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1.
Food Funct ; 11(2): 1547-1559, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32003372

RESUMO

Aging and overweight are involved in prostatic lesion development, due to their association with cell proliferation, hormonal imbalance and angiogenesis. The jaboticaba fruit is rich in bioactive compounds, showing potential chemopreventive action such as the capacity to modulate hormones and angiogenesis hallmarks. This study aimed to evaluate the jaboticaba extract (PJE) effect on the prostate morphology and on molecules related to hormone signaling and angiogenesis, during aging and/or high-fat diet (HFD) intake. Seventy FVB mice were distributed into experimental groups: YG group (young: 3 month old mice), AG group (aged: 11 month old mice), HfAG group (aged + HFD), JAGI group (aged + 2.9 g kg-1 PJE), JAGII group (aged + 5.8 g kg-1 PJE), HfJAGI group (aged + HFD + 2.9 g kg-1 PJE) and HfJAGII group (aged + HFD + 5.8 g kg-1 PJE). The ventral prostate was collected for morphological, immunohistochemistry and western-blotting analysis after 60 days of treatment. All PJE treatments promoted hormonal signaling balance and inhibited angiogenesis in the prostates of aged or HFD-fed aged mice, leading to the maintenance of healthy prostate morphology. A high dose of the PJE (JAGII and HfJAGII groups) led to the best capacity to reduce AR (58.40% and 74.42%; p = 0.0240 and p = 0.0023), ERα (30.29% and 45.12%; p = 0.0004 and p < 0.0001), aromatase (39.54% and 55.94%; p = 0.0038 and p = 0.0020), and VEGF (50.81% and 67.68%; p < 0.0001) and increase endostatin immunoexpression. Moreover, HFD intake intensified the hormonal and angiogenic alterations in the aged mouse prostates, contributing to the increase in premalignant lesion incidence. The PJE exerted a dose-dependent positive effect on aged or HFD-fed aged mouse prostates, contributing to the gland microenvironment recovery, mainly due to the hormonal and angiogenic balance. Therefore, we suggest that the PJE can be a potential candidate for prostatic lesion prevention.


Assuntos
Envelhecimento/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Myrtaceae/química , Extratos Vegetais/farmacologia , Próstata/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Masculino , Camundongos
2.
Life Sci ; 216: 10-21, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30414427

RESUMO

AIMS: Evidence suggests that administration of a high-fat diet (HFD) results in changes in the intestinal lumen environment. Gut dysbiosis associated with intestinal barrier disruption may be involved in type 2 diabetes mellitus (T2DM) development through increased intestinal permeability, which would trigger an inflammatory response leading to peripheral insulin resistance state and ultimately T2DM. In this study, we investigated the effect of the intestinal luminal content isolated from control or HFD-fed prediabetic mice upon the tight junction (TJ)-mediated epithelial barrier in Caco-2 and MDCK epithelial cell lines. METHODS/KEY FINDINGS: Exposure to small intestine luminal content (SI) isolated from HFD-fed prediabetic mice induced a more significant decrease in transepithelial electrical resistance (TEER), associated with higher paracellular flux in Caco-2 and MDCK cells after 6 h and 4 h respectively, as compared to the SI obtained from control mice. Such changes were accompanied by a significant decrease in TJ content of claudins, occludin, and ZO-1, indicative of disruption of the TJ barrier. Meanwhile, large intestine luminal content from control (Ctrl-LI) and prediabetic (HFD-LI) animals did not change TEER significantly, however, paracellular flux was significantly increased after 24 h, accompanied by a decrease in ZO-1 (after HFD-LI exposure) in Caco-2 and significant changes in the junctional distribution of claudins-1, -2, occludin and ZO-1 proteins in MDCK, particularly after HFD-LI exposure. SIGNIFICANCE: Luminal components of intestinal content, altered by HFD exposure, induce impairment of the TJ structure and function in vitro, corroborating the idea of a role of the intestinal paracellular barrier in the obesity-related T2DM pathogenesis.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Conteúdo Gastrointestinal , Mucosa Intestinal/fisiopatologia , Estado Pré-Diabético/fisiopatologia , Animais , Células CACO-2 , Claudinas/metabolismo , Modelos Animais de Doenças , Cães , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Permeabilidade , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1/metabolismo
3.
Life Sci ; 188: 149-157, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28882647

