effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

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BEHAVIORAL BIOLOGY 19, 417-423 (1977), Abstract No. 6255 BRIEF REPORT Effects of Estrogen and Prolactin on Ovariectomy- Induced Hyperphagia and Weight Gain in Female Rats 1 ALISON FLEMING 2 Department of Psychology, Erindale College, University of Toronto, Mississauga, Ontario L5L 1C6 The hypothesis that prolactin can antagonize the suppressive effects of estrogen on food intake and body weight was tested. In the first experiment, ovariec- tomized virgin females were injected with either estradiol benzoate (EB), EB and prolactin, or oil. Oil-injected animals gained significantly more weight than did the two EB-injected groups. Oil-treated animals and animals receiving both EB and prolactin ate more than animals injected with EB alone. In the second experiment, ovariectomized and sham-operated animals were injected with either prolactin or saline. Both ovariectomized groups gained weight as did the intact females in- jected with prolactin. Differences among these groups were not significant. These studies suggest that, while exogenous prolactin has weight enhancing properties in the intact animal, in ovariectomized and estrogen treated animals, prolactin ef- fects are not present. Nonlactating female rats respond to ovariectomy by elevating the body weight around which they regulate their energy balance (Kakolewski, Cox, and Valenstein, 1968; Wade, 1972, 1976). Exogenous estradiol ben- zoate (EB) precludes this elevation in the body weight set-point (Zucker, 1972; Wade, 1972; Tartellin and Gorski, 1973; Redick, Nussbaum, and Mook, 1973; Landau & Zucker, 1976). Lactating animals nursing large litters do not increase their body weight after ovariectomy (Fleming, in press). The absence of an effect of ovariec- tomy was explained by the fact that these animals secrete very low levels of estradiol (Rothchild, 1960), and the removal of the ovaries probably does not alter the endogenous levels of estradiol sufficiently to influence 1 This research was supported by a postdoctoral Fellowship MH-42175 from the National Institute of Mental Health awarded to A. Fleming and by a Faculty Research Grant from the University of Toronto. The first experiment was done at the University of California, Berkeley, California; the second experiment was done at the University of Toronto, On- tario, Canada. I am grateful to Darlene Frost and Eileen Foley for technical assistance and to Maryann Wells for her help with the manuscript. The prolactin was generously supplied by N.I.H. Endocrinology Study Section. The estradiol benzoate was obtained from the Schering Corp., Bloomfield, New Jersey. 2 To whom requests for reprints should be addressed. 417 Copyright © !977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0091-6773

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Page 1: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

BEHAVIORAL BIOLOGY 19, 417-423 (1977), Abstract No. 6255

BRIEF REPORT

Effects of Estrogen and Prolactin on Ovariectomy- Induced Hyperphagia and Weight Gain in Female Rats 1

ALISON FLEMING 2

Department of Psychology, Erindale College, University of Toronto, Mississauga, Ontario L5L 1C6

The hypothesis that prolactin can antagonize the suppressive effects of estrogen on food intake and body weight was tested. In the first experiment, ovariec- tomized virgin females were injected with either estradiol benzoate (EB), EB and prolactin, or oil. Oil-injected animals gained significantly more weight than did the two EB-injected groups. Oil-treated animals and animals receiving both EB and prolactin ate more than animals injected with EB alone. In the second experiment, ovariectomized and sham-operated animals were injected with either prolactin or saline. Both ovariectomized groups gained weight as did the intact females in- jected with prolactin. Differences among these groups were not significant. These studies suggest that, while exogenous prolactin has weight enhancing properties in the intact animal, in ovariectomized and estrogen treated animals, prolactin ef- fects are not present.

Nonlactating female rats respond to ovariectomy by elevating the body weight around which they regulate their energy balance (Kakolewski, Cox, and Valenstein, 1968; Wade, 1972, 1976). Exogenous estradiol ben- zoate (EB) precludes this elevation in the body weight set-point (Zucker, 1972; Wade, 1972; Tartellin and Gorski, 1973; Redick, Nussbaum, and Mook, 1973; Landau & Zucker, 1976).

