RESUMEN
Since its discovery in 2006 by Oh-I and colleagues, NUCB2/nesfatin-1 encoded by nucleobindin-2 (NUCB2) has drawn sustained attention as reflected in over 500 publications. Among those, more than half focused on the alterations of food intake, body weight and metabolism (glucose, fat) induced by nesfatin-1 and/or NUCB2/nesfatin-1. In the current review we discuss the existing literature focusing on NUCB2/nesfatin-1's influence on food intake, body weight and glucose as well as fat metabolism and highlight gaps in knowledge.
Asunto(s)
Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Nucleobindinas/metabolismo , Nucleobindinas/farmacología , Animales , Grasas/metabolismo , Glucosa/metabolismo , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismoRESUMEN
The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.
Asunto(s)
Enteritis/genética , Antagonistas de Estrógenos/toxicidad , Microbioma Gastrointestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Simportadores/metabolismo , Tamoxifeno/toxicidad , Envejecimiento , Animales , Biotina/metabolismo , Peso Corporal/efectos de los fármacos , Colon/patología , Citocinas/metabolismo , Diarrea/inducido químicamente , Diarrea/microbiología , Diarrea/patología , Enteritis/inducido químicamente , Enteritis/microbiología , Intestinos/microbiología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Simportadores/efectos de los fármacos , Simportadores/genéticaRESUMEN
Intestinal absorption of the water-soluble vitamins biotin and pantothenic acid is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene). We recently observed that intestinal-specific (conditional) knockout of the mouse Slc5a6 gene (SMVT-cKO) is associated with growth retardation, the development of spontaneous and severe inflammation, abnormal histology in the large intestine, altered gut permeability, and early death. Our aim in this study was to examine the possibility that biotin and pantothenic acid oversupplementation (BPS) of the SMVT-cKO mice could reverse the above-described abnormalities. BPS was provided in the drinking water to mice before conception, to dams during pregnancy and lactation, and to the SMVT-cKO mice throughout their life. Our findings showed that such a regimen prevents early death, as well as normalizes the growth rate, intestinal integrity, pathology, and inflammation in SMVT-cKO mice. These findings provide clear evidence for a role for biotin and/or pantothenic acid in the maintenance of normal intestinal integrity and health.
Asunto(s)
Biotina/farmacología , Mucosa Intestinal/efectos de los fármacos , Ácido Pantoténico/farmacología , Simportadores/metabolismo , Animales , Femenino , Inflamación/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Lactancia/efectos de los fármacos , Ratones , Ratones Noqueados , EmbarazoRESUMEN
Biotin (vitamin B7) is essential for human health because of its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and certain chronic inflammatory diseases are associated with biotin deficiency; however, the mechanisms that mediate the association between biotin status and inflammation are not well understood. In this study, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency-associated inflammation. Our investigations revealed that anti-CD3/CD28-stimulated CD4+ T cells cultured in biotin-deficient medium secreted significantly enhanced levels of the proinflammatory cytokines IFN-γ, TNF, and IL-17. Expression of the transcription factors T-bet and RORγt was increased, whereas Foxp3 expression was decreased, in biotin-deficient CD4+ T cells. The percentage of T regulatory cells was also decreased under biotin-deficient condition. A similar increase in T-bet, RORγt, and proinflammatory cytokine levels, as well as a decrease in Foxp3, was observed in inguinal lymph nodes of mice fed a biotin-deficient diet relative to pair-fed controls. Furthermore, differentiation of CD4+ T cells toward Th1 and Th17 cells was also enhanced. In vitro and in vivo investigations indicated that the increased inflammatory response was due to enhanced activation of the mammalian target of rapamycin signaling pathway in biotin-deficient CD4+ T cells. In summary, these results demonstrate that biotin deficiency enhances the inflammatory responses in CD4+ T cells, which may contribute to inflammation associated with biotin deficiency.
RESUMEN
Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) (SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.