RESUMO

AIMS: Type 2 diabetes mellitus (T2DM) is one of the most prevalent diseases worldwide. Diabetic nephropathy (DN) is a complication of diabetes and the mechanisms underlying onset and progression of this disease are not fully understood. It has been shown that hyperglycemia is an independent factor to predict the development of DN in individuals with T2DM, however, a link between high plasma glucose levels and renal tubular injuries in DN remains unknown. In this study, we investigated the effect of high levels of glucose (i.e. 180 or 360mg/dL) for up to 24h (acute) or over 72h (chronic) upon tight junction (TJ)-mediated epithelial barrier integrity of the kidney tubular cell line, MDCK. METHODS/KEY FINDINGS: High levels of glucose (180 and 360mg/dL) induced a decrease in transepithelial electrical resistance associated with an increase in TJ cation selectivity at 24h or in TJ permeability to a paracellular marker, Lucifer Yellow, at 72h-exposure when compared to control group (exposed to 100mg/dL glucose). Immunofluorescence analyses showed that glucose treatment induced a significant decrease in the tight junctional content of claudins-1 and -3 as well as a significant increase in claudin-2 (particularly at 24h-exposure) and a time-dependent change in occludin/ZO-1 junctional content. The analyses of total cell content of these junctional proteins by Western blot did not reveal significant changes, except in claudin-2 expression. SIGNIFICANCE: Our data suggest that high levels of glucose induce time-dependence changes in TJ structure in MDCK monolayers, suggesting a possible link between hyperglycemia-induced tubular epithelial barrier disruption and diabetic nephropathy.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glucose/efeitos adversos , Túbulos Renais/citologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologia , Animais , Células Cultivadas , Claudina-1/metabolismo , Claudina-2/metabolismo , Claudina-3/metabolismo , Cães , Células Madin Darby de Rim Canino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ocludina/metabolismo , Junções Íntimas/metabolismo
4.
Braz J Med Biol Res ; 50(10): e5948, 2017 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-28876363

RESUMO

Atropine (AT) and dipyrone (Dp) induce a delay of gastric emptying (GE) of liquids in rats by inhibiting muscarinic receptors and activating ß2-adrenergic receptors, respectively. The objective of the present study was to determine the effects of pretreatment with AT and Dp, given alone or in combination, on the effect of hypoglycemia in the liquid GE in rats. Male Wistar adult rats (280-310 g) were pretreated intravenously with AT, Dp, AT plus Dp or their vehicle and then treated 30 min later with iv insulin or its vehicle (n=8-10 animals/group). Thirty min after treatment, GE was evaluated by determining, in awake rats, the percent gastric retention (%GR) of a saline meal labeled with phenol red administered by gavage. The results indicated that insulin induced hypoglycemia in a dose-dependent manner resulting in a significant reduction in %GR of liquid only at the highest dose tested (1 U/kg). Pretreatment with AT significantly increased %GR in the rats treated with 1 U/kg insulin. Surprisingly, after pretreatment with AT, the group treated with the lowest dose of insulin (0.25 U/kg) displayed significantly lower %GR compared to its control (vehicle-treated group), which was not seen in the non-pretreated animals. Pretreatment with Dp alone at the dose of 40 mg/kg induced an increase in %GR in both vehicle and 0.25 U/kg-treated rats. A higher dose of Dp alone (80 mg/kg) significantly reduced the effect of a marked hypoglycemia induced by 1 U/kg of insulin on GE while in combination with AT the effect was completely abolished. The results with AT suggest that moderate hypoglycemia may render the inhibitory mechanisms of GE ineffective while Dp alone and in combination with AT significantly overcame the effect of hypoglycemia on GE.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Atropina/farmacologia , Dipirona/farmacologia , Esvaziamento Gástrico/efeitos dos fármacos , Hipoglicemia/fisiopatologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Atropina/administração & dosagem , Dipirona/administração & dosagem , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Masculino , Modelos Animais , Pré-Medicação , Ratos , Ratos Wistar
5.
Exp Biol Med (Maywood) ; 242(12): 1214-1226, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28504618

