Identifying neuronal molecular markers with restricted patterns of expression is a crucial step in dissecting the numerous pathways and functions of the brain. While the dorsomedial nucleus of the hypothalamus (DMH) has been implicated in a host of physiological processes, current functional studies have been limited by the lack of molecular markers specific for DMH. Identification of such markers would facilitate the development of mouse models with DMH-specific genetic manipulations. Here we used a combination of laser-capture microdissection (LCM) and gene expression profiling to identify genes that are highly expressed within the DMH relative to adjacent hypothalamic regions. Six of the most highly expressed of these genes, Gpr50, 4930511J11Rik, Pcsk5, Grp, Sulf1, and Rorβ, were further characterized by real-time polymerase chain reaction (PCR) analysis and in situ hybridization histochemistry. The genes identified in this article will provide the basis for future gene-targeted approaches for studying DMH function.
DMH; LCM; hypothalamus
Obesity has reached epidemic proportions in the United States, and obesity-related illnesses have become a leading preventable cause of death. Childhood obesity is also growing in frequency, and the impact of a lifetime spent in the overweight state is only beginning to emerge in the literature. In this issue of the JCI, Bumaschny et al. used a genetic mouse model to investigate the self-perpetuating nature of obesity and shed some light on why it can become increasingly difficult to lose weight over time.
Over the past century, prevalent models of energy and glucose homeostasis have been developed from a better understanding of the neural circuits underlying obesity and diabetes. From the early hypothalamic lesion reports to the more recent pharmacological and molecular/genetic studies, the hypothalamic melanocortin system has been shown to play a critical role in the regulation of metabolism. This review attempts to highlight contributions to our current understanding of how numerous neuromodulators (leptin, insulin, and serotonin) integrate with the central melanocortin system to coordinate alterations in energy and glucose balance.
leptin; insulin; serotonin; obesity; diabetes; patch-clamp
The ventral medial hypothalamic nucleus (VMH) regulates food intake and body weight homeostasis. The nuclear receptor NR5A1 (Steroidogenic factor 1; SF-1) is a transcription factor whose expression is highly restricted in the VMH and is required for the development of the nucleus. Neurons expressing SF-1 in the VMH have emerged as playing important roles in the regulation of body weight and energy homeostasis. Many of these studies have used site-specific gene KO approaches, providing insights into the molecular mechanisms underlying the regulation of energy homeostasis by the SF-1 neurons of the VMH. In this brief review, we will focus on recent studies defining the molecular mechanisms regulating energy homeostasis and body weight in the VMH, particularly stressing the SF-1 expressing neurons.
ARH, arcuate nucleus of the hypothalamus; Diet-induced obesity; DMH, dorsomedial hypothalamic nucleus; Energy homeostasis; SF-1, steroidogenic factor 1; VMH, ventral medial nucleus of the hypothalamus
Neurons within the central nervous system receive humoral and central (neurotransmitter or neuropeptide) signals that ultimately regulate ingestive behavior and metabolism. Recent advances in mouse genetics combined with neuroanatomical and electrophysiological techniques have contributed to a better understanding of these central mechanisms. This review integrates recently defined cellular mechanisms and neural circuits relevant to the regulation of feeding behavior, energy expenditure, and glucose homeostasis by metabolic signals.
electrophysiology; melanocortin receptor; agouti-related peptide; neuropeptide Y receptor; GABA; glutamate
Over the past two centuries, prevalent models of energy and glucose homeostasis have emerged from careful anatomical descriptions in tandem with an understanding of cellular physiology. More recent technological advances have culminated in the identification of peripheral and central factors that influence neural circuits regulating metabolism. This Review highlights contributions to our understanding of peripheral and central factors regulating food intake and energy expenditure.
Cloned in 1994, the ob gene encodes the protein hormone leptin, which is produced and secreted by white adipose tissue. Since its discovery, leptin has been found to have profound effects on behavior, metabolic rate, endocrine axes, and glucose fluxes. Leptin deficiency in mice and humans causes morbid obesity, diabetes, and various neuroendocrine anomalies, and replacement leads to decreased food intake, normalized glucose homeostasis, and increased energy expenditure. Here, we provide an update on the most current understanding of leptin-sensitive neural pathways in terms of both anatomical organization and physiological roles.
