is in the level o f v i t a m i n and mineral s u p p l e m e n t a t i o n o f the fat source. T h e c o m p o s i t i o n o f the Leibowitz fat source is lard s u p p l e m e n t e d with v i t a m i n s and minerals. H o w e v e r , the level o f s u p p l e m e n t a t i o n is b a s e d u p o n grams/kcal, w h e r e a s the v i t a m i n and mineral s u p p l e m e n t a t i o n o f the fat source used in these studies was b a s e d g r a m s o f s u p p l e m e n t s per g r a m o f food. T h u s , it is our s u g g e s t i o n that fat intake c a n be m o d u l a t e d in A D X rats b a s e d u p o n the mineral content o f the macronutrient sources. T h e salt-specific appetite o f the A D X rat h a s been thoroughly d o c u m e n t e d , starting with the pioneering work of Richter (16). Perhaps the drop in fat intake s u b s e q u e n t to adrenale c t o m y that is routinely o b s e r v e d in o u r laboratory m a y be c a u s e d by a shift in the hedonic value o f the protein and carbohydrate sources/kcal intake w h e n c o m p a r e d to the relatively dilute concentration o f s o d i u m in the s u p p l e m e n t e d fat source. T h e s e results are also in a g r e e m e n t with those reported by D e v e n p o r t et al. (5) w h o h a v e recently reported a selective decrease in fat intake s u b s e q u e n t to A D X in S p r a g u e - D a w l e y rats. W h e t h e r or not dietary differences determine all or only part o f this effect, it r e m a i n s a fact that w h e n the present sources are m a d e available to
2.3 3.9 2.4 40.8
± 0.7" + 0.7 b ± 0.6 b ___ 4.4 b
either Z u c k e r rats or to S p r a g u e - D a w l e y rats, fat intake is specifically attenuated by A D X . T h e s e results s u g g e s t that preference testing in A D X and s h a m - o p e r a t e d rats m a y resolve s o m e o f the c o n f u s i o n about the effects o f A D X on macronutrient selection. O n e of the m o s t intriguing aspects o f this e x p e r i m e n t is " W h y do food-restricted rats select f a t ? " S o m e insight into an a n s w e r to this question m a y be f o u n d in considering a food utilization strategy like that first proposed by F r i e d m a n and Stricker (7). T h e thesis proposed by these authors is that diabetic rats fail to maintain h y p e r p h a g i a w h e n put on a h i g h fat diet b e c a u s e fat utilization is the primary m e c h a n i s m u s e d by the diabetic rat to meet e n e r g y needs, since carbohydrate-derived glucose utilization is for the m o s t part insulin dependent. W e s u g g e s t that both the obese Z u c k e r rat and the food-restricted rat select fat in disproportionate a m o u n t s at least in part b e c a u s e they have relatively low levels o f glucose utilization. A l t h o u g h the o b e s e Z u c k e r rat h a s been reported to be mildly h y p e r g l y c e m i c , it h a s m o r e frequently been reported to be insulin insensitive (4, 15, 24). Like the Z u c k e r rat, the starved rat h a s very low levels o f circulating insulin resulting in low levels o f glucose utilization. Also note that coincident with these divergent m o d e l s is the finding that corticosterone is elevated (2, 3, 23). T h u s , both the obese rat and the starved rat c a n m o r e readily and effectively m e e t e n e r g y needs by selecting fat. O n e possible influence to account for the observation that a d r e n a l e c t o m y selectively attenuates the increase in fat intake is the w e l l - k n o w n increase in glucose utilization induced b y A D X first described H o u s s a y . A n o t h e r possible influence is an A D X induced increase in insulin sensitivity in the obese rat m o r e recently described by O h s h i m a et al. (14). This e m p h a s i s on
ADRENAL M O D U L A T I O N OF FAT INTAKE
381
selection guided by fuel utilization is similar to that fast proposed by Friedman and Stricker, but was formulated on the observations on obese or starved-refed rats instead of based around the feeding of diabetic rats. Nevertheless, the hypothesis that selection is guided by current metabolic conditions is supported by these observations. Future studies will be directed at determining the nature and location of the receptors that mediate fat intake. It remains plausible that dietary fat is selected in preference to carbohydrate
under some metabolic conditions because carbohydrate is not as readily used. In that sense, fat intake is increased out of default. It also remains possible that fat intake is actively monitored and not simply consumed as a second-best alternative to meeting energy needs. ACKNOWLEDGEMENT This work was supported by a grant from the Whitehall Foundation.
