Proopiomelanocortin Cells Research Articles

Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat.

Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterized. Our aim was to use two isogenic strains of a neurotropic virus (pseudorabies, Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague Dawley rats. Retrogradely labeled neurons in the CNS were characterized by immunohistochemistry and PCR. For the latter, laser capture of individual virally labeled neurons was used. All virally labeled brain regions contained neurons projecting to either and both WAT depots. Neurons to abdominal fat were the most abundant in males, whereas females contained a greater proportion of neurons to subcutaneous via private lines and collateral branches. Retrogradely labeled neurons directed to WAT expressed estrogen receptor-α (ERα), and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats, suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic.

Expression of GABAergic and glutamatergic phenotypic markers in hypothalamic proopiomelanocortin neurons

Hypothalamic proopiomelanocortin (POMC) neurons have traditionally been defined by their peptide transmitters, which are important regulators of energy balance and reward. Recent work shows that POMC neurons can also release the amino acid transmitters γ-aminobutyric acid (GABA) and glutamate, although studying GABAergic and glutamatergic populations of POMC neurons has been hindered by the difficulty in reliably identifying amino acid (AA) transmitter phenotypes. In the present study, fluorescent in situ hybridization and immunohistochemistry were used to identify POMC neurons and to detect the presence of mRNA for the transporters responsible for packaging either GABA (vesicular GABA transporter [vGAT]) or glutamate (vesicular glutamate transporter [vGLUT]) into vesicles, as well as the enzymes responsible for GABA synthesis, glutamic acid decarboxylase (GAD)65 and GAD67. Approximately 7% of POMC neurons expressed vGlut2 and the highest percentage of vGlut2-positive POMC cells were located in the rostral arcuate nucleus. Despite the reports of GABA release from POMC neurons, vGat was not detected in POMC neurons, although Gad65 and Gad67 were present in ~40% of POMC neurons. Approximately half of the vGlut2-expressing POMC cells also expressed Gad65. Markers of neurotransmitter phenotype were better detected by using in situ hybridization techniques rather than transgenic expression of fluorophores under the control of the vGat or Gad67 promoters. It is now clear that the expression of markers of AA phenotype provides a useful means to identify distinct subpopulations of POMC neurons. Additionally, the method described will be useful to explore the possibility that plasticity of AA phenotype is an important aspect of POMC neuron function.

Increased expression of miR-325-3p by urocortin 2 and its involvement in stress-induced suppression of LH secretion in rat pituitary

Urocortin 2 (Ucn2) is a member of the corticotropin releasing factor (CRF) peptide family, which binds to CRF type 2 receptor. We previously reported on expression of Ucn2 in proopiomelanocortin cells of rat pituitary and its inhibitory action on LH secretion. We also demonstrated that Ucn2 is involved in the mechanism underlying immobilization-induced suppression of LH secretion; the details remain unclear. Here, we found that Ucn2 increased the expression of miR-325-3p, one of three microRNAs with predicted sequence for binding to LH β-subunit 3'-untranslated region (3'-UTR) in monolayer cultured rat anterior pituitary cells, and that miR-325-3p was expressed in LH cells of the anterior pituitary. Immobilization also increased miR-325-3p expression in the anterior pituitary, and its increase was blocked by pretreatment with anti-Ucn2 IgG. Overexpression of miR-325-3p in cultured pituitary cells significantly suppressed intracellular contents and secretion of LH, while miR-325-3p knockdown blocked Ucn2-induced suppression of intracellular contents and secretion of LH. Coexpression of miR-325-3p with LH β-subunit 3'-UTR-fused luciferase vector significantly suppressed luciferase activity compared with that of mock transfectants. These results suggest that miR-325-3p is involved in immobilization-induced suppression of LH translation and secretion and that Ucn2 plays a role in the increase in miR-325-3p expression.

Relative Number and Distribution of Murine Hypothalamic Proopiomelanocortin Neurons Innervating Distinct Target Sites

Proopiomelanocortin (POMC) neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences.

