RESUMEN

Carbamates, most commonly used pesticides in agricultural practices, have been reported to produce free radicals causing deleterious effects in animals. The present study was designed to assess the carbofuran induced oxidative stress in rat liver slices in vitro and also to evaluate protective role of vitamin C by incubating them in Krebs-Ringer HEPES Buffer (KRHB) containing incubation media (Williams medium E (WME) supplemented with glucose and antibiotics) with different concentrations of carbofuran. The results demonstrated that carbofuran caused significant increase in lipid peroxidation and inhibition in the activity of hepatic superoxide dismutase (SOD) in concentration dependent manner. The data with incubation medium reflected that carbofuran at lowest concentration caused an increase in SOD activity followed by its inhibition at higher concentration. Carbofuran treatment caused inhibition in the activity of catalase in liver slices and WME incubation medium. Pre-incubation of liver slices and the WME media with vitamin C restored the values of biochemical indices tested. The results indicated that carbofuran might induce oxidative stress in hepatocytes. The pretreatment with vitamin C may offer hepatoprotection from toxicity of pesticide at low concentration only.

RESUMEN

OBJECTIVE: Glucose promotes lipid remodelling in pancreatic ß-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. METHODS: Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 ß-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in (3)H-sphinganine-labelled cells using TLC. RESULTS: Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. CONCLUSION: Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.

RESUMEN

Fermentation of dietary fibers by colonic microbiota generates short-chain fatty acids (SCFAs), e.g., propionic acid and butyric acid, which have been described to have "anti-obesity properties" by ameliorating fasting glycaemia, body weight and insulin tolerance in animal models. In the present study, we therefore investigate if propionic acid and butyric acid have effects on lipolysis, de novo lipogenesis and glucose uptake in primary rat adipocytes. We show that both propionic acid and butyric acid inhibit isoproterenol- and adenosine deaminase-stimulated lipolysis as well as isoproterenol-stimulated lipolysis in the presence of a phosphodiesterase (PDE3) inhibitor. In addition, we show that propionic acid and butyric acid inhibit basal and insulin-stimulated de novo lipogenesis, which is associated with increased phosphorylation and thus inhibition of acetyl CoA carboxylase, a rate-limiting enzyme in fatty acid synthesis. Furthermore, we show that propionic acid and butyric acid increase insulin-stimulated glucose uptake. To conclude, our study shows that SCFAs have effects on fat storage and mobilization as well as glucose uptake in rat primary adipocytes. Thus, the SCFAs might contribute to healthier adipocytes and subsequently also to improved energy metabolism with for example less circulating free fatty acids, which is beneficial in the context of obesity and type 2 diabetes.

RESUMEN

The development of mature adipocytes from pre-adipocytes is a highly regulated process. CD24 is a glycophosphatidylinositol-linked cell surface receptor that has been identified as a critical cell surface marker for identifying pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Here, we examined the role and regulation of CD24 during adipogenesis in vitro. We found that CD24 mRNA and protein expression is upregulated early during adipogenesis in the 3T3-L1 pre-adipocytes and in murine primary pre-adipocytes isolated from subcutaneous and visceral WAT, followed by downregulation in mature adipocytes. CD24 mRNA expression was found to be dependent on increased transcription due to increased promoter activity in response to activation of a pre-existing transcriptional regulator. Furthermore, either intracellular cAMP or dexamethasone were sufficient to increase expression in pre-adipocytes, while both additively increased CD24 expression. Preventing the increase in CD24 expression, by siRNA-mediated knock-down, resulted in fewer mature lipid-laden adipocytes and decreased expression of mature adipogenic genes. Therefore, conditions experienced during adipogenesis in vitro are sufficient to increase CD24 expression, which is necessary for differentiation. Overall, we conclude that the dynamic upregulation of CD24 actively promotes adipogenesis in vitro.

RESUMEN

The di-peptide Trp-His (WH) has vasorelaxant and anti-atherosclerotic functions. We hypothesized that WH has multiple biological functions and may aid AMP-activated protein kinase (AMPK) activation and affect the glucose transport system in skeletal muscle. First, we examined whether WH or His-Trp (HW) can activate AMPKα. Treatment of L6 myotubes with WH or HW significantly increased phosphorylation of AMPKα. WH activated AMPK independently of insulin and significantly increased glucose uptake into L6 myotubes following translocation of glucose transporter 4 (Glut4) to the plasma membrane. This activation was induced by the LKB1 pathway but was independent of changes in intracellular Ca(2+) levels and the Ca(2+)/calmodulin-dependent kinase pathway. L6 myotubes express only one type of oligopeptide transporter, peptide/histidine transporter 1 (PHT1, also known as SLC15a4), and WH is incorporated into cells and activates AMPKα following PHT1-mediated cell uptake. These findings indicate that (1) WH activates AMPK and insulin independently enhances glucose uptake following translocation of Glut4 to the plasma membrane, (2) activation of AMPKα by WH is mediated by the LKB1 pathway, without altering the Ca(2+)-dependent pathway, and (3) L6 myotubes express only one type of peptide transporter (PHT1; SLC15a4), which incorporates WH into cells to activate AMPKα.

