Neurotensin (NT) is implicated in the regulation of energy homeostasis, in addition to its many described physiological functions. NT is postulated to mediate, in part, the effects of leptin in the hypothalamus. We generated clonal, immortalized hypothalamic cell lines, N-39 and N-36/1, which are the first representative NT-expressing cell models available for the investigation of NT gene regulation and control mechanisms. The cell lines express the Ob-Rb leptin receptor neuropeptide Y (NPY)-Y1, Y2, Y4, Y5 receptors, melanocortin 4 receptor, insulin receptor, and the NT receptor. NT mRNA levels are induced by approximately 1.5-fold to twofold with leptin, insulin, and alpha-melanocyte stimulating hormone treatments but not by NPY. Leptin-mediated induction of NT gene expression was biphasic at 10(-11) and 10(-7) M. The leptin responsive region was localized to within -381 to -250 bp of the 5' regulatory region of the NT gene. Furthermore, we demonstrated direct leptin-mediated signal transducers and activators of transcription (STAT) binding to this region at 10(-11) m, but not 10(-7) m leptin, in chromatin precipitation assays. Leptin-induced NT regulation was attenuated by dominant-negative STAT3 protein expression. These data support the hypothesis that NT may have a direct role in the neuroendocrine control of feeding and energy homeostasis.

The melanocortin receptors, melanocortin-3-receptor (MC3-R) and melanocortin-4-receptor (MC4-R), are expressed in many discrete medial hypothalamic nuclei implicated in feeding regulation. The pro-opiomelanocortin product alpha-melanocyte stimulating hormone (alpha-MSH), an MC3/4-R agonist, decreases food intake following intracerebroventricular (ICV) injection in rats. MC4-R's involvement in feeding has been established although a function for the MC3-R is unclear. We investigated endogenous melanocortin ligand binding and activation at the MC3-R and MC4-R and their effects on feeding. We have shown that alpha-MSH, desacetyl-alpha-MSH and beta-MSH bound to the MC3-R and MC4-R with similar affinity and stimulated cAMP with similar potency in HEK 293 cells transfected with MC3-R and MC4-R. In contrast gamma(2)-MSH showed selectivity for the MC3-R over the MC4-R both in binding affinity and cAMP stimulation. alpha-MSH and beta-MSH injected ICV into fasted rats at doses of 1, 3 and 6 nmol resulted in a decrease in food intake, (2 h food intake: alpha-MSH 6 nmol, 1.7+/-0.3 g; beta-MSH 6 nmol, 1.5+/-0.3 g vs. saline 6.0+/-0.5 g, P<0.001). Desacetyl alpha-MSH did not reduce food intake at low doses but was significant at 25 nmol (2 h food intake: desacetyl-alpha-MSH 6.1+/-1.0 g vs. saline 9.5+/-1.4 g, P<0.05). In contrast, gamma(2)-MSH had no effect on food intake when administered ICV to fasted rats. We were unable to establish a role for the MC3-R in feeding regulation. Our evidence, however, strengthens the hypothesis that the melanocortin's effects on food intake are mediated via the MC4-R.

In goldfish, intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits feeding behavior, and fasting decreases hypothalamic MCH-like immunoreactivity. However, while MCH acts as an anorexigenic factor in goldfish, in rodents MCH has an orexigenic effect. Therefore, we examined the involvement of two anorexigenic neuropeptides, alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH), in the anorexigenic action of MCH in goldfish, using an alpha-MSH receptor antagonist, HS024, and a CRH receptor antagonist, alpha-helical CRH((9-41)). ICV injection of HS024, but not alpha-helical CRH((9-41)), suppressed MCH-induced anorexigenic action for a 60-min observation period. We then examined, using a real-time PCR method, whether MCH affects the levels of mRNAs encoding various orexigenic neuropeptides, including neuropeptide Y (NPY), orexin, ghrelin and Agouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for NPY and ghrelin, but not for orexin and AgRP. These results indicate that the anorexigenic action of MCH in the goldfish brain is mediated by the alpha-MSH signaling pathway and is accompanied by inhibition of NPY and ghrelin synthesis.

It is now well established that GPR139, a G-protein coupled receptor exclusively expressed in the brain and pituitary, is activated by the essential amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) via Gαq-coupling. The in vitro affinity and potency values of L-Trp and L-Phe are within the physiological concentration ranges of L-Trp and L-Phe. A recent paper suggests that adrenocorticotropic hormone (ACTH), α and β melanocyte stimulating hormones (α-MSH and β-MSH) and derivatives α-MSH1-9/α-MSH1-10 can also activate GPR139 in vitro. We tested this hypothesis using guanosine 5'-O-(3-[35S]thio)-triphosphate binding (GTPγS), calcium mobilization and [3H]JNJ-63533054 radioligand binding assays. In the GTPγS binding assay, α-MSH, α-MSH1-9/α-MSH1-10, and β-MSH had no effect on [35S]GTPγS incorporation in cell membranes expressing GPR139 up to 30 μM in contrast to the concentration dependent activation produced by L-Trp, JNJ-63533054, and TC-09311 (two small molecule GPR139 agonists). ACTH slightly decreased the basal level of [35S]GTPγS incorporation at 30 μM. In the GPR139 radioligand binding assay, a moderate displacement of [3H]JNJ-63533054 binding by ACTH and β-MSH was observed at 30 μM (40 and 30%, respectively); α-MSH, α-MSH1-9/α-MSH1-10 did not displace any specific binding at 30 μM. In three different host cell lines stably expressing GPR139, α-MSH, and β-MSH did not stimulate calcium mobilization in contrast to L-Trp, JNJ-63533054, and TC-09311. ACTH, α-MSH1-9/α-MSH1-10 only weakly stimulated calcium mobilization at 30 μM (<50% of EC100). We then co-transfected GPR139 with the three melanocortin (MC) receptors (MC3R, MC4R, and MC5R) to test the hypothesis that ACTH, α-MSH, and β-MSH might stimulate calcium mobilization through a MCR/GPR139 interaction. All three MC peptides stimulated calcium response in cells co-transfected with GPR139 and MC3R, MC4R, or MC5R. The MC peptides did not stimulate calcium response in cells expressing MC3R or MC5R alone consistent with the Gs signaling transduction pathway of these receptors. In agreement with the previously reported multiple signaling pathways of MC4R, including Gq transduction pathway, the MC peptides produced a calcium response in cells expressing MC4R alone. Together, our findings do not support that GPR139 is activated by ACTH, α-MSH, and β-MSH at physiologically relevant concentration but we did unravel an in vitro interaction between GPR139 and the MCRs.

A social signal transduction theory of depression has been proposed that states that exposure to social adversity alters the immune response and these changes mediate symptoms of depression such as anhedonia and impairments in social behavior The exposure of maternal rats to the chronic social stress (CSS) of a male intruder depresses maternal care and impairs social behavior in the F1 and F2 offspring of these dams. The objective of the present study was to characterize basal peripheral levels of several immune factors and related hormone levels in the adult F2 offspring of CSS exposed dams and assess whether changes in these factors are associated with previously reported deficits in allogrooming behavior. CSS decreased acid glycoprotein (α1AGP) and intercellular adhesion molecule-1 (ICAM-1) in F2 females, and increased granulocyte macrophage-colony stimulating factor (GM-CSF) in F2 males. There were also sex dependent changes in IL-18, tissue inhibitors of metalloproteinases 1 (TIMP-1), and vascular endothelial growth factor (VEGF). Progesterone was decreased and alpha melanocyte stimulating hormone (α-MSH) was increased in F2 males, and brain-derived neurotrophic factor (BDNF) was decreased in F2 females. Changes in α1AGP, GM-CSF, progesterone, and α-MSH were correlated with decreased allogrooming in the F2 offspring of stressed dams. These results support the hypothesis that transgenerational social stress affects both the immune system and social behavior, and also support previous studies on the adverse effects of early life stress on immune functioning and stress associated immunological disorders, including the increasing prevalence of asthma. The immune system may represent an important transgenerational etiological factor in disorders which involve social and/or early life stress associated changes in social behavior, such as depression, anxiety, and autism, as well as comorbid immune disorders. Future studies involving immune and/or endocrine assessments and manipulations will address specific questions of function and causation, and may identify novel preventative measures and treatments for the growing number of immune mediated disorders.

