RESUMEN
OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of ß-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.
Asunto(s)
Melanocortinas/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismo , Cromatografía Liquida , Femenino , Homeostasis/fisiología , Humanos , Hipotálamo , Leptina/metabolismo , Masculino , Espectrometría de Masas/métodos , Neuronas/metabolismo , Neuropéptidos/metabolismo , Células Madre Pluripotentes/metabolismo , Proopiomelanocortina/metabolismo , Receptores de Melanocortina/metabolismo , Espectrometría de Masas en TándemRESUMEN
GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 µM and 320 µM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle "similar targets bind similar ligands", we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and ß-melanocyte stimulating hormone (α-MSH and ß-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca2+-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low micromolar range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH1-9 was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, ß-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH1-9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Melanocitos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Cricetulus , Hormonas Estimuladoras de los Melanocitos/metabolismo , Proopiomelanocortina/metabolismo , Receptor de Melanocortina Tipo 4/metabolismoRESUMEN
Sequencing of candidate genes for obesity in Labrador retriever dogs identified a 14 bp deletion in pro-opiomelanocortin (POMC) with an allele frequency of 12%. The deletion disrupts the ß-MSH and ß-endorphin coding sequences and is associated with body weight (per allele effect of 0.33 SD), adiposity, and greater food motivation. Among other dog breeds, the deletion was only found in the closely related flat-coat retriever (FCR), where it is similarly associated with body weight and food motivation. The mutation is significantly more common in Labrador retrievers selected to become assistance dogs than pets. In conclusion, the deletion in POMC is a significant modifier of weight and appetite in Labrador retrievers and FCRs and may influence other behavioral traits.
Asunto(s)
Apetito/genética , Peso Corporal/genética , Eliminación de Gen , Obesidad/genética , Proopiomelanocortina/genética , Adiposidad/genética , Secuencia de Aminoácidos , Animales , Emparejamiento Base , Secuencia de Bases , Células COS , Chlorocebus aethiops , Perros , Conducta Alimentaria , Genotipo , Proopiomelanocortina/química , Proopiomelanocortina/metabolismo , Receptores de Melanocortina/metabolismo , beta-MSH/metabolismoRESUMEN
Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine(1), O-acetyl Serine(3)]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline(15)]-ß-MSH, together with [phosphoserine(15)]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.
Asunto(s)
Oryzias/metabolismo , Hipófisis/química , Proopiomelanocortina/metabolismo , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Oryzias/anatomía & histología , Hipófisis/metabolismo , Proopiomelanocortina/química , Proopiomelanocortina/genética , alfa-MSH/química , alfa-MSH/genética , alfa-MSH/metabolismo , beta-MSH/química , beta-MSH/genética , beta-MSH/metabolismoRESUMEN
Dogs have become one of the most important companion animals in modern society. However, it is estimated that 20% to 40% of owned dogs are obese, suggesting that obesity has become one of the most important canine health problem. In addition, obesity in dogs also leads to type II diabetes. Because the melanocortin-4 receptor (MC4R) has been shown to be essential in maintaining energy homeostasis in several different species, including rodents and humans, we initiated studies toward elucidating the roles of MC4R in obesity pathogenesis in dogs. Canine MC4R has been cloned, and a missense variant V213F was identified. We designed primers and successfully cloned canine MC4R and generated the variant V213F by site-directed mutagenesis. The objective of this study was to investigate the pharmacological properties of canine MC4R and its natural variant V213F. We measured ligand binding and signaling properties with the use of both natural and synthetic ligands. Human MC4R was also included in the experiments for comparison. Both wild-type canine MC4R and its natural variant V213F functioned normally in terms of binding and signaling. Of the ligands we used, [Nle(4), D-Phe(7)]-α-melanocyte-stimulating hormone is the most potent ligand. We conclude that the cloned canine MC4R is a functional receptor, and the natural variant V213F does not have any functional defect and therefore is not likely to cause obesity in dogs.
