RESUMO
Ghrelin and its mimetics have been shown to reduce cisplatin-induced emesis in preclinical studies using ferrets and shrews. This study investigated the effectiveness of ghrelin and des-acyl ghrelin (DAG) in antagonizing cisplatin-induced emesis and physiological changes indicative of nausea in Suncus murinus. Animals implanted with radiotelemetry devices were administered ghrelin (0.2, 1.0, and 5.0 µg/day), DAG (0.2, 1.0, and 5.0 µg/day), or saline (14 µL/day) intracerebroventricularly 4 days before and 3 days after treatment with cisplatin (30 mg/kg). At the end, the anti-apoptotic potentials of ghrelin and DAG were assessed by measuring Bax expression and cytochrome C activity. Neurotransmitter changes in the brain were evaluated using liquid chromatography-mass spectrometry analysis. Ghrelin and DAG reduced cisplatin-induced emesis in the delayed (24-72 h) but not the acute phase (0-24 h) of emesis. Ghrelin also partially reversed the inhibitory effects of cisplatin on food intake without affecting gastrointestinal myoelectrical activity or causing hypothermia; however, ghrelin or DAG did not prevent these effects. Ghrelin and DAG could attenuate the cisplatin-induced upregulation of Bax and cytochrome C in the ileum. Cisplatin dysregulated neurotransmitter levels in the frontal cortex, amygdala, thalamus, hypothalamus, and brainstem, and this was partially restored by low doses of ghrelin and DAG. Our findings suggest that ghrelin and DAG exhibit protective effects against cisplatin-induced delayed emesis. The underlying antiemetic mechanism may involve GHSR and/or unspecified pathways that modulate the neurotransmitters involved in emesis control in the brain and an action to attenuate apoptosis in the gastrointestinal tract.
Assuntos
Antieméticos , Antineoplásicos , Animais , Cisplatino/toxicidade , Grelina/farmacologia , Grelina/uso terapêutico , Vômito/induzido quimicamente , Vômito/tratamento farmacológico , Vômito/prevenção & controle , Citocromos c , Proteína X Associada a bcl-2 , Furões , Náusea/induzido quimicamente , Náusea/tratamento farmacológico , Náusea/prevenção & controle , Antieméticos/farmacologia , Antieméticos/uso terapêutico , Antineoplásicos/toxicidade , Neurotransmissores/efeitos adversosRESUMO
BACKGROUND: Colonic motility is regulated by various factors along the gut-brain axis; however, detailed mechanisms are unknown. This study aimed to examine the involvement of the autonomic nervous system in colonic motility. Suncus murinus (suncus) is a small laboratory mammal suitable for gastrointestinal motility studies. METHODS: Colonic motility and concomitant feeding and defecation behaviors in vagotomized and reserpine-administered suncus were recorded simultaneously for 24 h. Furthermore, we performed immunohistochemistry on tyrosine hydroxylase (TH) and in situ hybridization on corticotropin-releasing hormone (CRH) in suncus brain. Additionally, we examined c-Fos expression in the brain using immunohistochemistry in conscious suncus with colorectal distension. KEY RESULTS: In vagotomized suncus, clustered giant migrating contractions (GMCs), consisting of strong contractions occurring in a short time, were observed, and the percentage of GMCs without defecation increased. The frequency of GMCs in the reserpine-administered suncus increased during a light period (ZT0-4, 4-8) and decreased during a dark period (ZT16-20, 20-24) compared to a vehicle group. Additionally, the percentage of GMCs without defecation in the reserpine-administered suncus increased. Suncus TH-immunopositive neurons were found in the locus coeruleus (LC), as shown in rodents. In contrast, CRH mRNA-expressing cells were not observed in a region assumed to be the Barrington's nucleus (Bar). Furthermore, colorectal distension in conscious suncus induced c-Fos expression in LC TH neurons. CONCLUSIONS & INFERENCES: Our results suggest that the vagus and sympathetic nerves are not required for induction of GMCs in vivo. However, they are likely to exert a modulatory role in control of GMC frequency in Suncus murinus.
