Self-selection and the obese Zucker rat: The effect of dietary fat dilution

Physiology&Behavior,Voi. 33, pp. 119-126.Copyright©PergamonPress Ltd., 1984. Printedin the U.S.A.

0031-9384/84$3.1)0+ .00

Self-Selection and the Obese Zucker Rat: The Effect of Dietary Fat Dilution I T H O M A S W. C A S T O N G U A Y , S H E R I L. B U R D I C K , M A R I A A. G U Z M A N , G E O R G E H. C O L L I E R * A N D J U D I T H S. S T E R N

Food Intake Laboratory, Department of Nutrition, University of California, Davis, CA 95616 and *Department of Psychology, Ru.tgers, The State University, New Brunswick, N J 08903 R e c e i v e d 10 M a y 1982 CASTONGUAY, T. W., S. L. BURDICK, M. A. GUZMAN, G. H. COLLIER AND J. S. STERN. Self-selection and the obese Zucker rat: The effect of dietaryfat dilution. PHYSIOL BEHAV 33(1)119-126, 1984.--Adult female lean and obese Zucker rats were allowed to compose their own diets by giving them access to three macronutrient sources. After a baseline period, the fat source was serially diluted. In all, eight concentrations of fat were used. Dilution of the fat source promoted significant increases in the intake of fat by both lean and obese rats. The increased intake of fat was not simply compensatory in nature, but rather represented significant increases in fat consumption. These results suggest that the reported increased appetite for fat of the obese rat is not a unique trait of that genotype. Further, results from this experiment demonstrate that caloric and protein regulation mechanisms may not be controlling the intake of obese and lean rats as precisely as once believed. Diet selection

Dilution

Caloric regulation

Motivation

lean littermates. More recently, Castonguay et al. [9] have reported that obese rats, given three separate macronutrient sources, will compose a diet that provides more than twice the fat and 1.5 times the calories of the diet selected by lean littermates. The diet composed by lean rats provided 32% of the rats total calories as protein, 38% as carbohydrate and 30% as fat. By contrast, obese rats selected a diet that was only 12% protein and 24% carbohydrate but 64% fat. Differences in the composition of the self-selected diets of obese and lean rats serve as a starting point for the present experiment. We examined what effects diluting the fat source would have on the composition of diets selected by both obese and lean rats. To what extent is the elevated intake of fat that characterizes the obese rat resistant to the effects of dilution? Is the fat appetite of the obese rat motivated?

T H E mechanisms that control the food intake of the rat can be divided into two groups: factors that control caloric or energy needs, and factors that control dietary quality, such as macro or micro nutrient intake. One method of assessing how well a rat controls its caloric intake has been to dilute the diet with nonnutritive materials [17]. Adolph [1] was among the f'wst to demonstrate that rats will adjust their intake of calorically different diets so as to maintain a characteristic level of caloric intake. He demonstrated that adding non-nutritive material to a maintenance diet promoted increases in the intake of diluted food. Similarly, Carlisle and Stellar [6] have demonstrated that rats will decrease their consumption of diets that are made more calorically dense (see also [16]). The dilution paradigm has also been used in the study of how rats control their intake of particular macronutrients. For example, Rozin [29] has demonstrated that diluting a liquid protein source with water can promote compensat'ory increases in the intake of that protein source (see also [5]). Similarly, Collier and Bolles [13] have demonstrated that the intake of carbohydrate is also controlled. They showed that the dilution of a sucrose solution resulted in compensatory intake within a wide range of concentrations. Evidence from these and other studies [2, 3, 8] has led some authors to propose that, under certain conditions, the rat can regulate the composition of its diet [2,5]. Although most of these studies have used standard breeds of laboratory rats, there are a few reports of dietary component selection [22,26] by hyperphagic genetically obese Zucker rats. Using a choice paradigm, Anderson and his colleagues [2] have demonstrated that obese rats select a diet that is somewhat lower in protein than the diet composed by

EXPERIMENT 1 METHOD

Animals Twelve obese (fa/fa) and twelve lean (FaJ-) female Zucker rats, approximately 10 weeks old, were obtained from our breeding colony. The obese rats weighed an average of 295+--10 g and their lean littermates weighed an average of 201---5 g at the start of the experiment. They had been maintained on ad lib purina rat chow and water up until that time.

