The 5 HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT) increases the food intake of satiated Zucker rats, both lean and obese. Associated with this increased intake are changes in the hypothalamic content of serotonin and its metabolite, 5-HIAA (5-hydroxyindole-3-acetic acid); serotonin is increased while the level of 5-HIAA is decreased. Analysis of individual 5-HIAA/5-hydroxytryptamine (5-HT) ratios, a measure of serotonin turnover indicate that 8-OH DPAT affected serotonin turnover equally and dramatically in both phenotypes. This would be an expected physiological action of an autofeedback mechanism by a 5-HT1A receptor agonist. Dehydroepiandrosterone (DHEA) at doses as low as 10 mg/kg blocks the 8-OH-DPAT-induced increase in food intake but does not alter food intake of control satiated Zucker rats. The mechanism of DHEA's action was investigated by monitoring the steroid's effect on hypothalamic neurotransmitters in this satiated model. DHEA by itself induced some change in 5-HIAA in the obese satiated model but not the lean. 8-OH-DPAT, by itself, dramatically decreased serotonin turnover in either lean or obese rats, and DHEA combined with 8-OH-DPAT did not further change serotonin turnover, suggesting DHEA may work through mechanisms other than monoamines to cause its inhibition of 8-OH-DPAT-induced behavioral effects at such low doses.
- calorie intake
our laboratory explores the actions of dehydroepiandrosterone (DHEA) in Zucker rats in this study. This animal is a model of youth-onset, hyperphagic, genetic obesity. DHEA decreases food intake (23) and reduces the degree of obesity in the fatty phenotype (18, 19). Although this effect can be seen clearly in the chow-fed rat (14), it is even more apparent using animals that are consuming a macronutrient selection diet that contains high fat (12). Further, if Zucker rats are fed diets that contain only one major macronutrient at a time (i.e., fat, carbohydrate, or protein), DHEA is most effective at reducing the intake of rats presented the nearly pure fat selection (21).
We hypothesize that the effect of DHEA on food intake and macronutrient selection takes place in the hypothalamus, and much of our data points to a mechanism that involves serotonin (16, 18, 19). First, we found that DHEA increased levels of serotonin in the whole hypothalamus, whereas it decreased the caloric intake of obese female Zucker rats (1). Second, more detailed analysis showed that serotonin was elevated specifically in the lateral hypothalamus (LH) of such animals and decreased within 24 h of discontinuing DHEA (13). Third, when DHEA was given as an intraperitoneal injection to animals consuming chow, serotonin levels increased in the LH of the obese animal but not in other hypothalamic regions (26). An acute intraperitoneal injection of DHEA to obese female Zucker rats that had been consuming a macronutrient selection diet caused an elevation of serotonin and 5-hydroxyindole-3-acetic acid (5-HIAA) in the paraventricular nuclear (PVN) region (20). Finally, DHEA acted synergistically with fenfluramine, a serotonin-releasing drug, to decrease food intake in the Zucker rat (22).
To more clearly implicate and explore an action of DHEA in serotonin-regulated central neurons, we undertook an evaluation of the interaction of DHEA and 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT). It is proposed that the latter agent acts as a somatodendritic 5-hydroxytryptamine (5-HT)1A receptor agonist (5, 7, 27). It also acts at presynaptic receptors to alter serotonin release (27). In so doing, it lowers serotonin release and thus increases food intake. White et al. (25) showed that this effect was dependent upon the fat food preference of obese rats. Importantly, for our purposes, others have already shown that corticosterone, an adrenal steroid, modifies the actions of 8-OH-DPAT (2, 6). Hence, there is an experimental reason to believe that adrenal steroids can interact with this system. In this communication, we report the results of our studies of DHEA and 8-OH-DPAT in the satiated Zucker rat.
MATERIALS AND METHODS
Lean and obese, male and female Zucker rats aged 14–20 wk were obtained from our colony in the Animal Care Facility of Louisiana State University (LSU) Health Sciences Center in New Orleans. Growth and rat chow intake of lean and obese rats from this colony have been reported (14). In these protocols the weights of the lean rats used ranged from 326 to 436 g and that of the obese rats from 490 to 558 g. The rats were housed in a room with a 12:12-h light-dark cycle. The temperature was maintained at 22 ± 1 C°. Our facility is fully accredited by the American Association for Accreditation of Laboratory Animal Care. All experiments reported here were performed according to guidelines and principles of the National Institutes of Health Guidelines of the Use of Experimental Animals. Experimental protocols were approved by the LSU Health Science Center Institutional Animal Care and Use Committee.
