Activation of G protein-coupled estrogen receptor 1 (GPER-1) decreases fluid intake in female rats
Introduction
Estrogens, specifically estradiol (E2), have a profound effect on reducing fluid and food intake in females. These effects of E2 have a delayed onset, suggesting that genomic mechanisms are required for the changes in behavior (Graves et al., 2011, Kisley et al., 1999, Santollo et al., 2013). E2 acts at several receptor subtypes. The classically identified estrogen receptor (ER)α and ERβ proteins are nuclear receptors that have direct effects on gene expression. More recently, membrane-associated estrogen receptors (mER) have been identified that are capable of generating rapid effects on neuronal physiology (Levin, 2008). Although these rapid effects do not appear to require changes in gene expression, the receptors are capable of altering transcription indirectly by activating intracellular signaling cascades (Micevych and Dominguez, 2009, Micevych and Kelly, 2012).
Recent studies from our lab demonstrated that activation of mER decreases overnight water and food intake in female rats (Santollo et al., 2013), but which specific ER subtype(s) mediate these changes remains an open question. One testable hypothesis is that the effects are mediated by the G protein-coupled estrogen receptor 1 (GPER-1; previously referred to as the orphan receptor GPR30). Activation of GPER-1 by estrogens initiates a number of signaling cascades including activation of mitogen-activated protein (MAP) kinase family members (e.g., extracellular signal-related kinase 1/2; ERK1/2), activation of phosphatidylinositol-3-kinase (PI3K), generation of cAMP, and calcium mobilization (Meyer et al., 2011, Mizukami, 2010). These signaling cascades can produce rapid physiological changes and also can initiate gene transcription, the behavioral consequences of which may take hours or days to become observable.
Proper control of fluid intake is an essential component of blood pressure regulation (Daniels and Fluharty, 2009). In this sense, the known effects of GPER-1 on the maintenance of cardiovascular homeostasis (De Francesco et al., 2013, Lindsey et al., 2009, Martensson et al., 2009) make it tempting to speculate that GPER-1 affects fluid intake, perhaps more proximally than any potential effects on food intake. For example, GPER-1 activation decreases mean arterial blood pressure in hypertensive OVX mRen2.Lewis rats (Lindsey et al., 2009). Whether or not any blood pressure effects of GPER-1 are accompanied by or result from altered fluid intake remains an open question, but GPER-1 mRNA has been localized to nuclei involved in controlling fluid intake (Brailoiu et al., 2007). We therefore hypothesized that GPER-1 is at least in part responsible for fluid intake control in female rats. As an initial step toward testing this hypothesis, we evaluated the effect of all mER subtypes in acutely stimulated water intake and then tested the hypothesis that selective activation of GPER-1 is sufficient to decrease water intake. We also extended these studies to investigate the role of GPER-1 on saline intake because salt, in addition to water, is necessary for proper fluid balance, and estrogens exert an inhibitory effect on intake on both saline and water (Santollo and Daniels, 2015). In addition, we tested for delayed and rapid effects on fluid intake after GPER-1 activation to provide clues about the relevant intracellular actions of GPER-1.
Fluid and food intakes are often closely associated in rats, suggesting that any effects on fluid intake might generalize to food intake. In this respect, the paucity of relevant studies limits our understanding of any potential feeding effects of GPER-1 activation. There is a small number of relevant reports, however, the conclusions that can be drawn from these reports are clouded by discrepant results. For example, studies using GPER-1 knockout mice either found no relevant phenotype resulting from the knockout, or found changes in body weight and visceral adiposity (Liu et al., 2009, Wang et al., 2008). In addition to it being unclear if there is, in fact, an effect of GPER-1 on energy homeostasis, whether or not food intake underlies this potential effect remains underexplored, especially in rats. We therefore tested the hypothesis that GPER-1 activation altered food intake.
Section snippets
Animals and housing
Female Long Evans rats (Harlan Laboratories, Indianapolis, IN) weighing 175–200 g upon arrival into the facility were used in all of the studies described. Rats were singly housed in hanging wire-mesh stainless steel cages with ad libitum access to food (Teklad 2018; Harlan Laboratories) and tap water unless otherwise noted. Rats in double-bottle intake tests (Experiments 2A and 3A) had continuous access to an additional bottle containing a 1.5% saline solution. All testing occurred in the rat's
Experiment 1: Does activation of mER influence AngII-stimulated water intake?
Rats given E2-BSA drank less water in response to AngII than did rats given vehicle (t10 = 2.48, p < 0.05, d = .86; Fig. 1).
Experiment 2: Does activation of GPER-1 influence AngII-stimulated fluid intake?
After G1 treatment rats drank less saline in response to AngII than did rats given a vehicle treatment (F2,18 = 8.96, p < 0.01, η2 = 0.50; Fig. 2A). Both doses of G1 significantly decreased 30 min saline intake (p < 0.05). There was, however, no effect of G1 treatment on AngII-stimulated water intake (F2,18 = 1.84, p = n.s., η2 = 0.17; Fig. 2B). To rule out any possible confounding effects
Discussion
Decreased fluid intake by E2 has been well documented since the 1970s, but the relevant receptor populations underlying these changes remains unknown (Danielsen and Buggy, 1980, Findlay et al., 1979, Tarttelin and Gorski, 1971). The experiments described here provide the novel finding that non-selective activation of mER is sufficient to decrease acutely stimulated water intake in female rats. The observed effect was present when the treatment was given 8 h before the drinking stimulus,
Acknowledgments
We would like to thank Aniko Marshall, Megan Abman and Paul Errico for technical assistance. This work was supported by NIH grants HL-091911 (DD) and DK-098841 (JS).
Disclosures: None.
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