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Deficient wound healing is frequently observed in patients diagnosed with diabetes, a clinical complication that compromises mobility and leads to limb amputation, decreasing patient autonomy and family lifestyle. Fibroblasts are crucial for secreting the extracellular matrix (ECM) to pave the wound site for endothelial and keratinocyte regeneration. The biosynthetic pathways involved in collagen production and crosslinking are intimately related to fibroblast redox homeostasis. In this study, two sets of human dermic fibroblasts were cultured in normal (5 mM) and high (25 mM)-glucose conditions in the presence of 1 µM selenium, as sodium selenite (inorganic) and the two selenium amino acids (organic), Se-cysteine and Se-methionine, for ten days. We investigated the ultrastructural changes in the secreted ECM induced by these conditions using scanning electron microscopy (SEM). In addition, we evaluated the redox impact of these three compounds by measuring the basal state and real-time responses of the thiol-based HyPer biosensor expressed in the cytoplasm of these fibroblasts. Our results indicate that selenium compound supplementation pushed the redox equilibrium towards a more oxidative tone in both sets of fibroblasts, and this effect was independent of the type of selenium. The kinetic analysis of biosensor responses allowed us to identify Se-cysteine as the only compound that simultaneously improved the sensitivity to oxidative stimuli and augmented the disulfide bond reduction rate in high-glucose-cultured fibroblasts. The redox response profiles showed no clear association with the ultrastructural changes observed in matrix fibers secreted by selenium-treated fibroblasts. However, we found that selenium supplementation improved the ECM secreted by high-glucose-cultured fibroblasts according to endothelial migration assessed with a wound healing assay. Direct application of sodium selenite and Se-cysteine on purified collagen fibers subjected to glycation also improved cellular migration, suggesting that these selenium compounds avoid the undesired effect of glycation.
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BACKGROUND: The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a result of its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. RESULTS: Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P. canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24), including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. CONCLUSION: From an integrative physiological and anatomical perspective, P. canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Gastrópodes , Animais , Proteômica , Cisteína , Trato Gastrointestinal , Peptídeos , Metaloproteases , Serina Proteases , Serina Endopeptidases , SerinaRESUMO
Coronary artery endothelial cells (CAEC) exert an important role in the development of cardiovascular disease. Dysfunction of CAEC is associated with cardiovascular disease in subjects with type 2 diabetes mellitus (T2DM). However, comprehensive studies of the effects that a diabetic environment exerts on this cellular type are scarce. The present study characterized the molecular perturbations occurring on cultured bovine CAEC subjected to a prolonged diabetic environment (high glucose and high insulin). Changes at the metabolite and peptide level were assessed by Liquid Chromatography-Mass Spectrometry (LC-MS2) and chemoinformatics. The results were integrated with published LC-MS2-based quantitative proteomics on the same in vitro model. Our findings were consistent with reports on other endothelial cell types and identified novel signatures of DNA/RNA, amino acid, peptide, and lipid metabolism in cells under a diabetic environment. Manual data inspection revealed disturbances on tryptophan catabolism and biosynthesis of phenylalanine-based, glutathione-based, and proline-based peptide metabolites. Fluorescence microscopy detected an increase in binucleation in cells under treatment that also occurred when human CAEC were used. This multi-omics study identified particular molecular perturbations in an induced diabetic environment that could help unravel the mechanisms underlying the development of cardiovascular disease in subjects with T2DM.
