Fasting Modulates GABAergic Synaptic Transmission to Arcuate Kisspeptin Neurons in Female Mice.

It is well-established that the hypothalamic-pituitary-gonadal (HPG) axis is suppressed due to negative energy balance. However, less information is available on whether kisspeptin neuronal activity contributes to fasting-induced responses. In the present study, female and male mice were fasted for 24 hours or provided food ad libitum (fed group) to determine whether acute fasting is sufficient to modulate kisspeptin neuronal activity. In female mice, fasting attenuated luteinizing hormone (LH) and prolactin (PRL) serum levels and increased follicle-stimulating hormone levels compared with the fed group. In contrast, fasting did not affect gonadotropin or PRL secretion in male mice. By measuring genes related to LH pulse generation in micropunches obtained from the arcuate nucleus of the hypothalamus (ARH), we observed that fasting reduced Kiss1 mRNA levels in female and male mice. In contrast, Pdyn expression was upregulated only in fasted female mice, whereas no changes in the Tac2 mRNA levels were observed in both sexes. Interestingly, the frequency and amplitude of the GABAergic postsynaptic currents recorded from ARH kisspeptin neurons (ARHKisspeptin) were reduced in 24-hour fasted female mice but not in males. Additionally, neuropeptide Y induced a hyperpolarization in the resting membrane potential of ARHKisspeptin neurons of fed female mice but not in males. Thus, the response of ARHKisspeptin neurons to fasting is sexually dependent with a female bias, associated with changes in gonadotropins and PRL secretion. Our findings suggest that GABAergic transmission to ARHKisspeptin neurons modulates the activity of the HPG axis during situations of negative energy balance.

RFamide-related Peptide 3 Signaling via Neuropeptide FF Receptor Stimulates Prolactin Secretion in Female Rats.

The RF-amide peptides comprise a family of neuropeptides that includes the kisspeptin (Kp), the natural ligand of kisspeptin receptor (Kiss1r), and the RFamide-related peptide 3 (RFRP-3) that binds preferentially to the neuropeptide FF receptor 1 (Npffr1). Kp stimulates prolactin (PRL) secretion through the inhibition of tuberoinfundibular dopaminergic (TIDA) neurons. Because Kp also has affinity to Npffr1, we investigated the role of Npffr1 in the control of PRL secretion by Kp and RFRP-3. Intracerebroventricular (ICV) injection of Kp increased PRL and LH secretion in ovariectomized, estradiol-treated rats. The unselective Npffr1 antagonist RF9 prevented these responses, whereas the selective antagonist GJ14 altered PRL but not LH levels. The ICV injection of RFRP-3 in ovariectomized, estradiol-treated rats increased PRL secretion, which was associated with a rise in the dopaminergic activity in the median eminence, but had no effect on LH levels. The RFRP-3-induced increase in PRL secretion was prevented by GJ14. Moreover, the estradiol-induced PRL surge in female rats was blunted by GJ14, along with an amplification of the LH surge. Nevertheless, whole-cell patch clamp recordings showed no effect of RFRP-3 on the electrical activity of TIDA neurons in dopamine transporter-Cre recombinase transgenic female mice. We provide evidence that RFRP-3 binds to Npffr1 to stimulate PRL release, which plays a role in the estradiol-induced PRL surge. This effect of RFRP-3 is apparently not mediated by a reduction in the inhibitory tone of TIDA neurons but possibly involves the activation of a hypothalamic PRL-releasing factor.

Subacute high-refined carbohydrate diet leads to abnormal reproductive control of the hypothalamic-pituitary axis in female rats.

We previously reported that female rats placed on a diet containing refined carbohydrates (HCD) resulted in obesity and reproductive abnormalities, such as high serum LH concentration and abnormal ovarian function. However, the impacts at the hypothalamic-pituitary (HP) function, specifically regarding pathways linked to reproductive axis modulation are unknown. In this study, we assessed whether subacute feeding with HCD results in abnormal reproductive control in the HP axis. Female rats were fed with HCD for 15 days and reproductive HP axis morphophysiology was assessed. HCD reduced hypothalamic mRNA expression (Kiss1, Lepr, and Amhr2) and increased pituitary LHß+ cells. These changes likely contribute to the increase in serum LH concentration observed in HCD. Blunted estrogen negative feedback was observed in HCD, with increased kisspeptin protein expression in the arcuate nucleus of the hypothalamus (ARH), lower LHß+ cells and LH concentration in ovariectomized (OVX)+HCD rats. Thus, these data suggest that HCD feeding led to female abnormal reproductive control of HP axis.