A new optogenetics study finds that stimulation of pro-opiomelanocortin (POMC) and agouti-related peptide (AGRP) neurons acutely regulates feeding behavior. AGRP-induced hyperphagia is independent of melanocortin signaling.
Leptin regulates energy balance and glucose homeostasis. Shortly after leptin was identified, it was established that obesity is commonly associated with leptin resistance, though the molecular mechanisms remain to be identified. To explore potential mechanisms of leptin resistance, we employed organotypic brain slices to identify candidate signaling pathways that negatively regulate leptin sensitivity. We found that elevation of adenosine 3′, 5′-monophosphate (cAMP) levels impairs multiple signaling cascades activated by leptin within the hypothalamus. Notably, this effect is independent of protein kinase A activation. In contrast, activation of Epac, a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap1, was sufficient to impair leptin signaling with concomitant induction of SOCS-3 expression. Epac activation also blunted leptin-induced depolarization of hypothalamic POMC neurons. Finally, central infusion of an Epac activator blunted the anorexigenic actions of leptin. Thus, activation of hypothalamic cAMP-Epac pathway is sufficient to induce multiple indices of leptin resistance.
Reproductive function requires timely secretion of gonadotropin releasing hormone, which is controlled by a complex excitatory/inhibitory network influenced by sex steroids. Kiss1 neurons are fundamental players in this network, but it is currently unclear whether different conditions of circulating sex steroids directly alters Kiss1 neuronal activity. Here, we show that Kiss1 neurons in the anteroventral periventricular and anterior periventricular nuclei (AVPV/PeN) of males and females exhibit a bimodal resting membrane potential (RMP) influenced by KATP channels, suggesting the presence of two neuronal populations defined as Type I (irregular firing patterns) and Type II (quiescent). Kiss1 neurons in the arcuate nucleus (Arc) are also composed of firing and quiescent cells, but unlike AVPV/PeN neurons, the range of RMPs did not follow a bimodal distribution. Moreover, Kiss1 neuronal activity in the AVPV/PeN, but not in the Arc, is sexually dimorphic. In females, estradiol shifts the firing pattern of AVPV/PeN Kiss1 neurons and alters cell capacitance and spontaneous inhibitory postsynaptic potentials (IPSCs) amplitude of AVPV/PeN and Arc Kiss1 populations in an opposite manner. Notably, mice with selective deletion of estrogen receptor α (ERα) from Kiss1 neurons show cellular activity similar to that observed in ovariectomized females, suggesting that estradiol-induced changes in Kiss1 cellular properties require ERα. We also show that female prepubertal Kiss1 neurons are under higher inhibitory influence while all AVPV/PeN Kiss1 neurons are spontaneously active. Collectively, our findings indicate that changes in cellular activity may underlie Kiss1 action in pubertal initiation and female reproduction.
HPG axis; puberty; hypothalamus; neuroendocrine regulation; estrogen feedback
Melanocortin-4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R-GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R-expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid-thoracic level), the majority being small-sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R-expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C-fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities.
Dorsal root ganglion; green fluorescent protein; neuropeptide; nociceptor; nodose ganglion; vagus nerve
Leptin is an adipose-derived hormone that signals to inform the brain of nutrient status; loss of leptin signaling results in marked hyperphagia and obesity. Recent work has identified several groups of neurons that contribute to the effects of leptin to regulate energy balance, but leptin receptors are distributed throughout the brain, and the function of leptin signaling in discrete neuronal populations outside of the hypothalamus has not been defined. In the current study, we produced mice in which the long form of the leptin receptor (Lepr) was selectively ablated using Cre-recombinase selectively expressed in the hindbrain under control of the paired-like homeobox 2b (Phox2b) promoter (Phox2b Cre Leprflox/flox mice). In these mice, Lepr was deleted from glucagon-like 1 peptide–expressing neurons resident in the nucleus of the solitary tract. Phox2b Cre Leprflox/flox mice were hyperphagic, displayed increased food intake after fasting, and gained weight at a faster rate than wild-type controls. Paradoxically, Phox2b Cre Leprflox/flox mice also exhibited an increased metabolic rate independent of a change in locomotor activity that was dependent on food intake, and glucose homeostasis was normal. Together, these data support a physiologically important role of direct leptin action in the hindbrain.
