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1.
Oxid Med Cell Longev ; 2019: 7945983, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30805084

RESUMO

Background: Aurothioglucose- (ATG-) mediated inhibition of thioredoxin reductase-1 (TXNRD1) improves alveolarization in experimental murine bronchopulmonary dysplasia (BPD). Glutathione (GSH) mediates susceptibility to neonatal and adult oxidative lung injury. We have previously shown that ATG attenuates hyperoxic lung injury and enhances glutathione- (GSH-) dependent antioxidant defenses in adult mice. Hypothesis: The present studies evaluated the effects of TXNRD1 inhibition on GSH-dependent antioxidant defenses in newborn mice in vivo and lung epithelia in vitro. Methods: Newborn mice received intraperitoneal ATG or saline prior to room air or 85% hyperoxia exposure. Glutamate-cysteine ligase (GCL) catalytic (Gclc) and modifier (Gclm) mRNA levels, total GSH levels, total GSH peroxidase (GPx) activity, and Gpx2 expression were determined in lung homogenates. In vitro, murine transformed club cells (mtCCs) were treated with the TXNRD1 inhibitor auranofin (AFN) or vehicle in the presence or absence of the GCL inhibitor buthionine sulfoximine (BSO). Results: In vivo, ATG enhanced hyperoxia-induced increases in Gclc mRNA levels, total GSH contents, and GPx activity. In vitro, AFN increased Gclm mRNA levels, intracellular and extracellular GSH levels, and GPx activity. BSO prevented AFN-induced increases in GSH levels. Conclusions: Our data are consistent with a model in which TXNRD1 inhibition augments hyperoxia-induced GSH-dependent antioxidant responses in neonatal mice. Discrepancies between in vivo and in vitro results highlight the need for methodologies that permit accurate assessments of the GSH system at the single-cell level.


Assuntos
Antioxidantes/metabolismo , Displasia Broncopulmonar/enzimologia , Displasia Broncopulmonar/patologia , Glutationa/metabolismo , Tiorredoxina Redutase 1/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Aurotioglucose , Displasia Broncopulmonar/genética , Células Epiteliais/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/metabolismo , Hiperóxia/genética , Hiperóxia/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiorredoxina Redutase 1/metabolismo
2.
Lab Anim ; 53(1): 89-94, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30290722

RESUMO

This case report addresses the problem of underreporting negative results and adverse side effects in animal testing. We present our findings regarding a hyperphagic mouse model associated with unforeseen high mortality. The results outline the necessity of reporting detailed information in the literature to avoid duplication. Obese mouse models are essential in the study of obesity, metabolic syndrome and diabetes mellitus. An experimental model of obesity can be induced by the administration of gold thioglucose (GTG). After transcending the blood-brain barrier, the GTG molecule interacts with regions of the ventromedial hypothalamus, thereby primarily targeting glucose-sensitive neurons. When these neurons are impaired, mice become insensitive to the satiety effects of glucose and develop hyperphagia. In a pilot study for optimising dosage and body weight development, C57BL/6 mice were treated with GTG (0.5 mg/g body weight) or saline, respectively. Animals were provided a physiological amount of standard diet (5 g per animal) for the first 24 hours after treatment to prevent gastric dilatation. Within 24 hours after GTG injection, all GTG-treated animals died of gastric overload and subsequent circulatory shock. Animals developed severe attacks of hyperphagia, and as the amount of provided chow was restricted, mice exhibited unforeseen pica and ingested bedding material. These observations strongly suggest that restricted feeding is contraindicated concerning GTG application. Presumably, the impulse of excessive food intake was a strong driving force. Therefore, the actual degree of suffering in the GTG-induced model of hyperphagia should be revised from moderate to severe.


