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Objective: Somatostatin (SST) is secreted by islet delta-cells and by extraislet neuroendocrine cells. SST receptors have been identified on alpha- and beta-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating alpha- and beta-cell function. However, the specific intraislet function of delta-cell SST remains uncertain. We have used Sst(-/-) mice to investigate the role of delta-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo.Research Design and Methods: Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst(-/-) mice in vivo and from isolated islets in vitro.Results: Islet size and organization did not differ between Sst(-/-) and control islets, nor did islet glucagon or insulin content. Sst(-/-) mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst(-/-) islets compared with control islets and was inhibited by exogenous SST in Sst(-/-) but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst(-/-) islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst(-/-) islets.Conclusions: We suggest that delta-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, delta-cell SST is implicated in the nutrient-induced suppression of glucagon secretion. [ABSTRACT FROM AUTHOR]

Objective: Melanocyte-stimulating hormone (MSH) peptides processed from proopiomelanocortin (POMC) regulate energy homeostasis by activating neuronal melanocortin receptor (MC-R) signaling. Agouti-related peptide (AgRP) is a naturally occurring MC-R antagonist but also displays inverse agonism at constitutively active melanocortin-4 receptor (MC4-R) expressed on transfected cells. We investigated whether AgRP functions similarly in vivo using mouse models that lack all neuronal MSH, thereby precluding competitive antagonism of MC-R by AgRP.Research Design and Methods: Feeding and metabolic effects of the MC-R agonist melanotan II (MTII), AgRP, and ghrelin were investigated after intracerebroventricular injection in neural-specific POMC-deficient (Pomc(-/-)Tg/+) and global POMC-deficient (Pomc(-/-)) mice. Gene expression was quantified by RT-PCR.Results: Hyperphagic POMC-deficient mice were more sensitive than wild-type mice to the anorectic effects of MTII. Hypothalamic melanocortin-3 (MC3)/4-R mRNAs in POMC-deficient mice were unchanged, suggesting increased receptor sensitivity as a possible mechanism for the heightened anorexia. AgRP reversed MTII-induced anorexia in both mutant strains, demonstrating its ability to antagonize MSH agonists at central MC3/4-R, but did not produce an acute orexigenic response by itself. The action of ghrelin was attenuated in Pomc(-/-)Tg/+ mice, suggesting decreased sensitivity to additional orexigenic signals. However, AgRP induced delayed and long-lasting modifications of energy balance in Pomc(-/-)Tg/+, but not glucocorticoid-deficient Pomc(-/-) mice, by decreasing oxygen consumption, increasing the respiratory exchange ratio, and increasing food intake.Conclusions: These data demonstrate that AgRP can modulate energy balance via a mechanism independent of MSH and MC3/4-R competitive antagonism, consistent with either inverse agonist activity at MC-R or interaction with a distinct receptor. [ABSTRACT FROM AUTHOR]

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.

The realization of hybrid optics could be one of the best ways to fulfill the technological requirements of compact, light-weight, and multi-functional optical systems for modern industries. Planar diffractive lens (PDL) such as diffractive lenses, photonsieves, and metasurfaces can be patterned on ultra-thin flexible and stretchable substrates and be conformally attached on top of arbitrarily shaped surfaces. In this review, we introduce recent research works addressed to the design and manufacturing of ultra-thin graphene optics, which will open new markets in compact and light-weight optics for next-generation endoscopic brain imaging, space internet, real-time surface profilometry, and multi-functional mobile phones. To provide higher design flexibility, lower process complexity, and chemical-free process with reasonable investment cost, direct laser writing (DLW) of laser-induced-graphene (LIG) is actively being applied to the patterning of PDL. For realizing the best optical performances in DLW, photon-material interactions have been studied in detail with respect to different laser parameters; the resulting optical characteristics have been evaluated in terms of amplitude and phase. A series of exemplary laser-written 1D and 2D PDL structures have been actively demonstrated with different base materials, and then, the cases are being expanded to plasmonic and holographic structures. The combination of these ultra-thin and light-weight PDL with conventional bulk refractive or reflective optical elements could bring together the advantages of each optical element. By integrating these suggestions, we suggest a way to realize the hybrid PDL to be used in the future micro-electronics surface inspection, biomedical, outer space, and extended reality (XR) industries., (© 2023. The Author(s).)

Rationale: Obesity is a major health problem worldwide. An understanding of the factors that drive feeding behaviors is key to the development of pharmaceuticals to decrease appetite and consumption. Proopiomelanocortin (POMC), the melanocortin peptide precursor, is essential in the regulation of body weight and ingestive behaviors. Deletion of POMC or impairment of melanocortin signaling in the brain results in hyperphagic obesity. Neurons in the hypothalamic arcuate nucleus produce POMC and project to many areas including the nucleus accumbens (NAcc), which is well established in the rewarding and reinforcing effects of both food and drugs of abuse., Objective: These studies sought to determine the role of melanocortins in the NAcc on consumption of and motivation to obtain access to standard rodent chow., Methods: Male, C57BL/6J mice were microinjected bilaterally into the NAcc (100 nl/side) with the melanocortin receptor 3/4 agonist melanotan-II (MT-II; 0.1, 0.3, and 1 nmol), and ingestive behaviors were examined in both home cage and operant food self-administration experiments. In addition, the ability of MT-II in the NAcc to produce aversive properties or affect metabolic rate were tested., Results: MT-II injected into the NAcc significantly decreased consumption in both home cage and operant paradigms, and furthermore decreased appetitive responding to gain access to food. There was no development of conditioned taste avoidance or change in metabolic parameters following anorexic doses of MT-II., Conclusions: MT-II in the NAcc decreased both the motivation to eat and the amount of food consumed without inducing an aversive state or affecting metabolic rate, suggesting a role for melanocortin signaling in the NAcc that is selective for appetite and satiety without affecting metabolism or producing an aversive state., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

PCSK1 encodes an enzyme required for prohormone maturation into bioactive peptides. A striking number of SNPs and rare mutations in PCSK1 are associated with a range of clinical phenotypes. Infants bearing two copies of a catalytically inactivating mutation, such as G209R, exhibit life-threatening chronic diarrhea and subsequently develop systemic endocrinopathies. Using CRISPR/Cas9 technology, we have engineered a mouse model bearing a G209R missense mutation in exon 6 of the murine Pcsk1 locus. Most pups homozygous for the G209R mutation succumbed by day 2, and surviving pups were severely dwarfed. In homozygous (but not heterozygous) pups, blood glucose levels were significantly lower, accompanied by elevated plasma insulin-like immunoreactivity and accumulation of large quantities of unprocessed proinsulin in the pancreas. Peptide hormone processing was also aberrant in G209R mouse pituitary, with mature ACTH levels markedly reduced in homozygotes, accompanied by a significant accumulation of POMC. We also observed a significant reduction in PC1/3 protein in the brains of G209R homozygous mice by Western blotting, while PC2 levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired. Analysis of intestinal cell types indicated a modest reduction of enteroendocrine cells in G209R homozygotes. We suggest that the G209R Pcsk1 mouse model recapitulates many of the dramatic neonatal deficiencies of human patients with this homozygous mutation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, β-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, β-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, β-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of β-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of β-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Pro-opiomelanocortin (POMC) neurons form an integral part of the central melanocortin system regulating food intake and energy expenditure. Genetic and pharmacological studies have revealed that defects in POMC synthesis, processing, and receptor signaling lead to obesity. It is well established that POMC is extensively processed by a series of enzymes, including prohormone convertases PC1/3 and PC2, and that genetic insufficiency of both PC1/3 and POMC is strongly associated with obesity risk. However, whether PC1/3-mediated POMC processing is absolutely tied to body weight regulation is not known. To investigate this question, we generated a Pomc-CreER  T2; Pcsk1  lox/lox mouse model in which Pcsk1 is specifically and temporally knocked out in POMC-expressing cells of adult mice by injecting tamoxifen at eight weeks of age. We then measured the impact of Pcsk1 deletion on POMC cleavage to ACTH and α-MSH, and on body weight. In whole pituitary, POMC cleavage was significantly impacted by the loss of Pcsk1, while hypothalamic POMC-derived peptide levels remained similar in all genotypes. However, intact POMC levels were greatly elevated in Pomc-CreER  T2; Pcsk1  lox/lox mice. Males expressed two-fold greater levels of pituitary PC1/3 protein than females, consistent with their increased POMC cleavage. Past studies show that mice with germline removal of PC1/3 do not develop obesity, while mice expressing mutant PC1/3 forms do develop obesity. We conclude that obesity pathways are not disrupted by PC1/3 loss solely in POMC-expressing cells, further disfavoring the idea that alterations in POMC processing underlie obesity in PCSK1 deficiency., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We demonstrate a high repetition-rate upconversion green pulsed micro-laser, which is prepared by the fast thermal quenching of lanthanide-doped upconversion nanoparticles (UCNPs) via femtosecond-laser direct writing. The outer rim of the prepared upconversion hemi-ellipsoidal microstructure works as a whispering-gallery-mode (WGM) optical resonator for the coherent photon build-up of third-harmonic ultra-short seed pulses. When near-infrared (NIR) femtosecond laser pulses of wavelength 1545 nm are focused onto the upconversion WGM resonator, the optical third-harmonic is generated at 515 nm together with the upconversion luminescence. The weak third-harmonic (TH) seed pulses are coherently amplified in the hemi-ellipsoidal upconversion resonator as a result of the resonant interaction between the incident femtosecond laser field, the TH, the upconversion luminescence and the WGM. This upconversion lasing preserves the original repetition rate of the NIR pump laser and the output polarization state is also coherently aligned to the pump laser polarization. Because of the isotropic nature of the upconversion micro-ellipsoids, the upconversion lasing shows maximum intensity with a linearly polarized pump beam and minimum intensity with a circularly polarized pump beam. Our scheme devised for realizing high-repetition-rate lasing at higher photon energies in a compact micro platform will open up new ways for on-chip optical information processing, high-throughput microfluidic sensing, and localized micro light sources for optical memories.