Apparatus Animals were individually housed in standard, rack

JThis research was supported in part by grants T32AM07355 and AM 18899 from the National Institutes of Health.

119

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CASTONGUAY E T AL.

450

TABLE 1 COMPOSITIONOF DIETARYSOURCES

Cornstarch* Casein¢ DL-Methionine~ Cellulose§ Vitamin Mix Mineral Mix Corn Oil¶ Caloric Density

Source 88.50 -

-

-

-

4.00 1.50 6.00 -

-

3.80 kcal/g

(%) Source -87.76 0.74 4.00 1.50 6.00 -

-

3.80 kcal/g

100%

50%

25%

400 Source -

OB~~ESE

350

-

--

-

-

-

--

BW

(g)

300

25O

-

100.00 9.00 kcal/g

*National Starch and Chemical Corporation, Bridgewater, NJ 08807. tANRC Reference Casein (30 mesh), Sheffield Chemical, Norwich, NY 13815. ~:Fisher Scientific, Pittsburg, PA 15219. §Solka-Floc, Brown Company, Berlin, NH 03570. ¶Mazola, Best Foods, CPC International, Inc., Englewood Cliffs, NJ 07632.

mounted hanging cages in a temperature (22°C+_2) and lighting controlled laboratory. The lighting schedule was maintained on a 12 hour on/off cycle, with lights coming on at 0800 hr. The composition of the three dietary sources used during the experiment is presented in Table 1. Both carbohydrate and protein sources were held in ceramic food cups equipped with aluminum followers. Both cups were placed directly into each cage. Corn oil was used as a fat source, and was made accessible by hanging a corn oil-filled bottle on the front of each cage. A standard metal drinking spout extended into the cage from the fat source. Because of the tendency for some leakage to occur, a small petri dish was placed under each cage directly below the corn oil spout. Spillage of corn oil was weighed daily. Spillage of each solid diet, although minimal, was also collected and weighed. Drinking water and food sources were available ad lib. The dilution of corn oil was achieved by adding 0.5% Emplex (sodium stearoyl-2-1actylate) to the corn oil/water mixture. Emplex is a common low toxicity fat emulsifer that has been used extensively by the dairy and baking industries. It "conforms to the regulations of the federal Food and Drug Administrations pertaining to specifications for fats or fatty acids derived from edible sources" [14]. Smith and Dairiki [33] have shown that Emplex used in small quantities (< 1%) promotes better emulsification of milk fat than any of 35 other commonly used emulsifiers tested. Procedure

Everyday at 1000 hr each rat was weighed, and its food containers were weighed and refilled. Pilot work with the emulsifier revealed that the emulsion would remain stable for 3 to 4 days using the 0.5% concentration of emulsifier. However, to assure consistency, the emulsions were made up daily. The liquid was blended at low speed for two minutes in a Waring blender (Model 12BL18). Three concentrations of the fat source were initially used. At the start of the experiment all rats were given ad lib access to the three macronutrient sources for ten days. For day 11 through 20 the rats were given the same protein and carbohydrate sources, but were given a 50% corn oil emul-

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FIG. 1. Average daily body weights of lean and obese Zucker rats given access successively to 100%, 50% and 25% corn oil emulsions.