Asunto(s)
Mucosa Intestinal/fisiología , Simportadores/metabolismo , Animales , Biotina/administración & dosificación , Deficiencia de Biotinidasa/metabolismo , Células CACO-2 , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Humanos , Ratones , Ratones Noqueados , Permeabilidad , Simportadores/genética , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55-65 kg/m(2)), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40-50 kg/m(2), 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Mucosa Gástrica/metabolismo , Ghrelina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Obesidad Mórbida/metabolismo , Aciltransferasas/metabolismo , Adaptación Fisiológica , Adulto , Anciano , Western Blotting , Índice de Masa Corporal , Femenino , Mucosa Gástrica/patología , Humanos , Masculino , Persona de Mediana Edad , Nucleobindinas , Obesidad Mórbida/patología , Obesidad Mórbida/cirugíaRESUMEN
Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30-40, 40-50 and >50 were recruited (n=9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (-42%, p<0.01) whereas obese patients with BMI>50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62-78%, p<0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r=0.71, p<0.001) and a negative correlation with ghrelin (r=-0.60, p<0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.
Asunto(s)
Aciltransferasas/sangre , Índice de Masa Corporal , Ghrelina/metabolismo , Aciltransferasas/metabolismo , Femenino , Humanos , MasculinoRESUMEN
Ménétrier's disease is a rare hyperproliferative protein-losing gastropathy of the gastric foveolar epithelium. Most common symptoms include epigastric pain with fullness and vomiting, and generalized peripheral edema with hypoalbuminemia. Radiologically, the wall of the gastric body and fundus is diffusely thickened, often with antral sparing. Giant rugal edematous folds are seen on gastroscopy, and histology of biopsy material shows diffuse foveolar hyperplasia with cystic dilatation of the glandular portion of the gastric mucosa in the absence of significant inflammatory infiltrate. The recent discovery of transforming growth factor α overexpression opens the way of epidermal growth factor receptor blockade with cetuximab as first-line treatment modality in severe cases of Ménétrier's disease.
Asunto(s)
Gastritis Hipertrófica/diagnóstico , Gastritis Hipertrófica/etiología , Gastritis Hipertrófica/metabolismo , Gastritis Hipertrófica/terapia , Humanos , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100µg/kg) and blood was withdrawn before and at 2, 5, 7 and 24h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2h, 5h and 7h post injection respectively (p<0.05) and returned to baseline at 24h. The plasma NUCB2/nesfatin-1 increase at 2h was associated with increased corpus NUCB2 mRNA expression (p<0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p<0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Mucosa Gástrica/metabolismo , Lipopolisacáridos/farmacología , Proteínas del Tejido Nervioso/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/sangre , Proteínas de Unión al ADN/sangre , Mucosa Gástrica/efectos de los fármacos , Ghrelina/metabolismo , Inyecciones Intraperitoneales , Lipopolisacáridos/administración & dosificación , Masculino , Proteínas del Tejido Nervioso/sangre , Nucleobindinas , ARN Mensajero/metabolismo , Radioinmunoensayo , Ratas , Ratas Sprague-DawleyRESUMEN
Clinical studies are evaluating the efficacy of synthetic ghrelin agonists in postoperative ileus management. However, the control of ghrelin secretion under conditions of postoperative gastric ileus is largely unknown. Peripheral somatostatin inhibits ghrelin secretion in animals and humans. We investigated the time course of ghrelin changes postsurgery in fasted rats and whether somatostatin receptor subtype 2 (sst(2)) signaling is involved. Abdominal surgery (laparotomy and 1-min cecal palpation) induced a rapid and long-lasting decrease in plasma acyl ghrelin levels as shown by the 64, 67, and 59% reduction at 0.5, 2, and 5 h postsurgery, respectively, compared with sham (anesthesia alone for 10 min, P < 0.05). Levels were partly recovered at 7 h and fully restored at 24 h. The percentage of acyl ghrelin reduction was significantly higher than that of desacyl ghrelin at 2 h postsurgery and not at any other time point. This was associated with a 48 and 23% decrease in gastric and plasma ghrelin-O-acyltransferase protein concentrations, respectively (P < 0.001). Ghrelin-positive cells in the oxyntic mucosa expressed sst(2a) receptor and the sst(2) agonist S-346-011 inhibited fasting acyl ghrelin levels by 64 and 77% at 0.5 and 2 h, respectively. The sst(2) antagonist S-406-028 prevented the abdominal surgery-induced decreased circulating acyl ghrelin but not the delayed gastric emptying assessed 0.5 h postinjection. These data show that activation of sst(2) receptor located on gastric X/A-like cells plays a key role in the rapid inhibition of circulating acyl ghrelin induced by abdominal surgery while not being primarily involved in the early phase of postoperative gastric ileus.
