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1.
J Neuroendocrinol ; 35(11): e13354, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37946684

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the homologous peptide, vasoactive intestinal peptide (VIP), participate in glucose homeostasis using insulinotropic and counterregulatory processes. The role of VIP receptor 2 (VPAC2R) in these opposing actions needs further characterization. In this study, we examined the participation of VPAC2R on basal glycemia, fasted levels of glucoregulatory hormones and on glycemia responses during metabolic and psychogenic stress using gene-deleted (Vipr2-/- ) female mice. The mean basal glycemia was significantly greater in Vipr2-/- in the fed state and after an 8-h overnight fast as compared to wild-type (WT) mice. Insulin tolerance testing following a 5-h fast (morning fast, 0.38 U/kg insulin) indicated no effect of genotype. However, during a more intense metabolic challenge (8 h, ON fast, 0.25 U/kg insulin), Vipr2-/- females displayed significantly impaired insulin hypoglycemia. During immobilization stress, the hyperglycemic response and plasma epinephrine levels were significantly elevated above basal in Vipr2-/- , but not WT mice, in spite of similar stress levels of plasma corticosterone. Together, these results implicate participation of VPAC2R in upregulated counterregulatory processes influenced by enhanced sympathoexcitation. Moreover, the suppression of plasma GLP-1 levels in Vipr2-/- mice may have removed the inhibition on hepatic glucose production and the promotion of glucose disposal by GLP-1. qPCR analysis indicated deregulation of central gene markers of PACAP/VIP signaling in Vipr2-/- , upregulated medulla tyrosine hydroxylase (Th) and downregulated hypothalamic Vip transcripts. These results demonstrate a physiological role for VPAC2R in glucose metabolism, especially during insulin challenge and psychogenic stress, likely involving the participation of sympathoadrenal activity and/or metabolic hormones.


Assuntos
Receptores do Hormônio Hipofisário , Receptores de Peptídeo Intestinal Vasoativo , Camundongos , Feminino , Animais , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Deleção de Genes , Peptídeo Intestinal Vasoativo/metabolismo , Insulina/metabolismo , Glucose , Peptídeo 1 Semelhante ao Glucagon , Receptores do Hormônio Hipofisário/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética
2.
J Mol Neurosci ; 73(9-10): 724-737, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37646964

RESUMO

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.


Assuntos
Receptores do Hormônio Hipofisário , Traumatismos da Medula Espinal , Feminino , Ratos , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ratos Sprague-Dawley , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Traumatismos da Medula Espinal/metabolismo , Encéfalo/metabolismo
3.
Bioorg Med Chem Lett ; 84: 129194, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36813053

RESUMO

Melanin Concentrating Hormone (MCH) receptor is a G protein-coupled receptor (GPCR) with two subtypes R1 and R2. MCH-R1 is involved in the control of energy homeostasis, feeding behavior and body weight. Many studies have proved that administration of MCH-R1 antagonists significantly reduces food intake and causes weight loss in animal models. Herein, we report the optimization of our previously reported virtual screening hits into novel MCH-R1 ligands with chiral aliphatic nitrogen-containing scaffolds. The activity was improved from the micromolar range of the initial leads to 7 nM. We also disclose the first MCH-R1 ligands based on a diazaspiro[4.5]decane nucleus with sub-micromolar activity. A potent MCH-R1 antagonist with acceptable pharmacokinetic profile could represent a new hope for the management of obesity.


