RESUMEN
The g-protein coupled receptor GPR-160, recently identified as a putative receptor for the cocaine and amphetamine-regulated transcript (CART) peptide, shows abundant expression in the energy-balance control nuclei, including the dorsal vagal complex (DVC). However, its physiological role in the control of food intake has yet to be fully explored. Here, we performed a virally mediated, targeted knockdown (KD) of Gpr160 in the DVC of male rats to evaluate its physiological role in control of feeding. Our results indicate that DVC Gpr160 KD affects meal microstructure. Specifically, DVC Gpr160 KD animals consumed more frequent, but shorter meals during the dark phase and showed decreased caloric intake and duration of meals during the light phase. Cumulatively, however, these bidirectional effects on feeding resulted in no difference in body weight gain. We next tested the role of DVC GPR-160 in mediating the anorexigenic effects of exogenous CART. Our results show that DVC Gpr160 KD partially attenuates CART's anorexigenic effects. To further characterize Gpr160+ cells in the DVC, we utilized single-nucleus RNA sequencing data to uncover abundant GPR-160 expression in DVC microglia and only minimal expression in neurons. Altogether, our results suggest that DVC CART signaling may be mediated by Gpr160+ microglia, which in turn may be modulating DVC neuronal activity to control food intake.
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Núcleo Solitario , Nervio Vago , Ratas , Masculino , Animales , Ratas Sprague-Dawley , Nervio Vago/metabolismo , NeuronasRESUMEN
Connecting peptide, or C-peptide, is a part of the insulin prohormone and is essential for the proper folding and processing of the mature insulin peptide. C-peptide is released from the same beta cell secretory granules as insulin in equimolar amounts. However, due to their relative stabilities in plasma, the two peptides are detected in the circulation at ratios of approximately 4:1 to 6:1 (C-peptide to insulin), depending on metabolic state. C-peptide binds specifically to human cell membranes and induces intracellular signaling cascades, likely through an interaction with the G protein coupled receptor, GPR146. C-peptide has been shown to exert protective effects against the vascular, renal, and ocular complications of diabetes. The effects of C-peptide appear to be dependent upon the presence of insulin and the absolute, extracellular concentration of glucose. In this study, we employed HEK293 cells to further examine the interactive effects of C-peptide, insulin, and glucose on cell signaling. We observed that C-peptide's cellular effects are dampened significantly when cells are exposed to physiologically relevant concentrations of both insulin and C-peptide. Likewise, the actions of C-peptide on cFos and GPR146 mRNA expressions were affected by changes in extracellular glucose concentration. In particular, C-peptide induced significant elevations in cFos expression in the setting of high (25 mmol) extracellular glucose concentration. These data indicate that future experimentation on the actions of C-peptide should control for the presence or absence of insulin and the concentration of glucose. Furthermore, these findings should be considered prior to the development of C-peptide-based therapeutics for the treatment of diabetes-associated complications.
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Complicaciones de la Diabetes , Insulina , Glucemia , Péptido C , Glucosa/farmacología , Células HEK293 , Humanos , Insulina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) transmit the signals of a variety of hormones and neurotransmitters and are targets of more than 30% of all FDA-approved drugs. We developed an approach for identifying the endogenous ligands for a family of orphan GPCRs that enables the development of novel therapeutics for the potential treatment of a wide variety of disorders including pain, diabetes, appetitive behaviors, infertility and obesity. With this approach, we have deorphanized five previously orphaned GPCRs.
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Obesidad , Humanos , LigandosRESUMEN
In 2008, the first evidence of a new hormone called neuronostatin was published. The hormone was discovered using a bioinformatic method and found to originate from the same preprohormone as somatostatin. This small peptide hormone of 13 amino acids and a C-terminal amidation was soon found to exert pleiotropic physiological effects. In animal studies, neuronostatin has been shown to reduce food intake and delay gastric emptying and gastrointestinal transit. Furthermore, neuronostatin has been shown to affect glucose metabolism by increasing glucagon secretion during situations when glucose concentrations are low. Additionally, neuronostatin has been shown to affect neural tissue and cardiomyocytes by suppressing cardiac contractility. The effects of neuronostatin have not yet been delineated in humans, but if the effects found in animal studies translate to humans it could position neuronostatin as a promising target in the treatment of obesity, hypertension and diabetes. In this review, we describe the discovery of neuronostatin and the current understanding of its physiological role and potential therapeutic applicability.