It is now widely recognized that exposure to palatable foods engages reward circuits that promote over-eating and facilitate the development of obesity. While the melanocortin 4 receptor (MC4R) has previously been shown to regulate food intake and energy expenditure, little is known about its role in food reward. We demonstrate that MC4R is co-expressed with the dopamine 1 receptor (D1R) in the ventral striatum. While MC4R-null mice are hyperphagic and obese, they exhibit impairments in acquisition of operant responding for a high fat reinforcement. Restoration of MC4R signaling in D1R neurons normalizes procedural learning without affecting motivation to obtain high fat diet. MC4R signaling in D1R neurons is also required for learning in a non-food-reinforced version of the cued water maze. Finally, MC4R signaling in neostriatal slices increases phosphorylation of the Thr34 residue of DARPP-32, a protein phosphatase-1 inhibitor that regulates synaptic plasticity. These data identify a novel requirement for MC4R signaling in procedural memory learning.
melanocortin; dopamine; behavior; learning
Obesity, type 2 diabetes and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific over-expression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.
AMP-activated protein kinase (AMPK) has emerged as a metabolic “fuel gauge,” which oscillates between anabolic and catabolic processes that ultimately influence energy balance. A study in this issue of the JCI by Claret et al. now extends the role of AMPK in medial basal hypothalamic neurons (see the related article beginning on page 2325). These findings maintain AMPK signaling as a common cellular mechanism in proopiomelanocortin and neuropeptide Y/agouti-related protein neurons and links hypothalamic AMPK to coordinated energy and glucose homeostasis.
The differential expression and secretion of the neuropeptide kisspeptin from neurons in the arcuate (Arc) and anteroventral periventricular (AVPV) nuclei of the hypothalamus coordinate the temporal release of pituitary gonadotropins that control the female reproductive cycle. However, the molecular basis for this differential regulation is incompletely understood. Here, we report that liver receptor homolog-1 (LRH-1), a member of the nuclear receptor superfamily, is expressed in kisspeptin neurons in the Arc but not in the AVPV in female mice. LRH-1 binds directly to the kisspeptin (Kiss1) promoter and stimulates Kiss1 transcription. Deletion of LRH-1 from kisspeptin neurons in mice decreased Kiss1 expression in the Arc, leading to reduced plasma FSH levels, dysregulated follicle maturation, and prolongation of the estrous cycle. Conversely, overexpression of LRH-1 in kisspeptin neurons increased Arc Kiss1 expression and plasma FSH concentrations. These studies provide a molecular basis for the differential regulation of basal kisspeptin expression in Arc and AVPV neurons and reveal a prominent role for LRH-1 in hypothalamus in regulating the female reproductive axis.
The orexin/hypocretin system has the potential to significantly modulate affect, based on both the neuroanatomical projection patterns of these neurons and on the sites of orexin receptor expression. However, there is little data supporting the role of specific orexin receptors in the modulation of depression-like behavior. Here we report behavioral profiling of mice after genetic or pharmacologic inhibition of hcrtr1 and 2 receptor signaling. Hcrtr1 null mice displayed a significant reduction in behavioral despair in the forced swim test and tail suspension test. Wild-type mice treated with the hcrtr1 antagonist SB-334867 also displayed a similar reduction in behavioral despair. No difference in anxiety-like behavior was noted following hcrtr1 deletion. In contrast, hcrtr2-null mice displayed an increase in behavioral despair with no effect on measures of anxiety. These studies suggest that the balance of orexin action at either the hcrtr1 or the hcrtr2 receptor produces an anti-depressant or pro-depressant like effect, depending on the receptor subtype activated.