The preparation and composition of the three macronutrient selections used in this study have been described (12). One of the selections is 90% (calories) fat, another 90% (calories) protein, and the third is 90% (calories) carbohydrate. The remaining 10% of calories in each selection is made equally of the two other macronutrients. Each selection is supplemented with vitamins and salt. When not consuming the macronutrients, the rats were maintained on Purina Rodent Laboratory Chow no. 5001.
8-OH-DPAT and DHEA were obtained from Sigma. The 8-OH-DPAT was dissolved in 0.9% saline using a homogenizer. The DHEA was suspended in polypropylene glycol (PPG) by using a mortar and pestle and then treated with a sonicator until the DHEA was suspended evenly. Both drugs were administered in a volume of 1 ml/kg.
Protocol 1: Dose-response of 8-OH-DPAT on Zucker rat food intake.
Twelve lean male Zucker rats were placed on the three-bowl, macronutrient selection diet for 4 days. On the morning of the experiment, 1 h before the “lights-on” period, fresh bowls were given. Immediately before the lights-on period began, groups of three rats were given intraperitoneal injections of 8-OH-DPAT in the indicated doses or vehicle. Fresh preweighed bowls were placed in the cage, and the rats were allowed to eat for 4 h. At that point, the bowls were reweighed. The difference between the two weights was taken as the amount of food consumed. This was expressed as calories of each macronutrient. The next day the rats were allowed to eat the three-bowl, macronutrient selection diet undisturbed. On the following day, the experiment was repeated. The rats were randomized and given injections as above, and their food intake was monitored over 4 h, as before. Values from these two behavioral trials were combined (n = 6) and are shown in Table 1 and Fig. 1.
Protocol 2: Interaction of DHEA and 8-OH-DPAT on Zucker rat food intake.
Groups of 16 lean or obese Zucker rats were allowed to become accustomed to the macronutrient selection diet for 3–4 days. On the day of the experiment, fresh, filled bowls of food were placed in each cage 1 h before the lights-on period. Immediately before lights-on, bowls were removed and rats were given injections of vehicle, DHEA, 8-OH-DPAT, or DHEA and 8-OH-DPAT in combination intraperitoneally. The dose of 8-OH-DPAT was always 3 mg/kg body wt. The dose of DHEA in the first trial was 75 mg/kg in the lean rats. For the obese rats, the first dose tried was 25 mg/kg DHEA. All rats in each trial received two injections: either active agent or the appropriate vehicle. Rats were returned to their cages along with fresh, weighed bowls and allowed to eat for 4 h. At that point, bowls were removed and reweighed. The difference was used to calculate the amount of food consumed. Rats were returned to their home cages and allowed to consume the macronutrient diet until the next week when the experiment was repeated again using the next lower dose of DHEA. Rats were randomly assigned to groups. This entire experiment was repeated and the results pooled. Thus values for behavioral trials of each data point are of n = 8.
Protocol 3: Effect of DHEA and 8-OH-DPAT individually or in combination on Zucker hypothalamic neurotransmitters.
Twenty lean and 20 obese male Zucker rats were allowed to become accustomed to the macronutrient selection diet for 3–4 days. On the evening before the experiment, all bowls were checked to be sure that they were filled. On the morning of the experiment, bowls were removed. Rats received intraperitoneal injections of 25 mg/kg DHEA and/or 3 mg/kg 8-OH-DPAT, either individually or in combination. Controls received both vehicles, as did all rats. The rats were not given any food, but water was available. Two hours after the injection, the rats were decapitated rapidly with a guillotine. Brains were removed and hypothalamic regions and raphe identified. These regions were isolated, homogenized, centrifuged, and analyzed for neurotransmitters, according to protocols already reported (16, 17). Because 8-OH-DPAT is a selective serotonin 5-HT1A agonist, we also calculated a ratio of 5-HIAA/5-HT as an index of serotonin turnover. One would expect that 8-OH-DPAT should decrease turnover through its selective 5-HT1A action.