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Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Aminoácidos/metabolismo , Animais , Doenças Cardiovasculares/complicações , Bovinos , DNA/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Humanos , Metabolismo dos Lipídeos , Peptídeos/metabolismo , RNA/metabolismoRESUMO
Hydrogen sulfide (H2S) has been known for its toxicity. However, recent studies have focused on the mechanisms involved in endogenous production and function. To date, the H2S role in insulin signaling and glucose homeostasis is unclear. This uncertainty is even more evident in skeletal muscle, a physiological niche highly relevant for regulating glycemia in response to insulin. This study aimed to investigate the role of H2S on insulin signaling and glucose uptake in the L6 skeletal muscle cell line. We evaluated the endogenous synthesis with the fluorescent dye, 7-azido-4-methyl coumarin (7-AzMC). Glucose restriction-induced an increase in the endogenous levels of H2S, likely through stimulation of cystathionine γ-lyase activity, as its specific inhibitor, PAG (5 mM) prevented this increase, and mRNA levels of CSE decreased with glucose and amino acid restriction. Exogenous H2S reduced insulin-induced glucose uptake at 0.5 up to 24 h, an effect dissociated from the level of Akt phosphorylation. Our results show that glucose restriction induces endogenous production of H2S via CSE. In addition, H2S disrupts insulin-induced glucose uptake independent of the Akt pathway. These results suggest that H2S antagonism over insulin-induced glucose uptake could help maintain the plasmatic glucose levels in conditions that provoke hypoglycemia, which could serve as an H2S-regulated mechanism for maintaining glucose plasmatic levels through the inhibition of the skeletal muscle insulin-depended glucose uptake.
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Sulfeto de Hidrogênio , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Glucose/metabolismo , Sulfeto de Hidrogênio/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
In mammals, the daily variation in the ecology of the intestinal microbiota is tightly coupled to the circadian rhythm of the host. On the other hand, a close correlation between increased body weight and light pollution at night has been reported in humans and animal models. However, the mechanisms underlying such weight gain in response to light contamination at night remain elusive. In the present study, we tested the hypothesis that dim light pollution at night alters the colonic microbiota of mice, which could correlate with weight gain in the animals. By developing an experimental protocol using a mouse model that mimics light contamination at night in urban residences (dLAN, dim light at night), we found that mice exposed to dLAN showed a significant weight gain compared with mice exposed to control standard light/dark (LD) photoperiod. To identify possible changes in the microbiota, we sampled two stages from the resting period of the circadian cycle of mice (ZT0 and ZT10) and evaluated them by high-throughput sequencing technology. Our results indicated that microbial diversity significantly differed between ZT0 and ZT10 in both LD and dLAN samples and that dLAN treatment impacted the taxonomic composition, functions, and interactions of mouse colonic microbiota. Together, these results show that bacterial taxa and microbial metabolic pathways might be involved with the mechanisms underlying weight gain in mice subjected to light contamination at night.
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Colo/microbiologia , Microbioma Gastrointestinal , Poluição Luminosa/efeitos adversos , Aumento de Peso , Animais , CamundongosRESUMO
Metabolic syndrome is a condition whose incidence has been increasing around the world. It promotes a metabolic state of chronic systemic inflammation, correlated to cellular stress and genetic mutations, and subsequently with deadly chronic diseases, such as type 2 diabetes mellitus, cardiovascular diseases, and cancer. A randomized placebo-controlled study (n = 156) was conducted to determine the effects of consuming an enriched bread with 0.05% of a 1:1 mixture of (-)-epicatechin and quercetin on anthropometric and biochemical parameters of the participants. As a result, total cholesterol, LDL-cholesterol, total triglycerides, and fasting plasma glucose significantly decreased after three months of daily enriched bread consumption. Nuclear abnormalities in buccal epithelium cells also decreased (15.8 ± 3.2 down to 8.3 ± 1.0), showing a genoprotective effect. The antioxidant properties of these compounds were observed by monitoring changes in the cytoplasmic redox tone of intact Caco-2 cells expressing HyPer, a fluorescent redox biosensor. The combination of (-)-epicatechin and quercetin changes the cytoplasmic redox ambient in living cells and significantly improves biochemical parameters related to metabolic syndrome, and decreases the number of cell abnormalities in buccal epithelium cells of patients.
