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Objective: Proopiomelanocortin (POMC) neurons release potent anorexigenic neuropeptides, which suppress food intake and enhance energy expenditure via melanocortin receptors. Although the importance of central melanocortin in physiological regulation is well established, the underlying genetic mechanisms that define the functional identity of melanocortin neurons and maintain hypothalamic Pomc expression remain to be fully determined. In this study, we investigate the functional significance of Six3, a transcriptional regulator notably expressed in embryonic and adult mouse POMC neurons, in the regulation of hypothalamic Pomc expression and downstream physiological consequences., Methods: We first evaluated the expression of Six3 in the developing and adult hypothalamus by double fluorescence in situ hybridization. Next, we assessed POMC immunoreactivity in mutant mice selectively lacking Six3 from Pomc-expressing neurons and quantified Pomc mRNA levels in a tamoxifen-inducible Six3 knockout mouse model activated at embryonic E9.5 days. We also determined glucose and insulin sensitivity, daily food intake, body composition and body weight in adult male and female mice lacking Six3 specifically from POMC neurons. Lastly, we assessed the physiological consequences of ablating Six3 from POMC neurons in adult mice., Results: Six3 and Pomc were co-expressed in mouse hypothalamic neurons during development and adulthood. Mouse embryos deficient in Six3 showed reduced Pomc expression in the developing hypothalamus. Targeted deletion of Six3 specifically from POMC neurons resulted in decreased hypothalamic Pomc expression, increased daily food intake, enhanced glucose sensitivity and mild obesity in male but not in female mice. Finally, conditional removal of Six3 from POMC neurons in adult mice led to a reduction in hypothalamic POMC immunoreactivity with no significant effects in body weight or food intake., Conclusions: Altogether, our results demonstrate that Six3 plays an essential role in the early establishment of POMC neuron identity and the maintenance of physiological levels of hypothalamic Pomc expression. In addition, our study demonstrates that the functional significance of Six3 expression in POMC neurons is sexually dimorphic and age-dependent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Food intake and energy balance are tightly regulated by a group of hypothalamic arcuate neurons expressing the proopiomelanocortin ( POMC) gene. In mammals, arcuate-specific POMC expression is driven by two cis -acting transcriptional enhancers known as nPE1 and nPE2. Because mutant mice lacking these two enhancers still showed hypothalamic Pomc mRNA, we searched for additional elements contributing to arcuate Pomc expression. By combining molecular evolution with reporter gene expression in transgenic zebrafish and mice, here, we identified a mammalian arcuate-specific Pomc enhancer that we named nPE3, carrying several binding sites also present in nPE1 and nPE2 for transcription factors known to activate neuronal Pomc expression, such as ISL1, NKX2.1, and ERα. We found that nPE3 originated in the lineage leading to placental mammals and remained under purifying selection in all mammalian orders, although it was lost in Simiiformes (monkeys, apes, and humans) following a unique segmental deletion event. Interestingly, ablation of nPE3 from the mouse genome led to a drastic reduction (>70%) in hypothalamic Pomc mRNA during development and only moderate (<33%) in adult mice. Comparison between double (nPE1 and nPE2) and triple (nPE1, nPE2, and nPE3) enhancer mutants revealed the relative contribution of nPE3 to hypothalamic Pomc expression and its importance in the control of food intake and adiposity in male and female mice. Altogether, these results demonstrate that nPE3 integrates a tripartite cluster of partially redundant enhancers that originated upon a triple convergent evolutionary process in mammals and that is critical for hypothalamic Pomc expression and body weight homeostasis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use single-cell RNA-sequencing (scRNA-seq) to define the transcriptomes characterizing Pomc -expressing cells in the developing hypothalamus and translating ribosome affinity purification with RNA-sequencing (TRAP-seq) to analyze the subsequent translatomes of mature POMC neurons. Our data showed that Pomc -expressing neurons give rise to multiple developmental pathways expressing different levels of Pomc and unique combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts, represents the canonical arcuate POMC neurons. Additional cell clusters expressing medium or low levels of Pomc mature into different neuronal phenotypes featured by distinct sets of transcription factors, neuropeptides, processing enzymes, cell surface, and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to mature postnatally., Competing Interests: HY, MR, ML No competing interests declared, (© 2022, Yu et al.)

