RESUMEN
The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress.
Asunto(s)
Médula Suprarrenal , Células Cromafines , Médula Suprarrenal/inervación , Médula Suprarrenal/metabolismo , Animales , Células Cromafines/metabolismo , Epinefrina , Mamíferos/metabolismo , Norepinefrina , Ratas , Nervios Esplácnicos/metabolismoRESUMEN
Members of the Rab3 gene family are considered central to membrane trafficking of synaptic vesicles at mammalian central excitatory synapses. Recent evidence, however, indicates that the Rab27B-GTPase, which is highly homologous to the Rab3 family, is also enriched on SV membranes and co-localize with Rab3A and Synaptotagmin at presynaptic terminals. While functional roles of Rab3A have been well-established, little functional information exists on the role of Rab27B in synaptic transmission. Here we report on functional effects of Rab27B at SC-CA1 and DG-MF hippocampal synapses. The data establish distinct functional actions of Rab27B and demonstrate functions of Rab27B that differ between SC-CA1 and DG-MF synapses. Rab27B knockout reduced frequency facilitation compared to wild-type (WT) controls at the DG/MF-CA3 synaptic region, while increasing facilitation at the SC-CA1 synaptic region. Remarkably, Rab27B KO resulted in a complete elimination of LTP at the MF-CA3 synapse with no effect at the SC-CA1 synapse. These actions are similar to those previously reported for Rab3A KO. Specificity of action on LTP to Rab27B was confirmed as LTP was rescued in response to lentiviral infection and expression of human Rab27B, but not to GFP, in the DG in the Rab27B KO mice. Notably, the effect of Rab27B KO on MF-CA3 LTP occurred in spite of continued expression of Rab3A in the Rab27B KO. Overall, the results provide a novel perspective in suggesting that Rab27B and Rab3A act synergistically, perhaps via sequential effector recruitment or signaling for presynaptic LTP expression in this hippocampal synaptic region.
Asunto(s)
Hipocampo/metabolismo , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Potenciación a Largo Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de Unión al GTP rab3A/metabolismoRESUMEN
Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell-specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice.
Asunto(s)
Células Acinares/fisiología , Páncreas/fisiología , Proteínas de Transporte Vesicular/metabolismo , Células Acinares/citología , Células Acinares/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Ratones , Ratones Transgénicos , Páncreas/citología , Páncreas/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.
Asunto(s)
Páncreas Exocrino/enzimología , alfa-Amilasas Pancreáticas/metabolismo , Vesículas Secretoras/enzimología , Proteínas de Unión al GTP rab/fisiología , Células Acinares , Animales , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas Exocrino/ultraestructura , Vesículas Secretoras/ultraestructura , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP , Proteínas de Unión al GTP rab3/biosíntesisRESUMEN
Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic ß-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway.
Asunto(s)
Células Acinares/enzimología , Amilasas/metabolismo , AMP Cíclico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Islotes Pancreáticos/enzimología , Células Acinares/metabolismo , Animales , Forma de la Célula , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/deficiencia , Receptor del Péptido 1 Similar al Glucagón/genética , Incretinas/farmacología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Mensajero/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.
Asunto(s)
Amilasas/metabolismo , Páncreas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Exocitosis , Masculino , Ratones , Ratones Endogámicos ICR , Páncreas/citología , Fracciones Subcelulares/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
High levels of cholecystokinin (CCK) can stimulate pancreatic adaptive growth in which mature acinar cells divide, leading to enhanced pancreatic mass with parallel increases in protein, DNA, RNA, and digestive enzyme content. Prolonged release of CCK can be induced by feeding trypsin inhibitor (TI) to disrupt normal feedback control. This leads to exocrine growth in a CCK-dependent manner. The extracellular signal-related kinase (ERK) pathway regulates many proliferative processes in various tissues and disease models. The aim of this study was to evaluate the role of ERK signaling in pancreatic adaptive growth using the MEK inhibitors PD-0325901 and trametinib (GSK-1120212). It was determined that PD-0325901 given two times daily by gavage or mixed into powdered chow was an effective and specific inhibitor of ERK signaling in vivo. TI-containing chow led to a robust increase in pancreatic mass, protein, DNA, and RNA content. This pancreatic adaptive growth was blocked in mice fed chow containing the MEK inhibitors. PD-0325901 blocked TI-induced ERK-regulated early response genes, cell-cycle proteins, and mitogenesis by acinar cells. It was determined that ERK signaling is necessary for the initiation of pancreatic adaptive growth but not necessary to maintain it. PD-0325901 blocked adaptive growth when given before cell-cycle initiation but not after mitogenesis had been established. Furthermore, GSK-1120212, a chemically distinct inhibitor of the ERK pathway that is now approved for clinical use, inhibited growth similar to PD-0325901. These data demonstrate that the ERK pathway is required for CCK-stimulated pancreatic adaptive growth.
