MODULATORY INFLUENCES OF ESTRADIOL AND OTHER ANOREXIGENIC HORMONES ON METABOTROPIC, Gi/o-COUPLED RECEPTOR FUNCTION IN THE HYPOTHALAMIC CONTROL OF ENERGY HOMEOSTASIS
The appetite suppressant actions of estradiol are due to its ability to attenuate orexigenic signals and potentiate anorexigenic signals. The work from my laboratory has shown that male guinea pigs are more sensitive to the hyperphagic and hypothermic effects of cannabinoids than their female counterparts. Cannabinoid sensitivity is further dampened by the activational effects of estradiol. This occurs via the hypothalamic feeding circuitry, where estradiol rapidly attenuates the cannabinoid CB1 receptor-mediated presynaptic inhibition of glutamatergic input onto anorexigenic proopiomelanocortin (POMC) neurons in the arcuate nucleus. This disruption is blocked by the estrogen receptor antagonist ICI 182,780, and associated with increased expression of phosphatidylinositol-3-kinase (PI3K). Moreover, the ability of estradiol to reduce both the cannabinoid-induced hyperphagia and glutamate release onto POMC neurons is abrogated by the PI3K inhibitor PI 828. The peptide orphanin FQ/nociceptin (OFQ/N) activates opioid receptor-like (ORL)1 receptors to hyperpolarize and inhibit POMC neurons via the activation of postsynaptic G protein-gated, inwardly-rectifying (GIRK) channels. We have demonstrated that the fasting-induced hyperphagia observed in ORL1-null mice is blunted compared to wild type controls. In addition, the ORL1 receptor-mediated activation of GIRK channels in POMC neurons from ovariectomized female rats is markedly impaired by estradiol. The estrogenic attenuation of presynaptic CB1 and postsynaptic ORL1 receptor function may be part of a more generalized mechanism through which anorexigenic hormones suppress orexigenic signaling. Indeed, we have found that leptin robustly suppresses the OFQ/N-induced activation of GIRK channels in POMC neurons. Furthermore, its ability to augment excitatory input onto POMC neurons is blocked by PI 828. Thus, estradiol and other hormones like leptin reduce energy intake at least partly by activating PI3K to disrupt the pleiotropic functions of Gi/o-coupled receptors that inhibit anorexigenic POMC neurons.
1. Introduction<!>2.1 Sex Differences in the Cannabinoid Regulation of Energy Homeostasis<!>2.2 Estrogenic Modulation of Cannabinoid-Induced Changes in Energy Homeostasis<!>2.3 Anorexigenic Hormone Modulation of opioid receptor-like (ORL)1 receptor-mediated signaling in POMC Neurons<!>3. SIGNIFICANCE
<p>It has long been known that hypothalamic lesions produce obesity [1], and over the past several decades the scientific community has witnessed an explosion in the number of investigations into the hormonal and synaptic determinants underlying the hypothalamic control of energy homeostasis. The hypothalamic feeding circuitry comprises both orexigenic and anorexigenic components. The orexigenic components can be found in the lateral hypothalamic area (LHA) housing the orexin- and melanin-concentrating hormone (MCH)-containing somata [2], as well as the in the arcuate nucleus (ARC) where the neuropeptide Y (NPY)/agouti-related peptide (AgRP)- and ghrelin-containing somata reside [3–5]. Conversely, anorexigenic components can be found in the ARC as well as the hypothalamic paraventricular nucleus (PVN) that contain the cell bodies of the proopiomelanocortin (POMC) and corticotropin-releasing hormone (CRH) neurons, respectively [2,6]. The hypothalamic ventromedial nucleus (VMN) is also involved in satiety, as lesions of this region cause profound hyperphagia and obesity [7,8]. There is extensive synaptic reciprocity and interconnectivity between the nuclei comprising the hypothalamic feeding circuitry, which is also sensitive to circulating levels of nutrients and peripheral hormones such as glucose, insulin, ghrelin and leptin [9,10]. Interestingly, all of the afferent elements of the hypothalamic feeding circuitry project to the PVN, where they make synaptic contact with the CRH neurons residing there [9,10]. Thus, the CRH neurons serve as a point of convergence and synaptic integration that is important for generating an efferent response to changes in the prevailing energetic environment.