Objective: Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential regulators of energy balance. Selective loss of POMC production in these cells results in extreme obesity and metabolic comorbidities. Neurogenesis occurs in the adult hypothalamus, but it remains uncertain whether functional POMC neurons emerge in physiologically significant numbers during adulthood. Here, we tested whether Rax-expressing precursors generate POMC neurons in adult mice and rescue the metabolic phenotype caused by congenital hypothalamic POMC deficiency., Methods: Initially, we identified hypothalamic Rax-expressing cell types using wild-type and Rax-CreERT2:Ai34D mice. Then we generated compound Rax-CreERT2:ArcPomc loxTB/loxTB mice in which endogenous hypothalamic Pomc expression is silenced, but can be restored by tamoxifen administration selectively in neurons derived from Rax +  progenitors. The number of POMC neurons generated by Rax +  progenitors in adult mice and their axonal projections was determined. The metabolic effects of these neurons were assessed by measuring food intake, bodyweight, and body composition, along with glucose and insulin levels., Results: We found that Rax is expressed by tanycytes and a previously unrecognized cell type in the hypothalamic parenchyma of adult mice. Rax +  progenitors generated ~10% of the normal adult hypothalamic POMC neuron population within two weeks of tamoxifen treatment. The same rate and steady state of POMC neurogenesis persisted from young adult to aged mice. These new POMC neurons established terminal projections to brain regions that were involved in energy homeostasis. Mice with Rax +  progenitor-derived POMC neurons had reduced body fat mass, improved glucose tolerance, increased insulin sensitivity, and decreased bodyweight in proportion to the number of new POMC neurons., Conclusions: These data demonstrate that Rax +  progenitors generate POMC neurons in sufficient numbers during adulthood to mitigate the metabolic abnormalities of hypothalamic POMC-deficient mice. The findings suggest that adult hypothalamic neurogenesis is a robust phenomenon in mice that can significantly impact energy homeostasis., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Enhancer redundancy has been postulated to provide a buffer for gene expression against genetic and environmental perturbations. While work in Drosophila has identified functionally overlapping enhancers, work in mammalian models has been limited. Recently, we have identified two partially redundant enhancers, nPE1 and nPE2, that drive proopiomelanocortin gene expression in the hypothalamus. Here we demonstrate that deletion of nPE1 produces mild obesity while knockout of nPE2 has no discernible metabolic phenotypes. Additionally, we show that acute leptin administration has significant effects on nPE1 knockout mice, with food intake and body weight change significantly impacted by peripheral leptin treatment. nPE1 knockout mice became less responsive to leptin treatment over time as percent body weight change increased over 2 week exposure to peripheral leptin. Both Pomc and Agrp mRNA were not differentially affected by chronic leptin treatment however we did see a decrease in Pomc and Agrp mRNA in both nPE1 and nPE2 knockout calorie restricted mice as compared to calorie restricted PBS-treated WT mice. Collectively, these data suggest dynamic regulation of Pomc by nPE1 such that mice with nPE1 knockout become less responsive to the anorectic effects of leptin treatment over time. Our results also support our earlier findings in which nPE2 may only be critical in adult mice that lack nPE1, indicating that these neural enhancers work synergistically to influence metabolism., Competing Interests: The authors have declared that no competing interests exist.