RESUMO

In this study, we investigated the effect of diet supplementation with sodium butyrate (5% w/w), a short-chain fatty acid produced by the intestinal microbiota, on metabolic parameters, body adiposity, hepatic and pancreatic lipid accumulation, beta cell function/mass as well as on the structure and function of the tight junction-mediated intestinal epithelial barrier in both normal and obese/prediabetic C57 mice fed a regular (control) or high-fat diet for 60 days, respectively. Butyrate treatment significantly inhibited all the high-fat-induced metabolic dysfunctions evaluated, i.e. significantly reduced the weight gain and body adiposity as well as the insulin resistant state, hyperglycemia and hyperinsulinemia, without changing food intake. In addition, high-fat-fed mice treated with this short-chain fatty acid displayed no compensatory hyperplasia of pancreatic beta cells nor marked hepatic steatosis as seen in prediabetic mice after high-fat diet only. Isolated pancreatic islets from high-fat-fed mice treated with butyrate showed improvement of the insulin secretion, which was associated with a significant decrease in lipid accumulation within the pancreas. Butyrate enhanced the intestinal epithelial barrier, as revealed by the FITC-Dextran permeability assay, which was accompanied by a significant increase in the junctional content of the tight junction-associated claudin-1 in intestinal epithelia of jejunum, ileum, and colon of both control and high-fat mice. In conclusion, our results showed that diet supplementation with butyrate inhibits the deleterious effects of high-fat diet intake on metabolic parameters and structure/function of several tissues/organs associated with type 2 diabetes mellitus in a mouse model, suggesting a potential use of this short-chain fatty acid in the treatment of this endocrine-metabolic disorder. Impact statement Butyrate is a short-chain fatty acid produced by the intestinal microbiota through the fermentation of non-absorbable carbohydrates and proteins (e.g. fibers). Sodium butyrate incorporated into the diet displayed a protective action on metabolic, hepatic, pancreatic and intestinal alterations induced by high-fat diet in mice, resulting in significant inhibition of the development of a prediabetic state. Thus, our data suggest that butyrate may have a potential therapeutic use in the treatment of type 2 diabetes and related disorders.


Assuntos
Ácido Butírico/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Estado Pré-Diabético , Animais , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Fígado Gorduroso/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Junções Íntimas/efeitos dos fármacos
6.
Biochem Biophys Res Commun ; 478(4): 1534-40, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27576200

RESUMO

The Wnt/ß-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/ß-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active ß-catenin associated with significant increases in protein content and gene expression of ß-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/ß-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM.


Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/patologia , Via de Sinalização Wnt , Animais , Tamanho Celular , Dieta Hiperlipídica , Hiperplasia , Masculino , Camundongos Endogâmicos C57BL
7.
Ann Anat ; 200: 88-97, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25819502