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: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc -/- ) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc -/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels., Methods: Circulating adiponectin was measured in obese ArcPomc -/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc -/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc -/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc -/- mice. Finally, we generated compound Adiopoq -/- :ArcPomc -/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls., Results: Obese ArcPomc -/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc -/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc -/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc -/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc -/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc -/- mice did not exacerbate their metabolic abnormalities., Conclusion: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc -/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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.)

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

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.)

Common mutations in the human prohormone convertase (PC)1/3 gene (PCKSI) are linked to increased risk of obesity. Previous work has shown that the rs6232 single-nucleotide polymorphism (N221D) results in slightly decreased activity, although whether this decrease underlies obesity risk is not clear. We observed significantly decreased activity of the N221D PC1/3 enzyme at the pH of the trans-Golgi network; at this pH, the mutant enzyme was less stable than wild-type enzyme. Recombinant N221D PC1/3 also showed enhanced susceptibility to heat stress. Enhanced susceptibility to tunicamycin-induced endoplasmic reticulum stress was observed in AtT-20/PC2 cell clones in which murine PC1/3 was replaced by human N221D PC1/3, as compared with wild-type human PC1/3. However, N221D PC1/3-expressing AtT-20/PC2 clones processed proopiomelanocortin to α-MSH similarly to wild-type PC1/3. We also generated a CRISPR-edited mouse line expressing the N221D mutation in the PCKSI gene. When homozygous N221D mice were fed either a standard or a high-fat diet, we found no increase in body weight compared with their wild-type sibling controls. Sexual dimorphism was observed in pituitary ACTH for both genotypes, with females exhibiting lower levels of pituitary ACTH. In contrast, hypothalamic α-MSH content for both genotypes was higher in females compared with males. Hypothalamic corticotropin-like intermediate peptide content was higher in wild-type females compared with wild-type, but not N221D, males. Taken together, these data suggest that the increased obesity risk linked to the N221D allele in humans may be due in part to PC1/3-induced loss of resilience to stressors rather than strictly to decreased enzymatic activity on peptide precursors., (Copyright © 2019 Endocrine Society.)

