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Dietary self-selection in cycling and neonatally ovariectomized rats
Appetite 198 1, 2, 87- 101
Dietary Self-selection in Cycling and Neonatally Ovariectomized Rats PAULA J. GEISELMAN Department of Psychology and Brain Research Institute, University of California, Los Angeles
J. R. MARTIN Department of Behavioral Sciences, Swiss Federal Institute of Technology, Zurich
D. A. VANDERWEELE Department of Psychology, Occidental Col/ege, Los Angeles
D. NOVIN Department of Psychology and Brain Research Institute, University of California, Los Angeles
Dietary self-selection patterns were assessed in no-rmally cycling (four-day estrous cycle) rats with separate provisions of fa t (olive oil), carbohydrate (sucrose), and protein (casein). There was no fluctuation in daily caloric intake across the estrous cycle, but animals did demonstrate changes in consumption of specific macronutrients during the 24-h period in which estrus occurred . At estrus, animals exhibited increased carbohydra te intake and decreased fat intake in comparison with the remaining three days of the cycle. There was also a somewhat decreased (non-significant) protein consumption at estrus. Rats that had been ovariectomized on day 5 postnatally and then tested as adults in our paradigm ingested approximately the same quantity of daily kilocalories as observed in intact animals; but ovariectomy produced changes in consumption of specific macronutrients. In comparison with the intact animals, ova riectomized animals displayed increased fat intake and decreased carbohydrate intake. Thus, the fa t and carbohydrate self-selection patterns of our ovariectomized rats reflected the elimination of estrus behavior.
Quantity of food ingested by the female rat has been demonstrated to be of a cyclic nature and to correlate well with stages of the estrous cycle. _The peak in estradiol observed at proestrus (Yoshinaga, Hawkins & Stocker, 1969) is followed by a reduction in food intake (Brobeck, Wheatland & Strominger, 1947; Ota & Yokoyama, 1967; Tarttelin & Gorski, 1971 ; ter Haar, 1972; Wade, 1972). Con versely, the level of estradiol - - - - - - - - --
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This research was supported by grants MH1 5345 (PJG), NS 7687 (DN), and research development fu nds from Occident al College. PJG was supported by Alcohol, Drug Abuse and Ment al Health Administrati on National Research Service Award MH1 5345 from the National Institute of Mental Health, and JRM was supported by National Research Service ·Awa rd MH5101. We are grateful to Dwight M . Nance for providing helpful comments and suggestions both during the conduct of the experiments and the preparation of the manuscript. We are also grateful to Neil Rowland for his careful reading of the manuscript. Requests fo r re prints should be sent to Paula 1. Geiselman. Department of Psychology, Franz Hall, Uni versity of California, Los Angeles, Californ ia 90024, U.S .A. 0195- 6663/81/020087 + 15 S02'00/0
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198 1 Academic Press Inc. (London) Limited
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P. J. GEISELMAN ET AL.
is decreased at diestrus at which time the animal shows an increase in food intake (Brobeck et aI., 1947; Drewett, 1973; Tarttelin & Gorski, 1971; ter Harr, 1972). However, changes in the ingestive patterns ofthe various macronutrients in the cycling female rat have not been studied so extensively. In this regard, one might ask the question: Are there specific nutritional requirements that change with the physiological events characterizing the estrous cycle? One might anticipate that patterns of dietary self-selection would be cyclic and correlate with the stages of the estrous cycle. All calories are perhaps not equally important at any given time in the control of ingestive behavior, and more pertinent factors in the control of food intake may be the pre-existing metabolic condition of the animal (as defined by the estrous cycle) and the metabolic sequelae of ingested nutrients. Within this context, it is important to note that ovarian hormones have a multitude of effects on various metabolic processes [see Wade & Gray (1979) and Nance (in press) for review]. We are aware of no studies relating the estrous cycle to self-selection of each of the three primary macronutrients. Complex diets have been used to test self-selection patterns in normally cycling rats (Leshner, Siegel & Collier, 1972; Wurtman & Baum, 1980), but results of these studies cannot be treated as definitive since one cannot be sure of the relevance of each of the dietary components in the control of feeding behavior. Although Richter, Holt and Barelare (1937,1938) simultaneously presented separate rations offat, carbohydrate, and protein to normally cycling female rats, they did not relate their results to the estrous cycle. Likewise, Kanarek & Beck (1980) recently tested females using a similar paradigm, but it was not an intent of their study to examine self-selection patterns with regard to cyclicity. The purpose of Experiment 1 was to establish the patterns of self-selection of the three primary macronutrients across the estrous cycle in rats provided with discrete rations of fat, carbohydrate, and protein. Based upon preliminary experimentation, it was hypothesized that animals would show an increase in carbohydrate intake with an accompanying decrease in fat and protein intake at estrus. In addition, we attempted to determine whether or not the underlying mechanisms of self-selection responses involved the ovaries, the hormonal secretions of which define the stages of the estrous cycle. Certainly ovariectomy produces a dramatic increase in the quantity of food consumed (Drewett, 1973; Holt, Keeton, & Vennesland, 1936; Tarttelin & Gorski, 1973; Wade, 1972) and this effect has been attributed to the elimination of ovarian secretions of estradiol (see Wade, 1976; Nance, in press, for review). However, once again, studies of macro nutrient selection have not been prolific. Differences between intact and ovariectomized animals in the self-selection of complex diets have been studied (Leshner & Collier, 1973; Wurtman & Baum, 1980); but as previously discussed, such paradigms do not allow one to assess the role of the three macronutrients in ingestive behavior. Recently, however, Kanarek & Beck (1980) have reported that, when presented with separate provisions of fat, carbohydrate and protein, animals ovariectomized in adulthood did not display an alteration in dietary self-selection in comparison with their preoperative intakes. In testing for differences in measures of food intake in gonadally intact and ovariectomized adult animals, the age of the animal at the time when ovariectomy was performed has appeared to be inconsequential. This may be ascribed to data showing that ovariectomy performed prepubertally does not produce alterations in either food intake or body weight prior to the time at which puberty would normally occur (Grunt, 1964; Wade & Zucker, 1970a). Consistent with this finding, it has also been demonstrated that food intake is unresponsive to exogenous estradiol until after the
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89
time of puberty (Wade & Zucker, 1970 a). Thus, the conclusion that ovarian hormones do not influence food intake or body weight prepubertally is apparent. However, by subjecting more subtle feeding parameters to spectral analysis, Sieck, Nance, Ramaley, Taylor and Gorski (1977) have provided persuasive evidence that ovarian secretions influence prepubertal feeding behavior. The purpose of Experiment 2 was to use our paradigm to study the disruption of normal self-selection patterns in adult animals that had been ovariectomized neonatally. The surgery was performed on day 5 postnatally in an effort to minimize exposure to ovarian hormones and thus eliminate long-term experience with ovarian influences on feeding behavior. It was predicted that dietary self-selection patterns in our neonatally ovariectomized animals would largely reflect the elimination of estrus behavior. GENERAL METHOD
Animals and Maintenance Conditions
Animals serving as subjects were the female progeny of albino Sprague-Dawley strain rats (Holtzman). Nulliparous females were bred in our colony and then housed in group cages with ad lib. access to food (Purina Laboratory Chow) and water. Animals were always maintained under a 12: 12 light-dark regimen (light onset at 0800 hrs) in temperature-controlled room (22-25°C). Several days prior to delivery, mothers were placed in individual cages with metal floors. W oodchip bedding was supplied, and food and water remained available ad lib. Litter size was six to eight pups, and the initial 24-h period postpartum was regarded as day 1. With the exception of time required for surgery and recovery on day 5 (as described below), pups remained with their mothers until 21 days of age at which time they were weaned. Postweanling animals were housed individually in standard laboratory cages with food and water provided ad lib.
a
Surgery
At five days of age, pups were randomly chosen to undergo either ovariectomy or sham surgery. Pups were anesthetized by means of hypothermia in preparation for surgery. Bilateral ovariectomies were performed using two incisions, each of which was placed on the flank in a dorsolateral position posterior to the rib cage. Pups receiving sham ovariectomies were subjected to similar incision procedures. Following completion of surgery, wounds were sutured with sterile surgical gut and sealed with colloidin. Immediately postoperatively, pups were placed in a heated chamber (3033°C). After recovery and restoration to euthermic conditions, the pups were returned to their appropriate mothers. Apparatus and Procedure
For assessment of self-selection patterns, animals were placed in wire-mesh cages measuring 23 cm by 30·5 cm. Cages were suspended a minimum of 5 cm above woodchip shavings. Each of the three primary macronutrients (fat, carbohydrate, and protein) was presented as a separate food source in liquid form in a graduated 100-ml Richter tube attached to the front of the cage. The carbohydrate source was presented as a 30% (weight/volume) sucrose solution (Sigma Chemioal Company); the protein source was presented as a 15% (weight/volume) solution of casein hydrolysate (ICN Pharmaceuticals); and the fat source was presented as unadulterated olive oil (ICN
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Pharmaceuticals). In addition, a water bottle was placed on the front of each cage. Position of each of the three macronutrients and the water bottle was counterbalanced, although this control may not have been necessary as suggested by Young (1944). To provide a source of non-nutritive bulk, Alphacel (lCN Pharmaceuticals) was presented in a metal spill-proof cup secured to the cage floor. The Alphacel was prepared as a slurry by adding 2 g of Alphacel to 3 ml of a 1% solution of sodium chloride and potassium chloride. In addition, 1·5 ml ofliquid vitamins (Ross Laboratories, Vi-daylin M) was added to the mixture on each fourth day. The three macronutrients, nonnutritive bulk, and water were available ad lib. At 12-h intervals corresponding to light onset and light offset, the following variables were measured: body weight; water intake; and intake of fat, carbohydrate, and protein solutions. Animals were adapted to the test cage until reaching stabilization, which was defined as maintenance of body weight for an eight-day period during which time daily caloric intake varied less than 20% (Fox, Kipp & VanderWeele, 1976). Following stabilization, animals were tested for an additional4-day period, thus resulting in a 12day observation of self-selection behavior for each animal. Data collected during the final 12 days were grouped into three periods of 4 days each, and reliability estimates confirmed that the patterns of self-selection did not differ among these three periods. EXPERIMENT
1
DIET AR Y SILF-SELECTION PATTERNS MEASURED ACROSS THE ESTROUS CYCLE
Method Procedure
Seven female Sprague-Dawley strain (Holtzman) rats weighing 205 to 290 g with ages ranging from 60 to 90 days were tested in our self-selection paradigm. These animals had sustained sham ovariectomies on day 5. Vaginal cell samples confirmed that all animals had demonstrated a regular 4-day estrous cycle for at least 8 days (i.e., during the dietary self-selection stabilization period). Animals were then tested for an additional 4 days during which time we continued to take daily vaginal cell samples to verify the stage of the estrous cycle. These procedures resulted in a minimum observation of three estrous cycles for each animal. Young, Nance and Gorski (1978) have reported that vaginal-cell sampling procedures do not affect caloric intake, water intake or body weight. A standard nomenclature has not been establi'shed for the stages of the estrous cycle (Wade, 1976). In the present experiment, the 24-h period during which estrus occurred was recorded as day 1 of the estrous cycle. The remaining days of the cycle were simply recorded as days 2, 3, and 4. The stage of the cycle at which testing began was counterbalanced across animals. Design and Analysis
. Caloric intake (kcal/l00 g body weight), water intake (ml/IOO g body weight), and body weight (g) were grouped as follows and analyzed as a two-factor within-subjects design: estrous cycle (days 1,2,3, and 4) and nychthemeral period (12-h diurnal and 12-h nocturnal portions). Following the precedent of Kanarek and Beck (1980), percentage of 24-h kcal selected as fat, carbohydrate, and protein provided an assessment of dietary self-selection patterns. These data were grouped as follows and analyzed as a three-factor within-subjects design: estrous cycle (days 1, 2, 3, and 4), nychthemeral period (diurnal and nocturnal portions), and nutrient (fat, carbohydrate, and protein).
