Annals of Neurosciences, Vol 14, No 1 (2007)
Annals of Neurosciences, Volume 14, Issue 1 (January), 2007
STUDY OF INTERACTION OF ESTROGEN AND DOPAMINE INJECTED INTO VENTROMEDIAL HYPOTHALAMUS ON CONTROL OF OBESITY IN OVARIECTOMIZED ALBINO RATS
Corresponding author
Dr. GK Pal,
Professor of Physiology,
JIPMER, Pondicherry - 605 006
Phone: Res: 04132341627; Cell: 93446 28827
Office: 04132272381 Extn. 3142
E-mail:
Abstract
Dopamine and estrogen are known anorectic and body weight inhibiting agents. However, at their anorectic doses they also produce widespread metabolic actions. No study has been conducted till now to assess the interaction of these two anorectic neurotransmitters in brain to suggest whether they can be used in combination at low doses without causing significant side effects. Therefore, this study was conducted to assess the interaction of anorectic and body weight inhibiting effects of dopamine and estrogen injected into ventro-medial hypothalamus (VMH), through stereotaxically implanted cannula. Pre- and post-ovariectomy basal 24 h measurements of food intake, water intake and body weight were recorded in 24 female albino rats. Estrogen and dopamine were injected into VMH at different doses (0.5,1, 2 and 4 mg) separately in two different groups of rats to find out their minimum effective dose for production of significant anorexia and body weight loss. Then, they were co-injected at their minimum effective doses into VMH in another group of rats. Ovariectomy resulted in significant increase in food and water intake and body weight, which was reversed by intra-nuclear administration of dopamine and estrogen separately. The minimum dose for both was found to be 1 mg. Combined injection at this dose produced significant anorexia and body weight loss, which was similar to their individual highest effective dose. Thus, this preliminary study indicates that dopamine and estrogen can potentiate their effects even at a lower dose when administered in combination indicating a future implication of these two chemicals in obesity management.
Key Words: Dopamine, estrogen , Food intake, Water intake, Body weight, Ventromedial nucleus.
Introduction
In general population, prevalence of obesity is considerably more in females (36%) in comparison to males (15%) (1). One reason for the gender difference in obesity may be the variation in gonadal hormone concentration throughout a women's life which predisposes her to excess weight gain (2). Change in body composition at menopause may be due to decrease in circulating estrogen and a relative increase in the androgen estrogen ratio(3).
In ovariectomized female rats, food intake and body weight increases and substitution of estrogen either centrally (in brain directly) or peripherally, reverses these changes, suggesting that lack of estrogen was the major cause of body weight gain (4,5). Ovariectomy in rats provides a useful model to study the obesity changes in women. It has been reported that estradiol decreases food intake and body weight gain and adiposity by inhibiting the release of hypothalamic neuropeptide-Y, which is a potent stimulator of food intake (6). Studies have shown that estrogen also interacts with various neurotransmitters in different areas of the brain to elicit its anorectic effects (7). Basic and clinical researches have revealed that administration of D2 receptor antagonists induces obesity (8–10) and intracerebral injection of dopamine decreases feeding (11–15). Except in mesolimbic areas (16–20), dopamine has been documented to inhibit feeding profoundly (21,22). However, there has been no report available to suggest the actual site of estrogen action in the brain in the control of ingestive behaviours and body weight.
In human beings, many factors play a role in triggering obesity (23,24). It is likely that multiple neurotransmitter systems are involved in the genesis of obesity. Evidence suggests that dopamine is among the target neurotransmitter in the regulation of body weight gain (25). Hypothalamus possesses dopaminergic neurons, and dopamine inhibits hypothalamic expression of neuropeptide Y (NPY), which is a known orexigenic transmitter (6, 26, 27). Genetics studies in human on the involvement of dopamine system in obesity have mainly concentrated on the gene encoding D2 receptors. Positron emission tomography (PET) studies in human obese subjects with BMI between 40 to 60 have shown a significant reduction in D2 receptor availability in the brain (28). In human beings, anorexigenic drugs like amphetamine and cocaine have been tried in the treatment of obesity as they increase extracellular dopamine concentration in brain (29,30). However, the therapeutic benefit of these drugs is precluded by their addictive and psychoactive effects.