For food intake studies, the one-way ANOVA (Statview, a SAS program, MacIntosh version) was used. Fischer's protected least significant difference (LSD) test was used as a post hoc test. P values ≤0.05 were considered significant.
For the neurotransmitter experiments, a separate analysis was conducted for each neurotransmitter. The model applied was a three-level factorial ANOVA with concentration of neurotransmitter as the dependent variable and treatment, phenotype (obese or lean), and brain region as the three main factors. Initially, the full three-level factorial model was examined, considering all possible interactions for each neurotransmitter. To enhance interpretation and explanation of these factorial models, the models were simplified from the individual neurotransmitter three-level factorial ANOVAs. This simplification was done by reducing the model after inspection of the initial results to include only those effects and interactions that were significant (i.e., those with P < 0.05).
All statistical comparisons given in the results are from the P values of pair-wise protected t-tests conducted on least square means with alpha levels corrected (to a final level of 0.05) for the number of multiple comparisons conducted within the reduced design for each neurotransmitter. Turnover indexes were compared by one-way ANOVA followed by Fischer's LSD. P values less than 0.05 were considered significant.
Initial trials explored the effect of different doses of 8-OH-DPAT on Zucker food intake. The doses evaluated were similar to those used by White et al. (25). Rats that were accustomed to eating the three-bowl macronutrient selection diet had their bowls removed at lights-on on the morning of the experiment and were given an intraperitoneal injection of 8-OH-DPAT. Preweighed bowls were then returned, and the amount of food consumed over the next 4 h was measured. The data from macronutrient intake are shown in Table 1. Total caloric intake is shown in Fig. 1. There is a clear increase in total caloric intake in those rats receiving 1 or 3 mg/kg of 8-OH-DPAT; rats treated with these doses consumed nearly twice as many calories as vehicle-treated controls (Fig. 1). Protein consumption doubled and carbohydrate consumption increased nearly four times. Fat intake in these rats trended upward but missed being significantly different than control. Because it gave the most consistent increase in food intake in satiated rats, the 3 mg/kg dose was used in subsequent experiments.
Table 2 reveals the effect of 8-OH-DPAT and DHEA, alone or in combination, on the caloric intake of satiated lean Zucker rats. Controls received vehicle only. In each trial, 8-OH-DPAT (3 mg/kg) by itself significantly increased caloric intake over that of the control group. DHEA given by itself as an injection (0.025 to 75 mg/kg) under the same circumstances had no effect on basal satiated food intake. However, DHEA had an effect on the caloric intake of 8-OH-DPAT-treated lean male Zucker rats (combination). At 2.5 mg/kg, DHEA blunted the increment due to 8-OH-DPAT; it did not return the value to baseline, but the value of the combination was statistically lower than that of the 8-OH-DPAT-treated rats. At doses of DHEA greater than 2.5 mg DHEA/kg body wt, the amount of food consumed by 8-OH-DPAT/DHEA-treated rats returned to that of the vehicle-treated rats. At a very low dose, 0.025 mg/kg, DHEA had no statistical effect on the 8-OH-DPAT-induced increment.
Table 2 also reports the effects of DHEA and 8-OH-DPAT on the macronutrient selection of the satiated lean Zucker rats. The first column gives the total calories consumed. The second column shows that in these experiments 8-OH-DPAT did increase fat calories and the inclusion of 2.5 mg DHEA/kg body wt reduced the consumption of fat to that of control.
Similar results were found with obese rats, although the trials were conducted over a narrower dose range. Table 2 shows that obese rats treated with 3 mg/kg 8-OH-DPAT consume more calories than vehicle-treated controls. DHEA at a dose of 2.5 mg/kg does not effect this increase, but a dose of 10 or 25 mg/kg, given at the same time, blunts the increase in calories induced by 8-OH-DPAT. DHEA, by itself, at any dose tested did not affect caloric intake. Table 2 shows that 8-OH-DPAT increases carbohydrate consumption of the obese rats and that DHEA blocks this effect at doses of 10 mg/kg or greater. The fact that 8-OH-DPAT stimulated fat intake in lean and not obese rats suggests a basic difference in how these two phenotypes respond in the model of satiation.
