Agonist diversity in 5-HT2C receptor-mediated weight control in rats
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- Hayashi, A., Suzuki, M., Sasamata, M. et al. Psychopharmacology (2005) 178: 241. doi:10.1007/s00213-004-2019-z
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Food intake and energy expenditure are the two main determinants of body weight. Given that 5-HT2C receptor agonists are reported to have effects on both energy expenditure and food intake, this strongly suggests that 5-HT2C receptor agonists have excellent potential for development as antiobesitiy drugs. One important issue in antiobesity drug development is whether the effects of the compound are maintained during chronic drug treatment.
The purpose of the present study was to investigate the effect of repeated oral administration of three 5-HT2C receptor agonists, m-chlorophenylpiperazine (mCPP), d(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (RO60-0175) and (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348), on food intake and energy expenditure in rats.
In the food intake study, mCPP, RO60-0175 and YM348 decreased food intake in a dose-dependent manner on day 1 of administration. On day 14 of repeated administration, the hypophagic effect of YM348 was lost and that of mCPP was reduced. In contrast, the hypophagic effect of RO60-0175 was maintained even after repeated administration. The hypophagic effects of all agonists were significantly inhibited by a 5-HT2C receptor antagonist, SB242084. In contrast to the hypophagic effects, no drug tolerance developed with respect to the hyperthermic effects of mCPP, RO60-0175, and YM348. The hyperthermic effects of these drugs were also inhibited by SB242084.
Together, the difference between compounds in their hypophagic effects and the similarity in their hyperthermic effects suggest a diversity in agonists in 5-HT2C receptor-mediated weight control in rats.
Keywords(S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348)d(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (RO60-0175)m-Chlorophenylpiperazine (mCPP)Food intakeBody weightBody temperature
Serotonin (5-HT) has been implicated in a variety of behavioral and physiological functions through various 5-HT receptors. 5-HT mediates its effects through at least 14 different subtypes of 5-HT receptor, which are classified into seven major families termed 5-HT1 to 5-HT7 (Boess and Martin 1994; Martin and Humphrey 1994). The activation of one of these subtypes, 5-HT2C, has been reported to mediate various effects, including hypophagia and energy expenditure.
The involvement of 5-HT2C receptors in the control of food intake is suggested by many previous findings. The most commonly used 5-HT2C receptor agonist, m-chlorophenylpiperazine (mCPP), a metabolite of the antidepressant trazodone, is known to decrease food intake in rats (Kennett and Curzon 1988a,b) and mutant mice lacking functional 5-HT2C receptors develop obesity as a result of abnormal feeding behavior control (Tecott et al. 1995). In addition, these 5-HT2C receptor-mutant mice demonstrate a decreased metabolic rate (Nonogaki et al. 2002). Furthermore, mCPP is reported to increase body temperature in rats (Mazzola-Pomietto et al. 1996) and (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348), a novel 5-HT2C receptor agonist (Kimura et al. 2003), is reported to increase both oxygen consumption and rectal temperature in the same time course (Hayashi et al. 2004). Together, these findings suggest that 5-HT2C receptor agonism not only decreases food intake but also increases energy expenditure, which in turn leads to the hyperthermia. Given that food intake and energy expenditure are the two main determinants of body weight and its control, this strongly suggests that 5-HT2C receptor agonists have excellent potential for development as antiobesitiy drugs. One important issue in any such development, however, is whether the effects of the compound are maintained during chronic drug treatment.
It is generally well known that continuous exposure and repeated administration of an agonist can result in desensitization and tolerance. However, discrepancies have been noted in the development of tolerance to 5-HT2C receptor agonists. In in vitro studies, some reports that 5-HT2C receptor is susceptible to agonist-induced desensitization (Akiyoshi et al. 1995; Briddon et al. 1998), although, one report notes that the desensitization of 5-HT2C receptors is agonist-dependent (Stout et al. 2002). In in vivo studies, some reports have shown no reduction in hypophagic effect after the repeated administration of 5-HT2C receptor agonists (Vickers et al. 2003), whereas others have shown the development of partial tolerance after repeated administration of 5-HT2C receptor agonists (Fone et al. 1998; Vickers et al. 2000).
