Physiology & Behavior, Vol. 32, pp. 589-592.Copyright©PergamonPress Ltd., 1984.Printed in the U.S.A.
0031-9384/84$3.00 + .00
Effects of Stereoisomers of Estradiol on Food Intake, Body Weight and Hoarding Behavior in Female Rats T H O M A S P. D O N O H O E , R O B I N S T E V E N S , N E I L J. J O H N S O N A N D S A R A H B A R K E R
Department o f Psychology, University o f Nottingham, University Park Nottingham, U. K. N G 7 2RD Received 31 May 1983 DONOHOE, T. P., R. STEVENS, N. J. JOHNSON AND S. BARKER. Effects of stereoisomers of estradiol on food intake, body weight and hoarding behavior in female rats. PHYSIOL BEHAV 32(4)589-592, 1984.--The effects of 17-alpha and 17-beta estradiol on food intake, body weight and hoarding behavior in ovariectomised rats were investigated. For five days, ten animals received subcutaneous injections of both isomers (10 tzg/kg/day)in a counterbalanced design. Hoarding tests were conducted on the last three days of each 5-day injection period. 17-Alphaestradiol significantly reduced food intake but was without effect on body weight. 17-Betaestradiol reduced food intake significantly more than the alpha form and also significantly reduced body weight. These differential effects suggest that stereoisomers of estradiol may be acting on separate regulatory systems. The treatments did not change hoarding activity compared to pre-treatment levels. Stereoisomers
IN adult female rats, fluctuations of ovarian hormones, whether naturally-occurring or experimentally-induced, can alter bodyweight, body composition and other regulatory behaviors (e.g., food intake, locomotor activity and thermoregulation) . Johnson and Stevens (in press) investigated the effects of 17-alpha estradiol and 17-beta estradiol on locomotor activity and body weight. At a high dose (10 ~g/kg), the beta isomer significantly increased activity and decreased body weight, while the same dose of the alpha isomer increased activity to the same extent, but did not change body weight. It is possible that the differential effects on body weight were secondary to changes in food intake. It has been demonstrated that 17-alpha estradiol has much less affinity for the estrogen receptor than 17-beta estradiol , however little is known about the effects of stereoisomers of estradiol on regulatory behaviors. Thus the present study investigates their effects on both food intake and body weight. Coling and Herberg  have demonstrated that ovarian hormones can influence food-hoarding behavior in female rats. They found that body weights after ovariectomy rapidly increased for a month in paralell with a sharp increase in food hoarding. During the f'tfth and sixth weeks after ovariectomy, when body weights had stabilized at a higher level, hoarding activity reverted to preoperative levels. Daily intramuscular administration of estradiol benzoate (EB) (2.0 t~g or 25/xg/day for five days) significantly decreased body weight and hoarding activity. They concluded that the increased hoarding activity after ovariectomy is indicative of a rise in the defended level of body weight and supports earlier
suggestions that estrogen lowers the physiological setting about which body weight is regulated . Wade  suggests that the primary action of estrogen is to inhibit food intake via the "hypothalamic body weight regulating mechanism" where body weight is "the mass of adipose tissue," i.e., estrogen is thought to reduce food intake by resetting a "neural lipostat," causing a reduction in the mass of adipose tissue and thus a reduction in body weight. Wade and Gray  argue that ovariectomy in the rat causes obesity, and estrogen reduces this "adiposity" by respectively increasing or decreasing adipose tissue lipoprotein lipase (LPI) activity. However, Clark and Tartellin [1,2] think that the effects of estrogen on "adiposity" are more complex than suggested in the Wade and Gray  hypothesis, or in previous models . They suggest that the main effect of ovariectomy is not to increase fat levels, but to redistribute fat to the skin, while estrogen has the opposite effect. Chronic EB treatment increases fat reserves, but slows the growth of other body components, including the axial skeleton (c.f., [13,14]). Thus fat depots cannot be considered homogenous as far as basic metabolism and hormonal control are concerned. The findings suggest that Coling and Herberg's  hypothesis regarding the effects of ovarian hormones on foodhoarding behavior may be too simplistic. Thus the second aim of the present study is to examine whether the decrease in hoarding activity following estrogen treatment is caused by an estrogen-dependent "lowering of the regulated level of body weight." The effects of 17-alpha estradiol and 17-beta estradiol treatment on hoarding behaviour were investigated.