sion in place of the 100% corn oil fat source. This second concentration was then followed by a third, during which time a 25% corn oil emulsion was used for a final 10 days. RESULTS With the exception of the body weight loss observed after the first day of selection, all rats continued to gain weight throughout the remainder of the 30 day experimental period (see Fig. 1). Both genotype and dilution variables (but not their interaction) had significant impact upon the daily intake of the fat source. Obese rats typically ate more of the fat source than lean rats, F(1,66)=4.63, p<0.05. Further, both lean and obese rats consumed at least three times more of the 50% fat source than the 100% fat source. The average daily intake of the fat source is presented in the top panel of Fig. 2. Lean rats consumed an average of 2.17 ± 0.20 g/day of the 100% fat source. Obese rats drank an average of 5.20___0.88 g/day of the 100% fat source. Lean rats increased their consumption of fat to an average of 12.12±0.75 g/day and obese to an average of 16.30± 1.52 g/day subsequent to the 50% dilution. The 25% dilution promoted even further increases in fat source consumption. Lean rats drank an average of 25.7±2.36 g/day of the 25% fat source. Obese rats drank an average of 27.8±3.07 g/day of the 25% fat source. Of particular interest was the change in intake of each of the concentrations over the ten day period. Analysis of variance revealed a significant 3 way interaction (Genotype × Concentration x days, F(18,594)=2.99, p<0.01). Inspection of the average daily fat source intakes revealed considerable day to day variability at each concentration. However, a second comparison using the Duncan's New Multiple range test revealed that the consumption of fat during the first three days .of access to each of the dilutions was not significantly different from the average consumption during the last three days of that same period (see Table 2). Further, there was no overlap between a concentration distributions of daily averages. For example, when lean rats had access to the 100% source, their highest average dally intake of fat was 3.66±0.91 g. By comparison the lowest daily average intake of fat by these animals during access to the 50% concentra-

FAT SOURCE DILUTION

121 TABLE 2 CONSUMPTION OF FAT SOURCEAT EACH CONCENTRATION(GRAMS) Lean

Average 1st three days Average last three days Greatest daily average intake Smallest daily average intake

Obese

100

50

25

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10.0 +- 0.04 13.6 +_ 1.0 13.9 -+ 0.7 9.1 _+ 1.2

23.8 28.5 30.7 21.4

-+ 2.7 _+ 3.0 - 3,5 _+ 1.9

100 4.7 4.8 6.9 3.8

___0.9 _+ 0.9 _+ 1.2 _+ 0.8

50 12.7 -+ 1.9 19.2 +-- 1.4 22.3 _+ 1.3 10.4 + 2.2

25 31.5 25.6 35.3 23.0

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Values are means _+ S.E.M.

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g/day of the 25% emulsion in order to compensate for the dilutions. In contrast to the predicted outcome of a simple compensatory hypothesis, both obese and lean rats increased their consumption of fat subsequent to both dilutions. Protein and carbohydrate source consumption was not effected by varying the concentration of fat source. Further, no significant differences between genotypes in either protein or carbohydrate intake at any concentration condition were observed. Refer to the middle and bottom panels of Fig. 2.

9-

EXPERIMENT 2

8-

The results from the first experiment demonstrated that both lean and obese rats select diets that are high in fat. Further, both genotypes responded to the dilution of their fat sources by not only compensating for the dilution but in some cases actually increasing the fat content of their diets. These rats seem to be resistant to the effects of diluting their fat source. In an attempt to measure the strength of that resistance, a second experiment was conducted.

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Ten obese (fa/fa) and ten lean (Fa/-) female Zucker rats, approximately ten weeks old, were used. The obese rats weighed an average of 256--- 11.0 g ( m e a n _ S E M ) and their lean littermates weighed an average of 189.4---5.3 g at the start o f the experiment. All rats were maintained on ad lib rat chow and water up to that time.

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tion was 9.10---1.22 g - - a 2 1/2 fold increase. Similarly, the greatest daily average fat intake by lean rat during the 50% concentration condition was 13.85___0.73 g. The smallest average daily fat intake observed during the 25% concentration condition was 21.4___ 1.9 g. Similar non-overlapping distributions were observed in the obese data. The differences in consumption are summarized in Table 2. It is important to note that simple compensatory intake would have promoted much smaller increases in intake than those observed. For example, lean rats would have had to consume only 4.34 g/day of the 50% emulsion and only 8.68