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings.

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Pattern of gonadotropin secretion along the estrous cycle of C57BL/6 female mice.

The pattern of gonadotropin secretion along the estrous cycle was elegantly described in rats. Less information exists about the pattern of gonadotropin secretion in gonad-intact mice, particularly regarding the follicle-stimulating hormone (FSH). Using serial blood collections from the tail-tip of gonad-intact C57BL/6 mice on the first day of cornification (transition from diestrus to estrus; hereafter called proestrus), we observed that the luteinizing hormone (LH) and FSH surge cannot be consistently detected since only one out of eight females (12%) showed increased LH levels. In contrast, a high percentage of mice (15 out of 21 animals; 71%) exhibited LH and FSH surges on the proestrus when a single serum sample was collected. Mice that exhibited LH and FSH surges on the proestrus showed c-Fos expression in gonadotropin-releasing hormone- (GnRH; 83.4% of co-localization) and kisspeptin-expressing neurons (42.3% of co-localization) of the anteroventral periventricular nucleus (AVPV). Noteworthy, mice perfused on proestrus, but that failed to exhibit LH surge, showed a smaller, but significant expression of c-Fos in GnRH (32.7%) and AVPVKisspeptin (14.0%) neurons. Finally, 96 serial blood samples were collected hourly in eight regular cycling C57BL/6 females to describe the pattern of LH and FSH secretion along the estrous cycle. Small elevations in LH and FSH levels were detected at the time expected for the LH surge. In summary, the present study improves our understanding of the pattern of gonadotropin secretion and the activation of central components of the hypothalamic-pituitary-gonadal axis along the estrous cycle of C57BL/6 female mice.

Socs3 ablation in kisspeptin cells partially prevents lipopolysaccharide-induced body weight loss.

Many cytokines have been proposed to regulate reproduction due to their actions on hypothalamic kisspeptin cells, the main modulators of gonadotropin-releasing hormone (GnRH) neurons. Hormones such as leptin, prolactin and growth hormone are good examples of cytokines that lead to Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation, consequently exerting effects in kisspeptin neurons. Different studies have investigated how specific components of the JAK/STAT signaling pathway affect the functions of kisspeptin cells, but the role of the suppressor of cytokine signaling 3 (SOCS3) in mediating cytokine actions in kisspeptin cells remains unknown. Cre-Loxp technology was used in the present study to ablate Socs3 expression in kisspeptin cells (Kiss1/Socs3-KO). Then, male and female control and Kiss1/Socs3-KO mice were evaluated for sexual maturation, energy homeostasis features, and fertility. It was found that hypothalamic Kiss1 mRNA expression is significantly downregulated in Kiss1/Socs3-KO mice. Despite reduced hypothalamic Kiss1 mRNA content, these mice did not present any sexual maturation or fertility impairments. Additionally, body weight gain, leptin sensitivity and glucose homeostasis were similar to control mice. Interestingly, Kiss1/Socs3-KO mice were partially protected against lipopolysaccharide (LPS)-induced body weight loss. Our results suggest that Socs3 ablation in kisspeptin cells partially prevents the sickness behavior induced by LPS, suggesting that kisspeptin cells can modulate energy metabolism in mice in certain situations.

Vasoactive intestinal peptide exerts an excitatory effect on hypothalamic kisspeptin neurons during estrogen negative feedback.

Hypothalamic kisspeptin neurons are the primary modulators of gonadotropin-releasing hormone (GnRH) neurons. It has been shown that circadian rhythms driven by the suprachiasmatic nucleus (SCN) contribute to GnRH secretion. Kisspeptin neurons are potential targets of SCN neurons due to reciprocal connections with the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and the arcuate nucleus of the hypothalamus (ARH). Vasoactive intestinal peptide (VIP), a notable SCN neurotransmitter, modulates GnRH secretion depending on serum estradiol levels, aging or time of the day. Considering that kisspeptin neurons may act as interneurons and mediate VIP's effects on the reproductive axis, we investigated the effects of VIP on hypothalamic kisspeptin neurons in female mice during estrogen negative feedback. Our findings indicate that VIP induces a TTX-independent depolarization of approximately 30% of AVPV/PeN kisspeptin neurons in gonad-intact (diestrus) and ovariectomized (OVX) mice. In the ARH, the percentage of kisspeptin neurons that were depolarized by VIP was even higher (approximately 90%). An intracerebroventricular infusion of VIP leds to an increased percentage of kisspeptin neurons expressing the phosphoSer133 cAMP-response-element-binding protein (pCREB) in the AVPV/PeN. On the other hand, pCREB expression in ARH kisspeptin neurons was similar between saline- and VIP-injected mice. Thus, VIP can recruit different signaling pathways to modulate AVPV/PeN or ARH kisspeptin neurons, resulting in distinct cellular responses. The expression of VIP receptors (VPACR) was upregulated in the AVPV/PeN, but not in the ARH, of OVX mice compared to mice on diestrus and estradiol-primed OVX mice. Our findings indicate that VIP directly influences distinct cellular aspects of the AVPV/PeN and ARH kisspeptin neurons during estrogen negative feedback, possibly to influence pulsatile LH secretion.