DONOHOE, STEVENS, JOHNSON AND BARKER 25
If hoarding behavior was a result of an estrogen-induced change in body weight, only 17-beta estradiol should decrease hoarding behavior, since only this isomer has been found to reduce body weight.
Subjects and Housing Ten female Wistar rats weighing about 175 g were ovariectomized under ether anaesthesia. They were housed individually and Pilsbury (41B) rodent chow pellets and tap water was available ad lib. Food pellets could be eaten by the rat but not removed from the hopper. The rats were placed on a 12:12 hr reverse light-dark cycle (lights off at 0730 hr) and given tissue paper which they readily shredded to build nests. After a week, they were screened for hoarding for 6 days before the experimental period to ensure that each animal would reliably hoard.
Procedure Food intakes and body weights were measured daily to the nearest 0.1 g at 1500 hr throughout the experiment. Spillage was negligible. Hoarding tests were conducted between 1300 hr and 1500 hr on 3 consecutive days to provide baseline scores. A pyrex dish containing 30 pellets, each weighing 2 g, was placed in an open area of the cage for 10 minutes. Hoarding was scored by the number of pellets removed from the dish during this period. Testing was conducted in illumination from a low-intensity red lamp. After the observation period, the pellets were removed and weighed. The amount consumed was added to the daily food intake totals. Two days later, all animals received 5 daily injections of either 17-alpha estradiol or 17-beta estradiol (Sigma Chemicals, Poole, U.K.) dissolved in sunflower oil to a final concentration of 10 /xg/ml. Treatments were administered between 1600 hr and 1700 hr via subcutaneous injections in the flank to give 10/zg/kg body weight. On the last 3 days of the 5-day injection period, hoarding tests were conducted for each animal as described. Food intake and body weights were recorded for a further 4 days following the injection period. Four weeks later, the procedure was repeated with each animal receiving the alternate treatment. Thus all animals received both treatments but in a counterbalanced order. It is well documented that injections with sunflower oil alone do not affect food intake or body weight [4,7], thus control injections were not repeated in the present study.
Statistics Changes in food intake and body weight were analysed by comparing the means of the 5 day treatment period to the mean of the 3 day pre-treatment food intakes and body weights. Because of the variability of hoarding scores, medians were calculated instead of means . Statistical comparisons were made using Wilcoxon matched pairs tests. RESULTS Figure 1 shows the daily food intakes and body weights before, during and after treatment with 17-alpha estradiol and 17-beta estradiol. Only the beta-isomer reduced body weight over the treatment period compared to pre-treatment body weights (beta-estradiol: T=0, N=10, p<0.01, alphaestradioi: T=24, N = 10, n.s.). Both isomers significantly re-
FIG. 1. Shows the mean daily food intakes and body weights before, during and after treatment with 17-alpha estradiol and 17-beta estradiol. Treatments were administered on Days 4-8 inclusive. (Triangles = 17-alpha estradioi; Circles = 17-beta estradiol.)