Procedure For the first four days of the experiment the rats were given ad lib access to carbohydrate and protein sources as well as access to 100% corn oil as a fat source. On day 5, 0.5% Emplex (sodium stearoyl-2qactylate) was added to the 100% corn oil fat source referred to as 100% for three additional days: On days 8 through 10, a 75% corn oil emulsion was made available in place of the 100% source. Although the emulsifier was added to the 100% and 75% corn oil fat sources, the emulsifier had a tendency to separate from the fat at these concentrations. It was included for use in this series for its taste properties, and not for its value as an emulsifier. Because of the problems associated with the use of the 75% dilution, all measurements that were performed during that time are not included in this paper. Although obese and lean rats drank significant quantities of the 75% mixture (obese intake=5.66 g/day and lean intake--5.76

C A S T O N G U A Y ET AL.

122 TABLE 3 EXPERIMENTALDESIGN

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3.13

340

Days

(%) Fat Source Dilution

Fat Source Caloric Density (kcal/g)

1-4 5-8 9-11 12-14 15-17 18-20 21-23 24-26 27-29

100 100" 75 50 25 12.5 6.25 3.125 0

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g/day) it would be misleading to attempt to estimate the calories derived from the intake of that source. The fat source was further diluted every three days so that both obese and lean rats received access to 50%, 25%, 12.5%, 6.25%, 3.125% and 0% concentrations. A summary of the experimental design is presented in Table 3. Every day at 1230 hr each rat and its food and water sources were weighed. The food cups a n d water bottles were refilled as needed. The fat sources were weighed, the bottles were then emptied, washed thoroughly, and refilled with the appropriate dilution o f corn oil. Emulsions were made up no more than one hour prior to use, and were stable for the 24 hour experimental period. The data were analyzed with the use of a repeated measures analysis of variance. Where appropriate, Duncan's new multiple range test [34] was used to assess the statistical significance of difference between means. A p~<0.05 was considered statistically significant. RESULTS Just as in Experiment 1, body weights declined on the first day after the rats were placed on the self-selection regimen. After that initial loss, both lean and obese rats continuously gained weight throughout the remainder of the experiment (refer to Fig. 3). Addition of the emulsifier to the 100% concentration (100%+) did not alter the daily consumption of the fat source when compared to the 100% without emulsifier levels. Both lean and obese rats increased their consumption of the fat source by more than 10% when it was diluted to the 50% level (refer to the top panel of Fig. 4). Subsequent dilutions had different effects on the two genotypes, as revealed by a significant genotype × dilution interaction in an analysis of variance, F(8,136)=2.33, p=0.02. Obese rats drank significantly more of the 100% and 100%+ concentrations than did their lean littermates. Post hoc comparisons of interaction means revealed that although there was no significant difference between genotypes in the intake of fat during the 50% dilution, obese rats drank significantly more of the 25% fat source than did their lean littermates. When given access to

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FAT SOURCE DILUTION

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FIG. 5. Average Caloric Intake: Top Panel--Average daily Caloric intake (mean_+SEM) derived solely from fat consumption. Bottom panel--Average daily total caloric intake (mean+_SEM). Both measures are plotted as a function of the log concentration of the fat source.

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the 12.5% fat concentration, the difference between genotypes was once again statistically insignificant. As the dilution of the fat source increased beyond the 25% level, both obese and lean rats reduced their daily consumption of that source. At the 0% concentration, both groups of rats preferred the 0% fat (emulsifier + water) to their water source. The average protein consumption of both lean and obese rats is presented in the middle panel o f Fig. 4. Except at the 0% fat concentration, lean rats consistently ate only marginally more protein than did their obese littermates, F(1,77)=3.86, p=0.06. Post hoc comparisons of interaction means revealed two notable findings. The differences between genotypes attained statistical significance during access to all of the concentrations except the 3.12% and 0% fat concentrations. Secondly, a within genotype effect was found. Although lean rats ate no more protein during the 0% concentration than they did when they had access to the 100% concentration, obese rats significantly'increased their consumption of the protein source by 54% (from 3.66 g/day to 5.62 g/day). Unlike the almost constant intake of protein across a wide range of fat concentrations, the intake of the carbohydrate source wa~ greatly changed as the concentration of fat source decreased (Fig. 4, bottom panel). Both lean and obese rats increased their daily consumption of carbohydrate as the concentration of the fat source decreased. Except when given access to the 100% concentration, obese rats ate more of the carbohydrate source than did their lean controls during any one of the other eight concentrations used. The effect of increased consumption of the fat source subsequent to dilution is best described by considering the calories derived from fat source intake (refer to the top panel of Fig. 5). Obese rats consumed more calories as fat when given 100%, 100%+ and 25% concentrations when compared to the consumption of fat by lean rats at each concentration. All other differences between genotype were not statistically significant. Both lean and obese rats consumed significantly more fat when they had access to the 50% and 25% concentrations when compared to the fat intake levels observed with access to all other concentrations tested.