In the present study, the effects of repeated oral administration of three 5-HT2C receptor agonists, mCPP, d(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (RO60-0175) and YM348, on food intake and energy expenditure were examined in rats. mCPP and RO60-0175 are widely used 5-HT2C receptor agonists (Barnes and Sharp 1999; Martin et al. 1998). YM348 is a novel and orally active 5-HT2C receptor agonist, reported to have a high affinity for 5-HT2C receptors (Ki: 0.89 nM) which was 15 and 3 times higher than those for cloned human 5-HT2A and 5-HT2B receptors, respectively, and at least 100-fold selectivity over 46 different neurotransmitter binding sites including other 5-HT receptor subtypes (Kimura et al. 2003).
Materials and methods
Adult male Wistar rats (aged 5–8 weeks; Japan Charles River, Inc.) were housed in an animal room maintained at 21±1°C with a 12-h light cycle (lights on 0700 hours). The animals were maintained on a conventional pelleted stock diet, and fasted overnight before experiments. Each rat was moved to an individual cage for acclimation to the laboratory environment at least 2 h before experiments began. Animals were weighted before the experiment starts and divided evenly to the each group. Different animals were used for temperature and food intake study.
YM348 and RO60-0175 were synthesized at Yamanouchi Pharmaceutical Co., Ltd and mCPP was purchased from Tocris Co., Ltd. All compounds were orally administered once a day dissolved in sterile water at a concentration designed to provide the appropriate dose in 5 ml/kg body weight. Doses of YM348, RO60-0175, and mCPP were selected on the basis of previous studies describing their 5-HT2C-agonistic effects (Wozniak et al. 1988; Mazzola-Pomietto et al. 1996; Clifton et al. 2000; Kennett et al. 2000; Kimura et al. 2003) and our preliminary studies.
SB242084, a selective 5-HT2C receptor antagonist, was purchased from Sigma Co., Ltd, and prepared as a solution in physiological saline containing drops of Tween 80. SB242084 at a dose of 10 mg/kg was given intraperitoneally 0.5 h before agonist administration. The antagonist study was conducted before the tolerance development or the last day of the experiment if no tolerance developed. Doses of SB242084 were chosen primarily on the basis of previous studies (Clifton et al. 2000; Kennett et al. 2000; Vickers et al. 2001; Hayashi et al. 2004).
Measurement of food intake, rectal temperature and body weight
For the food intake study, all animals were fasted exactly 20 h before experiments and then moved to individual cages on the test days. The food pellets for each rat were weighed before administration as a pre-value, then made available at 0.5 h after the administration of 5-HT2C receptor agonists for 1 h. The difference in pellet weight from pre-administration to 1.5 h after administration was then calculated.
Rectal temperature was measured with a rectal thermometer probe for animals (Physitemp, Model BAT-12; Physitemp Instruments, Inc., USA). The probe was inserted approximately 3 cm and held until a plateau temperature was reached. Pre-administration temperature was measured 1 h before 5-HT2C receptor agonists or SB242084 administration and post-administration temperature was measured at 1 h after the administration of agonists. The difference between pre-administration and post-administration temperatures was calculated.
Body weight was measured before and 1 and 2 weeks after repeated oral administration of agonists.
The results are presented as the means±SEM. Rectal temperature and food intake in 2 weeks repeated administration study were analyzed by two-way analysis of variance (ANOVA) where drug was a between-subject factor and time a within-subject factor. Significant interactions were succeeded by the performance of one-way ANOVA at each level of time. Dunnett’s multiple range test was used where appropriate. Rectal temperature and food intake in the antagonist study, and body weight were analyzed by one-way ANOVA followed by Dunnett’s multiple range test or Student’s t-test. A P-value less than 0.05 was considered statistically significant.
Effects of repeated oral administration of mCPP, RO60-0175, and YM348 on food intake
Effect of SB242084 on mCPP-induced, RO60-0175-induced, and YM348-induced hypophagia
Effects on food intake of YM348 after 13 days’ administration of RO60-0175 and of RO60-0175 after 13 days’ administration of YM348
In contrast, animals treated with YM348 (1 mg/kg PO) for 14 days ate a similar amount of food to the vehicle group. RO60-0175 (10 mg/kg PO), administered in place of YM348 on day 14 to animals hitherto treated with YM348 for 13 days, did not decrease food intake in these animals [ANOVA: F(6,26)=25.8 P<0.01, Dunnett’s multiple range test: P=0.5], with this group eating a similar amount of food as those treated with YM348 for the full 14 days (Fig. 3).