RESUMO

In this study, we investigated a possible sexual dimorphism regarding metabolic response and structural and functional adaptations of the endocrine pancreas after exposure to a high-fat diet (HFd). On chow diet, male and female C57BL/6/JUnib mice showed similar metabolic and morphometric parameters, except that female islets displayed a relatively lower ß-cell:non-ß-cell ratio. After 30 days on HFd, both male and female mice showed increased weight gain, however only the males displayed glucose intolerance associated with high postprandial glycemia when compared to their controls. After 60 days on HFd, both genders became obese, hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, although the metabolic changes were more pronounced in males, while females displayed greater weight gain. In both genders, insulin resistance induced by HFd feeding was compensated by expansion of ß-cell mass without changes in islet cytoarchitecture. Interestingly, we found a strong correlation between the degree of ß-cell expansion and the levels of hyperglycemia in the fed state: male mice fed a 60d-HFd, showing higher glycemic levels also displayed a greater ß-cell mass increase in comparison with female mice. Additionally, sexual dimorphism was also observed regarding the source of ß-cell mass expansion following 60d-HFd: while in males, both hypertrophy and hyperplasia (revealed by morphometry and Ki67 immunoreaction) of ß-cells were observed, female islets displayed only a significant increase in ß-cell size. In conclusion, this study describes gender differences in metabolic response to high fat diet, paralleled by distinct compensatory morphometric changes in pancreatic islets.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Dieta , Ilhotas Pancreáticas/patologia , Doenças Metabólicas/patologia , Animais , Glicemia/metabolismo , Proliferação de Células , Tamanho Celular , Feminino , Intolerância à Glucose , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/ultraestrutura , Masculino , Doenças Metabólicas/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Caracteres Sexuais , Aumento de Peso
8.
Am J Physiol Endocrinol Metab ; 303(1): E144-51, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22569071

RESUMO

Gap junctional intercellular communication between ß-cells is crucial for proper insulin biosynthesis and secretion. The aim of this work was to investigate the expression of connexin (Cx)36 at the protein level as well as the function and structure of gap junctions (GJ) made by this protein in the endocrine pancreas of prediabetic mice. C57BL/6 mice were fed a high-fat (HF) or regular chow diet for 60 days. HF-fed mice became obese and prediabetic, as shown by peripheral insulin resistance, moderate hyperglycemia, hyperinsulinemia, and compensatory increase in endocrine pancreas mass. Compared with control mice, prediabetic animals showed a significant decrease in insulin-secretory response to glucose and displayed a significant reduction in islet Cx36 protein. Ultrastructural analysis further showed that prediabetic mice had GJ plaques about one-half the size of those of the control group. Microinjection of isolated pancreatic islets with ethidium bromide revealed that prediabetic mice featured a ß-cell-ß-cell coupling 30% lower than that of control animals. We conclude that ß-cell-ß-cell coupling mediated by Cx36 made-channels is impaired in prediabetic mice, suggesting a role of Cx36-dependent cell-to-cell communication in the pathogenesis of the early ß-cell dysfunctions that lead to type 2-diabetes.


Assuntos
Comunicação Celular , Conexinas/metabolismo , Regulação para Baixo , Junções Comunicantes/metabolismo , Células Secretoras de Insulina/metabolismo , Estado Pré-Diabético/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Junções Comunicantes/ultraestrutura , Imuno-Histoquímica , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/ultraestrutura , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Obesidade/complicações , Pâncreas/metabolismo , Pâncreas/patologia , Estado Pré-Diabético/complicações , Estado Pré-Diabético/etiologia , Estado Pré-Diabético/patologia , Proteína delta-2 de Junções Comunicantes
9.
Diabetologia ; 53(7): 1428-37, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20361177

RESUMO

AIMS/HYPOTHESIS: Cell-cell coupling mediated by gap junctions formed from connexin (CX) contributes to the control of insulin secretion in the endocrine pancreas. We investigated the cellular production and localisation of CX36 and CX43, and gap junction-mediated beta cell coupling in pancreatic islets from rats of different ages, displaying different degrees of maturation of insulin secretion. METHODS: The presence and distribution of islet connexins were assessed by immunoblotting and immunofluorescence. The expression of connexin genes was evaluated by RT-PCR and quantitative real-time PCR. The ultrastructure of gap junctions and the function of connexin channels were assessed by freeze-fracture electron microscopy and tracer microinjection, respectively. RESULTS: Young and adult beta cells, which respond to glucose, expressed significantly higher levels of Cx36 (also known as Gjd2) than fetal and newborn beta cells, which respond poorly to the sugar. Accordingly, adult beta cells also showed a significantly higher membrane density of gap junctions and greater intercellular exchange of ethidium bromide than newborn beta cells. Cx43 (also known as Gja1) was not expressed by beta cells, but was located in various cell types at the periphery of fetal and newborn islets. CONCLUSIONS/INTERPRETATION: These findings show that the pattern of connexins, gap junction membrane density and coupling changes in islets during the functional maturation of beta cells.


