Background: Mechanisms underlying overeating-induced obesity in post-menopausal woman include functional lack of 17 β-estradiol dysregulating dopamine D2 receptors, thereby inducing food addiction, glucose craving or alcohol dependence through reward circuitry. This study aimed at further understanding 17 β-estradiol and dopamine D2 receptors interferences in the etiology of woman obesity. Method: Seventy-two Wistar female rats weighing 200 - 205 g, individually-housed, were divided into non-ovariectomized control (C = 6 groups) and ovariectomized rats (OVX = 6 groups) which were concurrently subjected to the following treatments: Non-drug-treated (DMSO vehicle), 17 β-estradiol (E2, 5 μg/kg, s.c.), sulpiride (SUL, 20 mg/kg, i.p.), bromocriptine (BR, 0.1 mg/kg, i.p.), E2 + SUL or E2 + BR, designating the 6 constitutive groups of either control or ovariectomy. Within each experimental group, consumption of different solutions (10% alcohol, 10% sucrose and water) as well as food intake and body weight were daily measured, for 10 consecutive days. Results: This study indicated that D2S was a specific inducer of alcohol and food intakes, but reduced sugar consumption. In addition, 17 β- estradiol regulated the body weight set point, modulating D2S functions towards increased food intake at lower weights and decreased food intake at higher weights. D2S met the slow genomic actions induced by 17 β-estradiol. Conversely, D2L inhibited alcohol and food intakes, but induced specifically sugar consumption, thereby regulating blood glucose levels and promoting energy expenditure in reducing body weight. Indeed, 17 β-estradiol exerted a tonic inhibition on D2L which was released by OVX, exacerbating sugar intake and increasing body weight. D2L mediated the rapid metabolic effects of 17 β-estradiol. Conclusion: Our results supported physiological data reporting that activation of the mostly expressed presynaptically D2S-class autoreceptors decreased dopamine release stimulating food intake, whereas activation of the predominantly postsynaptic isoform D2L receptors increased dopamine activity inhibiting food intake. Our studies indicated that 17 β-estradiol acted on the two types of D2 receptors showing opposite functions to equilibrate energy intake vs. expenditure for weight set point regulation. Our data also supported biochemical findings reporting that 17 β-estradiol induced D2 genes transcriptional regulation, thereby involving both types of D2 receptors in the etiology of obesity. The combined dysregulated effects of D2L and D2S receptors, as 17 β-estradiol was lacking, would be causal factors underlying the etiology of obesity.
Obesity as a result of overeating and excessive weight gain, was defined as abnormal or excessive fat accumulation in adipose tissue, thereby becoming a major public health concern. It resulted from a dysfunction of the appetite normal regulation loop where an unbalance occurred between overeating and low energy expenditure, leading to fat accumulation [
One of the main mechanisms involved the dopamine-modulated reward circuits in both normal and pathologic eating behaviors [
Other mechanisms which influenced food consumption and body weight, related to steroidal hormones, in particular the 17β-estradiol (estradiol or E2) in females. There was tangible evidence that the 17β-estradiol decreased food consumption, body weight and fatty tissue [
From literature review, despite of sequential difference between the two isoforms (D2L and D2S), the specific function related to each isoform was poorly understood. Thus, mechanisms by which dopamine D2 receptors induced glucose craving or alcohol dependence were far from being clarified. In addition, mechanisms of 17β-estradiol-modulated D2 receptors to control fluid consumption (e.g. sugar, alcohol and water) and to regulate food intake and body weight were also unknown. For further understanding of these mechanisms, this study investigated the effects of 17β-estradiol without or with cotreatment by either dopamine receptor D2 agonist (bromocriptine), or antagonist (sulpiride) on: 1) daily consumption of different solutions (10% alcohol, 10% sucrose and tap water); 2) daily measurements of food intake and body weight gain, during a 10- day treatment period. Animals had free access to food and different solutions.
Nulliparous females of Wistar rats, bred in our colony, which were 12 weeks old and weighing 200 - 205 grams, were used in our experiments. They were maintained under standard laboratory conditions at an ambient temperature of 30 ± 2˚C, with light/dark cycles of 12 hours each and relative humidity reaching 85 ± 3%. Rats were individually housed in polypropylene cages (27 × 37 × 18 cm) with the floor covered by wood shavings and fed with pellet chow diet and water ad libitum. One week prior to the onset of the tests, they were acclimated to the experimental conditions. All experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.