Objective: Regulation of food intake and energy balance depends on a group of hypothalamic neurons that release anorexigenic melanocortins encoded by the Pomc gene. Although the physiological importance of central melanocortins is well appreciated, the genetic program that defines the functional identity of melanocortin neurons and assures high levels of hypothalamic Pomc expression is only beginning to be understood. This study assessed whether the transcriptional regulator PRDM12, identified as a highly expressed gene in adult mouse POMC neurons, plays an important role in the identity and function of melanocortin neurons., Methods: We first determined the cellular distribution of PRDM12 in the developing hypothalamus. Then we studied mutant mice with constitutively inactivated Prdm12 to evaluate possible changes in hypothalamic Pomc expression. In addition, we characterized conditional mutant mice specifically lacking Prdm12 in ISL1-positive or POMC neurons during development. Finally, we measured food intake, body weight progression up to 16 weeks of age, adiposity, and glucose tolerance in adult mice lacking Prdm12 selectively from POMC neurons., Results: PRDM12 co-expressed with POMC in mouse hypothalamic neurons from early development to adulthood. Mice lacking Prdm12 displayed greatly reduced Pomc expression in the developing hypothalamus. Selective ablation of Prdm12 from ISL1 neurons prevented hypothalamic Pomc expression. The conditional ablation of Prdm12 limited to POMC neurons greatly reduced Pomc expression in the developing hypothalamus and in adult mice led to increased food intake, adiposity, and obesity., Conclusions: Altogether, our results demonstrate that PRDM12 plays an essential role in the early establishment of hypothalamic melanocortin neuron identity and the maintenance of high expression levels of Pomc. Its absence in adult mice greatly impairs Pomc expression and leads to increased food intake, adiposity, and obesity., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Persistent alterations of proopiomelanocortin (Pomc) and mu-opioid receptor (Oprm1) activity and stress responses after alcohol are critically involved in vulnerability to alcohol dependency. Gene transcriptional regulation altered by alcohol may play important roles. Mice with genome-wide deletion of neuronal Pomc enhancer1 (nPE1 -/- ), had hypothalamic-specific partial reductions of beta-endorphin and displayed lower alcohol consumption, compared to wildtype littermates (nPE1 +/+ ). We used RNA-Seq to measure steady-state nuclear mRNA transcripts of opioid and stress genes in hypothalamus of nPE1 +/+ and nPE1 -/- mice after 1-day acute withdrawal from chronic excessive alcohol drinking or after water. nPE1 -/- had lower basal Pomc and Pdyn (prodynorphin) levels compared to nPE1 +/+ , coupled with increased basal Oprm1 and Oprk1 (kappa-opioid receptor) levels, and low alcohol drinking increased Pomc and Pdyn to the basal levels of nPE1 +/+ in the water group, without significant effects on Oprm1 and Oprk1. In nPE1 +/+ , excessive alcohol intake increased Pomc and Oprm1, with no effect on Pdyn or Oprk1. For stress genes, nPE1 -/- had lowered basal Oxt (oxytocin) and Avp (arginine vasopressin) that were restored by low alcohol intake to basal levels of nPE1 +/+ . In nPE1 +/+ , excessive alcohol intake decreased Oxt and Avpi1 (AVP-induced protein1). Functionally examining the effect of pharmacological blockade of mu-opioid receptor, we found that naltrexone reduced excessive alcohol intake in nPE1 +/+ , but not nPE1 -/- . Our results provide evidence relevant to the transcriptional profiling of the critical genes in mouse hypothalamus: enhanced opioid and reduced stress gene transcripts after acute withdrawal from excessive alcohol may contribute to altered reward and stress responses., (© 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Objective: Life-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation., Methods: To test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes., Results: Stimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice., Conclusion: In conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron-specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding.