Kisspeptin neurones in the arcuate nucleus play a pivotal role in the regulation of hypothalamic gonadotrophin-releasing hormone (GnRH) secretion in higher primates. To examine whether kisspeptin also influences the function of the primate pituitary directly, two experiments were performed in adult male rhesus monkeys. First, the distribution of kisspeptin-containing cells in the pituitary was described using fluorescence immunohistochemistry. Second, the secretion of non-gonadotrophin adenohypophysial hormones [growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH)] and cortisol in response to i.v. kisspeptin administration was examined. Eight animals were deeply anaesthetised and transcardially perfused with 4% paraformaldehyde. Fluorescence immunohistochemistry was performed on 25-microm thick free-floating pituitary sections to localise immunopositive kisspeptin cells and to examine their relationship with immunostaining for luteinising hormone (LH), follicle-stimulating hormone, GH, prolactin, alpha-melanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH) and GnRH. Kisspeptin cells were found in the intermediate lobe of all animals and, in four monkeys, this neuropeptide was also observed in cells scattered in the periphery of the anterior lobe. Kisspeptin colocalised with alpha-MSH-immunopositive cells in the intermediate lobe and, in 50% of the monkeys, with ACTH-immuunopositive cells in the periphery of the adenohypophysis. There was no evidence for colocalisation of kisspeptin with gonadotrophs, somatotrophs or lactotrophs. Beaded kisspeptin axons were observed in the neural lobe. In addition, assay of plasma samples that had been collected for a previous study documenting kisspeptin-10-induced LH release in male monkeys revealed that kisspeptin administration failed to influence circulating concentrations of GH, prolactin, TSH and cortisol. Release of all four of these non-gonadotrophic hormones, however, was stimulated markedly by NMDA, which is considered to act centrally. Although the morphological findings obtained in the present study are consistent with the notion that kisspeptin may act directly at the level of the pituitary, the nature of such an action remains to be defined.

Satiety and appetite signaling are accomplished by circulating peptide hormones. These peptide hormones require processing from larger precursors to become bioactive, often by the proprotein convertase 1/3 (PC1/3). Several subcellular maturation steps are necessary for PC1/3 to achieve its optimal enzymatic activity. Certain PC1/3 variants found in the general population slightly attenuate its enzymatic activity and are associated with obesity and diabetes. However, mutations that increase PC1/3 activity and/or affect its specificity could also have physiological consequences. We here present data showing that the known human Ser357Gly PC1/3 mutant (PC1/3(S357G)) represents a PC1/3 hypermorph. Conditioned media from human embryonic kidney-293 cells transfected with PC1/3(WT) and PC1/3(S357G) were collected and enzymatic activity characterized. PC1/3(S357G) exhibited a lower calcium dependence; a higher pH optimum (neutral); and a higher resistance to peptide inhibitors than the wild-type enzyme. PC1/3(S357G) exhibited increased cleavage to the C-terminally truncated form, and kinetic parameters of the full-length and truncated mutant enzymes were also altered. Lastly, the S357G mutation broadened the specificity of the enzyme; we detected PC2-like specificity on the substrate proCART, the precursor of the cocaine- and amphetamine regulated transcript neuropeptide known to be associated with obesity. The production of another anorexigenic peptide normally synthesized only by PC2, αMSH, was increased when proopiomelanocortin was coexpressed with PC1/3(S357G). Considering the aberrant enzymatic profile of PC1/3(S357G), we hypothesize that this enzyme possesses unusual processing activity that may significantly change the profile of circulating peptide hormones.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the CNS, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca(2+), which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.