Drugs and Chemicals used in these experiments were: 17β-estradiol (estradiol or E2), bromocriptine mesylate, sulpiride, dimethyl sulfoxide (DMSO) manufactured by Sigma-Aldrich Chemie GmbH (Eschenstrasse 5, 82,024 Taufkirchen, Germany). DMSO was the solvent used for all drug dilution. Bromocriptine mesylate and sulpiride were used as selective agonist and antagonist respectively, at central dopamine-D2 receptors [
Experiment 1: Control (non-ovariectomized rats)
Non-ovariectomized rats were treated for 10 days respectively with 17β-es- tradiol (5 µg/kg: [
- Group C (control): Six females non-drug-treated, injected with DMSO vehicle.
- Group C + E2 (17β-estradiol): Six females subcutaneously injected (s.c.) with 17β-estradiol (5 µg/kg body wt).
- Group C + BR (bromocriptine): Six females intraperitoneally injected (i.p.) with bromocriptine mesylate (0.1 mg/kg body wt), a D2 agonist.
- Group C + SUL (sulpiride): Six females treated with intraperitoneal injection (i.p.) of sulpiride (20 mg/kg body wt), a D2 antagonist.
- Group C + E2 + BR (17β-estradiol + bromocriptine): Six females treated with concomitant administration of 17β-estradiol (5 µg/kg body wt, s.c.) and bromocriptine (0.1 mg/kg body wt, i.p.).
- Group C + E2 + SUL (17β-estradiol + sulpiride): Six females treated with concomitant administration of 17β-estradiol (5 µg/kg body wt, s.c.) and sulpiride (20 mg/kg body wt, i.p.).
Within each experimental group, drinking solutions (water, 10% sucrose and 10% alcohol) were measured daily during an experimental period of 10 days. Food consumption and body weight were also daily measured in each cage. Drugs injections and the various measurements of weight or food and solutions intakes were performed every day at the same hour, 17:00 pm, corresponding to the beginning of activities, as the rats were nocturnal animals. Solution intake was measured by direct reading of the volume absorbed on graduated bottles, every 24 h. Fluids intake were measured in a four-bottle preference condition. After daily drug injection, the remaining content of the vial (one drug for a non- interchangeable 5 ml vial), as well as drinking solution contained in a 150 ml bottle were refreshed every day.
Experiment 2: Ovariectomy and 17β-estradiol (E2) replacement
Thirty-six (36) nulliparous female rats, individually housed, were divided into 6 treatment groups (6 rats/treatment group). For surgical procedure, rats were immersed in a glass jar and anesthetized by ethyl ether inhalation. Then, the rats underwent bilateral ovariectomy (OVX group) via midline dorsal incision. After ligation, the ovaries were excised from a longitudinal incision made on the dorsolateral body region. The risk of postoperative infection was eliminated by applying an antibiotic cream and betadine alcoholic solution twice weekly for 4 weeks. After 4 weeks of recovery period, all the OVX rats were divided into 6 groups (n = 6/group) including, OVX (six ovariectomized females, non-drug- treated, injected with DMSO vehicle), OVX + 17β-estradiol (E2, s.c.), OVX + Bromocriptine (BR, i.p.), OVX + Sulpiride (SUL, i.p.), OVX + E2 + BR and OVX + E2 + SUL, for 10 consecutive days. Drug administration pattern in OVX rats was identical to that of the control. During the 10-day treatment period, all animals were recorded for daily consumption of different solutions (10% alcohol, 10% sucrose and tap water) and daily measurements of food intake and body weight. Animals had free access to food and different solutions.
