RESUMEN
Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na(+)-K(+)-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, alpha(1)- and beta(1)-subunits of Na(+)-K(+)-ATPase, and ODC. Postnatal rats fed 6 mumol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na(+)-K(+)-ATPase activity and alpha(1)- and beta(1)-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Transportador de Glucosa de Tipo 2/biosíntesis , Intestino Delgado/metabolismo , Ornitina Descarboxilasa/biosíntesis , Transportador 1 de Sodio-Glucosa/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Espermidina/metabolismo , Complejo Sacarasa-Isomaltasa/biosíntesis , Administración Oral , Animales , Animales Recién Nacidos , Inducción Enzimática , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 2/genética , Intestino Delgado/crecimiento & desarrollo , Microvellosidades/metabolismo , Ornitina Descarboxilasa/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Transportador 1 de Sodio-Glucosa/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Espermidina/administración & dosificación , Complejo Sacarasa-Isomaltasa/genética , Factores de TiempoRESUMEN
The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/biosíntesis , Factor de Transcripción GATA4/fisiología , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/fisiología , Intestino Delgado/crecimiento & desarrollo , Lactasa-Florizina Hidrolasa/biosíntesis , Complejo Sacarasa-Isomaltasa/biosíntesis , Animales , Femenino , Glucocorticoides/farmacología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/embriología , Ratones , Embarazo , ARN Mensajero/metabolismo , DesteteRESUMEN
Sucrase-isomaltase (SI) gene expression is negatively regulated by glucose, but its molecular mechanism is not completely clear. The purpose of this study is to investigate whether HNF-1alpha and HNF-1beta contribute to glucose regulation of SI gene expression. To explore this question, we examined the association of gene expressions between SI and HNF-1alpha and HNF-1beta in Caco-2 cells cultured in medium containing 2.0 and 16.7 mM glucose. We found that gene expression of HNF-1alpha but not HNF-1beta exhibits a positive correlation with that of SI regulated by glucose. Moreover, to elucidate whether glucose regulation of SI gene expression is changed when HNF-1alpha and HNF-1beta are inhibited, we produced three stable cell lines, in which dominant-negative mutant HNF-1alphaT539fsdelC, mutant HNF-1betaR177X, and empty vector (as a control), respectively, were stably expressed. We found that the glucose regulation of SI gene expression was significantly attenuated in HNF-1alphaT539fsdelC cells, but it was well maintained in empty vector and HNF-1betaR177X cells. These results suggest that HNF-1alpha participates in glucose regulation of SI gene expression.
Asunto(s)
Glucosa/farmacología , Factor Nuclear 1-alfa del Hepatocito/fisiología , Factor Nuclear 1-beta del Hepatocito/fisiología , Complejo Sacarasa-Isomaltasa/biosíntesis , Células CACO-2 , Represión Enzimática , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/biosíntesis , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/biosíntesis , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Mutación , Complejo Sacarasa-Isomaltasa/genética , TransfecciónRESUMEN
In normal colon epithelium, cell proliferation is followed by cell differentiation. The purpose of this work was to investigate, in the HT29-D4 colon adenocarcinoma cell line, the occurrence of a temporal sequence of changes in cell proliferation and differentiation, the role of autocrine EGF family ligands and to determine which transduction pathway(s) are involved in these processes. In a medium lacking both growth factor and serum, HT29-D4 cells secreted amphiregulin (AR), which was shown to be strongly involved in cell adhesion, growth and differentiation. In the main, integrins alpha2beta1 and alphavbeta6 intervened in these processes. Using tyrphostins, it was demonstrated that AR involvement was mediated through the ErbB1/ERK1,2 and ErbB1/FAK pathways. These signalling molecules were directly involved in pRb inhibition and, thus, in cyclin A expression. Concomitantly, colon differentiation markers were also expressed. Furthermore, terminal cell maturation resulted in a colon absorptive cell with strong polarisation, the growth of which was inhibited by tyrphostin and an ERK1,2 inhibitor. It was concluded that in a colon adenocarcinoma, cell proliferation and differentiation can occur concomitantly and that these deregulated processes are controlled by autocrine secretion through the ErbB1/ERK1,2 and FAK pathways.