Diverse actions of estradiol on anorexigenic and orexigenic hypothalamic arcuate neurons.

Contribution to Special Issue on Fast effects of steroids. There is now compelling evidence for membrane-associated estrogen receptors in hypothalamic neurons that are critical for the hypothalamic control of homeostatic functions. It has been known for some time that estradiol (E2) can rapidly alter hypothalamic neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events. However, our understanding of how E2 signals via membrane-associated receptors and how these signals impact physiological functions is only just emerging. Thus, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell excitability and even gene transcription in hypothalamic neurons. One population of hypothalamic neurons, the anorexigenic proopiomelanocortin (POMC) neurons, has long been considered to be a target of E2's actions based on gene (Pomc) expression studies. However, we now know that E2 can rapidly alter POMC neuronal activity within seconds and activate several intracellular signaling cascades that ultimately affect gene expression, actions which are critical for maintaining sensitivity to insulin in metabolically stressed states. E2 also affects the orexigenic Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons in similarly rapid but antagonistic manner. Therefore, this review will summarize our current state of knowledge of how E2 signals via rapid membrane-initiated and intracellular signaling cascades in POMC and NPY/AgRP neurons to regulate energy homeostasis.

GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding.

Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.

Central and peripheral mechanisms involved in the control of GnRH neuronal function by metabolic factors.

Gonadotropin-releasing hormone (GnRH) neurons are the final output pathway for the brain control of reproduction. The activity of this neuronal population, mainly located at the preoptic area of the hypothalamus, is controlled by a plethora of metabolic signals. However, it has been documented that most of these signal impact on GnRH neurons through indirect neuronal circuits, Kiss1, proopiomelanocortin, and neuropeptide Y/agouti-related peptide neurons being some of the most prominent mediators. In this context, compelling evidence has been gathered in recent years on the role of a large range of neuropeptides and energy sensors in the regulation of GnRH neuronal activity through both direct and indirect mechanisms. The present review summarizes some of the most prominent recent advances in our understanding of the peripheral factors and central mechanisms involved in the metabolic control of GnRH neurons.

Melanocortin Signaling Connecting Systemic Metabolism With Mood Disorders.

Obesity and mood disorders are often overlapping pathologies that are prevalent public health concerns. Many studies have indicated a positive correlation between depression and obesity, although weight loss and decreased appetite are also recognized as features of depression. Accordingly, DSM-5 defines two subtypes of depression associated with changes in feeding: melancholic depression, characterized by anhedonia and associated with decreased feeding and appetite; and atypical depression, characterized by fatigue, sleepiness, hyperphagia, and weight gain. The central nervous system plays a key role in the regulation of feeding and mood, thus suggesting that overlapping neuronal circuits may be involved in their modulation. However, these circuits have yet to be completely characterized. The central melanocortin system, a circuitry characterized by the expression of specific peptides (pro-opiomelanocortins, agouti-related protein, and neuropeptide Y) and their melanocortin receptors, has been shown to be a key player in the regulation of feeding. In addition, the melanocortin system has also been shown to affect anxiety and depressive-like behavior, thus suggesting a possible role of the melanocortin system as a biological substrate linking feeding and depression. However, more studies are needed to fully understand this complex system and its role in regulating metabolic and mood disorders. In this review, we will discuss the current literature on the role of the melanocortin system in human and animal models in feeding and mood regulation, providing evidence of the biological interplay between anxiety, major depressive disorders, appetite, and body weight regulation.

Exercise increases NPY/AgRP and TH neuron activity in the hypothalamus of female mice.

Recent evidence identifies a potent role for aerobic exercise to modulate the activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low- (10 m/min), moderate- (14 m/min), and high (18 m/min)-intensity treadmill exercise in a randomized crossover design. Mice were fasted 10 h before exercise, and food intake was monitored for 48 h after bouts. Immunohistochemical detection of cFOS was performed 3 h post-exercise to determine the changes in hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), tyrosine hydroxylase (TH), and SIM1-expressing neuron activity concurrent with the changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate- and high-intensity exercises increased 24-h food intake by 5.9 and 19%, respectively, while low-intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and TH neuron activity were observed 3 h after high-intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3 h post-exercise. Overall, aerobic exercise increased the activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.

Energy Status Differentially Modifies Feeding Behavior and POMCARC Neuron Activity After Acute Treadmill Exercise in Untrained Mice.