REFERENCES 1. Bligh, M. E.; Castonguay, T. W. Glucocorticoid control of macronutrient choice subsequent to fasting; in preparation. 2. Bray, G. A.; York, D. A. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev. 59:719-809; 1979. 3. Castonguay, T. W.; Dallman, M. F.; Stern, J. S. Some metabolic and behavioral effects of adrenalectomy on obese Zucker rats. Am. J. Physiol. 251:R923-R933; 1986. 4. Chan, C. P.; Stern, J. S. Adipose lipoprotein lipase in insulin-treated diabetic lean and obese rats. Am. J. Physiol. 242:F_,445-E450; 1982. 5. Devenport, L.; Knehans, A.; Thomas, T.; Sundstrom, A. Macronutrient intake and utilization by rats: interactions with type I adrenocorticoid receptor stimulation. Am. J. Physiol.; in press. 6. Freedman, M. R.; Horwitz, B. A.; Stern, J. S. Effect of adrenalectomy and glucocorticoid replacement on development of obesity. Am. J. Physiol. 250:R595-R607; 1986. 7. Friedman, M. I.; Stricker, F. M. The physiological psychology of hunger: a physiological perspective. Psychol. Bull. 83:409-431; 1976. 8. Grogan, C. K.; Kim, H.; Romsos, D. R. Effects of adrenalectomy on energy balance in obese (ob/ob) mice fed high carbohydrate or high fat diets. J. Nutr. 117:1115-1120; 1987. 9. Kanarek, R. B.; Ho, L. Patterns of nutrient selection in rats with streptozotocin-induced diabetes. Physiol. Behav. 32:639--645; 1983. 10. King, B. M.; Banta, A. R.; Tharel, G. N.; Bruce, B. K.; Frohman, L. A. Hypothalamic hyperinsulinemia and obesity: role of adrenal glucocorticoids. Am. J. Physiol. 245:EI94-E199; 1983. 11. Kumar, B. A.; Leibowitz, S. F. Impact of acute corticosterone administration on feeding and macronutrient self-selection patterns. Am. J. Physiol. 254:R222-R228; 1988. 12. Langely, S. C.; York, D. A. The effects of the antiglucocorticoid RU-486 on the development of obesity in the obese 'fafa' Zucker rat. Am. J. Physiol.; in press.
13. Lucas, D. J.; Leibowitz, K. L.; Jhanwar, Y. S.; Leibowitz, S. F. Developmental patterns of macronutrient selection and body weight gain in male and female rats. Proc. Abstr. Annu. Meet. Eastern Psychological Assoc. 61:47; 1990. 14. Ohshima, K.; Shargill, N. S.; Chan, T. M.; Bray, G. A. Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice. Am. J. Physiol. 246:E193-E197; 1984. 15. Reaven, G. M.; Bernstein, R.; Davis, B.; Olefsky, J. M. Nonketotic diabetes mellitus: insulin deficiency or resistance. Am. J. Med. 60:80-84; 1976. 16. Richter, C. P. Decreased carbohydrate appetite of adrenalectomized rats. Proc. Soc. Exp. Biol. Med. 48:577-579; 1941. 17. Schiffer, F.; Wertheimer, E. Leanness in adrenalectomized rats. J. Endocrinol. 5:147-151; 1947. 18. Schutz, H. G.; Pilgrim, F. J. Changes in the self-selection pattern for purified dietary components by rats after starvation. J. Comp. Physiol. Psychol. 47:44A. a.~9; 1954. 19. Schwabedal, P. E.; Partenheimer, U. Uber vorkommen und strnktur accessorischer nebennieren bei der Wistar-Ratte. Leipzig 97:753-768; 1983. 20. Sklar, T. N.; Castonguay, T. W.; Horwitz, B. A.; Stem, J. S. Effect of adrenalectomy on rats fed a 32% sucrose solution. Am. J. Physiol.; submitted. 21. Solomon, J.; Mayer, J. The effect of adrenalectomy on the development of the obese-hyperglycemic syndrome in ob/ob mice. Endocrinology 93:510-513; 1973. 22. Wurdeman, R.; Berdanier, C. D.; Tobin, R. B. Enzyme overshoot in starved-refed rats: Role of the adrenal glucocorticoid. J. Nutr. 108:1457-1461; 1978. 23. Yukimura, Y.; Bray, G. A.; Wolfsen, A. R. Some effects of adrenalectomy in the fatty rat. Endocrinology 103:1924-1928; 1978. 24. Zucker, L. M.; Zucker, T. F. Fatty, a new mutation in the rat. J. Hered. 52:275-278; 1961.