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Antígenos de Neoplasias/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Integrina alfa2beta1/metabolismo , Integrinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Adenocarcinoma/enzimología , Fosfatasa Alcalina/biosíntesis , Anfirregulina , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Neoplasias del Colon/enzimología , Ciclinas/biosíntesis , Familia de Proteínas EGF , Activación Enzimática , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Células HT29 , Humanos , Proteínas de Microfilamentos/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinazolinas , Proteína de Retinoblastoma/biosíntesis , Complejo Sacarasa-Isomaltasa/biosíntesis , Tirfostinos/farmacologíaRESUMEN
We undertook a study of the mechanism by which rhesus monkey rotavirus (RRV) impairs the expression and enzyme activity of brush border-associated sucrase isomaltase (SI) in cultured, human, fully differentiated, intestinal Caco-2 cells. We provide evidence that the RRV-induced defects in the expression and enzyme activity of SI are not related to the previously observed, RRV-induced, Ca2+ -dependent, disassembly of the F-actin cytoskeleton. This conclusion is based on the facts that: (i) the intracellular Ca2+ blocker, BAPTA/AM, which antagonizes the RRV-induced increase in [Ca2+](i), fails to inhibit the RRV-induced decrease in SI expression and enzyme activity; and (ii) Jasplakinolide (JAS) treatment, known to stabilize actin filaments, had no effect on the RRV-induced decrease in SI expression. Results reported here demonstrate that the RRV-induced impairment in the expression and enzyme activity of brush border-associated SI results from a hitherto unknown mechanism involving PKA signalling. This conclusion is based on the observations that (i) intracellular cAMP was increased in RRV-infected cells and (ii) treatment of RRV-infected cells with PKA blockers resulted in the reappearance of apical SI expression, accompanied by the restoration of the enzyme activity at the brush border. In addition, in RRV-infected cells a twofold increase of phosphorylated form of cytokeratin 18 was observed after immunopurification and Western Blot analysis, which was antagonized by exposing the RRV-infected cells to the PKA blockers.
Asunto(s)
Células CACO-2/virología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Depsipéptidos , Rotavirus/fisiología , Complejo Sacarasa-Isomaltasa/metabolismo , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Humanos , Queratinas/metabolismo , Péptidos Cíclicos/farmacología , Fosforilación , Transducción de Señal , Complejo Sacarasa-Isomaltasa/antagonistas & inhibidores , Complejo Sacarasa-Isomaltasa/biosíntesisRESUMEN
The small intestine plays an important role in the digestion and absorption of many nutrients. To investigate the contribution of carbohydrate digestion to diabetes mellitus, we examined the morphological changes of the small intestine, and the expression of sucrase-isomaltase, which is one of the intestinal disaccharidases, in diabetic model rat, that is the streptozotocin-induced (STZ) diabetic rat (insulin-deficient model), and the Otsuka Long-Evans Tokushima Fatty (OLETF) rats and the Goto-Kakizaki (GK) rats (type 2 diabetic models). Intestinal hyperplasia was observed in STZ, OLETF, and GK rats. Moreover, in the small intestine of each diabetic strain, the proliferating cell nuclear antigen (PCNA)-labeling index, which is a marker of proliferation, was higher than in the respective control. Cdx1 and Cdx2, known to be transcriptional factors related to intestinal proliferation and differentiation, were more highly expressed in STZ, OLETF and GK rats than in the respective controls. These findings indicate that small intestinal hyperplasia, and thereby the resultant increase of total activity of disaccharidases such as sucrase and isomaltase in the entire small intestine, might be one of the reasons for postprandial hyperglycemia in diabetes mellitus.
