Physiology & Behavior, Vol. 22, pp. 627-629. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
The Effects of Estradiol on Body Weight and Food Intake in Normal Weight VMH-Lesioned Rats M I C H A E L E. T H O M P S O N A N D V E R N E C. C O X
University of Texas at Arlington, Arlington, TX 76019 (Received 3 N o v e m b e r 1978) THOMPSON, M. E. AND V. C. COX. The effects of estradiol on body weight and food intake in normal weight VMH-lesioned rats. PHYSIOL. BEHAV. 22(4) 627--629, 1979.--The effects of estradiol on food intake and body weight were examined in ovariectomized and VMH-lesioned, ovariectomized rats that were prevented from supranormal weight gain by food restriction. Estradiol injections that were effective in reducing weight in supranormal weight, ovariectomized rats had no effect on weight in normal weight, ovariectomized or VMH-lesioned, ovariectomized rats. Estradiol did not prevent hyperphagia and weight gain in VMH-lesioned, ovariectomized rats when they were provided with ad lib food.
Estradiol
Ventromedial nucleus
Hypothalamus
A NUMBER of studies have demonstrated that estrogens can have significant effects on body weight regulation and food intake in rats. Ovariectomy results in a limited period of elevated food intake and chronically elevated body weight [9, 11, 18, 22]. The anorexic and weight suppressant effects of estradiol are modulated by body weight level and are only evident at supranormal weight values [25]. Roy and Wade [18] and Blanstein, Gentry, Roy, and Wade [2] have demonstrated independent effects of estradiol on food intake and weight regulation. Consequently, the effects of estradiol on body weight are not necessarily due solely to the anorexic properties of estradiol. Several investigators have suggested that the effects of estrogens on weight and food intake are mediated via the ventromedial hypothalamic nucleus (VMH), [8, 16, 24, 25]. This area of the hypothalamus is a site of high estradiol uptake [1, 15, 21]. Direct application of estradiol to the VMH results in reduced food intake and body weight in ovariectomized rats [8, 24]. Redick, Nussbaum, and Mook [16] have suggested that estrogenic effects on feeding and weight are mediated by a VMH weight regulatory system. However, lesion studies in our own laboratory and elsewhere have demonstrated that ovariectomy-induced hyperphagia and weight gain can be obtained in VMH-lesioned rats and reversed by estradiol [10, 14, 17]. These findings indicate that the integrity of the VMH is not necessary for estrogenic suppression of food intake at supranormal body weight values. However, the possibility remains that the VMH might be necessary for weight modulation of estradiol-induced anorexia and therefore the attenuation of estradiol-induced
Body weight
anorexia at normal weight values. This hypothesis assumes that estradiol acts somewhere other than the VMH to suppress food intake and that this site is modulated by the VMH in relation to body weight. This assumption is consistent with previous work demonstrating estrogenic suppression of food intake in obese VMH-lesioned rats [10]. The hypothesis would predict that VMH lesions would permit estradiolinduced reduction in food intake and weight when administered to lesioned animals of normal or below normal body weight. Another possibility is suggested by previous work [20] which demonstrated that VMH-lesioned rats will main, tain normal body weight values when restricted to an unpalatable quinine-adulterated diet. If these results extend to another anorexic agent, estradiol, then normal weight VMHlesioned animals should maintain normal body weight values when administered estradiol. Such an outcome would be inconsistent with the position that weight modulation of estradiol-induced anorexia is mediated via the VMH. The validity of the hypothesis would be most easily examined by determining the effects of estradiol on food intake and weight in normal weight VMH-lesioned animals. The present study involves such an approach. METHOD
Animals The subjects were Holtzman female albino rats (245285g). They were housed in individual cages and maintained on powdered Purina lab chow and tap water. A 12-hr light .dark cycle was maintained in the animal quarters.
1This research was supported, in part, by the Organized Research Fund of the University of Texas at Arlington. We would like to thank Jan Stone Weissenburger for her assistance.