Asunto(s)
Abdomen/cirugía , Aciltransferasas/sangre , Vaciamiento Gástrico/fisiología , Ghrelina/sangre , Ileus/fisiopatología , ARN Mensajero/metabolismo , Receptores de Somatostatina/metabolismo , Gastropatías/fisiopatología , Aciltransferasas/metabolismo , Análisis de Varianza , Animales , Mucosa Gástrica/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/antagonistas & inhibidores , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100 microg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2, 5 and 7 h (p<0.01) post-injection compared to vehicle and recovered at 24 h. At 2 h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p<0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p<0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2 h period post-LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7 h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2 h is associated with reduced circulating GOAT.
Asunto(s)
Aciltransferasas/sangre , Aciltransferasas/metabolismo , Regulación hacia Abajo , Ghrelina/sangre , Lipopolisacáridos/toxicidad , Reacción de Fase Aguda/fisiopatología , Aciltransferasas/genética , Animales , Regulación del Apetito , Glucemia/análisis , Western Blotting , Temperatura Corporal , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/fisiopatología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.
Asunto(s)
Cuerpo Ciliar/metabolismo , Regulación de la Expresión Génica , Miocitos del Músculo Liso/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/fisiología , Células Cultivadas , Cuerpo Ciliar/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Análisis por Micromatrices , Miocitos del Músculo Liso/efectos de los fármacos , Prostaglandinas Sintéticas/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estudios de Validación como AsuntoRESUMEN
The enzyme that acylates ghrelin was recently identified in mice as the fourth member of the membrane-bound O-acyltransferases superfamily (MBOAT4) and named ghrelin-O-acyltransferase (GOAT). Only one report showed GOAT mRNA expression in ghrelin-expressing cells of the mouse stomach. We investigated the distribution of GOAT protein in peripheral tissues and co-expression with endocrine markers in the gastric mucosa using a custom-made anti-GOAT antibody. Tissues were collected from male Sprague-Dawley rats and C57BL/6 mice. Western blot revealed two immunoreactive bands in rat and mouse gastric corpus mucosal proteins, a 50 kDa band corresponding to the GOAT protein and a 100 kDa band likely corresponding to a dimer. Western blot also detected GOAT in the plasma and levels were strongly increased after 24-h fasting in mice and slightly in rats. GOAT-immunoreactive cells were located in the gastric corpus mucosa and the anterior pituitary gland, whereas other peripheral tissues of rats and mice examined were negative. In mice, GOAT-immunoreactive cells were mainly distributed throughout the middle portion of the oxyntic glands, whereas in rats they were localized mainly in the lower portion of the glands. Double labeling showed that 95+/-1% of GOAT-immunoreactive cells in mice co-labeled with ghrelin, whereas in rats only 56+/-4% of GOAT-positive cells showed co-expression of ghrelin. The remainder of the GOAT-immunopositive cells in rats co-expressed histidine decarboxylase (44+/-3%). No co-localization was observed with somatostatin in rats or mice. These data suggest species differences between rats and mice in gastric GOAT expression perhaps resulting in a different role of the MBOAT4 enzyme in the rat stomach. Detection of GOAT in the plasma raises the possibility that ghrelin octanoylation may occur in the circulation and the fasting-induced increase in GOAT may contribute to the increase of acylated ghrelin after fasting.
Asunto(s)
Aciltransferasas/metabolismo , Ayuno/metabolismo , Células Parietales Gástricas/enzimología , Aciltransferasas/sangre , Animales , Anticuerpos/inmunología , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
Nesfatin-1, derived from nucleobindin2, is expressed in the hypothalamus and reported in one study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of nesfatin-1 and whether gastric emptying (GE) is altered, we injected nesfatin-1 into the lateral brain ventricle (intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. Nesfatin-1 (0.05 microg/rat, icv) decreased 2-3 h and 3-6 h dark-phase FI by 87 and 45%, respectively, whereas ip administration (2 microg/rat) had no effect. The corticotropin-releasing factor (CRF)(1)/CRF(2) antagonist astressin-B or the CRF(2) antagonist astressin(2)-B abolished icv nesfatin-1's anorexigenic action, whereas an astressin(2)-B analog, devoid of CRF-receptor binding affinity, did not. Nesfatin-1 icv induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin(2)-B. Nesfatin-1 into the 4v (0.05 microg/rat) or ic (0.5 microg/rat) decreased cumulative dark-phase FI by 29 and 60% at 1 h and by 41 and 37% between 3 and 5 h, respectively. This effect was neither altered by ic astressin(2)-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of nesfatin-1 neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus solitarius. These data indicate that nesfatin-1 acts centrally to reduce dark phase FI through CRF(2)-receptor-dependent pathways after forebrain injection and CRF(2)-receptor-independent pathways after hindbrain injection. Activation of nesfatin-1 neurons by cholecystokinin at sites regulating food intake may suggest a role in gut peptide satiation effect.