Asunto(s)
Obesidad/veterinaria , Receptor de Melanocortina Tipo 4/metabolismo , Animales , Perros , Metabolismo Energético , Variación Genética , Concentración 50 Inhibidora , Mutagénesis Sitio-Dirigida , Obesidad/genética , Obesidad/metabolismo , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas , Receptor de Melanocortina Tipo 4/genética , Transducción de Señal , alfa-MSH/metabolismo , beta-MSH/metabolismoRESUMEN
The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.
Asunto(s)
Proteína Relacionada con Agouti/antagonistas & inhibidores , Proteína Relacionada con Agouti/fisiología , Polimorfismo Genético , Proopiomelanocortina/fisiología , Receptor de Melanocortina Tipo 4/genética , Proteína Relacionada con Agouti/biosíntesis , Proteína Relacionada con Agouti/metabolismo , Secuencia de Aminoácidos , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Obesidad/genética , Obesidad/metabolismo , Proopiomelanocortina/agonistas , Proopiomelanocortina/antagonistas & inhibidores , Proopiomelanocortina/biosíntesis , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/biosíntesis , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , alfa-MSH/farmacología , alfa-MSH/fisiología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
The current alarming spread of obesity in many parts of the world is caused by a sudden environmental shift characterized by replacement of a frugal diet with low cost, energy dense food, and little requests for physical activity during work and leisure time. Yet, not all people exposed to an obesogenic environment become obese, and individual differences in the propensity to gain weight as well as the occurrence of different obese phenotypes within the same environment indicate that the genetic heritage in this regard is significant and heterogeneous. The central melanocortin circuit has received much attention during the past decade, since mutations of genes expressing some key molecules in neurons of this system were discovered, which may cause monogenic forms of obesity in animals and humans. Within the arcuate nucleus of the hypothalamus the prohormone proopiomelanocortin is posttranslationally cleaved to produce the alpha-melanocyte stimulating hormone, a peptide with anorexigenic effects upon activation of the melanocortin-4 receptor (MC4R) expressed on the surface of target neurons. Studies regarding the frequency of MC4R mutations associated with human obesity have provided variable results (up to 6% of obese subjects). Various findings suggest an oligogenic and codominant mode of inheritance for MC4R deficiency, with modulation of expressivity and penetrance of the phenotype. The yield of MC4R testing in clinical diagnosis and treatment of obesity is at present undefined since the relatively low prevalence of MC4R pathogenic variants in the general population, along with the high number of sequence variants, has so far compromised the devising of systematic controlled intervention studies. Hopefully, in the future, MC4R testing will have practical implications for the development of new mechanism-based therapy of obesity as well as for the design of specific and more effective protocols, based on lifestyle intervention and current pharmacological or surgical approaches, for management of obesity in MC4R-mutated individuals.
Asunto(s)
Mutación , Obesidad/genética , Receptor de Melanocortina Tipo 4/genética , Proteína Relacionada con Agouti/metabolismo , Peso Corporal/genética , Peso Corporal/fisiología , Sistema Nervioso Central/metabolismo , Humanos , Leptina/metabolismo , Obesidad/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismoRESUMEN
To date, the principal receptor considered to regulate human pigmentation is the melanocortin-1 receptor (MC1-R) via induction of the cAMP/protein kinase A pathway by the melanocortins alpha-MSH and ACTH. In this context, it is noteworthy that beta-MSH can also induce melanogenesis, although it has a low affinity for the MC1-R, whereas the preferred receptor for this melanocortin is the MC4-R. Because beta-MSH is present in the epidermal compartment, it was of interest to ascertain whether functioning MC4-Rs are present in human epidermal keratinocytes and melanocytes. Our results provide evidence that the MC4-R is expressed in situ and in vitro throughout the human epidermis at the mRNA and protein level using RT-PCR, Western blotting, and double immunofluorescence staining. Moreover, radioligand binding studies yielded high-affinity receptors for beta-MSH on epidermal melanocytes (3600 receptors per cell), undifferentiated keratinocytes (7200 receptors per cell), and differentiated keratinocytes (72,700 receptors per cell), indicating that MC4-R expression correlates with epidermal differentiation. Importantly, increased melanogenesis after stimulation of the beta-MSH/cAMP/microphthalmia-associated transcription factor/tyrosinase cascade proved the functionality of this signal in melanocytes, which was attenuated in the presence of the specific MC4-R antagonist HS014. In summary, our results imply an important role for the beta-MSH/MC4-R cascade in human melanocyte biology, although the function and purpose of this signal in keratinocytes needs further elucidation.