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Catequina , Diabetes Mellitus Tipo 2 , Síndrome Metabólica , Antioxidantes/farmacologia , Células CACO-2 , Catequina/farmacologia , Humanos , Síndrome Metabólica/prevenção & controle , Quercetina/farmacologiaRESUMO
Most of the biological impacts of Vitamin E, including the redox effects, have been raised from studies with α-tocopherol only, despite the fact that tocopherol-containing foods carry mixed tocopherol isomers. Here, we investigated the cellular mechanisms involved in the immediate antioxidant responses evoked by α-, γ- and δ-tocopherol in Caco-2 cells. In order to track the cytosolic redox impact, we performed imaging on cells expressing HyPer, a fluorescent redox biosensor, while cytosolic calcium fluctuations were monitored by means of Fura-2 dye and imaging. With this approach, we could observe fast cellular responses evoked by the addition of α-, γ- and δ-tocopherol at concentrations as low as 2.5 µM. Each isomer induced rapid and consistent increases in cytosolic calcium with fast kinetics, which were affected by chelation of extracellular Ca2+, suggesting that tocopherols promoted a calcium entry upon the contact with the plasma membrane. In terms of redox effects, δ-tocopherol was the only isomer that evoked a significant change in the HyPer signal at 5 µM. By mimicking Ca2+ entry with ionomycin and monensin, a decline in the HyPer signal was induced as well. Finally, by silencing calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an intracellular Ca2+ chelator, none of the isomers were able to induce redox changes. Altogether, our data indicate that an elevation in cytoplasmic Ca2+ is necessary for the development of a tocopherol-induced antioxidant impact on the cytoplasm of Caco-2 cells reported by HyPer biosensor.
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Sucralose is a non-caloric artificial sweetener widely used in processed foods that reportedly affects energy homeostasis through partially understood mechanisms. Mitochondria are organelles fundamental for cellular bioenergetics that are closely related to the development of metabolic diseases. Here, we addressed whether sucralose alters mitochondrial bioenergetics in the enterocyte cell line Caco-2. Sucralose exposure (0.5-50 mM for 3-24 h) increased cellular reductive power assessed through MTT assay, suggesting enhanced bioenergetics. Low doses of sucralose (0.5 and 5 mM) for 3 h stimulated mitochondrial respiration, measured through oxygraphy, and elevated mitochondrial transmembrane potential and cytoplasmic Ca2+, evaluated by fluorescence microscopy. Contrary to other cell types, the increase in mitochondrial respiration was insensitive to inhibition of mitochondrial Ca2+ uptake. These findings suggest that sucralose alters enterocyte energy homeostasis, contributing to its effects on organismal metabolism.
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The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata. A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.
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Glicosídeo Hidrolases/genética , Proteômica , Caramujos/genética , Transcriptoma/genética , Animais , Cromatografia Líquida/métodos , Trato Gastrointestinal/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Espécies Introduzidas , Caramujos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Obesity is a public health problem worldwide, and especially in women in reproductive age where more than one in three have obesity. Maternal obesity is associated with an increased maternal, placental, and newborn oxidative stress, which has been proposed as a central factor in vascular dysfunction in large-for-gestational-age (LGA) newborn. However, cellular and molecular mechanisms behind this effect have not been elucidated. Untreated human umbilical artery endothelial cells (HUAEC) from LGA (LGA-HUAEC) presented higher O2- levels, superoxide dismutase activity and heme oxygenase 1 messenger RNA (mRNA) levels, paralleled by reduced GSH:GSSG ratio and NRF2 mRNA levels. In response to an oxidative challenge (hydrogen peroxide), only HUAEC from LGA exhibited an enhanced Glutathione Peroxidase 1 (GPX1) expression, as well as a more efficient antioxidant machinery measured by the biosensor probe, HyPer. An open state of chromatin in the TSS region of GPX1 in LGA-HUAEC was evidenced by the DNase-HS assay. Altogether, our data indicate that LGA-HUAEC have an altered cellular and molecular antioxidant system. We propose that a chronic pro-oxidant intrauterine milieu, as evidenced in pregestational obesity, could induce a more efficient antioxidant system in fetal vascular cells, which could be maintained by epigenetic mechanism during postnatal life.