Distinct effects of growth hormone deficiency and disruption of hypothalamic kisspeptin system on reproduction of male mice.

Growth hormone (GH) deficiency is a common cause of late sexual maturation and fertility issues. To determine whether GH-induced effects on reproduction are associated with alterations in hypothalamic kisspeptin system, we studied the male reproduction in two distinct GH deficiency mouse models. In the first model, mice present GH deficiency secondary to arcuate nucleus of the hypothalamus (ARH) lesions induced by posnatal monosodium glutamate (MSG) injections. MSG-induced ARH lesions led to significant reductions in hypothalamic Ghrh mRNA expression and consequently growth. Hypothalamic Kiss1 mRNA expression and Kiss1-expressing cells in the ARH were disrupted in the MSG-treated mice. In contrast, kisspeptin immunoreactivity remained preserved in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) of MSG-treated mice. Importantly, ARH lesions caused late sexual maturation and infertility in male mice. In our second mouse model, we studied animals profound GH deficiency due to a loss-of-function mutation in the Ghrhr gene (Ghrhrlit/lit mice). Interestingly, although Ghrhrlit/lit mice exhibited late puberty onset, hypothalamic Kiss1 mRNA expression and hypothalamic kisspeptin fiber density were normal in Ghrhrlit/lit mice. Despite presenting dwarfism, the majority of Ghrhrlit/lit male mice were fertile. These findings suggest that spontaneous GH deficiency during development does not compromise the kisspeptin system. Furthermore, ARH Kiss1-expressing neurons are required for fertility, while AVPV/PeN kisspeptin expression is sufficient to allow maturation of the hypothalamic-pituitary-gonadal axis in male mice.

Tumor Necrosis Factor α and Interleukin-1ß Acutely Inhibit AgRP Neurons in the Arcuate Nucleus of the Hypothalamus.

Obesity-associated low-grade inflammation favors weight gain, whereas systemic infection frequently leads to anorexia. Thus, inflammatory signals can either induce positive or negative energy balance. In this study, we used whole-cell patch-clamp to investigate the acute effects of three important proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-6, and interleukin-1ß (IL-1ß) on the membrane excitability of agouti-related peptide (AgRP)- or proopiomelanocortin (POMC)-producing neurons. We found that both TNF-α and IL-1ß acutely inhibited the activity of 35-42% of AgRP-producing neurons, whereas very few POMC neurons were depolarized by TNF-α. Interleukin-6 induced no acute changes in the activity of AgRP or POMC neurons. Our findings indicate that the effect of TNF-α and IL-1ß, especially on the activity of AgRP-producing neurons, may contribute to inflammation-induced anorexia observed during acute inflammatory conditions.

Effects of growth hormone in the central nervous system

ABSTRACT Growth hormone (GH) is best known for its effect stimulating tissue and somatic growth through the regulation of cell division, regeneration and proliferation. However, GH-responsive neurons are spread over the entire central nervous system, suggesting that they have important roles in the brain. The objective of the present review is to summarize and discuss the potential physiological importance of GH action in the central nervous system. We provide evidence that GH signaling in the brain regulates the physiology of numerous functions such as cognition, behavior, neuroendocrine changes and metabolism. Data obtained from experimental animal models have shown that disruptions in GH signaling in specific neuronal populations can affect the reproductive axis and impair food intake during glucoprivic conditions, neuroendocrine adaptions during food restriction, and counter-regulatory responses to hypoglycemia, and they can modify gestational metabolic adaptions. Therefore, the brain is an important target tissue of GH, and changes in GH action in the central nervous system can explain some dysfunctions presented by individuals with excessive or deficient GH secretion. Furthermore, GH acts in specific neuronal populations during situations of metabolic stress to promote appropriate physiological adjustments that restore homeostasis. Arch Endocrinol Metab. 2019;63(6):549-56

Growth hormone/STAT5 signaling in proopiomelanocortin neurons regulates glucoprivic hyperphagia.