Peroxisome proliferation–associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity

Previous studies have proposed roles for hypothalamic reactive oxygen species (ROS) in the modulation of circuit activity of the melanocortin system. Here we show that suppression of ROS diminishes pro-opiomelanocortin (POMC) cell activation and promotes the activity of neuropeptide Y (NPY)- and agouti-related peptide (AgRP)-co-producing (NPY/AgRP) neurons and feeding, whereas ROS-activates POMC neurons and reduces feeding. The levels of ROS in POMC neurons were positively correlated with those of leptin in lean and ob/ob mice, a relationship that was diminished in diet-induced obese (DIO) mice. High-fat feeding resulted in proliferation of peroxisomes and elevated peroxisome proliferator-activated receptor γ (PPAR-γ) mRNA levels within the hypothalamus. The proliferation of peroxisomes in POMC neurons induced by the PPAR-γ agonist rosiglitazone decreased ROS levels and increased food intake in lean mice on high-fat diet. Conversely, the suppression of peroxisome proliferation by the PPAR antagonist GW9662 increased ROS concentrations and c-fos expression in POMC neurons. Also, it reversed high-fat feeding-triggered elevated NPY/AgRP and low POMC neuronal firing, and resulted in decreased feeding of DIO mice. Finally, central administration of ROS alone increased c-fos and phosphorylated signal transducer and activator of transcription 3 (pStat3) expression in POMC neurons and reduced feeding of DIO mice. These observations unmask a previously unknown hypothalamic cellular process associated with peroxisomes and ROS in the central regulation of energy metabolism in states of leptin resistance.

Nicotine excites hypothalamic arcuate anorexigenic proopiomelanocortin neurons and orexigenic neuropeptide Y neurons: similarities and differences

Two of the biggest health problems facing us today are addiction to nicotine and the increased prevalence of obesity. Interestingly, nicotine attenuates obesity, but the underlying mechanism is not clear. Here we address the hypothesis that if weight-reducing actions of nicotine are mediated by anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus, nicotine should excite these cells. Nicotine at concentrations similar to those found in smokers, 100-1,000 nM, excited POMC cells by mechanisms based on increased spike frequency, depolarization of membrane potential, and opening of ion channels. This was mediated by activation of both α7 and α4β2 nicotinic receptors; by itself, this nicotine-mediated excitation could explain weight loss caused by nicotine. However, in control experiments nicotine also excited the orexigenic arcuate nucleus neuropeptide Y (NPY) cells. Nicotine exerted similar actions on POMC and NPY cells, with a slightly greater depolarizing action on POMC cells. Immunocytochemistry revealed cholinergic axons terminating on both cell types. Nicotine actions were direct in both cell types, with nicotine depolarizing the membrane potentials and reducing input resistance. We found no differences in the relative desensitization to nicotine between POMC and NPY neurons. Nicotine inhibited excitatory synaptic activity recorded in NPY, but not POMC, cells. Nicotine also excited hypocretin/orexin neurons that enhance cognitive arousal, but the responses were smaller than in NPY or POMC cells. Together, these results indicate that nicotine has a number of similar actions, but also a few different actions, on POMC and NPY neurons that could contribute to the weight loss associated with smoking.

Differential Expression and Sensitivity of Presynaptic and Postsynaptic Opioid Receptors Regulating Hypothalamic Proopiomelanocortin Neurons

Hypothalamic proopiomelanocortin (POMC) neurons release the endogenous opioid beta-endorphin and POMC neuron activity is inhibited by opioids, leading to the proposal that beta-endorphin acts to provide feedback inhibition. However, both intrinsic properties and synaptic inputs contribute to the regulation of POMC neurons such that attributing an autoregulatory role to opioids must include consideration of opioid receptor localization and sensitivity at both presynaptic and postsynaptic sites. In the present study, whole-cell recordings were made in POMC cells in mouse brain slices and the presynaptic and postsynaptic regulation of POMC neurons was examined using selective agonists for mu, kappa, and delta opioid receptors. Activation of mu, but not kappa or delta, receptors induced a direct postsynaptic outward current. Agonists for each of the receptors inhibited the frequency of spontaneous IPSCs. Mu and kappa, but not delta, agonists reduced the amplitude of evoked IPSCs and appeared to colocalize in a significant portion of GABAergic terminals onto POMC neurons. The presynaptic inhibition caused by the mu agonist DAMGO had an EC(50) of 80 nM, whereas the EC(50) was 350 nM when measuring the postsynaptic outward current. This differential sensitivity adds an unexpected component of opioid-dependent feedback regulation, where low levels of opioid receptor activation would likely disinhibit POMC neuron activity and higher concentrations would result in an overall inhibition. The results may help explain why it has been difficult to clearly discern the role that opioids play in the regulation of food intake and other processes involving POMC neurons.

