Membrane and Nuclear Initiated Estrogenic Regulation of Homeostasis

Abstract

Reproduction and energy balance are inextricably linked in order to optimize the evolutionary fitness of an organism. With insufficient or excessive energy stores a female is liable to suffer complications during pregnancy and produce unhealthy or obesity-prone offspring. The quintessential function of the hypothalamus is to act as a bridge between the endocrine and nervous systems, coordinating fertility and autonomic functions. Across the female reproductive cycle various motivations wax and wane, following levels of ovarian hormones. Estrogens, more specifically 17β-estradiol (E2), coordinate a triumvirate of hypothalamic neurons within the arcuate nucleus (ARH) that govern the physiological underpinnings of these behavioral dynamics. Arising from a common progenitor pool of cells, this triumvirate is composed of the kisspeptin (Kiss1^ARH), proopiomelanocortin (POMC), and neuropeptide Y/agouti-related peptide (AgRP) neurons. Although the excitability of these neuronal subpopulations is subject to genomic and rapid estrogenic regulation, kisspeptin neurons are the most sensitive, reflecting their integral function in female fertility. Based on the premise that E2 coordinates autonomic functions around reproduction, we will review the recent findings on the synaptic interactions between Kiss1, AgRP and POMC neurons and how the rapid membrane-initiated and intracellular signaling cascades activated by E2 in these neurons are critical for control of homeostatic functions supporting reproduction.

1. 17 β-Estradiol and reproduction

The hypothalamus regulates both puberty and fertility through secretion of gonadotropin-releasing hormone (GnRH) from neurons located primarily in the preoptic area in rodents [1] but extending caudally into the basal hypothalamus in sheep [2], guinea pigs [3], and primates including humans [4, 5]. GnRH stimulates the release of gonadotropins from the pituitary gland, controlling ovulation in females. This process is not linear, but rather relies on appropriately-timed GnRH pulses preceding a final surge to elicit luteinizing hormone (LH) release. Ovarian hormones are required for both negative and positive feedback actions that maintain a normal cycle. Classical estrogenic signaling is mediated by ERα [6, 7] and ERβ [8] receptors located in the cytosol which, upon binding E2, dimerize and enter the nucleus. These ERs interact with estrogen response elements (EREs) located within certain gene promotors to regulate transcription [9-11]. In addition, E2 may activate membrane-bound estrogen receptors (mERs) to mediate rapid, non-genomic actions [12, 13]. mERs can take the form of ERα/β [14, 15], G-protein coupled estrogen receptor (GPER1) [16-19], or an unidentified Gq-coupled receptor (Gq-mER) [20, 21]. However, GnRH neurons lack ERα [22, 23] suggesting the involvement of an extrinsic pulse generator or other estrogen signaling pathways (e.g. ERβ, GPER1, Gq-mER) [24, 25].

Neurons in the anteroventral periventricular (AVPV) and more caudal preoptic periventricular nucleus (PeN) co-express kisspeptin, a neuropeptide encoded by the Kissl gene, GABA [26], and tyrosine hydroxylase [27], Kisspeptin-54 is the endogenous ligand of G protein-coupled receptor 54 (GPR54, aka Kissl R) [28]. GPR54 is highly expressed in GnRH neurons [29], and mutations in GPR54 cause autosomal recessive idiopathic hypogonadism in humans and deletion of GPR54 or Kiss1 in mice results in defective sexual development and reproductive failure [30, 31]. Centrally administered kisspeptin robustly stimulates GnRH and gonadotropin secretion in both pre-pubertal and adult animals [32,33]. In vitro kisspeptin robustly excites GnRH neurons [34], and based on cell signaling studies, kisspeptin excites GnRH neurons primarily through activation of canonical transient receptor potential (TRPC) channels and to a lesser extent through inhibition of inwardly rectifying K⁺ channels [35-39].

