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LARGE CONTINUOUS DOSES OF ESTRADIOL LEAD TO CHANGES INLARGE CONTINUOUS DOSES OF ESTRADIOL LEAD TO CHANGES INFEMALE RATS’ APPETITE FOR COMPLEX, PALATABLE INGESTAFEMALE RATS’ APPETITE FOR COMPLEX, PALATABLE INGESTA

Karen J. Boswell, Christopher A. Caffalette, Stephanie A. Lamb, Karen T. Stitt, Meta L. Reid & Larry D. ReidLaboratory for Psychopharmacology, Siena College, Loudonville, NY and Rensselaer Polytechnic Institute, Troy, NY, UUSSAA

EV (1.5 mg/kg) and Intakes of Chocolate Cake Mix

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Fig. 1. Mean grams of chocolate cake mix batter per kilogram of bodyweight taken Fig. 1. Mean grams of chocolate cake mix batter per kilogram of bodyweight taken on each day it was presented are depicted. One group received an injection of 10 on each day it was presented are depicted. One group received an injection of 10 mg/kg of EV; the other group received carrier of EV (placebos). Ns = 10 females a mg/kg of EV; the other group received carrier of EV (placebos). Ns = 10 females a group. Bars are standard errors of the mean. The ordinate depicts the days after group. Bars are standard errors of the mean. The ordinate depicts the days after the day of injection. Notice that the females took inordinately large amounts of the the day of injection. Notice that the females took inordinately large amounts of the chocolate cake batter (across the first 4 days of opportunity to take batter, about chocolate cake batter (across the first 4 days of opportunity to take batter, about 10% of their bodyweights, daily). Despite what might appear to be a ceiling on 10% of their bodyweights, daily). Despite what might appear to be a ceiling on intake, the EV-treated females ate considerable more batter than placebo-controls. intake, the EV-treated females ate considerable more batter than placebo-controls. Notice that the enhanced intakes of the EV-treated females was evident even 2 Notice that the enhanced intakes of the EV-treated females was evident even 2 months after the injection, a time when estradiol was no longer being released from months after the injection, a time when estradiol was no longer being released from the EV injection.the EV injection.

Fig. 3. Mean grams of chocolate cake mix batter per Fig. 3. Mean grams of chocolate cake mix batter per kilogram of bodyweight taken on each of 8 days are kilogram of bodyweight taken on each of 8 days are depicted. One group received an injection of 1.5 mg/kg depicted. One group received an injection of 1.5 mg/kg of EV; the other group received carrier of EV of EV; the other group received carrier of EV (placebos). Ns = 10 females a group. Bars are standard (placebos). Ns = 10 females a group. Bars are standard errors of the mean. These females had the opportunity errors of the mean. These females had the opportunity to take cake batter before the injections. During their to take cake batter before the injections. During their first opportunities to take batter (and before injections) first opportunities to take batter (and before injections) their intakes were very similar to that of the placebo-their intakes were very similar to that of the placebo-controls of Fig. 1. Notice that the placebo-controls took controls of Fig. 1. Notice that the placebo-controls took large amounts of batter (about 8% of their bodyweight large amounts of batter (about 8% of their bodyweight daily). The EV-treated females took more (about 10% daily). The EV-treated females took more (about 10% of their bodyweight daily).of their bodyweight daily).

MethodsMethodsSubjectsSubjects The subjects in both experiments were female Sprague-Dawley rats (Taconic Farms, Germantown NY), weighing The subjects in both experiments were female Sprague-Dawley rats (Taconic Farms, Germantown NY), weighing about 200g on arrival. Twenty rats were tested in each experiment. They were housed individually in double-sized cages about 200g on arrival. Twenty rats were tested in each experiment. They were housed individually in double-sized cages in a windowless room with water and standard laboratory chow always available. The room was maintained at 23º C, with in a windowless room with water and standard laboratory chow always available. The room was maintained at 23º C, with a 12 h light/dark cycle, lights on at 800 h.a 12 h light/dark cycle, lights on at 800 h.