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Hormonas Peptídicas/fisiología , Animales , Regulación del Apetito/efectos de los fármacos , Regulación del Apetito/genética , Diabetes Mellitus/genética , Diabetes Mellitus/terapia , Vaciamiento Gástrico/efectos de los fármacos , Vaciamiento Gástrico/genética , Humanos , Hipertensión/genética , Hipertensión/terapia , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Obesidad/genética , Obesidad/terapia , Hormonas Peptídicas/farmacología , Transducción de Señal/efectos de los fármacos , Somatostatina/química , Somatostatina/farmacología , Somatostatina/fisiologíaRESUMEN
Cocaine- and amphetamine-regulated transcript encodes an eponymous peptide, CARTp, which exerts diverse pharmacologic actions in the central and peripheral nervous systems, as well as in several endocrine organs, including pancreas. Here we review those diverse actions, the physiological relevance of which had remained unestablished until recently. With the identification of a CARTp receptor, GPR160, the physiologic importance and therapeutic potential of CARTp or analogs are being revealed. Not only is the CARTp-GPR160 interaction essential for the circadian regulation of appetite and thirst but also for the transmission of nerve injury-induced pain. Molecular approaches now are uncovering additional physiologically relevant actions and the development of acute tissue-specific gene compromise approaches may reveal even more physiologically relevant actions of this pluripotent ligand/receptor pair.
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Proteínas del Tejido Nervioso/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Investigación Biomédica TraslacionalRESUMEN
The existence of the peptide encoded by the cocaine- and amphetamine-regulated transcript (Cartpt) has been recognized since 1981, but it was not until 1995, that the gene encoding CART peptide (CART) was identified. With the availability of the predicted protein sequence of CART investigators were able to identify sites of peptide localization, which then led to numerous approaches attempting to clarify CART's multiple pharmacologic effects and even provide evidence of potential physiologic relevance. Although not without controversy, a picture emerged of the importance of CART in ingestive behaviors, reward behaviors and even pain sensation. Despite the wealth of data hinting at the significance of CART, in the absence of an identified receptor, the full potential for this peptide or its analogs to be developed into therapeutic agents remained unrealized. There was evidence favoring the action of CART via a G protein-coupled receptor (GPCR), but despite multiple attempts the identity of that receptor eluded investigators until recently. Now with the identification of the previously orphaned GPCR, GPR160, as a receptor for CART, focus on this pluripotent neuropeptide will in all likelihood experience a renaissance and the potential for the development of pharmcotherapies targeting GPR160 seems within reach.
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Cocaína , Neuropéptidos , Conducta Alimentaria , Proteínas del Tejido Nervioso/genética , RecompensaRESUMEN
Recent work identified Gpr160 as a candidate receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp) and described its role in pain modulation. The aims of the present study were to determine if Gpr160 is required for the CARTp's ability to reduce food intake and water intake and to initially identify the distribution of Gpr160-like immunoreactivity (Gpr160ir) in the rat brain. A passive immunoneutralization approach targeting Gpr160 was used to block the behavioral effects of a pharmacological dose of CARTp in the fourth cerebroventricle (4V) of rats and to determine the importance of endogenously produced CARTp in the control of ingestive behaviors. Passive immunoneutralization of Gpr160 in the 4V blocked the actions of CARTp to inhibit food intake and water intake. Blockade of Gpr160 in the 4V, independent of pharmacological CART treatment, caused an increase in both overnight food intake and water intake. The decrease in food intake, but not water intake, caused by central injection of CARTp was demonstrated to be interrupted by prior administration of a glucagon-like peptide 1 (GLP-1) receptor antagonist. Gpr160ir was observed in several, distinct sites throughout the rat brain, where CARTp staining has been described. Importantly, Gpr160ir was observed to be present in both neuronal and nonneuronal cell types. These data support the hypothesis that Gpr160 is required for the anorexigenic actions of central CARTp injection and extend these findings to water drinking. Gpr160ir was observed in both neuronal and nonneuronal cell types in regions known to be important in the multiple pharmacological effects of CARTp, identifying those areas as targets for future compromise of function studies.