Asunto(s)
Intestino Delgado/enzimología , Intestino Delgado/patología , Complejo Sacarasa-Isomaltasa/metabolismo , Actinas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Factor de Transcripción CDX2 , Diabetes Mellitus Experimental/fisiopatología , Hemoglobina Glucada/metabolismo , Proteínas de Homeodominio/metabolismo , Hiperplasia/enzimología , Immunoblotting , Insulina/sangre , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Endogámicas OLETF , Ratas Endogámicas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Sacarasa-Isomaltasa/biosíntesis , TransactivadoresRESUMEN
Barrett's epithelium (BE), the esophageal columnar-lining with intestinal differentiation, is a premalignant condition predisposing to adenocarcinoma. Columnar cells are the prevalent element of BE, but the hallmark of intestinal differentiation is the goblet population that defines the specialised columnar epithelium (SCE). We have demonstrated that columnar cells adjacent to Barrett's adenocarcinoma (BA) exhibit enterocytic features in areas with and without SCE. Nevertheless, the relationship between malignancy and the presence of these elements is not established. To investigate whether intestinal differentiated cells, other than goblet cells, are associated to neoplasia we compared the prevalence of enterocytic features in columnar elements with and without associated BA through the use of sucrase-isomaltase (SI) immunoreactivity in 31 columnar esophageal segments (CLES) and 12 BA. In metaplasia, SI was only expressed at the columnar cells. Apical staining was exclusive of CLES with SCE. SI was present at the cytoplasm in 22.2% of CLES without SCE. Apical SI occurred in BE with and without carcinoma, similarly in areas with and without SCE (p = 0.11 and p = 0.50, respectively). In areas with SCE, columnar cells with apical SI were more frequent in cases of BE adjacent to carcinoma than in cases without neoplasia but the difference did not reach significance (p = 0.053). In areas without SCE, apical SI was significantly (p = 0.01) more frequent in cases with carcinoma. Apical SI was equally found in neoplastic as in metaplastic areas, with and without SCE, (p = 0.07 and p = 0.40, respectively). In conclusion this study on the frequency of SI on CLES with and without neoplasia demonstrated that additionally to SCE, metaplastic enterocytic cells are also associated with malignancy. It also confirmed that the presence of intestinal features are underestimated if only goblet elements are used for its identification, reinforcing the utility of the immunohistochemical recognition of enterocytic characteristics for establishing the diagnosis of BE.
Asunto(s)
Esófago de Barrett/patología , Adenocarcinoma/patología , Esófago de Barrett/metabolismo , Carcinoma/patología , Diferenciación Celular , Citoplasma/metabolismo , Células Epiteliales/metabolismo , Epitelio/patología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , Inmunohistoquímica , Membrana Mucosa/patología , Fenotipo , Complejo Sacarasa-Isomaltasa/biosíntesisRESUMEN
Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Mucosa Intestinal/metabolismo , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Proteínas Represoras/metabolismo , Complejo Sacarasa-Isomaltasa/genética , Envejecimiento/metabolismo , Animales , Células COS , Células CACO-2 , Colon/citología , Colon/crecimiento & desarrollo , Colon/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción GATA4 , Proteínas de Homeodominio , Humanos , Hibridación in Situ , Intestino Delgado/citología , Intestino Delgado/crecimiento & desarrollo , Intestino Delgado/metabolismo , Intestinos/citología , Intestinos/crecimiento & desarrollo , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Complejo Sacarasa-Isomaltasa/biosíntesis , Factores de Transcripción/metabolismo , TransgenesRESUMEN
Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.
Asunto(s)
Adhesión Bacteriana , Escherichia coli/metabolismo , Hidrolasas/metabolismo , Mucosa Intestinal/enzimología , Mucosa Intestinal/microbiología , Microvellosidades/enzimología , Microvellosidades/microbiología , Adhesinas de Escherichia coli/metabolismo , Apoptosis , Células CACO-2 , Diferenciación Celular , Niño , Diarrea/microbiología , Dipeptidil Peptidasa 4/biosíntesis , Dipeptidil Peptidasa 4/metabolismo , Escherichia coli/patogenicidad , Humanos , Hidrolasas/biosíntesis , Immunoblotting , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microvellosidades/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Complejo Sacarasa-Isomaltasa/biosíntesis , Complejo Sacarasa-Isomaltasa/metabolismo , VirulenciaRESUMEN