Assuntos
Neoplasias Colorretais , Reserpina , Animais , Nervo Vago/fisiologia , Motilidade Gastrointestinal/fisiologia , Estado de Consciência , Hormônio Liberador da Corticotropina , MamíferosRESUMO
Ghrelin is a multifunctional gut peptide with a unique structure, which is modified by a medium chain fatty acid at the third serine by ghrelin O-acyl transferase (GOAT). It is well known that the major source of plasma ghrelin is the stomach, but the transcriptional regulation of gastric ghrelin and GOAT is incompletely understood. Here, we studied the involvement of the nuclear receptors REV-ERBα and REV-ERBß on ghrelin and GOAT gene expression in vivo and in vitro. Reverse-transcriptase polymerase chain reaction analysis showed that REV-ERBα and REV-ERBß mRNAs were expressed in the stomach and a stomach-derived ghrelin cell line (SG-1 cells). In vivo experiments with mice revealed the circadian rhythm of ghrelin, GOAT, and REV-ERBs. The peak expression of ghrelin and GOAT mRNAs occurred at Zeitgeber time (ZT) 4, whereas that of REV-ERBα and REV-ERBß was observed at ZT8 and ZT12, respectively. Treatment of SG-1 cells with SR9009, a REV-ERB agonist, led to a significant reduction in ghrelin and GOAT mRNA levels. Overexpression of REV-ERBα and REV-ERBß decreased ghrelin and GOAT mRNA levels in SG-1 cells. In contrast, small-interfering RNA (siRNA)-mediated double-knockdown of REV-ERBα and REV-ERBß in SG-1 cells led to the upregulation in the expression of ghrelin and GOAT mRNAs. These results suggest that REV-ERBs suppress ghrelin and GOAT mRNA expression.
Assuntos
Aciltransferases/biossíntese , Grelina/metabolismo , Grelina/farmacologia , Proteínas de Membrana/biossíntese , Receptor ErbB-2/genética , Estômago/metabolismo , Aciltransferases/genética , Animais , Linhagem Celular , Ritmo Circadiano , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Pirrolidinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Estômago/efeitos dos fármacos , Tiofenos/farmacologiaRESUMO
Neuromedin U (NMU) shows circadian expression in the rat pars tuberalis (PT), and is known to be suppressed by melatonin. Here we examined the involvement of adenosine in the regulation of Nmu expression. We found that the rat PT expressed adenosine receptor A2b and that an adenosine receptor agonist, NECA, stimulated Nmu expression in brain slice cultures. In vitro promoter assays revealed that NECA stimulated Nmu promoter activity via a cAMP response element (CRE) in the presence of adenosine receptor A2b. NECA also increased the levels of phosphorylated CRE-binding protein. These findings suggest that adenosine stimulates Nmu expression by activating the cAMP signaling pathway through adenosine receptor A2b in the rat PT. This is the first report to demonstrate that Nmu expression in the PT is regulated by adenosine, which acts as an intravital central metabolic signal, in addition to melatonin, which acts as an external photoperiodic environmental signal.
Assuntos
Adenosina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neuropeptídeos/biossíntese , Hipófise/metabolismo , RNA Mensageiro/biossíntese , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , AMP Cíclico/metabolismo , Masculino , Hipófise/citologia , Ratos , Ratos Endogâmicos F344 , Receptor A2B de Adenosina/metabolismoRESUMO
Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by ß-oxidation of long-chain fatty acids provided from the circulation.