Assuntos
Conexinas/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Animais Recém-Nascidos , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Feminino , Imunofluorescência , Junções Comunicantes/metabolismo , Immunoblotting , Células Secretoras de Insulina/ultraestrutura , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/ultraestrutura , Masculino , Microscopia Eletrônica , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína delta-2 de Junções Comunicantes
10.
Cell Biol Int ; 30(2): 101-13, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16458027

RESUMO

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.


Assuntos
Dexametasona/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Prolactina/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Impedância Elétrica , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Fatores de Tempo
11.
Can J Physiol Pharmacol ; 83(2): 142-51, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15791287

RESUMO

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.


Assuntos
Conexina 43/biossíntese , Conexinas/biossíntese , Ilhotas Pancreáticas/metabolismo , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Células Cultivadas , Conexina 43/genética , Conexinas/genética , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para Cima , Proteína delta-2 de Junções Comunicantes
12.
Diabetes Metab ; 28(6 Pt 2): 3S25-8; discussion 3S108-12, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12688630

RESUMO

The presence of thioredoxin peroxidase (TPx), also known as thiol specific antioxidant (TSA), was investigated in neonatal and adult rat islets, and in the beta-cell line HIT-T15. Western blotting of extracts from neonatal and adult pancreatic islets and from the tumoral cell line HIT-T15 revealed the presence of a 25 kDa protein that comigrated with purified yeast TPx. Endocrine pancreatic TPx accounted for approximately 0.01% of the total protein content. Treatment with H2O2 for 3 h increased the expression of TPx in HIT-T15 cells. The distribution of TPx throughout the islet cells was confirmed by immunocytochemistry. Since pancreatic beta-cells possess a weak antioxidant enzyme defense system, especially with regard to hydrogen peroxidase-decomposing enzymes, the presence of a TPx analog in islets suggests that this enzyme may play a role in protecting pancreatic cells against reactive oxygen species.


Assuntos
Ilhotas Pancreáticas/enzimologia , Proteínas de Neoplasias , Peroxidases/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Células Cultivadas , Insulinoma , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Estresse Oxidativo/fisiologia , Neoplasias Pancreáticas , Peroxirredoxinas , Ratos , Ratos Wistar , Células Tumorais Cultivadas
13.
Pancreas ; 23(2): 177-85, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11484920

RESUMO

Fetal and neonatal pancreatic islets have lower insulin secretory responses compared with adult islets. In culture conditions and after treatment with mammosomatotropic hormones, neonatal islets undergo maturation of the secretory machinery that might involve regulation of cell-cell contacts within the islet. This study is an investigation of the effect of prolonged culturing and in vitro treatment with prolactin on the expression of the gap junction-associated connexin 43 and the adherens junction-associated beta-catenin in cultured neonatal rat islets. Pancreatic islets from neonatal Wistar rats were cultured for 24 hours or 7 days, and the treated group was exposed to 2 microg/mL prolactin daily for 7 days. Connexin 43 and beta-catenin were barely detected at the cell-cell contacts in 24-hour-cultured islets, as revealed by immunocytochemical analysis. Nevertheless, both junctional proteins were well expressed at the junctional region in islet cells cultured for 7 days and showed even greater staining in islets after long-term prolactin treatment. In accordance with the morphologic data, neonatal islets cultured for 24 hours displayed a relatively low level of connexin 43, as determined by Western blot analysis. Culturing for 7 days or combined prolactin treatment induced a significant increase in connexin 43 expression; this was 40% greater in the prolactin-treated group than in the control group. Furthermore, an enhancement of the expression of beta-catenin and translocation of this protein to the cell-cell contact site was also observed in neonatal islets cultured for 7 days compared with those cultured for 24 hours. In vitro prolactin treatment induced even greater expression of beta-catenin in islet cells. A correlation was observed between the increased expression of these junctional proteins and an increase in insulin secretion in cultured neonatal islets. In conclusion, prolonged culturing and in vitro treatment with prolactin induce the modulation of gap and adherens junctional proteins in pancreatic islets, which may be an important event in the in vitro maturation process of neonatal islet cells.