DIET SELECTION IN CYCLING AND SPA YEO RATS
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Results Caloric Int(/ke, Water Intake, and Body Weight
Caloric intake expressed as kcal/lOO g body weight is depicted in Table I as a function ofthe estrous cycle. There was neither a significant main effect for cycle nor a significant interaction of this factor with the nychthemeral period. Because the interaction was non-significant and, further, since cycle was the factor of interest, the data in Table 1 were collapsed across the nychthemeral period and are presented as 24h mean values for each of the four days of the estrous cycle. These data are consistent with the daily caloric intake reported by Richter et al. (1938) for normally cycling females given the present nutrients. Neither water intake (ml/100 g body weight) nor body weight (g) varied across either the diurnal or nocturnal portions of the four days ofthe estrous cycle. For each of these two variables, there was neither a significant main effect for cycle nor a significant cycle by nychthemeral period interaction. It is noteworthy that water intake was negligible at all times. This result was anticipated since the animals were obtaining water in food solutions. (Mean 24-h water intake across the four-day estrous cycle: 2·2, 1·7,2·5, and 2·6 ml/100 g body weight, respectively. Mean body weight across the cycle: 245, 247, 246, and 247 g, respectively.) The expected nychthemeral period main effects were obtained with the present data (caloric intake, water intake, and body weight) but will not be reported since diurnal and nocturnal differences in ingestive behavior are already well established (Morimoto, Arisue & Yamamura, 1977; Siegel, 1961; Zucker, 1971). Further, the nychthemeral period factor was of interest in the present study only in interaction with the cycle factor. Percent ()f Caloric Intake Selected as Fat, Carbohydrate, or Protein
The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period is shown in Table 2. A significant nutrient by nychthemeral period interaction was obtained (F 2.12 = 5·3, p < 0·023). Therefore, simple main effects were computed to determine the variation ascribable to the nutrient factor occurring within each of the two levels of the nychthemeral period. A non-significant effect was obtained for the diurnal period. This result shows that, collapsing across all four days of the estrous cycle, there was no difference in the percentage of daily caloric intake chosen as fat, carbohydrate, or protein during the diurnal period. The effect for the nocturnal period, however, was significant (F 2.24 = 8·08, p < 0·01), indicating tha t animals ingested less protein than fat during the nocturnal period collapsed across all four days of the estrous cycle (Tukey's TARLE 1 Twenty-four hour measures of caloric intake across the estrous cycle (mean ± s.e.)
Estrous cycle Caloric intake
Day I"
Day 2
Day 3
Day 4
kcal/IOO g body weight
21·1 ±S·I
20·0±2·t
2t·2±3·6
IS·7±3·1
"Day 1 of the estrous cycle represents the 24-hour period during which estrus occurred.
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P. J . GEISELMAN ET AL.
TABLE 2 Percentage of daily caloric intake selected as fat , carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in intact rats (mean± s.e .)
Nutrient
Nychthemeral period Diurnal Nocturnal
Fat
Carbohydrate
Protein
5'2±1'2 44·0±9·7
8·0± 1·7 26'6±6'6
5·8± 1·1 1O·3±O·9
post-test, p
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FIGURE 1. Mean percentage of24-h caloric intake selected as fat, carbohydrate, and protein during each of the four days of the estrous cycle.
DIET SELECTION IN CYCLING AND SPAYED RATS EXPERIMENT
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2
DIETARY SELF-SELECTION PATTERNS IN NEONATALLY OVARIECTOMIZED RATS
Method Procedure Ten Sprague-Dawley strain (Holtzman) rats that were ovariectomized on day 5 were tested in our dietary self-selection paradigm. The animals weighed 223 to 300 g and ranged from 60 to 80 days of age when tested. Animals in the ovariectomized group were subjected to the same stabilization and additional testing procedures as previously described. These animals were tested in our self-selection cages at the same time that intact animals were being tested. Design and Analysis Data collected from the ovariectomized animals were compared with data collected from the sham-operated, intact females. Days of the estrous cycle, which was the factor of interest in Experiment 1, was no longer of concern in the present analysis. The factors of interest in Experiment 2 were: group (ovariectomized or intact; between ,factor); nychthemeral period (diurnal and nocturnal portions; within factor); and nutrient (fat, carbohydrate, and protein; within factor). Caloric intake (kcal/lOO g body weight), water intake (ml/lOO g body weight ), and body weight (g) were analyzed as a two-factor (group and nychthemeral period) splitplot design. Percentage of24-h kcal selected as fat, carbohydrate, and protein provided an assessment of dietary self-selection patterns. These data were analyzed as a threefactor (group, nychthemeral period, and nutrient) split-plot design. Results Caloric Intake, Water Intake, and Body Weight Caloric intake expressed as kcal/lOO g body weight is depicted in Table 3 for intact and ovariectomized rats. There was neither a significant main effect for group nor a significant interaction of this factor with the nychth-emeral period. Because the interaction was non-significant, the data in Table 3 were collapsed across the nychthemeral period and are presented as 24-h mean values for each of the two groups. Neither water intake (ml/lOO g body weight) nor body weight (g) differed between the two groups during either the diurnal or nocturnal portions of the nychthemeral period. For each of these two variables, there was neither a significant main effect for group nor a significant group by nychthemeral period interaction. It is again noteworthy that water intake was negligible, as expected, for both groups. (Mean 24-h water intake for ovariectomized and intact animals: 3·3 and 2·3 mljlOO g body weight, respectively. Mean body weight for ovariectomized and intact animals: 267 and 246 g respectively.) TABLE 3 Twentylour hour measures of caloric intake in intact and ovariectomized rats (mean ±s.e.)
Group Caloric intake
Ovariectomized
Intact
kcalj100g body weight
no ± 1·0
20·3 ± 2·5
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P. J. GEISELMAN ET AL.
The expected nychthemerill'period main effects were obtained with the present data (caloric intake, water intake, and body weight) but will not be reported. Percent of Caloric Intake Selected as Fat, Carbohydrate, or Protein
The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period is shown in Table 4. (For comparison, these data are depicted in Table 5 for ovariectomized rats only,) A significant nutrient by nychthemeral period interaction was obtained (F 2,30 = 26'46, p < 0'00001), and simple main effects were computed to determine the variation due to the nutrient factor occurring within the diurnal and nocturnal periods. A non-significant effect was obtained for the diurnal period. Thus, collapsing across groups, there was no difference in the percentage of daily caloric intake chosen as fat, carbohydrate, or protein during the diurnal perioO. The effect for the nocturnal period, however, was significant (F 2.60 = 53'73, P <0·00001), indicating that the intact and the ovariectomized rats ingested less protein than fat during the nocturnal period (Tukey's post-test, p < 0,01). The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein by the intact and the ovariectomized rats is depicted in Figure 2. A significant nutrient by group interaction was obtained (F 2.30 = 4· 34, p < 0'022), and simple main effects were conducted to determine the variation due to the group factor occurring at each level of the nutrient factor. A significant effect was obtained for fat (F 1,45 = 7'58, p < 0'01); the ovariectomized rats ingested a significantly greater percentage of their daily caloric intake as fat than did the intact rats. The effect for carbohydrate was significant (F 1,45 = 5'2, P < 0'04), indicating that the ovariectomized rats ingested less of their daily calories as carbohydrate than did the intact rats. Finally, percentage of daily caloric intake ingested as protein was observed to be lower in the ovariectomized than in the intact animals, but this effect did not reach significance. TABLE
4
Percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in intact and ovariectomized rats (mean±s.e.) Nychthemeral period Diurnal Nocturnal
Nutrient Fat
Carbohydrate
Protein
6·0±I·5 57'8±6'I
4·9± 1·1 17·8 ± 4'5
4'8±O'5 8·9±O·9
TABLE 5 Percentage of daily calotic intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in ovariectomized rats only (mean±s.e.)
Nutrient Nychthemeral period Diurnal Nocturnal
Fat
Carbohydrate
Protein
6·6±2-4 67'5±7'O
2·7±1·0 11'6±5'5
4·1 ±O'3 7'9± 1-4
DIET SELECTION IN CYCLING AND SPAYED RATS
95
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FIGURE 2. Mean percentage of24-h caloric intake selected as fat, carbohydrate, and protein in intact (0) and ovariectomized (.) animals.