Effects of repeated oral administration of mCPP, RO60-0175, and YM348 on rectal temperature
Effect of SB242084 on the mCPP-induced, RO60-0175-induced, and YM348-induced hyperthermia
Effects of repeated oral administration of mCPP, RO60-0175, and YM348 on body weight
In this study, we examined the effects of repeated oral administration of three 5-HT2C receptor agonists, mCPP, RO60-0175, and YM348, on food intake and energy expenditure in rats. Although the single administration of these 5-HT2C receptor agonists inhibited food intake, on repeated administration they differed with regard to the tolerance development of their hypophagic effects.
In the case of mCPP, a significant hypophagic effect was seen on day 1 of administration, but this effect gradually weakened over the 2-week administration period. This apparent partial tolerance for mCPP has been observed previously (Vickers et al. 2000). Repeated administration of RO60-0175 and YM348 showed apparently different effects on food intake: RO60-0175 (10 mg/kg) maintained its hypophagic effect without any tolerance throughout administration whereas that of YM348 had effectively been lost by the end of administration. The hypophagic effect of all three 5-HT2C receptor agonists was significantly inhibited by the 5-HT2C receptor antagonist SB242084, suggesting that the difference in the tolerance development of effect may not have derived from any difference in the subtype of receptor to which they bind.
One possible explanation is a difference in the desensitization mechanism of the 5-HT2C receptor to the three tested agonists. Stout et al. demonstrated that desensitization of the 5-HT2C receptor is agonist-dependent and that the capacity of agonists to elicit desensitization is not related to their efficacy in activating signaling (Stout et al. 2002). Further, a difference has been shown in the relative efficacies of agonists in their activation of two signal transduction pathways, phospholipase C-mediated inositol phosphate accumulation and phospholipase A2-mediated arachidonic acid release (Berg et al. 1998). This dependence of relative efficacy on effector pathway has been assumed to be due to ligand-specific receptor conformations that have differing capacities in their coupling to or activation of individual signaling systems (Kenakin 1995; Clarke and Bond 1998). It is therefore possible that these ligand-specific receptor conformations also differ in their capacity to activate desensitization mechanisms. In the present study, RO60-0175 showed a hypophagic effect in animals chronically treated with RO60-0175, but no effect in animals chronically treated with YM348. It is conceivable that this was because the receptor was already down regulated or decreased by repeated administration of YM348. In any case, these results indicate that YM348 and RO60-0175 bind to the same receptor but that the mechanism by which they develop tolerance is different.
The involvement of other receptors cannot be perfectly eliminated. 5-HT2A receptor activation is reported to show hypophagia (Aulakh et al. 1994) and 5-HT2B receptor activation is reported to increase or decrease food intake (Kennett et al. 1997; Schreiber et al. 2000). However, the involvement of other receptors in this hypophagic effect is less likely, since our result in antagonist study and many previous studies suggest that the 5-HT2C receptor is the main receptor mediating the hypophagic effect of YM348, RO60-0175, and mCPP (Tecott et al. 1995; Clifton et al. 2000; Hayashi et al. 2004). The possibility of a change in pharmacokinetics by repeated administration or the induction of drug metabolizing enzyme can also be rejected since no tolerance was seen in the effect of any of the agonists on energy expenditure. In addition, results of our preliminary study showed that all three agonists showed similar duration of hyperthermic effect when acutely administrated (Significant effect duration: YM348; 3 h, mCPP; 1 h, RO60-0175; 6 h). Therefore, it is likely that pharmacokinetic character of each compound does not account for the differential tolerance. The possible effect on the behavior should also be considered since activation of 5-HT2C receptor is reported to induce hypolocomotion when 5-HT2C receptor agonists at high dose are administrated (Vickers et al. 2000; Grottick et al. 2001). However, this hypolocomotion induced by 5-HT2C receptor agonists is reported to diminish when agonists are repeatedly or chronically administered (Fone et al. 1998; Vickers et al. 2000). From these reports, it is less likely that hypophagia of RO60-0175 on day 14 of repeated administration is affected by RO60-0175’s possible effect on locomotion.