Drugs and ingestaDrugs and ingesta Estradiol valerate (EV) (Sigma-Aldrich, St. Louis, MO, USA). The carrier of EV was sesame oil (Sigma-Aldrich). All Estradiol valerate (EV) (Sigma-Aldrich, St. Louis, MO, USA). The carrier of EV was sesame oil (Sigma-Aldrich). All injections were 0.2 ml, given intramuscularly. In Experiment 1, rats received wither 2.0 mg EV (about 10 mg/kg) or a injections were 0.2 ml, given intramuscularly. In Experiment 1, rats received wither 2.0 mg EV (about 10 mg/kg) or a placebo injection. In Experiment 2, the dose of EV was 0.3 mg (about 1.5 mg/kg). The ingesta was chocolate cake mix placebo injection. In Experiment 2, the dose of EV was 0.3 mg (about 1.5 mg/kg). The ingesta was chocolate cake mix (Duncan Hines Devil’s Food). A standard package of mix was combined with 320 ml water, forming a thick batter.(Duncan Hines Devil’s Food). A standard package of mix was combined with 320 ml water, forming a thick batter.

ProcedureProcedure The basic procedures for both experiments were similar. In each, all measurements (body weight, batter weight) were The basic procedures for both experiments were similar. In each, all measurements (body weight, batter weight) were taken between 1200 and 1300 h).taken between 1200 and 1300 h).

Experiment 1:Experiment 1: Two weeks after arrival in the laboratory, two groups of rats (N = 10 each) were formed, equated for Two weeks after arrival in the laboratory, two groups of rats (N = 10 each) were formed, equated for weight. One group was injected with 2.0 mg EV and carrier, and the other received just carrier. Chocolate cake batter weight. One group was injected with 2.0 mg EV and carrier, and the other received just carrier. Chocolate cake batter was introduced 10 days post-injection. To present batter, glass cups (11.2 cm high, 6.2 cm diameter at top) were filled and was introduced 10 days post-injection. To present batter, glass cups (11.2 cm high, 6.2 cm diameter at top) were filled and placed in the cages, held in place by a pair of springs. There was no evidence of spillage throughout the course of the placed in the cages, held in place by a pair of springs. There was no evidence of spillage throughout the course of the study. To assess loss due to evaporation, 4 additional containers were filled and left on the top of the cage racks. Rate of study. To assess loss due to evaporation, 4 additional containers were filled and left on the top of the cage racks. Rate of evaporation was stable at about 1.5 g/day. Containers of batter were weighed before being placed in cages. During each evaporation was stable at about 1.5 g/day. Containers of batter were weighed before being placed in cages. During each subsequent day of the procedures, containers were removed from cages, weighed, additional batter added to replace what subsequent day of the procedures, containers were removed from cages, weighed, additional batter added to replace what was consumed, and reweighed prior to being returned to the cages.was consumed, and reweighed prior to being returned to the cages.

For 4 weeks, rats were provided with cake batter for 4 consecutive days followed by 3 days of no batter. During the For 4 weeks, rats were provided with cake batter for 4 consecutive days followed by 3 days of no batter. During the following week, all rats received chocolate cake batter for 4 days, then for the next 4 days half of the rats continued to following week, all rats received chocolate cake batter for 4 days, then for the next 4 days half of the rats continued to receive chocolate cake batter while the other half was given white cake batter (the data for these 4 days are not reported receive chocolate cake batter while the other half was given white cake batter (the data for these 4 days are not reported here). For the next 19 days no batter was provided. Finally, all rats were given chocolate cake batter for 4 more days.here). For the next 19 days no batter was provided. Finally, all rats were given chocolate cake batter for 4 more days.

Experiment 2:Experiment 2: The data of interest were obtained after rats had several weeks of exposure to either white or chocolate cake The data of interest were obtained after rats had several weeks of exposure to either white or chocolate cake batter (see prior history, below). Fifty-one days after arriving in the laboratory, rats were assigned to 2 groups (N = 10 batter (see prior history, below). Fifty-one days after arriving in the laboratory, rats were assigned to 2 groups (N = 10 each), equated for weight, and injected with 0.3 mg EV and carrier or just carrier. 11 days post-injection, all rats were each), equated for weight, and injected with 0.3 mg EV and carrier or just carrier. 11 days post-injection, all rats were presented with chocolate cake batter for 8 consecutive days.presented with chocolate cake batter for 8 consecutive days.Prior history:Prior history: 3 days after arriving in the laboratory, rats were divided evenly into 2 groups (equated for weight) and 3 days after arriving in the laboratory, rats were divided evenly into 2 groups (equated for weight) and received either white or chocolate cake batter. For 2 weeks, batter was provided in a 4 days on, 3 days off cycle. Then rats received either white or chocolate cake batter. For 2 weeks, batter was provided in a 4 days on, 3 days off cycle. Then rats were given batter for 8 consecutive days, followed by 13 days of no batter. Following this, all rats received chocolate cake were given batter for 8 consecutive days, followed by 13 days of no batter. Following this, all rats received chocolate cake batter for 4 consecutive days. Then 10 days of no batter elapsed before rats were given injections. The data for these batter for 4 consecutive days. Then 10 days of no batter elapsed before rats were given injections. The data for these earlier (pre-injection) weeks of white and chocolate batter are not presented here.earlier (pre-injection) weeks of white and chocolate batter are not presented here.