Sucrase-alpha-dextrinase (S-D), a glycoprotein hydrolase in the border surface of the enterocyte, is altered in congenitally diabetic BioBreed Wistar (BB(d)) rats. Its intracellular assembly was examined in the current studies. Following pulse-chase experiments, S--D was specifically immuno-isolated from ER-Golgi and brush border membranes, and examined by SDS-PAGE and autoradiography. While synthesis and co-translational glycosylation of an immature species, P(i), in the ER proceeded normally, post-translational maturation of the N-linked carbohydrate chains was altered in the BB(d) rat. The mature species, P(m), was 10 kDa larger in BB(d) than in normal rats, and approximately 25% of its N-linked chains remained immature. The difference in mass was attributed to an appreciably larger mass of the O-linked chains of P(m) in BB(d) rats. In vivo kinetics of intracellular assembly displayed a delay in the appearance of P(m) in Golgi (Wistar, 15 min; BB(d), 30--60 min). These experiments, revealing structural alterations in congenital diabetes suggest an important role for intracellular glycosylation in the orderly assembly and processing of brush border S-D in the enterocyte.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Complejo Sacarasa-Isomaltasa/biosíntesis , Animales , Diabetes Mellitus Experimental , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Yeyuno/metabolismo , Cinética , Masculino , Microvellosidades/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas , Ratas , Ratas Mutantes , Ratas Wistar , Complejo Sacarasa-Isomaltasa/química , Factores de TiempoRESUMEN
Barrett's esophagus develops with the following 2 distinct types of lining mucosa: with and without specialized intestinal metaplasia (SIM). Goblet cells found only in SIM areas identify an intestinal phenotype, recognized as the histological hallmark diagnosing Barrett's metaplasia, and selecting high-risk patients for endoscopic surveillance. The columnar non-goblet cells are the major component of the heterogeneous Barrett's metaplastic cell population and are present in areas either with or without SIM. Their significance in the differentiation of columnar-lined esophagus, and their relationship to malignancy, is still unclear. This immunohistochemical study used two markers of enterocytic differentiation, to explore the intestinal phenotype of the non-goblet cell population of Barrett's epithelium and Barrett's-associated adenocarcinoma cells. Sucrase-isomaltase (SI) and dipeptidilpeptidase IV (DPP) immunoexpression was assessed in paraffin-embedded samples of 12 surgical specimens containing Barrett's esophageal mucosa in association with adenocarcinoma/high grade dysplasia. Ileal mucosa and mucosa from normal gastric and esophageal segments of the surgical specimen were used as positive and negative controls, respectively. SI and DPP were expressed by the neoplastic cells and the columnar non-goblet, being negative in goblet cells. The localization of the enzymes was predominantly apical for SI and cytoplasmatic for DPP. There was immunoreactivity for SI in 58.3% of the carcinomas and in 66.6% of Barrett's mucosa, with equal frequency in areas with and without SIM. DPP was identified in 66.6% of the carcinomas, in 50% of the cases of Barrett's metaplasia with SIM, and in 75% of those without SIM. The columnar non-goblet cell components of Barrett's metaplasia contain small intestine enzymes in the areas either with or without SIM, which suggests that they identify an "incomplete form" of intestinal metaplasia. The demonstration that the two enzymes, SI and DPP, are produced by the columnar non-goblet cell metaplastic population and by the neoplastic cells of the associated adenocarcinoma, indicates that, in addition to the goblet cells, the non-goblet elements may also be involved in the malignant transformation of Barrett's esophagus.
Asunto(s)
Adenocarcinoma/patología , Esófago de Barrett/patología , Neoplasias Esofágicas/patología , Intestinos/patología , Adenocarcinoma/enzimología , Esófago de Barrett/enzimología , Dipeptidil Peptidasa 4/biosíntesis , Neoplasias Esofágicas/enzimología , Humanos , Inmunohistoquímica , Intestinos/enzimología , Metaplasia , Membrana Mucosa/enzimología , Membrana Mucosa/patología , Estudios Retrospectivos , Complejo Sacarasa-Isomaltasa/biosíntesisRESUMEN
To clarify the relationship between diabetes mellitus and carbohydrate digestion, the activities of sucrase and isomaltase, which form a complex enzyme (SI complex) on the brush border membranes, were compared in the progression of diabetes mellitus in Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus with insulin resistance, and Long-Evans Tokushima Otsuka (LETO) rats as non-diabetic controls. Until 40 weeks of age, OLETF rats were obese and had a high plasma glucose level, compared to age-matched LETO rats, but the sucrase and isomaltase activities showed no significant differences between the two strains. Oral glucose tolerance test revealed that during 40-48 weeks of age, NIDDM became very severe with advancing insulin resistance in OLETF rats. In OLETF rats, in contrast to LETO rats, at 48 weeks of age, abnormal increases in the sucrase and isomaltase activities occurred, along with a remarkable decrease in body weight and a further great increase in the plasma glucose level in the non-fasting state. Hyperinsulinemia occurred in 20-week-old OLETF rats; however, at 40 and 48 weeks of age, the plasma insulin level in the non-fasting state in OLETF rats was not significantly different from that in LETO rats. The level of mRNA encoding the SI complex increased abnormally in 48-week-old OLETF rats. These results suggest that the advance of insulin resistance leads to an increase in the expression of the SI complex on the transcriptional level.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina , Intestino Delgado/enzimología , Complejo Sacarasa-Isomaltasa/biosíntesis , Factores de Edad , Animales , Glucemia/análisis , Peso Corporal , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Modelos Animales de Enfermedad , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Masculino , Ratas , Ratas Endogámicas OLETF , Ratas Long-Evans , Triglicéridos/sangreRESUMEN