Emerging evidence identifies a potent role for aerobic exercise to modulate activity of neurons involved in regulating appetite; however, these studies produce conflicting results. These discrepancies may be, in part, due to methodological differences, including differences in exercise intensity and pre-exercise energy status. Consequently, the current study utilized a translational, well-controlled, within-subject, treadmill exercise protocol to investigate the differential effects of energy status and exercise intensity on post-exercise feeding behavior and appetite-controlling neurons in the hypothalamus. Mature, untrained male mice were exposed to acute sedentary, low (10m/min), moderate (14m/min), and high (18m/min) intensity treadmill exercise in a randomized crossover design. Fed and 10-hour-fasted mice were used, and food intake was monitored 48h. post-exercise. Immunohistochemical detection of cFOS was performed 1-hour post-exercise to determine changes in hypothalamic NPY/AgRP, POMC, tyrosine hydroxylase, and SIM1-expressing neuron activity concurrent with changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Results demonstrated that fasted high intensity exercise suppressed food intake compared to sedentary trials, which was concurrent with increased anorexigenic POMC neuron activity. Conversely, fed mice experienced augmented post-exercise food intake, with no effects on POMC neuron activity. Regardless of pre-exercise energy status, tyrosine hydroxylase and SIM1 neuron activity in the paraventricular nucleus was elevated, as well as NPY/AgRP neuron activity in the arcuate nucleus. Notably, these neuronal changes were independent from changes in pSTAT3tyr705 and pERKthr202/tyr204 signaling. Overall, these results suggest fasted high intensity exercise may be beneficial for suppressing food intake, possibly due to hypothalamic POMC neuron excitation. Furthermore, this study identifies a novel role for pre-exercise energy status to differentially modify post-exercise feeding behavior and hypothalamic neuron activity, which may explain the inconsistent results from studies investigating exercise as a weight loss intervention.

Chronic Isolation Stress Affects Central Neuroendocrine Signaling Leading to a Metabolically Active Microenvironment in a Mouse Model of Breast Cancer.

Social isolation is a powerful stressor capable of affecting brain plasticity and function. In the case of breast cancer, previous data indicate that stressful experiences may contribute to a worse prognosis, activating neuroendocrine and metabolism pathways, although the mechanisms underlying these effects are still poorly understood. In this study, we tested the hypothesis that chronic isolation stress (IS) may boost hypothalamic-pituitary-adrenal (HPA) axis activity, leading to changes in the hypothalamic expression of genes modulating both mood and metabolism in an animal model of breast cancer. This centrally activated signaling cascade would, in turn, affect the mammary gland microenvironment specifically targeting fat metabolism, leading to accelerated tumor onset. MMTVNeuTg female mice (a model of breast cancer developing mammary hyperplasia at 5 months of age) were either group-housed (GH) or subjected to IS from weaning until 5 months of age. At this time, half of these subjects underwent acute restraint stress to assess corticosterone (CORT) levels, while the remaining subjects were characterized for their emotional profile in the forced swimming and saccharin preference tests. At the end of the procedures, all the mice were sacrificed to assess hypothalamic expression levels of Brain-derived neurotrophic factor (Bdnf), Neuropeptide Y (NpY), Agouti-Related Peptide (AgRP), and Serum/Glucocorticoid-Regulated Protein Kinase 1 (SgK1). Leptin and adiponectin expression levels, as well as the presence of brown adipose tissue (BAT), were assessed in mammary fat pads. The IS mice showed higher CORT levels following acute stress and decreased expression of NpY, AgRP, and SgK1, associated with greater behavioral despair in the forced swimming test. Furthermore, they were characterized by increased consumption of saccharin in a preference test, suggesting an enhanced hedonic profile. The IS mice also showed an earlier onset of breast lumps (assessed by palpation) accompanied by elevated levels of adipokines (leptin and adiponectin) and BAT in the mammary fat pads. Overall, these data point to IS as a pervasive stressor that is able to specifically target neuronal circuits, mastered by the hypothalamus, modulating mood, stress reactivity and energy homeostasis. The activation of such IS-driven machinery may hold main implications for the onset and maintenance of pro-tumorigenic environments.

Time and metabolic state-dependent effects of GLP-1R agonists on NPY/AgRP and POMC neuronal activity in vivo.

OBJECTIVE: Long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest. Thus, whether liraglutide or semaglutide modifies the activity of POMC and NPY/AgRP neurons in vivo as well as mechanisms required for any changes in cellular activity remains undefined. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to examine changes in the activity of POMC and NPY/AgRP neurons after injection of either liraglutide or semaglutide (intraperitoneal - I.P. and subcutaneous - S·C.). POMC and NPY/AgRP neurons were targeted for patch-clamp electrophysiology as well as in vivo fiber photometry. RESULTS: We found that liraglutide and semaglutide directly activate and increase excitatory tone to POMC neurons in a time-dependent manner. This increased activity of POMC neurons required GLP-1Rs in POMC neurons as well as a downstream mixed cation channel comprised of TRPC5 subunits. We also observed an indirect upregulation of excitatory input to POMC neurons originating from glutamatergic cells that also required TRPC5 subunits. Conversely, GLP-1Ra's decreased excitatory input to and indirectly inhibited NPY/AgRP neurons through activation of K-ATP and TRPC5 channels in GABAergic neurons. Notably, the temporal activation of POMC and inhibition of NPY/AgRP neuronal activity after liraglutide or semaglutide was injected [either intraperitoneal (I.P.) or subcutaneous (S·C.)] was dependent upon the nutritional state of the animals (fed vs food-deprived). CONCLUSIONS: Our results support a mechanism of liraglutide and semaglutide in vivo to activate POMC while inhibiting NPY/AgRP neurons, which depends upon metabolic state and mirrors the pharmacokinetic profile of these compounds in vivo.

Direct and indirect effects of liraglutide on hypothalamic POMC and NPY/AgRP neurons - Implications for energy balance and glucose control.

OBJECTIVE: The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, stimulates insulin secretion and efficiently suppresses food intake to reduce body weight. As such, liraglutide is growing in popularity in the treatment of diabetes and chronic weight management. Within the brain, liraglutide has been shown to alter the activity of hypothalamic proopiomelanocortin (POMC) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. Moreover, the acute activities of POMC and NPY neurons have been directly linked to feeding behavior, body weight, and glucose metabolism. Despite the increased usage of liraglutide and other GLP-1 analogues as diabetic and obesity interventions, the cellular mechanisms by which liraglutide alters the activity of metabolically relevant neuronal populations are poorly understood. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY neurons for patch-clamp electrophysiology experiments. RESULTS: We found that liraglutide directly activated arcuate POMC neurons via TrpC5 channels, sharing a similar mechanistic pathway to the adipose-derived peptide leptin. Liraglutide also indirectly increases excitatory tone to POMC neurons. In contrast, liraglutide inhibited NPY/AgRP neurons through post-synaptic GABAA receptors and enhanced activity of pre-synaptic GABAergic neurons, which required both TrpC5 subunits and K-ATP channels. In support of an additive role of leptin and liraglutide in suppressing food intake, leptin potentiated the acute effects of liraglutide to activate POMC neurons. TrpC5 subunits in POMC neurons were also required for the intact pharmacological effects of liraglutide on food intake and body weight. Thus, the current study adds to recent work from our group and others, which highlight potential mechanisms to amplify the effects of GLP-1 agonists in vivo. Moreover, these data highlight multiple sites of action (both pre- and post-synaptic) for GLP-1 agonists on this circuit. CONCLUSIONS: Taken together, our results identify critical molecular mechanisms linking GLP-1 analogues in arcuate POMC and NPY/AgRP neurons with metabolism.

An overview of energy and metabolic regulation.

The physiology and behaviors related to energy balance are monitored by the nervous and humoral systems. Because of the difficulty in treating diabetes and obesity, elucidating the energy balance mechanism and identifying critical targets for treatment are important research goals. Therefore, the purpose of this article is to describe energy regulation by the central nervous system (CNS) and peripheral humoral pathway. Homeostasis and rewarding are the basis of CNS regulation. Anorexigenic or orexigenic effects reflect the activities of the POMC/CART or NPY/AgRP neurons within the hypothalamus. Neurotransmitters have roles in food intake, and responsive brain nuclei have different functions related to food intake, glucose monitoring, reward processing. Peripheral gut- or adipose-derived hormones are the major source of peripheral humoral regulation systems. Nutrients or metabolites and gut microbiota affect metabolism via a discrete pathway. We also review the role of peripheral organs, the liver, adipose tissue, and skeletal muscle in peripheral regulation. We discuss these topics and how the body regulates metabolism.

New insight into GABAergic neurons in the hypothalamic feeding regulation.

Several lines of study have suggested that GABA in the hypothalamic feeding center plays a role in promoting food intake. Recent studies revealed that not only NPY/AgRP neurons in the hypothalamic arcuate nucleus (ARC) that co-express GABA but also other GABAergic neurons act as an orexigenic. Here, we review the progress of studies on hypothalamic GABAergic neurons distributed in ARC, dorsomedial hypothalamus (DMH), and lateral hypothalamus (LH). Three advanced technologies have been applied and greatly contributed to the recent progress. Optogenetic (and chemogenetic) approaches map input and output pathways of particular subpopulations of GABAergic neurons. In vivo Ca2+ imaging using GRIN lens and GCaMP can correlate the activity of GABAergic neuron subpopulations with feeding behavior. Single-cell RNA-seq approach clarifies precise transcriptional profiles of GABAergic neuron subpopulations. These approaches have shown diversity of GABAergic neurons and the subpopulation-dependent role in feeding regulation.

Prolyl carboxypeptidase in Agouti-related Peptide neurons modulates food intake and body weight.

OBJECTIVE: Prolyl carboxypeptidase (PRCP) plays a role in the regulation of energy metabolism by inactivating hypothalamic α-melanocyte stimulating hormone (α-MSH) levels. Although detected in the arcuate nucleus, limited PRCP expression has been observed in the arcuate POMC neurons, and its site of action in regulating metabolism is still ill-defined. METHODS: We performed immunostaining to assess the localization of PRCP in arcuate Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons. Hypothalamic explants were then used to assess the intracellular localization of PRCP and its release at the synaptic levels. Finally, we generated a mouse model to assess the role of PRCP in NPY/AgRP neurons of the arcuate nucleus in the regulation of metabolism. RESULTS: Here we show that PRCP is expressed in NPY/AgRP-expressing neurons of the arcuate nucleus. In hypothalamic explants, stimulation by ghrelin increased PRCP concentration in the medium and decreased PRCP content in synaptic extract, suggesting that PRCP is released at the synaptic level. In support of this, hypothalamic explants from mice with selective deletion of PRCP in AgRP neurons (PrcpAgRPKO) showed reduced ghrelin-induced PRCP concentration in the medium compared to controls mice. Furthermore, male PrcpAgRPKO mice had decreased body weight and fat mass compared to controls. However, this phenotype was sex-specific as female PrcpAgRPKO mice show metabolic differences only when challenged by high fat diet feeding. The improved metabolism of PrcpAgRPKO mice was associated with reduced food intake and increased energy expenditure, locomotor activity, and hypothalamic α-MSH levels. Administration of SHU9119, a potent melanocortin receptor antagonist, selectively in the PVN of PrcpAgRPKO male mice increased food intake to a level similar to that of control mice. CONCLUSIONS: Altogether, our data indicate that PRCP is released at the synaptic levels and that PRCP in AgRP neurons contributes to the modulation of α-MSH degradation and related metabolic control in mice.

Amylin and Leptin: Co-Regulators of Energy Homeostasis and Neuronal Development.

While the regulation of energy homeostasis by amylin is already well-characterized, emerging data suggest that amylin is also crucial for the development of neural pathways in the hypothalamus and caudal hindbrain (area postrema, AP; nucleus tractus solitarius, NTS). Exciting new findings demonstrate crucial amylin-leptin interactions in altering the activity of specific hypothalamic and AP neurons, and a role for amylin as a novel class of 'leptin sensitizers' which enhance leptin signaling in both leptin-sensitive and -resistant individuals, in part by stimulating IL-6 production by hypothalamic microglia. This review summarizes these findings and provides a hypothetical framework for future studies to elucidate the mechanisms by which amylin and leptin act individually and as co-conspirators to alter energy homeostasis and neuronal development.

Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus.

Postnatal undernutrition delays a key step in the maturation of hypothalamic feeding circuits.

OBJECTIVE: Humans and animals exposed to undernutrition (UN) during development often experience accelerated "catch-up" growth when food supplies are plentiful. Little is known about the mechanisms regulating early growth rates. We previously reported that actions of leptin and presynaptic inputs to orexigenic NPY/AgRP/GABA (NAG) neurons in the arcuate nucleus of the hypothalamus are almost exclusively excitatory during the lactation period, since neuronal and humoral inhibitory systems do not develop until after weaning. Moreover, we identified a critical step that regulates the maturation of electrophysiological responses of NAG neurons at weaning - the onset of genes encoding ATP-dependent potassium (KATP) channel subunits. We explored the possibility that UN promotes subsequent catch-up growth, in part, by delaying the maturation of negative feedback systems to neuronal circuits driving food intake. METHODS: We used the large litter (LL) size model to study the impacts of postnatal UN followed by catch-up growth. We evaluated the maturation of presynaptic and postsynaptic inhibitory systems in NAG neurons using a combination of electrophysiological and molecular criteria, in conjunction with leptin's ability to suppress fasting-induced hyperphagia. RESULTS: The onset of KATP channel subunit expression and function, the switch in leptin's effect on NAG neurons, the ingrowth of inhibitory inputs to NAG neurons, and the development of homeostatic feedback to feeding circuits were delayed in LL offspring relative to controls. The development of functional KATP channels and the establishment of leptin-mediated suppression of food intake in the peri-weaning period were tightly linked and were not initiated until growth and adiposity of LL offspring caught up to controls. CONCLUSIONS: Our data support the idea that initiation of KATP channel subunit expression in NAG neurons serves as a molecular gatekeeper for the maturation of homeostatic feeding circuits.

Gq-mER signaling has opposite effects on hypothalamic orexigenic and anorexigenic neurons.

Two populations of cells within the hypothalamus exert opposite actions on food intake: proopiomelanocortin (POMC) neurons decrease it, while neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase it. 17ß-Estradiol (E2) is a potent anorexigenic hormone that exerts both genomic and non-genomic, rapid actions on these metabolic neurons. This review focuses on the rapid membrane effects of E2 in both POMC and NPY/AgRP neurons and how these combined effects mediate the anorexigenic effects of this steroid.