Assuntos
Aurotioglucose/administração & dosagem , Modelos Animais de Doenças , Dilatação Gástrica/etiologia , Hiperfagia/fisiopatologia , Pica/fisiopatologia , Animais , Glicemia/metabolismo , Ingestão de Alimentos , Evolução Fatal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Pica/induzido quimicamente , Projetos Piloto
3.
Am J Physiol Lung Cell Mol Physiol ; 314(5): L736-L742, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29368550

RESUMO

We previously showed that the thioredoxin reductase-1 (TrxR1) inhibitor aurothioglucose (ATG) improves alveolarization in hyperoxia-exposed newborn C3H/HeN mice. Our data supported a mechanism by which the protective effects of ATG are mediated via sustained nuclear factor E2-related factor 2 (Nrf2) activation in hyperoxia-exposed C3H/HeN mice 72 h after ATG administration. Given that inbred mouse strains have differential sensitivity and endogenous Nrf2 activation by hyperoxia, the present studies utilized two C57BL/6 exposure models to evaluate the effects of ATG on lung development and Nrf2 activation. The first model (0-14 days) was used in our C3H/HeN studies and the 2nd model (4-14 days) is well characterized in C57BL/6 mice. ATG significantly inhibited lung TrxR1 activity in both models; however, there was no effect on parameters of alveolarization in C57BL/6 mice. In sharp contrast to C3H/HeN mice, there was no effect of ATG on pulmonary NADPH quinone oxidoreductase-1 ( Nqo1) and heme oxygenase-1 ( Hmox1) at 72 h in either C57BL/6 model. In conclusion, although ATG inhibited TrxR1 activity in the lungs of newborn C57BL/6 mice, effects on lung development and sustained Nrf2-dependent pulmonary responses were blunted. These findings also highlight the importance of strain-dependent hyperoxic sensitivity in evaluation of potential novel therapies.


Assuntos
Aurotioglucose/farmacologia , Displasia Broncopulmonar/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/citologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Alvéolos Pulmonares/citologia , Tiorredoxina Redutase 1/metabolismo , Animais , Animais Recém-Nascidos , Antirreumáticos/farmacologia , Displasia Broncopulmonar/tratamento farmacológico , Displasia Broncopulmonar/metabolismo , Células Cultivadas , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Tiorredoxina Redutase 1/genética
4.
Int J Radiat Biol ; 93(4): 407-415, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27921518

RESUMO

PURPOSE: Gold nanoparticles modified by thio-glucose are believed to increase the toxicity of radiotherapy in human malignant cells. We report the effect of thio-glucose bound gold nanoparticles (Glu-G nanoparticles), 16 nm in size, on two human lung (QU-DB) and breast (MCF7) cancer cell lines combined with kilo and megavoltage X-rays. MATERIALS AND METHODS: The shape and surface characteristics, the size distribution and light absorption spectrum of the prepared nanoparticles were measured by transmission electron microscopy, dynamic light scattering, and ultraviolet-visible spectrophotometry, respectively. The cell uptake was assayed using the atomic absorption spectrometry. Mitochondrial activity, colony formation, and comet assays were applied to assess and compare the enhanced radiotoxicity of 100 KV and 6 MV X-rays, when combined with Glu-G nanoparticles. RESULTS: Glu-G nanoparticles had no significant toxicity for MCF7 and QU-DB cells up to 100 micromolar concentration. Compared to radiation alone, the intracellular uptake of Glu-G nanoparticles resulted in increased inhibition of cell proliferation by 64.1% and 38.7% for MCF7 cells, and 64.4% and 32.4% for QU-DB cells by 100 kVp and 6 MV X-rays, respectively. Comet assay confirmed an increase of DNA damage as a result of combination of 6 MV photons with Glu-G nanoparticles. CONCLUSION: Glu-G nanoparticles have remarkable potential for enhancing radiotoxicity of both low and high energy photons in MCF7 and QU-DB cells.


Assuntos
Aurotioglucose/administração & dosagem , Sobrevivência Celular/efeitos da radiação , Nanopartículas Metálicas/administração & dosagem , Neoplasias Experimentais/radioterapia , Radiossensibilizantes/administração & dosagem , Radioterapia de Alta Energia/métodos , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Células MCF-7 , Neoplasias Experimentais/patologia , Fótons/uso terapêutico , Tolerância a Radiação/efeitos dos fármacos , Dosagem Radioterapêutica , Resultado do Tratamento
5.
Am J Respir Cell Mol Biol ; 55(3): 419-28, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27089175