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/040627-03502.00/0
628
THOMPSON AND COX 400
Procedure
The experiment employed three groups of animals, ovariectomized (OVX) N = 8 , ovariectomized plus VMH lesions (OVX+VMH) N = 6 , and intact normal animals (CONTROL) N=8. In addition, a group of five animals were ovariectomized prior to the experiment and allowed to reach asymptotic supranormal weights. These animals were used to document the weight suppressant effects of the estradiol dosage used in the experiment. Preoperative measurements of food intake and daily weight gain were obtained for 14 days prior to the day of surgery. On the day of surgery the two surgical groups were anesthetized with Chloropent (Fort Dodge Laboratories) and ovariectomized via bilateral flank incisions. The lesion group also received bilateral lesions of the VMH. With the skull level between bregma and lambda, the tip of the lesion probe was placed 3.0 mm posterior to bregma, 0.5 mm lateral to the midline, and 9.3 mm below the skull surface. The anodal current used was 2.0 ma applied for twenty seconds. Following the day of surgery food rations for the OVX and OVX + VMH animals were re stricted to their presurgical food intake values. These values were then adjusted for all group members to obtain group mean body weight values comparable to those observed in the CONTROL group which continued to have ad lib food. Estradiol benzoate injections (1.5/xg) in 0.1 cc sesame oil were begun at the end of the sixth post-surgical week when the OVX and O V X + V M H groups had mean body weight values within two grams of the CONTROL group for five consecutive days. Once estradiol injections began food rations for the OVX and O V X + V M H animals were fixed at the values employed in the last five pre-estradiol days. Estradiol injections continued for twelve days and during this period CONTROL animals received oil injections and the supranormal weight ovariectomized animals received estradiol injections as well. Following twelve days of food restriction and estradiol injections the OVX and O V X + V M H groups were given ad lib access to food and estradiol injections continued for twelve days. At the conclusion of the experiment OVX + V M H animals were sacrificed with an overdose of anesthetic and perfused intracardially with 0.9% saline and 10% Formalin solutions. Their brains were removed and frozen sections, 60/xm thick, were stained with cresylecht violet and examined microscopically to determine the extent and location of lesions. Six O V X + V M H animals met histological criteria for complete bilateral symmetrical destruction of the cross-sectional area of the VMH at the center of its anterior-posterior extent with minimal damage to adjacent tissue[4] see Fig. 2). RESULTS Figure 1 illustrates the changes in body weight observed in all three groups during the period of estradiol injections with restricted and ad lib food. Data analyses and Fig. 1 include only the six O V X + V M H animals that met histological criteria. During twelve days of estradiol injections and restricted food rations no decline in body weight was observed in the OVX, O V X + V M H , or CONTROL groups, F(2,19)=2.635, p>0.05. All animals in the OVX and OVX + VMH groups consumed all of their restricted food rations during this period. The estradiol benzoate dosage of 1.5/xg was effective in reducing the body weight in the supranormal weight animals that had been ovariectomized prior
m ka.a
3¢: m-~VMH&OVX N-.OVX
200
.,..CONTROL
pTmn*lnl U
mllllll*mml*n,Jlmmn,,m,, RESTRICTED+EB I AD LIB+EB DAYS
FIG. 1. Body weight for five pre-estradiol baseline days, 12 days of restricted food plus estradiol, and 12 days of ad lib food plus estradiol. to the experiment and allowed to achieve asymptotic supranormal weight. This group had a mean weight of 368 g at the beginning of estradiol injections and displayed a significant decline of 29.8 g during the twelve days of estradiol injections (t= 12.78, df = 4, p < 0.05). Following twelve days of food restriction, ad lib access to food was provided for the OVX and O V X + V M H groups and they received estradiol injections for twelve additional days. During this period the O V X + V M H animals displayed a substantial gain in mean weight of 76 g. This weight gain is comparable to that observed in the same period of time in previous studies in our laboratory with otherwise intact VMH-lesioned female rats [10]. An A N O V A of body weight on the last day of ad lib food yielded a significant F value which reflected the weight gain of the O V X + V M H group relative to the other two groups, F(2,19)= 46.78, p
The Effects of Estradiol on Body Weight and Food Intake in Normal Weight VMH-Lesioned Rats M I C H A E L E. T H O M P S O N A N D V E R N E C. C O X
University of Texas at Arlington, Arlington, TX 76019 (Received 3 N o v e m b e r 1978) THOMPSON, M. E. AND V. C. COX. The effects of estradiol on body weight and food intake in normal weight VMH-lesioned rats. PHYSIOL. BEHAV. 22(4) 627--629, 1979.--The effects of estradiol on food intake and body weight were examined in ovariectomized and VMH-lesioned, ovariectomized rats that were prevented from supranormal weight gain by food restriction. Estradiol injections that were effective in reducing weight in supranormal weight, ovariectomized rats had no effect on weight in normal weight, ovariectomized or VMH-lesioned, ovariectomized rats. Estradiol did not prevent hyperphagia and weight gain in VMH-lesioned, ovariectomized rats when they were provided with ad lib food.