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Antioxidantes/metabolismo , Células Endoteliais/citologia , Regulação da Expressão Gênica , Idade Gestacional , Artérias Umbilicais/citologia , Cromatina/metabolismo , Células Endoteliais/enzimologia , Feminino , Fluorescência , Dissulfeto de Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Recém-Nascido , Cinética , Modelos Biológicos , Obesidade/patologia , Oxirredução , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Aerobic metabolism brings inexorably the production of reactive oxygen species (ROS), which are counterbalanced by intrinsic antioxidant defenses avoiding deleterious intracellular effects. Redox balance is the resultant of metabolic functioning under environmental inputs (i.e. diet, pollution) and the activity of intrinsic antioxidant machinery. Monitoring of intracellular hydrogen peroxide has been successfully achieved by redox biosensor advent; however, to track the intrinsic disulfide bond reduction capacity represents a fundamental piece to understand better how redox homeostasis is maintained in living cells. In the present work, we compared the informative value of steady-state measurements and the kinetics of HyPer, a H2O2-sensitive fluorescent biosensor, targeted at the cytosol, mitochondrion and endoplasmic reticulum. From this set of data, biosensor signal recovery from an oxidized state raised as a suitable parameter to discriminate reducing capacity of a close environment. Biosensor recovery was pH-independent, condition demonstrated by experiments on pH-clamped cells, and sensitive to pharmacological perturbations of enzymatic disulfide reduction. Also, ten human cell lines were characterized according their H2O2-pulse responses, including their capacity to reduce disulfide bonds evaluated in terms of their migratory capacity. Finally, cellular migration experiments were conducted to study whether migratory efficiency was associated with the disulfide reduction activity. The migration efficiency of each cell type correlates with the rate of signal recovery measured from the oxidized biosensor. In addition, HyPer-expressing cells treated with N-acetyl-cysteine had accelerated recovery rates and major migratory capacities, both reversible effects upon treatment removal. Our data demonstrate that the HyPer signal recovery offers a novel methodological tool to track the cellular impact of redox active biomolecules.
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Antioxidantes/isolamento & purificação , Técnicas Biossensoriais , Peróxido de Hidrogênio/química , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
Mifepristone is the only FDA-approved drug for glycaemia control in patients with Cushing's syndrome and type 2 diabetes. Mifepristone also has beneficial effects in animal models of diabetes and patients with antipsychotic treatment-induced obesity. However, the mechanisms through which Mifepristone produces its beneficial effects are not completely elucidated. PURPOSE: To determine the effects of mifepristone on insulin-stimulated glucose uptake on a model of L6 rat-derived skeletal muscle cells. RESULTS: Mifepristone enhanced insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane and Akt Ser473 phosphorylation in L6 myotubes. In addition, mifepristone reduced oxygen consumption and ATP levels and increased AMPK Thr172 phosphorylation. The knockdown of AMPK prevented the effects of mifepristone on insulin response. CONCLUSIONS: Mifepristone enhanced insulin-stimulated glucose uptake through a mechanism that involves a decrease in mitochondrial function and AMPK activation in skeletal muscle cells.