The molecular mechanisms that regulate the proliferation and differentiation of intestinal mucosal epithelial cells are not well understood. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth factor that may be involved in the maintenance of mucosal epithelial populations and in mediating epithelial repair after injury. The sucrase-isomaltase (SI) gene, which encodes an enterocyte brush border disaccharidase, has served as a model for study of intestinal-specific gene expression and differentiation. KGF down-regulated SI mRNA and protein expression in Caco-2 intestinal epithelial cells but not the expression of other brush border enzymes. The down-regulation was dose- and time-dependent and specifically blocked by anti-KGF antibodies. Transfection experiments using SI promoter constructs demonstrated that KGF decreased SI gene transcription. In contrast, the stability of SI mRNA was not affected by incubation of Caco-2 cells with KGF. Electrophoretic mobility shift analysis demonstrated that binding of nuclear proteins to the SI footprint (SIF) 3 and SIF4 regulatory elements within the SI promoter region was increased in Caco-2 cells that had been incubated with KGF. In transfection experiments using a construct in which tandem copies of the SIF4-binding site were inserted upstream of the SV40 promoter and luciferase gene, incubation with KGF resulted in a significant decrease in luciferase activity. However, transfection with a similar construct containing tandem copies of SIF3 had no significant effect on SV40 promoter activity following KGF treatment. SIF4 may bind E4BP4, a previously identified transcriptional repressor protein. This factor may in part mediate the decrease in SI transcription by KGF in Caco-2 cells.
Asunto(s)
Regulación hacia Abajo/fisiología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/fisiología , Mucosa Intestinal/enzimología , Receptores de Factores de Crecimiento de Fibroblastos , Complejo Sacarasa-Isomaltasa/genética , Células CACO-2 , Diferenciación Celular , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/genética , Complejo Sacarasa-Isomaltasa/biosíntesisRESUMEN
Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268-8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.
Asunto(s)
Rotavirus/fisiología , Complejo Sacarasa-Isomaltasa/metabolismo , Transporte Biológico , Células CACO-2 , Membrana Celular/metabolismo , Citoesqueleto/fisiología , Humanos , Mucosa Intestinal/metabolismo , Microvellosidades , Complejo Sacarasa-Isomaltasa/biosíntesisRESUMEN
Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation and adaptation processes in the mammalian intestinal epithelium. Insight into these mechanisms has been gained by the study of transcriptional regulation of the sucrase-isomaltase gene. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes such as sucrase-isomaltase will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.
Asunto(s)
Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Complejo Sacarasa-Isomaltasa/biosíntesis , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Mucosa Intestinal/crecimiento & desarrollo , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Mamíferos , Transcripción GenéticaRESUMEN
During postnatal maturation of the rat small intestine, glucocorticoid hormones (GC) and thyroxine (T4) act synergistically to elicit a precocious increase of sucrase activity. The current work shows that the synergistic effect on sucrase activity is paralleled by increased steady-state levels of sucrase-isomaltase mRNA. The enhancing effects of T4 on dexamethasone (DEX)-induced sucrase activity was seen even after prolonged treatment (9 days). Moreover, when the location of sucrase-bearing cells was examined after 2 days of hormone treatment, there was distinctly stronger immunostaining of sucrase in the presence of T4, and the sucrase-bearing cells were located on the lower quarter of the intestinal villi regardless of whether the animals received DEX or T4 plus DEX. Thus, despite predictions from the literature, there was no evidence for increased migration in the presence of T4. Instead, we conclude that the synergism between the two hormones is due to greater accumulation of sucrase-isomaltase per epithelial cell.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Yeyuno/enzimología , Complejo Sacarasa-Isomaltasa/biosíntesis , Tiroxina/farmacología , alfa-Glucosidasas/biosíntesis , Animales , Proteínas Bacterianas , Movimiento Celular , Sinergismo Farmacológico , Yeyuno/anatomía & histología , Yeyuno/crecimiento & desarrollo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Complejo Sacarasa-Isomaltasa/genética , Factores de Tiempo , alfa-Glucosidasas/genéticaRESUMEN