Neurotransmitters in three hypothalamic regions, the LH, ventromedial hypothalamus (VMH), and PVN, as well as the raphe, were determined in rats treated with DHEA and 8-OH-DPAT alone or in combination. Control rats were treated with vehicle. These rats, like the ones in the trials used to generate Tables 1 and 2, had been allowed to eat overnight. However, after their injections, these rats were fasted until the end of the experiment when they were killed.
Table 3 gives the P values for the parameters investigated in the ANOVA of the neurotransmitters. As would be expected, there were regional differences in the levels of each neurotransmitter. Phenotype made a difference as well for each of the species analyzed, even when expressed over all treatments, with the exception of the level of serotonin (5-HT). The metabolite of serotonin, 5-HIAA, was very significantly changed across phenotype.
Table 3 also evaluates the effect of treatment on hypothalamic neurotransmitters over all regions and in all rats. DHEA given at a dose of 25 mg/kg does not cause any global change in neurotransmitters compared with vehicle, but 8-OH-DPAT (3 mg/kg) does. The latter affects all analytes except dopamine. The combination of DHEA and 8-OH-DPAT causes differences in all analytes except dopamine compared with vehicle control, but the values overall are not different from those of 8-OH-DPAT alone. Therefore, most of the treatment effect is due to 8-OH-DPAT.
Results in Table 4 reveal the absolute values of each of the neurotransmitters in each specific region for each treatment in both lean and obese rats. Several clear effects of 8-OH-DPAT and some suggestive trends are found. As might be predicted from Table 3, 8-OH-DPAT altered the serotonergic system [i.e., the content of serotonin (5-HT; and its metabolite 5-HIAA)] in most regions (treatment effect). There was a statistically significant difference in either 5-HT or 5-HIAA in the raphe, PVN, and LH (Table 4) in lean rats and in three of the four regions of the obese rats. Further, even in other regions, there was a trend for the values to be higher in the treated rats. At the same time, 8-OH-DPAT caused a fall in the content of 5-HIAA in three of the eight region/phenotype comparisons (Table 4) and caused a similar trend in three others. DHEA, by itself, caused a minor increase in dopamine in the lateral hypothalamus of the lean rat, and in the obese rat, DHEA caused a rise in the levels of 5-HIAA that was significant compared with control in three of four regions. The effect of the combination of 8-OH-DPAT and DHEA on neurotransmitters in many ways is most comparable to the results induced by the former alone. For example, if the value for serotonin was raised by 8-OH-DPAT by itself, then the combination had a raised level. These findings were clearly seen in the VMH, PVN, and raphe of obese rats. There was only one statistically significant difference between the combination and the 8-OH-DPAT alone value: a lower norepinephrine value in the former compared with the latter in the raphe of the obese rat.
Data for 5-HIAA/5-HT ratios, an index of serotonin turnover is observed in Fig. 2. 8-OH-DPAT resulted in dramatic decreases in serotonin turnover compared with vehicle as judged by the ratio of 5-HIAA/5-HT. In all cases, combination of DHEA with 8-OH-DPAT was not different from DPAT alone. DHEA (25 mg/kg) by itself did not cause any consistent changes in serotonin turnover compared with vehicle in lean rats, but it did significantly increase serotonin turnover in the raphe and PVN of obese rats. These data also suggest that changes in serotonin turnover do not mediate DHEA's inhibition of the 8-OH-DPAT behavioral changes.
DHEA is the dominant steroid hormone in the serum of young healthy human adults. In the obese Zucker rat, a model of human childhood onset obesity, exogenous DHEA has a myriad of salutary actions: it reduces insulin resistance, lowers food intake, lowers lipids, and improves immune function (19). There is evidence that DHEA may be important in human disease (17). DHEA is made in the central nervous system of rats and is found in the cerebrospinal fluid of humans (10). It is thus important to understand where and how this steroid works in the central nervous system and its mechanism of action. This paper is one step toward these goals. The approach is to ascertain whether DHEA interferes with a centrally acting agent that enhances food intake in a satiated model and whether there are predictable changes in hypothalamic neurotransmitters that can explain this behavioral interaction.