Several hypothalamic neuronal populations are directly responsive to growth hormone (GH) and central GH action regulates glucose and energy homeostasis. However, the potential role of GH signaling in proopiomelanocortin (POMC) neurons has not been studied yet. Thus, we investigated whether POMC neurons are responsive to GH and if ablation of GH receptor (GHR) or STAT5 in POMC cells leads to metabolic imbalances. Approximately 60% of POMC neurons of the arcuate nucleus exhibited STAT5 phosphorylation after intracerebroventricular GH injection. Ablation of GHR or STAT5 in POMC cells did not affect energy or glucose homeostasis. However, glucoprivic hyperphagia was blunted in male and female GHR knockout mice, and in male POMC-specific STAT5 knockout mice. Additionally, the absence of GHR in POMC neurons decreased glycemia during prolonged food restriction in male mice. Thus, GH action in POMC neurons regulates glucoprivic hyperphagia as well as blood glucose levels during prolonged food restriction.

Acute effects of somatomammotropin hormones on neuronal components of the hypothalamic-pituitary-gonadal axis.

Growth hormone (GH) and prolactin (PRL) are known as pleiotropic hormones. Accordingly, the distribution of their receptors comprises several organs and tissues, including the central nervous system. The appropriate secretion of both hormones is essential for sexual maturation and maintenance of reproductive functions, while defects in their secretion affect puberty onset and can cause infertility. Conversely, GH therapy at a prepubertal age may accelerate puberty. On the other hand, hyperprolactinemia is a frequent cause of infertility. While the action of PRL in some central components of the Hypothalamic-Pituitary-Gonadal (HPG) axis, such as the kisspeptin neurons, has been well documented, the possible effects of GH in the hypothalamus are still elusive. Thus, the present study was designed to investigate whether somatomammotropin hormones are able to modulate the activity of critical neuronal components of the HPG axis, including kisspeptin neurons and cells of the ventral premammillary nucleus (PMv). Our results revealed that GH effects in kisspeptin neurons of the anteroventral periventricular and rostral periventricular nuclei or in PMv neurons relies predominantly on the recruitment of the signal transducer and activator of transcription 5 (STAT5) rather than through acute changes in resting membrane potential. Importantly, kisspeptin neurons located at the arcuate nucleus were not directly responsive to GH. Additionally, our findings further identified PMv neurons as potential targets of PRL, since PRL induces the phosphorylation of STAT5 and depolarizes PMv neurons. Combined, our data provide evidence that GH and PRL may affect the HPG axis via specific hypothalamic neurons.

Conspecific odor exposure predominantly activates non-kisspeptin cells in the medial nucleus of the amygdala.

A small neuronal subpopulation in the medial nucleus of the amygdala (MeA), expressing the Kiss1 gene, is now considered an important mediator that integrates socio-sexual behavior and odor information in order to modulate the Hypothalamic-Pituitary-Gonadal (HPG) axis. Previous studies demonstrated that exogenous kisspeptin administration or selective activation of Kiss1-expressing neurons in the MeA modulates the onset of puberty, LH secretion and sexual behavior. These functions are supported by the known MeA neuronal connections. In the MeA, as well as in the hypothalamus, Kiss1 mRNA expression mostly depends on sex steroids levels. However, the percentage of Kiss1-expressing cells that co-express estrogen receptor α (ERα) in the MeA is currently unknown. Additionally, whether MeA kisspeptin neurons show Fos expression due to pheromone exposure is still undisclosed. In the present study, we used adult male and female mice that express a reporter protein under the Kiss1 promoters to determine the percentage of Kiss1-expressing neurons that co-express the ERα in the MeA and, whether those cells are activated by olfactory cues. We found a high percentage of Kiss1-expressing neurons in the MeA co-expressing the ERα. The proportion of co-expression was similar between male and female mice in diestrus. Interestingly, a low percentage of Kiss1-expressing neurons in the MeA co-express Fos after conspecific odor exposure, despite a significant increase of Fos positive cells in the MeA. Additionally, odor exposition leads to a sexually dimorphic change in Kiss1 expression in the posterior subdivision of the MeA. Our findings suggest that olfactory signals predominantly activate non-kisspeptin cells in the MeA to modulate responses to pheromones and therefore the HPG axis.

STAT5 signaling in kisspeptin cells regulates the timing of puberty.