Lactating animals nursing large litters do not increase their body weight after ovariectomy (Fleming, in press). The absence of an effect of ovariec- tomy was explained by the fact that these animals secrete very low levels of estradiol (Rothchild, 1960), and the removal of the ovaries probably does not alter the endogenous levels of estradiol sufficiently to influence

1 This research was supported by a postdoctoral Fellowship MH-42175 from the National Institute of Mental Health awarded to A. Fleming and by a Faculty Research Grant from the University of Toronto. The first experiment was done at the University of California, Berkeley, California; the second experiment was done at the University of Toronto, On- tario, Canada. I am grateful to Darlene Frost and Eileen Foley for technical assistance and to Maryann Wells for her help with the manuscript. The prolactin was generously supplied by N.I.H. Endocrinology Study Section. The estradiol benzoate was obtained from the Schering Corp., Bloomfield, New Jersey.

2 To whom requests for reprints should be addressed.

417 Copyright © !977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0091-6773

Page 2: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

418 ALISON FLEMING

further the rate of body weight gain. If this explanation is correct, one would expect EB to prevent the body weight gain as it does after ovariec- tomy in the nonlactating animal. However, lactating animals nursing large litters also show no reduction in their rate of weight gain during treatment with EB (Fleming, in press).

Nursing animals secrete high levels of prolactin which may compete with or antagonize the effects of estradiol on body weight (Grosvenor and Turner, 1957, 1958). Prolactin is known to stimulate an increase in food intake and body weight in nonlactating rats (Pfaff, 1969; Leon, 1974). Also, Wade and Zucker (1970) report that the hyperphagia and rapid weight gain of prepubertal female rats are also not depressed by EB. In this case, however, the presence of high endogenous levels of another metabolic pituitary hormone, growth hormone, was found to prevent the estrogen effect (Wade, 1976).

The present study was designed to test the hypothesis that, during lactation, prolactin could be antagonizing the suppressive action of ex- ogenously administered estrogen on body weight gain. In the first study, food intake and body weight were measured in ovariectomized virgin animals treated with either EB alone, EB in combination with prolactin, or saline and oil. In the second study, body weights were measured in ovariectomized and intact virgin animals injected with either prolactin or saline.

Taken together, these studies show, that while intact animals injected with prolactin undergo a weight gain, prolactin is neither able to prevent the EB suppression of weight gain nor to significantly enhance weight gain in ovariectomized animals.

Virgin female Sprague-Dawley rats were used. Unless otherwise indi- cated, they weighed between 250-300 g at the time of mating.

On arrival from the Holtzman Company (Madison, Wisconsin), animals were housed individually in 8 x 8 x 12-in. cages in a room maintained on an LD 12:12 cycle (lights on at 0900 hr daily) and at a temperature of 23°C. Cages were equipped with a Wahmann feeder (LC-278) and a graduated Richter water tube, and animals were permitted ad lib access to both food and water. A nutritionally balanced powdered diet (S/L white diet, 24% protein, 6.7% fat) obtained from the Simonsen Laboratories was used in all experiments. Measurements of body weight (to the nearest 1.0 g) and food intake (to the nearest 0.1 g) were obtained daily between 0900 to 1100 hr.

Effects of prolactin in combination with EB on food consumption and body weight in ovariectomized animals. After 7 days of collecting baseline food intake and body weight data, virgin rats were ovariec- tomized under ether anesthesia, and, beginning 2 days after surgery, they were injected (sc) daily for 14 days with either EB (1/xg/0.1 ml) in sesame oil vehicle (n = 6), EB and ovine prolactin (0.65 mg(15 IU)/0.1 ml)

Page 3: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

ESTROGEN, PROLACTIN, AND ENERGY BALANCE 419

dissolved in saline (n = 8), or oil and saline (0.1-ml volume) (n = 7). Prolactin and saline were injected twice daily, between 0900 and 1100 hr and between 1700 and 1900 hr [daily prolactin dose = 1.3 mg(30 IU)]. EB and oil were injected once daily, between 0900 and 1100 hr.

For each group, Wilcoxen matched-pairs signed-ranks tests were com- puted comparing body weights recorded on the day before and on the fifteenth day after the initiation of the 14-day injection schedule. Differ- ences between groups in weight gain over the 14-day period were analyzed using the Mann-Whi tney U test.

For each group, Wilcoxen tests were used to compare mean food intake scores obtained on the 2 days prior to ovar iectomy and both Days 9 and 10 and 13 and 14 after initiation of the injection series. Differences between groups in food intake on Days 9 and 10 and 13 and 14 of the injection regimen were analyzed using the Mann-Whi tney U test. All differences achieved significance at the P < 0.05.