Asunto(s)
Epidermis/metabolismo , Melanocitos/metabolismo , Receptor de Melanocortina Tipo 4/fisiología , Pigmentación de la Piel/genética , beta-MSH/fisiología , Sitios de Unión , Diferenciación Celular/fisiología , Células Cultivadas , AMP Cíclico/metabolismo , Células Epidérmicas , Humanos , Queratinocitos/metabolismo , Melanocitos/fisiología , Péptidos Cíclicos/farmacología , Unión Proteica , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , beta-MSH/metabolismoRESUMEN
BACKGROUND: The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. RESULTS: We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. CONCLUSION: This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.
Asunto(s)
Evolución Molecular , Petromyzon/genética , Receptor de Melanocortina Tipo 1/genética , Receptor de Melanocortina Tipo 4/genética , Hormona Adrenocorticotrópica/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Línea Celular Transformada , Cosintropina/metabolismo , AMP Cíclico/metabolismo , Duplicación de Gen , Biblioteca de Genes , Anguila Babosa/genética , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/metabolismo , Filogenia , Proopiomelanocortina/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Receptores de Melanocortina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sistemas de Mensajero Secundario , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Piel/metabolismo , Especificidad de la Especie , Vísceras/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , beta-MSH/metabolismo , gamma-MSH/metabolismoRESUMEN
The CNS melanocortin pathway plays an important role in the control of body weight. Two papers in this issue of Cell Metabolism, Lee et al., 2006 and Biebermann et al., 2006, suggest that beta MSH--a product of POMC processing--plays an unanticipated role in this pathway in humans.
Asunto(s)
Metabolismo Energético/fisiología , Homeostasis/fisiología , Obesidad/genética , Proopiomelanocortina/metabolismo , Transducción de Señal/fisiología , beta-MSH/metabolismo , Sistema Nervioso Central/metabolismo , Metabolismo Energético/genética , Homeostasis/genética , Humanos , Modelos Biológicos , Mutación/genética , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , beta-MSH/genéticaRESUMEN
The melanocortin-4 receptor (MC4R) plays a critical role in the control of energy balance. Of its two pro-opiomelanocortin (POMC)-derived ligands, alpha- and beta-MSH, the majority of attention has focused on alpha-MSH, partly reflecting the absence of beta-MSH in rodents. We screened the POMC gene in 538 patients with severe, early-onset obesity and identified five unrelated probands who were heterozygous for a rare missense variant in the region encoding beta-MSH, Tyr221Cys. This frequency was significantly increased (p < 0.001) compared to the general UK Caucasian population and the variant cosegregated with obesity/overweight in affected family members. Compared to wild-type beta-MSH, the variant peptide was impaired in its ability to bind to and activate signaling from the MC4R. Obese children carrying the Tyr221Cys variant were hyperphagic and showed increased linear growth, both of which are features of MC4R deficiency. These studies support a role for beta-MSH in the control of human energy homeostasis.