Asunto(s)
Ingestión de Alimentos , Vaciamiento Gástrico , Proteínas del Tejido Nervioso/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ingestión de Alimentos/efectos de la radiación , Vaciamiento Gástrico/efectos de la radiación , Luz , Masculino , Neuronas/metabolismo , Nucleobindinas , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
Nesfatin-1 is one of the peptide products of posttranslational processing of the nucleobindin-2 (NUCB2) gene, suggested to have physiological relevance to suppress food intake and body weight gain in rats. Nesfatin-1-immunoreactive cells have been found in distinct nuclei in the rat brain related to circuitries regulating food intake. Here, we report novel yet undescribed localization of NUCB2/nesfatin-1 at the mRNA and protein level in the rat central nervous system. Immunohistochemical staining revealed the localization of NUCB2/nesfatin-1 in the piriform and insular cortex, endopiriform nucleus, nucleus accumbens, lateral septum, bed nucleus of stria terminalis, central amygdaloid nucleus, medial preoptic area, dorsal raphe nucleus, ambiguus nucleus, ventrolateral medulla and gigantocellular reticular nucleus, as well as Purkinje-cells of the cerebellum. In the spinal cord, nesfatin-1 immunoreactivity (IR) was found in both sympathetic and parasympathetic preganglionic neuronal groups and in the dorsal area X from lower thoracic to sacral segments. The immunohistochemical results were confirmed by RT-PCR in the central amygdaloid nucleus, nucleus accumbens, cerebellum and lumbar spinal cord microdissected by punch technique. The features and distributions of nesfatin-1 IR and mRNA expression in the brain and spinal cord suggest that NUCB2/nesfatin-1 could play a wider role in autonomic regulation of visceral-endocrine functions besides food intake.
Asunto(s)
Sistema Nervioso Autónomo/metabolismo , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Médula Espinal/metabolismo , Animales , Proteínas de Unión al Calcio , Proteínas de Unión al ADN , Expresión Génica , Inmunohistoquímica , Masculino , Proteínas del Tejido Nervioso/genética , Nucleobindinas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hypothalamic nesfatin-1, derived from the nucleobindin2 (NUCB2) precursor, inhibits nocturnal food intake and body weight gain in rats. Nesfatin-1 is able to cross the blood-brain barrier, suggesting a peripheral source of nesfatin-1. Many centrally acting food intake regulatory neuropeptides are also produced in the periphery, especially in the gastrointestinal tract. Therefore, we investigated the gene expression of NUCB2 and distribution of nesfatin-1-immunoreactive cells in the stomach. Microarray mRNA expression profiles in purified small endocrine cells of the gastric mucosa substantiated by quantitative RT-PCR showed significantly higher NUCB2 mRNA expression compared with brain and heart. Western blot confirmed the expression of NUCB2 protein and its transport into a secretory soluble fraction of gastric mucosal endocrine cell homogenates. Immunohistochemical colabeling for nesfatin-1 and ghrelin, histidine decarboxylase, or somatostatin revealed two subtypes of nesfatin-1-positive endocrine cells. Cells in the midportion of the glands coexpressed nesfatin-1 and ghrelin, whereas few cells in the glandular base coexpressed nesfatin-1 and somatostatin or histidine decarboxylase. High-resolution three-dimensional volume imaging revealed two separate populations of intracytoplasmic vesicles in these cells, one containing nesfatin-1 and the other ghrelin immunoreactivity. Microarray rat genome expression data of NUCB2 in small gastric endocrine cells confirmed by quantitative RT-PCR showed significant down-regulation of NUCB2 after 24 h fasting. In summary, NUCB2 mRNA expression as well as protein content is present in a specific subset of gastric endocrine cells, most of which coexpress ghrelin. NUCB2 gene expression is significantly regulated by nutritional status, suggesting a regulatory role of peripheral nesfatin-1 in energy homeostasis.