RESUMO

Oxygen toxicity and antioxidant deficiencies contribute to the development of bronchopulmonary dysplasia. Aurothioglucose (ATG) and auranofin potently inhibit thioredoxin reductase-1 (TrxR1), and TrxR1 disruption activates nuclear factor E2-related factor 2 (Nrf2), a regulator of endogenous antioxidant responses. We have shown previously that ATG safely and effectively prevents lung injury in adult murine models, likely via Nrf2-dependent mechanisms. The current studies tested the hypothesis that ATG would attenuate hyperoxia-induced lung developmental deficits in newborn mice. Newborn C3H/HeN mice were treated with a single dose of ATG or saline within 12 hours of birth and were exposed to either room air or hyperoxia (85% O2). In hyperoxia, ATG potently inhibited TrxR1 activity in newborn murine lungs, attenuated decreases in body weight, increased the transcription of Nrf2-regulated genes nicotinamide adenine dinucleotide phosphate reduced quinone oxidoreductase-1 (NQO1) and heme oxygenase 1, and attenuated alterations in alveolar development. To determine the impact of TrxR1 inhibition on Nrf2 activation in vitro, murine alveolar epithelial-12 cells were treated with auranofin, which inhibited TrxR1 activity, enhanced Nrf2 nuclear levels, and increased NQO1 and heme oxygenase 1 transcription. Our novel data indicate that a single injection of the TrxR1 inhibitor ATG attenuates hyperoxia-induced alterations in alveolar development in newborn mice. Furthermore, our data support a model in which the effects of ATG treatment likely involve Nrf2 activation, which is consistent with our findings in other lung injury models. We conclude that TrxR1 represents a novel therapeutic target to prevent oxygen-mediated neonatal lung injury.


Assuntos
Hiperóxia/complicações , Hiperóxia/enzimologia , Lesão Pulmonar/complicações , Lesão Pulmonar/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Auranofina/farmacologia , Aurotioglucose/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Hiperóxia/patologia , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C3H , Morfogênese/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo
6.
Molecules ; 20(7): 12732-56, 2015 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-26184149

RESUMO

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide disulfide oxidoreductases playing a central role in cellular redox homeostasis and signaling pathways. Recently, these selenoproteins have emerged as promising therapeutic targets for anticancer drug development, often being overexpressed in tumor cells and contributing to drug resistance. Herein, we summarize the current knowledge on metal- and semimetal-containing molecules capable of hampering mammalian TrxRs, with an emphasis on compounds reported in the last decade.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Auranofina/síntese química , Auranofina/farmacologia , Aurotioglucose/síntese química , Aurotioglucose/farmacologia , Inibidores Enzimáticos/síntese química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Modelos Moleculares , Proteínas de Neoplasias/química , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/farmacologia , Estresse Oxidativo , Fosfinas/síntese química , Fosfinas/farmacologia , Compostos de Rutênio/síntese química , Compostos de Rutênio/farmacologia , Tiorredoxina Dissulfeto Redutase/química
7.
J Cutan Pathol ; 42(8): 568-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25950356

RESUMO

Localized chrysiasis is rare and can occur in two settings: after localized or traumatic implantation of elemental gold or gold salts or after localized laser or light therapy in someone who has been previously exposed to systemic gold therapy. We report a unique case of localized chrysiasis with associated aluminum salt deposition and sclerosing lipogranulomas because of previous injections of aurothioglucose (Solganal®). The unique histopathologic findings seen in this case have not been previously reported.


Assuntos
Alumínio/metabolismo , Aurotioglucose/efeitos adversos , Calcinose/induzido quimicamente , Granuloma/induzido quimicamente , Idoso , Antirreumáticos/administração & dosagem , Antirreumáticos/efeitos adversos , Artrite Reumatoide/tratamento farmacológico , Aurotioglucose/administração & dosagem , Calcinose/metabolismo , Calcinose/patologia , Feminino , Granuloma/metabolismo , Granuloma/patologia , Humanos , Síndrome de Sjogren/tratamento farmacológico
8.
Breast Cancer ; 22(4): 413-20, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24114595