Assuntos
Receptores do Hormônio Hipofisário , Animais , Ligantes , Receptores Acoplados a Proteínas G , Obesidade/tratamento farmacológico , Melaninas
4.
Int J Mol Sci ; 23(7)2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35409167

RESUMO

Melanin-concentrating hormone receptor 1 (MCHR1) has been a target for appetite suppressants, which are helpful in treating obesity. However, it is challenging to develop an MCHR1 antagonist because its binding site is similar to that of the human Ether-à-go-go-Related Gene (hERG) channel, whose inhibition may cause cardiotoxicity. Most drugs developed as MCHR1 antagonists have failed in clinical development due to cardiotoxicity caused by hERG inhibition. Machine learning-based prediction models can overcome these difficulties and provide new opportunities for drug discovery. In this study, we identified KRX-104130 with potent MCHR1 antagonistic activity and no cardiotoxicity through virtual screening using two MCHR1 binding affinity prediction models and an hERG-induced cardiotoxicity prediction model. In addition, we explored other possibilities for expanding the new indications for KRX-104130 using a transcriptome-based drug repositioning approach. KRX-104130 increased the expression of low-density lipoprotein receptor (LDLR), which induced cholesterol reduction in the gene expression analysis. This was confirmed by comparison with gene expression in a nonalcoholic steatohepatitis (NASH) patient group. In a NASH mouse model, the administration of KRX-104130 showed a protective effect by reducing hepatic lipid accumulation, liver injury, and histopathological changes, indicating a promising prospect for the therapeutic effect of NASH as a new indication for MCHR1 antagonists.


Assuntos
Reposicionamento de Medicamentos , Hepatopatia Gordurosa não Alcoólica , Animais , Cardiotoxicidade , Humanos , Aprendizado de Máquina , Camundongos , Receptores do Hormônio Hipofisário , Receptores de Somatostatina/metabolismo , Transcriptoma
5.
PLoS Genet ; 18(3): e1010044, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35271580

RESUMO

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal (GI) motility disorder of dogs in which reduced peristaltic activity and dilation of the esophagus prevent the normal transport of food into the stomach. Affected puppies regurgitate meals and water, fail to thrive, and experience complications such as aspiration pneumonia that may necessitate euthanasia. The German shepherd dog (GSD) has the highest disease incidence, indicative of a genetic predisposition. Here, we discover that male GSDs are twice as likely to be affected as females and show that the sex bias is independent of body size. We propose that female endogenous factors (e.g., estrogen) are protective via their role in promoting relaxation of the sphincter between the esophagus and stomach, facilitating food passage. A genome-wide association study for CIM revealed an association on canine chromosome 12 (P-val = 3.12x10-13), with the lead SNPs located upstream or within Melanin-Concentrating Hormone Receptor 2 (MCHR2), a compelling positional candidate gene having a role in appetite, weight, and GI motility. Within the first intron of MCHR2, we identified a 33 bp variable number tandem repeat (VNTR) containing a consensus binding sequence for the T-box family of transcription factors. Across dogs and wolves, the major allele includes two copies of the repeat, whereas the predominant alleles in GSDs have one or three copies. The single-copy allele is strongly associated with CIM (P-val = 1.32x10-17), with homozygosity for this allele posing the most significant risk. Our findings suggest that the number of T-box protein binding motifs may correlate with MCHR2 expression and that an imbalance of melanin-concentrating hormone plays a role in CIM. We describe herein the first genetic factors identified in CIM: sex and a major locus on chromosome 12, which together predict disease state in the GSD with greater than 75% accuracy.


Assuntos
Acalasia Esofágica , Repetições Minissatélites , Animais , Cães , Acalasia Esofágica/veterinária , Feminino , Estudo de Associação Genômica Ampla , Íntrons/genética , Masculino , Receptores do Hormônio Hipofisário
6.
Molecules ; 26(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673598

RESUMO

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.


Assuntos
Fármacos Antiobesidade/química , Obesidade/tratamento farmacológico , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Tecido Adiposo/efeitos dos fármacos , Alcinos/química , Aminobutiratos/química , Animais , Fármacos Antiobesidade/farmacologia , Apetite/efeitos dos fármacos , Ácido Aspártico/química , Modelos Animais de Doenças , Descoberta de Drogas , Ácido Glutâmico/química , Glicina/análogos & derivados , Glicina/química , Células HEK293 , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Insulina/metabolismo , Lactamas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Triazóis/química
7.
Ann N Y Acad Sci ; 1494(1): 70-86, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33502798

RESUMO

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin-concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via ß-adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT-qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [18 F]FDG and the MCHR1-tracer [11 C]SNAP-7941. We found that the ß3-adrenoceptor (ADRB3) agonist CL316,243 increased [11 C]SNAP-7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP-7941-a low-affinity ADRB3 ligand-stimulated [18 F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the ß3-adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity.