As would be expected, 8-OH-DPAT increased caloric intake in both lean and obese satiated Zucker rats; at doses greater than 1 mg/kg, there was a clear, statistically significant increment in total calories (Table 1 and Fig. 2). In some trials, fat was increased significantly, while in others, it was only a trend. There appeared to be a fairly consistent increase in the consumption of carbohydrate chow.
8-OH-DPAT by itself also caused changes in the levels of hypothalamic neurotransmitters. There was an increment in serotonin content (Tables 3 and 4), a change that was significant in several regions in both the lean and the obese Zucker rats (Tables 4). At the same time that serotonin is increased by 8-OH-DPAT, there is a trend of decreasing 5-HIAA in the hypothalamus, suggesting that the increment of serotonin is caused by a decreased release into the synaptic clefts and a resultant decrease in metabolism. Voigt et al. (24) reported that at 0.3 mg/kg 8-OH-DPAT reduces serotonin release from the lateral hypothalamus in rats as judged by microdialysis. Shimizu et al. (15) used the same technique to show that 1 mg/kg 8-OH-DPAT reduced stress-induced serotonin release from the same area.
DHEA at a dose of 25 mg/kg did not significantly alter food consumption when given alone to satiated rats. In other experiments, we have reported that DHEA decreases food intake, but those trials utilized fasted rats, 24-h food intake models, or 1-h feeding models. In this study, the rats are satiated, having just finished eating fresh macronutrient. Under these conditions, rats eat little over the next 4 h during the lights-on period (unless they are given 8-OH-DPAT). Using such satiated rats, DHEA blocked the behavioral effect of 8-OH-DPAT. Effective doses are as low as 2.5 to 10 mg/kg. This, in itself, is important, for when evaluated in fasted rats, doses of at least 50–100 mg/kg are needed to see an effect. Those doses are so high, that many question the relevance of such findings. Doses of 10 mg/kg, however, are in the range given to humans (11). Thus the approach used in this study may be one of the most sensitive assays of DHEA's central actions on food intake and may provide clues as to the most effective times to use DHEA for its appetite-suppressing potential.
DHEA's effect on hypothalamic neurotransmitters is much less impressive than that of 8-OH-DPAT. Table 3 shows that across all rats, there was a significant treatment effect; however, most of this was due to the action of 8-OH-DPAT. Table 4 shows that this relatively small dose of DHEA does have some limited effects, however. It causes an isolated increase in lateral hypothalamic dopamine in the lean rat (compared with vehicle-treated control), and in the obese rats, an increase in 5-HIAA in three of the four hypothalamic regions was examined. As there is no statistically significant change in serotonin content, our hypothesis for this increase in 5-HIAA is that serotonin production is increased at the same time there is an increased release.
DHEA at very low concentrations blocks the increased food intake caused by 8-OH-DPAT. If monoamine neurotransmitters are responsible for this effect, then one might find a region in which 8-OH-DPAT induces a change, and the addition of DHEA blocks or alters that change. Table 4 shows that there are no overall differences between the values of the 8-OH-DPAT group and the group that received DHEA and 8-OH-DPAT. The only exception might be the rise in serotonin in the lateral hypothalamic region that is seen with 8-OH-DPAT treatment of the obese Zucker rat. This increment is blocked by the administration of DHEA. This site of action (LH) is consistent with some of our past results where we found that DHEA altered serotonin in the LH (26) and with the findings of Voigt et al. (24) and Shimizu et al. (15). Importantly, serotonin levels were not altered in the same area in lean rats, although the trends with the 8-OH-DPAT and DHEA were the same.
One clear argument against DHEA's effect through the 5-HT1A receptor mechanism is revealed in the turnover index in Fig. 2. 8-OH-DPAT, by itself, altered serotonin turnover as measured by the ratio of 5-HIAA/5-HT in both phenotypes and in each area measured. This is exactly what one would expect a 5-HT1A agonist to do. Lower serotonin turnover is compatible with the feedback action of this drug and its combination with a 5-HT1A receptor. In each case, in both lean and obese rats, the combination of DHEA and DPAT did not alter further the turnover index of serotonin compared with DPAT alone. These data are also consistent across phenotype. Therefore, turnover data of serotonin strongly argue for a mechanism, other than monoamines, to explain the behavioral effect of DHEA when combined with DPAT in both lean and obese rats.