SIENA COLLEGESIENA COLLEGE

Results and DiscussionResults and Discussion

The injections of EV led to no gain in bodyweights across a period of about 5 days. The placebo-treated females gained weight The injections of EV led to no gain in bodyweights across a period of about 5 days. The placebo-treated females gained weight across these days. Consequently, the females of EV-treatment weighed less during the time of presentation of the chocolate batter. across these days. Consequently, the females of EV-treatment weighed less during the time of presentation of the chocolate batter. The results, in terms of grams of batter taken per kilogram of bodyweight, are presented in Figs. 1 and 3. The results, across all The results, in terms of grams of batter taken per kilogram of bodyweight, are presented in Figs. 1 and 3. The results, across all days of batter presentation, in terms of total grams taken, are presented in Figs. 2 and 4. days of batter presentation, in terms of total grams taken, are presented in Figs. 2 and 4.

The data clearly indicate that both the 10.0 and the 1.5 mg/kg doses led to heightened intakes of chocolate cake batter. During The data clearly indicate that both the 10.0 and the 1.5 mg/kg doses led to heightened intakes of chocolate cake batter. During the initial days of presentation of the batter (Fig. 1), all females took inordinate amounts of the batter and quickly escalated intakes the initial days of presentation of the batter (Fig. 1), all females took inordinate amounts of the batter and quickly escalated intakes to amounts that might be considered a ceiling on intakes. The placebo-treated females then reduced their intakes as the novelty of to amounts that might be considered a ceiling on intakes. The placebo-treated females then reduced their intakes as the novelty of the ingesta waned. The EV-treated females continued to take large amounts. Recall that the females of the lower dose had the ingesta waned. The EV-treated females continued to take large amounts. Recall that the females of the lower dose had previously been given batter prior to injections. This habituation to the ingesta did not lead to maximal intakes by the placebo-previously been given batter prior to injections. This habituation to the ingesta did not lead to maximal intakes by the placebo-treated females. The EV-treated females did take extraordinarily large amounts of batter (Fig 3).treated females. The EV-treated females did take extraordinarily large amounts of batter (Fig 3).

These doses of EV induce very high levels of circulating estradiol that wane across days. The 10 mg/kg dose probably induced a These doses of EV induce very high levels of circulating estradiol that wane across days. The 10 mg/kg dose probably induced a disturbance in the processes of the arcuate n. inducing some enduring lesion (x), which is probably related to the enduring disturbance in the processes of the arcuate n. inducing some enduring lesion (x), which is probably related to the enduring enhancement of intake. It is not known whether the smaller dose induces the same permanent toxicity.enhancement of intake. It is not known whether the smaller dose induces the same permanent toxicity.

It is widely believed that estradiol reduces intake of ingesta (e.g., x, x). Estradiol, however, may reduce intakes for only a few It is widely believed that estradiol reduces intake of ingesta (e.g., x, x). Estradiol, however, may reduce intakes for only a few days after being administered. When given continuously at doses that probably saturate the receptors, a change occurs that is days after being administered. When given continuously at doses that probably saturate the receptors, a change occurs that is manifest as an enhanced appetite for “luxury” ingesta (alcohol, cake, and saccharin-sweetened water).manifest as an enhanced appetite for “luxury” ingesta (alcohol, cake, and saccharin-sweetened water).

This enhanced appetite was not seen previously because, for a short time directly following injection of EV, rats experience body This enhanced appetite was not seen previously because, for a short time directly following injection of EV, rats experience body weight loss and reduced appetite. Thus, any testing done during these few days has contributed to the conclusion that estrogens weight loss and reduced appetite. Thus, any testing done during these few days has contributed to the conclusion that estrogens were anorectic. Further work with EV has led to the conclusions that heightened appetite only emerges with sustained large doses were anorectic. Further work with EV has led to the conclusions that heightened appetite only emerges with sustained large doses of estradiol and may only be related to “luxury” ingesta (which are not the circumstances of the usual experiment using rats). It of estradiol and may only be related to “luxury” ingesta (which are not the circumstances of the usual experiment using rats). It remains to be seen whether this enhanced appetite occurs with the estrogenic preparations that are currently being taken by remains to be seen whether this enhanced appetite occurs with the estrogenic preparations that are currently being taken by women. Nevertheless, sustained dosing with estrogenic compounds in an environment of nearly continuous presentation of luxury women. Nevertheless, sustained dosing with estrogenic compounds in an environment of nearly continuous presentation of luxury ingesta is the circumstance of many women.ingesta is the circumstance of many women.