Assuntos
Tecido Adiposo Marrom/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Fluordesoxiglucose F18/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-Dawley
8.
Artigo em Inglês | MEDLINE | ID: mdl-32849267

RESUMO

Lactation is a complex physiological process, depending on orchestrated central and peripheral events, including substantial brain plasticity. Among these events is a novel expression of pro-melanin-concentrating hormone (Pmch) mRNA in the rodent hypothalamus, such as the ventral part of the medial preoptic area (vmMPOA). This expression reaches its highest levels around postpartum day 19 (PPD19), when dams transition from lactation to the weaning period. The appearance of this lactation-related Pmch expression occurs simultaneously with the presence of one of the Pmch products, melanin-concentrating hormone (MCH), in the serum. Given the relevance of the MPOA to maternal physiology and the contemporaneity between Pmch expression in this structure and the weaning period, we hypothesized that MCH has a role in the termination of lactation, acting as a mediator between central and peripheral changes. To test this, we investigated the presence of the MCH receptor 1 (MCHR1) and its gene expression in the mammary gland of female rats in different stages of the reproductive cycle. To that end, in situ hybridization, RT-PCR, RT-qPCR, nucleotide sequencing, immunohistochemistry, and Western blotting were employed. Although Mchr1 expression was detected in the epidermis and dermis of both diestrus and lactating rats, parenchymal expression was exclusively found in the functional mammary gland of lactating rats. The expression of Mchr1 mRNA oscillated through the lactation period and reached its maximum in PPD19 dams. Presence of MCHR1 was confirmed with immunohistochemistry with preferential location of MCHR1 immunoreactive cells in the alveolar secretory cells. As was the case for gene expression, the MCHR1 protein levels were significantly higher in PPD19 than in other groups. Our data demonstrate the presence of an anatomical basis for the participation of MCH peptidergic system on the control of lactation through the mammary gland, suggesting that MCH could modulate a prolactation action in early postpartum days and the opposite role at the end of the lactation.


Assuntos
Lactação , Glândulas Mamárias Animais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Animais , Feminino , Imuno-Histoquímica , Masculino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Ratos , Ratos Long-Evans
9.
Gen Comp Endocrinol ; 293: 113474, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32240710

RESUMO

GPCRs are the largest family of receptors accounting for about 30% of the current drug targets. However, it is difficult to fully elucidate the mechanisms regulating intracellular GPCR signal regulation. It is thus important to consider and investigate GPCRs with respect to endogenous situations. Our group has been investigating GPCRs involved in body color (teleost and amphibian) and eating (vertebrate). Here, I review two independent GPCR systems (heterodimer formation and primary ciliated GPCR) that can be breakthroughs in GPCR research. In teleosts, MCRs form heterodimers, which significantly reduce their affinity for acetylated ligands. In mammals, MCHR1 is localized in the ciliary membrane and shortens the length of the primary cilia through a unique signal from the ciliary membrane. Considering these two new GPCR concepts is expected to advance the overall view of the GPCR system.


Assuntos
Cílios/metabolismo , Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Melanocortina/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Membrana Celular/metabolismo , Humanos
10.
Peptides ; 126: 170249, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31911169