Previous studies have shown that kisspeptin neurons are important mediators of prolactin's effects on reproduction. However, the cellular mechanisms recruited by prolactin to affect kisspeptin neurons remain unknown. Using whole-cell patch-clamp recordings of brain slices from kisspeptin reporter mice, we observed that 20% of kisspeptin neurons in the anteroventral periventricular nucleus was indirectly depolarized by prolactin via an unknown population of prolactin responsive neurons. This effect required the phosphatidylinositol 3-kinase signaling pathway. No effects on the activity of arcuate kisspeptin neurons were observed, despite a high percentage (70%) of arcuate neurons expressing prolactin-induced STAT5 phosphorylation. To determine whether STAT5 expression in kisspeptin cells regulates reproduction, mice carrying Stat5a/b inactivation specifically in kisspeptin cells were generated. These mutants exhibited an early onset of estrous cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the timing of puberty.

Interactions between prolactin and kisspeptin to control reproduction.

Prolactin is best known for its effects of stimulating mammary gland development and lactogenesis. However, prolactin is a pleiotropic hormone that is able to affect several physiological functions, including fertility. Prolactin receptors (PRLRs) are widely expressed in several tissues, including several brain regions and reproductive tract organs. Upon activation, PRLRs may exert prolactin's functions through several signaling pathways, although the recruitment of the signal transducer and activator of transcription 5 causes most of the known effects of prolactin. Pathological hyperprolactinemia is mainly due to the presence of a prolactinoma or pharmacological effects induced by drugs that interact with the dopamine system. Notably, hyperprolactinemia is a frequent cause of reproductive dysfunction and may lead to infertility in males and females. Recently, several studies have indicated that prolactin may modulate the reproductive axis by acting on specific populations of hypothalamic neurons that express the Kiss1 gene. The Kiss1 gene encodes neuropeptides known as kisspeptins, which are powerful activators of gonadotropin-releasing hormone neurons. In the present review, we will summarize the current knowledge about prolactin's actions on reproduction. Among other aspects, we will discuss whether the interaction between prolactin and the Kiss1-expressing neurons can affect reproduction and how kisspeptins may become a novel therapeutic approach to treat prolactin-induced infertility.

Interactions between prolactin and kisspeptin to control reproduction

ABSTRACT Prolactin is best known for its effects of stimulating mammary gland development and lactogenesis. However, prolactin is a pleiotropic hormone that is able to affect several physiological functions, including fertility. Prolactin receptors (PRLRs) are widely expressed in several tissues, including several brain regions and reproductive tract organs. Upon activation, PRLRs may exert prolactin’s functions through several signaling pathways, although the recruitment of the signal transducer and activator of transcription 5 causes most of the known effects of prolactin. Pathological hyperprolactinemia is mainly due to the presence of a prolactinoma or pharmacological effects induced by drugs that interact with the dopamine system. Notably, hyperprolactinemia is a frequent cause of reproductive dysfunction and may lead to infertility in males and females. Recently, several studies have indicated that prolactin may modulate the reproductive axis by acting on specific populations of hypothalamic neurons that express the Kiss1 gene. The Kiss1 gene encodes neuropeptides known as kisspeptins, which are powerful activators of gonadotropin-releasing hormone neurons. In the present review, we will summarize the current knowledge about prolactin’s actions on reproduction. Among other aspects, we will discuss whether the interaction between prolactin and the Kiss1-expressing neurons can affect reproduction and how kisspeptins may become a novel therapeutic approach to treat prolactin-induced infertility.

Changes in Leptin Signaling by SOCS3 Modulate Fasting-Induced Hyperphagia and Weight Regain in Mice.

Weight regain frequently follows interventions that reduce body weight, leading to a failure in long-term obesity treatment. Inhibitory proteins of the leptin signaling pathway, such as the suppressor of cytokine signaling 3 (SOCS3), have been studied in conditions that predispose animals to obesity. However, whether SOCS3 modulates postrestriction hyperphagia and weight regain remains unknown. Mice lacking SOCS3 protein specifically in leptin receptor (LepR)-expressing cells (LepR SOCS3 knockout [KO]) were generated and studied in fasting and refeeding conditions. LepR SOCS3 KO mice exhibited increased leptin sensitivity in the hypothalamus. Notably, LepR SOCS3 KO males and females showed attenuated food intake and weight regain after 48 hours of fasting. Postrestriction hyperleptinemia was also prevented in LepR SOCS3 KO mice. Next, we studied possible mechanisms and neural circuits involved in the SOCS3 effects. SOCS3 deletion did not prevent fasting- or refeeding-induced c-Fos expression in the arcuate nucleus of the hypothalamus (ARH) nor fasting-induced increased excitability of ARH LepR-expressing cells. On the other hand, SOCS3 ablation reduced the mRNA levels of hypothalamic orexigenic neuropeptides during fasting (neuropeptide Y, agouti-related protein, orexin, and melanin-concentrating hormone). In summary, our findings suggest that increased leptin sensitivity contributes to the maintenance of a reduced body weight after food deprivation. In addition, the attenuated postrestriction food intake observed in mutant mice was not explained by fasting-induced changes in the activity of ARH neurons but exclusively by a lower transcription of orexigenic neuropeptides during fasting. These results indicate a partial dissociation between the regulation of neuronal activity and gene expression in ARH LepR-expressing cells.