A fixed dose combination of bupropion (BPP) and naltrexone (NTX), Contrave®, is an FDA approved pharmacotherapy for the treatment of obesity. A recent study found that combining BPP with low-dose NTX reduced alcohol drinking in alcohol-preferring male rats. To explore potential pharmacological effects of the BPP + NTX combination on alcohol drinking, both male and female C57Bl/6J mice were tested on one-week drinking-in-the dark (DID) and three-week intermittent access (IA) models. Neuronal proopiomelanocortin (POMC) enhancer knockout (nPE -/- ) mice with hypothalamic-specific deficiency of POMC, and its bioactive peptides melanocyte stimulating hormone and beta-endorphin, were used as a genetic control for the effects of the BPP + NTX. A single administration of BPP + NTX (10 mg/kg + 1 mg/kg) decreased alcohol intake after DID in C57Bl/6J males, but not females. Also in C57Bl/6J males, BPP + NTX reduced intake of the caloric reinforcer sucrose, but not the non-caloric reinforcer saccharin. In contrast, BPP + NTX had no effect on alcohol DID in nPE -/- males. Pretreatment with the selective melanocortin 4 receptor (MC4R) antagonist HS014 reversed the anti-dipsogenic effect of BPP + NTX on alcohol DID in C57Bl/6J males. In the 3-week chronic IA model, single or repeated administrations for four days of BPP + NTX reduced alcohol intake and preference in C57Bl/6J males only. The behavioral measures observed in C57Bl/6J mice provide clear evidence that BPP + NTX profoundly reduced alcohol drinking in males, but the doses tested were not effective in females. Furthermore, our results suggest a hypothalamic POMC/MC4R-dependent mechanism for the observed BPP + NTX effects on alcohol drinking in male mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Food intake is tightly regulated by a group of neurons present in the arcuate nucleus of the hypothalamus, which release Pomc -encoded melanocortins, the absence of which induces marked hyperphagia and early-onset obesity. Although the relevance of hypothalamic POMC neurons in the regulation of body weight and energy balance is well appreciated, little is known about the transcription factors that establish the melanocortin neuron identity during brain development and its phenotypic maintenance in postnatal life. Here, we report that the transcription factor NKX2.1 is present in mouse hypothalamic POMC neurons from early development to adulthood. Electromobility shift assays showed that NKX2.1 binds in vitro to NKX binding motifs present in the neuronal Pomc enhancers nPE1 and nPE2 and chromatin immunoprecipitation assays detected in vivo binding of NKX2.1 to nPE1 and nPE2 in mouse hypothalamic extracts. Transgenic and mutant studies performed in mouse embryos of either sex and adult males showed that the NKX motifs present in nPE1 and nPE2 are essential for their transcriptional enhancer activity. The conditional early inactivation of Nkx2.1 in the ventral hypothalamus prevented the onset of Pomc expression. Selective Nkx2.1 ablation from POMC neurons decreased Pomc expression in adult males and mildly increased their body weight and adiposity. Our results demonstrate that NKX2.1 is necessary to activate Pomc expression by binding to conserved canonical NKX motifs present in nPE1 and nPE2. Therefore, NKX2.1 plays a critical role in the early establishment of hypothalamic melanocortin neuron identity and participates in the maintenance of Pomc expression levels during adulthood. SIGNIFICANCE STATEMENT Food intake and body weight regulation depend on hypothalamic neurons that release satiety-inducing neuropeptides, known as melanocortins. Central melanocortins are encoded by Pomc , and Pomc mutations may lead to hyperphagia and severe obesity. Although the importance of central melanocortins is well appreciated, the genetic program that establishes and maintains fully functional POMC neurons remains to be explored. Here, we combined molecular, genetic, developmental, and functional studies that led to the discovery of NKX2.1, a transcription factor that participates in the early morphogenesis of the developing hypothalamus, as a key player in establishing the early identity of melanocortin neurons by activating Pomc expression. Thus, Nkx2.1 adds to the growing list of genes that participate in body weight regulation and adiposity., (Copyright © 2019 the authors.)

Cocaine is a highly abused drug, and cocaine addiction affects millions of individuals worldwide. Cocaine blocks normal uptake function at the dopamine transporter (DAT), thus increasing extracellular dopamine. Currently, no chemical therapies are available to treat cocaine abuse. Previous works showed that the selective inhibitors of protein kinase Cβ (PKCβ), enzastaurin and ruboxistaurin, attenuate dopamine overflow and locomotion stimulated by another psychostimulant drug, amphetamine. We now test if ruboxistaurin similarly affects cocaine action. Perfusion of 1 μM ruboxistaurin directly into the core of the nucleus accumbens via retrodialysis reduced cocaine-stimulated increases in dopamine overflow, measured using microdialysis sampling, with simultaneous reductions in locomotor behavior. Because cocaine activity is highly regulated by dopamine autoreceptors, we examined whether ruboxistaurin was acting at the level of the D2 autoreceptor. Perfusion of 5 μM raclopride, a selective D2-like receptor antagonist, before addition of ruboxistaurin, abrogated the effect of ruboxistaurin on cocaine-stimulated dopamine overflow and hyperlocomotion. Further, ruboxistaurin was inactive against cocaine-stimulated locomotor activity in mice with a genetic deletion in D2 receptors as compared to wild-type mice. In contrast, blockade or deletion of dopamine D2 receptors did not abolish the attenuating effect of ruboxistaurin on amphetamine-stimulated activities. Therefore, the inhibition of PKCβ reduces dopamine overflow and locomotor activity stimulated by both cocaine and amphetamine, but the mechanism of action differs for each stimulant. These data suggest that inhibition of PKCβ would serve as a target to reduce the abuse of either amphetamine or cocaine.