RESUMO

Serotonergic neurons of the median raphe nucleus (MnR) and hypothalamic melanin-concentrating hormone (MCH)-containing neurons, have been involved in the control of REM sleep and mood. In the present study, we examined in rats and cats the anatomical relationship between MCH-containing fibers and MnR neurons, as well as the presence of MCHergic receptors in these neurons. In addition, by means of in vivo unit recording in urethane anesthetized rats, we determined the effects of MCH in MnR neuronal firing. Our results showed that MCH-containing fibers were present in the central and paracentral regions of the MnR. MCHergic fibers were in close apposition to serotonergic and non-serotonergic neurons. By means of an indirect approach, we also analyzed the presence of MCHergic receptors within the MnR. Accordingly, we microinjected MCH conjugated with the fluorophore rhodamine (R-MCH) into the lateral ventricle. R-MCH was internalized into serotonergic and non-serotonergic MnR neurons; some of these neurons were GABAergic. Furthermore, we determined that intracerebroventricular administration of MCH induced a significant decrease in the firing rate of 53 % of MnR neurons, while the juxtacellular administration of MCH reduced the frequency of discharge in 67 % of these neurons. Finally, the juxtacellular administration of the MCH-receptor antagonist ATC-0175 produced an increase in the firing rate in 78 % of MnR neurons. Hence, MCH produces a strong regulation of MnR neuronal activity. We hypothesize that MCHergic modulation of the MnR neuronal activity may be involved in the promotion of REM sleep and in the pathophysiology of depressive disorders.


Assuntos
Hormônios Hipotalâmicos/farmacologia , Hipotálamo/efeitos dos fármacos , Melaninas/farmacologia , Fibras Nervosas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Núcleos da Rafe/efeitos dos fármacos , Receptores do Hormônio Hipofisário/metabolismo , Animais , Gatos , Hipotálamo/metabolismo , Hipotálamo/fisiologia , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Núcleos da Rafe/metabolismo , Núcleos da Rafe/fisiologia , Ratos , Ratos Wistar
11.
Bioorg Med Chem Lett ; 29(24): 126741, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678007

RESUMO

MCH receptor is a G protein-coupled receptor with two subtypes R1 and R2. Many studies have demonstrated the role of MCH-R1 in feeding and energy homeostasis. It has been proven that oral administration of small molecule MCH-R1 antagonists significantly reduces food intake and causes a dose-dependent weight loss. In this study, two ligand-based pharmacophores were developed and validated based on recently published MCH-R1 antagonists with diverse structures. Successful pharmacophores had one hydrogen bond acceptor, one positive ionizable, one ring aromatic and two or three hydrophobic groups. These 3D-QSAR models were used for virtual screening of the ZINC chemical database resulting in the identification of nine compounds with more than 50% displacement of radiolabeled MCH at a 20 µM concentration. Moreover, four of these compounds showed antagonistic activities in Aequorin functional assay, including MH-3 which is the first MCH-R1 antagonist based on a diazaspiro[4.5]decane scaffold. The most active compounds were also docked into our previously published MCH-R1 homology model to gain insights into their binding determinants. These compounds could represent a viable starting scaffold for the design of potent MCH-R1 antagonists with improved pharmacokinetic properties as an effective treatment for obesity.


Assuntos
Fármacos Antiobesidade/uso terapêutico , Obesidade/tratamento farmacológico , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Fármacos Antiobesidade/farmacologia , Humanos , Relação Estrutura-Atividade
12.
Nihon Yakurigaku Zasshi ; 154(4): 179-185, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597896

RESUMO

G-protein-coupled receptors (GPCRs), which constitute a highly diverse family of seven transmembrane receptors, respond to external signals and regulate a variety of cellular and physiological processes. GPCRs are encoded by about 800 different genes in human and they represent the largest family of drug targets in clinical trials, which accounts for about 30% of approved drugs acting on 108 unique GPCRs. Signaling through GPCRs can be optimized by enriching receptors, selective binding partners, and downstream effectors in discrete cellular environment. The primary cilium is a ubiquitous organelle that functions as a sensory antenna for surrounding physical and chemical stimuli. Primary cilium's compartment is as little as 1/10,000th of the total cell volume. Therefore, the ciliary membrane is highly enriched for specific signaling molecules, allowing the primary cilium to organize signaling in a highly ordered microenvironment. Recently, a set of non-olfactory GPCRs such as somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1) have been found to be selectively targeted to cilia on several mammalian cell types including neuronal cells both in vitro and in vivo approaches. Moreover, investigations into the pathophysiology have implicated GPCR ciliary signaling in a number of developmental and cellular pathways. Thus, cilia are now considered as an increasingly important connection for GPCR signaling. This review summarizes our current understanding of the signaling pathways though ciliary GPCR, especially feeding- and mood-related GPCR MCHR1, along with specific biological phenomenon as cilia length shortening.