The two-way ANOVA was used to compare the effects of treatments on ovariectomy (6 factors) and control (6 factors) × 10-day treatment period (10 factors). Post hoc testing (p’s = 0.05) was carried out using the Protected Least Significant Difference (PLSD) for means comparison [
A two-way ANOVA on water intake (
4.128, p < 0.01], showing water consumption collapse in all ovariectomized groups relative to the control ones. However, there were no treatment x day interactions [F(99, 600) = 0.315, p > 0.99]. Indeed, Post hoc comparisons, using Fisher’s PLSD test (p’s = 0.05) showed a drastic inhibition of water consumption in ovariectomized rats (OVX: 0.68 ± 0.36 ml/rat/day), compared to the control rats (C: 7.78 ± 0.55 ml/rat/day), (p < 0.01), suggesting that 17β-estradiol (E2) exerted a tonic action activating water consumption. Supporting that hypothesis, administration of E2 to the females doubled the mean volume of water ingested daily (C + E2: 13.93 ± 0.68 ml/rat/day), compared to the control (p < 0.01). Likewise, the average daily water intake was amplified in female rats co-administered with either “17β-estradiol + sulpiride” (C + E2 + SUL: 19.55 ± 0.81 ml/rat/day) or “17β-estradiol + bromocriptine” (C + E2 + BR: 16.97 ± 0.77 ml/rat/day) compared to the controls (p < 0.01, respectively). However, water consumption was strongly inhibited in ovariectomized rats, and none of the treatments carried out with 17β-estradiol (OVX + E2: 2.29 ± 0.41 ml/rat/day), sulpiride (OVX + SUL: 0.96 ± 0.39 ml/rat/day) or bromocriptine (OVX + BR: 2.34 ± 0.45 ml/rat/day), could restore water consumption relative to their respective controls (p < 0.01 in the three cases), showing the inability of these drug-mechanisms to support the observed tonic activation of water consumption in OVX rats. The most credible drug-mechanisms supporting the tonic activation of water intake were related to hormone-D2 receptors interactions effects, while comparing the C + E2 + BR group (16.97 ± 0.77 ml/rat/day) to the OVX + E2 + BR group (5.17 ± 0.50 ml/rat/day), (p < 0.01) showing that the E2 replacement partially restored water intake by means of bromocriptine-activated D2 receptors (D2/BR) residual activities, after ovariectomy. Likewise, the C + E2 + SUL group (19.55 ± 0.81 ml/rat/day) compared to the OVX + E2 + SUL group (5.31 ± 0.51 ml/rat/day), (p < 0.01), also indicated that E2 replacement in ovariectomized rats partially restored water intake by means of sulpiride-activated D2 receptors (D2/SUL) residual activities, after ovariectomy. The results indicated that E2 controlled both D2/BR and D2/SUL receptors for activating tonic water intake.
A two-way ANOVA test on daily sucrose solution intake, comparing OVX versus control groups, yielded the main treatment effects [F(11, 600) = 213.03, p < 0.01], with significant changes over days [F(9, 600) = 3.13, p = 0.001], without any significant treatment × day interactions [F(99, 600) = 0.22, p > 0.99], showing an exacerbation of sucrose intake in the six ovariectomized groups relative to their corresponding control groups (
Overall analysis on alcohol intake (
D2/BR receptors only. The last results were confirmed when comparing OVX + BR (0.095 ± 0.12 ml/rat/day) with OVX + E2 + BR (1.07 ± 0.19 ml/rat/day), revealing no synergized actions between BR and E2 (p = 0.533). These observations showed that after ovariectomy, a residual basal activity of the D2/BR receptors persisted, which continued to have a direct and tonic inhibitory action on alcohol consumption, without any synergy with E2. Conversely, the C + SUL group significantly increased its alcohol consumption (8.2 ± 0.33 ml/rat/day) compared to the OVX + SUL group (0.579 ± 0.19 ml/rat/day), (p < 0.01), suggesting that sulpiride receptors functioning necessitated a baseline tone of E2. In addition, alcohol consumption was efficiently inhibited in the OVX + E2 + SUL group (0.18 ± 0.13 ml/rat/day) compared to the OVX + SUL group (0.579 ± 0.19 ml/rat/day), (p < 0.01), showing a residual activity of the D2/SUL receptors which acted synergistically with E2 to inhibit the persisting alcohol intake in OVX rats. Furthermore, control and ovariectomized rats treated respectively with either E2 + SUL or E2 + BR, compared with control and ovariectomized treated with either SUL or BR (p < 0.01 in most cases) indicated that E2 modulated more efficiently D2/SUL receptors in reducing alcohol intake than D2/BR receptors.