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Glucose/metabolismo , Insulina/farmacologia , Mifepristona/farmacologia , Células Musculares/metabolismo , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenilato Quinase/metabolismo , Animais , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Células Musculares/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RatosRESUMO
RESUMEN Las legumbres han sido históricamente consideradas una apetecible fuente de proteínas y fibra. En estos tiempos caracterizados por una tendencia epidemiológica al sobrepeso y a la obesidad, corregir hábitos poco saludables es prioritario. La incorporación de legumbres al menú cotidiano como sustituto de carnes podría significar, a mediano y largo plazo, aminorar la prevalencia de enfermedades crónicas no-transmisibles. Este trabajo ofrece una mirada actualizada de algunos constituyentes claves presentes en las semillas de legumbres frecuentemente consumidas por la población chilena: porotos (Phaseolus vulgaris L.), lentejas (Lens culinaris L.); garbanzos (Cicer arietinum L.) y arvejas (Pisum sativum L.). Con una perspectiva realista, se expone el efecto del remojo y la cocción, ambos procesos simples utilizados frecuentemente en su preparación. Además, se ha considerado la digestión y fermentación como procesos claves en la liberación de compuestos bioactivos y su interacción con la microbiota residente en la porción distal del tracto gastrointestinal. Finalmente, estudios epidemiológicos en conjunto con datos experimentales permiten obtener una idea de los mecanismos que subyacen al impacto nutricional que tiene el consumo habitual de legumbres.
ABSTRACT Legumes have historically been considered a good source of proteins and fiber. Currently, there is an undeniable epidemiologic trend towards overweight and obesity; therefore, it is a priority to correct unhealthy habits. The incorporation of legumes to the daily menu, perhaps as a replacement for meat, could lead, in the short- or longterm, to a slowing in the prevalence of non-communicable chronic diseases. This work offers an updated perspective to some key constituents present in the legumes frequently consumed in the Chilean diet: beans (Phaseolus vulgaris L.), lentils (Lens culinaris L.), chickpeas (Cicer arietinum L.) and green peas (Pisum sativum L.). The effects of soaking and cooking, both simple processes widely used during its home preparation, along with digestion and fermentation, are crucial in the release of bioactive compounds at the distal portion of gastrointestinal tract, which determines its interaction with resident microbiota. In conclusion, epidemiological studies together with experimental data offer an integral view of subjacent mechanisms of nutritional impact by regular consumption of legumes.
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Humanos , Disponibilidade Biológica , Microbiota , Fabaceae , Dieta SaudávelRESUMO
Chronic inflammation and metabolic reprogramming have been proposed as hallmarks of cancer development. Currently, many of the functional clues between these two phenomena are studied under the integrative view of functional stroma-epithelia interaction. It has been proposed that stromal cells, due to their abundance and avidity for glucose, are able to modify the metabolic behavior of an entire solid tumor. In the present study, using a mammary stromal cell line derived from healthy tissue subjected to long-term culture in low (5 mM) or high (25 mM) glucose, we found that the hyperglycemic condition favors the establishment of a pro-inflammatory and pro-oxidant environment characterized by the induction of the COX-2/PGE2 axis. In this condition, epithelial migration was stimulated. Moreover, we also found that stromal-derived PGE2, acting as a stimulator of IL-1 epithelial expression was one of the factors that promote the acquisition of motile properties by epithelial cells and the maintenance of a COX-2/PGE2-dependent inflammatory condition. Overall, our work provides experimental evidence that glucose stimulates a tumor inflammatory environment that, as a result of a functional cross-talk between stroma and epithelia, may be responsible for tumor progression. J. Cell. Biochem. 118: 994-1002, 2017. © 2016 Wiley Periodicals, Inc.