Assuntos
Cílios/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores do Hormônio Hipofisário/fisiologia , Transdução de Sinais , Animais , Humanos
13.
Nat Commun ; 10(1): 2505, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175285

RESUMO

Brain signals that govern memory formation remain incompletely identified. The hypothalamus is implicated in memory disorders, but how its rapidly changing activity shapes memorization is unknown. During encounters with objects, hypothalamic melanin-concentrating hormone (MCH) neurons emit brief signals that reflect object novelty. Here we show that targeted optogenetic silencing of these signals, performed selectively during the initial object encounters (i.e. memory acquisition), prevents future recognition of the objects. We identify an upstream inhibitory microcircuit from hypothalamic GAD65 neurons to MCH neurons, which constrains the memory-promoting MCH cell bursts. Finally, we demonstrate that silencing the GAD65 cells during object memory acquisition improves future object recognition through MCH-receptor-dependent pathways. These results provide causal evidence that object-associated signals in genetically distinct but interconnected hypothalamic neurons differentially control whether the brain forms object memories. This gating of memory formation by hypothalamic activity establishes appropriate behavioral responses to novel and familiar objects.


Assuntos
Glutamato Descarboxilase/metabolismo , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/fisiologia , Melaninas/metabolismo , Memória/fisiologia , Neurônios/metabolismo , Hormônios Hipofisários/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Hipotálamo/citologia , Hipotálamo/metabolismo , Memória/efeitos dos fármacos , Camundongos , Inibição Neural/fisiologia , Vias Neurais , Optogenética , Piperidinas/farmacologia , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Reconhecimento Psicológico/efeitos dos fármacos
14.
Brain Res ; 1719: 71-76, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31121161

RESUMO

Melanin concentrating hormone (MCH) is involved in the initiation of maternal behavior during the postpartum period. Virgin females also display some aspects of maternal care independent of the hormonal and neurochemical changes associated with pregnancy and parturition. Maternal behavior in virgin females is triggered by pups-generated chemosensory signals. We therefore examined the role of MCH in maternal-related behaviors in virgin mice and whether it involves chemosensory mechanisms. We used mice with germline knock-out of MCH receptor (MCHR1 KO) and mice with conditional ablation of MCH neurons (MCH cKO) using Cre-inducible diphtheria toxin (iDTR) system. We report that germline deletion of MCHR1 and ablation of MCH neurons impair spontaneous maternal behavior that is induced upon pups' exposure. The latency and duration to retrieve pups by MCHR1 KO and MCH cKO mice are longer than their control littermate mice. In support of this finding, we found that in the three-chamber social test, both MCHR1 KO and MCH cKO mice display a lack of interest in interacting with pups. Strikingly, however, we found that while MCHR1 KO mice were unable to detect pups' chemosensory signals and displayed impairment in general olfactory discrimination, MCH cKO mice exhibited normal olfactory function. Our findings indicate that the lack of MCHR1 or of normal MCH levels causes defects in maternal behavior in non-sensitized virgin mice, and that disruption of the olfactory signaling might not count for these defects.