A two-way ANOVA on food intake (FI) and body weight (BW), (
D2/BR receptors which increased both FI and BW. Conversely, sulpiride maintained or increased efficiently FI and BW in both control and OVX rats compared to their respective control non-drug treated rats (p < 0.01 for any comparison). In control rats, treatment with E2 + SUL increased exponentially FI and BWT comparatively to SUL treatment (p < 0.01). Inversely, in OVX rats, treatment with E2 + SUL decreased exponentially FI and BWT comparatively to SUL alone (p < 0.01). These results indicated a tonic activation of E2 on the D2/SUL receptor which appeared to be a specific inducer of FI and BW. Indeed, D2/SUL receptors appeared to be more severely inhibited by E2 than D2/BR receptors, while comparing effects of E2 + SUL and E2 + BR with individual treatments of SUL and BR respectively on control and OVX rats (p < 0.01 for any comparison).
Our results indicated that administration of 17β-estradiol doubled the mean volume of water ingested daily in control rats, while sulpiride or bromocriptine administrations did not show any significant effects. However, the average daily water intake was amplified by concomitant administration of either “17β-estradiol + sulpiride” or “17β-estradiol + bromocriptine” in control rats, indicating that 17β-estradiol activated D2 receptors to induce water intake. Conversely, ovariectomy collapsed water intake. Replacement of 17β-estradiol partially restored water intake, demonstrating that 17β-estradiol (E2) exerted a tonic action activating water consumption.
In control rats, 17β-estradiol administration reduced the average volume of a 10% sucrose solution consumed per day. However sulpiride or bromocriptine administrations had no effect on sucrose intake. Under hormonal induction of 17β-estradiol, sulpiride reduced more significantly sucrose intake than bromocroptine in the controls. Indeed, sulpiride actions on sucrose intake were E2-dependent, because the drug administered to OVX rats had no effect on sugar appetite. Conversely, ovariectomy exacerbated sucrose consumption, expressing a 17β-estradiol inhibitory tone on sugar intake. Likewise, bromocriptine alone significantly increased sugar consumption in OVX rats, demonstrating that bromocriptine is a specific inducer of sugar intake. However, 17β-estradiol replacement in OVX rats did not restore sugar consumption to a normal level. Indeed, E2 exerted a tonic inhibition on the D2/BR receptors which was released by OVX.
In our studies, administration of 17β-estradiol alone did not influence significantly alcohol intake in control female rats. However, ovariectomy collapsed alcohol intake indicating that 17β-estradiol exerted an activating tonus on baseline alcohol consumption. Replacement of 17β-estradiol generated a residual alcohol intake in OVX rats. In control female rats, sulpiride administration induced excessive alcohol intake, while bromocriptine treatment reduced efficiently alcohol consumption. However, E2 controlled both sulpiride and bromocriptine activities to reduce alcohol intake in physiological conditions. Indeed, our results indicated that E2 modulated more efficiently sulpiride activities in reducing alcohol intake than bromocriptine, in both control and OVX rats. For instance, E2 administered to OVX rats revealed no synergized actions with BR to reduce alcohol intake, while such a synergy was exhibited with SUL. Rather, bromocriptine inhibited specifically alcohol intake in both control and ovariectomized rats independently from E2.
Our results indicated that OVX increased food intake (FI) and body weight (BW) relative to the control. Conversely, E2 administration to both OVX and control females decreased efficiently FI and BW. Similarly, bromocriptine administered to the female rats reduced severely FI and BW in both control and OVX rats. OVX abolished the tonic inhibition of E2 on D2/BR receptors which increased both FI and BW. Conversely, sulpiride increased efficiently FI and BW in both control and OVX rats. In control rats, treatment with E2 + SUL increased exponentially FI and BWT. Inversely, in OVX rats, treatment with E2 + SUL decreased exponentially FI and BWT. These results assigned to E2 a main modulatory role on D2/SUL receptors regulating both FI and BW.