Estrogen is a steroid hormone. Steroid hormones have genomic and nongenomic effects. Many of the nongenomic effects are mediated via G proteins in the membrane or via generation of a second messenger (31). It has been investigated that many steroid actions require co-participation of other neurotransmitters and one such possible chemical could be the dopamine (32). Estrogen directly potentiates the dopamine release in the mesolimbic structures in rats (33).
Ventromedial hypothalamus (VMH) is known as the satiety center that inhibits feeding behaviour. Presence of estrogen-sensitive receptors and dopaminergic neurons has been reported earlier in the hypothalamic areas (34). However, no studies have demonstrated the existence of possible interaction between estrogen and dopamine in mediating the ingestive behaviors and body weight in VMH. Understanding these interactions is likely to help us to develop the therapeutic targets for prevention and early intervention for obesity in women. Therefore, in our present study we hypothesized that dopamine might play a significant role in the estrogenic control of food intake and body weight in VMH.
Materials and Methods
A total number of institute-bred 24 adult healthy female albino rate of Wistar strain weighing between 200–300g were taken for the study. All the experiments were performed after obtaining approval of the Research Council and Animal Ethics Committee of Jawaharlal Institute of Post-graduate Medical Education and Research (JIPMER). Animals were housed individually in plastic cages on a 12 h light-dark cycle. They were fed standard rodent chow and fresh tap water available ad lib. Rats were allowed 10 days for habituation in the individual cages before basal measurements were made. Daily food and water intake and body weight were measured for one week to determine their mean 24 h basal recordings.
Ovariectomy and Implantation of Cannula
Following basal measurements, all the rats were ovariectomized bilaterally through a single midline abdominal incision, using sterile surgical technique, under the mixture of ketamine and xylazine anesthesia. 22 G stainless steel cannulae of desired length were implanted on the right side VMH in all groups by stereotaxy using the coordinates of Konig and Klippel, following the standard procedures. After surgery, animals were allowed one week for recovery and post-recovery basal measurements were recorded.
Experimental Protocol
Eighteen rats were divided into 3 experimental groups (6 rats in each) depending on the nature of intervention. Six rats served as control for all experiments that were performed sequentially.
a) Estrogen group: Four different dosages (0.5, 1.0, 2.0 and 4.0 μg; each dose dissolved in 0.5 μl of sesame oil) of estrogen (Sigma, USA) were injected into VMH of all rate on different days and post-injection 24 h food intake and body weight were recorded. The gap between each injection was 48 hours. The minimum effective dose of estrogen was noted. Control rats received 0.5 μl of sesame oil without drug.
b) Dopamine group: Four different dosages (0.5,1.0,2.0 and 4.0 μg; each dose dissolved in 0.5 μl of ethanol) of dopamine (Sigma, USA) were injected into VMH of rate on different days and post-injection 24 h food intake and body weight were recorded. The gap between each injection was 48 hours. The minimum effective dose of dopamine was noted. Control rats received 0.5 μl of ethanol without drug.
c) Coinjection group: The lowest-effective dosages of estrogen and dopamine as noted from above experiments were injected in combination into VMH of all rate. Five such injections were given to each rat on different days and post-injection 24 h food intake and body weight were recorded. The control rate received vehicles (0.5 μl of sesame oil and 0.5 μl of ethanol) without any drug.
After completion of above-mentioned experimental procedures, rats were sacrificed, their brains were fixed, removed and processed for histological study to confirm cannula placement in the VMH, following standard procedures as used in our earlier studies (18, 19). Data were analyzed by t test and one way ANOVA.