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Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/farmacologia , Glucose/farmacologia , Interleucina-1/metabolismo , Células Estromais/citologia , Neoplasias da Mama/imunologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Feminino , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Microambiente TumoralRESUMO
Oxidative stress (OS) derived from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and lifespan. It has also been associated with several age-related disorders, like postmenopausal osteoporosis of Mesenchymal stem cells (MSCs). MSCs are the common precursors for osteoblasts and adipocytes; appropriate commitment and differentiation of MSCs into a specific phenotype is modulated, among other factors, by ROS balance. MSCs have shown more resistance to ROS than differentiated cells, and their redox status depends on complex and abundant anti-oxidant mechanisms. The purpose of this work was to analyze in real time, H2 O2 signaling in individual h-MSCs, and to compare the kinetic parameters of H2 O2 management by cells derived from both control (c-) and osteoporotic (o-) women. For these purposes, cells were infected with a genetically encoded fluorescent biosensor named HyPer, which is specific for detecting H2 O2 inside living cells. Subsequently, cells were sequentially challenged with 50 and 500 µM H2 O2 pulses, and the cellular response was recorded in real time. The results demonstrated adequate expression of the biosensor allowing registering fluorescence from HyPer at a single cell level. Comparison of the response of c- and o-MSCs to the oxidant challenges demonstrated improved antioxidant activity in o-MSCs. This was further corroborated by measuring the relative expression of mRNAs for catalase, superoxide dismutase-1, thioredoxine, and peroxiredoxine, as well as by cell-surviving capacity under short-term H2 O2 treatment. We conclude that functional differences exist between healthy and osteoporotic human MSCs. The mechanism for these differences requires further study. J. Cell. Biochem. 118: 585-593, 2017. © 2016 Wiley Periodicals, Inc.
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Antioxidantes/metabolismo , Células da Medula Óssea/metabolismo , Peróxido de Hidrogênio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Oxirredutases/metabolismo , Transdução de Sinais , Idoso , Células da Medula Óssea/patologia , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/patologiaRESUMO
Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc.
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Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Glucose/metabolismo , Células Estromais/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Ácido Láctico/metabolismo , Glândulas Mamárias Humanas/metabolismo , Mitocôndrias/metabolismoRESUMO
Brain cell metabolism is intimately associated with intracellular oxidation-reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 µM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca(2+) abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.
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Citoplasma/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Citoplasma/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oxirredução , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
AIMS: To assess the mechanisms involved in lipopolysaccharide (LPS)-induced neuronal cell death, we examined the cellular consequences of LPS exposure in differentiated PC12 neurons and primary hippocampal neurons. RESULTS: Our data show that LPS is able to induce PC12 neuronal cell death without the participation of glial cells. Neuronal cell death was mediated by an increase in cellular reactive oxygen species (ROS) levels. Considering the prevalent role of specific ion channels in mediating the deleterious effect of ROS, we assessed their contribution to this process. Neurons exposed to LPS showed a significant intracellular Ca(2+) overload, and nonselective cationic channel blockers inhibited LPS-induced neuronal death. In particular, we observed that both LPS and hydrogen peroxide exposure strongly increased the expression of the transient receptor protein melastatin 7 (TRPM7), which is an ion channel directly implicated in neuronal cell death. Further, both LPS-induced TRPM7 overexpression and LPS-induced neuronal cell death were decreased with dithiothreitol, dipheniliodonium, and apocynin. Finally, knockdown of TRPM7 expression using small interference RNA technology protected primary hippocampal neurons and differentiated PC12 neurons from the LPS challenge. INNOVATION: This is the first report showing that TRPM7 is a key protein involved in neuronal death after LPS challenge. CONCLUSION: We conclude that LPS promotes an abnormal ROS-dependent TRPM7 overexpression, which plays a crucial role in pathologic events, thus leading to neuronal dysfunction and death.
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Lipopolissacarídeos/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Citometria de Fluxo , Neurônios/citologia , Células PC12 , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM/genéticaRESUMO
Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by L-threo-beta-benzyloxyaspartate or Na(+) replacement with NMDG(+), showing the participation of the Na(+)-glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na(+)/K(+) ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na(+) rise, glutamate and D-aspartate triggered a Ca(2+)signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca(2+) signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na(+) and Ca(2+) signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling.
Assuntos
Astrócitos/fisiologia , Sinalização do Cálcio/fisiologia , Transportador de Glucose Tipo 1/fisiologia , Transdução de Sinais/fisiologia , Sódio/fisiologia , Animais , Encéfalo/fisiologia , Tamanho Celular , Células Cultivadas , Corantes Fluorescentes , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/agonistas , Receptores de Glutamato Metabotrópico/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , TemperaturaRESUMO
The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P-mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.