Following our experimental design, female rats had free access to three conditions of solutions intakes (e.g. 10% sucrose, 10% alcohol and water). In these conditions, administration of 17β-estradiol to control females substantially increased water intake. It appeared that direct activation of dopamine D2 receptors by bromocriptine or blockade of these D2 receptors by sulpiride did not influence significantly water intake in control rats. However, 17β-estradiol powerfully amplified water intake (2 - 2.5 times) in co-administration with either sulpiride or bromocriptine which individually had no effect on water consumption in control rats. Previous studies reported that both systemic [
Indeed, previous studies involved estrogen in genomic mechanisms underlying the cyclicity of water intake in female rodents [
While ovariectomy collapsed water intake, in the contrary, it exacerbated sucrose intake, showing a 17β-estradiol inhibitory tone on sugar consumption. The drugs mechanisms supporting the 17β-estradiol inhibitory tone on sugar consumption were related to hormone-D2 receptors interactions effects. Indeed, bromocriptine increased 3-fold sugar consumption in ovariectomized rats, indicating that 17β-estradiol exerted a tonic inhibition on the D2/BR receptors which was released by ovariectomy. Thus, D2 receptors-binding bromocriptine (D2/BR) appeared to be specific inducers of sugar intake. Conversely, sulpiride failed to show any significant effect on sucrose intake, either in control or ovariectomized rats. In addition to the permanent inhibitory role played by 17β-estradiol on sucrose intake, it appeared also that under hormonal induction of 17β-estradiol, both D2/BR and D2/SUL receptors reduced efficiently sucrose intake. 17β-estradiol involvement in reduced sucrose intake was currently reported previously. Thus, Kenney and Redick [
Moreover, alcohol intake was powerfully increased in control rats, following sulpiride administration, contrary to bromocriptine that reduced it. The 17β-estradiol which had no effect on alcohol consumption, specifically induced both D2/BR and D2/SUL isoforms enhancing inhibition of alcohol intake in non-ovariectomized rats. In particular, 17β-estradiol counteracted efficiently the effects of D2/SUL-induced excessive alcohol drinking, while it acted synergistically with D2/BR to inhibit alcohol intake in control females. In the same control females, D2/SUL isoform primarily decreasing sugar consumption, is going to amplify alcohol consumption; D2/BR receptors were acting exactly in the opposite way. These observations highlighted the divergent nature of the two isoforms. Our results were consistent with the findings of Bulwa et al. [
Our results also showed that the consumption of alcohol collapsed in the ovariectomized group showing that 17β-estradiol exerted a tonic action facilitating the basic consumption of alcohol. Priddy et al. [
Investigating body weight and food intake, daily administration of sulpiride and bromocriptine to female rats showed opposite effects. In control rats, sulpiride significantly increased food intake and body weight, while bromocriptine caused a significant decrease in food intake and body weight. These results demonstrated the direct involvement of dopamine D2 receptors in the regulation of food intake and body weight. Previous studies indicated that D2 receptor KO mice showed reduced food intake and body weight along with an increased basal energy expenditure level compared to their wild type littermates [
Ovariectomy increased food consumption and body weight in the present study. Subsequent 17β-estradiol replacement in ovariectomized rats blockaded food consumption and reduced their weight, confirming that 17β-estradiol regulated food intake and body weight. Literature has recurrently involved the 17β-estradiol in reduced food intake and body weight [
Collectively, our studies highlighted a direct involvement of 17β-estradiol in the regulation of body weight set point, either controlling sucrose intake via D2/BR receptors (D2L) for glucose homeostasis and energy expenditure, or modulating food intake via D2/SUL receptors (D2S) for regulating body weight. From these observations, the effects of D2/SUL receptors (D2S) appeared to meet the slow genomic actions induced by 17β-estradiol, whereas D2/BR receptors (D2L) effects met the rapid non-genomic actions of 17β-estradiol, thereby involving both types of D2 receptors in the etiology of obesity. Indeed, removal of the ovaries produced an efficient and lasting elevation in food reward and food-seeking behavior, suggesting that ovarian sex steroids were critical for the maintenance of normal food reward behavior [
A.B designed the study and wrote the manuscript; S.S., B.B., L.B and S.-V.G. performed experiments.
The authors have no conflict of interest to declare relating this work.
Bâ, A., Silué, S., Bamba, B., Bamba, L. and Gahié, S.-V. (2018) Effects of Ovariectomy and 17β- Estradiol Replacement on Dopamine D2 Receptors in Female Rats: Consequences on Sucrose, Alcohol, Water Intakes and Body Weight. Journal of Behavioral and Brain Science, 8, 1-25. https://doi.org/10.4236/jbbs.2018.81001