</p><p>The neurons within the hypothalamic feeding circuitry express signaling molecules such as phosphatidylinositol-3-kinase (PI3K) and AMP-activated protein kinase (AMPK) that mediate the glucose sensing and changes in neuronal activity that occur upon leptin and insulin receptor activation [11–14]. These and other hormones can also influence the levels of orexigenic endocannabinoids such as 2-arachidonoylglycerol (2-AG) within the nuclei comprising the hypothalamic feeding circuitry. For example, the orexigenic effect of ghrelin, as well as its ability to presynaptically inhibit glutamatergic input onto parvocellular neurons in the PVN, is due to augmented biosynthesis of 2-AG in the hypothalamus that then increases hypothalamic AMPK activity [15] via CB1 receptor activation. Conversely, anorexigenic leptin decreases hypothalamic endocannabinoid levels, which are elevated in obese Zucker (fa/fa) rats [16,17]. In addition, leptin diminishes the glucocorticoid-stimulated, endocannabinoid-mediated decrease in glutamatergic input onto parvocellular PVN neurons, an effect that is absent in fa/fa rats [17]. Moreover, CB1 receptors are expressed in the LHA [18], PVN [2] and the VMN [19], and administration of CB1 receptor agonists like tetrahydrocannabinol (THC) and anandamide into these hypothalamic nuclei elicits a hyperphagic response [20,21]. Furthermore, in the LHA endocannabinoids retrogradely inhibit GABAergic input onto MCH neurons [22], and the elevated endocannabinoid biosynthetic capacity seen with diet-induced obesity and in ob/ob mice enhances the retrograde inhibition of GABAergic input onto orexin neurons [18]. While it is clear that cannabinoids can impact the homeostatic control of energy balance, they can also influence hedonic aspects of consumption; particularly in relation to highly palatable foods. For example, cannabinoids influence synaptic transmission in the nucleus accumbens, a structure containing the terminal fields of the mesolimbic dopamine neurons mediating natural and drug-induced reward [23,24]. In addition, diet-induced obesity is associated with a down-regulation of CB1 receptors in extrahypothalamic regions like the nucleus accumbens; suggesting that endogenous cannabinoids provide the initial impetus for the ingestion of palatable food [25]. Indeed, administration of the endogenous cannabinoid agonists 2-AG and anandamide into the nucleus accumbens stimulate feeding and augment the "liking" of intraoral sucrose [26,27]. By contrast, blockade of CB1 receptors with Rimonabant diminishes dopamine release in the nucleus accumbens caused by ingestion of highly palatable food [28]. Interestingly, the cell bodies of mesolimbic dopamine neurons from peripubertal female rats are more sensitive to endocannabinoid-mediated depolarization-induced suppression of GABAA receptor-mediated inhibition (DSI) than those from their male counterparts [29]. However, given the burgeoning wealth of information on the neuroendocrine regulation of the hypothalamic feeding circuitry, we focus here on the modulation of orexigenic, metabotropic Gi/o-coupled receptors such the CB1 and opioid receptor-like (ORL)1 receptors by the anorexigenic hormones estradiol and leptin.</p><!><p>The literature is replete with examples of sex differences in cannabinoid-regulated biology. For example, female rodents are more sensitive to the antinociceptive effect of cannabinoids than their male counterparts [30,31]. They are more sensitive to the locomotor actions of cannabinoids as well [30–32]. In humans, women are more responsive to the cannabinoid-induced orthostatic hypotension that occurs when transitioning from the supine to the standing position [33]. They are more susceptible to the visuospatial memory impairment caused by cannabinoids as well [34]. Self-administration studies have revealed that female rats more rapidly acquire stable cannabinoid intake than males, and maintain a higher rate of responding [35]. Likewise, developmental studies have uncovered that females rats are more vulnerable to the effects of adolescent cannabinoid exposure on subsequent behavioral and biochemical changes (e.g., anhedonia, CB1 receptor downregulation) occurring in adulthood [36]. Similar studies have shown sexually disparate, behavioral interactions between subchronic nicotine exposure during adolescence and acute cannabinoid administration in adulthood [37].</p><p>While the considerable majority of the evidence presented above indicates that females are more responsive to cannabinoids, the converse is true with regard to energy homeostasis. For example, CB1 receptor agonists administered into the fourth ventricle of male rats are ~10X more potent in stimulating the consumption of sweetened condensed milk than they are in females [38]. In addition, we have demonstrated that CB1 receptor agonists produce a greater increase in cumulative energy intake in orchidectomized male guinea pigs than they do in their ovariectomized female counterparts [39]. They also produce a reduction in core body temperature that is ~0.5 °C greater in orchidectomized males than that observed in ovariectomized females [39].