Hypothalamic POMC deficiency leads to obesity and metabolic deficiencies, largely due to the loss of melanocortin peptides. However, POMC neurons in the arcuate nucleus (ARC) are comprised of glutamatergic and GABAergic subpopulations. The developmental program, relative proportion and function of these two subpopulations are unresolved. To test whether glutamatergic POMC neurons serve a distinct role in maintaining energy homeostasis, we activated Pomc expression Cre- dependently in Vglut2 -expressing neurons of mice with conditionally silenced Pomc alleles. The Vglut2 - Pomc restored mice had normal ARC Pomc mRNA levels, POMC immunoreactivity, as well as body weight and body composition at age 12 weeks. Unexpectedly, the cumulative total of Vglut2 + glutamatergic- and Gad67 + GABAergic - Pomc neurons detected by in situ hybridization (ISH) exceeded 100% in both Vglut2 - Pomc restored and control mice, indicating that a subpopulation of Pomc neurons must express both neuronal markers. Consistent with this hypothesis, triple ISH of C57BL/6J hypothalami revealed that 35% of ARC Pomc neurons were selectively Gad67 + , 21% were selectively Vglut2 + , and 38% expressed both Gad67 and Vglut2 . The single Gad67 + and Vglut2 + Pomc neurons were most prevalent in the rostral ARC, while the Vglut2/Gad67 + dual-phenotype cells predominated in the caudal ARC. A lineage trace using Ai9-tdTomato reporter mice to label fluorescently all Vglut2 -expressing neurons showed equal numbers of tdTomato + and tdTomato - POMC immunoreactive neurons. Together, these data suggest that POMC neurons exhibit developmental plasticity in their expression of glutamatergic and GABAergic markers, enabling re-establishment of normal energy homeostasis in the Vglut2 - Pomc restored mice.

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.)

Background: The role of the kidney in glucose homeostasis has gained global interest. Kidneys are innervated by renal nerves, and renal denervation animal models have shown improved glucose regulation. We hypothesized that stimulation of renal nerves at kilohertz frequencies, which can block propagation of action potentials, would increase urine glucose excretion. Conversely, we hypothesized that low frequency stimulation, which has been shown to increase renal nerve activity, would decrease urine glucose excretion., Methods: We performed non-survival experiments on male rats under thiobutabarbital anesthesia. A cuff electrode was placed around the left renal artery, encircling the renal nerves. Ureters were cannulated bilaterally to obtain urine samples from each kidney independently for comparison. Renal nerves were stimulated at kilohertz frequencies (1-50 kHz) or low frequencies (2-5 Hz), with intravenous administration of a glucose bolus shortly into the 25-40-min stimulation period. Urine samples were collected at 5-10-min intervals, and colorimetric assays were used to quantify glucose excretion and concentration between stimulated and non-stimulated kidneys. A Kruskal-Wallis test was performed across all stimulation frequencies (α = 0.05), followed by a post-hoc Wilcoxon rank sum test with Bonferroni correction (α = 0.005)., Results: For kilohertz frequency trials, the stimulated kidney yielded a higher average total urine glucose excretion at 33 kHz (+ 24.5%; n  = 9) than 1 kHz (- 5.9%; n  = 6) and 50 kHz (+ 2.3%; n  = 14). In low frequency stimulation trials, 5 Hz stimulation led to a lower average total urine glucose excretion (- 40.4%; n  = 6) than 2 Hz (- 27.2%; n  = 5). The average total urine glucose excretion between 33 kHz and 5 Hz was statistically significant ( p  < 0.005). Similar outcomes were observed for urine flow rate, which may suggest an associated response. No trends or statistical significance were observed for urine glucose concentrations., Conclusion: To our knowledge, this is the first study to investigate electrical stimulation of renal nerves to modulate urine glucose excretion. Our experimental results show that stimulation of renal nerves may modulate urine glucose excretion, however, this response may be associated with urine flow rate. Future work is needed to examine the underlying mechanisms and identify approaches for enhancing regulation of glucose excretion., Competing Interests: Competing interestsRJS has received research support from and/or has served as an advisor or consultant to Ethicon Endo-Surgery/Johnson & Johnson, Orexigen, Novo Nordisk, Daiichi Sankyo, Janssen/Johnson & Johnson, Novartis, Paul Hastings Law Firm, Zafgen, MedImmune, Sanofi, Kallyope, and Scohia., (© The Author(s) 2018.)

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.)

Background: A recent clinical trial found that pharmacological blockade of V1b receptors reduces alcohol relapse in alcohol-dependent patients. SSR149415 is a selective V1b receptor antagonist that has potential for development as an alcohol dependency treatment. In this study, we investigated whether SSR149415 alone or in combination with the mu-opioid receptor (MOP-r) antagonist naltrexone (NTN) would alter excessive alcohol drinking in mice., Methods: Both sexes of C57BL/6J (B6) mice were subjected to a chronic intermittent access (IA) drinking paradigm (2-bottle choice, 24-hour access every other day) for 3 weeks. Sucrose and saccharin drinking were used as controls for alcohol-specific drug effects. Neuronal proopiomelanocortin (POMC) enhancer (nPE) knockout mice with hypothalamic-specific loss of POMC (including beta-endorphin, the main endogenous ligand of MOP-r) were used as a genetic control for the effects of NTN., Results: Acute administration of SSR149415 (1 to 30 mg/kg) reduced alcohol intake and preference in a dose-dependent manner in both male and female B6 mice after IA. To investigate potential synergistic effects between NTN and SSR149415, we tested 6 different combination doses of SSR149415 and NTN, and found that a combination of SSR149415 (3 mg/kg) and NTN (1 mg/kg) reduced alcohol intake profoundly at doses lower than the individual effective doses in both sexes of B6 mice. We confirmed the effect of SSR149415 on reducing alcohol intake in nPE-/- male mice, consistent with independent mechanisms by which SSR149415 and NTN decrease alcohol drinking., Conclusions: The combination of V1b antagonist SSR149415 with NTN at individual subthreshold doses shows potential in alcoholism treatment, possibly with less adverse effects., (Copyright © 2017 by the Research Society on Alcoholism.)