Assuntos
Comportamento Materno/fisiologia , Receptores de Somatostatina/genética , Olfato/genética , Animais , Comportamento Animal/fisiologia , Feminino , Mutação em Linhagem Germinativa , Hormônios Hipotalâmicos/genética , Masculino , Melaninas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Hormônios Hipofisários/genética , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Somatostatina/metabolismo , Transdução de Sinais/fisiologia , Olfato/fisiologia
15.
Gen Comp Endocrinol ; 281: 91-104, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31121165

RESUMO

Two structurally related peptides, arginine vasotocin (AVT) and mesotocin (MT), are reported to regulate many physiological processes, such as anti-diuresis and oviposition in birds, and their actions are likely mediated by four AVT/MT receptors (AVPR1A, AVPR1B, MTR and AVPR2b), which are orthologous/paralogous to human AVPR1A, AVPR1B, OXTR and AVPR2 respectively. However, our knowledge regarding the functions of these avian AVT/MT receptors has been limited. Here, we examined the functionality and expression of these receptors in chickens and investigated the roles of AVT in the anterior pituitary. Our results showed that 1) AVPR1A, AVPR1B and AVPR2b could be preferentially activated by AVT, monitored by cell-based luciferase reporter assays and/or Western blot, indicating that they are AVT-specific receptors (AVPR1A; AVPR1B) or AVT-preferring receptor (AVPR2b) functionally coupled to intracellular calcium, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, MTR could be activated by AVT and MT with similar potencies, indicating that MTR is a receptor common for both peptides; 2) Using qPCR, differential expression of the four receptors was found in chicken tissues including the oviduct and anterior pituitary. In particular, only AVPR1A is abundantly expressed in the uterus, suggesting its involvement in mediating AVT-induced oviposition. 3) In cultured chick pituitary cells, AVT could stimulate ACTH and PRL expression and secretion, an action likely mediated by AVPR1B and/or AVPR1A abundantly expressed in anterior pituitary. Collectively, our data helps to elucidate the roles of AVT/MT in birds, such as the 'oxytocic action' of AVT, which induces uterine muscle contraction during oviposition.


Assuntos
Oviposição/fisiologia , Hipófise/metabolismo , Prolactina/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Vasotocina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Galinhas/metabolismo , Patos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Pró-Opiomelanocortina/farmacologia , Prolactina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Vasotocina/química
16.
Neuropharmacology ; 139: 238-256, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29981758

RESUMO

The rat nucleus incertus (NI) contains GABA/peptide-projection neurons responsive to orexin (hypocretin)/orexin receptor-2 (OX2) signalling. Melanin-concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor-1 (MCH1) expression, and neurophysiological and behavioural effects of MCH c.f. orexin-A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub-zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH-containing fibres in NI. Single-cell RT-PCR detected MCH1 and OX2 mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co-expression patterns of MCH1 and OX2 mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch-clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1 µM), representing a distinct population from OXA-sensitive NI neurons (35%). Intra-NI OXA infusion (600 pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra-NI MCH infusion (600 pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre-fasted rats tested during the dark/active phase, intra-NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide-immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours.


Assuntos
Neurônios/citologia , Neurônios/metabolismo , Receptores de Orexina/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Nível de Alerta/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Ingestão de Alimentos/efeitos dos fármacos , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/metabolismo , Hormônios Hipotalâmicos/metabolismo , Masculino , Melaninas/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Orexinas/metabolismo , Hormônios Hipofisários/metabolismo , RNA Mensageiro/metabolismo , Núcleos da Rafe/efeitos dos fármacos , Ratos Sprague-Dawley , Ratos Wistar , Técnicas de Cultura de Tecidos , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Ácido gama-Aminobutírico/metabolismo
17.
Obes Res Clin Pract ; 12(2): 158-166, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29066024