</p><!><p>The sex differences that we observed in the cannabinoid regulation of energy balance occurred in castrated animals. This begged the question: what are the activational effects of estradiol in modulating cannabinoid-induced changes in energy balance in the female? Estradiol is a well-documented anorexigenic hormone that, in humans, diminishes energy intake during the periovulatory phase of the menstrual cycle [40] and, in rodents, decreases consumption during the estrus phase of the estrous cycle [41]. It does so, in general, by attenuating orexigenic signaling and potentiating anorexigenic signaling within the hypothalamic feeding circuitry [42]. Using an established treatment paradigm schematically represented by the timeline shown in Figure 1, we found that estradiol per se increased the expression of the PI3K p85α regulatory subunit in the ARC (Figure 2). Western blotting of ARC tissue microdissected from two to three 1mm-thick coronal slices spanning its rostral-caudal extent as described previously [43–45] revealed clear phosphorylated Akt (pAkt) and total Akt bands measuring 60 KDa in weight (Figure 3). Estradiol administered two hours prior to tissue harvesting increased the amount of pAkt in the ARC compared to vehicle-treated controls (Figure 3). As we have previously reported [43–46], estradiol also decreased cumulative energy intake, as well as several measures of energy expenditure such as O2 consumption, CO2 production and metabolic heat production (Figure 4). The estradiol-induced decreases in energy intake, but not energy expenditure, were blocked by the PI3K inhibitor PI 828 (Figure 4). Most importantly, we have found that estradiol rapidly attenuates cannabinoid-induced hyperphagia and hypothermia [46], the former of which is restored when PI 828 is co-administered along with the cannabinoid receptor agonist WIN 55,212-2 into the third ventricle of ovariectomized, estradiol-treated female guinea pigs (Figure 5). These findings are in keeping with the fact that estradiol decreases the hypothalamic expression of the CB1 receptor [47], and that levels of anandamide and 2-AG in the rodent hypothalamus are reduced during proestrus relative to diestrus [48].</p><p>We then endeavored to resolve this estrogenic diminution of the cannabinoid-induced increase in energy intake down to the level of the hypothalamic feeding circuitry. Our electrophysiological studies revealed that the cannabinoid agonist WIN 55,212-2, upon activation of a metabotropic, Gi/o-coupled receptor like the CB1 receptor, decreased the number of excitatory synaptic events impinging on anorexigenic POMC neurons per unit time in vehicle-treated hypothalamic slices (Figure 6). This was evident upon examination of either the representative traces of membrane current, or the cumulative distribution plots of the interval between contiguous miniature excitatory postsynaptic currents (mEPSCs), under baseline control conditions and in the presence of WIN 55,212-2. By contrast, bath application of estradiol for a mere 10 minutes markedly blunted the ability of WIN 55,212-2 to decrease the number of excitatory synaptic events per unit time (Figure 6). However, co-perfusion of the estrogen receptor antagonist ICI 182,780 along with estradiol enabled WIN 55,212-2 to significantly decrease the number of excitatory synaptic events per unit time to a level observed in recordings from vehicle-treated slices (Figures 6 and 7). Likewise, PI 828 abrogated the estrogenic attenuation of the ability of WIN 55,212-2 to decrease in mEPSC frequency (Figure 7). Collectively, this demonstrates that estradiol activates PI3K to diminish the cannabinoid-induced increase in energy intake that occurs, at least in part, via a reduction in the cannabinoid-induced presynaptic inhibition of excitatory input onto POMC neurons. In view of this rapid estrogenic attenuation of the cannabinoid-induced decrease in glutamate release onto POMC neurons, it therefore should not be surprising that estradiol per se increases basal mEPSC frequency in POMC neurons as well as POMC expression in the ARC [43,49,50]. We and others have shown that these rapid effects of estradiol on POMC neurons are due to activation of estrogen receptor (ER)α and a Gq-coupled membrane ER (Gq-mER)[42,44,51]. These ERs can be trafficked and tethered to the plasma membrane via caveolins and palmitoylation, as has been shown in the ARC, hippocampus and vascular endothelial cells [52–54].</p><!><p>The question arises: can estradiol negatively modulate the coupling of other orexigenic, Gi/o-coupled receptors to their effector systems in POMC neurons? One such likely candidate is the ORL1 receptor, which is found in abundance in the mediobasal hypothalamus [55–58]. The ORL1 receptor is the cognate receptor of the endogenous peptide orphanin FQ (aka nociceptin; OFQ/N) [57,59]. When activated by OFQ/N, the ORL1 receptor hyperpolarizes and thereby inhibits POMC neurons via activation of a G protein-gated, inwardly-rectifying K+ (GIRK) channel [60]. This observation is consistent with the fact that: 1) OFQ/N stimulates energy intake in rats [61,62], 2) OFQ/N prolongs deprivation-induced consumption and decreases c-fos expression in POMC neurons at the time of meal termination [63], and 3) ORL1 knockout blunts fasting-induced hyperphagia [64]. In electrophysiological recordings of POMC neurons from vehicle-treated, ovariectomized female rats, we found that OFQ/N produced a robust outward current that returned to baseline levels of holding current upon its clearance from the slice (Figure 8A). This outward current was associated with an increase in conductance, and reversed polarity around the Nernst equilibrium potential for K+ (Figure 8B and 8C). These are characteristic features associated with activation of GIRK channels. However, in recordings of POMC neurons from estradiol-treated animals, this OFQ/N-induced activation of GIRK channels was markedly diminished (Figure 8).</p><p>Do other anorexigenic hormones negatively modulate the function of orexigenic, Gi/o-coupled receptors? One likely candidate is the adipokine leptin that is the product of the ob gene [65]. Once released by adipocytes, it activates its cognate receptor that is expressed in several hypothalamic appetite-regulating centers to suppress energy intake via negative feedback [22,65–70]. Rodents and humans lacking either the leptin receptor or leptin itself exhibit several features that are characteristic of perturbed energy homeostasis; including hyperphagia, increased adiposity, hyperglycemia, hyperinsulinemia and dyslipidemia [65,71–73]. In addition, obesity seen under a variety of pathophysiological conditions is most often associated with hyperleptinemia and leptin resistance [72,74–76].</p><p>Leptin receptor activation elicits a complex signal transduction cascade, one that shares several features in common with estrogen receptor signaling [77]. The receptor has a cytoplasmic domain that, following activation and subsequent dimerization, allows for the association of Janus kinases (JAKs). This interaction leads to the activation of JAKs that, in turn, facilitates the recruitment and tyrosine phosphorylation of other molecules like signal transducer and activator of transcription (STAT)-3, insulin receptor substrate, forkhead box protein, mammalian target of rapamycin, extracellular signal-regulated protein kinase (ERK), phospholipase C (PLC), and PI3K/Akt [12,13,78–81].</p><p>In POMC neurons, and in steroidogenic factor (SF)1-containing neurons in the VMN, this signaling pathway leads to a membrane depolarization that increases the probability of generating action potentials [13,68,82,83]. This increase in firing rate ultimately is due to an inward current caused by the leptin-induced activation of postsynaptic transient receptor potential C (TRPC) channels [81]. On the other hand, leptin hyperpolarizes NPY/AgRP neurons [84] and attenuates the fasting-induced increase in neuronal firing [85], which may be attributed to the activation of postsynaptic KATP channels in these cells [86]. Leptin also disrupts endocannabinoid-mediated DSI in MCH [22] and orexin [18] neurons in the LHA. Conversely, it has been reported to decrease inhibitory GABAergic input onto POMC neurons [87]. Leptin receptors are also expressed in glutamatergic neurons in the VMN that play an important role in the leptin-induced changes in energy balance [19,88,89]. Similar to estradiol, we found that a brief exposure to leptin, in the presence of 2-APB to block the depolarizing effect of TRPC channel activation [81], markedly attenuated the reversible, OFQ/N-induced outward current in POMC neurons from ovariectomized female rats, as well as the associated increase in conductance (Figure 9). We also found that leptin increased the number of excitatory synaptic events per unit time impinging on POMC neurons from ovariectomized female rats, which can be readily ascertained upon examination of both the representative traces of membrane current and the quantile plot depicting the significant leptin-induced shift in the cumulative probability distribution for the interval between contiguous mEPSCs (Figure 10A and 10C). Importantly, this leptin-induced increase in mEPSC frequency is completely reversed by PI 828 (Figure 10B and 10C). Thus, both estradiol and leptin can negatively modulate the coupling of orexigenic, Gi/o-coupled receptors to their effector systems. The resultant increase in POMC neuronal activity occurs through facilitation of excitatory neurotransmission, antagonism of GIRK channel currents, and activation of TRPC channels in these cells via stimulation of PI3K.