Verification of Ovariectomy At the conclusion oftesting, animals that had been ovariectomized neonatally were examined for confirmation of the surgical procedure. At sacrifice, it was verified that none of the animals in this group had ovaries and that vaginal canalization had not occurred. Also, all animals in the ovariectomized group had infantile uteri. DISC USSION
Experiment 1 provided an answer to the question: Are there specific nutritional requirements that change across the estrous cycle? Patterns exhibited during days 2, 3, and 4 were indistinguishable from one another; but at estrus animals exhibited increased carbohydrate intake and de.creased fat intakl!: Interestingly, the fat and carbohydrate self-selection patterns of our ovariectomized rats reflected the elimination of estrus behavior. These changes in intake at estrus and following ovariectomy are consistent with Richter's (1954) observation that an inverse relationship exists between fat and carbohydrate intake; i.e., when intake of one of these two macronutrients changes in one direction, intake of the other changes in the opposite direction. The fat-intake results in both our intact and our ovariectomized animals could be interpreted as being consistent with Nance's (in press) demonstration that a fat load suppressed subsequent food intake in intact but not in ovariectomized rats. Also, Young, N ance and Gorski (1978) showed that estradiol produced a greater depression of food intake in female rats maintained on a high-fat diet than in those fed chow or a high-dextrose diet. These results are all predictable from Wade & Gray's (1979) hypothesis that "estradiol may decrease food intake in female rats by increasing the
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P. 1. GEISELMAN ET AL.
availability of triglyceride as a metabolic fuel." In this context, our results further suggest that, rather than merely decreasing caloric intake in general, the high levels of estradiol occurring at proestrus may subsequently suppress the specific appetite fo r fat. Moreover, when ovariectomy was performed and the suppressive influence was eliminated, the animals demonstrated an enhanced fat intake. It is well documented that estradiol increases circulating levels of triglycerides (Ferreri & Naito, 1978 ; Hazzard, Spiger, Bagdade & Bierman, 1969; Kim & Kalkhoff, 1975; Schillinger & Gerhards, 1973; Watkins, Fizette & Heimberg, 1972). Thus, the present results might suggest that dietary intake of fat varies inversely with blood levels of triglycerides across the estrous cycle. In contrast to our fat-intake results, carbohydrate intake was increased at estrus; consistent with this, the ovariectomized animals showed a decrease in carbohydrate consumption. These data are compatible with ovarian influences on insulin and carbohydrate metabolism. Our intact animals may have had an increased carbohydrate appetite in response to estradiol-induced hypoglycemia (Goodman & Hazelwood, 1974). At estrus, animals are hyperinsulinemic and show enhanced glucose tolerance (Bailey & Matty, 1972). Following ovariectomy, however, a reduction in plasma insulin is observed and glucose tolerance is impaired; but these responses can be restored with ovarian hormones (Bailey & Matty, 1972). Moreover, it has been shown that estradiol enhances hepatic glycogen levels (McKerns, Coulomb, Kaleita & de Renzo, 1958; Matute & Kalkhoff, 1973) as well as facilitating glucose uptake by the uterus (Roskoski & Steiner, 1967; Smith & Gorski, 1968), muscle (McKerns et al., 1958), and adipose tissue (Gilmour & McKerns, 1966). The estradiol-induced increase in insulin (Bailey & Matty, 1972; Basabe, Chieri & Foglia, 1969) may be ascribable to both direct and indirect actions of the hormone on the pancreas (Costrini & Kalkhoff, 1971; Goodman & Hazelwood, 1974). The increase in carbohydrate intake observed in our animals at estrus is consistent with Richter's (1954) demonstration that exogenous insulin produces an increase in carbohydrate intake. Moreover, our ovariectomized animals exhibited dietary self-selection patterns (decreased carbohydrate and increased fat intakes) quite similar to those seen in pancreatectomized animals (Richter & Schmidt, 1941; Richter, Schmidt & Malone, 1945). The increase in carbohydrate intake found at estrus in animals serving as subjects in Experiment 1 was also observed in preliminary investigations. Using the present selfselection paradigm, animals having access to activity wheels showed an increase in both activity level and carbohydrate intake a t estrus. Considered together, these results indicate that an increase in carbohydrate intake occurs at estrus regardless of whether the animal has a high level of activity . However, animals with restricted activity levels display greater energy output as heat loss at estrus than do active animals (McLean & Coleman, 1971), and ovariectomized animals show substantial heat loss in response to estradiol treatment regardless of whether or not activity levels are restricted (Laudenslager, Wilkinson, Carlisle & Hammel, 1980). Therefore, expenditure of energy (as heat loss and/or muscular activity) cannot be eliminated as a contributing cause of increased ingestion of a nutrient providing readily available energy. Our intact animals ingested approximately 16% of their daily caloric intake as protein across the four days of the estrous cycle.- Both the absolute and relative amounts of protein ingested, which were consistent with that reported by Richter et al. (1938) and Fox et al. (1976), were certainly sufficient to maintain hea lth. This was evidenced by the general appearance of the animals, their maintenance of body weight , and the fact that all animals serving as subjects exhibited normal estrous cyclicity,
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which has been shown to be disrupted when nutrition is inadequate (Leathem, 1961). The present nutritional sources were chosen on the basis of previous reports indicating that animals thrive when placed in self-selection paradigms using these three specific nutrients (F ox et aI., 1976; Richter et al., 1938). When maintained for prolonged periods with separate sources of sucrose, casein, and olive oil, Richter et al. (1938) reported that self-selecting females displayed body-weight curves essentially identical to those of the control animals that were ingesting standard laboratory diets (Richter et al., 1938). More importantly, their self-selecting rats were able to maintain these comparable body weights even though they were ingesting 20% fewer kilocalories than the control group. Thus, when given the above choices, ingestive patterns of selfselecting animals were more efficient than those of the control animals given standard laboratory diets. Consistent with this conclusion, Richter et al. (1938) also reported that their self-selecting animals showed more regularity in estrous cyclicity than did the control animals, another indication of greater nutritional efficiency in the self-selecting animals (Leathem, 1961). Forty years later, using the same three macronutrients as those used by Richter et al. (1938), Fox et al. (1976) also found that female rats were able to maintain their body weights even though they were ingesting fewer calories than typically observed with laboratory chow. Kanarek & Beck's (1980) results indicating that intact females chose one-third of their daily calories from each of the three macronutrients and that these patterns did not change following ovariectomy are not consistent with the present results. There are procedual differences that may account for this. Their animals were not ovariectomized until adulthood, and their dietary provisions differed from ours, both of which are pertinent factors to consider when comparing results across studies (Lat, 1967; Sieck et al., 1977). When presented with specific dietary sources identical to those used by Richter et al. (1938), our intact animals ingested approximately 49% of their daily caloric intake as fat, 35% as carbohydrate, and 16% as protein across the four-day estrous cycle. Thus, our results are consistent with those obtained by Richter et al. (1938) with normally cycling animals. The gonadally intact animals maintained in our self-selection paradigm did not exhibit hypophagia at estrus. It can be assumed that this was not a floor effect since all animals exhibited stable weight maintenance. The estradiol-induced anorexia occurring at estrus in animals maintained with laboratory chow is somewhat difficult to reconcile with the present results due to the very high proportion of carbohydrate and the very low proportion of fat in the laboratory chow. It is interesting, however, that Richter et al. (1938) have demonstrated that normally cycling female rats are actually hyperphagic when maintained on standard diets. In consideration of the low-fat content oflaboratory chow, our results suggest that the hyperphagia observed when an animal ingests laboratory chow may be attributable to an effort to increase fat intake and that the anorexia shown at estrus with laboratory chow may be an attempt to decrease fat intake. This interpretation is compatible with Young et al.'s (1978) demonstration that a high-fat diet (two parts chow and one part fat) enhances the estradiol-induced anorexic effect. However, the influence of ovarian hormones on feeding obviously are not entirely attributable to one macronutrient only. Wade & Gray (1979) have developed a convincing argument for the importance of the role offat metabolism in the mediation of estradol's influence on feeding, but this argument does not provide an apparent explanation for the observation that both estradiol and food deprivation have essentially the same effect on adipose tissue yet have opposing effects on ingestive behavior (Nance, in press). While a number of discrepancies have been
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pointed out in the literature of ovarian influences on insulin and carbohydrate metabolism (Wade & Gray, 1979), one cannot dismiss the importance of carbohydrates in the control of ingestive behavior (Nance, in press). With the interrelationship between fat and carbohydrate metabolism, it would be difficult to consider one to the exclusion of the other. Moreover, an explanation of ovarian influences on ingestive behavior is further complicated by data suggesting that, in addition to producing an elevation in insulin levels, estradiol also may result in increases in somatotrophic hormone (Frantz & Rabkin, 1965; Goodman & Hazelwood, 1974; Maw & Wynn, 1972) and corticosteroids (Critchlow, Liebelt, Bar-Sela, Mountcastle & Lipscomb, 1963; Goodman & Hazelwood, 1974), both of which influence the metabolism of macro nutrients in a manner that is essentially antagonistic to the action of insulin. Actually these additional hormonal increases may provide at least a partial explanation for the seemingly paradoxical increase in plasma insulin levels and concurrent decrease in adipose tissue lipoprotein lipase activity in response to estradiol (Hamosh & Hamosh, 1975; Patten, 1970; Wade & Gray, 1979). While the present results might suggest that dietary intakes of fat and carbohydrate vary inversely with circulating levels of triglyceride and glucose, respectively, it is obvious that the hormonal influences on these selection patterns are quite complex. The alterations in self-selection patterns in our intact animals are probably not due primarily to changes in taste responsiveness. Taste preferences do not vary across the estrous cycle (Wade, 1976), while our results and those of previous studies show feeding fluctuations that are related to cyclicity (Brobeck et al., 1947; Drewett, 1973; Ota & Yokoyama, 1967; Tarttelin & Gorski, 1971; ter Haar, 1972). Also, ovariectomized animals show a decrease in food intake following administration of estradiol (see Wade, 1976; and Nance, in press), yet treatment of ovariectomized animals with varying doses of estradiol alone is ineffective in restoring saccharin preference (Zucker, 1969). Wade (1976) has pointed out that taste responsivity is negatively correlated with selection of dietary protein. Since ovariectomized animals show decreased taste responsivity (Wade & Zucker, 1970 b; Zucker, 1969), one would predict that our ovariectomized animals would have chosen more of the protein source than the intact animals if the choice of nutrients had been made on the basis of a taste factor alone. In actuality, however, there was a tendency for our ovariectomized rats to ingest less protein than the intact rats. Finally, since fat has greater caloric density than either carbohydrate or protein, it might appear that rather than specifically increasing carbohydrate intake and decreasing fat intake at estrus, animals may have made their selections in an effort to obtain more dilute sources of calories at that stage of the cycle. However, the reader is reminded that, if caloric density had been the primary basis of selection, one would not have expected the animals to discriminate between the dilute sources of carbohydrate and protein at estrus, as they clearly did: At estrus, our animals reliably increased their intake of the 30% carbohydrate solution yet tended to decrease their intake of the 15% protein solution. While we cannot conclusively eliminate caloric density as a possible contributing cause of changes in ingestive behavior, we feel that our argument based on metabolic and hormonal factors provides a more parsimonious explanation of changes in intake across the estrous cycle. REFERENCES
Bajley, C. J., & Matty, A. J. Glucose tolerance and plasma insulin of the rat in relation to the oestrous cycle and sex hormones. Hormone and Metabolic Research, 1972,4,266-270.