Asunto(s)
Metabolismo Energético/genética , Homeostasis/genética , Obesidad/genética , beta-MSH/genética , Preescolar , Cromatografía Líquida de Alta Presión , Femenino , Tamización de Portadores Genéticos , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Mutación Missense/genética , Receptor de Melanocortina Tipo 4/metabolismo , Análisis de Secuencia de ADN , Reino Unido , Población Blanca , beta-MSH/metabolismoRESUMEN
Human beta-MSH(1-22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human beta-MSH(5-22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human beta-MSH(5-22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, beta-MSH(7-22) (GPYRMEHFRWGSPPKD) and beta-MSH(9-22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of beta-MSH(7-22) (Ki=4.6 nM, EC50=0.6 nM) and beta-MSH(9-22) (Ki=5.7 nM, EC50=0.6 nM) are almost an order of magnitude greater than those of their parent peptide, beta-MSH(5-22) (MC4R, Ki=23 nM, EC50= 3nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, beta-MSH(9-22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, beta-MSH(5-22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous beta-MSH(5-22) to more potent peptides that regulate energy homeostasis in the hypothalamus.
Asunto(s)
Catepsina C/fisiología , Dipeptidil Peptidasa 4/fisiología , Péptidos/agonistas , Receptor de Melanocortina Tipo 3/agonistas , Receptor de Melanocortina Tipo 4/agonistas , beta-MSH/metabolismo , Animales , Catepsina C/química , Línea Celular , Dipeptidil Peptidasa 4/química , Metabolismo Energético/fisiología , Homeostasis/fisiología , Humanos , Hipotálamo/metabolismo , Hipotálamo/fisiología , Masculino , Péptidos/metabolismo , Ratas , Ratas Long-Evans , Receptor de Melanocortina Tipo 3/química , Receptor de Melanocortina Tipo 3/metabolismo , Receptor de Melanocortina Tipo 4/química , Receptor de Melanocortina Tipo 4/metabolismoRESUMEN
The pro-opiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) mediates many diverse physiological actions, including anti-inflammatory and immunomodulatory effects. However, little is known about the physiological roles of the other melanocortins, beta- and gamma-MSH. Here, we investigated the effects of melanocortin peptides in an in vivo neuroinflammation model. Six hours following intracisternal (i.c.) administration of 10 microg lipopolysaccharide (LPS) to mice a five-fold increase in the nitric oxide (NO) level was seen in the animals' brains, when detected by electron paramagnetic resonance (EPR). All tested melanocortins, alpha-, beta-, gamma1- and gamma2-MSH (0.001-10 nmol/mouse i.c.), dose dependently reduced the LPS induced increases in brain NO, with an order of effectiveness: beta-MSH > or = gamma1-MSH=gamma2-MSH>alpha-MSH. Our results suggest specialized functions of beta- and gamma-MSH melanocortins in inflammatory signal modulation in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Óxido Nítrico/metabolismo , beta-MSH/metabolismo , gamma-MSH/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Masculino , Ratones , Ratones Endogámicos ICR , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/farmacología , gamma-MSH/farmacologíaRESUMEN
Ethanol is a caloric compound, and ethanol drinking and food intake are both appetitive and consummatory behaviors. Furthermore, both ethanol and food have rewarding properties. It is therefore possible that overlapping central pathways are involved with uncontrolled eating and excessive ethanol consumption. A growing list of peptides has been shown to regulate food intake and/or energy homeostasis. Peptides such as the melanocortins, corticotropin releasing factor, and cholecystokinin promote reductions of food intake while others such as galanin and neuropeptide Y stimulate feeding. The present review highlights research aimed at determining if ingestive peptides also regulate voluntary ethanol intake, with an emphasis on the melanocortins and neuropeptide Y. It is suggested that research directed at ingestive peptides may expand our understanding of the neurobiological mechanisms that drive ethanol self-administration, and may reveal new therapeutic candidates for treating alcohol abuse and alcoholism.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Alcoholismo/metabolismo , Encéfalo/fisiopatología , Ingestión de Alimentos , Neuropéptido Y/metabolismo , Obesidad/metabolismo , Alcoholismo/fisiopatología , Animales , Colecistoquinina/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Etanol/efectos adversos , Galanina/metabolismo , Humanos , Narcóticos/metabolismo , Obesidad/fisiopatología , Receptores de Melanocortina/metabolismo , alfa-MSH/metabolismo , beta-MSH/metabolismo , gamma-MSH/metabolismoRESUMEN