Asunto(s)
Proliferación Celular , Colecistoquinina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Páncreas/enzimología , Animales , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Replicación del ADN , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos ICR , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Páncreas/efectos de los fármacos , Páncreas/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , ARN/biosíntesis , Factores de Tiempo , Inhibidores de Tripsina/farmacologíaRESUMEN
The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells.
Asunto(s)
Células Acinares/metabolismo , Amilasas/metabolismo , Exocitosis/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Páncreas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Células Acinares/citología , Amilasas/genética , Animales , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/genética , Masculino , Ratones , Ratones Endogámicos ICR , Mutación , Páncreas/citología , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.
Asunto(s)
Hígado Graso/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Porfirias Hepáticas/metabolismo , Animales , Modelos Animales de Enfermedad , Hígado Graso/etiología , Hígado Graso/genética , Ferroquelatasa/genética , Proteínas de Unión al GTP/antagonistas & inhibidores , Células Hep G2 , Humanos , Cuerpos de Mallory/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Estrés Oxidativo , Porfirias Hepáticas/complicaciones , Porfirias Hepáticas/genética , Proteína Glutamina Gamma Glutamiltransferasa 2 , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Protoporfirinas/farmacología , Piridinas/toxicidad , Transglutaminasas/antagonistas & inhibidoresRESUMEN
Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, ß-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of ß-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.
Asunto(s)
Células Acinares/metabolismo , Desdiferenciación Celular/fisiología , Colecistoquinina/farmacología , Replicación del ADN/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Células Acinares/efectos de los fármacos , Amilasas/metabolismo , Animales , Cadherinas/metabolismo , Desdiferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/metabolismo , Replicación del ADN/efectos de los fármacos , Ratones , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , beta Catenina/metabolismoRESUMEN
Feeding mice with protease inhibitor (PI) leads to increased endogenous cholecystokinin (CCK) release and results in pancreatic growth. This adaptive response requires calcineurin (CN)-NFAT and AKT-mTOR pathways, but the genes involved, the dynamics of their expression, and other regulatory pathways remain unknown. Here, we examined the early (1-8 h) transcriptional program that underlies pancreatic growth. We found 314 upregulated and 219 downregulated genes with diverse temporal and functional profiles. Several new identifications include the following: stress response genes Gdf15 and Txnip, metabolic mediators Pitpnc1 and Hmges2, as well as components of growth factor response Fgf21, Atf3, and Egr1. The genes fell into seven self-organizing clusters, each with a distinct pattern of expression; a representative gene within each of the upregulated clusters (Egr1, Gadd45b, Rgs2, and Serpinb1a) was validated by qRT-PCR. Genes up at any point throughout the time course and CN-dependent genes were subjected to further bioinformatics-based networking and promoter analysis, yielding STATs as potential transcriptional regulators. As shown by PCR, qPCR, and Western blots, the active phospho-form of STAT3 and the Jak-STAT feedback inhibitor Socs2 were both increased throughout early pancreatic growth. Moreover, immunohistochemistry showed a CCK-dependent and acinar cell-specific increase in nuclear localization of p-STAT3, with >75% nuclear occupancy in PI-fed mice vs. <0.1% in controls. Thus, the study identified novel genes likely to be important for CCK-driven pancreatic growth, characterized and biologically validated the dynamic pattern of their expression and investigated STAT-Socs signaling as a new player in this trophic response.
Asunto(s)
Colecistoquinina/farmacología , Regulación de la Expresión Génica , Páncreas/efectos de los fármacos , Páncreas/crecimiento & desarrollo , Factores de Transcripción STAT/fisiología , Animales , Colecistoquinina/metabolismo , Análisis por Conglomerados , Ayuno/metabolismo , Ayuno/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes , Masculino , Ratones , Ratones Endogámicos ICR , Análisis por Micromatrices , Páncreas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de TiempoRESUMEN
Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Regulación de la Expresión Génica , Intestino Delgado/citología , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Caliciformes/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Células de Paneth/metabolismo , Regiones Promotoras Genéticas , Receptor Notch1/antagonistas & inhibidores , Receptor Notch2/antagonistas & inhibidores , Transducción de Señal , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Adaptive exocrine pancreatic growth is mediated primarily by dietary protein and the gastrointestinal hormone cholecystokinin (CCK). Feeding trypsin inhibitors such as camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response. Our aim was to test the role of secretin in pancreatic development and adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor. The lack of secretin in the intestine or the secretin receptor in the pancreas was confirmed by RT-PCR. Other related components, such as vasoactive intestinal polypeptide (VIP) receptors (VPAC(1) and VPAC(2)), were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor-deleted mice, whereas VIP and forskolin still induced a normal response. Secretin in vivo failed to induce fluid secretion in receptor-deficient mice. The pancreas of secretin or secretin receptor-deficient mice was of normal size and histology, indicating that secretin is not necessary for normal pancreatic differentiation or maintenance. When WT mice were fed 0.1% camostat in powdered chow, the pancreas doubled in size in 1 wk, accompanied by parallel increases in protein and DNA. Camostat-fed littermate secretin and secretin receptor-deficient mice had similar pancreatic mass to WT mice. These results indicate that secretin is not required for normal pancreatic development or adaptive growth mediated by CCK.