Assuntos
Ácidos Graxos/metabolismo , Grelina/metabolismo , Processamento de Proteína Pós-Traducional , Acilação , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Animais , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Oxirredução , RNA Mensageiro/metabolismoRESUMO
Ghrelin is a peptide hormone with a unique structure comprising a medium chain fatty acid modification. Ghrelin cells are known to be abundantly localized in the gastric mucosa and are released into the blood stream to exert their multifunctional physiological effects. To elucidate the regulatory mechanisms of ghrelin secretion and acyl-modification, we developed novel ghrelin-producing cell lines. Using ghrelinoma cell lines, we focused on the mechanisms of ghrelin secretion and found that several GPCRs were highly expressed in ghrelin cells. Then, we showed that noradrenaline treatment stimulated ghrelin secretion via ß1-adrenergic receptor, and fasting-induced ghrelin elevation was completely inhibited by the ß1-adrenergic receptor antagonist in mice. In addition, we demonstrated that long chain fatty acids, glucose, and L-glutamate significantly inhibited ghrelin secretion. Furthermore, we recently revealed that the genes involved in fatty acid synthesis and long chain fatty acid metabolism were expressed in ghrelin cells, and that CPT-1 inhibitor treatment dramatically decreased the levels of acyl-modified ghrelin. Here, we introduce the current knowledge of the mechanisms involving ghrelin secretion and its acyl-modification.
Assuntos
Grelina/metabolismo , Acilação , Animais , Linhagem Celular Tumoral , Mucosa Gástrica/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
AIM: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action. METHODS: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. RESULTS: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. CONCLUSIONS: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.
Assuntos
Tecido Adiposo/patologia , Glucose/metabolismo , Leptina/farmacologia , Lipase/fisiologia , Lipólise/efeitos dos fármacos , Esterol Esterase/fisiologia , Tecido Adiposo/efeitos dos fármacos , Animais , Feminino , Leptina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.
Assuntos
Contagem de Células , Hipotálamo/citologia , Neurônios/citologia , Caracteres Sexuais , Androgênios/farmacologia , Animais , Callithrix , Di-Hidrotestosterona/farmacologia , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Orquiectomia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores Androgênicos/metabolismo , Comportamento Sexual Animal/efeitos dos fármacos , Comportamento Sexual Animal/fisiologia , Testosterona/farmacologiaRESUMO
The adenohypophysis is formed from the oral ectoderm and consists of the pars distalis (PD), pars intermedia, and pars tuberalis (PT). The mechanisms of PD development have been extensively studied, and the cellular differentiation of the PD is well understood. However, the morphogenesis and differentiation of the PT are still unclear, and the genes expressed during PT development remain largely unknown. We have explored genes specifically expressed in the PT during embryonic development and analyzed their spatiotemporal expression patterns. Microarray analysis of laser-captured PT and PD tissues obtained from chick embryos on embryonic day 10 (E10.0) has shown high expression of Cytokine-like 1 (CYTL1) and Gap junction protein alpha 5 (GJA5) genes in the PT. Detailed analysis of these spatiotemporal expression patterns during chick embryo development by in situ hybridization has revealed that CYTL1 mRNA first appears in the lateral head ectoderm and ventral head ectoderm at E1.5. The expression of CYTL1 moves into Rathke's pouch at E2.5 and is then localized in the PT primordium where it is continuously expressed until E12.0. GJA5 mRNA is transiently detected in the PT primordium from E6.0 to E12.0, whereas its expression is not detected in the PD during development. Thus, these genes might be involved in the regulation mechanisms of PT development and could be useful markers for PT development.
Assuntos
Biomarcadores/metabolismo , Conexinas/genética , Citocinas/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Animais , Embrião de Galinha , Conexinas/metabolismo , Citocinas/metabolismo , Desenvolvimento Embrionário/genética , Estudos de Associação Genética , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.
Assuntos
Caspase 3/análise , Caspase 9/análise , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Apoptose/efeitos dos fármacos , Caspase 3/química , Caspase 9/química , Cicloeximida/toxicidade , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by â¼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.
Assuntos
Grelina/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ácidos Graxos/farmacologia , Alimentos , Mucosa Gástrica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina/farmacologia , Pró-Proteína Convertase 1/genética , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Estômago/química , Neoplasias Gástricas/metabolismo , Ácido alfa-Linolênico/farmacologiaRESUMO
Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to ß1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.
Assuntos
Cálcio/metabolismo , Grelina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Norepinefrina/metabolismo , Agonistas alfa-Adrenérgicos/química , Animais , Cloreto de Cádmio/química , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Ácido Egtázico/química , Imuno-Histoquímica , Camundongos , Nifedipino/química , Transdução de Sinais , Sinaptotagminas/metabolismoRESUMO
The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the ß1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.