Bringing all these results together leads to the following hypothesis: 8-OH-DPAT, through a 5-HT1A receptor-mediated mechanism, decreases serotonin release and turnover in multiple regions of the hypothalamus and raphe. By an action in the hypothalamus or raphe, this leads to an increase in food intake in the present experiments. When DHEA and 8-OH-DPAT are used together in the behavioral paradigm, DHEA at low doses counteracts this effect.
Many alternative possibilities exist to explain the discordance of neurotransmitter action and behavioral action of DHEA. First, it should be noted that 8-OH-DPAT and DHEA both work on the behavior of the lean rat, but such changes in the levels of neurotransmitter content are not seen. There is no clear explanation for this DHEA behavior effect in the lean rat, nor is there a clear difference between the two phenotypes in neurotransmitters under these experimental conditions.
This brings the discussion to potential limitations of the technique used in this paper. Measuring regional content gives important clues as to which neurotransmitters might be important and which regions need to be evaluated. For example, Tables 3 and 4 show that there are differences between the levels of hypothalamic neurotransmitters in the lean and obese rats. However, the technique of measuring content may not be sensitive enough to detect all differences. Perhaps, techniques that measure acute release such as microdialysis may be more appropriate. Likewise, in situ hybridization techniques may provide a better tool for evaluating levels of key synthetic enzymes, and thus production of neurotransmitters. Furthermore, other brain or gut sites may also be affected by DHEA and/or 8-OH DPAT, and this could contribute to the effect on feeding behavior (4, 9). Currently, we are exploring the use of these approaches and other brain areas in our research with DHEA. Alternatively, observation of the turnover data (Fig. 2) derived from individual content data of these monoamines would argue that this assay is sensitive enough. Turnover data suggest that DHEA is working through a mechanism different from monoamines to cause such a profound behavioral effect in the satiated model. The advantage of doing the content studies first is that they provide guidance as to which enzymes and which regions may be most promising first targets. The final limitation of this approach is that it only takes into account biogenic amine neurotransmitters. Because of the profound behavioral effect of DHEA in this paradigm of satiated feeding, we may have to explore alternative mechanisms.
Other modulators of satiety and macronutrient selection like neuropeptide Y (NPY), leptin, or cyclo-His Pro may be affected by DHEA and 8-OH-DPAT, and they would not be seen by this approach. These possibilities need to be evaluated separately. One possible mechanism of DHEA's ability to alter increased food intake in the satiated model could relate to the reciprocal relationship in NPY, which, in turn, inhibits the biosynthesis of the neurosteroid DHEA in the hypothalamus by activating y1 receptors (3). NPY is clearly connected to the serotonergic systems in the brain (8), and thus the drug 8-OH-DPAT may decrease serotonin, which, in turn, may cause in increase in NPY and increase food intake. Perhaps some of this increase in food intake is related to the ability of the NPY to suppress endogenous neurosteroid DHEA SO4. Therefore, when DHEA is administered exogenously, it reverses this balance between endogenous NPY and endogenous DHEA and results in a suppression of the 8-OH-DPAT-induced increase in food intake.
In conclusion, experiments in which the effects of 8-OH-DPAT are evaluated in the presence and absence of DHEA are consistent with the hypothesis that in the obese Zucker rat, DHEA blocks caloric intake by eventually interacting with the serotonergic system (induced by 8-OH-DPAT) in the raphe or hypothalamic regions. That the profound effect of DHEA on the 8-OH-DPAT-induced behavioral assay is not reflected in further individual changes in neurotransmitter content when DHEA is combined with 8-OH-DPAT suggests that DHEA may be working by another mechanism to suppress the feeding behavior induced by 8-OH-DPAT.
This work was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund Gant HEF (2000–05)-04 and American Heart Association Grant Southern Consortium 9808326V. H. Thompson is supported by NEI-EY02377.
We acknowledge the help of S. Yien Oie, a visiting fellow from the Netherlands.
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- Copyright © 2005 the American Physiological Society