RESUMO

BACKGROUND: Gold nanoparticles (GNPs) have been conceived to cause increased cytotoxicity of radiotherapy in human malignant cells. Greater uptake of GNPs by cells may induce increased radiation effects. Here we report the radiosensitization effect of glucose-capped GNPs (Glu-GNPs) with different sizes (16 nm and 49 nm) on MDA-MB-231 cells in the presence of megavoltage X-rays. METHODS: Transmission electron microscopy (TEM) was used to observe the distribution of Glu-GNPs in cells. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to measure the quantities of Glu-GNPs absorbed by cells. After treatment of Glu-GNPs with a series of concentrations, we used the MTT and clonogenic assays to confirm the radiation enhancement effect of Glu-GNPs on MDA-MB-231 cells. The cell cycle distribution was analyzed by flow cytometry to further explore the mechanisms of enhanced radiosensitivity of Glu-GNPs. RESULTS: TEM showed that Glu-GNPs are mainly distributed in the cytoplasm of cells, including endosomes and lysosomes. ICP-AES indicates that MDA-MB-231 cells absorb more 49-nm Glu-GNPs than 16-nm Glu-GNPs in number (P < 0.05). Glu-GNPs have little cytotoxicity toward MDA-MB-231 cells with a concentration below 20 nM. In the clonogenic assay, the combination of Glu-GNPs with radiation induced a significant growth inhibition, compared with radiation alone (P < 0.05). Moreover 49-nm Glu-GNPs induced much greater radiation effects than 16-nm Glu-GNPs (P < 0.05). Flow cytometry shows that Glu-GNPs can help radiation arrest more cells in the G2/M phase, with greater effect with 49-nm Glu-GNPs than 16-nm Glu-GNPs. CONCLUSIONS: Glu-GNPs can increase the cytotoxicity of radiation toward MDA-MB-231 cells, probably by regulating the distribution of the cell cycle, with more cells in the G2/M phase. The effect of radiation enhancement may be related to the quantities of Glu-GNPs in the cells.


Assuntos
Aurotioglucose/farmacologia , Nanopartículas/química , Radiossensibilizantes/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/radioterapia , Aurotioglucose/química , Aurotioglucose/farmacocinética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Feminino , Citometria de Fluxo , Humanos , Microscopia Eletrônica de Transmissão , Neoplasias de Mama Triplo Negativas/patologia
9.
J Gastroenterol ; 49(6): 1065-73, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23800945

RESUMO

BACKGROUND: Obesity-induced liver disease (nonalcoholic fatty liver disease, NAFLD) is now the commonest cause of chronic liver disease in affluent nations. There are presently no proven treatments for NAFLD or its more severe stage, nonalcoholic steatohepatitis (NASH). Bofutsushosan (BTS), a Japanese herbal (Kampo) medicine, long used as an anti-obesity medicine in Japan and other Asian countries, has been shown to reduce body weight and improve insulin resistance (IR) and hepatic steatosis. The precise mechanism of action of BTS, however, remains unclear. To evaluate the ability of BTS to prevent the development of NASH, and determine the mediators and pathways involved. METHODS: C57BL/6 mice were injected intra-peritoneally with gold-thioglucose and fed a high-fat diet (HF) or HF diet admixed with either 2 or 5 % BTS for 12 weeks. The effectiveness of BTS in attenuating features of NASH and the mechanisms through which BTS attenuated NASH were then assayed through an assessment of the anthropometric, radiological, biochemical and histological parameters. RESULTS: BTS attenuated the progression of NASH through induction of adiponectin and its receptors along with an induction of PPAR-α and PPAR-γ, decreased expression of SREBP-1c, increased hepatic fatty acid oxidation and increased hepatic export of triglycerides. BTS moreover, reduced IR through phosphorylation of the protein kinase, Akt. CONCLUSIONS: BTS through induction of adiponectin signaling and Akt attenuated development of NASH. Identification of the active entity in BTS should allow development of novel treatments for NASH.


Assuntos
Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicina Kampo/métodos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/metabolismo , Animais , Aurotioglucose/farmacologia , Western Blotting , Dieta Hiperlipídica , Progressão da Doença , Teste de Tolerância a Glucose , Resistência à Insulina , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Tomografia Computadorizada por Raios X
10.
Antioxid Redox Signal ; 20(17): 2681-91, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24295151