Gonadotropin-Releasing Hormone Fibers Contact POMC Neurons in the Hypothalamic Arcuate Nucleus

The metabolic state has long been shown to affect reproduction. Peripheral signals and hormones from the reproductive organs are also known to regulate energy metabolism and feeding and energy expenditure. Much attention has been paid to determine the signaling flow from key hypothalamic neuronal populations, including those producing the anorexigenic proopiomelanocortin (POMC) derivate, α-melanocyte stimulating hormone (α-MSH), to the medial preoptic area gonadotropin-releasing hormone (GnRH) neurons, cells that are the drivers of ovulation and reproduction in general. In this study, the authors explored whether a reverse signaling modality may also exist. Specifically, the authors analyzed GnRH efferents in the arcuate nucleus with particular emphasis on their anatomical proximity to arcuate nucleus melanocortin perikarya. Using correlated light and electron microscopy, the authors observed direct apposition between GnRH-containing axon terminals and POMC cell bodies. These data provide the first experimental evidence to suggest that GnRH may have a direct influence on feeding, energy expenditure, and glucose homeostasis, independent of the activity of the gonadal axis.

Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity

The neuronal circuits involved in the regulation of feeding behavior and energy expenditure are soft-wired, reflecting the relative activity of the postsynaptic neuronal system, including the anorexigenic proopiomelanocortin (POMC)-expressing cells of the arcuate nucleus. We analyzed the synaptic input organization of the melanocortin system in lean rats that were vulnerable (DIO) or resistant (DR) to diet-induced obesity. We found a distinct difference in the quantitative and qualitative synaptology of POMC cells between DIO and DR animals, with a significantly greater number of inhibitory inputs in the POMC neurons in DIO rats compared with DR rats. When exposed to a high-fat diet (HFD), the POMC cells of DIO animals lost synapses, whereas those of DR rats recruited connections. In both DIO rats and mice, the HFD-triggered loss of synapses on POMC neurons was associated with increased glial ensheathment of the POMC perikarya. The altered synaptic organization of HFD-fed animals promoted increased POMC tone and a decrease in the stimulatory connections onto the neighboring neuropeptide Y (NPY) cells. Exposure to HFD was associated with reactive gliosis, and this affected the structure of the blood-brain barrier such that the POMC and NPY cell bodies and dendrites became less accessible to blood vessels. Taken together, these data suggest that consumption of an HFD has a major impact on the cytoarchitecture of the arcuate nucleus in vulnerable subjects, with changes that might be irreversible due to reactive gliosis.

Kisspeptin Directly Excites Anorexigenic Proopiomelanocortin Neurons but Inhibits Orexigenic Neuropeptide Y Cells by an Indirect Synaptic Mechanism

The neuropeptide kisspeptin is necessary for reproduction, fertility, and puberty. Here, we show strong kisspeptin innervation of hypothalamic anorexigenic proopiomelanocortin (POMC) cells, coupled with a robust direct excitatory response by POMC neurons (n > 200) to kisspeptin, mediated by mechanisms based on activation of a sodium/calcium exchanger and possibly opening of nonselective cation channels. The excitatory actions of kisspeptin on POMC cells were corroborated with quantitative PCR data showing kisspeptin receptor GPR54 expression in the arcuate nucleus, and the attenuation of excitation by the selective kisspeptin receptor antagonist, peptide 234. In contrast, kisspeptin inhibits orexigenic neuropeptide Y (NPY) neurons through an indirect mechanism based on enhancing GABA-mediated inhibitory synaptic tone. In striking contrast, gonadotropin-inhibiting hormone (GnIH and RFRP-3) and NPY, also found in axons abutting POMC cells, inhibit POMC cells and attenuate the kisspeptin excitation by a mechanism based on opening potassium channels. Together, these data suggest that the two central peptides that regulate reproduction, kisspeptin and GnIH, exert a strong direct action on POMC neurons. POMC cells may hypothetically serve as a conditional relay station downstream of kisspeptin and GnIH to signal the availability of energy resources relevant to reproduction.