All three groups underwent a significant weight gain over the 14-day injection period; however , ovariectomized oil-injected (ovx-oil) animals gained significantly more than did either ovariectomized EB-injected (ovx + EB) or ovar iectomized EB + prolactin-injected (ovx + EB + prolactin) groups. Although animals injected with both estradiol and prolactin gained more weight than animals receiving estradiol alone, these differ- ences were not significant (Fig. 1).

Ovx-oil animals ate significantly more food on Days 9 and 10 of the injection period than they did prior to ovariectomy. Increments in food intake over this same interval were not significant in the two hormone injected groups. By Days 13 and 14, food intake had declined somewhat, and differences in food consumption prior to ovar iectomy and on Days 13 and 14 were not significant.

When the three groups were compared for food intake on Days 9 and 10 of the injection schedule, ovx-oil animals ate significantly more than the ovx-EB group, but did not differ from the ovx + EB + prolactin females. Also ovx + EB + prolactin animals ate significantly more than animals receiving only EB (Fig. 2). Comparisons among the three groups for food intake on Days 13 and 14 were no longer significant.

Effects of prolactin on body weight gain in intact and ovariectomized animals. Virgin female Sprague-Dawley rats obtained from the Sprague-Dawley breeding farms, Madison, Wisconsin were used. Ani- mals weighed between 250-300 g at the start of the experiment. General procedures are the same as described before, with the following excep- tions. The room was maintained on a reverse LD 12:12 cycle (lights on at 1900 hr daily), and measurements of body weights were obtained daily at 1300 hr. Food intake measurements were not taken. Virgin rats were either ovariectomized (n = 10) or sham-ovariectomized (n = 11). Begin- ning 2 days after surgery, five ovariectomized (ovx ÷ prolactin) and six

Page 4: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

420 ALISON FLEMING

5O

45

,,5 "~ 30

25

'~ 20

.m 10

5 IE

OVX OVX OVX + + +

OIL EB + ProL EB

Groups

FIG. I. Mean body weight gain over a 14-day injection period after ovariectomy.

sham-operated (sham + prolactin) animals began a 9-day series of daily subcutaneous injections of 1 mg of prolactin in a 0.1-ml volume, injected between 1200 and 1300 hr. Five ovariectomized females (ovx + saline) and five sham-operated females (sham + saline) received a saline vehicle injected subcutaneously in a 0. l-ml volume.

A

s

4

i 3 ._= g 2

1

0

- 1 OVX OVX OVX + O~ EB + Prol. EB

Groups

FIG. 2. Mean change in food intake during the first 10 days of a 14-day injection period after ovariectomy.

Page 5: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

ESTROGEN, PROLACTIN, AND ENERGY BALANCE 421

Daily body weight measurements were taken throughout the 9-day injection period. For each group, Wilcoxen matched-pairs signed-ranks tests were computed comparing body weight on the first and tenth days after the initiation of the daily injections. Differences in weight gain between groups were analyzed using the Mann-Whitney U test. Results were considered significant when P values were less than 0.05.

Both ovariectomized groups showed a significant weight gain over the 10-day period. Differences between the two groups in the extent of weight gain were not significant. However, by the tenth day after the initiation of injections, both ovariectomized groups had gained significantly more

• weight than had saline-injected sham controls. Sham-prolactin animals also underwent a significant weight gain during this period. By Day 10, these animals had gained more weight than had the sham-saline females; however, this difference only approached significance (P < 0.07). Al- though sham-prolactin animals gained less than the two ovariectomized groups, these differences were not significant. Also, when the two prolac- tin injected groups were compared for their weight gain, no differences were apparent.

While prolactin itself has weight enhancing properties, it is not able to antagonize the suppressive effects of EB on body weight. The weight enhancing effects of prolactin also do not seem to be additive with the ovariectomy-induced weight gains. Ovariectomized animals receiving prolactin gained no more weight than did ovariectomized animals receiv- ing saline. From these data, it seems that prolactin is able to stimulate a weight gain if animals are intact and cycling; however, in animals who are either ovariectomized and/or who have been injected with estrogen, pro- lactin effects are not in evidence.

If prolactin is not conferring a refractiveness to EB during lactation, why then is estradiol ineffective in suppressing weight gain in the lactating

FIG. 3.

40

35 m v

+ .~ 2E

21]

| OVX SHAM OVX SHAM

+ + + +

Saline Saline Prol. Prol,

Groups

Mean body weight gain over a 9-day injection period after surgery.