RESUMO

AIMS: Inflammation and oxygen toxicity increase free radical production and contribute to the development of acute respiratory distress syndrome (ARDS), which is a significant cause of morbidity and mortality in intensive care patients. We have previously reported increased glutathione (GSH) levels in lung epithelial cells in vitro and attenuated adult murine hyperoxic lung injury in vivo after pharmacological thioredoxin reductase-1 (TrxR1) inhibition. Using a murine ARDS model, we tested the hypothesis that aurothioglucose (ATG) treatment increases pulmonary GSH levels, attenuates lung injury, and decreases mortality in a GSH-dependent manner. RESULTS: Adult mice received a single intratracheal dose of 0.375 µg/g lipopolysaccharide (LPS) 12 h before a single intraperitoneal injection of 25 mg/kg ATG. Control mice received intratracheal and/or intraperitoneal saline. Mice were then exposed to room air or hyperoxia (>95% O2). Lung injury was assessed by bronchoalveolar lavage protein concentrations. Expression of glutamate-cysteine ligase modifier subunit (GCLM), GSH, cytokines, and chemokines was determined. Exposure to LPS/hyperoxia induced inflammation and lung injury. ATG treatment significantly attenuated lung injury, increased lung GCLM expression and GSH levels, and decreased mortality. GSH depletion completely prevented the protective effects of ATG in LPS/hyperoxia-exposed mice. INNOVATION: ATG treatment significantly attenuates lung injury and enhances survival in a clinically relevant murine model of ARDS. The protective effects of ATG are GSH dependent. CONCLUSION: Augmentation of GSH systems by TrxR1 inhibition could represent a promising therapeutic approach to attenuate oxidant-mediated lung injury and improve patient outcomes.


Assuntos
Aurotioglucose/administração & dosagem , Lesão Pulmonar/tratamento farmacológico , Síndrome do Desconforto Respiratório do Adulto/tratamento farmacológico , Tiorredoxina Redutase 1/metabolismo , Animais , Modelos Animais de Doenças , Radicais Livres/toxicidade , Glutationa/metabolismo , Humanos , Hiperóxia/metabolismo , Hiperóxia/patologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Camundongos , Oxigênio/toxicidade , Síndrome do Desconforto Respiratório do Adulto/etiologia , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Síndrome do Desconforto Respiratório do Adulto/patologia , Tiorredoxina Redutase 1/antagonistas & inibidores
11.
Neuropharmacology ; 71: 164-73, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23603200

RESUMO

The ventromedial nucleus of the hypothalamus (VMH) plays an important role in feeding and energy homeostasis. Electroconvulsive seizure (ECS) therapy is highly effective in the treatment of several psychiatric diseases, including depression, but may also have beneficial effects in other neurological diseases. Although it has been reported that the neurons of the VMH are strongly activated by ECS stimulation, the specific effects of ECS in this hypothalamic subnucleus remain unknown. To address this issue, we investigated the changes in gene expression in microdissected-VMH samples in response to ECS in mice, and examined the behavioral effects of ECS on feeding behavior. ECS significantly induced the expression of immediate-early genes such as Fos, Fosb, and Jun, as well as Bdnf, Adcyap1, Hrh1, and Crhr2 in the VMH. Given that signals of these gene products are suggested to have anorexigenic roles in the VMH, we also examined the effect of ECS on food intake and body weight. Repeated ECS had a suppressive effect on food intake and body weight gain under both regular and high-fat diet conditions. Furthermore, gold-thioglucose-induced hypothalamic lesions, including the VMH and the arcuate nucleus, abolished the anorexigenic effects of ECS, indicating the requirement for the activation of the hypothalamus. Our data show an effect of ECS on increased expression of anorexigenic factors in the VMH, and suggest a role in the regulation of energy homeostasis by ECS.


Assuntos
Regulação do Apetite , Eletroconvulsoterapia , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Obesidade/terapia , Regulação para Cima , Núcleo Hipotalâmico Ventromedial/metabolismo , Animais , Regulação do Apetite/efeitos dos fármacos , Comportamento Apetitivo/efeitos dos fármacos , Aurotioglucose/toxicidade , Comportamento Animal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Comportamento Alimentar/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Microdissecção , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Síndromes Neurotóxicas/complicações , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Regulação para Cima/efeitos dos fármacos , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos , Núcleo Hipotalâmico Ventromedial/patologia , Perda de Peso/efeitos dos fármacos
12.
J Neuroendocrinol ; 25(4): 340-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23253150

RESUMO

Systemic administration of gold thioglucose (GTG) causes a hypothalamic lesion that extends from the ventral part of the ventromedial hypothalamus (VMH) to the dorsal part of the arcuate nucleus (ARC), resulting in hyperphagia and obesity in mice. In the present study, we used in situ hybridisation histochemistry to explore the effects of GTG on the central corticotrophin-releasing hormone (CRH) system, which regulates feeding and energy homeostasis. Type 2 CRH receptor (CRHR-2) mRNA expression decreased by 40% at 8 weeks in the VMH and by 40-60% at 2 and 8 weeks in the ARC after GTG injection. By contrast, CRHR-2 mRNA expression in the hypothalamic paraventricular nucleus (PVN) and lateral septum was unchanged. Urocortin (Ucn) 3 mRNA expression in the perifornical area and medial amygdala decreased, whereas CRH mRNA expression in the PVN increased at 2 and 8 weeks after GTG injection. Ucn 1 mRNA expression in the Edingher-Westphal nucleus and Ucn 2 mRNA expression in the PVN were unchanged. Because Ucn 3 is an anorexigenic and a possible endogenous ligand for VMH CRHR-2, our results suggest that decreased Ucn 3 expression and decreased VMH CRHR-2 expression contribute, in part, to GTG-induced hyperphagia and obesity. To determine whether VMH CRHR-2 mediates the anorexigenic effects of Ucn 3, Ucn 3 was administered i.c.v. and food intake was measured 8 weeks after GTG treatment. Ucn 3 decreased cumulative food intake on days 4-7 after surgery compared to i.c.v. administration of vehicle in control mice. By contrast, the anorexigenic effects of i.c.v. Ucn 3 were abolished in GTG-treated mice. Taken together, our results indicate that the Ucn 3 pathway, which innervates the VMH, is involved in appetite regulation via CRHR-2. It remains to be determined whether CRHR-2 in the ARC has additional roles in appetite regulation by Ucn 3.


Assuntos
Aurotioglucose/farmacologia , Hormônio Liberador da Corticotropina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hormônio Liberador da Corticotropina/genética
13.
J Biol Chem ; 287(45): 38210-9, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22977247

RESUMO

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.


Assuntos
Aurotioglucose/farmacologia , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/metabolismo , Animais , Auranofina/farmacologia , Azóis/farmacologia , Western Blotting , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Glutationa/antagonistas & inibidores , Células HCT116 , Células HeLa , Humanos , Modelos Biológicos , Mutação , Compostos Organosselênicos/farmacologia , Oxirredução/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética , Fatores de Tempo
14.
Antioxid Redox Signal ; 17(10): 1407-16, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22607006

RESUMO

AIMS: Pulmonary oxygen toxicity contributes to lung injury in newborn and adult humans. We previously reported that thioredoxin reductase (TrxR1) inhibition with aurothioglucose (ATG) attenuates hyperoxic lung injury in adult mice. The present studies tested the hypothesis that TrxR1 inhibition protects against the effects of hyperoxia via nuclear factor E2-related factor 2 (Nrf2)-dependent mechanisms. RESULTS: Both pharmacologic and siRNA-mediated TrxR1 inhibition induced robust Nrf2 responses in murine-transformed Clara cells (mtCC). While TrxR1 inhibition did not alter the susceptibility of cells to the effects of hyperoxia, glutathione (GSH) depletion after TrxR1 inhibition markedly enhanced the hyperoxic susceptibility of cultured mtCCs. Finally, in vivo data revealed dose-dependent increases in the expression of the Nrf2 target gene NADPH:quinone oxidoreductase 1 (NQO1) in the lungs of ATG-treated adult mice. INNOVATION: TrxR1 inhibition activates Nrf2-dependent antioxidant responses in mtCCs in vitro and in adult murine lungs in vivo, providing a plausible mechanism for the protective effects of TrxR1 inhibition in vivo. CONCLUSION: GSH-dependent enzyme systems in mtCCs may be of greater importance for protection against hyperoxic exposure than are TrxR-dependent systems. The induction of Nrf2 activation via TrxR1 inhibition represents a novel therapeutic strategy that attenuates oxidant-mediated lung injury. Similar expression levels of TrxR1 in newborn and adult mouse or human lungs broaden the potential clinical applicability of the present findings to both neonatal and adult oxidant lung injury.