Role of urocortin 2 secreted by the pituitary in the stress-induced suppression of luteinizing hormone secretion in rats

We have previously shown that urocortin 2 (Ucn 2), a member of the corticotropin-releasing factor (CRF) peptide family that binds to CRF type 2 receptor, is expressed in proopiomelanocortin (POMC) cells of rat pituitary and that its secretion and expression are increased by CRF in both the anterior and intermediate lobes and suppressed by glucocorticoids in the anterior lobe. We have also shown that Ucn 2 secreted by POMC cells acts on gonadotrophs expressing CRF type 2 receptors and inhibits the expression and secretion of gonadotropins. In the present study, we examined whether pituitary Ucn 2 is involved in stress-induced inhibition of gonadotropin secretion. A 90-min period of immobilization stress increased POMC mRNA expression without influencing Ucn 2 mRNA expression and suppressed luteinizing hormone (LH) β-subunit mRNA expression in the anterior lobe and plasma LH levels, while it increased both POMC and Ucn 2 mRNA expression in the intermediate lobe of the pituitary. Pretreatment with anti-CRF IgG blocked immobilization-induced increases in plasma ACTH and corticosterone and in POMC mRNA expression in both pituitary lobes and Ucn 2 mRNA expression in the intermediate pituitary. It also blocked immobilization-induced suppression of plasma LH and LH β-subunit mRNA expression. Pretreatment with anti-Ucn 2 IgG blocked immobilization-induced suppression of plasma LH and LH β-subunit expression without affecting immobilization-induced ACTH and corticosterone release and POMC or Ucn 2 mRNA expression. These results suggest that CRF suppresses the secretion and expression of LH probably through pituitary Ucn 2 in stress.

Cooperation between Cyclin E and p27Kip1 in Pituitary Tumorigenesis

Cushing's disease is caused by glucocorticoid-resistant pituitary corticotroph adenomas. We have previously identified the loss of nuclear Brg1 as one mechanism that may lead to partial glucocorticoid resistance: this loss is observed in about 33% of human corticotroph adenomas. We now show that Brg1 loss of function correlates with cyclin E expression in corticotroph adenomas and with loss of the cell cycle inhibitor p27(Kip1) expression. Because Brg1 is thought to have tumor suppressor activity, the present study was undertaken to understand the putative contribution of cyclin E derepression produced by loss of Brg1 expression on adenoma development. Overexpression of cyclin E in pituitary proopiomelanocortin cells leads to abnormal reentry into cell cycle of differentiated proopiomelanocortin cells and to centrosome instability. These alterations are consistent with the intermediate lobe hyperplasia and anterior lobe adenomas that were observed in these pituitaries. When combined with the p27(Kip1) knockout, overexpression of cyclin E increased the incidence of pituitary tumors, their size, and their proliferation index. These results suggest that cyclin E up-regulation and p27(Kip1) loss-of-function act cooperatively on pituitary adenoma development.

Kisspeptin Cells in the Ewe Brain Respond to Leptin and Communicate with Neuropeptide Y and Proopiomelanocortin Cells

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 microg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 microg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

Segregation of Acute Leptin and Insulin Effects in Distinct Populations of Arcuate Proopiomelanocortin Neurons

Acute leptin administration results in a depolarization and concomitant increase in the firing rate of a subpopulation of arcuate proopiomelanocortin (POMC) cells. This rapid activation of POMC cells has been implicated as a cellular correlate of leptin effects on energy balance. In contrast to leptin, insulin inhibits the activity of some POMC neurons. Several studies have described a "cross talk" between leptin and insulin within the mediobasal hypothalamus via the intracellular enzyme, phosphoinositol-3-kinase (PI3K). Interestingly, both insulin and leptin regulate POMC cellular activity by activation of PI3K; however, it is unclear whether leptin and insulin effects are observed in similar or distinct populations of POMC cells. We therefore used dual label immunohistochemistry/in situ hybridization and whole-cell patch-clamp electrophysiology to map insulin and leptin responsive arcuate POMC neurons. Leptin-induced Fos activity within arcuate POMC neurons was localized separate from POMC neurons that express insulin receptor. Moreover, acute responses to leptin and insulin were largely segregated in distinct subpopulations of POMC cells. Collectively, these data suggest that cross talk between leptin and insulin occurs within a network of cells rather than within individual POMC neurons.

PACAP Neurons in the Hypothalamic Ventromedial Nucleus Are Targets of Central Leptin Signaling

The adipose-derived hormone, leptin, was discovered over 10 years ago, but only now are we unmasking its downstream pathways which lead to reduced energy intake (feeding) and increased energy expenditure (thermogenesis). Recent transgenic models have challenged the long-standing supposition that the hypothalamic arcuate nucleus (Arc) is omnipotent in the central response to leptin, and research focus is beginning to shift to examine roles of extra-arcuate sites. Dhillon et al. (2006) demonstrated that targeted knock out of the signaling form of the leptin receptor (lepr-B) in steroidogenic factor 1 (SF-1) cells of the hypothalamic ventromedial nucleus (VMN) produces obesity of a similar magnitude to the pro-opiomelanocortin (POMC)-driven lepr-B deleted mouse, via a functionally distinct mechanism. These findings reveal that SF-1 cells of the VMN could be equally as important as POMC cells in mediating leptin's anti-obesity effects. However, the identification of molecular and cellular correlates of this relationship remains tantalizingly unknown. Here, we have shown that mRNA expression of the VMN-expressed neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated according to energy status and that it exerts catabolic effects when administered centrally to mice. Furthermore, we have shown that SF-1 and PACAP mRNAs are colocalized in the VMN, and that leptin signaling via lepr-B is required for normal PACAP expression in these cells. Finally, blocking endogenous central PACAP signaling with the antagonist PACAP(6-38) markedly attenuates leptin-induced hypophagia and hyperthermia in vivo. Thus, it appears that PACAP is an important mediator of central leptin effects on energy balance.

Enhanced Stat3 Activation in POMC Neurons Provokes Negative Feedback Inhibition of Leptin and InsulinSignaling in Obesity

Leptin-stimulated Stat3 activation in proopiomelanocortin (POMC)-expressing neurons of the hypothalamus plays an important role in maintenance of energy homeostasis. While Stat3 activation in POMC neurons is required for POMC expression, the role of elevated basal Stat3 activation as present in the development of obesity has not been directly addressed. Here, we have generated and characterized mice expressing a constitutively active version of Stat3 (Stat3-C) in POMC neurons (Stat3-C(POMC) mice). On normal chow diet, these animals develop obesity as a result of hyperphagia and decreased POMC expression accompanied by central leptin and insulin resistance. This unexpected finding coincides with POMC-cell-specific, Stat3-mediated upregulation of SOCS3 expression inhibiting both leptin and insulin signaling as insulin-stimulated PIP(3) (phosphatidylinositol-3,4,5 triphosphate) formation and protein kinase B (AKT) activation in POMC neurons as well as with the fact that insulin's ability to hyperpolarize POMC neurons is largely reduced in POMC cells of Stat3-C(POMC) mice. These data indicate that constitutive Stat3 activation is not sufficient to promote POMC expression but requires simultaneous PI3K (phosphoinositide 3-kinase)-dependent release of FOXO1 repression. In contrast, upon exposure to a high-fat diet, food intake and body weight were unaltered in Stat3-C(POMC) mice compared with control mice. Taken together, these experiments directly demonstrate that enhanced basal Stat3 activation in POMC neurons as present in control mice upon high-fat feeding contributes to the development of hypothalamic leptin and insulin resistance.

Neuroendocrine Proopiomelanocortin Neurons Are Excited by Hypocretin/Orexin

Hypocretin/orexin, produced by a group of neurons in the lateral hypothalamus/perifornical area, enhances cognitive arousal and also may play a crucial role in modulating the neuroendocrine system. How hypocretin modulates the endocrine system remains an open question. Hypocretin cells innervate the mediobasal hypothalamus where they can potentially influence the activity of specific cell populations within the arcuate nucleus. Here, we examine whether hypocretin modulates the median eminence-projecting proopiomelanocortin (POMC) neurons identified by selective green fluorescent protein expression and antidromic stimulation or retrograde Evans blue dye tracing in transgenic mice. We find that POMC neurons, in general, and, in addition, those that project their axons to the median eminence, were robustly activated by hypocretin in a dose-dependent manner. These excitatory actions included a threefold increase in spike frequency and direct membrane depolarization of up to 22 mV (mean, 17.9+/-7.2 mV). Direct postsynaptic depolarization was decreased at more positive membrane potentials, inhibited by the sodium-calcium exchanger antagonist KB-R7943, and reduced by lowering the bath temperature, or by buffering the postsynaptic calcium with BAPTA, suggesting that the primary mechanism for hypocretin-mediated excitation is the activation of the sodium-calcium exchanger. Hypocretin also enhanced excitatory inputs to POMC cells via a presynaptic mechanism and indirectly increased the release of GABA onto these cells in a spike-dependent manner. However, these synaptic actions were not necessary to cause postsynaptic membrane depolarization and spiking. Thus, in contrast to previous suggestions that hypocretin inhibited POMC cells, our results demonstrate robust direct excitation of POMC neurons by hypocretin.