Weight loss is an effective intervention for diminishing disease burden in obese older adults. Pharmacological interventions that reduce food intake and thereby promote weight loss may offer effective strategies to reduce age-related disease. We previously reported that 17α-estradiol (17α-E2) administration elicits beneficial effects on metabolism and inflammation in old male mice. These observations were associated with reduced calorie intake. Here, we demonstrate that 17α-E2 acts through pro-opiomelanocortin (Pomc) expression in the arcuate nucleus (ARC) to reduce food intake and body mass in mouse models of obesity. These results confirm that 17α-E2 modulates appetite through selective interactions within hypothalamic anorexigenic pathways. Interestingly, some peripheral markers of metabolic homeostasis were also improved in animals with near complete loss of ARC Pomc transcription. This suggests that 17α-E2 might have central and peripheral actions that can beneficially affect metabolism cooperatively or independently., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Objective: Hypothalamic arcuate nucleus-specific pro-opiomelanocortin deficient (ArcPomc -/- ) mice exhibit improved glucose tolerance despite massive obesity and insulin resistance. We demonstrated previously that their improved glucose tolerance is due to elevated glycosuria. However, the underlying mechanisms that link glucose reabsorption in the kidney with ArcPomc remain unclear. Given the function of the hypothalamic melanocortin system in controlling sympathetic outflow, we hypothesized that reduced renal sympathetic nerve activity (RSNA) in ArcPomc -/- mice could explain their elevated glycosuria and consequent enhanced glucose tolerance., Methods: We measured RSNA by multifiber recording directly from the nerves innervating the kidneys in ArcPomc -/- mice. To further validate the function of RSNA in glucose reabsorption, we denervated the kidneys of WT and diabetic db/db mice before measuring their glucose tolerance and urine glucose levels. Moreover, we performed western blot and immunohistochemistry to determine kidney GLUT2 and SGLT2 levels in either ArcPomc -/- mice or the renal-denervated mice., Results: Consistent with our hypothesis, we found that basal RSNA was decreased in ArcPomc -/- mice relative to their wild type (WT) littermates. Remarkably, both WT and db/db mice exhibited elevated glycosuria and improved glucose tolerance after renal denervation. The elevated glycosuria in obese ArcPomc -/- , WT and db/db mice was due to reduced renal GLUT2 levels in the proximal tubules. Overall, we show that renal-denervated WT and diabetic mice recapitulate the phenotype of improved glucose tolerance and elevated glycosuria associated with reduced renal GLUT2 levels observed in obese ArcPomc -/- mice., Conclusion: Hence, we conclude that ArcPomc is essential in maintaining basal RSNA and that elevated glycosuria is a possible mechanism to explain improved glucose tolerance after renal denervation in drug resistant hypertensive patients., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The hypothalamus is a region of the anterior forebrain that controls basic aspects of vertebrate physiology, but the genes involved in its development are still poorly understood. Here, we investigate the function of the homeobox gene Rax/Rx in early hypothalamic development using a conditional targeted inactivation strategy in the mouse. We found that lack of Rax expression prior to embryonic day 8.5 (E8.5) caused a general underdevelopment of the hypothalamic neuroepithelium, while inactivation at later timepoints had little effect. The early absence of Rax impaired neurogenesis and prevented the expression of molecular markers of the dorsomedial hypothalamus, including neuropeptides Proopiomelanocortin and Somatostatin. Interestingly, the expression domains of genes expressed in the ventromedial hypothalamus and infundibulum invaded dorsal hypothalamic territory, showing that Rax is needed for the proper dorsoventral patterning of the developing medial hypothalamus. The phenotypes caused by the early loss of Rax are similar to those of eliminating the expression of the morphogen Sonic hedgehog (Shh) specifically from the hypothalamus. Consistent with this similarity in phenotypes, we observed that Shh and Rax are coexpressed in the rostral forebrain at late head fold stages and that loss of Rax caused a downregulation of Shh expression in the dorsomedial portion of the hypothalamus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.

Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+), EPSC(-), and EPSC(+/-)) based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+) neurons, but increased it in EPSC(-) neurons. Unlike EPSC(+) and EPSC(-) neurons, EPSC(+/-) neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/-) neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals.