Estradiol
Ventromedial nucleus
Hypothalamus
A NUMBER of studies have demonstrated that estrogens can have significant effects on body weight regulation and food intake in rats. Ovariectomy results in a limited period of elevated food intake and chronically elevated body weight [9, 11, 18, 22]. The anorexic and weight suppressant effects of estradiol are modulated by body weight level and are only evident at supranormal weight values [25]. Roy and Wade [18] and Blanstein, Gentry, Roy, and Wade [2] have demonstrated independent effects of estradiol on food intake and weight regulation. Consequently, the effects of estradiol on body weight are not necessarily due solely to the anorexic properties of estradiol. Several investigators have suggested that the effects of estrogens on weight and food intake are mediated via the ventromedial hypothalamic nucleus (VMH), [8, 16, 24, 25]. This area of the hypothalamus is a site of high estradiol uptake [1, 15, 21]. Direct application of estradiol to the VMH results in reduced food intake and body weight in ovariectomized rats [8, 24]. Redick, Nussbaum, and Mook [16] have suggested that estrogenic effects on feeding and weight are mediated by a VMH weight regulatory system. However, lesion studies in our own laboratory and elsewhere have demonstrated that ovariectomy-induced hyperphagia and weight gain can be obtained in VMH-lesioned rats and reversed by estradiol [10, 14, 17]. These findings indicate that the integrity of the VMH is not necessary for estrogenic suppression of food intake at supranormal body weight values. However, the possibility remains that the VMH might be necessary for weight modulation of estradiol-induced anorexia and therefore the attenuation of estradiol-induced
Body weight
anorexia at normal weight values. This hypothesis assumes that estradiol acts somewhere other than the VMH to suppress food intake and that this site is modulated by the VMH in relation to body weight. This assumption is consistent with previous work demonstrating estrogenic suppression of food intake in obese VMH-lesioned rats [10]. The hypothesis would predict that VMH lesions would permit estradiolinduced reduction in food intake and weight when administered to lesioned animals of normal or below normal body weight. Another possibility is suggested by previous work [20] which demonstrated that VMH-lesioned rats will main, tain normal body weight values when restricted to an unpalatable quinine-adulterated diet. If these results extend to another anorexic agent, estradiol, then normal weight VMHlesioned animals should maintain normal body weight values when administered estradiol. Such an outcome would be inconsistent with the position that weight modulation of estradiol-induced anorexia is mediated via the VMH. The validity of the hypothesis would be most easily examined by determining the effects of estradiol on food intake and weight in normal weight VMH-lesioned animals. The present study involves such an approach. METHOD
Animals The subjects were Holtzman female albino rats (245285g). They were housed in individual cages and maintained on powdered Purina lab chow and tap water. A 12-hr light .dark cycle was maintained in the animal quarters.
1This research was supported, in part, by the Organized Research Fund of the University of Texas at Arlington. We would like to thank Jan Stone Weissenburger for her assistance.
Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/040627-03502.00/0
628
THOMPSON AND COX 400
Procedure
The experiment employed three groups of animals, ovariectomized (OVX) N = 8 , ovariectomized plus VMH lesions (OVX+VMH) N = 6 , and intact normal animals (CONTROL) N=8. In addition, a group of five animals were ovariectomized prior to the experiment and allowed to reach asymptotic supranormal weights. These animals were used to document the weight suppressant effects of the estradiol dosage used in the experiment. Preoperative measurements of food intake and daily weight gain were obtained for 14 days prior to the day of surgery. On the day of surgery the two surgical groups were anesthetized with Chloropent (Fort Dodge Laboratories) and ovariectomized via bilateral flank incisions. The lesion group also received bilateral lesions of the VMH. With the skull level between bregma and lambda, the tip of the lesion probe was placed 3.0 mm posterior to bregma, 0.5 mm lateral to the midline, and 9.3 mm below the skull surface. The anodal current used was 2.0 ma applied for twenty seconds. Following the day of surgery food rations for the OVX and OVX + VMH animals were re stricted to their presurgical food intake values. These values were then adjusted for all group members to obtain group mean body weight values comparable to those observed in the CONTROL group which continued to have ad lib food. Estradiol benzoate injections (1.5/xg) in 0.1 cc sesame oil were begun at the end of the sixth post-surgical week when the OVX and O V X + V M H groups had mean body weight values within two grams of the CONTROL group for five consecutive days. Once estradiol injections began food rations for the OVX and O V X + V M H animals were fixed at the values employed in the last five pre-estradiol days. Estradiol injections continued for twelve days and during this period CONTROL animals received oil injections and the supranormal weight ovariectomized animals received estradiol injections as well. Following twelve days of food restriction and estradiol injections the OVX and O V X + V M H groups were given ad lib access to food and estradiol injections continued for twelve days. At the conclusion of the experiment OVX + V M H animals were sacrificed with an overdose of anesthetic and perfused intracardially with 0.9% saline and 10% Formalin solutions. Their brains were removed and frozen sections, 60/xm thick, were stained with cresylecht violet and examined microscopically to determine the extent and location of lesions. Six O V X + V M H animals met histological criteria for complete bilateral symmetrical destruction of the cross-sectional area of the VMH at the center of its anterior-posterior extent with minimal damage to adjacent tissue[4] see Fig. 2). RESULTS Figure 1 illustrates the changes in body weight observed in all three groups during the period of estradiol injections with restricted and ad lib food. Data analyses and Fig. 1 include only the six O V X + V M H animals that met histological criteria. During twelve days of estradiol injections and restricted food rations no decline in body weight was observed in the OVX, O V X + V M H , or CONTROL groups, F(2,19)=2.635, p>0.05. All animals in the OVX and OVX + VMH groups consumed all of their restricted food rations during this period. The estradiol benzoate dosage of 1.5/xg was effective in reducing the body weight in the supranormal weight animals that had been ovariectomized prior
m ka.a
3¢: m-~VMH&OVX N-.OVX
200
.,..CONTROL
pTmn*lnl U
mllllll*mml*n,Jlmmn,,m,, RESTRICTED+EB I AD LIB+EB DAYS
FIG. 1. Body weight for five pre-estradiol baseline days, 12 days of restricted food plus estradiol, and 12 days of ad lib food plus estradiol. to the experiment and allowed to achieve asymptotic supranormal weight. This group had a mean weight of 368 g at the beginning of estradiol injections and displayed a significant decline of 29.8 g during the twelve days of estradiol injections (t= 12.78, df = 4, p < 0.05). Following twelve days of food restriction, ad lib access to food was provided for the OVX and O V X + V M H groups and they received estradiol injections for twelve additional days. During this period the O V X + V M H animals displayed a substantial gain in mean weight of 76 g. This weight gain is comparable to that observed in the same period of time in previous studies in our laboratory with otherwise intact VMH-lesioned female rats [10]. An A N O V A of body weight on the last day of ad lib food yielded a significant F value which reflected the weight gain of the O V X + V M H group relative to the other two groups, F(2,19)= 46.78, p