The possibility that estrogenic compounds could enhance appetite for food and drink opens the question of whether the many The possibility that estrogenic compounds could enhance appetite for food and drink opens the question of whether the many circumstances of increased exposure to high levels of circulating estrogenic compounds (e.g., those induced by medicines, circumstances of increased exposure to high levels of circulating estrogenic compounds (e.g., those induced by medicines, pregnancy, liver malfunction due to excessive intake of alcohol, environmental pollutants or even body fat itself) could contribute pregnancy, liver malfunction due to excessive intake of alcohol, environmental pollutants or even body fat itself) could contribute to the epidemic rise in obesity seen among Americans.to the epidemic rise in obesity seen among Americans.

EV (10 mg/kg) and Intakes of Chocolate Cake Mix

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Estradiol valerate (EV) is a preparation that delivers Estradiol valerate (EV) is a preparation that delivers estradiol for an extended period (1). Large doses of EV estradiol for an extended period (1). Large doses of EV (e.g., 2 mg a female rat) have been shown to produce (e.g., 2 mg a female rat) have been shown to produce selective lesions of the arcuate n. (2). When these doses selective lesions of the arcuate n. (2). When these doses are given to female rats, they dramatically change the are given to female rats, they dramatically change the females’ appetite for alcoholic beverages. During the first females’ appetite for alcoholic beverages. During the first few days after injections, intakes of alcoholic beverages few days after injections, intakes of alcoholic beverages are suppressed. Subsequently, intake is increased (3). are suppressed. Subsequently, intake is increased (3). The single, large dose of EV enhanced intakes for many The single, large dose of EV enhanced intakes for many kinds of alcoholic beverage; enhanced intake endured for kinds of alcoholic beverage; enhanced intake endured for months after the single injection (4,5).months after the single injection (4,5).

To test whether enhanced intakes were specific to To test whether enhanced intakes were specific to alcoholic beverages, we provided EV- and placebo-treated alcoholic beverages, we provided EV- and placebo-treated females an opportunity to take one of two saccharin females an opportunity to take one of two saccharin solutions. One solution (0.25% solution) was taken in solutions. One solution (0.25% solution) was taken in large amounts and the other (a 2.0% solution) was taken large amounts and the other (a 2.0% solution) was taken in only small amounts. The 2.0% solution is presumably in only small amounts. The 2.0% solution is presumably somewhat bitter and, hence, is taken in smaller amounts. somewhat bitter and, hence, is taken in smaller amounts. Rats given EV increased their intakes of the more Rats given EV increased their intakes of the more palatable solution (0.25% solution) and decreased their palatable solution (0.25% solution) and decreased their intakes of less palatable solution (2.0% solution). As with intakes of less palatable solution (2.0% solution). As with the alcoholic beverages, the changed intakes endured for the alcoholic beverages, the changed intakes endured for months after the injections (xx). months after the injections (xx).

The results from the study of saccharin solutions The results from the study of saccharin solutions indicated that EV-treatment did not uniformly increase indicated that EV-treatment did not uniformly increase appetite. Also, EV-treatment does not increase intake of appetite. Also, EV-treatment does not increase intake of ordinary laboratory chow (based on the fact that EV-ordinary laboratory chow (based on the fact that EV-treated rats do not gain weight at a higher rate than treated rats do not gain weight at a higher rate than placebo-controls).placebo-controls).

The present study addressed whether EV-treatment The present study addressed whether EV-treatment modifies intake of a highly palatable, complex ingesta: modifies intake of a highly palatable, complex ingesta: chocolate cake mix batter. chocolate cake mix batter.

Presented at Soc. for Neuroscience meeting Nov., 2003, New Orleans

Fig. 2. Total intake of batter, Fig. 2. Total intake of batter, in terms of grams, for the in terms of grams, for the two groups (Fig. 1). The EV-two groups (Fig. 1). The EV-treated rats took about 11% treated rats took about 11% more grams of batter even more grams of batter even though they weighed less.though they weighed less.

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Fig. 4.Total intake of batter, Fig. 4.Total intake of batter, in grams, for the two groups in grams, for the two groups (Fig. 3), summed across the (Fig. 3), summed across the 8 days of batter presenta-8 days of batter presenta-tion. The females of EV-tion. The females of EV-treatment took more batter. treatment took more batter.