Asunto(s)
Páncreas Exocrino/crecimiento & desarrollo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/metabolismo , Células Acinares/metabolismo , Animales , Colecistoquinina/metabolismo , AMP Cíclico/metabolismo , Masculino , Ratones , Ratones Noqueados , Páncreas Exocrino/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/genética , Secretina/genética , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a ß-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K(+)-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas R-SNARE/química , Empalme Alternativo , Secuencias de Aminoácidos , Animales , Membrana Celular/metabolismo , Exocitosis , Hormona de Crecimiento Humana/metabolismo , Humanos , Células PC12 , Estructura Terciaria de Proteína , Ratas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sintaxina 1/químicaRESUMEN
Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK-independent growth. To identify mechanisms whereby protein stimulates pancreatic growth in the absence of CCK release, C57BL/6 control and CCK-null male mice were fed normal-protein (14% casein) or high-protein (75% casein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 26% in CCK-null mice fed high-protein chow. Changes in pancreatic weight in control mice were due to both cell hypertrophy and hyperplasia since there was an increase in protein-to-DNA ratio, total DNA content, and DNA synthesis. In CCK-null mice pancreatic growth was almost entirely due to hypertrophy with both protein-to-DNA ratio and cell size increasing without significant increases in DNA content or DNA synthesis. ERK, calcineurin, and mammalian target of rapamycin complex 1 (mTORC1) are activated in models of CCK-induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK-null mice. In contrast, mTORC1 activation was increased after feeding and the duration of activation was prolonged in mice fed high-protein chow compared with normal-protein chow. Changes in pancreatic weight and RNA content were completely inhibited, and changes in protein content were partially abated, when the mTORC1 inhibitor rapamycin was administered during high-protein chow feeding. Prolonged mTORC1 activation is thus required for dietary protein-induced pancreatic growth in the absence of CCK.
Asunto(s)
Colecistoquinina/metabolismo , Proteínas en la Dieta/metabolismo , Páncreas Exocrino/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Análisis de Varianza , Animales , Western Blotting , Calcineurina/metabolismo , Colecistoquinina/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos , Tamaño de los Órganos , Páncreas Exocrino/metabolismo , Fosforilación , Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Insulina/metabolismo , Microscopía Fluorescente/métodos , Mapeo de Interacción de Proteínas/métodos , Vesículas Secretoras/metabolismo , Animales , Calibración , Línea Celular , Colorantes Fluorescentes/metabolismo , Secreción de Insulina , Ratones , Unión Proteica , Solubilidad , Transfección , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
BACKGROUND & AIMS: Growth of exocrine pancreas is regulated by gastrointestinal hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates Nuclear Factor of Activated T cells (NFATs), but the genetic underpinnings and feedback mechanisms that regulate this response are not known. METHODS: Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI stimulation was performed on Affymetrix 430A chips, and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with "flox-on" Rcan1 mice. RESULTS: CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1-8 hours course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice. CONCLUSIONS: We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator, and firmly establish that CN-NFAT signaling is required for this response.