Assuntos
Conexina 43/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ilhotas Pancreáticas/metabolismo , Transativadores , Animais , Animais Recém-Nascidos , Células Cultivadas , Imuno-Histoquímica , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Prolactina/farmacologia , Ratos , Fatores de Tempo , beta Catenina
14.
Infect Immun ; 68(12): 7202-8, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11083857

RESUMO

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.


Assuntos
Salmonella typhimurium/patogenicidade , Junções Íntimas/fisiologia , Animais , Linhagem Celular , Difusão , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Permeabilidade , Esfingomielinas/metabolismo , Estaurosporina/farmacologia
15.
Eur J Cell Biol ; 76(2): 85-92, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9696347

RESUMO

Polarized monolayers of strain II Madin-Darby canine kidney cells (MDCK II) were treated with vanadate/H2O2, known inhibitors of protein tyrosine phosphatase activity. Vanadate/H2O2 treatment resulted in a rapid increase in paracellular permeability as revealed by decreased transepithelial resistance and increased permeability to inulin. These alterations in epithelial barrier function coincided with increased phosphotyrosine immunofluorescence in the vicinity of intercellular junctions and with redistribution of F-actin, the adherens junction protein E-cadherin and the tight junction protein ZO-1. The effects of vanadate/H2O2 on intercellular junction permeability and protein distribution were completely blocked by the specific protein tyrosine kinase (PTK) inhibitor tyrphostin 25 and partially inhibited by the alternative PTK inhibitor genistein. The relative potency of these two inhibitors in blocking the effects of vanadate/H2O2 on intercellular junctions correlated with their abilities to inhibit tyrosine phosphorylation. The potent ser/thr protein kinase inhibitor staurosporine had only a small influence on the vanadate/H2O2-induced increase in paracellular permeability and did not affect the observed redistribution of intercellular junction proteins or phosphotyrosine immunofluorescence. The relative potencies of these distinct protein kinase inhibitors in reversing the effects of vanadate/H2O2 indicate that these effects are directly related to tyrosine phosphorylation. In conclusion, our data provide evidence that enhanced tyrosine phosphorylation of intercellular junction proteins in MDCK epithelia increases paracellular permeability and can also induce prominent reorganization of the junctional complex.


Assuntos
Fosfotirosina/metabolismo , Junções Íntimas/metabolismo , Tirfostinas , Actinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cães , Genisteína/farmacologia , Imuno-Histoquímica , Inulina/metabolismo , Rim , Proteínas de Membrana/metabolismo , Nitrilas/farmacologia , Peróxidos/farmacologia , Faloidina/análogos & derivados , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Rodaminas , Estaurosporina/farmacologia , Junções Íntimas/efeitos dos fármacos , Vanadatos/farmacologia , Proteína da Zônula de Oclusão-1
16.
Cell Tissue Res ; 291(2): 267-76, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9426313

RESUMO

Distinct epithelial MDCK cell strains displaying extremes in transepithelial electrical resistance (paracellular permeability) have been established in co-culture and the subsequent cellular behaviour and formation of junctional complexes investigated. After high-density seeding, MDCK strain I and II cells in co-culture are initially randomly distributed but subsequently sort themselves out in a time-dependent manner to form separate homotypic aggregates. The final pattern of cell arrangement of homotypic aggregates depends on the relative seeding proportion of each cell type. Immunostaining of established marker proteins for junctional complexes has revealed that MDCK I and II cells differ in the degree of expression of the zonula-adherens-associated protein, E-cadherin, their cytoskeletal architecture and the junctional distribution of a desmosomal protein, and by showing subtle differences in tight junction staining for the zona-occludens-associated proteins, ZO-1 and occludin. The distinct pattern of junctional protein expression is maintained when the two MDCK strains are co-cultured; however, morphologically atypical intercellular junctions between heterotypic cells at the boundary of homotypic cell aggregates have been observed. It has been suggested that cell sorting, a phenomenon yet to be completely understood, is involved in important morphogenetic processes. We propose that co-culture of strains of the well-characterised MDCK cell line may be a novel but well-defined cell system for studying epithelial cell rearrangement and sorting in intact epithelial sheets.