Hypothalamic proopiomelanocortin (POMC) neurons constitute a critical anorexigenic node in the central nervous system (CNS) for maintaining energy balance. These neurons directly affect energy expenditure and feeding behavior by releasing bioactive neuropeptides but are also subject to signals directly related to nutritional state such as the adipokine leptin. To further investigate the interaction of diet and leptin on hypothalamic POMC peptide levels, we exposed 8- to 10-wk-old male POMC-Discosoma red fluorescent protein (DsRed) transgenic reporter mice to either 24-48 h (acute) or 2 wk (chronic) food restriction, high-fat diet (HFD), or leptin treatment. Using semiquantitative immunofluorescence and radioimmunoassays, we discovered that acute fasting and chronic food restriction decreased the levels of adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-endorphin in the hypothalamus, together with decreased DsRed fluorescence, compared with control ad libitum-fed mice. Furthermore, acute but not chronic HFD or leptin administration selectively increased α-MSH levels in POMC fibers and increased DsRed fluorescence in POMC cell bodies. HFD and leptin treatments comparably increased circulating leptin levels at both time points, suggesting that transcription of Pomc and synthesis of POMC peptide products are not modified in direct relation to the concentration of plasma leptin. Our findings indicate that negative energy balance persistently downregulated POMC peptide levels, and this phenomenon may be partially explained by decreased leptin levels, since these changes were blocked in fasted mice treated with leptin. In contrast, sustained elevation of plasma leptin by HFD or hormone supplementation did not significantly alter POMC peptide levels, indicating that enhanced leptin signaling does not chronically increase Pomc transcription and peptide synthesis.

Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity.

The gene encoding the prohormone proopiomelanocortin (POMC) is mainly expressed in two regions in vertebrates, namely corticotrophs and melanotrophs in the pituitary and a small population of neurons in the arcuate nucleus of the hypothalamus. In this latter region, POMC-derived peptides participate in the control of energy balance and sensitivity to pain. Neuronal expression of POMC is conferred by two enhancers, nPE1 and nPE2, which are conserved in most mammals, but no transcription factors are yet known to bind to these enhancers. In this work, by means of a one-hybrid screening, we identify that nPE2 possesses an element recognized by transcription factors of the nuclear receptor superfamily. This element, named NRBE, is conserved in all known nPE2 enhancers and is necessary to confer full enhancer strength to nPE2-driven reporter gene expression in transgenic mice assays, indicating that the phylogenetic conservation of the element is indicative of its functional importance. In a search for candidate nuclear receptors that might control POMC we observed that estrogen receptor alpha (ESR1) - a known regulator of energy balance at the hypothalamic level - can bind to the NRBE element in vitro. In addition we observed by immunofluorescence that ESR1 is coexpressed with POMC in around 25-30% of hypothalamic neurons of males and females during late embryonic stages and adulthood. Thus, our results indicate that hypothalamic expression of POMC is controlled by nuclear receptors and establish ESR1 as a candidate regulator of POMC., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In 5-month-old male and female dopamine receptor 2 (D2R) knockout mice food intake per animal was unaltered while food per g BW was increased. We wished to evaluate the effect of D2R disruption on different components of energy balance and food intake regulation. We determined hypothalamic orexin precursor (PPO) expression, its receptor OX1, serum leptin levels, hypothalamic leptin receptor (OBR), circulating and pituitary alpha MSH levels, as well as central MC3 and MC4 receptors and NPY mRNA in wildtype and D2R knockout mice (KO). Loss of D2R caused a marked increase in serum prolactin levels, to higher levels in females compared to male KO mice. On the other hand, it produced a female-specific increase in circulating alphaMSH, and hypothalamic alphaMSH content, while neurointermediate alphaMSH content was decreased in both sexes. No differences were found in hypothalamic NPY, MC3R or MC4R concentration. Hypothalamic PPO mRNA expression was significantly decreased only in female KOs, while OX1 mRNA was not different between genotypes. Serum leptin levels were also similar in both genotypes. Our results show that in female and not in male mice disruption of the D2R produces two potentially anorexigenic events: an increase in serum and hypothalamic alphaMSH, and a decrease in hypothalamic orexin expression. Very high prolactin levels, which are orexigenic, probably counterbalance these effects, so that food intake is slightly altered. In males, on the other hand, hypothalamic PPO, and serum or hypothalamic alphaMSH are not modified, and increased prolactin levels may account for increased food intake per g BW. These results suggest a sexually dimorphic participation of the D2R in food intake regulation.