Glucose regulates the effects of leptin on hypothalamic POMC neurons

Leptin is believed to exert its potent appetite-suppressing effects via stimulation of hypothalamic anorexigenic proopiomelanocortin (POMC)-containing neurons and inhibition of orexigenic agouti-related protein (AgRP) neurons. We show here that at 11 mM glucose leptin excites POMC cells. At 5 mM glucose, however, leptin hyperpolarizes POMC neurons and suppresses action potential firing, by producing a greater decrease in excitatory synaptic tone than inhibitory tone. These results argue that when glucose is low (5 mM or less) AgRP neurons will be more important for mediating the anorectic effects of leptin than POMC cells. However, at high glucose concentrations (11 mM), activation of POMC cells may contribute to the appetite-suppressing effects of leptin. Our data also suggest the regulation of neuropeptide efficacy as a novel function of hypothalamic glucose sensing.

Acute effects of leptin require PI3K signaling in hypothalamic proopiomelanocortin neurons in mice

Normal food intake and body weight homeostasis require the direct action of leptin on hypothalamic proopiomelanocortin (POMC) neurons. It has been proposed that leptin action requires PI3K activity. We therefore assessed the contribution of PI3K signaling to leptin's effects on POMC neurons and organismal energy balance. Leptin caused a rapid depolarization of POMC neurons and an increase in action potential frequency in patch-clamp recordings of hypothalamic slices. Pharmacologic inhibition of PI3K prevented this depolarization and increased POMC firing rate, indicating a PI3K-dependent mechanism of leptin action. Mice with genetically disrupted PI3K signaling in POMC cells failed to undergo POMC depolarization or increased firing frequency in response to leptin. Insulin's ability to hyperpolarize POMC neurons was also abolished in these mice. Moreover, targeted disruption of PI3K blunted the suppression of feeding elicited by central leptin administration. Despite these differences, mice with impaired PI3K signaling in POMC neurons exhibited normal long-term body weight regulation. Collectively, these results suggest that PI3K signaling in POMC neurons is essential for leptin-induced activation and insulin-induced inhibition of POMC cells and for the acute suppression of food intake elicited by leptin, but is not a major contributor to the regulation of long-term organismal energy homeostasis.

In vivo time-lapse imaging delineates the zebrafish pituitary proopiomelanocortin lineage boundary regulated by FGF3 signal

The anterior pituitary gland (adenohypophysis) comprises anterior and intermediate lobes (the pars distalis and pars intermedia) arising from placodal ectoderm at the anterior neural ridge. Signaling molecules including SHH, FGF, WNT, BMP and Notch are involved in regulating primordial pituitary proliferation and lineage determination. However, morphogenic events and molecular mechanisms governing anterior and intermediate lobe specification are not clear. Pituitary expression of proopiomelanocortin (POMC), the common precursor for adrenocorticotropin (ACTH) of pars distalis corticotropes and α-melanocyte-stimulating hormone (α-MSH) of pars intermedia melanotropes, provides a unique marker for anterior and intermediate lobe morphogenesis. We performed time-lapse confocal microscopy lineage tracing in live zebrafish embryos expressing GFP driven by the pomc promoter and show distinct migration pathways of POMC cells destined to the anterior and intermediate lobes. Using morpholino oligonucleotides, we show that hypomorphic FGF3 down-regulation induces specific defects of pars intermedia POMC cells while pomc, growth hormone and prolactin expression remain intact in the pars distalis. This lineage-specific process is independent of the FGF3 effect on early pituitary specifying transcription factors as indicated by normal Lim3 and Pit1 expression in hypomorphic FGF3 morphants. These findings suggest that the FGF3 signal, in addition to its previously described role of regulating progenitor proliferation and survival, delineates the melanotrope and corticotrope lineage boundary, contributing to establishment of the pituitary pars distalis and pars intermedia.