20"]~-'w.~..~a~ 10 1 ~ - - ' ' - ~ • , , s 100 5'0 2'5 12.5 625 3J3 % FAT CONCENTRATION FIG. 6. Average percentageof totai daily caloric intake derived from fat (top panel) protein (middle panel) and carbohydrate (bottom panel) plotted as a function of the log concentration of the fat source (mean_+SEM).

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The pattern of total dally caloric intake in response to dilution was very similar to the pattern of daily caloric intake from fat. More specifically, the net effect of the changes in the intake of each of the food sources was to change daily caloric intake (genotype x dilution interaction F(8,136) =2.70, p <0.01; refer to Fig. 5, lower panel). Access to the 50% concentration significantly increased average daily total caloric intake in both genotypes. Obese rats consuming the 25% emulsion further increased their total caloric intake. Lean rats did not increase consumption when provided with the 25% concentration. When t h e y were given access to the 50%, 25%, and 12.5% concentrations, both lean and obese rats significantly increased total caloric intake above their average levels during the 100% concentration. Lean rats ate significantly fewer calories when given access to the 0% fat source than they did when given access to the 10{)% fat source. Finally, the percentage of calories derived from any one of the three macronutrient sources is presented in Fig. 6. The top panel of the Fig. 6 illustrates the effect of varying the concentration of the fat source upon the percentage of calories derived from fat. Obese rats initially reduced percent of calories from dietary fat when the emulsifier was added to the fat source. Compared to the diet they composed during the 100% concentration condition, obese rats selected a diet with significantly fewer calories from fat during all but the 25% concentration condition. In contrast, lean rats increased the proportion of calories consumed as fat during the 50% and 25% concentration conditions. Obese rats selected a diet that

124

CASTONGUAY ET AL.

was higher in fat than the diet composed by lean rats during the 100% and 100%+ concentration conditions but significantly lower at the 50% conditions. At no other concentration was a genotype difference observed. The middle panel of Fig. 6 illustrated the effect of varying the fat source concentration upon relative protein intake. Lean rats given access to the 50% concentration significantly decreased the percentage o f calories taken as protein when compared to 101)% concentration levels. Each additional dilution resulted in gradual increases in relative protein consumption so that no differences between the 100% and 25%, 12.5%, 6.25% and 3.125% conditions were observed, Lean rats selected diets that were significantly higher in percent protein during the 0% concentration than they did during the 100%. By contrast, obese rats did not decrease percent protein consumption when given access to the 50% emulsion. The small drop in relative protein intake that was observed during the 25% condition was significantly different from the level of protein intake observed during the 50% condition, but not different from the level observed during the 100% condition. F a t concentrations of 12.5% or less promoted increased percent protein intake when compared to the level of protein selected during the 10(~ condition. Lean rats composed diets that were higher in percent protein than the diets composed by obese rats during all but the 50% and 12.5% conditions. Finally, the percentage of total calories derived from carbohydrate is presented in the bottom of Fig. 6. Lean rats composed a diet that was significantly higher in percent of calories as carbohydrate than the diet composed by obese rats during the 100% condition. With that exception, obese rats selected a diet that was significantly higher in percent carbohydrate than the diet selected by lean rats. The difference between groups was statistically significant in all but the 25% condition. From the graph, it is clear that as the caloric role of fat decreased, dietary carbohydrate increased. GENERAL DISCUSSION The results from this experiment demonstrate that dilution of a liquid fat source, and not simply the addition of an emulsifier, can promote significant increases in the intake of fat by lean rats as well as obese rats. These increases are not simply compensatory in nature, but represent increases in fat calories. The increased consumption of the fat source is maintained through a wide range of concentrations. The concentrations of fat ranging from 25% to 50% promote the most fat consumption. Obese rats select diets that are higher in fat content if the fat source was more concentrated than 50%. Lean and obese rats do not select different amounts of fat if the fat source is more dilute than the 25% concentration. Finally, total daily caloric intake and the daily intake of calories from fat were closely related. As the number of calories derived from the consumption of fat increased (as the fat source was diluted to 50% and then 25%) total caloric intake was also increased. As the consumption of fat calories decreased (with subsequent dilutions) total caloric intake returned to " n o r m a l " levels. We have previously reported that when obese Zucker rats were given access to a carbohydrate, protein and liquid fat source they compose a diet that is significantly higher in fat and lower in protein than the diet composed by lean Zucker rats [9]. That original finding, using male, adult rats has been extended to include female adult rats by the experiment re-