The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes [sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Insulina/análogos & derivados , Insulina/farmacología , Mucosa Intestinal/enzimología , Receptor de Insulina/fisiología , Transducción de Señal/fisiología , Complejo Sacarasa-Isomaltasa/biosíntesis , Transcripción Genética , Envejecimiento/metabolismo , Aminopeptidasas/metabolismo , Animales , Duodeno/efectos de los fármacos , Duodeno/crecimiento & desarrollo , Humanos , Íleon/efectos de los fármacos , Íleon/crecimiento & desarrollo , Factor I del Crecimiento Similar a la Insulina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/crecimiento & desarrollo , Lactasa , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptor de Insulina/efectos de los fármacos , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Sacarasa/metabolismo , alfa-Glucosidasas/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
An abnormally high level of the sucrase-isomaltase (SI) complex in the small intestine of rats with streptozotocin-induced insulin-dependent diabetes mellitus (IDDM) was normalized in 11 h by the administration of insulin, in addition to normalization of the blood glucose level. Phlorizin, an inhibitor of renal glucose reabsorption, also caused normalization of the blood glucose level in the IDDM rats; however, the level of the SI complex was barely changed. When mucosa explants were cultured in a medium, the SI complex synthesized during the cultivation was accumulated as its precursor protein without maturation, owing to the absence of pancreatic proteases, and the amount of the precursor protein that accumulated in the explants was decreased by the addition of insulin into the medium. Further, the mRNA level of the SI complex in the explants incubated with insulin was obviously lower than that in the absence of insulin. These results indicate that insulin has a suppressive effect on the synthesis of the SI complex, presumably by decreasing the transcriptional level of the gene encoding the complex, in small-intestinal epithelial cells. Thus the synthesis of the SI complex might exceed normal levels in the epithelial cells as a direct result of the depletion of insulin under IDDM conditions.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Células Epiteliales/enzimología , Insulina/farmacología , Intestino Delgado/enzimología , Complejo Sacarasa-Isomaltasa/antagonistas & inhibidores , Complejo Sacarasa-Isomaltasa/biosíntesis , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Humanos , Inyecciones Intravenosas , Insulina/administración & dosificación , Mucosa Intestinal/enzimología , Intestino Delgado/efectos de los fármacos , Yeyuno , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , EstreptozocinaRESUMEN
The intestinal sucrase-isomaltase (SI) complex is a glycoprotein of the small intestine brush border membrane that plays an important role in the final degradation of carbohydrate. To clone the chicken SI, we employed reverse transcriptase polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products exhibited one amplified band of approximately 800 bp. The fragment was extracted from the gel and sequenced. The cDNA sequence of the chicken SI is 786 bp in length and exhibits 99% identity at the nucleotide level to the Homo sapiens SI mRNA. Using our cDNA as a probe, Northern analysis revealed a transcript of approximately 6.0 kb in chicken jejunum and ileum tissues.
Asunto(s)
Pollos/genética , Complejo Sacarasa-Isomaltasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Electroforesis en Gel de Agar , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conejos , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Complejo Sacarasa-Isomaltasa/biosíntesis , Complejo Sacarasa-Isomaltasa/químicaRESUMEN
Developmental expression of the sucrase-isomaltase (SI) gene in the mouse intestine involves two major transitions that correspond to critical developmental events. Low levels of SI mRNA were first identified in day 16.5 fetal mouse intestine, immediately after the transition from stratified endoderm to a columnar epithelium organized in nascent villi. Low levels were maintained until the third week of life, when induction of SI mRNA to adult levels was observed coincident with the time of weaning. The mechanism of this pattern of SI gene expression was studied in transgenic mice using a reporter gene construct containing an SI gene promoter that is evolutionarily conserved between mouse and human (nucleotides -201 to +54 of the mouse SI gene). This promoter included the necessary regulatory information to direct transcription to enterocytes in developmental and differentiation-dependent patterns that recapitulated the expression of the endogenous SI gene. However, transgenes lacked the ability to direct induction of precocious expression in suckling animals after administration of corticosteroids. These findings define a short SI gene promoter that contains cis-acting elements that are responsible for developmental and differentiation-dependent transcriptional regulation.