In our study, the hypophagic effect of YM348 was weakened by the end of 2 weeks’ repeated administration, indicating that the hypophagic effect of YM348 is diminished in this model. However, given reports that animals ate significantly more than the vehicle group after the withdrawal of mCPP (Vickers et al. 2000, 2003), it is possible that the present animals would have eaten more than the control if YM348 had had no effect in week 2 of administration. On this basis, YM348 may have a hypophagic effect, which was nevertheless difficult to recognize under the conditions of the present study.
The difference in the development of tolerance to hypophagia with that to thermogenesis may arise from differences in the localization of the same 5-HT2C receptors. In contrast to the hypophagic effects, repeated administration of the three agonists did not result in any decrease in their effect on body temperature. Although the site of action of 5-HT2C receptor agonists in the control of food intake is structurally well defined, with the VMH and LHA in the hypothalamus and parabrachial nucleus in the caudal brain stem representing the major neuroendocrine effector systems (Shiraishi and Simpson 1982; Grossman and Grossman 1989; Kasser et al. 1989; Tuomisto et al. 1995; Mystkowski et al. 2000; Heisler et al. 2002), that of energy expenditure is largely unknown. Moreover, the possible difference in the isoforms of 5-HT2C receptor in these regions are worthwhile to notice. Porter et al. demonstrated that the degree of desensitization is differ between two isoforms of 5-HT2C receptor (Porter et al. 2001). The difference in the density of these isoforms in different region may cause different degree of desensitization. A further complication is that the involvement of the peripheral adipose tissue in the control of energy expenditure is not negligible (Collins et al. 1994; Grujic et al. 1997; Evans et al. 1998; Collins et al. 1999). In this regard, Nonogaki et al. (2002) have reported that the expression of genes involved in energy expenditure in the peripheral adipose tissue is altered in 5-HT2C receptor-mutant mice. From these findings, it is possible that the peripheral tissue plays a role in the differing development of tolerance to the effect on food intake and that on energy expenditure.
Repeated administration of mCPP and RO60-0175 significantly decreased body weight gain. YM348 also reduced body weight gain but not significantly so. Since all these three agonists did not show tolerance development to their hyperthermic effects, it is very likely that the different effect on body weight arises primarily through their different hypophagic effects, considering that food intake and energy expenditure are the two primary determinants of weight control. However, the hyperthermic effects of 5-HT2C receptor agonists on body weight gain are not negligible. It has been previously suggested that hyperthermia may be an important factor in reducing body weight gain when hypophagic effects are reduced after mCPP chronic infusion (Vickers et al. 2000). In addition, chronic SC infusion of YM348 reduces body weight gain with tolerance being evident for the hypophagic effect but not the hyperthermic effect in Zucker rats (Hayashi et al. 2004). Therefore, it may also be possible that hyperthermic effects play an important role in the inhibition of rebound weight gain after the loss of hypophagic effect.
Obesity is one of the most common metabolic pathologies in contemporary society. Pharmacotherapy with antiobesity agents is an important management strategy. However, current marketed antiobesity agents are known to have side effects such that sibutramine is known to have cardiovascular side effects and orlistat has a gastrointestinal effects (Fujioka et al. 2000; McMahon et al. 2000; Leung et al. 2003). These findings from 5-HT2C receptor agonists study introduce a new approach for therapy of obesity.
The present study showed both different characteristics in hypophagic actions and similar characteristics in hyperthermic actions on repeated oral administration of the 5-HT2C receptor agonists mCPP, RO60-0175, and YM348 in rats. Although the development of tolerance on repeated administration of an agonist might seem obvious, as far as we are aware this is the first report to conclusively demonstrate an agonist-dependent tolerance in the hypophagic component of 5-HT2C agonism. The possibility that agonists may differ in their effects emphasizes the importance of assessing drug potency by defining a compound’s effect on repeated administration.