ported here. Additional statistical comparisons of the two groups have shown that there is no statistically significant difference in the diets composed by male and female obese or male and female lean rats when given the 100% corn oil source and the previously described protein and carbohydrate sources. The results from the present experiment demonstrate that the seemingly aberrant selection of fat that has been observed only in obese rats can also be promoted in lean rats if the fat source is modified so as to reduce its caloric density. Both lean and obese rats compose diets with equivalent fat levels if the fat source is diluted by 50% or more. The suggestion from this experiment is that the rat's appetite for fat is not a unique trait. Obese rats select a diet that is higher in fat than the diet composed by lean rats only when both groups are given a concentrated fat source (refer to the top panel o f Fig. 6). To account for these findings we have hypothesized that the intake of fat at higher concentrations is elevated in obese rats due to their tendency to consume larger meals than are consumed by lean rats [10,24]. Our suggestion is that obese rats may fail to respond to the same satiety cues that are used by lean rats to determine the size of individual bouts of corn oil drinking. If this hypothesis is correct, then the increase in fat consumption observed in obese rats given access to concentrated fat sources would not be defended subsequent to dilution. With only one qualification, the present experiment supports that hypothesis. No difference between genotypes was observed subsequent to diluting the fat source, except during the 25% concentration condition. Obese rats drank significantly more of the 25% concentration than did their lean littermates. It should be noted that although a difference in intake was observed, the proportion of calories derived from fat at the 25% concentration by lean and obese rats did not significantly differ compared to fat calories consumed during the 100% concentration. Both lean and obese rats increased their intake of fat during the 50% and 25% concentrations and calories from fat with subsequent fat dilutions. These similarities in response to the fat source dilution suggest that there is little or no difference in the mechanisms that guide the selection and consumption of fat. Although the differences observed during the 25% concentration were significant statistically, the remaining measures lead to the suggestion that increased motivation does not play a major role in determining the fat appetite of the obese rat. In addition to qualifying the selection data that has already been published, the present experiment provides more evidence that caloric regulation [23] is more fragile than once thought. Although there have been several demonstrations that rats will adjust their intake of foods so as to preserve a characteristic level of caloric intake [l, 6, 16, 17, 21, 22], the results from this study and others [4, 8, 16, 18, 19, 25, 27, 29, 30, 31, 32, 35] demonstrate that the behavioral tendency to maintain a characteristic level of caloric intake is not comparable to the precision observed in other regulatory systems (e.g., blood glucose levels, electrolyte composition of extracellular fluid, or cerebrospinal fluid composition). Caloric intakes may be controlled behaviorally, but the mechanisms controlling intake are subject to influences that make it imprecise. The results from Experiment l showed that even if these rats were given each concentration for ten days (instead of the three days of access used in this experiment) the elevation in total caloric intake was maintained. Taken together, these results show that although a characteristic level of caloric intake is maintained at any one concentration, total caloric intake can be manipulated by changing the concen-

FAT SOURCE D I L U T I O N

125

tration of a fat source. The term regulation implies that a feedback loop exerts some inhibition on those factors that control intake [21]. That the metabolic and/or taste factors that promote fat ingestion can influence that feedback loop suggests that its influence over intake when several food items are freely available is extremely weak. This qualification is not new. For example, small errors in daily caloric intake may ultimately contribute to significant increases in body weight. Kanarek and colleagues [19,20] have used just that argument in proposing their model of sucrose-induced obesity. The present results have demonstrated that the intake of calories can be increased by 50% or more by changing the density of a presumably good tasting fat source. Although the size of the increase was much more modest, we have observed increases in total caloric intake in Sprague Dawley rats that are given access to highly palatable sugar solutions in addition to their maintenance diet [8]. Further, Sclafani and Springer have demonstrated that highly palatable food sources can promote excessive weight gains in rats [32]. Although several investigators have shown that high fat diets can promote over-eating and weight gain [4, 25, 30], results from this study show that the rat will elect to overeat, despite access to dietary fractions that will permit "normal" caloric intake and adequate nutrient intake. Similarly, results from this experiment qualify the claim that there is no difference between genotypes in the regulation of protein intake [2]. In comparison to selection with access to the 100% fat source, obese rats increased both the absolute amount and the percent of total daily calories derived from both carbohydrate and protein when they were given access to the 0% concentration. On the other hand, both lean and obese rats maintained a characteristic level of protein diet intake as the concentration of the fat source was varied from 50% to 3.125%. These results are in agreement with the observation that rats will adjust their intakes of protein in response to environmental variables such as the availability of the protein source [7], and the availability of other nutrient sources [8], and are in marked contrast to the report that obese rats do not differ from lean rats in their regulation of protein intake [2]. Lean and obese rats selected diets that were significantly higher in percent protein during the 0% concentration condition than the diets they selected

in the presence of a fat source (refer to the middle of panel of Fig. 6). Finally, the results from this experiment also challenge the theory that obese Zucker rats eat more than their lean littermates in response to a stimulus arising from their impaired protein deposition [28]. Obese rats that have access to three macronutrient sources select diets that are lower rather than higher in protein than the diets selected by their lean littermates. The suggestion from the present work is that obese rats eat more than their lean littermates in response to one or several other metabolic anomalies that have been reported, such as elevated insulin levels [11,36] or elevated lipoprotein lipase levels [12,15]. In an attempt to further investigate the differences between genotypes we have initiated two additional experiments. In the first we have given weanling rats access to the original three macronutrient sources. With the exception of only two out of more than a dozen rats already tested, obese Zucker rats fail to survive on the selection regimen for more than a few days. By contrast, all lean weanling Zucker rats have thrived on the same dietary regimen. We are currently studying not only the developmental aspects of the differences in selection, but are also manipulating insulin levels in diabetic obese and lean rats, in an attempt to learn more about the factors that promote differences in selection patterns. As of this writing, we have learned that reducing the insulin levels in obese rats to levels normally found in lean rats reduces the intake of fat that characterizes the obese rats selection habits. Much more work needs to be completed before an accurate assessment of the role of insulin on the fat appetite of obese rats can be made. ACKNOWLEDGEMENTS We would like to thank Dr. Lloyd Smith of the Food Science and Technology Department, UC Davis for his help in selecting the emulsifier used in this study. We also thank the Patco Products Division of the C. J. Paterson Co., Kansas City, MO for providing the Emplex used in this research. Finally, we would like to thank Mr. Paul Schneeman for the assistance in the statistical treatment of the data reported in this paper. Portion of this report was presented at the 53rd Annual Meetings of the Eastern Psychological Association, Baltimore, MD 1982.

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