We report cloning, pharmacological characterization, tissue distribution, detailed brain mapping, and role in control of food intake of melanocortin 4 receptor in goldfish (gMC4R). The gMC4R protein has 68% identity with the human ortholog and is conserved in important functional domains. Pharmacological profiling showed similar affinities and potency order to hMC4R for MSH peptides, whereas MTII and HS024 were identified as high-affinity agonist and antagonist analogs, respectively. The gMC4R-mRNA was found in brain and some peripheral tissues including the ovary, gill, and spleen. Detailed MC4R-mRNA mapping showed expression in main neuroendocrine and food intake-controlling areas. High expression levels were found in the telencephalon, preoptic area, ventral thalamus, tuberal hypothalamus, and hypothalamic inferior lobe. By RT-PCR, low levels were also detected in the cerebellum, medulla, and spinal cord. Intracerebroventricular MTII administration inhibited food intake in 24-h fasted animals in a dose-dependent manner, whereas HS024 stimulated food intake in fed animals, suggesting that melanocortins exert a tonic inhibitory effect on food intake, which is mediated through central MC4R signaling. The conserved central expression pattern and physiological role in regulation of food intake for the MC4R suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates.
Asunto(s)
Química Encefálica/fisiología , Ingestión de Alimentos/fisiología , Carpa Dorada/fisiología , Receptores de Corticotropina/genética , alfa-MSH/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Mapeo Encefálico , Clonación Molecular , Femenino , Expresión Génica/fisiología , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , ARN Mensajero/análisis , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Especificidad de la Especie , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.4+/-0.4 nmol/l versus alpha-MSH, 324+/-16 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.0+/-0.4 nmol/l versus alpha-MSH, 22.5+/-2.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.
Asunto(s)
Metabolismo Energético , Hipotálamo/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , beta-MSH/metabolismo , Animales , Células CHO , Cricetinae , Privación de Alimentos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Homeostasis , Humanos , Insulina/sangre , Ligandos , Masculino , Obesidad/metabolismo , Ratas , Ratas Wistar , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/genéticaRESUMEN
The functional loss of both alleles of the human pro-opiomelanocortin (POMC) gene leads to a very rare syndrome of hypoadrenalism, red hair and early-onset obesity. In order to examine whether more subtle genetic variants in POMC might contribute to early-onset obesity, the coding region of the gene was sequenced in 262 Caucasian subjects with a history of severe obesity from childhood. Two children were found to be heterozygous for a missense mutation, R236G, which disrupts the dibasic cleavage site between beta melanocyte-stimulating hormone (beta-MSH) and beta-endorphin. Beta-TC3 cells transfected with the mutant POMC cDNA produced a mutant beta-MSH/beta-endorphin fusion protein. This fusion protein bound to the human melanocortin-4 receptor (hMC4R) with an affinity similar to its natural ligands, but had a markedly reduced ability to activate the receptor. This variant co-segregated with early-onset obesity over three generations in one family and was absent in 412 normal weight UK Caucasian controls. Combining the results in UK Caucasians with a new case-control study in French subjects and three previously published reports, mutations disrupting this processing site were present in 0.88% of subjects with early-onset obesity and 0.22% of normal-weight controls. These results suggest that the R236G mutation may confer an inherited susceptibility to obesity through the production of an aberrant fusion protein that has the capacity to interfere with central melanocortin signalling.