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K(+) current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective micro -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 +/- 1.2 pA), which reversed at K(+) equilibrium potential (E(K+)), in the majority (85%) of POMC neurons with an EC(50) of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with an inhibition constant of 3.1 nM. In addition, the gamma-aminobutyric acid(B) receptor agonist baclofen (40 micro M) caused an outward current (21.6 +/- 4.0 pA) that reversed at E(K+) in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K(+) current (maximum of 18.7 +/- 2.2 pA) in the majority (92%) of POMC neurons with an EC(50) of 61 micro M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal.

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.

The pro-opiomelanocortin (POMC) gene is expressed in a subset of hypothalamic and hindbrain neurons and in pituitary melanotrophs and corticotrophs. POMC neurons release the potent opioid beta-endorphin and several active melanocortins that control homeostasis and feeding behavior. POMC gene expression in the CNS is believed to be controlled by distinct cis-acting regulatory sequences. To analyze the transcriptional regulation of POMC in neuronal and endocrine cells, we produced transgenic mice carrying POMC27*, a transgene containing the entire 6 kb of the POMC transcriptional unit together with 13 kb of 5' flanking regions and 8 kb of 3' flanking regions. POMC27* was tagged with a heterologous 30 bp oligonucleotide in the third exon. In situ hybridization studies showed an accurate cell-specific pattern of expression of POMC27* in the arcuate nucleus and the pituitary. Hypothalamic mRNA-positive neurons colocalized entirely with beta-endorphin immunoreactivity. No ectopic transgenic expression was detected in the brain. Deletional analyses demonstrated that neuron-specific expression of POMC transgenes required distal 5' sequences localized upstream of the pituitary-responsive proximal cis-acting elements that were identified previously. POMC27* exhibited a spatial and temporal pattern of expression throughout development that exactly paralleled endogenous POMC. RNase protection assays revealed that POMC27* expression mimicked that of POMC in different areas of the CNS and most peripheral organs with no detectable ectopic expression. Hormonal regulation of POMC27* and POMC was identical in the hypothalamus and pituitary. These results show that distal 5' sequences of the POMC gene located between -13 and -2 kb target expression into the CNS of transgenic mice in a precise neuron-specific, developmentally and hormonally regulated manner.

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.

The proopiomelanocortin (POMC) gene is expressed predominantly in corticotrophs of the pituitary anterior lobe, melanotrophs of the intermediate lobe and neurons of the arcuate nucleus of the hypothalamus. The different ontogeny of POMC mRNA as well as the complicated hormonal regulation of POMC gene expression in the three different cell types suggests a concerted interaction between several cis-acting elements in the POMC gene and transcription factors located in each of the three cell types. To investigate cell-specific elements in the POMC gene we tested two different constructs in transgenic mice. The construct -4000rPOMCLacZ, carrying 4 kb of the rat POMC promoter fused to the Escherichia coli beta-galactosidase gene, showed appropriate expression in melanotrophs in 50% of the mice analyzed. beta-Galactosidase activity was less evident in corticotrophs under basal environmental conditions. In brain, 7 out of 15 independently derived transgenic founders had ectopic expression of the transgene in different areas; however, none of the animals analyzed expressed beta-galactosidase in neurons of the arcuate nucleus. The construct HAL*, a 'tagged' 10.2-kb mouse genomic fragment, was more efficiently targeted to the pituitary. Using in situ hybridization, we detected uniform expression of HAL* in melanotrophs in 100% of the 6 pedigrees analyzed and transgenic mRNA levels paralleled those of the endogenous POMC mRNA. In corticotrophs, basal expression was low but after adrenalectomy HAL* mRNA levels were comparable to those of POMC. None of the 6 pedigrees had appropriate expression of HAL* in the brain; however, 2 lines had ectopic expression in the dentate gyrus of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E. coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)