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Proliferación Celular , Colecistoquinina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Páncreas/metabolismo , Animales , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Proteínas de Unión al Calcio , Dieta , Inhibidores Enzimáticos/farmacología , Ésteres , Retroalimentación Fisiológica , Gabexato/administración & dosificación , Gabexato/análogos & derivados , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Guanidinas , Integrasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas Musculares/genética , Factores de Transcripción NFATC/metabolismo , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos , Páncreas/efectos de los fármacos , Páncreas/crecimiento & desarrollo , Elastasa Pancreática/genética , Inhibidores de Proteasas/administración & dosificación , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina A/metabolismo , Receptores de Estrógenos/genética , Transducción de Señal/efectos de los fármacos , Tacrolimus/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Cholecystokinin (CCK) has been shown to activate RhoA and Rac1, as well as reorganize the actin cytoskeleton and, thereby, modify acinar morphology and amylase secretion in mouse pancreatic acini. The aim of the present study was to determine which heterotrimeric G proteins activate RhoA and Rac1 upon CCK stimulation. Galpha(13), but not Galpha(12), was identified in mouse pancreatic acini by RT-PCR and Western blotting. Using specific assays for RhoA and Rac1 activation, we showed that only active Galpha(13) activated RhoA. By contrast, active Galpha(13) and Galpha(q), but not Galpha(s), slightly increased GTP-bound Rac1 levels. A greater increase in Rac1 activation was observed when active Galpha(13) and active Galpha(q) were coexpressed. Galpha(i) was not required for CCK-induced RhoA or Rac1 activation. The regulator of G protein signaling (RGS) domain of p115-Rho guanine nucleotide exchange factor (p115-RGS), a specific inhibitor of Galpha(12/13)-mediated signaling, abolished CCK-stimulated RhoA activation. By contrast, both RGS-2, an inhibitor of Galpha(q), and p115-RGS abolished CCK-induced Rac1 activation, which was PLC pathway-independent. Active Galpha(q) and Galpha(13), but not Galpha(s), induced morphological changes and actin redistribution similar to 1 nM CCK. CCK-induced actin cytoskeletal reorganization was inhibited by RGS-2, but not by p115-RGS, whereas CCK-induced amylase secretion was blocked by both inhibitors. Together, these findings indicate that, in mouse pancreatic acini, Galpha(13) links CCK stimulation to the activation of RhoA, whereas both Galpha(13) and Galpha(q) link CCK stimulation to the activation of Rac1. CCK-induced actin cytoskeletal reorganization is mainly mediated by Galpha(q). By contrast, Galpha(13) and Galpha(q) signaling are required for CCK-induced amylase secretion.
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Amilasas/metabolismo , Colecistoquinina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Neuropéptidos/metabolismo , Páncreas Exocrino/enzimología , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Forma de la Célula , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Páncreas Exocrino/metabolismo , Estructura Terciaria de Proteína , Proteínas RGS/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Fosfolipasas de Tipo C/metabolismo , Proteína de Unión al GTP rac1 , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The rough endoplasmic reticulum (RER) is a central organelle for synthesizing and processing digestive enzymes and alteration of ER functions may participate in the pathogenesis of acute pancreatitis (AP). To comprehensively characterize the normal and diseased RER subproteome, this study quantitatively compared the protein compositions of pancreatic RER between normal and AP animals using isobaric tags (iTRAQ) and 2D LC-MALDI-MS/MS. A total of 469 unique proteins were revealed from four independent experiments using two different AP models. These proteins belong to a large number of functional categories including ribosomal proteins, translocon subunits, chaperones, secretory proteins, and glyco- and lipid-processing enzymes. A total of 37 RER proteins (25 unique in arginine-induced, 6 unique in caerulein-induced and 6 common in both models of AP) showed significant changes during AP including translational regulators and digestive enzymes, whereas only mild changes were found in some ER chaperones. The six proteins common to both AP models included a decrease in pancreatic triacylglycerol lipase precursor, Erp27, and prolyl 4-hydroxylase beta polypeptide as well as a dramatic increase in fibrinogen alpha, beta and gamma chains. These results suggest that the early stages of AP involve changes of multiple RER proteins that may affect the synthesis and processing of digestive enzymes.
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Retículo Endoplásmico/metabolismo , Páncreas/metabolismo , Pancreatitis/metabolismo , Proteómica , Enfermedad Aguda , Animales , Cromatografía Liquida , Inmunohistoquímica , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
BACKGROUND & AIMS: Dietary protein deficiency results in diminished capacity of the pancreas to secrete enzymes needed for macronutrient digestion. Previous work has suggested that modulation of the mammalian target of rapamycin (mTOR) pathway by the hormone cholecystokinin (CCK) plays an important role in normal digestive enzyme synthesis after feeding. The purpose of this study was to elucidate the role of mTOR in protein deficiency-induced pancreatic dysfunction. METHODS: Wild-type and CCK-null mice were fed protein-deficient chow for 4 days and then allowed to recover on control chow in the presence or absence of the mTOR inhibitor rapamycin. RESULTS: The size and secretory capacity of the pancreas rapidly decreased after feeding protein-deficient chow. Refeeding protein-replete chow reversed these changes in both wild-type and CCK-null mice. Changes in the size of the pancreas were paralleled by changes in the content and secretion of digestive enzymes, as well as the phosphorylation of downstream targets of mTOR. Administration of the mTOR inhibitor rapamycin decreased regrowth of the pancreas but did not affect digestive enzyme content or secretory capacity. CONCLUSIONS: These studies demonstrate that dietary protein modulates pancreatic growth, but not digestive enzyme synthesis, via CCK-independent activation of the mTOR pathway.