Asunto(s)
Mutación Missense/genética , Obesidad/genética , Proopiomelanocortina/genética , Adolescente , Insuficiencia Suprarrenal/genética , Animales , Células CHO , Estudios de Casos y Controles , Cricetinae , Análisis Mutacional de ADN , Cartilla de ADN/química , Susceptibilidad a Enfermedades , Femenino , Color del Cabello/genética , Humanos , Masculino , Obesidad/etiología , Linaje , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Pruebas de Precipitina , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , betaendorfina/metabolismo , beta-MSH/metabolismoRESUMEN
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.
Asunto(s)
Encéfalo/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Hormonas Estimuladoras de los Melanocitos/farmacología , Receptores de Corticotropina/efectos de los fármacos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Ingestión de Alimentos/fisiología , Masculino , Hormonas Estimuladoras de los Melanocitos/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/metabolismo , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides beta-endorphin, ACTH, beta-MSH, and alpha-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, beta-endorphin, beta-MSH, and alpha-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.
Asunto(s)
Péptidos/metabolismo , Proopiomelanocortina/metabolismo , Proproteína Convertasa 1 , Piel/metabolismo , Hormona Adrenocorticotrópica/biosíntesis , Hormona Adrenocorticotrópica/genética , Hormona Adrenocorticotrópica/metabolismo , Animales , Ácido Aspártico Endopeptidasas/biosíntesis , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Femenino , Cabello/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Proopiomelanocortina/biosíntesis , Proopiomelanocortina/genética , Proproteína Convertasa 2 , Proproteína Convertasas , ARN Mensajero/metabolismo , Piel/citología , Subtilisinas/biosíntesis , Subtilisinas/genética , Subtilisinas/metabolismo , Factores de Tiempo , Transcripción Genética , alfa-MSH/biosíntesis , alfa-MSH/genética , alfa-MSH/metabolismo , betaendorfina/biosíntesis , betaendorfina/genética , betaendorfina/metabolismo , beta-MSH/biosíntesis , beta-MSH/genética , beta-MSH/metabolismoRESUMEN
Crossbridge dynamics underlying the acute and chronic inotropic effects of doxorubicin (Dox) were studied by application of releasing length steps (amplitude, 0.5-10%) to skinned cardiac trabeculae. Acute incubation of trabeculae with 20 microM Dox for 30 min resulted in a decrease of the velocity of unloaded shortening (V(0), from 9.3 +/- 1.1 to 7.7 +/- 0.7 microm/s, P <.05) and in an increase of the rate of force redevelopment (tau(r), from 56 +/- 4 to 65 +/- 3 ms, P <.05) in response to step amplitudes ranging from 5 to 10%. In contrast, chronic Dox treatment in rats (2 mg/kg/week for 4 weeks) significantly impaired trabecular crossbridge dynamics after step releases of 0.5%. This was reflected by an increase of all time constants describing tension recovery: tau(1), from 10 +/- 1 to 14 +/- 1 ms; tau(2), from 65 +/- 6 to 82 +/- 6 ms; tau(3), from 92 +/- 7 to 293 +/- 67 ms; P <.05. In addition, V(0) was decreased (from 8.6 +/- 0.6 to 6.8 +/- 0.3 microm/s, P <.05) and tau(r) was increased (from 67 +/- 4 to 89 +/- 3 ms; P <.05) in the slack-test. We found that chronic Dox treatment resulted in a shift from the "high ATPase" alpha-myosin heavy chain (MHC) isoform toward the "low-ATPase" beta-MHC isoform in the ventricles (control: alpha-MHC 79 +/- 2% and beta-MHC 21 +/- 2%; Dox-treated: alpha-MHC 53 +/- 2% and beta-MHC 47 +/- 2%; P <.05). The present results show that acute Dox incubation affects the detachment rate of crossbridges, which leads to a delayed relaxation and an arrest of crossbridges in strongly bound states. In contrast, chronic Dox treatment leads to an impairment of both the attachment and detachment rates in the crossbridge cycle, which may be explained by an altered MHC isoform composition in ventricular myocardium. Interfering with Dox-induced alterations of crossbridge kinetics may provide a new strategy to prevent Dox-associated cardiotoxicity.