Alzheimer-associated Aß oligomers impact the central nervous system to induce peripheral metabolic deregulation.

Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aß oligomers (AßOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AßOs failed to induce glucose intolerance, suggesting AßOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AßOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AßOs further induced eIF2α-P and activated pro-inflammatory IKKß/NF-κB signaling in the hypothalamus of mice and macaques. AßOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AßOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AßOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

Leptin resistance is not the primary cause of weight gain associated with reduced sex hormone levels in female mice.

Several studies have shown that estrogens mimic leptin's effects on energy balance regulation. However, the findings regarding the consequences of reduced sex hormone levels on leptin sensitivity are divergent. In the present study, we employed different experimental paradigms to elucidate the interaction between estrogens, leptin, and energy balance regulation. We confirmed previous reports showing that ovariectomy caused a reduction in locomotor activity and energy expenditure leading mice to obesity and glucose intolerance. However, the acute and chronic anorexigenic effects of leptin were preserved in ovariectomized (OVX) mice despite their increased serum leptin levels. We studied hypothalamic gene expression at different time points after ovariectomy and observed that changes in the expression of genes involved in leptin resistance (suppressors of cytokine signaling and protein-tyrosine phosphatases) did not precede the early onset of obesity in OVX mice. On the contrary, reduced sex hormone levels caused an up-regulation of the long form of the leptin receptor (LepR), resulting in increased activation of leptin signaling pathways in OVX leptin-treated animals. The up-regulation of the LepR was observed in long-term OVX mice (30 d or 24 wk after ovariectomy) but not 7 days after the surgery. In addition, we observed a progressive decrease in the coexpression of LepR and estrogen receptor-α in the hypothalamus after the ovariectomy, resulting in a low percentage of dual-labeled cells in OVX mice. Taken together, our findings suggest that the weight gain caused by reduced sex hormone levels is not primarily caused by induction of a leptin-resistance state.

Estradiol modulates Kiss1 neuronal response to ghrelin.

Ghrelin is a metabolic signal regulating energy homeostasis. Circulating ghrelin levels rise during starvation and fall after a meal, and therefore, ghrelin may function as a signal of negative energy balance. Ghrelin may also act as a modulator of reproductive physiology, as acute ghrelin administration suppresses gonadotropin secretion and inhibits the neuroendocrine reproductive axis. Interestingly, ghrelin's effect in female metabolism varies according to the estrogen milieu predicting an interaction between ghrelin and estrogens, likely at the hypothalamic level. Here, we show that ghrelin receptor (GHSR) and estrogen receptor-α (ERα) are coexpressed in several hypothalamic sites. Higher levels of circulating estradiol increased the expression of GHSR mRNA and the coexpression of GHSR mRNA and ERα selectively in the arcuate nucleus (ARC). Subsets of preoptic and ARC Kiss1 neurons coexpressed GHSR. Increased colocalization was observed in ARC Kiss1 neurons of ovariectomized estradiol-treated (OVX + E2; 80%) compared with ovariectomized oil-treated (OVX; 25%) mice. Acute actions of ghrelin on ARC Kiss1 neurons were also modulated by estradiol; 75 and 22% of Kiss1 neurons of OVX + E2 and OVX mice, respectively, depolarized in response to ghrelin. Our findings indicate that ghrelin and estradiol may interact in several hypothalamic sites. In the ARC, high levels of E2 increase GHSR mRNA expression, modifying the colocalization rate with ERα and Kiss1 and the proportion of Kiss1 neurons acutely responding to ghrelin. Our findings indicate that E2 alters the responsiveness of kisspeptin neurons to metabolic signals, potentially acting as a critical player in the metabolic control of the reproductive physiology.