Orexin; Depression; Anxiety
d-Fenfluramine (d-Fen) increases serotonin (5-HT) content in the synaptic cleft and exerts anorexigenic effects in animals and humans. However, the neural circuits that mediate these effects are not fully identified. To address this issue, we assessed the efficacy of d-Fen-induced hypophagia in mouse models with manipulations of several genes in selective populations of neurons. Expectedly, we found that global deletion of 5-HT 2C receptors (5-HT2CRs) significantly attenuated d-Fen-induced anorexia. These anorexigenic effects were restored in mice with 5-HT2CRs expressed only in pro-opiomelanocortin (POMC) neurons. Further, we found that deletion of melanocortin 4 receptors (MC4Rs), a downstream target of POMC neurons, abolished anorexigenic effects of d-Fen. Reexpression of MC4Rs only in SIM1 neurons in the hypothalamic paraventricular nucleus and neurons in the amygdala was sufficient to restore the hypophagic property of d-Fen. Thus, our results identify a neurochemically defined neural circuit through which d-Fen influences appetite and thereby indicate that this 5-HT2CR/POMC-MC4R/SIM1 circuit may yield a more refined target to exploit for weight loss.
Studies have suggested that manipulations of the central melanocortin circuitry by pharmacological agents produce robust effects on the regulation of body weight and glucose homeostasis. In this review, we discuss recent findings from genetic mouse models that have further established the physiological relevance of this circuitry in the context of glucose and energy balance. In addition, we will discuss distinct neuronal populations that respond to central melanocortins to regulate food intake, energy expenditure, insulin sensitivity, and insulin secretion, respectively. Finally, multiple hormonal and neural cues (e.g., leptin, estrogen, and serotonin) that use the melanocortin systems to regulate energy and glucose homeostasis will be reviewed. These findings suggest that targeting the specific branches of melanocortin circuits may be potential avenues to combat the current obesity and diabetes epidemics.
melanocortins; leptin; estrogen; serotonin; body weight
Estrogens regulate body weight and reproduction primarily through actions on estrogen receptor-α (ERα). However, ERα-expressing cells mediating these effects are not identified. We demonstrate that brain-specific deletion of ERα in female mice causes abdominal obesity stemming from both hyperphagia and hypometabolism. Hypometabolism and abdominal obesity, but not hyperphagia, are recapitulated in female mice lacking ERα in hypothalamic steroidogenic factor-1 (SF1) neurons. In contrast, deletion of ERα in hypothalamic pro-opiomelanocortin (POMC) neurons leads to hyperphagia, without directly influencing energy expenditure or fat distribution. Further, simultaneous deletion of ERα from both SF1 and POMC neurons causes hypometabolism, hyperphagia and increased visceral adiposity. Additionally, female mice lacking ERα in SF1 neurons develop anovulation and infertility, while POMC-specific deletion of ERα inhibits negative feedback regulation of estrogens and impairs fertility in females. These results indicate that estrogens act on distinct hypothalamic ERα neurons to regulate different aspects of energy homeostasis and reproduction.
Serotonin 2C receptors (5-HT2CRs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT2CRs have not yet been identified. In the present study, we found the putative transient receptor potential C (TRPC) channels mediate the activation of a subpopulation of POMC neurons by mCPP (a 5-HT2CR agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT2CR and leptin receptors are expressed by distinct subpopulations of arcuate POMC neurons and that both 5-HT and leptin exert their actions in POMC neurons via TRPC channels.
Calorie restriction induces long-term changes in motivation to eat highly palatable food, and in body weight regulation, through an unknown mechanism.
Following a period of calorie restriction and re-feeding, mice were assessed by behavioral and metabolic studies and for levels of the transcription factor ΔFosB. ΔFosB levels were then increased specifically in nucleus accumbens (NAc) using viral-mediated gene transfer, and behavioral and metabolic studies were conducted.
We show that accumulation of ΔFosB in the NAc shell after calorie restriction in mice corresponds to a period of increased motivation for high fat reward and reduced energy expenditure. Furthermore, ΔFosB over-expression in this region increases instrumental responding for a high fat reward via an orexin-dependent mechanism, while also decreasing energy expenditure and promoting adiposity.
These results suggest that ΔFosB signaling in NAc mediates adaptive responses to calorie restriction.
Feeding; Metabolism; Nucleus Accumbens; Appetite; Orexin
SF-1-expressing neurons of the ventromedial hypothalamus (VMH) control energy homeostasis, but the role of insulin action in these cells remains undefined. We show that insulin activates PI3-kinase (PI3k) signaling in SF-1 neurons and reduces firing frequency in these cells via activation of KATP-channels. These effects are abrogated in mice with insulin receptor (IR) deficiency restricted to SF-1 neurons (SF-1ΔIR-mice). While body weight and glucose homeostasis remain unaltered in SF-1ΔIR-mice under normal chow diet, they exhibit protection from diet-induced leptin resistance, weight gain, adiposity and impaired glucose tolerance. High-fat feeding activates PI3k signaling in SF-1 neurons of control mice, and this response is attenuated in the VMH of SF-1ΔIR-mice. Mimicking diet-induced overactivation of PI3k signaling by disruption of the PIP3-phosphatase PTEN leads to increased body weight and hyperphagia under normal chow diet. Collectively, our experiments reveal a critical role for HFD-induced, insulin-dependent PI3k activation in VMH neurons to control energy homeostasis.
The transcription factor FOXO1 plays a central role in metabolic homeostasis by regulating leptin and insulin activity in many cell types, including neurons. However, the neurons mediating these effects and the identity of the molecular targets through which FOXO1 regulates metabolism remain to be defined. Here, we show that the ventral medial nucleus of the hypothalamus (VMH) is a key site of FOXO1 action. We found that mice lacking FOXO1 in steroidogenic factor 1 (SF-1) neurons of the VMH are lean due to increased energy expenditure. The mice also failed to appropriately suppress energy expenditure in response to fasting. Furthermore, these mice displayed improved glucose tolerance due to increased insulin sensitivity in skeletal muscle and heart. Gene expression profiling and sequence analysis revealed several pathways regulated by FOXO1. In addition, we identified the nuclear receptor SF-1 as a direct FOXO1 transcriptional target in the VMH. Collectively, our data suggest that the transcriptional networks modulated by FOXO1 in VMH neurons are key components in the regulation of energy balance and glucose homeostasis.
Evidence suggests that the role played by the adipocyte-derived hormone leptin in female reproductive physiologyis mediated in part by neurons located within the ventral premammillary nucleus (PMV). Leptin activates PMV neurons; however, the intracellular signaling pathway and channel(s) involved remain undefined. Notably, leptin's excitatory and inhibitory effects within hypothalamic and brainstem nuclei share the intracellular signaling cascade phosphoinositide 3 kinase (PI3K). Therefore, we assessed whether PI3K signaling is required for the acute effect of leptin to alter cellular activity of PMV neurons that express leptin receptors (LepR PMV neurons). Leptin caused a rapid depolarization in the majority of LepR PMV neurons in patch-clamp recordings of hypothalamic slices, while a subset of LepR PMV neurons were hyperpolarized in response to leptin. Data were obtained from both male and female mice and results demonstrate that the acute effect of leptin on LepR PMV neurons was identical for both sexes. Pharmacological inhibition of PI3K prevented the acute leptin-induced change in neuronal activity of LepR PMV neurons, indicating a PI3K-dependent mechanism of leptin action. Similarly, mice with genetically disrupted PI3K signaling in LepR PMV neurons failed to alter cellular activity in response to leptin. Moreover, the leptin-induced depolarization was dependent on a putative TRPC channel. In contrast, the leptin-induced-hyperpolarization required the activation of a putative Katp channel. Collectively, these results suggest that PI3K signaling in LepR PMV neurons is essential for leptin-induced alteration in cellular activity, and these data may suggest a cellular correlate in which leptin contributes to the initiation of reproductive development.