Background & Aims: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia., Methods: Primary enteric glial cultures were generated from the VillinCre:Men1 FL/FL :Sst -/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom., Results: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas., Conclusions: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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.)

Objective: The increasing prevalence of type 2 diabetes (T2D) and associated morbidity and mortality emphasizes the need for a more complete understanding of the mechanisms mediating glucose homeostasis to accelerate the identification of new medications. Recent reports indicate that the obesity medication lorcaserin, a 5-hydroxytryptamine (5-HT, serotonin) 2C receptor (5-HT 2C R) agonist, improves glycemic control in association with weight loss in obese patients with T2D. Here we evaluate whether lorcaserin has an effect on glycemia without body weight loss and how this effect is achieved., Methods: Murine models of common and genetic T2D were utilized to probe the direct effect of lorcaserin on glycemic control., Results: Lorcaserin dose-dependently improves glycemic control in mouse models of T2D in the absence of reductions in food intake or body weight. Examining the mechanism of this effect, we reveal a necessary and sufficient neurochemical mediator of lorcaserin's glucoregulatory effects, brain pro-opiomelanocortin (POMC) peptides. To clarify further lorcaserin's therapeutic brain circuit, we examined the receptor target of POMC peptides. We demonstrate that lorcaserin requires functional melanocortin4 receptors on cholinergic preganglionic neurons (MC4R ChAT ) to exert its effects on glucose homeostasis. In contrast, MC4R ChAT signaling did not impact lorcaserin's effects on feeding, indicating a divergence in the neurocircuitry underpinning lorcaserin's therapeutic glycemic and anorectic effects. Hyperinsulinemic-euglycemic clamp studies reveal that lorcaserin reduces hepatic glucose production, increases glucose disposal and improves insulin sensitivity., Conclusions: These data suggest that lorcaserin's action within the brain represents a mechanistically novel treatment for T2D: findings of significance to a prevalent global disease., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A specter is haunting the world, the specter of obesity. During the last decade, this pandemia has skyrocketed threatening children, adolescents and lower income families worldwide. Although driven by an increase in the consumption of ultraprocessed edibles of poor nutritional value, the obesogenic changes in contemporary human lifestyle affect people differently, revealing that some individuals are more prone to develop increased adiposity. During the last years, we performed a variety of genetic, evolutionary, biochemical and behavioral experiments that allowed us to understand how a group of neurons present in the arcuate nucleus of the hypothalamus regulate the expression of the proopiomelanocortin (Pomc) gene and induce satiety. We disentangled the neuronal transcriptional code of Pomc by identifying the cis-acting regulatory elements and primary transcription factors controlling hypothalamic Pomc expression and determined their functional importance in the regulation of food intake and adiposity. Altogether, our studies reviewed here shed light on the power and limitations of the mammalian central satiety pathways and may contribute to the development of individual and collective strategies to reduce the debilitating effects of the self-induced obesity pandemia., (© 2017 Federation of European Biochemical Societies.)