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Basabe, J. c., Chiere, R. A., & Foglia, V. C. Action of sex hormones on the insulinemia of castrated female rats. Proceedings of the Society for Experimental BioLogy and Medicine, 1969,130, 1159-1161.
Brobeck, J. R., Wheatland, M., & Strominger, J. L. Variations in regulation of energy exchange associated with estrus, diestrus, and pseudopregnancy in rats. Endocrinology, 1947, 40, 65- 72. Costrini, N. V., & Kalkhoff, R. K. Relative effects of pregnancy, estradiol, and progesterone on pl asma insulin and pancreatic islet insulin secretion. Journal q{Clinicallllvestigation, 1971, 50, 992- 999.
Critchlow, V., Liebelt, R. A., Bar-Sela, M., Mountcastle, W., & Lipscomb, H. S. Sex difference in resting pituitary-adrenal function in the rat. American Journal of Physiology, 1963, 205, 807-815. Drewett, R. F. Oestrous and dioestrous components of the ovarian inhibition of hunger in the rat. Animal Behavior, 1973, 21, 772- 780. Ferreri, L. F., & Naito, H . K . Effects of estrogens on rat serum cholesterol concentrations: Consideration of dose, type of estrogen and treatment duration. Endocrinology, 1978,102, 1621-1627. Fox, K. A., Kipp, S. c., & VanderWeele, D. A. Dietary self-selection following subdiaphragmatic vagotomy in the white rat. American Journal of Physiology, 1976,231,1790-1793. Frantz, A. G., & Rabkin, M. T. Effects of estrogen and sex differences on secretion of human growth hormones. Journal of Clinical Endocrinology and Metabolism, 1965,25,1470-1480. Gilmour, K. E., & McKerns, K. W. Insulin and estrogen regulation of lipid synthesis in adipose tissue. Biochimica et Biophysica Acta, 1966, 116, 220-228. Goodman, M. N., & Hazelwood, R. L. Short-term effects of oestradiol benzoate in normal, hypophysectomized and alloxan-diabetic male rats. Journal of Endocrinology, 1974, 62, 439-449. Grunt, J. A. Effects of neonatal gonadectomy on skeletal growth and development in the rat. Endocrinology, 1964, 75, 805-808. H azzard, W. R., Spiger, M. J ., Bagdade, J. P., & Bierman, E. L. Studies of the mechanism of increased plasma triglyceride levels induced by oral contraceptives. New England Journal of Medicine, 1969,280,471-474.
Holt, H., Keeton, R. W., & Vennesland, B. The effect of gonadectomy on body structure and body weight in albino rats. American Journal of Physiology, 1963,114,515-525. Kanarek, R. B., & Beck, J. M. Role of gonadal hormones in diet selection and food utilization in female rats . Physiology and B ehavior, 1980,24, 381- 386. Kim, H.-J., & Kalkhoff, R. K . Sex steroid influence on triglyceride metabolism . Journal of Clinical Investigations, 1975, 56, 888- 896. Lat, J. Self-selection of dietary components. In C. F. Code & W. Heidel (Eds.), Handbook of physiology, Section 6, Alimentary canal, (Vol. 1), Control of food and water intake. Pp.367-386. Washington, D . c.: American Physiological Society, 1967. Laudenslager, M. L., Wilkinson, C. W., Carlisle, H. J., & Hammel, H. T. Energy balance in ovariectomized rats with and without estrogen replacement. American Journal of Physiology, 1980, 238, R400-R405 . _Leathem. J. H. Nutritional effects on endocrine secretions. In W. C. Young (Ed.), Sex and internal secretions. Baltimore:Williams & Wilkins, 1961. Leshner, A. 1., & Collier, G. The effects of gonadectomy on the sex differences in dietary selfselection patterns and carcass compositions of rats . Physiology and Behavior, 1973, 11, 671- 676. Leshner, A. 1., Siegel, H. £., & Collier, G . Dietary self-selection by pregnant and lactating rats . Physiology and Behavior, 1972, 8, 151-154. Matute, M. L., & Kalkhoff, R. K . Sex steroid influence on hepatic gluconeogenesis and glycogen formation. Endocrinology, 1973,92, 762-768. Maw, D. S. J., & Wynn, V. The relation of growth hormone to altered carbohydrate metabolism in women taking oral contraceptives. Journal of Clinical Pathology, 1972,25, 354-358. McKerns, K. W., Coulomb, B., Kaleita, E., & deRenzo, E. C. Some effects of in vivo administered estrogens on glucose metabolism and adrenal cortical secretion in vitro. Endocrinology, 1958,63, 709-72. McLean, J. H. & Coleman, W. P. Temperature variation during the estrous cycle: active vs. restricted rats. Psychonomic Science, 1971,22, 179-180.
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Morimoto, Y., Arisue, K., & Yamamura, Y. Relationship between circadian rhythm of food intake and that of plasma corticosterone and effect of food restriction on circadian adrenocortical rhythm in the rat. Neuroendocrinology, 1977,23,212-222. Nance, D. M. Neuroestrogenic control of feeding behavior in the rat: its development and regulation. In R. B. Tobin & M. A. Mehlman (Eds.), Advances in modern human nutrition (Vol. 2). Park Forest South, Illinois: Pathotox Publishers, Inc., in press. Ota, K., & Yokoyama, A. Body weight and food consumption of lactating rats: effects of ovariectomy and of arrest and resumption of suckling. Journal of Endocrinology, 1967,38, 251-261. Patten, R. L. The reciprocal regulation of lipoprotein lipase activity and hormone-sensitive lipase activity in rat adipocytes. Journal of Biological Chemistry, 1970,245, 5577-5584. Richter, C. P. Behavioral regulators of carbohydrate homeostasis. Acta Neurovegetativa, Vienna, 1954,247-259. Richter, C, Holt, L., & Barelare, B. The elTect of self-selection of diet food (protein, carbohydrates, and fat), minerals, and vitamins-on growth, activity and reproduction of rats. American Journal of Physiology, 1937, 119, 388-389. Richter, c., Holt, L., & Barelare, B. Nutritional requirements for normal growth and reproduction in rats studied by the self-selection method. American Journal of Physiology, 1938, 122, 734-744. Richter, C., Schmidt, E. Increased fat and decreased carbohydrate appetite of pancreatectomized rats. Endocrinology, 1941,28,179-192. Richter, C., Schmidt, E., & Malone, P. Further observations of the self-regulatory dietary selection of rats made diabetic by pancreatectomy. Bulletin ofJohns Hopkins H ospUal, 1945, 76,192-219. Roskoski, R., & Steiner, D. F. The effect of estrogen on sugar transport in the rat uterus. Biochimica et Biophysica Acta, 1967, 135, 717-726. Schillinger, L., & Gerhards, E. Influence of hormonal contraceptives on carbohydrate and lipid metabolism in the rat. Biochemical Pharmacology, 1973, 22, 1835-1844. Sieck, G. c., Nance, D. M., Ramaley, J. A., Taylor, A. N., & Gorski, R. A. Prepubertal cyclicity in feeding behavior and' body weight regulation in the female rat. Physiology and Behavior, 1977,18,299-305. Siegel, P. S. Food intake in the rat in relation to the dark-light cycle. Journal of Comparative and Physiological Psychology, 1961, 54, 294-30l. Smith, D. E., & Gorski, J. Estrogen control of uterine glucose metabolism. Journal of Biological Chemistry, 1968, 243, 4169-4174. Tarttelin, M. F., & Gorski, R. A. Variations in food and water intake in normal and acyclic female rats. PhYSiology and Behavior, 1971, 7, 847-852. Tarttelin, M. F., & Gorski, R. A. The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinologica, 1973, 72,551-568. ter Haar, M. B. Circadian and estrual rhythms in food intake in the rat. Hormones and Behavior, 1972, 3, 213-219. Wade, G. N. Gonadal hormones and behavioral regulation of body weight. Physiology and Behavior, 1972, 8, 523-534. Wade, G. N. Sex hormones, regulatory behaviors, and body weight. In J. S. Rosenblatt, R. A. Hinde, E. Shaw, & c. G. Beer (Eds.), Advances in the study of behavior (Vol. 6). New York: Academic Press, 1976. Wade, G. N., & Gray, J. M. Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiology and Behavior, 1979, 22, 583-593. Wade, G. N., & Zucker, I. Development of hormonal control over food intake and body weight in female rats. Journal of Comparative and Physiological Psychology, 1970 a, 40, 213-220. Wade, G. N., & Zucker, I. H~rmonal modulation of responsiveness to an aversive taste stimulus in rats. Psychology and Behavior, 1970 b, 5, 269-273. Watkins, M. L., Fizette, N., & Heimberg, M. Sexual influences on hepatic secretion of triglyceride. Biochimica et Biophysica Acta, 1972, 280, 82-85. Wurtman, J. J., & Baum, M. J. Estrogen reduces total food and carbohydrate intake, but not protein intake in female rats. Physiology and Behavior, 1980, 24, 823-827. Yoshinaga, K., Hawkins, W., & Stocker, J. F. Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endocrinology, 1969,85, 103-112.
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Young, J. K., Nance, J. M., & Gorski, R. A. Dietary effects upon food and water intake and responsiveness to estrogen, 2-deoxy-glucose and glucose in female rats. Physiology and Behavior, 1978, 21, 395-403. Young, P. Studies of food preference, appetite, and dietary habit. II. Group self-selection maintenance as a method in the study of food preferences. Journal of Comparative Psychology, 1944,37, 371 391. Zucker, I. Hormonal determinants of sex differences in saccharin preference, food intake, and body weight. Physiology and Behavior, 1969, 4, 595602. Zucker, I. Light-dark rhythms in rat eating and drinking behavior. Physiology and Behavior, 1971,6, 115-126.