The AVPV/PeN expresses high levels of ERα and ERβ, and the actions of the gonadal steroids on kisspeptin neurons are mediated, at least in part, via nuclear-initiated signaling (transcriptional) mechanisms [22, 40, 41], Kiss1 mRNA expression is increased in the AVPV/PeN following E2 treatment, while decreased in the more caudally located arcuate nucleus [42]. Importantly, endogenous currents involved in regulating neuronal excitability, including h-, T-type calcium and a persistent sodium current (l_NaP), are all expressed in AVPV/PeN Kiss1 (Kiss1^AVPV/PeN) neurons, and both currents and corresponding ion channels are highly upregulated by proestrus levels of E2 [43-46]. These findings combined with previous observations that lesions of or implants of ER antagonists into the AVPV/PeN in rodents abrogate the positive feedback effects of E2 [47-50] have led to the hypothesis that E2 acts on Kiss1^AVPV/PeN neurons to induce positive feedback on GnRH and LH secretion. Finally, more recent experiments have shown that high frequency optogenetic stimulation of Kiss1^AVPV/PeN neurons releases kisspeptin that excites (depolarizes) GnRH neurons through activation of TRPC channels [51].

2. Links between reproduction and energy homeostasis

Kiss1^ARH, POMC and AgRP neurons arise from a common precursor [52, 53] and together these neurons govern both reproduction and energy homeostasis. The ARH stands well-positioned at the “headwaters” of the natural reward circuit. By virtue of close apposition to the median eminence, a circumventricular organ, ARH neurons act as first-order neurons, able to sense and respond to indicators of the energy state of the animal [54, 55]. ERs are densely expressed in this region [56-58], granting ARH neurons sensitivity to circulating steroid hormones. The ARH was also proposed to be the center of the GnRH “pulse generator” based on in vivo recordings of multi-unit activity [59-62]. More recently Kiss1^ARH neurons have been identified as the source of this patterned activity [51, 63, 64]. Furthermore, the cellular underpinnings of synchronization, a network of reciprocal connections between Kiss1^ARH neurons, has been elucidated [51]. When E2 levels are low, Kiss1^ARH neurons produce and co-release the neuropeptides neurokinin B (NKB) and dynorphin. Through a network of reciprocal connections, Kiss1^ARH neurons excite each other through NKB release. Dynorphin then presynaptically inhibits further release [51]. This sequence of events repeats, giving rise to synchronized oscillations in activity. As E2 rises in preparation of ovulation, mRNA expression of the peptide neurotransmitters NKB, dynorphin, and kisspeptin falls [42, 64, 65] with glutamate emerging as the dominant neurotransmitter [66]. This progression from peptidergic to amino acid transmission affects not just synapses with Kiss1^ARH and Kiss1^AVPV/PeN neurons, but two antagonistic ARH cell types (see below).