Observations and Results
Ovariectomy (Ovx) resulted in a significant increase in food and water intake, and body weight as compared with their pre-Ovx basal (Table 1).
| FI (g) | WI (ml) | BW(g) | |
|---|---|---|---|
| Pre-Ovx basal | 12.20 ± 0.50 | 22.80 ± 0.80 | 218.10 + 3.60 |
| Post-Ovx basal | 20.14 ±0.56** | 26.12 ± 0.74* | 262.80± 4.20** |
* P < 0.05; ** P < 0.0001
Estrogen group: Injection of estrogen dosages (0.5, 1.0, 2.0 and 4.0 μg) into VMH of rats resulted in a dose dependent inhibition of food intake, water intake and body weight. However, the minimum effective dose for significant change in food intake and body weight was found to be 1 μg (Table 2). Water intake changed significantly only at 4 μg dose. Control rats did not show any significant change from their post-Ovx basal level.
Dopamine group: Injection of dopamine dosages into VMH had similar effects (Table 3) as that of estrogen injection as noted in Table 2. However, the effects on body weight were less than the estrogen effects.
Coinjection group: Coadministration of lowest-effective doses of estrogen and dopamine into VMH resulted in very significant decrease in food intake and body weight compared with their individual inhibitory effects on these parameters at the same doses (Table 4).
| EB doses (μg) | FI (g) | WI (ml) | BW(g) |
|---|---|---|---|
| Ovx-basal (0 μg) | 20.40 ± 0.78 | 26.90 ± 0.68 | 268.50 ± 3.40 |
| 0.5 μg | 19.20 ± 0.67 | 26.10 ± 0.82 | 260.30 ± 4.50 |
| 1 μg | 17.16 ± 0.70* | 25.42 ± 0.64 | 248.46± 4.10** |
| 2 μg | 14.80 ± 0.84*** | 24.80 ± 0.69 | 241.10 ± 3.50*** |
| 4 μg | 14.76 ± 0.80*** | 24.04 ± 0.50* | 238.80 ± 4.05*** |
| dF/F | 4,25/11.206 | 4,25/2.815 | 4,25/10.679 |
| P value of entire group | < 0.0001 | 0.0468 | < 0.0001 |
Analysis of data was done by one way ANOVA and Tukey Kramer multiple comparison test.
* P < 0.05; ** P < 0.01; *** P < 0.001. Asterisks indicate the level of difference from the value of Ovx-basal. Ovx-basal means basal 24 hour mean recordings after ovariectomy. There was no statistically significant difference between 2 μg and 4 μg doses. Differences among other doses have not been highlighted, to avoid complexity. No significant change was observed in control rats.
| DA doses (μg) | FI (g) | WI (ml) | BW (g) |
|---|---|---|---|
| Ovx basal (0 μg) | 19.76 ± 0.60 | 25.98 ± 0.72 | 265.40 ± 3.80 |
| 0.5 μg | 18.50 ± 0.64 | 25.30 ± 0.64 | 260.20 ± 4.10 |
| 1 μg | 16.90 ± 0.56* | 24.94 ± 0.68 | 249.40 ± 3.65* |
| 2 μg | 15.20 ± 0.68*** | 23.37 ± 0.78 | 244.64 ± 3.70** |
| 4 μg | 15.08 ± 0.75*** | 23.30 ± 0.70* | 242.42 ± 3.96** |
| dF/F | 4,25/9.941 | 4,25/2.868 | 4,25/8.565 |
| P value of entire group | < 0.0001 | 0.0439 | 0.0009 |
Analysis of data was done by one way ANOVA and Tukey Kramer multiple comparison test.