Based upon similarities between the urge to move and sensory discomfort of restless legs syndrome (RLS) and properties of melanocortin hormones, including their incitement of movement and hyperalgesia, we assessed plasma and cerebrospinal fluid (CSF) α-melanocyte-stimulating hormone (α-MSH) and β-endorphin in RLS patients and controls.

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

We studied whether myo-inositol supplementation throughout lactation, alone and combined with leptin, may reverse detrimental effects on hypothalamic structure and function caused by gestational calorie gestation (CR) in rats. Candidate early transcript-based biomarkers of metabolic health in peripheral blood mononuclear cells (PBMC) were also studied. Offspring of dams exposed to 25% gestational CR and supplemented during lactation with physiological doses of leptin (CR-L), myo-inositol (CR-M), the combination (CR-LM), or the vehicle (CR-V) as well as control rats (CON-V) were followed and sacrificed at postnatal day 25. Myo-inositol and the combination increased the number of neurons in arcuate nucleus (ARC) (only in females) and paraventricular nucleus, and myo-inositol (alone) restored the number of αMSH+ neurons in ARC. Hypothalamic mRNA levels of Lepr in CR-M and Insr in CR-M and CR-LM males were higher than in CR-V and CON-V, respectively. In PBMC, increased expression levels of Lrp11 and Gls in CR-V were partially normalized in all supplemented groups (but only in males for Gls). Therefore, myo-inositol supplementation throughout lactation, alone and combined with leptin, reverts programmed alterations by fetal undernutrition on hypothalamic structure and gene expression of potential early biomarkers of metabolic health in PBMC, which might be attributed, in part, to increased leptin sensitivity.

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.

The endogenous melanocortin, alpha-melanocyte-stimulating hormone (alpha-MSH), is a neurohormone secreted by the neurointermediate lobe of the pituitary. Alpha-MSH promotes intermale aggression in mice by influencing pheromone secretion, but the role of specific melanocortin receptors has not been determined. We assessed mice made deficient in the gene for the melanocortin-5 receptor (MC5R) to determine its role in pheromone-regulated behavior. In heterotypic pairs assessed in the social interaction test (SIT), MC5R-deficient mice exhibited less aggressive behavior and more defensive behavior than their wild-type opponents. By contrast, when assessed in homotypic pairs and against stimulus animals in the SIT, MC5R-deficient and wild-type mice behaved similarly. Moreover, urine from MC5R deficient mice stimulated more aggression than did urine from wild-type mice. The results suggest that MC5R deficiency disinhibits an aggression-suppressing pheromonal signal.

Radioimmunoassay (RIA) detected the presence of beta-endorphin in the intraglandular colloid (IGC) of bovine pituitary intermediate lobe origin. The amount of beta-endorphin recovered in each of twelve samples ranged from 0.15 to 218.30 pmol/mg protein. A second group of assays [amino acid analysis, high performance liquid chromatography (HPLC) and mass spectral analysis] confirmed the RIA findings in another series of colloid samples. Approximately 75 pmol was collected from eight pooled glands. beta-Endorphin is an addition to the list of peptide hormones (e.g., methionine-enkephalin, adrenocorticotropin, arginine-vasopressin, alpha-melanocyte-stimulating hormone, beta-lipotropin and somatostatin) previously discovered in IGC by this laboratory.

E. coli releases a 33 amino acid peptide melanocortin-like peptide of E. coli (MECO-1) that is identical to the C-terminus of the E. coli elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.