Neuropeptide Y/agouti related peptide (AgRP) neurons are considered orexigenic with their activation instigating robust food consumption within minutes, regardless of the energy state of the animal [67-70]. AgRP neurons do not synapse on GnRH neurons, but AgRP neurons synapse on Kiss1 neurons and inhibit their activity via GABA release [71]. Ablation of AgRP neurons markedly decreases presynaptic inhibition of Kiss1 projections [71] and fasting reduces fertility and expression of Kissl in the ARH [72]. The opposing, anorexigenic POMC neurons decrease food intake with sustained stimulation [67, 73, 74] and, in addition to GABA [75, 76] and glutamate [75], release a diverse complement of neuropeptides. The POMC precursor peptide is processed to produce α-melanocyte stimulating hormone (α-MSH, excitatory) [77] and β-endorphin (inhibitory) [78]. GnRH neurons receive POMC inputs and are hyperpolarized by the μ-opioid receptor agonist DAMGO ([D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin) through activation of a K⁺ conductance [79-82]. Naloxone block of opioid signaling stimulates GnRH release [83-87] and increases LH production [88, 89]. This would suggest POMC signaling inhibits GnRH activity [90]. Therefore, excess opioid tone is inhibitory to reproductive function. However, selective activation of the α-MSH pathway can be stimulatory [90]. More straightforward are Kiss1 to POMC projections. Kisspeptin administered icv reduces food intake [91], optogenetic stimulation of Kiss1^ARH neurons elicits glutamatergic excitation [66, 92], and kisspeptin depolarizes POMC neurons [93]. Though POMC neurons make reciprocal projections to Kissl neurons, these circuits are poorly understood [66, 90, 94, 95]. Perhaps POMC signaling uses Kiss1 neurons as an intermediary with GnRH neurons considering a subpopulation of Kiss1^AVPV/PeN neurons expresses melanocortin 4 receptor (MC4R) [26]. Blockade of melanocortin signaling in peripubertal females selectively decreases Kiss1 mRNA expression in the Kiss1^AVPV/PeN neurons, and while MC4R knockout mice can reproduce, they are subfertile [96]. Overexpression of AgRP, an inverse agonist for MCRs, causes infertility [90, 97, 98]. Therefore, while the content and functional significance of POMC inputs to Kiss1 neurons remains unclear, AgRP neurons unmistakably act to inhibit reproduction during an energy deficit [71].

AgRP and POMC neurons are inversely regulated by glucose and metabolic hormones including leptin and insulin [99-101]. The involvement of ARH neurons in reproduction is predictable since pregnancy is metabolically demanding. A lack of sufficient energy stores increases the risk of miscarriage in underweight females [102], and women suffering from anorexia often display amenorrhea [103-105]. Leptin, a hormone produced by white adipocytes [106], signals the total body energy stores. Mutations in leptin production and signaling result in an infertile, obese phenotype [107, 108]; however, GnRH neurons lack leptin receptors [109, 110]. As this phenotype can be reversed by ablating AgRP neurons [97], leptin regulation of GnRH neurons is potentially indirect. Kiss1^ARH neurons express leptin receptors [111, 112], and like with POMC neurons, leptin depolarizes and increase their firing [99, 112]. Without the hyperpolarizing influence of leptin [113], AgRP neurons become highly active, inhibiting Kiss1^ARH and Kiss1^AVPV/PeN neurons (Figure 1) [71]. In lean mice overexpression or injection of leptin accelerates puberty onset [114, 115], and obesity is associated with precocious puberty in women [116, 117]. Therefore, leptin levels and by proxy, adiposity affects not just fertility but pubertal development.

Figure 1.

Circuit diagram of neurons of the arcuate nucleus of the hypothalamus. Fasted State (left panel): When a female experiences an energy deficit, AgRP neurons become highly active and release GABA onto Kiss1 and POMC neurons, inhibiting fertility and satiety. Fed State (right panel): When the female is fed and/or in a high 17β-estradiol state (e.g., proestrus), AgRP neurons become less active and their inhibitory input onto Kiss1 and POMC neurons is diminished. Concurrently, the probability of glutamate release is enhanced in both Kiss1 and POMC neurons. While glutamate released onto AgRP neurons will activate excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, the influence will be an overall inhibitory effect through Group II/III metabotropic glutamate channels. Conversely, POMC neurons express excitatory Group I metabotropic glutamate receptors and their increased activity may cause the release of neuropeptides such as β-endorphin, which will further inhibit AgRP neurons, decreasing food intake. POMC neurons also excite Kiss1 neurons via glutamate release.