Opioid receptor antagonist naltrexone reduces alcohol consumption and relapse in both humans and rodents. This study investigated whether hypothalamic proopiomelanocortin (POMC) neurons (producing beta-endorphin and melanocortins) play a role in alcohol drinking behaviors. Both male and female mice with targeted deletion of two neuronal Pomc enhancers nPE1 and nPE2 (nPE-/-), resulting in hypothalamic-specific POMC deficiency, were studied in short-access (4-h/day) drinking-in-the-dark (DID, alcohol in one bottle, intermittent access (IA, 24-h cycles of alcohol access every other day, alcohol vs. water in a two-bottle choice) and alcohol deprivation effect (ADE) models. Wild-type nPE+/+ exposed to 1-week DID rapidly established stable alcohol drinking behavior with more intake in females, whereas nPE-/- mice of both sexes had less intake and less preference. Although nPE-/- showed less saccharin intake and preference than nPE+/+, there was no genotype difference in sucrose intake or preference in the DID paradigm. After 3-week IA, nPE+/+ gradually escalated to high alcohol intake and preference, with more intake in females, whereas nPE-/- showed less escalation. Pharmacological blockade of mu-opioid receptors with naltrexone reduced intake in nPE+/+ in a dose-dependent manner, but had blunted effects in nPE-/- of both sexes. When alcohol was presented again after 1-week abstinence from IA, nPE+/+ of both sexes displayed significant increases in alcohol intake (ADE or relapse-like drinking), with more pronounced ADE in females, whereas nPE-/- did not show ADE in either sex. Our results suggest that neuronal POMC is involved in modulation of alcohol 'binge' drinking, escalation and 'relapse', probably via hypothalamic-mediated mechanisms, with sex differences., (© 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Objective: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice., Methods: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc (-/-)) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc (-/-) mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc (-/-) mice to mimic the super-elevated leptin levels of obese mice., Results: ArcPomc (-/-) mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc (-/-) mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc (-/-) mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc (-/-) mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc (-/-) mice., Conclusions: Pomc reactivation in previously obese, calorie-restricted ArcPomc (-/-) mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc (-/-) mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc (-/-) mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc (-/-) mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight.

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 proopiomelanocortin (POMC) is essential for the physiological regulation of energy balance; however, its role in glucose homeostasis remains less clear. We show that hypothalamic arcuate nucleus (Arc)POMC-deficient mice, which develop severe obesity and insulin resistance, unexpectedly exhibit improved glucose tolerance and remain protected from hyperglycemia. To explain these paradoxical phenotypes, we hypothesized that an insulin-independent pathway is responsible for the enhanced glucose tolerance. Indeed, the mutant mice demonstrated increased glucose effectiveness and exaggerated glycosuria relative to wild-type littermate controls at comparable blood glucose concentrations. Central administration of the melanocortin receptor agonist melanotan II in mutant mice reversed alterations in glucose tolerance and glycosuria, whereas, conversely, administration of the antagonist Agouti-related peptide (Agrp) to wild-type mice enhanced glucose tolerance. The glycosuria of ArcPOMC-deficient mice was due to decreased levels of renal GLUT 2 (rGLUT2) but not sodium-glucose cotransporter 2 and was associated with reduced renal catecholamine content. Epinephrine treatment abolished the genotype differences in glucose tolerance and rGLUT2 levels, suggesting that reduced renal sympathetic nervous system (SNS) activity is the underlying mechanism for the observed glycosuria and improved glucose tolerance in ArcPOMC-deficient mice. Therefore, the ArcPOMC-SNS-rGLUT2 axis is potentially an insulin-independent therapeutic target to control diabetes., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified., Methods: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT 2C R (CRE) mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable Pomc (NEO) mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight., Results: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity., Conclusions: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual.

The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 μM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.

Somatostatin is a neuropeptide hormone that inhibits pituitary growth hormone (GH) release. Using microarray analysis of gene expression in the livers of wildtype control and somatostatin knockout mice, we have previously identified a panel of genes whose GH-dependent and sexually dimorphic expression patterns are significantly altered by the absence of somatostatin (1). Here, we provide methodological and analytical details of that study, the raw data of which is deposited in the Gene Expression Omnibus as data set GSE56520. In addition, we performed further gene ontology analysis of the data and found that the differential expression of a second subset of genes in the livers of somatostatin-knockout mice versus wildtype controls is likely independent of GH signaling and involved in the innate immune response.

Food intake and body weight regulation depend on proper expression of the proopiomelanocortin gene (Pomc) in a group of neurons located in the mediobasal hypothalamus of all vertebrates. These neurons release POMC-encoded melanocortins, which are potent anorexigenic neuropeptides, and their absence from mice or humans leads to hyperphagia and severe obesity. Although the pathophysiology of hypothalamic POMC neurons is well understood, the genetic program that establishes the neuronal melanocortinergic phenotype and maintains a fully functional neuronal POMC phenotype throughout adulthood remains unknown. Here, we report that the early expression of the LIM-homeodomain transcription factor Islet 1 (ISL1) in the developing hypothalamus promotes the terminal differentiation of melanocortinergic neurons and is essential for hypothalamic Pomc expression since its initial onset and throughout the entire lifetime. We detected ISL1 in the prospective hypothalamus just before the onset of Pomc expression and, from then on, Pomc and Isl1 coexpress. ISL1 binds in vitro and in vivo to critical homeodomain binding DNA motifs present in the neuronal Pomc enhancers nPE1 and nPE2, and mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to hypothalamic POMC neurons in transgenic mice and zebrafish. ISL1 is necessary for hypothalamic Pomc expression during mouse and zebrafish embryogenesis. Furthermore, conditional Isl1 inactivation from POMC neurons impairs Pomc expression, leading to hyperphagia and obesity. Our results demonstrate that ISL1 specifies the identity of hypothalamic melanocortin neurons and is required for melanocortin-induced satiety and normal adiposity throughout the entire lifespan.