α- and β-melanocyte-stimulating hormones (MSH) are derived from pro-opiomelanocortin (POMC) and are the natural agonist ligands of the melanocortin 4 receptor, a key regulator of energy homeostasis. Recent rodent and human data have implicated the MAGEL2 gene, which may regulate activation of POMC neurons, as a significant contributor to the metabolic symptoms observed in Prader-Willi Syndrome (PWS). Firstly, patients with protein truncating mutations in MAGEL2 exhibit numerous clinical characteristics of PWS. Secondly, Magel2-null mice may not normally activate MC4 receptors, as they are defective in the activation of their POMC neurons and hence may fail to normally release the POMC-derived MC4 receptor agonist ligands α- and β-MSH. Magel2-null mice represent a tractable animal model for the metabolic and appetitive imbalance seen in patients with PWS.

As part of the study of their bioluminescence, the deep-sea lanternshark Etmopterus spinax and Etmopterus molleri (Chondrichthyes, Etmopteridae) received growing interest over the past ten years. These mesopelagic sharks produce light thanks to a finely tuned hormonal control involving melatonin, adrenocorticotropic hormone and α-melanocyte-stimulating hormone. Receptors of these hormones, respectively the melatonin receptors and the melanocortin receptors, are all members of the G-protein coupled receptor family i.e. coupled with specific G proteins involved in the preliminary steps of their transduction pathways. The present study highlights the specific localization of the hormonal receptors, as well as of their associated G-proteins within the light organs, the so-called photophores, in E. spinax and E. molleri through immunohistofluorescence technic. Our results allow gaining insight into the molecular actors and mechanisms involved in the control of the light emission in Etmopterid sharks.

Cystic fibrosis (CF) belongs to the most common inherited diseases. The severity of the disease and chronic bacterial infections are associated with a lower body index, undernutrition, higher number of pulmonary exacerbations, more hospital admissions, and increased mortality. The aim of our study was to determine the impact of the severity of the disease and the type of bacterial infection in 38 CF patients on the serum level of appetite-regulating hormones including leptin, ghrelin, neuropeptide Y, agouti-signaling protein, proopiomelanocortin, kisspeptin, putative protein Y, and α-melanocyte-stimulating hormone. The patients were divided according to the severity of the disease according to spirometry and the type of chronic bacterial infection. We found that leptin level was significantly higher in patients with severe CF than in patients with mild disease (20.02 ± 8.09 vs. 12.38 ± 6.03 ng/mL, p = 0.028). Furthermore, leptin level was elevated in patients with chronic infection with Pseudomonas aeruginosa compared to uninfected participants (15.74 ± 7.02 vs. 9.28 ± 1.72 ng/mL, p = 0.043). The severity of the disease and the type of bacterial infection did not affect the levels of other appetite-regulating hormones. Moreover, we found a positive correlation between pro-inflammatory interleukin-6 and leptin level (p = 0.0426, R = 0.333). Taken together, our results indicate that both the severity of the disease and the type of bacterial infection are associated with elevated leptin levels in CF patients. Future CF treatment strategies should consider possible disturbances in the hormones that regulate appetite and the factors that influence their levels.

Hyperpigmentation is a type of pigmentary disorder induced by overexpression of melanin content activated severe esthetic problems as melasma, freckle, ephelides, lentigo and other forms on human skin. Several whitening agents have restricted use because of their side effects or stability such as kojic acid, ascorbic acid and hydroquinone can act as cytotoxic substance which associated to dermatitis and skin cancer. To find for the safe substance, this study aimed to find for the ability of several components in Sucrier banana peel (SBP) extracts to inhibit melanogenesis process through p38 signaling pathway in B16F10 mouse melanoma cells. Tyrosinase activity and the cellular melanin content were dose dependent manner decreasing after SBP treatment. Furthermore, SBP decreased the expression of melanogenesis relate protein as microphthalmia-associated transcription factor (MITF) and tyrosinase protein after 24 hours incubation with α-melanocyte stimulating hormones (MSH) stimulating. The findings demonstrated that SBP contained an effective agent for hyperpigmentation inhibitor through p38 signaling pathways without any effect to ERK pathway, and subsequent down-regulate MITF expression and tyrosinase enzyme family production.

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60. 1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken.