Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.     

A variable cytoplasmic domain segment is necessary for γ-protocadherin trafficking and tubulation in the endosome/lysosome pathway

Clustered protocadherins (Pcdhs) are arranged in gene clusters (α, β, and γ) with variable and constant exons. Variable exons encode cadherin and transmembrane domains and ~90 cytoplasmic residues. The 14 Pcdh-αs and 22 Pcdh-γs are spliced to constant exons, which, for Pcdh-γs, encode ~120 residues of an identical cytoplasmic moiety. Pcdh-γs participate in cell-cell interactions but are prominently intracellular in vivo, and mice with disrupted Pcdh-γ genes exhibit increased neuronal cell death, suggesting nonconventional roles. Most attention in terms of Pcdh-γ intracellular interactions has focused on the constant domain. We show that the variable cytoplasmic domain (VCD) is required for trafficking and organelle tubulation in the endolysosome system. Deletion of the constant cytoplasmic domain preserved the late endosomal/lysosomal trafficking and organelle tubulation observed for the intact molecule, whereas deletion or excision of the VCD or replacement of the Pcdh-γA3 cytoplasmic domain with that from Pcdh-α1 or N-cadherin dramatically altered trafficking. Truncations or internal deletions within the VCD defined a 26-amino acid segment required for trafficking and tubulation in the endolysosomal pathway. This active VCD segment contains residues that are conserved in Pcdh-γA and Pcdh-γB subfamilies. Thus the VCDs of Pcdh-γs mediate interactions critical for Pcdh-γ trafficking.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.     

Effects of hypothalamic neurodegeneration on energy balance

Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.     

Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

Serotonin 2C receptors (5-HT(2C)Rs) 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-HT(2C)Rs 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-HT(2C)R agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT(2C)R 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. VIDEO ABSTRACT:     

Critical Role of Arcuate Y4 Receptors and the Melanocortin System in Pancreatic Polypeptide-Induced Reduction in Food Intake in Mice

Background

Pancreatic polypeptide (PP) is a potent anti-obesity agent known to inhibit food intake in the absence of nausea, but the mechanism behind this process is unknown.

Methodology/Principal Findings

Here we demonstrate that in response to i.p. injection of PP in wild type but not in Y4 receptor knockout mice, immunostaining for the neuronal activation marker c-Fos is induced specifically in neurons of the nucleus tractus solitarius and the area postrema in the brainstem, notably in cells also showing immunostaining for tyrosine hydroxylase. Importantly, strong c-Fos activation is also detected in the arcuate nucleus of the hypothalamus (ARC), particularly in neurons that co-express alpha melanocyte stimulating hormone (α-MSH), the anorexigenic product of the proopiomelanocortin (POMC) gene. Interestingly, other hypothalamic regions such as the paraventricular nucleus, the ventromedial nucleus and the lateral hypothalamic area also show c-Fos induction after PP injection. In addition to c-Fos activation, PP injection up-regulates POMC mRNA expression in the ARC as detected by in situ hybridization. These effects are a direct consequence of local Y4 signaling, since hypothalamus-specific conditional Y4 receptor knockout abolishes PP-induced ARC c-Fos activation and blocks the PP-induced increase in POMC mRNA expression. Additionally, the hypophagic effect of i.p. PP seen in wild type mice is completely absent in melanocortin 4 receptor knockout mice.

Conclusions/Significance

Taken together, these findings show that PP reduces food intake predominantly via stimulation of the anorexigenic α-MSH signaling pathway, and that this effect is mediated by direct action on local Y4 receptors within the ARC, highlighting a potential novel avenue for the treatment of obesity.     

Neuropeptide Y-mediated inhibition of proopiomelanocortin neurons in the arcuate nucleus shows enhanced desensitization in ob/ob mice

NPY and alphaMSH are expressed in distinct neurons in the arcuate nucleus of the hypothalamus, where alphaMSH decreases and NPY increases food intake and body weight. Here we use patch-clamp electrophysiology from GFP-labeled POMC and NPY neurons to demonstrate that NPY strongly hyperpolarized POMC neurons through the Y1R-mediated activation of GIRK channels, while the alphaMSH analog, MTII, had no effect on activity of NPY neurons. While initially NPY had similar effects on POMC neurons derived from ob/ob mice, further studies revealed a significant increase in desensitization of the NPY-induced currents in POMC neurons from ob/ob mice. This increase in desensitization was specific to NPY, as GABA(B) and microOR agonists showed unaltered desensitization in POMC neurons from ob/ob mice. These data reveal an intricate and asymmetric interplay between NPY and POMC neurons in the hypothalamus and have important implications for the delineation of the neural circuits that regulate feeding behavior.     

Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis

Neuroanatomical and electrophysiological studies have shown that hypothalamic POMC neurons are targets of the adipostatic hormone leptin. However, the physiological relevance of leptin signaling in these neurons has not yet been directly tested. Here, using the Cre/loxP system, we critically test the functional importance of leptin action on POMC neurons by deleting leptin receptors specifically from these cells in mice. Mice lacking leptin signaling in POMC neurons are mildly obese, hyperleptinemic, and have altered expression of hypothalamic neuropeptides. In summary, leptin receptors on POMC neurons are required but not solely responsible for leptin's regulation of body weight homeostasis.     

神经元稳态失衡和饮食诱导肥胖症的相关性研究进展

近年来,因肥胖症所造成的社会问题和医疗负担越发严重。肥胖主要是由于机体能量的摄入与消耗不平衡所致,而中枢神经系统以及相关神经元在机体能量代谢平衡的调控中发挥着重要作用。下丘脑弓状核含有抑食性阿黑皮素原(Proopiomelanocortin,POMC)神经元和促食性神经肽Y (Neuropeptid Y,NPY)/刺鼠相关蛋白(Agouti-related protein,AgRP)神经元,是调控机体摄食行为的主要神经元。研究显示,高脂饮食会诱导POMC神经元中的Rb蛋白发生磷酸化修饰并失活,导致POMC神经元从静息状态重新进入细胞周期循环,进而迅速转向细胞凋亡。高脂饮食也会引起神经元再生抑制,并诱导炎症发生和神经元损伤,使神经元稳态失衡,引发瘦素抵抗,最终导致肥胖症的发生。文中就神经元稳态失衡以及肥胖症等疾病之间的关系进行了综述,希望能为饮食诱导肥胖症等疾病的治疗和预防提供新的方向和思路。     

Identification of the cluster control region for the protocadherin-beta genes located beyond the protocadherin-gamma cluster

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.     

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.     

Apelin and the proopiomelanocortin system: a new regulatory pathway of hypothalamic α-MSH release

Neuronal networks originating in the hypothalamic arcuate nucleus (Arc) play a fundamental role in controlling energy balance. In the Arc, neuropeptide Y (NPY)-producing neurons stimulate food intake, whereas neurons releasing the proopiomelanocortin (POMC)-derived peptide α-melanocyte-stimulating hormone (α-MSH) strongly decrease food intake. There is growing evidence to suggest that apelin and its receptor may play a role in the central control of food intake, and both are concentrated in the Arc. We investigated the presence of apelin and its receptor in Arc NPY- and POMC-containing neurons and the effects of apelin on α-MSH release in the hypothalamus. We showed, by immunofluorescence and confocal microscopy, that apelin-immunoreactive (IR) neuronal cell bodies were distributed throughout the rostrocaudal extent of the Arc and that apelin was strongly colocalized with POMC, but weakly colocalized with NPY. However, there were numerous NPY-IR nerve fibers close to the apelin-IR neuronal cell bodies. By combining in situ hybridization with immunohistochemistry, we demonstrated the presence of apelin receptor mRNA in Arc POMC neurons. Moreover, using a perifusion technique for hypothalamic explants, we demonstrated that apelin-17 (K17F) increased α-MSH release, suggesting that apelin released somato-dendritically or axonally from POMC neurons may stimulate α-MSH release in an autocrine manner. Consistent with these data, hypothalamic apelin levels were found to be higher in obese db/db mice and fa/fa Zucker rats than in wild-type animals. These findings support the hypothesis that central apelin is involved in regulating body weight and feeding behavior through the direct stimulation of α-MSH release.     

Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction

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.     

Rapamycin Ameliorates Age-Dependent Obesity Associated with Increased mTOR Signaling in Hypothalamic POMC Neurons

The prevalence of obesity in older people is the leading cause of metabolic syndromes. Central neurons serving as homeostatic sensors for body-weight control include hypothalamic neurons that express pro-opiomelanocortin (POMC) or neuropeptide-Y (NPY) and agouti-related protein (AgRP). Here, we report an age-dependent increase of mammalian target of rapamycin (mTOR) signaling in POMC neurons that elevates the ATP-sensitive potassium (K(ATP)) channel activity cell-autonomously to silence POMC neurons. Systemic or intracerebral administration of the mTOR inhibitor rapamycin causes weight loss in old mice. Intracerebral rapamycin infusion into old mice enhances the excitability and neurite projection of POMC neurons, thereby causing?a reduction of food intake and body weight. Conversely, young mice lacking the mTOR-negative regulator TSC1 in POMC neurons, but not those lacking TSC1 in NPY/AgRP neurons, were obese. Our study reveals that an increase in mTOR signaling in hypothalamic POMC neurons contributes to age-dependent obesity. VIDEO ABSTRACT: