RESUMEN
Selenium is an essential micronutrient derived from daily diet to maintain the normal growth and development of vertebrates. Excessive selenium intake will induce cardiovascular toxicity, reproductive toxicity and neurotoxicity. However, there have been few studies of the toxic effects of selenium on neural development and locomotor behavior. In this study, newly fertilized zebrafish embryos were treated with selenium. As a result, selenium treatment at the concentration of 0.5 µM decreased the moving speed and distance and blunted the touch response of zebrafish embryos. TUNEL assay and immunofluorescence analysis revealed that selenium induced nervous system impairment including promoted cell apoptosis, proliferation and neuroinflammation, and decreased neurons in zebrafish embryos. RNA-seq and RT-PCR results indicated that selenium treatment significantly decreased the expression of the dopaminergic neuron, motor neuron, GABAergic neuron and neurotransmitter transport marker genes in zebrafish embryos. The expression of PPAR signaling pathway marker genes was significantly down-regulated in selenium-treated embryos. Two PPAR agonists (rosiglitazone and bezafibrate) and an anti-cancer drug (cisplatin) were tested for their effects to alleviate selenium-induced locomotor defects. Rosiglitazone and bezafibrate could restore the expression of some neural marker genes but could not fully rescue the selenium-induced locomotor behavior defects. The supplementation of cisplatin could restore the dysfunctional locomotor behavior and the abnormal expression of the PPAR and neural marker genes to almost the normal levels. In conclusion, the results of this study reveal that selenium-induced neural development and locomotor behavior defects are caused by multiple complex factors including PPAR signaling, and all the factors might be recovered by cisplatin through unknown mechanisms.
Asunto(s)
Selenio , Pez Cebra , Animales , Bezafibrato/metabolismo , Bezafibrato/farmacología , Cisplatino , Embrión no Mamífero , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Rosiglitazona/farmacología , Selenio/metabolismo , Selenio/farmacología , Pez Cebra/metabolismoRESUMEN
BACKGROUND: We previously demonstrated the in vitro killing of AML cells by the combination of the lipid-lowering agent bezafibrate (BEZ) and the contraceptive hormone medroxyprogesterone acetate (MPA). A phase II trial demonstrated in vivo safety and efficacy of BEZ and MPA (BaP) in elderly, relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS) patients. However, we observed dose-limiting toxicities in a second trial that attempted to improve outcomes via escalation of BaP doses. Thus we sought to identify a third repurposed drug that potentiates activity of low dose BaP (BaP 0.1 mM). METHODS AND RESULTS: We demonstrate that addition of a commonly used anti-epileptic, valproic acid (VAL) to low dose BaP (BaP 0.1 mM)(VBaP) enhanced killing of AML cell lines/primary AML cells to levels similar to high dose BaP (BaP 0.5 mM). Similarly, addition of VAL to BaP 0.1 mM enhanced reactive oxygen species (ROS), lipid peroxidation and inhibition of de novo fatty acid synthesis. Overexpression of Nrf2 in K562 and KG1a completely inhibited ROS production and rescued cells from VAL/BaP 0.1 mM/VBaP killing. CONCLUSIONS: Given the good safety data of low-dose BaP in elderly/relapsed/refractory AML patients, and that VAL alone is well-tolerated, we propose VBaP as a novel therapeutic combination for AML.
Asunto(s)
Antioxidantes/metabolismo , Bezafibrato/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Acetato de Medroxiprogesterona/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Valproico/farmacología , Anticonvulsivantes/farmacología , Línea Celular Tumoral , Agentes Anticonceptivos Hormonales/farmacología , Humanos , Hipolipemiantes/farmacología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Dosis Máxima ToleradaRESUMEN
Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear. The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial. We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.
Asunto(s)
Bezafibrato/farmacología , Síndromes Congénitos de Insuficiencia de la Médula Ósea/tratamiento farmacológico , Metabolismo Energético , Fibroblastos/efectos de los fármacos , Hipolipemiantes/farmacología , Errores Innatos del Metabolismo Lipídico/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Musculares/tratamiento farmacológico , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Síndromes Congénitos de Insuficiencia de la Médula Ósea/metabolismo , Síndromes Congénitos de Insuficiencia de la Médula Ósea/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Estrés Oxidativo , Receptores Activados del Proliferador del Peroxisoma/genéticaRESUMEN
Non-ketotic hyperglycinemia (NKH) is a severe neurological disorder caused by defects in glycine (GLY) catabolism and characterized by a high cerebrospinal fluid/plasma GLY ratio. Treatment is often ineffective and limited to the control of symptoms and detoxification of GLY. In the present work, we investigated the in vivo effects of GLY intracerebroventricular administration on oxidative stress parameters in rat striatum, cerebral cortex, and hippocampus. In vitro effects of GLY were also evaluated in striatum. The effects of bezafibrate (BEZ), a potential neuroprotective agent, on the possible alterations caused by GLY administration were further evaluated. Our in vivo results showed that GLY increased the activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) in striatum. Furthermore, GLY decreased the concentrations of total glutathione and reduced glutathione (GSH), as well as GSH/oxidized glutathione ratio in vivo in hippocampus. In vitro data also showed that GLY induced lipid peroxidation and decreased GSH in striatum. Regarding the effects of BEZ, we found that GLY-induced increase of GPx, SOD, and GR activities was attenuated or prevented by this compound. However, BEZ did not alter GLY-induced decrease of GSH in hippocampus. We hypothesize that GLY-induced increase of the activities of antioxidant enzymes in striatum occurs as a mechanism to avoid accumulation of reactive oxygen species and consequent oxidative damage. Furthermore, since BEZ prevented GLY-induced alterations, it might be considered as an adjuvant therapy for NKH.
Asunto(s)
Antioxidantes/metabolismo , Bezafibrato/farmacología , Cuerpo Estriado/enzimología , Glicina/toxicidad , Animales , Cuerpo Estriado/efectos de los fármacos , Glutatión/metabolismo , Glicina/administración & dosificación , Inyecciones Intraventriculares , Malondialdehído/metabolismo , Ratas WistarRESUMEN
INTRODUCTION: We evaluated the effects of bezafibrate (BEZ) on ß-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. METHODS: Cultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96h in the presence of 0-800µM BEZ using tandem mass spectrometry. RESULTS: The IVP assay showed that 100µM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via ß-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100µM BEZ, the level of C2 remained low. At concentrations higher than 100µM, BEZ further decreased the level of ACs including C16, but a concentration over 400µM decreased the level of C2 in most cases. DISCUSSION: BEZ at 100µM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of ß-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.
Asunto(s)
Bezafibrato/farmacología , Carnitina/análogos & derivados , Fibroblastos/efectos de los fármacos , Reguladores del Metabolismo de Lípidos/farmacología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/tratamiento farmacológico , Adolescente , Adulto , Edad de Inicio , Carnitina/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Activadores de Enzimas/farmacología , Femenino , Fibroblastos/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , Palmitoilcarnitina/metabolismo , Receptores Activados del Proliferador del Peroxisoma/agonistas , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
OBJECTIVE: Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD) is a rare mitochondrial fatty acid ß-oxidation disorder. We aimed to explore the clinical, biochemical, and genetic findings, treatments and outcomes in eight Chinese VLCADD patients. METHODS: Eight patients from six unrelated Chinese families with symptomatic VLCADD were diagnosed in the past 4 years. The clinical features and ACADVL gene mutations were analyzed. RESULTS: One patient underwent newborn screening and has been treated timely, she hardly had any symptoms. The remaining seven patients were found because of edema, diarrhea, coma, liver damage and psychomotor retardation. Seven patients had fatty liver. Five had myopathy. All patients had elevated blood tetradecanoylcarnitine. Nine heterozygous mutations of the ACADVL gene were found. Three (c.1102C > T, c.1795G > A and IVS10, +6T > A) were novel. Seven patients completely recovered after treatment. One patient died before diagnosis due to cardiomyopathy. His mother underwent amniocentesis for prenatal diagnosis. The fetus had the same gene mutation of the proband and markedly elevated tetradecanoylcarnitine in amniotic fluid. The boy has been treated after birth and he is healthy now. CONCLUSIONS: Dietary treatment usually leads to good outcomes to VLCADD patients. Amniocytes ACADVL mutations and amniotic fluid tetradecanoylcarnitine analysis are useful for the prenatal diagnosis.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Pueblo Asiatico/genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Tamizaje Neonatal , Diagnóstico Prenatal , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Acil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasas/metabolismo , Líquido Amniótico/química , Ácido Ascórbico/farmacología , Bezafibrato/farmacología , Carnitina/análogos & derivados , Carnitina/sangre , Carnitina/farmacología , Estudios de Casos y Controles , China , Cromatografía Liquida , Síndromes Congénitos de Insuficiencia de la Médula Ósea , ADN Complementario , Exones , Femenino , Pruebas Genéticas , Heterocigoto , Humanos , Lactante , Fórmulas Infantiles/química , Recién Nacido , Errores Innatos del Metabolismo Lipídico/dietoterapia , Masculino , Enfermedades Mitocondriales/dietoterapia , Enfermedades Musculares/dietoterapia , Mutación Missense , Alineación de Secuencia , Análisis de Secuencia de ADN , Espectrometría de Masas en Tándem , Resultado del Tratamiento , Triglicéridos/farmacología , Complejo Vitamínico B/farmacologíaRESUMEN
Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Ambroxol/farmacología , Activadores de Enzimas/farmacología , Lisosomas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , alfa-Galactosidasa/genética , alfa-Glucosidasas/genética , 1-Desoxinojirimicina/farmacología , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Bezafibrato/farmacología , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Expresión Génica , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Células HEK293 , Humanos , Leupeptinas/farmacología , Lisosomas/metabolismo , Pioglitazona , Plásmidos/química , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazolidinedionas/farmacología , Transfección , alfa-Galactosidasa/metabolismo , alfa-Glucosidasas/metabolismoRESUMEN
AIM: The aim of the present study was to evaluate whether activation of peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma by Bezafibrate (BZ) could attenuate hepatic and white adipose tissue (WAT) abnormalities in male offspring from diet-induced obese dams. MATERIALS AND METHODS: C57BL/6 female mice were fed a standard chow (SC; 10% lipids) diet or a high-fat (HF; 49% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring received SC diet at weaning and were subdivided into four groups: SC, SC/BZ, HF and HF/BZ. Treatment with BZ (100 mg/Kg diet) started at 12 weeks of age and was maintained for three weeks. RESULTS: The HF diet resulted in an overweight phenotype and an increase in oral glucose intolerance and fasting glucose of dams. The HF offspring showed increased body mass, higher levels of plasmatic and hepatic triglycerides, higher levels of pro-inflammatory and lower levels of anti-inflammatory adipokines, impairment of glucose metabolism, abnormal fat pad mass distribution, higher number of larger adipocytes, hepatic steatosis, higher expression of lipogenic proteins concomitant to decreased expression of PPARalpha and carnitine palmitoyltransferase I (CPT-1) in liver, and diminished expression of PPARgamma and adiponectin in WAT. Treatment with BZ ameliorated the hepatic and WAT abnormalities generated by diet-induced maternal obesity, with improvements observed in the structural, biochemical and molecular characteristics of the animals' livers and epididymal fat. CONCLUSION: Diet-induced maternal obesity lead to alterations in metabolism, hepatic lipotoxicity and adverse liver and WAT remodeling in the offspring. Targeting PPAR with Bezafibrate has beneficial effects reducing the alterations, mainly through reduction of WAT inflammatory state through PPARgamma activation and enhanced hepatic beta-oxidation due to increased PPARalpha/PPARgamma ratio in liver.
Asunto(s)
Bezafibrato/farmacología , Hipolipemiantes/farmacología , Obesidad/complicaciones , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Efectos Tardíos de la Exposición Prenatal/tratamiento farmacológico , Adipocitos/fisiología , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Bezafibrato/uso terapéutico , Peso Corporal , Metabolismo de los Hidratos de Carbono , Tamaño de la Célula , Dieta Alta en Grasa/efectos adversos , Evaluación Preclínica de Medicamentos , Ingestión de Energía , Metabolismo Energético , Hígado Graso/tratamiento farmacológico , Hígado Graso/etiología , Femenino , Expresión Génica , Hipolipemiantes/uso terapéutico , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR alfa/genética , PPAR gamma/genética , Embarazo , Complicaciones del Embarazo/etiología , Efectos Tardíos de la Exposición Prenatal/etiología , Fenómenos Fisiologicos de la Nutrición Prenatal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Multienzyme branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) catalyzes the regulatory step of oxidative catabolism of indispensable branched-chain amino acids (BCAA). The activity of the BCKDH complex is regulated by a reversible phosphorylation, end-product inhibition and by changes in the gene expression of BCKDH component enzymes. It has been shown previously that a high dose of bezafibrate (an agent added to rat chow at final concentration of 0.5%) changes mRNA levels of BCKDH-related enzymes and increases dephosphorylation of the complex leading to stimulation of liver BCKDH activity and the enhanced BCAA catabolism. The aim of the present study was to determine an in vivo effect of low, clinically relevant doses of bezafibrate on BCKDH activity in rat liver. Bezafibrate was administrated for 14 days by gastric gavage to Wistar male rats (fed low-protein chow; 8% protein) at one of the following daily doses of 5, 10 and 20mg/kgb.wt. The control group was given the vehicle (0.3% methylcellulose) only. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically before and after incubation with a broad-specificity phosphatase, respectively. The mRNA levels of the selected genes (BCKDH catalytic subunits and regulatory enzymes) were quantified by means of semi-quantitative RT-PCR. Current catalytic activity of BCKDH (described as BCKDH activity state - the proportion of the BCKDH complex in its active dephosphorylated form) increased by 2.1 ± 0.2, 2.3 ± 0.2 and 2.7 ± 0.2 fold (p<0.01). Changes in BCKDH activity did not correspond with changes in mRNA levels of the complex catalytic subunits. Moreover, mRNA levels of regulatory enzymes remained unaltered. Initially bezafibrate caused a transient insignificant reduction in body weight, but it had no effect on the final body weight. The highest dose of bezafibrate induced hepatomegaly. In conclusion, these data indicate that under conditions of dietary protein restriction low, clinically relevant doses of bezafibrate have a similar adverse effect on rat liver BCKDH activity and BCAA degradation rate as the high experimental dose. Up-regulation of liver BCKDH activity by low doses of bezafibrate appears to result mainly from changes in phosphorylation status of the complex (increased dephosphorylation) and is not associated with elevations in mRNA levels of BCKDH enzymatic components.
Asunto(s)
3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Bezafibrato/farmacología , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/biosíntesis , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Fosforilación/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Postprandial lipidemia is a risk factor for cardiovascular diseases. Thus, the suppression of postprandial lipidemia is valuable for disease management. Peroxisome proliferator-activated receptor- (PPAR ) is a key regulator in the lipid metabolism of peripheral tissues such as the liver and skeletal muscle, whose activation enhances fatty acid oxidation and decreases circulating lipid level. Recently, we have shown that bezafibrate, an agonistic compound for PPAR , suppresses post-prandial lipidemia by enhancing fatty acid oxidation in intestinal epithelial cells under physiological conditions. However, it was not elucidated whether the effect of PPAR on postprandial lipidemia is also observed under obese conditions, which change lipid metabolisms in various tissues and cells. Here, we observed that bezafibrate enhanced fatty acid oxidation in intestinal epithelial cells of obese diabetic KK-Ay mice. Bezafibrate treatment increased the mRNA expression levels of fatty acid oxidation-related genes, which are targets of PPAR , and enhanced CO2 production from [14C]-palmitic acid. The bezafibrate-treated mice showed the suppression of increasing serum triacylglyceride level after the oral administration of olive oil. Moreover, the effects of bezafibrate on mRNA expression and fatty acid oxidation were shown in only the proximal intestinal epithelial cells. These findings indicate that PPAR activation suppresses postprandial lipidemia under obese conditions through the enhancement of fatty acid oxidation, and that only the proximal intestine con-tributes to the effects in mice, suggesting that intestinal PPAR can be a target for prevention of obese-induced postprandial lipidemia.
Asunto(s)
Hiperlipidemias/prevención & control , Mucosa Intestinal/metabolismo , PPAR alfa/genética , Periodo Posprandial/efectos de los fármacos , Animales , Bezafibrato/farmacología , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Intestinos/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Obesos , Aceite de Oliva , PPAR alfa/metabolismo , Aceites de Plantas/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/sangreRESUMEN
Long-term alcohol exposure sensitizes hepatocytes to tumor necrosis factor-α (TNF) cytotoxicity. 4-Hydroxynonenal (4-HNE) is one of the most abundant and reactive lipid peroxides. Increased hepatic 4-HNE contents present in both human alcoholics and alcohol-fed animals. In the present study, we investigated the effects of intracellular 4-HNE accumulation on TNF-induced hepatotoxicity and its potential implication in the pathogenesis of alcoholic liver disease. Male C57BL/6 mice were fed an ethanol-containing or a control diet for 5 weeks. Long-term alcohol exposure increased hepatic 4-HNE and TNF levels. Cell culture studies revealed that 4-HNE, at nontoxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-κB transactivity. Further investigation demonstrated that 4-HNE prevented TNF-induced inhibitor of κBα phosphorylation without affecting upstream IκB kinase activity. An immunoprecipitation assay revealed that increased 4-HNE content was associated with increased formation of 4-HNE-inhibitor of κBα adduction in both 4-HNE-treated hepatocytes and in the livers of alcohol-fed mice. Prevention of intracellular 4-HNE accumulation by bezafibrate, a peroxisome proliferator-activated receptor-α agonist, protected hepatocytes from TNF killing via NF-κB activation. Supplementation of N-acetylcysteine, a glutathione precursor, conferred a protective effect on alcohol-induced liver injury in mice, was associated with decreased hepatic 4-HNE formation, and improved hepatic NF-κB activity. In conclusion, increased 4-HNE accumulation represents a potent and clinically relevant sensitizer to TNF-induced hepatotoxicity. These data support the notion that removal of intracellular 4-HNE can serve as a potential therapeutic option for alcoholic liver disease.
Asunto(s)
Aldehídos/toxicidad , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Hígado/lesiones , FN-kappa B/metabolismo , Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Animales , Bezafibrato/farmacología , Bezafibrato/uso terapéutico , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Etanol/efectos adversos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Hepatopatías Alcohólicas/tratamiento farmacológico , Hepatopatías Alcohólicas/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients' cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. METHODS: We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. RESULTS: Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 µM BZF, and peaked at 400 µM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 µM CuCl2 and 200 µM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. CONCLUSIONS: These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.
Asunto(s)
Bezafibrato/farmacología , Proteínas Portadoras/genética , Cobre/farmacología , Deficiencia de Citocromo-c Oxidasa/genética , Fibroblastos/efectos de los fármacos , Proteínas Mitocondriales/genética , Mutación , Adenosina Trifosfato/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Deficiencia de Citocromo-c Oxidasa/tratamiento farmacológico , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Mitocondriales/metabolismo , Chaperonas MolecularesRESUMEN
This study aimed to compare the effects of ω-3 fatty acids and fibrate treatment on plasma levels and activities of hemostatic risk factors on glucose and lipid metabolism in subjects with isolated hypertriglyceridemia. Seventy-three subjects with elevated triglyceride levels were allocated into one of the following treatment options: bezafibrate (200 mg twice daily), ω-3 fatty acids (1 g twice daily) or placebo. Plasma lipids, glucose homeostasis markers (fasting and 2-h post-glucose load plasma glucose levels and HOMA), as well as plasma levels/activities of fibrinogen, factor VII and PAI-1 were determined at baseline, on the day of randomization, and after 4 and 12 weeks of the treatment. Not only did bezafibrate improve plasma lipids, but it also increased glucose sensitivity and tended to reduce post-glucose loads of plasma glucose. Except for the reduction in plasma triglycerides, ω-3 fatty acids produced no effect on the lipid profile and insulin sensitivity. Both treatment options reduced, to similar extents, plasma levels of fibrinogen and PAI-1 and factor VII coagulant activity. Our study indicates that, although fibrates exhibit more-pronounced metabolic effects than do ω-3 fatty acids, both these treatment options are equipotent in producing a complex beneficial effect on hemostasis in isolated hypertriglyceridemic subjects.
Asunto(s)
Bezafibrato/farmacología , Ácidos Grasos Omega-3/farmacología , Hipertrigliceridemia/tratamiento farmacológico , Hipolipemiantes/farmacología , Glucemia/efectos de los fármacos , Femenino , Hemostasis/efectos de los fármacos , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Persona de Mediana EdadRESUMEN
Fibrates and omega-3 fatty acids have been used for many years in the treatment of increased triglyceride levels. Unfortunately, pleiotropic effects of these agents in patients with isolated hypertriglyceridaemia are poorly understood. The aim of this study was to compare the effect of bezafibrate and omega-3 fatty acids on monocyte cytokine release and systemic inflammation in this type of dyslipidaemia. The study included eighty-seven hypertriglyceridaemic subjects randomly allocated to one of three groups, treated respectively with bezafibrate (200 mg twice daily), omega-3 fatty acids (1 g twice daily) or placebo for 90 days. Eighty-three subjects completed the study. Apart from improvement in lipid profile, bezafibrate treatment reduced plasma high-sensitivity C-reactive protein (hsCRP) levels and inhibited monocyte release of interleukin-6, interleukin-1ß, monocyte chemoattractant protein-1 and tumour necrosis factor-α. Bezafibrate action on plasma hsCRP and monocyte cytokine release was lipid-independent but correlated with drug-induced improvement in insulin sensitivity. Omega-3 fatty acids reduced plasma triglycerides, but did not induce any significant changes in monocyte secretory function and plasma hsCRP. Our study suggests that bezafibrate is superior to omega-3 fatty acids in reducing systemic inflammation and in producing monocyte-suppressing effects. Anti-inflammatory actions of bezafibrate may contribute to the clinical effectiveness of fibrates in the prevention and treatment of isolated hypertriglyceridaemia.
Asunto(s)
Bezafibrato/farmacología , Ácidos Grasos Omega-3/farmacología , Hipertrigliceridemia/tratamiento farmacológico , Hipolipemiantes/farmacología , Proteína C-Reactiva/metabolismo , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Femenino , Humanos , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
Several rodent studies have demonstrated that fibrate drugs can activate peroxisome proliferator-activated receptor alpha (PPARalpha) and increase reactive oxygen species (ROS) production. The persistence of strong PPARalpha activation is considered to be a possible mechanism related to the adverse effects of these agents in humans. We recently found that bezafibrate-treated mice at clinically-relevant doses (10 mg/kg/day) exhibited similar pharmacokinetics to humans, but were different from previous rodent data (> 50 mg/kg/day). To examine whether clinical doses of bezafibrate do in fact activate PPARalpha and increase hepatic oxidative stress in mice, we administered bezafibrate to wild-type and Ppara-null mice at high (100 mg/kg/day) or low (10 mg/kg/day) doses and assessed ROS-related pathways in the liver. High-dose bezafibrate increased hepatic lipid peroxides in a PPARalpha-dependent manner, likely from discordant induction of PPARalpha-regulated ROS-generating enzymes (acyl-CoA oxidase, cytochrome P450 4A, and NADPH oxidase) and enhancement of mitochondrial beta-oxidation. The treatment also activated protein kinase C and phosphatidylinositol-3-kinase in wild-type mice only, suggesting an association between strong PPARalpha activation and an altered cell signaling cascade. Meanwhile, low-dose bezafibrate reduced serum/liver triglycerides in both genotypes without activating PPARalpha or enhancing hepatic oxidative stress. These results may support the safety of bezafibrate treatment at clinically-relevant doses.
Asunto(s)
Bezafibrato/farmacología , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Bezafibrato/farmacocinética , Humanos , Hipolipemiantes/farmacocinética , Ratones , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The optimal dietary n-6/n-3 ratio has not been fully elucidated. To investigate the influence of the dietary n-6/n-3 ratio on this ratio in the body and on liver beta-oxidation peroxisomal activity, rats were fed diets containing fat at an n-6/n-3 ratio of 1 to 16 for 4 weeks. To investigate whether elevation of the liver peroxisomal beta-oxidation activity increases the n-6/n-3 ratio in the body, rats were fed a diet containing a peroxisome-activating agent, bezafibrate, for 2 weeks, and its influence on the liver n-6/n-3 ratio was examined. The slope of the regression line between the dietary and liver total lipid n-6/n-3 ratios was significantly smaller when the dietary n-6/n-3 ratio was 4 or greater than when it was smaller than 4. Peroxisomal beta-oxidation and acyl CoA oxidase activities were significantly lower in rats fed a diet with an n-6/n-3 ratio of 16 than in those fed a diet with a ratio of 1. The peroxisomal beta-oxidation activity in the bezafibrate-supplemented group was significantly higher than that in the control group. The serum and liver total lipid n-6/n-3 ratios were significantly higher in the 0.015% bezafibrate-supplemented group than in the control group. These findings suggest that the liver n-6/n-3 ratio might be controlled via peroxisomal beta-oxidation in rats.
Asunto(s)
Grasas de la Dieta/farmacología , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-6/farmacología , Hígado/efectos de los fármacos , Peroxisomas/efectos de los fármacos , Animales , Bezafibrato/farmacología , Peso Corporal/efectos de los fármacos , Grasas de la Dieta/sangre , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-6/sangre , Hipolipemiantes/farmacología , Hígado/metabolismo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Peroxisomas/metabolismo , Ratas , Ratas Endogámicas BBRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs play a critical role in lipid and glucose metabolism. We examined whether chronic treatment with bezafibrate, a PPAR agonist, would alter sleep and body temperature (BT). Mice fed with a control diet were monitored for BT, electroencephalogram (EEG), and electromyogram for 48 h under light-dark conditions. After obtaining the baseline recording, the mice were provided with bezafibrate-supplemented food for 2 wk, after which the same recordings were performed. Two-week feeding of bezafibrate decreased BT, especially during the latter half of the dark period. BT rhythm and sleep/wake rhythm were phase advanced about 2-3 h by bezafibrate treatment. Bezafibrate treatment also increased the EEG delta-power in nonrapid eye movement sleep compared with the control diet attenuating its daily amplitude. Furthermore, bezafibrate-treated mice showed no rebound of EEG delta-power in nonrapid eye movement sleep after 6 h sleep deprivation, whereas values in control mice largely increased relative to baseline. DNA microarray, and real-time RT-PCR analysis showed that bezafibrate treatment increased levels of Neuropeptide Y mRNA in the hypothalamus at both Zeitgeber time (ZT) 10 and ZT22, and decreased proopiomelanocortin-alpha mRNA in the hypothalamus at ZT10. These findings demonstrate that PPARs participate in the control of both BT and sleep regulation, which accompanied changes in gene expression in the hypothalamus. Activation of PPARs may enhance deep sleep and improve resistance to sleep loss.
Asunto(s)
Bezafibrato/farmacología , Temperatura Corporal/efectos de los fármacos , Ritmo Delta/efectos de los fármacos , Hipolipemiantes/farmacología , Fases del Sueño/efectos de los fármacos , Animales , Regulación de la Temperatura Corporal/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos ICR , Neuropéptido Y/genética , Neuropéptido Y/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Activados del Proliferador del Peroxisoma/agonistas , Proopiomelanocortina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vigilia/efectos de los fármacosRESUMEN
To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.
Asunto(s)
Luciferasas/genética , PPAR delta/agonistas , PPAR delta/genética , Proteínas Recombinantes de Fusión/genética , Células 3T3 , Animales , Bezafibrato/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Vectores Genéticos/química , Vectores Genéticos/genética , Células HeLa , Humanos , Ácido Linoleico/farmacología , Lípidos/química , Luciferasas/metabolismo , Ratones , Proteínas Recombinantes de Fusión/metabolismo , Transfección/métodosRESUMEN
Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARalpha and PPARbeta from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.
Asunto(s)
Monitoreo del Ambiente/métodos , Receptores Activados del Proliferador del Peroxisoma/agonistas , Acetil-CoA C-Aciltransferasa/genética , Alitretinoína , Inhibidores de la Angiogénesis/farmacología , Animales , Bezafibrato/farmacología , Clonación Molecular , ADN/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Gemfibrozilo/farmacología , Genes Reporteros , Humanos , Hipolipemiantes/farmacología , Técnicas In Vitro , Hígado/enzimología , Luciferasas/metabolismo , Oncorhynchus mykiss , PPAR alfa/genética , PPAR-beta/genética , Proliferadores de Peroxisomas/farmacología , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Pirimidinas/farmacología , Ratas , Receptores X Retinoide/metabolismo , Transcripción Genética , Transfección , Tretinoina/farmacología , Contaminantes Químicos del Agua/análisisRESUMEN
The effect of short-term bezafibrate (BF) administration over time on the expression of UCP mRNA in the tissues was examined in Otsuka Long Evans Tokushima Fatty (OLETF) rats. Eight-week-old rats were divided into a high-dose (100 mg/kg) BF group (n = 15), a low-dose (10 mg/kg) BF group (n = 15) and a control group (n = 15), and followed for 14 days. Feed intake by the high-dose BF group increased significantly between days 10 and 14 of administration. Triglyceride, free fatty acid, and T(4) levels decreased significantly in a dose-dependent manner in the high-dose BF group. Leptin and insulin levels significantly decreased on days 3 and 7. Throughout the study period, liver UCP2 mRNA increased in the high-dose BF group. On day 3 of BF administration, the levels of UCP2 mRNA expression in the skeletal muscles as well as UCP3 mRNA expression in the WAT were significantly increased in the high-dose BF group. PPAR-alpha mRNA significantly increased in the liver on day 3 of BF administration. We thus conclude that the PPAR-alpha-mediated effects of BF on the expression of liver UCP2 may be one of the factors that helped to decrease insulin levels.