RESUMO

BACKGROUND: Prader Willi Syndrome (PWS) is a syndromic form of obesity caused by a chromosomal aberration on chromosome 15q11.2-q13. Patients with a comparable phenotype to PWS not carrying the 15q11.2-q13 defect are classified as Prader Willi like (PWL). In literature, PWL patients do frequently harbor deletions at 6q16, which led to the identification of the single-minded 1 (SIM1) gene as a possible cause for the presence of obesity in these patients. However, our previous work in a PWL cohort showed a rather limited involvement of SIM1 in the obesity phenotype. In this paper, we investigated the causal role of the melanin-concentrating hormone receptor 2 (MCHR2) gene in PWL patients, as most of the reported 6q16 deletions also encompass this gene and it is suggested to be active in the control of feeding behavior and energy metabolism. METHODS: Copy number variation analysis of the MCHR2 genomic region followed by mutation analysis of MCHR2 was performed in a PWL cohort. RESULTS: Genome-wide microarray analysis of 109 patients with PWL did not show any gene harboring deletions on chromosome 6q16. Mutation analysis in 92 patients with PWL demonstrated three MCHR2 variants: p.T47A (c.139A>G), p.A76A (c.228T>C) and c.*16A>G. We identified a significantly higher prevalence of the c.228T>C C allele in our PWL cohort compared to previously published results and controls of the ExAC Database. CONCLUSION: Overall, our results are in line with some previously performed studies suggesting that MCHR2 is not a major contributor to human obesity and the PWL phenotype.


Assuntos
Variações do Número de Cópias de DNA/genética , Obesidade/genética , Síndrome de Prader-Willi/genética , Receptores Acoplados a Proteínas G/genética , Receptores do Hormônio Hipofisário/genética , Análise de Sequência de DNA , Adolescente , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Criança , Análise Mutacional de DNA , Metabolismo Energético/genética , Comportamento Alimentar/fisiologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Análise em Microsséries , Obesidade/etiologia , Fenótipo , Síndrome de Prader-Willi/complicações
18.
Reprod Sci ; 25(8): 1218-1223, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29141508

RESUMO

OBJECTIVE: To test whether recombinant anti-Müllerian hormone (AMH) can inhibit ovarian cortex function by modulating the expression of other hormone receptors. MATERIALS AND METHODS: Pilot experimental study with ovarian cortex obtained from 5 patients. Immediately after explant, the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (uncultured) and 4 were incubated for 48 hours at 37°C in a pH-adjusted gamete buffer medium with increasing AMH concentrations of 0, 5, 25, and 50 ng/mL. After incubation, all specimens were rinsed and flash-frozen for polymerase chain reaction (PCR) executed in triplicates. We utilized real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine messenger RNA (mRNA) levels of AMH and its receptor Anti-Müllerian Hormone-Receptor 2 (AMH-R2), follicle stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R), inhibin B, and insulin-like growth factor 1 receptor 1 (IGF1-R1) in ovarian cortex tissue. In addition, we performed Ki-67 immunostaining to evaluate cell proliferation in the treatment groups. RESULTS: Absence of recombinant human AMH (rAMH) caused upregulation of all markers. Exposure to increasing rAMH concentrations caused tissue AMH expression downregulation ( P = .024), while AMH-R2 ( P = .005), FSH-R ( P = .009), LH-R ( P = .003), and inhibin B ( P = .001) mRNA expression followed a bell-shaped response with an increased expression at low dose, followed by a decreased expression at higher doses. Expression of IGF1-R1 was independent ( P = .039) of rAMH exposure. The Ki-67 immunostaining showed an increased cell proliferation in the media control compared to the uncultured and the tissue cultured with rAMH. CONCLUSIONS: Culture with increasing rAMH concentrations caused downregulation of its own, as well as other hormone receptors, and a decreased ovarian cortex cell proliferation. These results help understanding the inhibitory effects of AMH on follicular development.


Assuntos
Hormônio Antimülleriano/metabolismo , Ovário/metabolismo , Receptores de Peptídeos/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Adulto , Hormônio Antimülleriano/administração & dosagem , Feminino , Regulação da Expressão Gênica , Humanos , Inibinas/metabolismo , Ovário/efeitos dos fármacos , Projetos Piloto , Pré-Menopausa , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores do FSH/metabolismo , Receptores do LH/metabolismo , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes
19.
Gen Comp Endocrinol ; 264: 138-150, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28647318