Received 18 July, 1980; revision received 6 October, 1980
Dietary Self-selection in Cycling and Neonatally Ovariectomized Rats PAULA J. GEISELMAN Department of Psychology and Brain Research Institute, University of California, Los Angeles
J. R. MARTIN Department of Behavioral Sciences, Swiss Federal Institute of Technology, Zurich
D. A. VANDERWEELE Department of Psychology, Occidental Col/ege, Los Angeles
D. NOVIN Department of Psychology and Brain Research Institute, University of California, Los Angeles
Dietary self-selection patterns were assessed in no-rmally cycling (four-day estrous cycle) rats with separate provisions of fa t (olive oil), carbohydrate (sucrose), and protein (casein). There was no fluctuation in daily caloric intake across the estrous cycle, but animals did demonstrate changes in consumption of specific macronutrients during the 24-h period in which estrus occurred . At estrus, animals exhibited increased carbohydra te intake and decreased fat intake in comparison with the remaining three days of the cycle. There was also a somewhat decreased (non-significant) protein consumption at estrus. Rats that had been ovariectomized on day 5 postnatally and then tested as adults in our paradigm ingested approximately the same quantity of daily kilocalories as observed in intact animals; but ovariectomy produced changes in consumption of specific macronutrients. In comparison with the intact animals, ova riectomized animals displayed increased fat intake and decreased carbohydrate intake. Thus, the fa t and carbohydrate self-selection patterns of our ovariectomized rats reflected the elimination of estrus behavior.
Quantity of food ingested by the female rat has been demonstrated to be of a cyclic nature and to correlate well with stages of the estrous cycle. _The peak in estradiol observed at proestrus (Yoshinaga, Hawkins & Stocker, 1969) is followed by a reduction in food intake (Brobeck, Wheatland & Strominger, 1947; Ota & Yokoyama, 1967; Tarttelin & Gorski, 1971 ; ter Haar, 1972; Wade, 1972). Con versely, the level of estradiol - - - - - - - - --
-
-
This research was supported by grants MH1 5345 (PJG), NS 7687 (DN), and research development fu nds from Occident al College. PJG was supported by Alcohol, Drug Abuse and Ment al Health Administrati on National Research Service Award MH1 5345 from the National Institute of Mental Health, and JRM was supported by National Research Service ·Awa rd MH5101. We are grateful to Dwight M . Nance for providing helpful comments and suggestions both during the conduct of the experiments and the preparation of the manuscript. We are also grateful to Neil Rowland for his careful reading of the manuscript. Requests fo r re prints should be sent to Paula 1. Geiselman. Department of Psychology, Franz Hall, Uni versity of California, Los Angeles, Californ ia 90024, U.S .A. 0195- 6663/81/020087 + 15 S02'00/0
T~;
198 1 Academic Press Inc. (London) Limited
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is decreased at diestrus at which time the animal shows an increase in food intake (Brobeck et aI., 1947; Drewett, 1973; Tarttelin & Gorski, 1971; ter Harr, 1972). However, changes in the ingestive patterns ofthe various macronutrients in the cycling female rat have not been studied so extensively. In this regard, one might ask the question: Are there specific nutritional requirements that change with the physiological events characterizing the estrous cycle? One might anticipate that patterns of dietary self-selection would be cyclic and correlate with the stages of the estrous cycle. All calories are perhaps not equally important at any given time in the control of ingestive behavior, and more pertinent factors in the control of food intake may be the pre-existing metabolic condition of the animal (as defined by the estrous cycle) and the metabolic sequelae of ingested nutrients. Within this context, it is important to note that ovarian hormones have a multitude of effects on various metabolic processes [see Wade & Gray (1979) and Nance (in press) for review]. We are aware of no studies relating the estrous cycle to self-selection of each of the three primary macronutrients. Complex diets have been used to test self-selection patterns in normally cycling rats (Leshner, Siegel & Collier, 1972; Wurtman & Baum, 1980), but results of these studies cannot be treated as definitive since one cannot be sure of the relevance of each of the dietary components in the control of feeding behavior. Although Richter, Holt and Barelare (1937,1938) simultaneously presented separate rations offat, carbohydrate, and protein to normally cycling female rats, they did not relate their results to the estrous cycle. Likewise, Kanarek & Beck (1980) recently tested females using a similar paradigm, but it was not an intent of their study to examine self-selection patterns with regard to cyclicity. The purpose of Experiment 1 was to establish the patterns of self-selection of the three primary macronutrients across the estrous cycle in rats provided with discrete rations of fat, carbohydrate, and protein. Based upon preliminary experimentation, it was hypothesized that animals would show an increase in carbohydrate intake with an accompanying decrease in fat and protein intake at estrus. In addition, we attempted to determine whether or not the underlying mechanisms of self-selection responses involved the ovaries, the hormonal secretions of which define the stages of the estrous cycle. Certainly ovariectomy produces a dramatic increase in the quantity of food consumed (Drewett, 1973; Holt, Keeton, & Vennesland, 1936; Tarttelin & Gorski, 1973; Wade, 1972) and this effect has been attributed to the elimination of ovarian secretions of estradiol (see Wade, 1976; Nance, in press, for review). However, once again, studies of macro nutrient selection have not been prolific. Differences between intact and ovariectomized animals in the self-selection of complex diets have been studied (Leshner & Collier, 1973; Wurtman & Baum, 1980); but as previously discussed, such paradigms do not allow one to assess the role of the three macronutrients in ingestive behavior. Recently, however, Kanarek & Beck (1980) have reported that, when presented with separate provisions of fat, carbohydrate and protein, animals ovariectomized in adulthood did not display an alteration in dietary self-selection in comparison with their preoperative intakes. In testing for differences in measures of food intake in gonadally intact and ovariectomized adult animals, the age of the animal at the time when ovariectomy was performed has appeared to be inconsequential. This may be ascribed to data showing that ovariectomy performed prepubertally does not produce alterations in either food intake or body weight prior to the time at which puberty would normally occur (Grunt, 1964; Wade & Zucker, 1970a). Consistent with this finding, it has also been demonstrated that food intake is unresponsive to exogenous estradiol until after the
DIET SELECTION IN CYCLING AND SPAYED RATS
89
time of puberty (Wade & Zucker, 1970 a). Thus, the conclusion that ovarian hormones do not influence food intake or body weight prepubertally is apparent. However, by subjecting more subtle feeding parameters to spectral analysis, Sieck, Nance, Ramaley, Taylor and Gorski (1977) have provided persuasive evidence that ovarian secretions influence prepubertal feeding behavior. The purpose of Experiment 2 was to use our paradigm to study the disruption of normal self-selection patterns in adult animals that had been ovariectomized neonatally. The surgery was performed on day 5 postnatally in an effort to minimize exposure to ovarian hormones and thus eliminate long-term experience with ovarian influences on feeding behavior. It was predicted that dietary self-selection patterns in our neonatally ovariectomized animals would largely reflect the elimination of estrus behavior. GENERAL METHOD
Animals and Maintenance Conditions
Animals serving as subjects were the female progeny of albino Sprague-Dawley strain rats (Holtzman). Nulliparous females were bred in our colony and then housed in group cages with ad lib. access to food (Purina Laboratory Chow) and water. Animals were always maintained under a 12: 12 light-dark regimen (light onset at 0800 hrs) in temperature-controlled room (22-25°C). Several days prior to delivery, mothers were placed in individual cages with metal floors. W oodchip bedding was supplied, and food and water remained available ad lib. Litter size was six to eight pups, and the initial 24-h period postpartum was regarded as day 1. With the exception of time required for surgery and recovery on day 5 (as described below), pups remained with their mothers until 21 days of age at which time they were weaned. Postweanling animals were housed individually in standard laboratory cages with food and water provided ad lib.
a
Surgery
At five days of age, pups were randomly chosen to undergo either ovariectomy or sham surgery. Pups were anesthetized by means of hypothermia in preparation for surgery. Bilateral ovariectomies were performed using two incisions, each of which was placed on the flank in a dorsolateral position posterior to the rib cage. Pups receiving sham ovariectomies were subjected to similar incision procedures. Following completion of surgery, wounds were sutured with sterile surgical gut and sealed with colloidin. Immediately postoperatively, pups were placed in a heated chamber (3033°C). After recovery and restoration to euthermic conditions, the pups were returned to their appropriate mothers. Apparatus and Procedure
For assessment of self-selection patterns, animals were placed in wire-mesh cages measuring 23 cm by 30·5 cm. Cages were suspended a minimum of 5 cm above woodchip shavings. Each of the three primary macronutrients (fat, carbohydrate, and protein) was presented as a separate food source in liquid form in a graduated 100-ml Richter tube attached to the front of the cage. The carbohydrate source was presented as a 30% (weight/volume) sucrose solution (Sigma Chemioal Company); the protein source was presented as a 15% (weight/volume) solution of casein hydrolysate (ICN Pharmaceuticals); and the fat source was presented as unadulterated olive oil (ICN
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Pharmaceuticals). In addition, a water bottle was placed on the front of each cage. Position of each of the three macronutrients and the water bottle was counterbalanced, although this control may not have been necessary as suggested by Young (1944). To provide a source of non-nutritive bulk, Alphacel (lCN Pharmaceuticals) was presented in a metal spill-proof cup secured to the cage floor. The Alphacel was prepared as a slurry by adding 2 g of Alphacel to 3 ml of a 1% solution of sodium chloride and potassium chloride. In addition, 1·5 ml ofliquid vitamins (Ross Laboratories, Vi-daylin M) was added to the mixture on each fourth day. The three macronutrients, nonnutritive bulk, and water were available ad lib. At 12-h intervals corresponding to light onset and light offset, the following variables were measured: body weight; water intake; and intake of fat, carbohydrate, and protein solutions. Animals were adapted to the test cage until reaching stabilization, which was defined as maintenance of body weight for an eight-day period during which time daily caloric intake varied less than 20% (Fox, Kipp & VanderWeele, 1976). Following stabilization, animals were tested for an additional4-day period, thus resulting in a 12day observation of self-selection behavior for each animal. Data collected during the final 12 days were grouped into three periods of 4 days each, and reliability estimates confirmed that the patterns of self-selection did not differ among these three periods. EXPERIMENT
1
DIET AR Y SILF-SELECTION PATTERNS MEASURED ACROSS THE ESTROUS CYCLE
Method Procedure
Seven female Sprague-Dawley strain (Holtzman) rats weighing 205 to 290 g with ages ranging from 60 to 90 days were tested in our self-selection paradigm. These animals had sustained sham ovariectomies on day 5. Vaginal cell samples confirmed that all animals had demonstrated a regular 4-day estrous cycle for at least 8 days (i.e., during the dietary self-selection stabilization period). Animals were then tested for an additional 4 days during which time we continued to take daily vaginal cell samples to verify the stage of the estrous cycle. These procedures resulted in a minimum observation of three estrous cycles for each animal. Young, Nance and Gorski (1978) have reported that vaginal-cell sampling procedures do not affect caloric intake, water intake or body weight. A standard nomenclature has not been establi'shed for the stages of the estrous cycle (Wade, 1976). In the present experiment, the 24-h period during which estrus occurred was recorded as day 1 of the estrous cycle. The remaining days of the cycle were simply recorded as days 2, 3, and 4. The stage of the cycle at which testing began was counterbalanced across animals. Design and Analysis
. Caloric intake (kcal/l00 g body weight), water intake (ml/IOO g body weight), and body weight (g) were grouped as follows and analyzed as a two-factor within-subjects design: estrous cycle (days 1,2,3, and 4) and nychthemeral period (12-h diurnal and 12-h nocturnal portions). Following the precedent of Kanarek and Beck (1980), percentage of 24-h kcal selected as fat, carbohydrate, and protein provided an assessment of dietary self-selection patterns. These data were grouped as follows and analyzed as a three-factor within-subjects design: estrous cycle (days 1, 2, 3, and 4), nychthemeral period (diurnal and nocturnal portions), and nutrient (fat, carbohydrate, and protein).