RESUMO
The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Melatonina/farmacologia , Neuropeptídeos/genética , Adeno-Hipófise/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de Neurotransmissores/genética , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Ritmo Circadiano/fisiologia , Epêndima/citologia , Epêndima/efeitos dos fármacos , Epêndima/metabolismo , Regulação da Expressão Gênica , Microdissecção e Captura a Laser , Masculino , Melatonina/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/metabolismo , Fotoperíodo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10(-10) M), ghrelin also induces gastric contractions at levels of 10(-10) M to 10(-7) M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.
Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Grelina/farmacologia , Neurônios Aferentes/metabolismo , Estômago/inervação , Estômago/fisiologia , Animais , Feminino , Técnicas In Vitro , Neurônios Aferentes/efeitos dos fármacos , MusaranhosRESUMO
The pars tuberalis (PT) is a part of the anterior pituitary gland that is located as a thin cell layer surrounding the median eminence. The characteristics of PT, including cell shape and cell composition, differ from those of the pars distalis (PD), suggesting that PT has unique physiological functions and different morphogenesis compared to PD. In this study, we used chicken embryos and showed for the first time that most hormone-producing cells in PT at embryonic day (E) 20.0 were only glycoprotein α subunit (αGSU)-positive staining cells. Then, using serial frontal and sagittal sections, we examined the detailed distribution of the αGSU mRNA-expressing region, as a marker of PT in the chicken embryonic pituitary gland during the E3.0-20.0 period. This three-dimensional expression pattern analysis clarified that αGSU mRNA expression initially appeared only in the bilateral regions of the Rathke's recess (RR) at E3.5, and this region expanded and showed a ring-like structure on RR. Subsequently, this αGSU mRNA-expressing region gradually expanded upward and reached the diencephalon at E8.0. This region became thinner as it surrounded the base of the diencephalon from E12.0 to E20.0. In this study, we demonstrated the detailed morphological changes of the chicken PT primordium by detecting αGSU mRNA, and we also showed that PT is a unique region in the early developmental stage.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Morfogênese , Hipófise/embriologia , Hipófise/metabolismo , Animais , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa de Hormônios Glicoproteicos/genética , Hipófise/anatomia & histologia , Hipófise/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.
Assuntos
Colecistocinina/biossíntese , Células Enteroendócrinas/citologia , Polipeptídeo Inibidor Gástrico/biossíntese , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Neurotensina/biossíntese , Peptídeo YY/metabolismo , Animais , Linhagem da Célula , Separação Celular , Diabetes Mellitus/prevenção & controle , Células Enteroendócrinas/metabolismo , Citometria de Fluxo , Grelina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica/métodos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Transgênicos , Obesidade/prevenção & controle , Regiões Promotoras GenéticasRESUMO
The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have examined the roles of ghrelin receptor (GHSR) expression in the mouse hindbrain. Notably, selective hindbrain ghrelin receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action.
Assuntos
Glicemia/metabolismo , Jejum/sangue , Receptores de Grelina/metabolismo , Rombencéfalo/metabolismo , Transdução de Sinais , Animais , Ingestão de Alimentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/farmacologia , Proteínas de Homeodomínio/metabolismo , Hipoglicemia/sangue , Hipoglicemia/genética , Hipoglicemia/patologia , Integrases/metabolismo , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Grelina/genética , Rombencéfalo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismoRESUMO
Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9 ± 1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0 ± 0.3%, 12.6 ± 0.3%, and 2.5 ± 0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.
Assuntos
Cálcio/metabolismo , Grelina/farmacologia , Adeno-Hipófise/metabolismo , Hormônios Adeno-Hipofisários/metabolismo , Animais , Corantes Fluorescentes , Hormônio Foliculoestimulante/metabolismo , Fura-2 , Grelina/fisiologia , Hormônio do Crescimento/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Espaço Intracelular/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Prolactina/metabolismo , Ratos , Ratos Wistar , Receptores de Grelina/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca(2+) in vitro (IC(50) = approximately 10 µM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.