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Depresores del Apetito/farmacología , Tronco Encefálico/efectos de los fármacos , Conducta de Ingestión de Líquido/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Proteínas del Tejido Nervioso/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Tronco Encefálico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
There are examples of physiological conditions under which thirst is inappropriately exaggerated, and the mechanisms for these paradoxical ingestive behaviors remain unknown. We are interested in thirst mechanisms across the female life cycle and have identified a novel mechanism through which ingestive behavior may be activated. We discovered a previously unrecognized endogenous hypothalamic peptide, phoenixin (PNX), identified physiologically relevant actions of the peptide in brain and pituitary gland to control reproductive hormone secretion in female rodents, and in the process identified the previously orphaned G protein-coupled receptor Gpr173 to be a potential receptor for the peptide. Labeled PNX binding distribution in brain parallels areas known to be important in ingestive behaviors as well in areas where gonadal steroids feedback to control estrous cyclicity (Stein LM, Tullock CW, Mathews SK, Garcia-Galiano D, Elias CF, Samson WK, Yosten GLC, Am J Physiol Regul Integr Comp Physiol 311: R489-R496, 2016). We have demonstrated upregulation of Gpr173 during puberty, fluctuations across the estrous cycle, and, importantly, upregulation during the last third of gestation. It is during this hypervolemic, hyponatremic state that both vasopressin secretion and thirst are inappropriately elevated in humans. Here, we show that central administration of PNX stimulated water drinking in both males and females under ad libitum conditions, increased water drinking after overnight fluid deprivation, and increased both water and 1.5% NaCl ingestion under fed and hydrated conditions. Importantly, losartan pretreatment blocked the effect of PNX on water drinking, and knockdown of Gpr173 by use of short interfering RNA constructs significantly attenuated water drinking in response to overnight fluid deprivation. These actions, together with the stimulatory action of PNX on vasopressin secretion, suggest that this recently discovered neuropeptide may impact the recruitment of critically important neural circuits through which ingestive behaviors and endocrine mechanisms that maintain fluid and electrolyte homeostasis are regulated.
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Conducta de Ingestión de Líquido/fisiología , Hipotálamo/metabolismo , Hormonas Peptídicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sed/fisiología , Animales , Ciclo Estral/metabolismo , Femenino , Homeostasis/fisiología , Masculino , Hormonas Peptídicas/genética , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Treating neuropathic pain is challenging and novel non-opioid-based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence, and in situ hybridization, we found that the expression of the orphan GPCR Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element-binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights into its signaling pathways. CARTp is involved in many diseases including depression and reward and addiction; de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease.
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Neuralgia/etiología , Neuralgia/fisiopatología , Receptores Acoplados a Proteínas G/fisiología , Animales , Línea Celular , Femenino , Humanos , Ligandos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/genética , Células PC12 , ARN Interferente Pequeño/genética , Ratas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Nervio Ciático/lesiones , Nervio Ciático/fisiopatología , Transducción de Señal , Médula Espinal/metabolismo , Regulación hacia ArribaRESUMEN
Chemotherapy-induced neuropathic pain (CINP) in both sexes compromises many current chemotherapeutics and lacks an FDA-approved therapy. We recently identified the sphingosine-1-phosphate receptor subtype 1 (S1PR1) and A3 adenosine receptor subtype (A3AR) as novel targets for therapeutic intervention. Our work in male rodents using paclitaxel, oxaliplatin, and bortezomib showed robust inhibition of CINP with either S1PR1 antagonists or A3AR agonists. The S1PR1 functional antagonist FTY720 (Gilenya) is FDA-approved for treating multiple sclerosis, and selective A3AR agonists are in advanced clinical trials for cancer and inflammatory disorders, underscoring the need for their expedited trials in patients with CINP as chemotherapy adjuncts. Our findings reveal that S1PR1 antagonists and A3AR agonists mitigate paclitaxel and oxaliplatin CINP in female and male rodents, but failed to block or reverse bortezomib-induced neuropathic pain (BINP) in females. Although numerous mechanisms likely underlie these differences, we focused on receptor levels. We found that BINP in male rats, but not in female rats, was associated with increased expression of A3AR in the spinal cord dorsal horn, whereas S1PR1 levels were similar in both sexes. Thus, alternative mechanisms beyond receptor expression may account for sex differences in response to S1PR1 antagonists. Morphine and duloxetine, both clinical analgesics, reversed BINP in female mice, demonstrating that the lack of response is specific to S1PR1 and A3AR agents. Our findings suggest that A3AR- and S1PR1-based therapies are not viable approaches in preventing and treating BINP in females and should inform future clinical trials of these drugs as adjuncts to chemotherapy.