RESUMO

To evaluate the association of the melanotropic peptides and their receptors for morphological color change, we investigated the effects of changes in background color, between white and black, on xanthophore density in the scales and expression levels of genes for hormonal peptides and corresponding receptors (MCH-R2, MC1R, and MC5R) in goldfish (Carassius auratus). The xanthophore density in both dorsal and ventral scales increased after transfer from a white to black background. However, xanthophore density in dorsal scales increased after transfer from a black to white background, and that of ventral scales decreased after transfer from a black to black background, which served as the control. In the white-reared fish, melanin-concentrating hormone (mch) mRNA content in the brain was higher than that in black-reared fish, whereas proopiomelanocortin a (pomc-a) mRNA content in the pituitary was lower than that in the black-reared fish. Agouti-signaling protein (asp) mRNA was detected in the ventral skin but not in the dorsal skin. No difference was observed in the asp mRNA content between fish reared in white or black background, suggesting that ASP might not be associated with background color adaptation. In situ hybridization revealed that both mc1r and mc5r were expressed in the xanthophores in scales. The mRNA content of mc1r in scales did not always follow the background color change, whereas those of mc5r decreased in the white background and increased in the black background, suggesting that mc5r might be a major factor reinforcing the function of MSH in morphological color changes. White backgrounds increased mch mRNA content in the brain, but decreased mch-r2 mRNA content in the scales. These altered expression levels of melanotropin receptors might affect reactivity to melanotropins through long-term adaptation to background color.


Assuntos
Regulação da Expressão Gênica , Carpa Dourada/genética , Hormônios Estimuladores de Melanócitos/genética , Pigmentação/genética , Receptores do Hormônio Hipofisário/genética , Escamas de Animais/metabolismo , Animais , Encéfalo/metabolismo , Cor , Carpa Dourada/metabolismo , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/metabolismo , Melaninas/genética , Melaninas/metabolismo , Hormônios Estimuladores de Melanócitos/metabolismo , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Pele/metabolismo
20.
Gen Comp Endocrinol ; 253: 44-52, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28842217

RESUMO

Primary cilia are specialized microtubule-based organelles. Their importance is highlighted by the gamut of ciliary diseases associated with various syndromes including diabetes and obesity. Primary cilia serve as signaling hubs through selective interactions with ion channels and conventional G-protein-coupled receptors (GPCRs). Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a key regulator of feeding, is selectively expressed in neuronal primary cilia in distinct regions of the mouse brain. We previously found that MCH acts on ciliary MCHR1 and induces cilia shortening through a Gi/o-dependent Akt pathway with no cell cycle progression. Many factors can participate in cilia length control. However, the mechanisms for how these molecules are relocated and coordinated to activate cilia shortening are poorly understood. In the present study, we investigated the role of cytoskeletal dynamics in regulating MCH-induced cilia shortening using clonal MCHR1-expressing hTERT-RPE1 cells. Pharmacological and biochemical approaches showed that cilia shortening mediated by MCH was associated with increased soluble cytosolic tubulin without changing the total tubulin amount. Enhanced F-actin fiber intensity was also observed in MCH-treated cells. The actions of various pharmacological agents revealed that coordinated actin machinery, especially actin polymerization, was required for MCHR1-mediated cilia shortening. A recent report indicated the existence of actin-regulated machinery for cilia shortening through GPCR agonist-dependent ectosome release. However, our live-cell imaging experiments showed that MCH progressively elicited cilia shortening without exclusion of fluorescence-positive material from the tip. Short cilia phenotypes have been associated with various metabolic disorders. Thus, the present findings may contribute toward better understanding of how the cytoskeleton is involved in the GPCR ligand-triggered cilia shortening with cell mechanical properties that underlies clinical manifestations such as obesity.


Assuntos
Cílios/metabolismo , Citoesqueleto/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Corpo Celular/metabolismo , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Cílios/efeitos dos fármacos , Citosol/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hormônios Hipotalâmicos/farmacologia , Ligantes , Melaninas/farmacologia , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Hormônios Hipofisários/farmacologia , Polimerização , Solubilidade , Tubulina (Proteína)/metabolismo
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