DIET SELECTION IN CYCLING AND SPA YEO RATS
91
Results Caloric Int(/ke, Water Intake, and Body Weight
Caloric intake expressed as kcal/lOO g body weight is depicted in Table I as a function ofthe estrous cycle. There was neither a significant main effect for cycle nor a significant interaction of this factor with the nychthemeral period. Because the interaction was non-significant and, further, since cycle was the factor of interest, the data in Table 1 were collapsed across the nychthemeral period and are presented as 24h mean values for each of the four days of the estrous cycle. These data are consistent with the daily caloric intake reported by Richter et al. (1938) for normally cycling females given the present nutrients. Neither water intake (ml/100 g body weight) nor body weight (g) varied across either the diurnal or nocturnal portions of the four days ofthe estrous cycle. For each of these two variables, there was neither a significant main effect for cycle nor a significant cycle by nychthemeral period interaction. It is noteworthy that water intake was negligible at all times. This result was anticipated since the animals were obtaining water in food solutions. (Mean 24-h water intake across the four-day estrous cycle: 2·2, 1·7,2·5, and 2·6 ml/100 g body weight, respectively. Mean body weight across the cycle: 245, 247, 246, and 247 g, respectively.) The expected nychthemeral period main effects were obtained with the present data (caloric intake, water intake, and body weight) but will not be reported since diurnal and nocturnal differences in ingestive behavior are already well established (Morimoto, Arisue & Yamamura, 1977; Siegel, 1961; Zucker, 1971). Further, the nychthemeral period factor was of interest in the present study only in interaction with the cycle factor. Percent ()f Caloric Intake Selected as Fat, Carbohydrate, or Protein
The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period is shown in Table 2. A significant nutrient by nychthemeral period interaction was obtained (F 2.12 = 5·3, p < 0·023). Therefore, simple main effects were computed to determine the variation ascribable to the nutrient factor occurring within each of the two levels of the nychthemeral period. A non-significant effect was obtained for the diurnal period. This result shows that, collapsing across all four days of the estrous cycle, there was no difference in the percentage of daily caloric intake chosen as fat, carbohydrate, or protein during the diurnal period. The effect for the nocturnal period, however, was significant (F 2.24 = 8·08, p < 0·01), indicating tha t animals ingested less protein than fat during the nocturnal period collapsed across all four days of the estrous cycle (Tukey's TARLE 1 Twenty-four hour measures of caloric intake across the estrous cycle (mean ± s.e.)
Estrous cycle Caloric intake
Day I"
Day 2
Day 3
Day 4
kcal/IOO g body weight
21·1 ±S·I
20·0±2·t
2t·2±3·6
IS·7±3·1
"Day 1 of the estrous cycle represents the 24-hour period during which estrus occurred.
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P. J . GEISELMAN ET AL.
TABLE 2 Percentage of daily caloric intake selected as fat , carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in intact rats (mean± s.e .)
Nutrient
Nychthemeral period Diurnal Nocturnal
Fat
Carbohydrate
Protein
5'2±1'2 44·0±9·7
8·0± 1·7 26'6±6'6
5·8± 1·1 1O·3±O·9
post-test, p
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FIGURE 1. Mean percentage of24-h caloric intake selected as fat, carbohydrate, and protein during each of the four days of the estrous cycle.
DIET SELECTION IN CYCLING AND SPAYED RATS EXPERIMENT
93
2
DIETARY SELF-SELECTION PATTERNS IN NEONATALLY OVARIECTOMIZED RATS
Method Procedure Ten Sprague-Dawley strain (Holtzman) rats that were ovariectomized on day 5 were tested in our dietary self-selection paradigm. The animals weighed 223 to 300 g and ranged from 60 to 80 days of age when tested. Animals in the ovariectomized group were subjected to the same stabilization and additional testing procedures as previously described. These animals were tested in our self-selection cages at the same time that intact animals were being tested. Design and Analysis Data collected from the ovariectomized animals were compared with data collected from the sham-operated, intact females. Days of the estrous cycle, which was the factor of interest in Experiment 1, was no longer of concern in the present analysis. The factors of interest in Experiment 2 were: group (ovariectomized or intact; between ,factor); nychthemeral period (diurnal and nocturnal portions; within factor); and nutrient (fat, carbohydrate, and protein; within factor). Caloric intake (kcal/lOO g body weight), water intake (ml/lOO g body weight ), and body weight (g) were analyzed as a two-factor (group and nychthemeral period) splitplot design. Percentage of24-h kcal selected as fat, carbohydrate, and protein provided an assessment of dietary self-selection patterns. These data were analyzed as a threefactor (group, nychthemeral period, and nutrient) split-plot design. Results Caloric Intake, Water Intake, and Body Weight Caloric intake expressed as kcal/lOO g body weight is depicted in Table 3 for intact and ovariectomized rats. There was neither a significant main effect for group nor a significant interaction of this factor with the nychth-emeral period. Because the interaction was non-significant, the data in Table 3 were collapsed across the nychthemeral period and are presented as 24-h mean values for each of the two groups. Neither water intake (ml/lOO g body weight) nor body weight (g) differed between the two groups during either the diurnal or nocturnal portions of the nychthemeral period. For each of these two variables, there was neither a significant main effect for group nor a significant group by nychthemeral period interaction. It is again noteworthy that water intake was negligible, as expected, for both groups. (Mean 24-h water intake for ovariectomized and intact animals: 3·3 and 2·3 mljlOO g body weight, respectively. Mean body weight for ovariectomized and intact animals: 267 and 246 g respectively.) TABLE 3 Twentylour hour measures of caloric intake in intact and ovariectomized rats (mean ±s.e.)
Group Caloric intake
Ovariectomized
Intact
kcalj100g body weight
no ± 1·0
20·3 ± 2·5
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P. J. GEISELMAN ET AL.
The expected nychthemerill'period main effects were obtained with the present data (caloric intake, water intake, and body weight) but will not be reported. Percent of Caloric Intake Selected as Fat, Carbohydrate, or Protein
The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period is shown in Table 4. (For comparison, these data are depicted in Table 5 for ovariectomized rats only,) A significant nutrient by nychthemeral period interaction was obtained (F 2,30 = 26'46, p < 0'00001), and simple main effects were computed to determine the variation due to the nutrient factor occurring within the diurnal and nocturnal periods. A non-significant effect was obtained for the diurnal period. Thus, collapsing across groups, there was no difference in the percentage of daily caloric intake chosen as fat, carbohydrate, or protein during the diurnal perioO. The effect for the nocturnal period, however, was significant (F 2.60 = 53'73, P <0·00001), indicating that the intact and the ovariectomized rats ingested less protein than fat during the nocturnal period (Tukey's post-test, p < 0,01). The mean percentage of daily caloric intake selected as fat, carbohydrate, or protein by the intact and the ovariectomized rats is depicted in Figure 2. A significant nutrient by group interaction was obtained (F 2.30 = 4· 34, p < 0'022), and simple main effects were conducted to determine the variation due to the group factor occurring at each level of the nutrient factor. A significant effect was obtained for fat (F 1,45 = 7'58, p < 0'01); the ovariectomized rats ingested a significantly greater percentage of their daily caloric intake as fat than did the intact rats. The effect for carbohydrate was significant (F 1,45 = 5'2, P < 0'04), indicating that the ovariectomized rats ingested less of their daily calories as carbohydrate than did the intact rats. Finally, percentage of daily caloric intake ingested as protein was observed to be lower in the ovariectomized than in the intact animals, but this effect did not reach significance. TABLE
4
Percentage of daily caloric intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in intact and ovariectomized rats (mean±s.e.) Nychthemeral period Diurnal Nocturnal
Nutrient Fat
Carbohydrate
Protein
6·0±I·5 57'8±6'I
4·9± 1·1 17·8 ± 4'5
4'8±O'5 8·9±O·9
TABLE 5 Percentage of daily calotic intake selected as fat, carbohydrate, or protein during the diurnal and nocturnal portions of the nychthemeral period in ovariectomized rats only (mean±s.e.)
Nutrient Nychthemeral period Diurnal Nocturnal
Fat
Carbohydrate
Protein
6·6±2-4 67'5±7'O
2·7±1·0 11'6±5'5
4·1 ±O'3 7'9± 1-4
DIET SELECTION IN CYCLING AND SPAYED RATS
95
80 c-----~~-------------------,
70 ~ o
C 6 u
~o
~, 50
<;t
N
o ~4 0
o
C
~ ~ 30
c
o :2'
'" 20 16 14
10
8'---~
Ma c r on ut rien ts
FIGURE 2. Mean percentage of24-h caloric intake selected as fat, carbohydrate, and protein in intact (0) and ovariectomized (.) animals.