Asunto(s)
Mucosa Gástrica/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Parietales Gástricas/citología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al ADN/genética , Ingestión de Energía , Ghrelina/metabolismo , Histidina Descarboxilasa/metabolismo , Inmunohistoquímica , Masculino , Nucleobindinas , Análisis de Secuencia por Matrices de Oligonucleótidos , Páncreas/citología , Hipófisis/citología , ARN/genética , ARN/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somatostatina/metabolismo , Aumento de PesoRESUMEN
Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.
Asunto(s)
Células Enterocromafines/metabolismo , Ayuno/metabolismo , Regulación de la Expresión Génica/fisiología , Liberación de Histamina/genética , Sistemas de Transporte de Aminoácidos Neutros/biosíntesis , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Recuento de Células , Inducción Enzimática , Perfilación de la Expresión Génica , Histamina/metabolismo , Liberación de Histamina/fisiología , Histidina Amoníaco-Liasa/biosíntesis , Histidina Amoníaco-Liasa/genética , Histidina Descarboxilasa/metabolismo , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/genética , Transcripción Genética , Urocanato Hidratasa/biosíntesis , Urocanato Hidratasa/genética , Proteínas de Transporte Vesicular de Monoaminas/biosíntesis , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
The gastric entero-chromaffin-like (ECL) cell plays a key regulatory role in peripheral regulation of acid secretion due to the release of histamine that stimulates acid secretion by the parietal cell. Studies in intact animals, gastric glands, and isolated cells after short-term culture have shown expression of stimulatory CCK2 and PAC1 and inhibitory SST2 and Gal1 receptors as well as histidine decarboxylase. However, the pattern of its gene expression as a neuroendocrine cell has not been explored. Comparison of gene expression by 95% pure ECL cells obtained by density gradient, elutriation, and fluorescence-assisted cell sorting with isolates of the intact fundic gastric epithelium (i.e., "subtractive hybridization") identified a variety of additional expressed gene families characteristic of this neuroendocrine cell. These include genes 1) involved in neuropeptide synthesis and secretory vesicle exocytosis, 2) involved in control of inflammation, 3) implicated in healing of the epithelium, 4) encoding inhibitory Gi protein-coupled receptors, 5) playing a role in neuroendocrine regulation of food intake, and 6) encoding proteins likely involved in maintenance of circadian rhythm, in addition to the ECL cell-specific genes histidine decarboxylase and monoamine transporter. Particularly, the inhibitory apelin receptor gene, APJ, was highly expressed in the ECL cell preparation. Because parietal cells express apelin, immunohistochemical and functional studies showed that there is an inhibitory feed back loop between the parietal and ECL cell during gastrin stimulation, providing evidence for a novel pathway of downregulation of acid secretion due to interaction between these two cell types.
Asunto(s)
Células Enterocromafines/metabolismo , Ácido Gástrico/metabolismo , Regulación de la Expresión Génica , Células Parietales Gástricas/metabolismo , Transcripción Genética , Animales , Apelina , Receptores de Apelina , Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Células Enterocromafines/efectos de los fármacos , Retroalimentación Fisiológica , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Gastrinas/farmacología , Perfilación de la Expresión Génica , Inmunohistoquímica , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina , Células Parietales Gástricas/efectos de los fármacos , ARN Mensajero/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Genomic microarray analysis of genes specifically expressed in a pure cell isolate from a heterocellular organ identified the likely K efflux channel associated with the gastric H-K-ATPase. The function of this channel is to supply K to the luminal surface of the pump to allow H for K exchange. KCNQ1-KCNE2 was the most highly expressed and significantly enriched member of the large variety of K channels expressed in the gastric epithelium. The function of this K channel in acid secretion was then shown by inhibition of secretion in isolated gastric glands with specific KCNQ inhibitors and by colocalization of the channel with the H-K-ATPase in the secretory canaliculus of the parietal cell. KCNQ1-KCNE2 appears to be the K efflux channel that is essential for gastric acid secretion.