Page 6: Effects of estrogen and prolactin on ovariectomy induced hyperphagia and weight gain in female rats

422 ALISON FLEMING

female? The observation that lactating animals nursing small litters gain weight more rapidly than those nursing large litters suggests an alternate mechanism for the lactation-induced refractoriness to estrogen effects (Fleming, in press). According to this, the lactating animal, who secretes very low levels of estrogen, undergoes an elevation in her body weight set-point in much the same way as does the virgin ovariectomized animal (see Wade, 1976). However, unlike her virgin counterpart, the animal nursing a large litter expends a lot of energy in the form of milk produc- tion, and so is not able to lay down sufficient fat to appreciably elevate body weight. Thus, she remains chronically below her elevated set-point. Virgin ovariectomized animals who are prevented from gaining weight also do not respond to estradiol with a decline in body weight (Mook, Kenney, Roberts, Nussbaum, and Rodier, 1972; Zucker, 1972; Redick et al., 1973; Landau and Zucker, 1976). Apparently, estradiol is only effec- tive in animals facing either real or impending obesity (see Wade, 1976). However, in the case of the lactating animal nursing a small, as opposed to a large litter, the demands of lactation are reduced and animals may gain considerably more weight.

If lactating animals do undergo an elevation in body weight set-point and if animals with small, as opposed to large, litters more easily ap- proach that set-point, one would expect EB to effectively reduce the rate of weight gain in the animal with a small litter. There is some evidence suggesting that this may be the case (Fleming, in press). However, to specifically test the hypothesis that lactating animals undergo an elevation in body weight set-point, it would be necessary to demonstrate that lactating animals forcibly elevated above the hypothesized set-point would reduce their ad lib food consumption so as to realign their body weights with their set-point level.

REFERENCES Fleming, A. Ovarian influences on food intake and body weight regulation in lactating rats.

Physiol. Behav., in press. Grosvenor, C., and Turner, C. (1957). Release and restoration of pituitary lactogen in

response to nursing a stimuli in lactating rats. Proc. Soc. Exp. Biol. Med. 96, 723-725. Grosvenor, C., and Turner, C. (1958). Pituitary lactogenic hormone concentration and milk

secretion in lactating rats. Endocrinology 63, 535-539. Kakolewski, J., Cox, V., and Valenstein, E. (1968). Sex differences in body weight changes

following gonadectomy of rats. Psychol. Rep. 22, 547-554. Landau, T., and Zucker, I. (1976). Estrogenic regulation of body weight in the female rat.

Horm. Behav. 7, 29-39. Leon, M. (1974). Maternal Pheromone. Physiol. Behav. 13, 441-453. Mook, D., Kenney, N., Roberts, S., Nussbaum, A., and Rodier, W. (1972). Ovarian-

adrenal interactions in regulation of body weight by female rats. J. Cornp. Physiol. Psychol. 81, 198-211.

Pfaff, D. (1969). Sex differences in food intake changes following pituitary growth hormone or prolactin injections. "Proceedings, 77 Annual Convention, A,P.A.," pp. 211-212.

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ESTROGEN, PROLACTIN, AND ENERGY BALANCE 423

Redick, J., Nussbaum, A., and Mook, D. (1973). Estradiol induced suppression of feeding in the female rat: Dependence on body weight. Physiol. Behav. 10, 543-547.

Rothchild, I. (1960). The corpus luteum-pituitary relationship. The association between the cause of luteotrophic secretion and the cause of follicular quiescence during lactation. Endocrinology 67, 9-41.

Tartellin, M., and Gorski, R. (1973). Effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinol. 72, 551-568.

Wade, G. (1972). Gonadal hormones and behavioral regulation of body weight. Physiol. Behav. 8, 47-57.

Wade, G. (1974). Interaction between estradiol-17 and growth hormone in control of food intake in weanling rats. J. Comp. Physiol. Psychol. 86, 359-362.

Wade, G. (1976). Sex hormones, regulatory behaviors and body weight. In J. Rosenblatt, R. Hinde, E. Shaw, and C. Beer (Eds.), "Advances in the Study of Behavior," Vol. 5, 1976, pp. 201-279.

Wade, G., and Zucker, I. (1970). Development of hormonal control over tood intake and body weight in female rats. J. Comp. Physiol. Psychol. 70, 213-220.

Zucker, I, (1972). Body weight and age factors determining estrogen responsiveness in the rat feeding system. Behav. Biol. 7, 527-542.