Assuntos
Lesão Pulmonar/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxidantes/toxicidade , Tiorredoxina Dissulfeto Redutase/metabolismo , Animais , Aurotioglucose/farmacologia , Humanos , Camundongos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores
15.
Cell Metab ; 14(4): 516-27, 2011 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-21982711

RESUMO

Under conditions of obesity and insulin resistance, the serine/threonine protein kinase Akt/PKB is required for lipid accumulation in liver. Two forkhead transcription factors, FoxA2 and FoxO1, have been suggested to function downstream of and to be negatively regulated by Akt and are proposed as key determinants of hepatic triglyceride content. In this study, we utilize genetic loss of function experiments to show that constitutive activation of neither FoxA2 nor FoxO1 can account for the protection from steatosis afforded by deletion of Akt2 in liver. Rather, another downstream target positively regulated by Akt, the mTORC1 complex, is required in vivo for de novo lipogenesis and Srebp1c expression. Nonetheless, activation of mTORC1 and SREBP1c is not sufficient to drive postprandial lipogenesis in the absence of Akt2. These data show that insulin signaling through Akt2 promotes anabolic lipid metabolism independent of Foxa2 or FoxO1 and through pathways additional to the mTORC1-dependent activation of SREBP1c.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Antirreumáticos/farmacologia , Aurotioglucose/farmacologia , Dieta Hiperlipídica , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR , Triglicerídeos/metabolismo
16.
Liver Int ; 31(4): 542-51, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21382165

RESUMO

BACKGROUND: The search for effective treatments of non-alcoholic steatohepatitis (NASH), now the most common chronic liver disease in affluent countries, is hindered by a lack of animal models having the range of anthropometric and pathophysiological features as human NASH. AIMS: To examine if mice treated with gold thioglucose (GTG) - known to induce lesions in the ventromedial hypothalamus, leading to hyperphagia and obesity - and then fed a high-fat diet (HF) had a comprehensive histological and dysmetabolic phenotype resembling human NASH. METHODS: C57BL/6 mice were injected intraperitoneally with GTG and then fed HF for 12 weeks (GTG+HF). The extent of abdominal adiposity was assayed by CT scanning. A glucose tolerance test and an insulin tolerance test were performed to evaluate insulin resistance (IR). Histological, molecular and biochemical analyses were also performed. RESULTS: Gold thioglucose+HF induced dysmetabolism, with hyperphagia, obesity with increased abdominal adiposity, IR and consequent steatohepatitis, with hepatocyte ballooning, Mallory-Denk bodies, perivenular and pericellular fibrosis as seen in adult NASH, paralleled by an increased expression of the profibrogenic factors, transforming growth factor-ß1 and TIMP-1. Plasma adiponectin and the expression of adiponectin receptor 1 and receptor 2 were decreased, while PPAR-γ and FAS were increased in the livers of GTG+HF mice. In addition, GTG+HF mice showed glucose intolerance and severe IR. CONCLUSIONS: Treatment with GTG and HF diet induce, in mice, a comprehensive model of human NASH, with the full range of dysmetabolic and histological abnormalities.


Assuntos
Aurotioglucose/toxicidade , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Adiponectina/sangue , Tecido Adiposo/diagnóstico por imagem , Animais , Aurotioglucose/administração & dosagem , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Teste de Tolerância a Glucose , Hipotálamo/efeitos dos fármacos , Injeções Intraperitoneais , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , PPAR gama/metabolismo , Receptores de Adiponectina/metabolismo , Estatísticas não Paramétricas , Tomografia Computadorizada por Raios X
17.
Obesity (Silver Spring) ; 19(3): 514-21, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20706204