Assuntos
Caderinas/fisiologia , Células Epiteliais/fisiologia , Junções Intercelulares/química , Rim/citologia , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Animais , Caderinas/análise , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Agregação Celular/fisiologia , Permeabilidade da Membrana Celular , Técnicas de Cocultura , Cães , Células Epiteliais/química , Junções Intercelulares/fisiologia , Potenciais da Membrana , Proteínas de Membrana/análise , Microscopia Eletrônica , Microscopia de Fluorescência , Morfogênese , Ocludina , Fosfoproteínas/análise , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteína da Zônula de Oclusão-1
17.
Infect Immun ; 63(1): 356-9, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7806378

RESUMO

Short-term infection of MDCK II monolayers with Salmonella typhimurium SL1344 caused a progressive decrease in transepithelial electrical resistance concomitant with decreased cation permselectivity and increased paracellular inulin flux. Cytochemical staining of F-actin, E-cadherin, and ZO-1 revealed the concentration of each junctional protein in invaded cells as a result of contraction at their apical poles and resultant distortion of adjacent uninvaded cells.


Assuntos
Permeabilidade da Membrana Celular , Junções Intercelulares/ultraestrutura , Potenciais da Membrana , Salmonella typhimurium/crescimento & desenvolvimento , Actinas/isolamento & purificação , Animais , Caderinas/isolamento & purificação , Polaridade Celular , Células Cultivadas , Cães , Impedância Elétrica , Epitélio/microbiologia , Imunofluorescência , Rim/citologia , Proteínas de Membrana/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Proteína da Zônula de Oclusão-1
18.
Biochim Biophys Acta ; 1222(2): 147-58, 1994 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-8031850

RESUMO

The polarised nature of the increase in paracellular permeability induced by Ca(2+)-chelation with EGTA was investigated in several cultured epithelial cell lines. In strain I MDCK cells (canine kidney cells), a marked decrease (> 90%) in transepithelial electrical resistance (RT) and increase in mannitol and inulin permeabilities were only observed after addition of EGTA (for 4 h) to either basolateral (basal) or both (apical+basal) bathing solutions; apical Ca(2+)-chelation resulted in significant smaller changes (approximately 30%) in these variables. The increase in paracellular permeability upon basal EGTA addition was significantly lower than that produced by simultaneous apical and basal addition of 2 mM EGTA. A higher concentration of EGTA (20 mM) did not significantly eliminate this difference in potency between basal and apical+basal Ca(2+)-chelation. The polarised Ca(2+)-dependence of the paracellular barrier was associated with polarised effects on the junctional/cytoskeletal protein distribution. Basal or apical+basal EGTA addition induced substantial internalisation of uvomorulin with some cellular redistribution of the perijunctional actin ring and desmosomes and gaps in ZO-1 location between adjacent cells. In addition, polarised Ca(2+)-dependence of the paracellular barrier (assessed by measuring RT) was observed also in strain II MDCK and two human adenocarcinoma intestinal cell lines, Caco-2 and HCT-8, demonstrating generality of the phenomenon. Therefore, the data show a polarity in the ability of EGTA to enhance epithelial permeability and induce cellular redistribution of cytoskeletal/junctional proteins in several epithelia. The basolateral membrane sensitivity to Ca(2+)-chelation might be explained by the polarised distribution of uvomorulin.


Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular , Animais , Caderinas/metabolismo , Cálcio/química , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Cães , Ácido Egtázico/química , Ácido Egtázico/farmacologia , Condutividade Elétrica , Impedância Elétrica , Inulina/metabolismo , Manitol/metabolismo , Potássio/metabolismo
19.
Histochemistry ; 101(3): 185-94, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7520032

RESUMO

Strains I and II of Madin-Darby canine kidney (MDCK) cells, which differ markedly in transepithelial resistance (RT) and paracellular permeability, have been used to investigate whether differences in the cellular content of uvomorulin/E-cadherin and phosphotyrosine may be correlated with junctional properties. Using immunocytochemistry, the strain I "tight" epithelia showed significantly stronger uvomorulin staining at regions of cell-cell contact compared with strain II "leaky" MDCK epithelia. In contrast, strain I MDCK cells showed a relatively faint phosphotyrosine staining, distributed evenly throughout the cytoplasm, while strain II MDCK cells displayed intense staining for phosphotyrosine residues in the junctional region and the lateral cell membrane with additional labelling of the cytoplasm. Exposure to vanadate in conjunction with H2O2 (which are potent inhibitors of protein tyrosine phosphatases) resulted in a dramatic increase in phosphotyrosine staining at the intercellular area and, concomitantly, induced changes in cell morphology, a significant decrease in RT, increase in paracellular inulin permeability, and time-dependent disappearance of uvomorulin from the cell-cell contact sites. Moreover, the effects of vanadate/H2O2 treatment were more dramatic in strain II compared with strain I cells, consistent with greater generation of tyrosine-modified protein in strain II cells. An inverse relationship was demonstrated between membrane-associated uvomorulin/E-cadherin and cellular phosphotyrosine content, which varied between the two strains of MDCK cells and when phosphotyrosine was directly manipulated. These data support the hypothesis that regulation of paracellular permeability may result from specific tyrosine phosphorylation of protein components of the junctional complex.


Assuntos
Caderinas/metabolismo , Permeabilidade da Membrana Celular , Membrana Celular/fisiologia , Junções Intercelulares/fisiologia , Fosfoproteínas/metabolismo , Tirosina/análogos & derivados , Animais , Caderinas/análise , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Cães , Condutividade Elétrica/efeitos dos fármacos , Eletrofisiologia/métodos , Epitélio/fisiologia , Epitélio/ultraestrutura , Peróxido de Hidrogênio/farmacologia , Junções Intercelulares/ultraestrutura , Rim , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Fosfoproteínas/análise , Fosfotirosina , Tirosina/análise , Vanadatos/farmacologia
20.
Braz J Med Biol Res ; 26(9): 1009-14, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8298525

RESUMO

The effects of cold restraint stress on gastric emptying (GE) and the involvement of the hypothalamic paraventricular nucleus (PVN) were investigated in male Wistar rats (200-250 g body weight). Electrolytic lesions were produced stereotaxically in the nucleus by passing a 2.0-mA current for 10 s through stainless steel electrodes. GE was measured by means of a liquid test meal of 5% (w/v) glucose solution plus phenol red (6 mg/dl) dye as marker, given by orogastric infusion. Cold restraint stress induces a significant increase (43.7%, N = 11) in gastric retention of a 5% glucose solution in rats, i.e., a delay in GE of this solution. However, restraint stress alone does not produce any change. Both truncal vagotomy and electrolytic lesion of the PVN completely block the cold restraint-induced delay in GE. However, PVN lesion per se results in a decrease of GE (30.6%, N = 10) when compared to nonoperated controls. In addition, PVN-lesioned rats exposed to cold restraint present a slightly faster GE (14.7%, N = 11) than controls, demonstrating an opposite response to that initially observed without lesion. These data suggest an important role for PVN efferents, probably influencing medullary vagal preganglionic neurons, in the development of this gastric motor impairment under stress conditions.


Assuntos
Esvaziamento Gástrico/fisiologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Estresse Fisiológico/fisiopatologia , Animais , Temperatura Baixa , Glucose , Masculino , Núcleo Hipotalâmico Paraventricular/lesões , Ratos , Restrição Física , Técnicas Estereotáxicas , Vagotomia
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