duced food intake during the treatment period (T= 1, N = I 0, p<0.01 for alpha-estradiol; T=0. N=10, p<0.01 for betaestradiol). 17-Beta estradiol was significantly more effective than the alpha isomer in reducing food intake (T=3. N = 10. p =0.01). There was'considerable intra- and inter-individual variation in hoarding activity, however neither 17-alpha estradiol nor 17-beta estradioi were found to affect hoarding behavior compared to pre-treatment hoarding scores (T= 16.5, T=25 respectively). The mean daily hoarding scores (derived from each set of median scores) were 4.95 pellets for the pretreatment period, 4.90 pellets during treatment with 17-alpha estradiol and 5.15 pellets during treatment with 17-beta estradiol. DISCUSSION The two isomers of estradiol have differential effects on energy regulation in ovariectomized rats. Although both
STEREOISOMERS OF ESTRADIOL
significantly reduced food intake during the treatment period, 17-beta estradiol was significantly more effective in reducing food intake and only the beta form reduced body weight. Earley and Leonard  concluded that there may be different mechanisms by which estrogens affect food intake and body weight regulation in male rats. The present study suggests that the same is true in female rats. Under physiological conditions, estrogens may influence feeding behavior and energy regulation via multiple and apparently redundant mechanisms. The differential effects of the two isomers of estradiol may be a useful tool in the investigation of what are the primary effects of estrogen on energy regulation. It has been suggested that estrogens may act on central and peripheral sites to reduce food intake. Wade and Gray  proposed a metabolic hypothesis in which the primary influence of gonadal steroids is on enzymatic mechanisms that control fat storage and breakdown. However, our studies have shown that estrogen may act directly on specific neural loci to reduce food intake and body weight . In particular, estradiol can reduce food intake by acting on the ventromedial nucleus of the hypothalamus [VMH] in ovariectomised rats . There are also extrahypothalamic sites where estrogen may act to reduce food intake and body weight since estradiol may decrease food intake and body weight in rats with hypothalamic lesions (but see ). We have demonstrated that estradiol may also act on the corticomedial nuclei of the amygdala to reduce food intake without involving the VMH-Arcuate region or producing vaginal cornification . The effects of beta-estradiol on regulatory behaviors are well documented [4,15], however little is known about the alpha isomer. The present study suggests that 17-alpha estradiol affects the "feeding" mechanism but not the "weight regulating" mechanism, while 17-beta estradiol acts on both systems. 17-Alpha estradiol has much less affinity for the estrogen receptor than 17-beta estradiol and this specificity extends to the central nervous system . Sexual receptivity in rodents is also differentially sensitive to the two isomers  with the beta form possessing greater potency. No conclusions can be drawn from the present study regarding the differential effects of the stereoisomers. It is possible that
17-beta estradiol may act on both central and peripheral sites to reduce food intake and body weight. However, 17-alpha estradiol may act on central or peripheral sites to reduce food intake without affecting the sites involved in weight regulation. To answer these questions further studies are required to investigate the effects of stereoisomers of estradiol on adipose tissue L P L activity and their ability to bind to the V M H and corticomedial nuclei of the amygdala. Only tentative conclusions concerning the effects of stereoisomers of estradiol on hoarding behavior can be drawn since neither treatment produced any change in hoarding behavior compared with the baseline period. Variable hoarding scores are commonly reported  and this variance, even in a within subject design, precluded a significant finding. However, if a reduction in hoarding behavior is a reflection of decreased body weight as suggested by Coling and Herberg , then only the beta isomer should have reduced hoarding behavior. The reduction in hoarding behavior in Coling and Herberg's  study could be attributed to "competing responses" produced by estrogen-induced hyperactivity , particularly since their second does was 25/xg EB per day. Similarly, the increased hoarding behavior after ovariectomy could perhaps be a result of decreased locomotor activity . However, it is unlikely that hoarding behavior is related to levels of " a c t i v i t y " since the doses of each isomer used in the present study increase locomotor activity to the same extent (Johnson and Stevens, in press). It is also possible that hoarding behavior is related to the amount of food ingested in that the pattern of food intake parallels that for hoarding over the estrous cycle, after ovariectomy and following estrogen treatment [3,11]. The present study would not support this hypothesis since both isomers reduced food intake but neither reduced hoarding behavior. However, it is possible that the treatments could not reduce hoarding behavior below the low levels found. Further studies are required to test these hypotheses using a range of doses.
ACKNOWLEDGEMENTS We would like to acknowledge the excellent technical help of Lynne Easom and Teresa Sharp.
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