Short-term indicators of energy balance affect ARH function through both genomic and rapid mechanisms (Figure 2). In lean animals, insulin is released in response to higher blood glucose levels after meal consumption. Circulating insulin easily reaches the ARH neurons adjacent to the median eminence, and perfusion of insulin in vitro rapidly depolarizes POMC neurons through activation of TRPC5 channels [118]. In addition, insulin significantly increases Pomc mRNA expression in 72 hours following icv administration [119]. Both the canonical PI3K/AKT and calcium-activated signaling cascades, as a result of influx through the TRPC5 channels, can cause new gene (Pomc) transcription [120]. TPRC channels can function as both receptor- or store-operated channels opened, respectively, by membrane delimited receptors or depletion of Ca²⁺ stores [121, 122]. Obesity contributes to the development of insulin resistance, a core feature of metabolic disorders. Central nervous system (CNS) neurons, like other cells throughout the body, become insulin resistant. In the obese state, TRPC channels associate with the endoplasmic reticulum protein stromal-interaction molecule (STIM1) to function as store-operated and, hence, are no longer opened by insulin [122]. E2 protects females from developing CNS insulin resistance by downregulating STIM1 in POMC neurons, increasing their excitability, and preventing TRPC conversion to store-operated channels [123]. E2 also helps by preventing high-fat diet related upregulation of SOCS-3 (suppressor of cytokine signaling 3), preserving insulin signaling in females [123, 124]. Therefore, circulating estrogens are vital for maintaining insulin sensitivity throughout the female reproductive cycle and are neuroprotective against insulin resistance in obese states.

Figure 2.

E2 and insulin signaling cascades in POMC neurons. Insulin through its cognate receptor can activate phospholipase C (PLCγ) to cleave Phosphotidylinositol 4,5 biphosphate (PIP₂) into Diacylglycerol (DAG) and Inositol triphosphate (IP₃) opening TRPC5 channels and generating an inward cationic current to depolarize POMC neurons. Binding of E2 to Gαq-coupled mERs activates phospholipase C (PLCβ) – protein kinase C (PKCδ) – adenylyl cyclase (ACVII) – protein kinase A (PKA) signaling cascade to decouple GABA_B (and μ-opioid) receptors from inhibitory G protein-coupled inwardly rectifying K⁺(GIRK) channels. PKA can also phosphorylate cAMP response element binding protein (pCREB) to generate new gene transcription through CRE’s. In addition, E2 binds to estrogen receptor α (ERα) in POMC neurons to increase Pomc, Vglut2, TRPC5 and CaV3.1 gene expression through ERE’s. On the other hand, stromal interacting molecule 1 (Stim1) expression is decreased, preserving TRPC5 channels as receptor operated channels for transmitting insulin’s (and leptin’s) effects. Note: E2 has similar actions in kisspeptin neurons with the exception that E2 downregulates the expression of the peptide.

3. E2 regulation of ARH neurons

More subtle influences are present during normal healthy reproductive cycles. Food intake, specifically sweet foods, decreases during the follicular phase (high E2) of the menstrual cycle [125-127]. The degeneration of AgRP neurons, induced via cell-specific deletion of the mitochondrial transcription factor A gene, eliminates cyclic changes in ingestive behavior [128]. Ovariectomy leads to a decrease in motor activity [129-131] and increased food intake [21, 132-137], but E2 replacement is sufficient to restore normal energy balance [21, 129- 131, 133, 138]. On the receptor side of estrogenic signaling, ERα knockout mice develop an obese phenotype similar to that seen following ovariectomy [139]. Metabolic deficits are reversed by restoration of ERα, despite lacking the ERE targeting domain, which emphasizes the importance of non-classical signaling [140]. Interestingly, POMC-specific deletion of ERα is sufficient to induce hyperphagia and increased heat production [54]. This phenotype could be due to higher circulating levels of E2, which is suggested by blunted negative feedback of E2 on LH release that produces abnormal estrous cycles. Therefore, estrogenic signaling is of tantamount importance to the anorexigenic function of POMC neurons in females. Perhaps, Kiss1 and POMC neurons set the tone of homeostatic circuits based on the reproductive state of the female. The neuronal activity of AgRP neurons is suppressed until energy reserves reach critical low levels. For example, fasting enhances AgRP activity and signaling by rapidly rewiring circuits and affecting gene transcription [141, 142]. However, under normal physiological conditions rapid E2 signaling in AgRP neurons may be a more appropriate means of adjusting their activity. For example, E2 quickly alters AgRP excitability, enhancing or attenuating the excitability (i.e., coupling of GABA_B receptors to activate G protein inwardly rectifying K⁺ (GIRK) channels), possibly depending on the relative expression of ERα to mER [14,, 15]. These findings suggest that estrogenic signaling uses both genomic and rapid mechanisms to regulate POMC and Kiss1 function but relies more on membrane-initiated signaling for AgRP neurons.