RESUMEN

NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2 (.-) and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

RESUMEN

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca(2+)- and Mg(2+)-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca(2+) and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca(2+) and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca(2+)]i and insulin secretion in INS-1E cells.

Asunto(s)

Calcio/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Canales Catiónicos TRPV/metabolismo , Línea Celular , Humanos , Secreción de Insulina

RESUMEN

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50µM of each tetracycline-derived compound 20 min prior to exposure to 500µM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca(2+) uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca(2+) uptake and suppressed the Ca(2+)-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca(2+) uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.

Asunto(s)

Canales de Calcio/efectos de los fármacos , Doxiciclina/farmacología , Hepatocitos/efectos de los fármacos , Minociclina/farmacología , Daño por Reperfusión/prevención & control , Tetraciclina/farmacología , Animales , Antibacterianos/farmacología , Calcio/farmacocinética , Canales de Calcio/metabolismo , Hepatocitos/metabolismo , Hipoxia/prevención & control , Hierro/farmacocinética , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Ratas , Ratas Sprague-Dawley

RESUMEN

Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na(+)/H(+) exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na(+))i] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 µM) abolished short-term Na(+) influx, but was unable to prevent the accumulation of (Na(+))i observed at 6 and 24h after LPS exposure. The NHE inhibitor cariporide (1 µM) significantly reduced accumulation of (Na(+))i 6 and 24h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H2O2 and expression of gp91(phox), an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX+cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation.

Asunto(s)

Inflamación/metabolismo , Inflamación/patología , Microglía/metabolismo , Intercambiadores de Sodio-Hidrógeno/fisiología , Canales de Sodio Activados por Voltaje/fisiología , Animales , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/patología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología

RESUMEN

BACKGROUND & AIMS: Dual oxidases (DUOX) are conserved reduced nicotinamide adenine dinucleotide phosphate oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOX maturation factor (DUOXA) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1/DUOXA1 and DUOX2/DUOXA2). Expression of these genes is up-regulated during bacterial infections and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. METHODS: Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection--the most common human chronic infection. RESULTS: Infection with H. felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H. felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H. felis colonization also was increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H. felis that colonized Duoxa(-/-) mice, compared with that found in controls (P = .0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H. felis failed to prevent a direct bacteriostatic effect at sustained levels of H2O2 as low as 30 µmol/L. CONCLUSIONS: Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H. felis and the inflammatory response. These findings indicate the nonredundant function of epithelial production of H2O2 in restricting microbial colonization.

Asunto(s)

Mucosa Gástrica/metabolismo , Gastritis/prevención & control , Infecciones por Helicobacter/prevención & control , Helicobacter felis , Peróxido de Hidrógeno/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epitelio/metabolismo , Epitelio/microbiología , Femenino , Mucosa Gástrica/microbiología , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter felis/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Técnicas In Vitro , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Estómago/microbiología , Regulación hacia Arriba

RESUMEN

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting ß-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by ß-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on ß-arrestin 2 mediated internalization.

Asunto(s)

Endocitosis/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Arrestinas/antagonistas & inhibidores , Arrestinas/genética , Arrestinas/metabolismo , Western Blotting , Células Cultivadas , Concanavalina A/farmacología , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas para Inmunoenzimas , Riñón/citología , Riñón/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Adrenérgicos alfa 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Arrestina beta 2 , beta-Arrestinas

RESUMEN

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia-reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe(2+), ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe(2+). Desferal and starch-desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch-desferal, and the inhibitor of the mitochondrial Ca(2+) uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch-desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe(2+) during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.

Asunto(s)

Hepatocitos/metabolismo , Hierro/metabolismo , Lisosomas/metabolismo , Mitocondrias Hepáticas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Hepatocitos/citología , Quelantes del Hierro/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismo

RESUMEN

We studied adipocytes from 8-week-old control rat offspring (CON) or rat offspring subjected to maternal low (8%) protein (MLP) feeding during pregnancy/lactation, a procedure predisposing to obesity. Acute exposure to isoproterenol or adenosine enhanced PDK4 and PPARγ mRNA gene expression in CON and MLP adipocytes. Enhanced adipocyte Pdk4 expression correlated with increased PPARγ expression. Higher levels of PDK4 and PPARγ were observed in MLP adipocytes. SCD1 is a PPARγ target. Isoproterenol enhanced adipocyte PDK4 and SCD1 gene expression in parallel. This could reflect augmented PPARγ expression together with enhanced lipolytic stimulation to supply endogenous PPARγ ligands, allowing enhanced adipocyte PDK4 and SCD1 expression via PPARγ activation. In contrast, the effect of adenosine to increase PDK4 expression is independent of stimulation of lipolysis and, as SCD1 expression was unaffected by adenosine, unlikely to reflect PPARγ activation. Increased adipocyte expression of both PDK4 and SCD1 in the MLP model could participate as components of a "thrifty" phenotype, favouring the development of obesity.