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Antineoplásicos/efectos adversos , Bortezomib/efectos adversos , Neuralgia/tratamiento farmacológico , Receptor de Adenosina A3/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Antagonistas del Receptor de Adenosina A3/administración & dosificación , Antagonistas del Receptor de Adenosina A3/uso terapéutico , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/uso terapéutico , Animales , Clorhidrato de Duloxetina/administración & dosificación , Clorhidrato de Duloxetina/uso terapéutico , Femenino , Clorhidrato de Fingolimod/administración & dosificación , Clorhidrato de Fingolimod/uso terapéutico , Masculino , Morfina/administración & dosificación , Morfina/uso terapéutico , Neuralgia/inducido químicamente , Oxaliplatino/efectos adversos , Paclitaxel/efectos adversos , Ratas , Factores Sexuales , Moduladores de los Receptores de fosfatos y esfingosina 1/administración & dosificación , Moduladores de los Receptores de fosfatos y esfingosina 1/uso terapéutico , Asta Dorsal de la Médula Espinal/efectos de los fármacosRESUMEN
Nesfatin-1 is a peptide derived from the nucleobindin 2 (Nucb2) precursor protein that has been shown to exert potent effects on appetite and cardiovascular function in male animals. Sex hormones modulate the expression of Nucb2 in several species, including goldfish, mouse, and rat, and human studies have revealed differential expression based on male or female sex. We therefore hypothesized that the ability of nesfatin-1 to increase mean arterial pressure (MAP) would be influenced by stage of the estrous cycle. Indeed, we found that in cycling female Sprague-Dawley rats, nesfatin-1 induced an increase in MAP on diestrus, when both estrogen and progesterone levels are low but not on proestrus or estrus. The effect of nesfatin-1 on MAP was dependent on functional central melanocortin receptors, because the nesfatin-1-induced increase in MAP was abolished by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119. We previously reported that nesfatin-1 inhibited angiotensin II-induced water drinking in male rats but found no effect of nesfatin-1 in females in diestrus. However, nesfatin-1 enhanced angiotensin II-induced elevations in MAP in females in diestrus but had no effect on males. Finally, in agreement with previous reports, the expression of Nucb2 mRNA in hypothalamus was significantly reduced in female rats in proestrus compared with rats in diestrus. From these data we conclude that the function and expression of nesfatin-1 are modulated by sex hormone status. Further studies are required to determine the contributions of chromosomal sex and individual sex hormones to the cardiovascular effects of nesfatin-1.
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Ciclo Estral/metabolismo , Hormonas/metabolismo , Nucleobindinas/metabolismo , Animales , Proteínas de Unión al ADN/genética , Femenino , Hipotálamo/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Hormonas Peptídicas/metabolismo , Ratas Sprague-Dawley , Receptores de Melanocortina/metabolismoRESUMEN
Using a series of classical protein purification techniques, coupled with more modern molecular approaches, a family of neuropeptides, the Phoenixins, was identified to be produced in brain and heart, and to bind selectively in pituitary gland, ovary and brain. These same binding sites were revealed, using a novel receptor identification strategy, to express the orphan G protein-coupled receptor, GPR173, the expression of which was required for the actions of phoenixin both in vivo and in vitro. In fact, studies using small interfering RNA molecules to compromise GPR173 expression revealed the physiologic relevance of the initially reported pharmacologic actions of the peptides. Those include not only the reproductive actions of the peptides in brain and pituitary gland, but also a CNS site of action in the maintenance of fluid and electrolyte homeostasis. Additional pharmacologic actions of the phoenixins have been described and the race is on to establish the physiologic relevance of those actions as well as the therapeutic potential of phoenixin analogs.