For some time now, it has been accepted that AgRP neurons send inhibitory GABAergic projections to POMC neurons [70, 99]. More recently evidence has emerged that POMC neurons send reciprocal projections to AgRP neurons, primarily releasing β-endorphin and glutamate (Figure 1) [78]. A previous report found GABAergic inhibitory currents to be the most common response in unidentified ARH neurons following optogenetic stimulation of POMC neurons [75]. Surprisingly, optogenetic activation of POMC neurons rarely elicits a fast GABAergic or a slow excitatory (e.g. α-MSH) postsynaptic current in AgRP neurons [67, 70, 78]. While very few ARH NPY neurons express MC4R, nearly half express MC3R [143]. Therefore, the infrequency of GABA and melanocortin mediated responses suggests segregated neurotransmission [144-147]. The predominance of POMC glutamatergic, not GABAergic, inputs is counterintuitive as one would not expect a satiety neuron to excite a hunger neuron. However, glutamatergic input from POMC neurons would still be inhibitory in a high E2 state since Group II/III mGluRs are the most highly expressed in AgRP neurons [66]. High E2 will directly enhance the efficacy of POMC signaling through greater precursor peptide processing [148, 149] and increased expression of Vglut2 [78]. Furthermore, acutely applied E2 or STX increases the probability of glutamate release [78], possibly by decoupling GABA_B and G protein-coupled inwardly rectifying K⁺ (GIRK) channels in POMC nerve terminals (Figure 2) [21, 150]. Together these effects will support POMC inhibition of AgRP neurons. Kiss1^ARH neurons also display increased expression of Vglut2 and glutamate release probability onto POMC and AgRP neurons [66]. Therefore when E2 is low, POMC activity will be minimal, generating a trickle of glutamate release onto AgRP neurons (Figure 1). When an animal is fed, AgRP neurons become less active, reducing inhibitory input onto POMC neurons [151]. POMC activity will increase to higher (20 Hz) frequencies [152], which are capable of eliciting β-endorphin release to inhibit AgRP neurons via activation of μ-opioid receptors [78]. In a high-E2 state, POMC and Kiss1 neurons will also have enhanced glutamate release, inhibiting AgRP neurons through Group II/III metabotropic glutamate receptors [66, 78]. Concurrently, Kiss1^ARH neurons will excite POMC neurons through Group I mGluRs [66]. This arrangement between ARH neurons prevents minor fluctuations in the energy state of the animal from triggering drastic changes in ARH function [151], but also allows E2 to bias the system towards reduced food intake. Rapid estrogenic signaling will smooth the state transitions as slower, transcriptional mechanisms are engaged. The functional relevance of these circuit dynamics could be realized in shifts in food motivation. Assuming the female has sufficient energy stores, ovulation is induced, and reward salience is shifted from food to potential mates. Without estrogen ovariectomized female rodents retain food motivation and find sucrose more rewarding than E2-treated females [153]. When Vglut2 is deleted from Kiss1^ARH neurons, this protective effect of E2 is abrogated [66], suggesting glutamatergic inhibition of AgRP neurons and excitation of POMC neurons may be an underlying mechanism (Figure 1). Therefore, there is little doubt that estrogenic signaling is necessary for Kiss1 neurons to control GnRH and LH release. However, it is also becoming clear that estrogens orchestrate communication between Kiss1, AgRP, and POMC neurons to optimize reproductive success.