Verification of Ovariectomy At the conclusion oftesting, animals that had been ovariectomized neonatally were examined for confirmation of the surgical procedure. At sacrifice, it was verified that none of the animals in this group had ovaries and that vaginal canalization had not occurred. Also, all animals in the ovariectomized group had infantile uteri. DISC USSION
Experiment 1 provided an answer to the question: Are there specific nutritional requirements that change across the estrous cycle? Patterns exhibited during days 2, 3, and 4 were indistinguishable from one another; but at estrus animals exhibited increased carbohydrate intake and de.creased fat intakl!: Interestingly, the fat and carbohydrate self-selection patterns of our ovariectomized rats reflected the elimination of estrus behavior. These changes in intake at estrus and following ovariectomy are consistent with Richter's (1954) observation that an inverse relationship exists between fat and carbohydrate intake; i.e., when intake of one of these two macronutrients changes in one direction, intake of the other changes in the opposite direction. The fat-intake results in both our intact and our ovariectomized animals could be interpreted as being consistent with Nance's (in press) demonstration that a fat load suppressed subsequent food intake in intact but not in ovariectomized rats. Also, Young, N ance and Gorski (1978) showed that estradiol produced a greater depression of food intake in female rats maintained on a high-fat diet than in those fed chow or a high-dextrose diet. These results are all predictable from Wade & Gray's (1979) hypothesis that "estradiol may decrease food intake in female rats by increasing the
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availability of triglyceride as a metabolic fuel." In this context, our results further suggest that, rather than merely decreasing caloric intake in general, the high levels of estradiol occurring at proestrus may subsequently suppress the specific appetite fo r fat. Moreover, when ovariectomy was performed and the suppressive influence was eliminated, the animals demonstrated an enhanced fat intake. It is well documented that estradiol increases circulating levels of triglycerides (Ferreri & Naito, 1978 ; Hazzard, Spiger, Bagdade & Bierman, 1969; Kim & Kalkhoff, 1975; Schillinger & Gerhards, 1973; Watkins, Fizette & Heimberg, 1972). Thus, the present results might suggest that dietary intake of fat varies inversely with blood levels of triglycerides across the estrous cycle. In contrast to our fat-intake results, carbohydrate intake was increased at estrus; consistent with this, the ovariectomized animals showed a decrease in carbohydrate consumption. These data are compatible with ovarian influences on insulin and carbohydrate metabolism. Our intact animals may have had an increased carbohydrate appetite in response to estradiol-induced hypoglycemia (Goodman & Hazelwood, 1974). At estrus, animals are hyperinsulinemic and show enhanced glucose tolerance (Bailey & Matty, 1972). Following ovariectomy, however, a reduction in plasma insulin is observed and glucose tolerance is impaired; but these responses can be restored with ovarian hormones (Bailey & Matty, 1972). Moreover, it has been shown that estradiol enhances hepatic glycogen levels (McKerns, Coulomb, Kaleita & de Renzo, 1958; Matute & Kalkhoff, 1973) as well as facilitating glucose uptake by the uterus (Roskoski & Steiner, 1967; Smith & Gorski, 1968), muscle (McKerns et al., 1958), and adipose tissue (Gilmour & McKerns, 1966). The estradiol-induced increase in insulin (Bailey & Matty, 1972; Basabe, Chieri & Foglia, 1969) may be ascribable to both direct and indirect actions of the hormone on the pancreas (Costrini & Kalkhoff, 1971; Goodman & Hazelwood, 1974). The increase in carbohydrate intake observed in our animals at estrus is consistent with Richter's (1954) demonstration that exogenous insulin produces an increase in carbohydrate intake. Moreover, our ovariectomized animals exhibited dietary self-selection patterns (decreased carbohydrate and increased fat intakes) quite similar to those seen in pancreatectomized animals (Richter & Schmidt, 1941; Richter, Schmidt & Malone, 1945). The increase in carbohydrate intake found at estrus in animals serving as subjects in Experiment 1 was also observed in preliminary investigations. Using the present selfselection paradigm, animals having access to activity wheels showed an increase in both activity level and carbohydrate intake a t estrus. Considered together, these results indicate that an increase in carbohydrate intake occurs at estrus regardless of whether the animal has a high level of activity . However, animals with restricted activity levels display greater energy output as heat loss at estrus than do active animals (McLean & Coleman, 1971), and ovariectomized animals show substantial heat loss in response to estradiol treatment regardless of whether or not activity levels are restricted (Laudenslager, Wilkinson, Carlisle & Hammel, 1980). Therefore, expenditure of energy (as heat loss and/or muscular activity) cannot be eliminated as a contributing cause of increased ingestion of a nutrient providing readily available energy. Our intact animals ingested approximately 16% of their daily caloric intake as protein across the four days of the estrous cycle.- Both the absolute and relative amounts of protein ingested, which were consistent with that reported by Richter et al. (1938) and Fox et al. (1976), were certainly sufficient to maintain hea lth. This was evidenced by the general appearance of the animals, their maintenance of body weight , and the fact that all animals serving as subjects exhibited normal estrous cyclicity,
DIET SELECTION IN CYCLING AND SPA YEO RATS
97
which has been shown to be disrupted when nutrition is inadequate (Leathem, 1961). The present nutritional sources were chosen on the basis of previous reports indicating that animals thrive when placed in self-selection paradigms using these three specific nutrients (F ox et aI., 1976; Richter et al., 1938). When maintained for prolonged periods with separate sources of sucrose, casein, and olive oil, Richter et al. (1938) reported that self-selecting females displayed body-weight curves essentially identical to those of the control animals that were ingesting standard laboratory diets (Richter et al., 1938). More importantly, their self-selecting rats were able to maintain these comparable body weights even though they were ingesting 20% fewer kilocalories than the control group. Thus, when given the above choices, ingestive patterns of selfselecting animals were more efficient than those of the control animals given standard laboratory diets. Consistent with this conclusion, Richter et al. (1938) also reported that their self-selecting animals showed more regularity in estrous cyclicity than did the control animals, another indication of greater nutritional efficiency in the self-selecting animals (Leathem, 1961). Forty years later, using the same three macronutrients as those used by Richter et al. (1938), Fox et al. (1976) also found that female rats were able to maintain their body weights even though they were ingesting fewer calories than typically observed with laboratory chow. Kanarek & Beck's (1980) results indicating that intact females chose one-third of their daily calories from each of the three macronutrients and that these patterns did not change following ovariectomy are not consistent with the present results. There are procedual differences that may account for this. Their animals were not ovariectomized until adulthood, and their dietary provisions differed from ours, both of which are pertinent factors to consider when comparing results across studies (Lat, 1967; Sieck et al., 1977). When presented with specific dietary sources identical to those used by Richter et al. (1938), our intact animals ingested approximately 49% of their daily caloric intake as fat, 35% as carbohydrate, and 16% as protein across the four-day estrous cycle. Thus, our results are consistent with those obtained by Richter et al. (1938) with normally cycling animals. The gonadally intact animals maintained in our self-selection paradigm did not exhibit hypophagia at estrus. It can be assumed that this was not a floor effect since all animals exhibited stable weight maintenance. The estradiol-induced anorexia occurring at estrus in animals maintained with laboratory chow is somewhat difficult to reconcile with the present results due to the very high proportion of carbohydrate and the very low proportion of fat in the laboratory chow. It is interesting, however, that Richter et al. (1938) have demonstrated that normally cycling female rats are actually hyperphagic when maintained on standard diets. In consideration of the low-fat content oflaboratory chow, our results suggest that the hyperphagia observed when an animal ingests laboratory chow may be attributable to an effort to increase fat intake and that the anorexia shown at estrus with laboratory chow may be an attempt to decrease fat intake. This interpretation is compatible with Young et al.'s (1978) demonstration that a high-fat diet (two parts chow and one part fat) enhances the estradiol-induced anorexic effect. However, the influence of ovarian hormones on feeding obviously are not entirely attributable to one macronutrient only. Wade & Gray (1979) have developed a convincing argument for the importance of the role offat metabolism in the mediation of estradol's influence on feeding, but this argument does not provide an apparent explanation for the observation that both estradiol and food deprivation have essentially the same effect on adipose tissue yet have opposing effects on ingestive behavior (Nance, in press). While a number of discrepancies have been
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P. J. GEISELMAN ET AL.
pointed out in the literature of ovarian influences on insulin and carbohydrate metabolism (Wade & Gray, 1979), one cannot dismiss the importance of carbohydrates in the control of ingestive behavior (Nance, in press). With the interrelationship between fat and carbohydrate metabolism, it would be difficult to consider one to the exclusion of the other. Moreover, an explanation of ovarian influences on ingestive behavior is further complicated by data suggesting that, in addition to producing an elevation in insulin levels, estradiol also may result in increases in somatotrophic hormone (Frantz & Rabkin, 1965; Goodman & Hazelwood, 1974; Maw & Wynn, 1972) and corticosteroids (Critchlow, Liebelt, Bar-Sela, Mountcastle & Lipscomb, 1963; Goodman & Hazelwood, 1974), both of which influence the metabolism of macro nutrients in a manner that is essentially antagonistic to the action of insulin. Actually these additional hormonal increases may provide at least a partial explanation for the seemingly paradoxical increase in plasma insulin levels and concurrent decrease in adipose tissue lipoprotein lipase activity in response to estradiol (Hamosh & Hamosh, 1975; Patten, 1970; Wade & Gray, 1979). While the present results might suggest that dietary intakes of fat and carbohydrate vary inversely with circulating levels of triglyceride and glucose, respectively, it is obvious that the hormonal influences on these selection patterns are quite complex. The alterations in self-selection patterns in our intact animals are probably not due primarily to changes in taste responsiveness. Taste preferences do not vary across the estrous cycle (Wade, 1976), while our results and those of previous studies show feeding fluctuations that are related to cyclicity (Brobeck et al., 1947; Drewett, 1973; Ota & Yokoyama, 1967; Tarttelin & Gorski, 1971; ter Haar, 1972). Also, ovariectomized animals show a decrease in food intake following administration of estradiol (see Wade, 1976; and Nance, in press), yet treatment of ovariectomized animals with varying doses of estradiol alone is ineffective in restoring saccharin preference (Zucker, 1969). Wade (1976) has pointed out that taste responsivity is negatively correlated with selection of dietary protein. Since ovariectomized animals show decreased taste responsivity (Wade & Zucker, 1970 b; Zucker, 1969), one would predict that our ovariectomized animals would have chosen more of the protein source than the intact animals if the choice of nutrients had been made on the basis of a taste factor alone. In actuality, however, there was a tendency for our ovariectomized rats to ingest less protein than the intact rats. Finally, since fat has greater caloric density than either carbohydrate or protein, it might appear that rather than specifically increasing carbohydrate intake and decreasing fat intake at estrus, animals may have made their selections in an effort to obtain more dilute sources of calories at that stage of the cycle. However, the reader is reminded that, if caloric density had been the primary basis of selection, one would not have expected the animals to discriminate between the dilute sources of carbohydrate and protein at estrus, as they clearly did: At estrus, our animals reliably increased their intake of the 30% carbohydrate solution yet tended to decrease their intake of the 15% protein solution. While we cannot conclusively eliminate caloric density as a possible contributing cause of changes in ingestive behavior, we feel that our argument based on metabolic and hormonal factors provides a more parsimonious explanation of changes in intake across the estrous cycle. REFERENCES
Bajley, C. J., & Matty, A. J. Glucose tolerance and plasma insulin of the rat in relation to the oestrous cycle and sex hormones. Hormone and Metabolic Research, 1972,4,266-270.