</p><!><p>The results gathered by our laboratory over the past few years have uncovered sex differences in, and modulatory effects of anorexigenic hormones on, the neuroendocrine regulation of energy homeostasis. Male guinea pigs are more responsive than females to cannabinoid-induced increases in energy intake and decreases in core body temperature, and estradiol further dampens these effects in females. These findings are consistent with those observed in the rat [38], and have clear clinical relevance. In the phase II trial and randomized, placebo-controlled studies touting the efficacy of THC and dronabinol to ameliorate cachexic symptoms and promote appetite in patients with cancer [90] and HIV/AIDS [91,92], the considerable majority of study participants were men. However, in another clinical trial in which the gender ratio of the cancer patients was more equitably distributed, the results were not as compelling [93]. This reinforces the idea that gender and endocrine status needs to be taken into account in developing rational therapies for the treatment of HIV/AIDS- or cancer-related cachexia. Thus, it is predicted that CB1 receptor agonists would be less effective in normally cycling women, especially around the time of ovulation, and more effective under hypoestrogenic conditions such as anorexia nervosa, in which dronabinol promotes weight gain [94], and menopause. Regarding the latter, ~80% of the participants in the RIO-North America obesity trial were women with an average age of ~45 years that showed significant improvement in body weight, waist circumference and blood lipid profile following long-term treatment with the CB1 receptor antagonist Rimonabant [95]. Were it not for an increase in the incidence of dysphoric side effects (e.g., nausea, anxiety and, to a lesser extent, depression) relative to that experienced in placebo-treated controls, this drug undoubtedly would still be on the market to this day. While baseline female reproductive status was not specifically evaluated in this study, it is quite reasonable to assume that a substantial fraction of the study participants were women who were either poised to enter into their climacteric or fully menopausal. With this in mind, the Rimonabant-induced amelioration of the perturbed energy balance observed in the RIO-North America obesity trial might be explained, at least in part, by its ability to block the enhanced endocannabinoid tone resulting from the hypoestrogenic state experienced by a large percentage of the study participants.</p><p>The sex difference in, and estrogenic influence on, the cannabinoid regulation of energy homeostasis observed at the behavioral level is accompanied by parallel underlying changes in the cellular physiology at POMC synapses. In females, CB1 receptors couple to and thereby activate Kv4.2 channels associated with a transient A-type K+ current in POMC neurons by reducing the amount of hyperpolarization required to transition the channels from the inactivated to the closed state [96], whereas in males they couple to GIRK channels that elicit a sizable outward current and membrane hyperpolarization [97]. Estradiol antagonizes the cannabinoid-induced activation of the Kv4.2 channels in POMC neurons, and attenuates the cannabinoid-induced presynaptic inhibition of glutamatergic input impinging upon these cells [46,98]. It does so by activating ERα and the Gq-mER [44]. This ER-mediated disruption of CB1 receptor/effector coupling in and around POMC neurons can be attributed to activation of PI3K, which is an upstream signaling molecule responsible for a variety of estradiol-mediated effects ranging from synaptogenesis [99] to endothelial nitric oxide production [100]. The pathway though which estradiol impairs CB1 receptor-mediated signaling also includes protein kinase C (PKC) and neuronal nitric oxide synthase (nNOS), as inhibitors of these enzymes rescue both the cannabinoid-induced hyperphagia and presynaptic inhibition of glutamate release onto POMC neurons in the presence of the steroid [44,45]. These synaptic dynamics are illustrated in Figure 11. Finally, the estradiol-induced activation of PI3K is involved along with PLC, PKC and protein kinase A in uncoupling other metabotropic, Gi/o-coupled receptors like the GABAB and μ-opioid receptors from their GIRK channels in POMC neurons [51,101].</p><p>In addition, estradiol diminishes the coupling of ORL1 receptors from their postsynaptic GIRK channels in POMC neurons, and reduces the OFQ/N-induced presynaptic inhibition of glutamatergic input onto these cells [102]. Activation of ARC ORL1 receptors by OFQ/N elicits orexigenesis [62], due in large part to reduced excitability of POMC neurons involved in meal termination [63]. This reduced POMC neuronal activity is the result of the ORL1 receptor-mediated hyperpolarization caused by activation of postsynaptic GIRK channels as well as the decreased glutamate release onto these cells [60,64,102]. Moreover, fasting-induced hyperphagia, as well as the pleiotropic actions of OFQ/N on POMC neurons, are appreciably blunted in ORL1 receptor null mice [64]. Thus, the disruption of the function of the orexigenic, Gi/o-coupled ORL1 receptor expressed in POMC neurons and its excitatory synaptic inputs represents another physiologically relevant example of how estradiol exerts its anorexigenic actions. Furthermore, it is consistent with the estrogenic attenuation of the ORL1 receptor-mediated antinociception that occurs via an ERK-dependent mechanism [103].</p><p>Most interestingly, leptin can also impair ORL1 receptor/GIRK channel coupling in POMC neurons. This observation cannot be attributed simply to a matter of synaptic integration because TRPC channels, whose activation by leptin would exert opposing influences on membrane current (or potential) [81], were blocked by 2-APB. Therefore, the most parsimonious explanation is that leptin hampers the ability of OFQ/N to activate GIRK channels in POMC neurons. The signaling pathways of estradiol and leptin share many components in common including, but not limited to, STAT-3 and PI3K [77]. Both hormones also increase excitatory synaptic input onto POMC neurons [43,77]. These excitatory input arises from SF1-containing, leptin receptor- and CB1 receptor-bearing neurons in the VMN that are sensitive to fasting and reduced in ob/ob mice [10,19,88,89,104]. We have shown previously that this excitatory input is obliterated following application of ionotropic glutamate receptor antagonists [64,98]. In addition, we found that the leptin-induced increase in glutamatergic neurotransmission received by POMC neurons is due to the activation of a PI3K pathway. This same signaling molecule is also critical for the leptin-induced activation of TRPC channels and the subsequent increase in the excitability of these cells [13,81]. Moreover, OFQ/N hyperpolarizes and thereby inhibits these same leptin receptor-expressing VMN neurons via activation of GIRK channels [105]. Collectively, these findings illustrate the anatomical convergence and dynamic regulatory interplay between the SF1-containing, leptin receptor- and CB1 receptor-bearing VMN neurons and the anorexigenic POMC neurons, and how their activity can be profoundly influenced by estradiol, leptin, cannabinoids and OFQ/N. In conclusion, our findings lend considerable insight into the pleiotropic mechanisms through which these anorexigenic hormones modulate the function of orexigenic, Gi/o-coupled receptors. Given that they also help coordinate the timing of sexual behavior with ovulation, fertilization and implantation [42,106–110], they help to establish the inexorable link between energy homeostasis and reproduction. Thus, during times of heightened female sexual receptivity, as seen during behavioral estrus around the time of ovulation, the primary focus is on finding an appropriate male counterpart with which to mate. Other appetitive behaviors related to energy intake are secondary. If successful fertilization has been achieved, then the converse would be true in the luteal phase, during which time an increased energy demand is created by the blastocyst being implanted into estradiol-primed, progesterone-stimulated uterine endometrium. On the other hand, primary amenorrheic individuals like those suffering from anorexia nervosa are both hypoestrogenemic and hypoleptinemic [111,112]. Refeeding in these individuals increases body weight, body mass index, leptin and luteinizing hormone, which is associated with increased estradiol levels and a resumption of menstruation [112–114].</p>