RESUMO

An obesity-induced diabetes model using genetically normal mouse strains would be invaluable but remains to be established. One reason is that several normal mouse strains are resistant to high-fat diet-induced obesity. In the present study, we show the effectiveness of gold thioglucose (GTG) in inducing hyperphagia and severe obesity in mice, and demonstrate the development of obesity-induced diabetes in genetically normal mouse strains. GTG treated DBA/2, C57BLKs, and BDF1 mice gained weight rapidly and exhibited significant increases in nonfasting plasma glucose levels 8-12 weeks after GTG treatment. These mice showed significantly impaired insulin secretion, particularly in the early phase after glucose load, and reduced insulin content in pancreatic islets. Interestingly, GTG treated C57BL/6 mice did not become diabetic and retained normal early insulin secretion and islet insulin content despite being as severely obese and insulin resistant as the other mice. These results suggest that the pathogenesis of obesity-induced diabetes in GTG-treated mice is attributable to the inability of their pancreatic ß-cells to secrete enough insulin to compensate for insulin resistance. Mice developing obesity-induced diabetes after GTG treatment might be a valuable tool for investigating obesity-induced diabetes. Furthermore, comparing the genetic backgrounds of mice with different susceptibilities to diabetes may lead to the identification of novel genetic factors influencing the ability of pancreatic ß-cells to secrete insulin.


Assuntos
Diabetes Mellitus Experimental/etiologia , Modelos Animais de Doenças , Hiperfagia/induzido quimicamente , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Obesidade Mórbida/complicações , Animais , Aurotioglucose , Glicemia/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Predisposição Genética para Doença , Intolerância à Glucose , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos , Obesidade Mórbida/induzido quimicamente , Obesidade Mórbida/fisiopatologia , Especificidade da Espécie
18.
Biochemistry ; 48(26): 6213-23, 2009 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-19366212

RESUMO

Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox-active tetrapeptide of sequence Gly-Cys(1)-Sec(2)-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulysin, HIV Tat protein, H(2)O(2), lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys(2) mutant enzyme or the N-terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild-type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175-550-fold reduction in k(cat). Our data support the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, or (iii) that are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC(50) for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83-142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.


Assuntos
Selênio/química , Selenocisteína/química , Tiorredoxina Dissulfeto Redutase/química , Substituição de Aminoácidos , Animais , Auranofina/química , Aurotioglucose/química , Biocatálise , Caenorhabditis elegans/enzimologia , Cistina/análogos & derivados , Cistina/química , Dinitrobenzenos/química , Ditiotreitol/química , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/química , Deleção de Genes , Glutationa/química , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Modelos Químicos , Compostos Organosselênicos/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Selenocisteína/genética , Selenito de Sódio/química , Especificidade por Substrato , Ácido Tióctico/química , Tiorredoxina Redutase 2/antagonistas & inibidores , Tiorredoxina Redutase 2/química , Tiorredoxina Redutase 2/genética , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/genética
20.
J Comp Neurol ; 499(1): 120-31, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16958086

RESUMO

The effect of gold thioglucose (GTG) administration on neurons containing feeding-related peptides in the hypothalamic arcuate nucleus was examined in mice. Intraperitoneal GTG injection increased the body weight and produced a hypothalamic lesion that extended from the ventral part of the ventromedial nucleus to the dorsal part of the arcuate nucleus. Neurons containing proopiomelanocortin (POMC) and neuropeptide Y (NPY) present in the dorsal part of the arcuate nucleus were destroyed by GTG. In addition, the peptide-containing fibers that extended from the remaining arcuate neurons were degenerated at the lesion site. The number of POMC-containing fibers in the paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamus was found to have decreased significantly when examined at 2 days and 2 weeks after the GTG treatment. In contrast, the number of NPY-containing fibers in the lateral hypothalamus remained unchanged after the GTG treatment, probably because of the presence of an unaffected NPY-containing fiber pathway passing through the tuberal region and projecting onto the lateral hypothalamus. The number of NPY-immunoreactive fibers in the paraventricular and dorsomedial nuclei showed a moderate but significant decrease at 2 days after the GTG treatment, but it recovered to the normal levels 2 weeks later. The NPY-containing fibers were found to have regenerated across the lesion site 2 weeks later, and this might contribute to the recovery of the NPY-immunoreactive fibers in these regions. The present results first demonstrate that POMC- and NPY-containing neurons in the arcuate nucleus respond differently to the lesion produced by the GTG treatment.


Assuntos
Antirreumáticos/administração & dosagem , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Aurotioglucose/administração & dosagem , Neurônios/efeitos dos fármacos , Neuropeptídeo Y/metabolismo , Pró-Opiomelanocortina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/lesões , Contagem de Células/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fibras Nervosas/metabolismo , Neurônios/metabolismo , Fatores de Tempo
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