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Basabe, J. c., Chiere, R. A., & Foglia, V. C. Action of sex hormones on the insulinemia of castrated female rats. Proceedings of the Society for Experimental BioLogy and Medicine, 1969,130, 1159-1161.
Brobeck, J. R., Wheatland, M., & Strominger, J. L. Variations in regulation of energy exchange associated with estrus, diestrus, and pseudopregnancy in rats. Endocrinology, 1947, 40, 65- 72. Costrini, N. V., & Kalkhoff, R. K. Relative effects of pregnancy, estradiol, and progesterone on pl asma insulin and pancreatic islet insulin secretion. Journal q{Clinicallllvestigation, 1971, 50, 992- 999.
Critchlow, V., Liebelt, R. A., Bar-Sela, M., Mountcastle, W., & Lipscomb, H. S. Sex difference in resting pituitary-adrenal function in the rat. American Journal of Physiology, 1963, 205, 807-815. Drewett, R. F. Oestrous and dioestrous components of the ovarian inhibition of hunger in the rat. Animal Behavior, 1973, 21, 772- 780. Ferreri, L. F., & Naito, H . K . Effects of estrogens on rat serum cholesterol concentrations: Consideration of dose, type of estrogen and treatment duration. Endocrinology, 1978,102, 1621-1627. Fox, K. A., Kipp, S. c., & VanderWeele, D. A. Dietary self-selection following subdiaphragmatic vagotomy in the white rat. American Journal of Physiology, 1976,231,1790-1793. Frantz, A. G., & Rabkin, M. T. Effects of estrogen and sex differences on secretion of human growth hormones. Journal of Clinical Endocrinology and Metabolism, 1965,25,1470-1480. Gilmour, K. E., & McKerns, K. W. Insulin and estrogen regulation of lipid synthesis in adipose tissue. Biochimica et Biophysica Acta, 1966, 116, 220-228. Goodman, M. N., & Hazelwood, R. L. Short-term effects of oestradiol benzoate in normal, hypophysectomized and alloxan-diabetic male rats. Journal of Endocrinology, 1974, 62, 439-449. Grunt, J. A. Effects of neonatal gonadectomy on skeletal growth and development in the rat. Endocrinology, 1964, 75, 805-808. H azzard, W. R., Spiger, M. J ., Bagdade, J. P., & Bierman, E. L. Studies of the mechanism of increased plasma triglyceride levels induced by oral contraceptives. New England Journal of Medicine, 1969,280,471-474.
Holt, H., Keeton, R. W., & Vennesland, B. The effect of gonadectomy on body structure and body weight in albino rats. American Journal of Physiology, 1963,114,515-525. Kanarek, R. B., & Beck, J. M. Role of gonadal hormones in diet selection and food utilization in female rats . Physiology and B ehavior, 1980,24, 381- 386. Kim, H.-J., & Kalkhoff, R. K . Sex steroid influence on triglyceride metabolism . Journal of Clinical Investigations, 1975, 56, 888- 896. Lat, J. Self-selection of dietary components. In C. F. Code & W. Heidel (Eds.), Handbook of physiology, Section 6, Alimentary canal, (Vol. 1), Control of food and water intake. Pp.367-386. Washington, D . c.: American Physiological Society, 1967. Laudenslager, M. L., Wilkinson, C. W., Carlisle, H. J., & Hammel, H. T. Energy balance in ovariectomized rats with and without estrogen replacement. American Journal of Physiology, 1980, 238, R400-R405 . _Leathem. J. H. Nutritional effects on endocrine secretions. In W. C. Young (Ed.), Sex and internal secretions. Baltimore:Williams & Wilkins, 1961. Leshner, A. 1., & Collier, G. The effects of gonadectomy on the sex differences in dietary selfselection patterns and carcass compositions of rats . Physiology and Behavior, 1973, 11, 671- 676. Leshner, A. 1., Siegel, H. £., & Collier, G . Dietary self-selection by pregnant and lactating rats . Physiology and Behavior, 1972, 8, 151-154. Matute, M. L., & Kalkhoff, R. K . Sex steroid influence on hepatic gluconeogenesis and glycogen formation. Endocrinology, 1973,92, 762-768. Maw, D. S. J., & Wynn, V. The relation of growth hormone to altered carbohydrate metabolism in women taking oral contraceptives. Journal of Clinical Pathology, 1972,25, 354-358. McKerns, K. W., Coulomb, B., Kaleita, E., & deRenzo, E. C. Some effects of in vivo administered estrogens on glucose metabolism and adrenal cortical secretion in vitro. Endocrinology, 1958,63, 709-72. McLean, J. H. & Coleman, W. P. Temperature variation during the estrous cycle: active vs. restricted rats. Psychonomic Science, 1971,22, 179-180.
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Morimoto, Y., Arisue, K., & Yamamura, Y. Relationship between circadian rhythm of food intake and that of plasma corticosterone and effect of food restriction on circadian adrenocortical rhythm in the rat. Neuroendocrinology, 1977,23,212-222. Nance, D. M. Neuroestrogenic control of feeding behavior in the rat: its development and regulation. In R. B. Tobin & M. A. Mehlman (Eds.), Advances in modern human nutrition (Vol. 2). Park Forest South, Illinois: Pathotox Publishers, Inc., in press. Ota, K., & Yokoyama, A. Body weight and food consumption of lactating rats: effects of ovariectomy and of arrest and resumption of suckling. Journal of Endocrinology, 1967,38, 251-261. Patten, R. L. The reciprocal regulation of lipoprotein lipase activity and hormone-sensitive lipase activity in rat adipocytes. Journal of Biological Chemistry, 1970,245, 5577-5584. Richter, C. P. Behavioral regulators of carbohydrate homeostasis. Acta Neurovegetativa, Vienna, 1954,247-259. Richter, C, Holt, L., & Barelare, B. The elTect of self-selection of diet food (protein, carbohydrates, and fat), minerals, and vitamins-on growth, activity and reproduction of rats. American Journal of Physiology, 1937, 119, 388-389. Richter, c., Holt, L., & Barelare, B. Nutritional requirements for normal growth and reproduction in rats studied by the self-selection method. American Journal of Physiology, 1938, 122, 734-744. Richter, C., Schmidt, E. Increased fat and decreased carbohydrate appetite of pancreatectomized rats. Endocrinology, 1941,28,179-192. Richter, C., Schmidt, E., & Malone, P. Further observations of the self-regulatory dietary selection of rats made diabetic by pancreatectomy. Bulletin ofJohns Hopkins H ospUal, 1945, 76,192-219. Roskoski, R., & Steiner, D. F. The effect of estrogen on sugar transport in the rat uterus. Biochimica et Biophysica Acta, 1967, 135, 717-726. Schillinger, L., & Gerhards, E. Influence of hormonal contraceptives on carbohydrate and lipid metabolism in the rat. Biochemical Pharmacology, 1973, 22, 1835-1844. Sieck, G. c., Nance, D. M., Ramaley, J. A., Taylor, A. N., & Gorski, R. A. Prepubertal cyclicity in feeding behavior and' body weight regulation in the female rat. Physiology and Behavior, 1977,18,299-305. Siegel, P. S. Food intake in the rat in relation to the dark-light cycle. Journal of Comparative and Physiological Psychology, 1961, 54, 294-30l. Smith, D. E., & Gorski, J. Estrogen control of uterine glucose metabolism. Journal of Biological Chemistry, 1968, 243, 4169-4174. Tarttelin, M. F., & Gorski, R. A. Variations in food and water intake in normal and acyclic female rats. PhYSiology and Behavior, 1971, 7, 847-852. Tarttelin, M. F., & Gorski, R. A. The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinologica, 1973, 72,551-568. ter Haar, M. B. Circadian and estrual rhythms in food intake in the rat. Hormones and Behavior, 1972, 3, 213-219. Wade, G. N. Gonadal hormones and behavioral regulation of body weight. Physiology and Behavior, 1972, 8, 523-534. Wade, G. N. Sex hormones, regulatory behaviors, and body weight. In J. S. Rosenblatt, R. A. Hinde, E. Shaw, & c. G. Beer (Eds.), Advances in the study of behavior (Vol. 6). New York: Academic Press, 1976. Wade, G. N., & Gray, J. M. Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiology and Behavior, 1979, 22, 583-593. Wade, G. N., & Zucker, I. Development of hormonal control over food intake and body weight in female rats. Journal of Comparative and Physiological Psychology, 1970 a, 40, 213-220. Wade, G. N., & Zucker, I. H~rmonal modulation of responsiveness to an aversive taste stimulus in rats. Psychology and Behavior, 1970 b, 5, 269-273. Watkins, M. L., Fizette, N., & Heimberg, M. Sexual influences on hepatic secretion of triglyceride. Biochimica et Biophysica Acta, 1972, 280, 82-85. Wurtman, J. J., & Baum, M. J. Estrogen reduces total food and carbohydrate intake, but not protein intake in female rats. Physiology and Behavior, 1980, 24, 823-827. Yoshinaga, K., Hawkins, W., & Stocker, J. F. Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endocrinology, 1969,85, 103-112.
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Young, J. K., Nance, J. M., & Gorski, R. A. Dietary effects upon food and water intake and responsiveness to estrogen, 2-deoxy-glucose and glucose in female rats. Physiology and Behavior, 1978, 21, 395-403. Young, P. Studies of food preference, appetite, and dietary habit. II. Group self-selection maintenance as a method in the study of food preferences. Journal of Comparative Psychology, 1944,37, 371 391. Zucker, I. Hormonal determinants of sex differences in saccharin preference, food intake, and body weight. Physiology and Behavior, 1969, 4, 595602. Zucker, I. Light-dark rhythms in rat eating and drinking behavior. Physiology and Behavior, 1971,6, 115-126.
Received 18 July, 1980; revision received 6 October, 1980