* P < 0.05; ** P < 0.01; *** P < 0.001. Asterisks indicate the level of difference from values of Ovx-basal. Ovx-basal means basal 24 hour recordings after ovariectomy. There was no statistically significant difference between 2 μg and 4 μg doses. Differences among other doses have not been highlighted, to avoid complexity. No significant change was observed in control rats.
| FI(g) | WI (ml) | BW(g) | |
|---|---|---|---|
| Control | 20.45 ± 0.70 | 26.30 ± 0.76 | 263.20 ± 4.60 |
| 1 μg EB + 1 μg DA | 13.64 ± 0.46** | 23.10 ± 0.64* | 228.80 ± 4.10** |
*P< 0.01; **P< 0.0001
Discussion
A significant increase in food intake, water intake and body weight following bilateral ovariectomy indicates the strong inhibitory effects of ovarian hormones on these physiological parameters. As the degree of inhibition of water intake was less in comparison to food intake and body weight, it is well derived that effect of ovarian hormones is more on feeding and body weight gain than on dipsogenesis. These results are consistent with the results of previous studies (4,5,32).
Injection of estrogen resulted in a dose dependent decrease in food intake and body weight, which indicates that estrogen is a strong anorectic neurotransmitter and there are adequate estrogen receptors in this nucleus. Change in water intake was significant only at the highest dose, indicating the weak estrogenic inhibition of water intake. Injection of dopamine resulted in a similar inhibition in these parameters, indicating that dopamine is equally potent anorectic neurotransmitter in this brain area. However, estrogen effect was more than dopamine effect for inhibition of body weight. Thus, estrogen has a stronger body weight inhibiting effect.
Acting locally within the ventromedial hypothalamus (VMH), perifomical area and arcuate nucleus dopamine has been reported to inhibit feeding (26). Thus, our result is consistent with the results of previous studies. Neuropeptide Y (NPY) is a potent stimulator of food intake. Dopamine inhibits food intake by hypothalamic expression and release of NPY (6). The nongenomic effects of estrogen have been reported to be mediated via G proteins in the membrane or via generation of second messengers (31) that are facilitated by dopamine. It is also clearly suggested that many important steroid actions require co-participation of other neurotransmitters involving hormone actions on cells and one such possible neurotransmitter could be dopamine (32). Also, reports indicate that, estrogen directly potentiates the dopamine release in the mesolimbic structures in rats (33).
Thus, previous studies clearly indicate that dopamine and estrogen share some of the second messengers for their intracellular actions. Though estrogen is a steroid hormone, for some of its nongenomic actions, it utilizes second messengers generated by dopamine. However, to our knowledge, no work has been done to assess whether the effects of estrogen on food intake and body weight are partly mediated through dopamine receptors or second messengers. Also, it has not been clearly defined whether the effects of estrogen on food intake and body weight are partly nongenomic. What appears from the observation of our present study is that effects of estrogen on food intake and body weight are partly nongenomic and partly mediated through dopamine actions.
From the previous discussion, one may possibly conclude that estrogen and dopamine facilitate the anorectic and body weight reducing properties of each other. In the present experiment, coadministration of minimum effective doses of estrogen and dopamine resulted in an extreme degree of inhibition of food intake and body weight, which was even more than the maximum inhibition by their individual highest doses. The changes following coinjections were almost close to their preovariectomy values. These observations indicate that coinjections have more inhibitory effects than the individual injections.
In humans, anorexigenic drugs like amphetamine and cocaine have been tried in the treatment of obesity as they increase extracellular dopamine concentration in brain (29,30). However, the therapeutic benefit of these dopamine agonists is precluded by their addictive and psychoactive effects. Similarly, estrogen at its maximum anorectic and body weight inhibiting dose has severe metabolic effects, therefore, estrogen has not been prescribed for management of obesity. Result of the present study, which highlights the additive effects of dopamine and estrogen in producing a significant inhibition of food intake and body weight at lower doses promises further research to investigate its application in obesity management. Also, work should be done to assess the magnitude of side effects of these chemicals at these doses.
Any centrally acting anorectic agent has to act on VMH, satiety center. As the present study indicates potentiation of the effects of dopamine and estrogen on decreasing body weight, in future studies we plan to assess the effects of these drugs administered peripherally, and whether they can achieve obesity control without perilous side effects. Also, we plan to assess in our future works the receptor subtypes of estrogen and dopamine in mediation of hypophagia and body weight reduction.
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