Activation of ERα is necessary for estradiol's anorexigenic effect in female rats
Research Highlights
►Estradiol decreases food intake in female rats. ►Estradiol's anorexigenic effect is blocked by the ERα antagonist, MPrP. ►Estradiol's anorexigenic effect was not influenced by the ERβ antagonist, PHTPP. ►Activation of ERα is necessary for estradiol's anorexigenic effect.
Introduction
The ovarian hormone estradiol appears to play a physiological role in the control of food intake in female rats. The best evidence in support of this statement comes from studies in which ovariectomy has been shown to cause hyperphagia and weight gain (Wade and Gray, 1979), both of which can be prevented by estradiol treatment alone (Asarian and Geary, 2002). Additionally, the pre-ovulatory rise in circulating estrogens in cycling rats promotes a reduction in food intake during the estrous stage of the ovarian reproductive cycle (Drewett, 1973, Blaustein and Wade, 1976, Eckel et al., 2000). This estrous-related decrease in food intake appears to be mediated by estradiol, with minimal involvement of estriol or estrone, since estradiol treatment alone can reinstate this cyclic reduction in food intake in OVX rats (Asarian and Geary, 2002).
Many of the behavioral effects of estradiol are not apparent until hours or days following the rise in circulating estradiol in female rats. For example, the decrease in food intake observed in estrous rats does not occur until about 60 h after the initial rise in circulating estradiol (Becker et al., 2005) and treatment with exogenous estradiol takes ~ 36 h before any behavioral change in food intake is detected in ovariectomized (OVX) rats (Asarian and Geary, 2002). Thus, the anorexigenic effect of estradiol likely involves a genomic mechanism that is initiated by activation of one or both of the nuclear estrogen receptors (ERs), ERα and ERβ.
Studies involving either activation or blockade of ERα/ERβ signaling have been used to investigate the relative contribution of each ER subtype to estradiol's anorexigenic effect. Multiple groups have shown that treatment with an ERα agonist decreases food intake in OVX rats and mice (Roesch, 2006, Santollo et al., 2007, Thammacharoen et al., 2009). In comparison, treatment with an ERβ agonist fails to alter either food intake or the ability of an ERα agonist to reduce food intake (Roesch, 2006, Santollo et al., 2007). While these studies suggest that activation of ERα alone is sufficient to reduce food intake in OVX rats, other studies involving disruptions in ERα/ERβ signaling have provided equivocal evidence regarding the necessity of each ER subtype in mediating estradiol's anorexigenic effect. For example, an examination of the feeding behavior of female mice with a null mutation of the ERα subtype (i.e., αERKO mice) revealed that they were insensitive to the effects of estradiol treatment on several feeding-related measures (Geary et al., 2001). This finding suggests that estradiol's anorexigenic effect requires ERα and extends previous demonstrations that ERβ alone is not sufficient. In another study, however, the anorexigenic effect of estradiol was blocked by intracerebroventricular administration of an ERβ-selective, but not an ERα-selective, antisense oligodeoxynucleotide in the OVX rat (Liang et al., 2002). This finding suggests that estradiol's anorexigenic effect in the rat requires a functional ERβ.
Progress in determining the relative necessity of ERα and/or ERβ signaling in mediating estradiol's anorexigenic effect has been further hampered by the lack of suitable ER-selective antagonists. For example, methyl-piperidino-pyrazole (MPP), a non-steroidal, pyrazole compound (Sun et al., 2002), was originally classified as an ERα antagonist based on in vitro tests of its ability to antagonize estrogen-regulated genes (Harrington et al., 2003). However, in subsequent tests of its in vivo actions, MPP increased uterine weight in mice and rats (Davis et al., 2006, Santollo and Eckel, 2009), reduced food intake in OVX rats (Santollo and Eckel, 2009), and failed to attenuate the anorexigenic effects of estradiol and an ERα agonist in OVX rats (Santollo and Eckel, 2009). Despite these multiple, estradiol-like effects, MPP did attenuate the estrous-related decrease in food intake in cycling rats (Santollo and Eckel, 2009). Taken together, these studies suggest that MPP acts as an ERα antagonist following in vitro applications, but exerts mixed ERα agonist/antagonist actions following in vivo applications. Thus, MPP, which was initially categorized as an ERα antagonist, better resembles a selective ER modulator (SERM; a compound that exerts mixed agonist/antagonist activities at ERs). Indeed, MPP's structure, which is comprised of a core, non-steroidal ER ligand with a basic side chain, is similar to the structure of most SERMs. Moreover, it appears that under certain conditions MPP's basic side chain may be metabolically cleaved resulting in a compound with agonist, rather than antagonist, qualities (Zhou et al., 2008). In light of these findings, Katzenellenbogen's group developed a novel MPP analog, called methyl-piperidinopropyl pyrazole (MPrP), which contains a basic side chain that cannot undergo metabolic cleavage. While this novel compound is highly selective for ERα in binding affinity assays and exerts potent ER antagonist activity in transcription activation assays (Zhou et al., 2008), its in vivo actions have yet to be evaluated.
Currently, there is no evidence that ERβ-selective compounds, like the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), exert the mixed agonist/antagonist properties that are often seen in ERα-selective compounds. For example, PHTPP displays 36-fold selectivity for ERβ over ERα and displays complete antagonism for ERβ in reporter gene assays in co-transfected endometrial cells (Compton et al., 2004). The goal of the present study was to use these ER-selective antagonists (MPrP and PHTPP) to evaluate the relative necessity of ERα- and ERβ-activation in mediating estradiol's anorexigenic effect in OVX and cycling rats.
Section snippets
Animals and housing
Female, Long–Evans rats (Charles River Breeding Laboratory, Raleigh, NC), weighing ~ 250 g at study onset, were individually housed in custom, shoebox cages. Each cage contained a spill-proof food cup, a water bottle, and a sleeping niche. Rats had free access to powdered chow (Purina 5001) and tap water. The colony room was maintained at 20 °C with a 12:12 h light/dark cycle (dark onset = 1300 h). Animal usage and all procedures were approved by the Florida State University Institutional Animal Care
Experiment 1
Food intake during the 24-h period following drug treatment was not influenced by acute administration of 25 μg MPrP in OVX rats, t(5) = 1.29, n.s. (Fig. 1A). During this same period, neither dose of PHTPP influenced food intake in OVX rats, F(2,7) = 1.85, n.s. (Fig. 1B). In addition, food intake was not influenced by either drug during the second and third days following drug treatment (data not shown).
Experiment 2
The anorexigenic effect of EB was blocked in OVX rats that were treated with the ERα antagonist
Discussion
Studies involving disruptions in ERα/ERβ signaling have provided equivocal evidence regarding the necessity of each ER subtype in mediating estradiol's anorexigenic effect (e.g., (Geary et al., 2001, Liang et al., 2002). Progress in answering this question has also been hampered by the limited availability of selective ER antagonists that are devoid of tissue- or species-specific estrogenic activity (Wade and Heller, 1993, Davis et al., 2006, Santollo and Eckel, 2009). The goal of the present
Acknowledgments
This work was supported by grants from the NIH: NS-062667 (JS), CA-018119 (BSK), DK-015556 (JAK), and DK-073936 (LAE).
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