Peptides derived from the proopiomelanocortin (POMC) precursor are critical for the normal regulation of many physiological parameters, and POMC deficiency results in severe obesity and metabolic dysfunction. Conversely, augmentation of central nervous system melanocortin function is a promising therapeutic avenue for obesity and diabetes but is confounded by detrimental cardiovascular effects including hypertension. Because the hypothalamic population of POMC-expressing neurons is neurochemically and neuroanatomically heterogeneous, there is interest in the possible dissociation of functionally distinct POMC neuron subpopulations. We used a Cre recombinase-dependent and hypothalamus-specific reactivatable PomcNEO allele to restrict Pomc expression to hypothalamic neurons expressing leptin receptor (Lepr) in mice. In contrast to mice with total hypothalamic Pomc deficiency, which are severely obese, mice with Lepr-restricted Pomc expression displayed fully normal body weight, food consumption, glucose homeostasis, and locomotor activity. Thus, Lepr+ POMC neurons, which constitute approximately two-thirds of the total POMC neuron population, are sufficient for normal regulation of these parameters. This functional dissociation approach represents a promising avenue for isolating therapeutically relevant POMC neuron subpopulations.

Distinct male and female patterns of pituitary GH secretion produce sexually differentiated hepatic gene expression profiles, thereby influencing steroid and xenobiotic metabolism. We used a fully automated system to obtain serial nocturnal blood samples every 15 minutes from cannulated wild-type (WT) and somatostatin knockout (Sst-KO) mice to determine the role of SST, the principal inhibitor of GH release, in the generation of sexually dimorphic GH pulsatility. WT males had lower mean and median GH values, less random GH secretory bursts, and longer trough periods between GH pulses than WT females. Each of these parameters was feminized in male Sst-KO mice, whereas female Sst-KO mice had higher GH levels than all other groups, but GH pulsatility was unaffected. We next performed hepatic mRNA profiling with high-density microarrays. Male Sst-KO mice exhibited a globally feminized pattern of GH-dependent mRNA levels, but female Sst-KO mice were largely unaffected. Among the differentially expressed female-predominant genes was Serpina6, which encodes corticosteroid-binding globulin (CBG). Increased CBG was associated with elevated diurnal peak plasma corticosterone in unstressed WT females and both sexes of Sst-KO mice compared with WT males. Sst-KO mice also had exaggerated ACTH and corticosterone responses to acute restraint stress. However, consistent with their lack of phenotypic signs of excess glucocorticoids, cerebrospinal fluid concentrations of free corticosterone in Sst-KO mice were not elevated. In summary, SST is necessary for the prolonged interpulse troughs that define masculinized pituitary GH secretion. SST also contributes to sexual dimorphism of the hypothalamic-pituitary-adrenal axis via GH-dependent regulation of hepatic CBG production.

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

The phenomenon commonly described as the middle-age spread is the result of elevated adiposity accumulation throughout adulthood until late middle-age. It is a clinical imperative to gain a greater understanding of the underpinnings of age-dependent obesity and, in turn, how these mechanisms may impact the efficacy of obesity treatments. In particular, both obesity and aging are associated with rewiring of a principal brain pathway modulating energy homeostasis, promoting reduced activity of satiety pro-opiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus (ARC). Using a selective ARC-deficient POMC mouse line, here we report that former obesity medications augmenting endogenous 5-hydroxytryptamine (5-HT) activity d-fenfluramine and sibutramine require ARC POMC neurons to elicit therapeutic appetite-suppressive effects. We next investigated whether age-related diminished ARC POMC activity therefore impacts the potency of 5-HT obesity pharmacotherapies, lorcaserin, d-fenfluramine, and sibutramine and report that all compounds reduced food intake to a comparable extent in both chow-fed young lean (3-5 months old) and middle-aged obese (12-14 months old) male and female mice. We provide a mechanism through which 5-HT anorectic potency is maintained with age, via preserved 5-HT-POMC appetitive anatomical machinery. Specifically, the abundance and signaling of the primary 5-HT receptor influencing appetite via POMC activation, the 5-HT2CR, is not perturbed with age. These data reveal that although 5-HT obesity medications require ARC POMC neurons to achieve appetitive effects, the anorectic efficacy is maintained with aging, findings of clinical significance to the global aging obese population.