RESUMEN
Oxidative stress increases with age and is postulated to be a major causal factor for sarcopenia in aging. Here, we examined whether the administration of a cystine-based antioxidant (F1) can alleviate/delay age-specific changes in skeletal muscles. C57BL6 male mice aged 17 months (middle aged) were fed with normal diet with or without supplementation of F1 (3 mg/kg food) for 6 months. Compared with young (5 months old) mice old mice exhibited increased markers of oxidative stress, inflammation, and muscle cell apoptosis and decreased muscle weight. These age-related changes were further associated with inactivation of adenosine-5'-monophosphate-activated protein kinase (AMPK), increased lipogenesis, activation of c-Jun NH2-terminal kinase, and decreased expression of Delta 1, phospho-Akt, and proliferating cell nuclear antigen in aged skeletal muscle. Such alterations were significantly prevented by F1. These results demonstrate the beneficial effects of F1 to attenuate loss of muscle mass associated with aging.
Asunto(s)
Envejecimiento , Antioxidantes/uso terapéutico , Apoptosis/efectos de los fármacos , Cistina/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sarcopenia/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Cistina/metabolismo , Modelos Animales de Enfermedad , Proteínas Quinasas JNK Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Resultado del TratamientoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver pathologies and is associated with obesity and the metabolic syndrome. Here, we investigated the molecular mechanisms by which a novel cystine based glutathione precursor with added selenomethionine (F1) prevents hepatic steatosis in a moderate high fat dietary model of NAFLD. Adult (8 weeks old), male apolipoprotein E (ApoE)-/- mice were fed with a normal diet (ND) or high fat diet (HFD), consisting of 21% fat and 0.21% cholesterol, with or without dietary supplementation of F1 (3 g/kg food) for 16 weeks. Compared with ApoE-/- mice fed with ND with or without F1, ApoE-/- mice fed with HFD exhibited significant weight gain, hepatomegaly, and increased serum cholesterol and triglycerides levels with no change in serum albumin levels. High resolution light and electron microscopy revealed micro-and macro-vesicular steatosis in ApoE-/- mice fed on a HFD. HFD-induced obesity also led to increased lipogenesis, oxidative stress, activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), perturbation of the BAX/BCL-2 rheostat, hepatocyte apoptosis, and activation of caspases 9 and 3. F1 fully prevented the adverse effects of HFD on serum triglyceride levels, body and liver weights, and hepatic steatosis and substantially attenuated HFD-induced increase in lipogenesis, oxidative stress, kinase activation, apoptotic signaling, and hepatocyte ultrastructural abnormalities. These results demonstrate that administration of F1, a glutathione precursor, ameliorates HFD-induced hepatic steatosis in ApoE-/- mice and emphasizes the role of oxidative stress in diet-induced obesity and hepatic steatosis.
Asunto(s)
Acetilcisteína/farmacología , Grasas de la Dieta/efectos adversos , Hígado Graso/prevención & control , Depuradores de Radicales Libres/farmacología , Obesidad/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Modelos Animales de Enfermedad , Quimioterapia Combinada , Hígado Graso/complicaciones , Hígado Graso/metabolismo , Hígado Graso/patología , Hepatocitos , Lipogénesis/efectos de los fármacos , MAP Quinasa Quinasa 4/biosíntesis , Masculino , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico , Obesidad/complicaciones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Selenometionina/farmacología , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15-21 (E15-E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15-E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.
Asunto(s)
Apoptosis/efectos de los fármacos , Cocaína/toxicidad , Regulación hacia Abajo , Exposición Materna/efectos adversos , Minociclina/farmacología , Miocitos Cardíacos/citología , Estrés Oxidativo/efectos de los fármacos , Trastornos Relacionados con Sustancias/metabolismo , Animales , Femenino , Corazón/efectos de los fármacos , Corazón/embriología , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Trastornos Relacionados con Sustancias/fisiopatologíaRESUMEN
Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and cardiovascular disease, which is largely mediated by oxidative stress. We investigated the effect of three glutathione (GSH) precursors: N-acetyl-cysteine (NAC), cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1), and NAC fortified with selenomethionine (F2) on oxidative stress induced by spermine (a uremic toxin) in cultured human aortic vascular smooth muscle cells (VSMC). VSMC were exposed to spermine (15 microM) with or without the given antioxidants (dose 50, 100, 200, and 500 microg/ml) or vehicle (control) and assessed for intracellular GSH levels, 4-hydroxy-trans-2-nonenal (4-HNE), and incorporation of 13C from glucose into alanine and protein. Spermine exposure reduced intracellular GSH levels, increased 4-HNE, and impaired glucose metabolism through reduction in pyruvate generation and/or transamination. Treatment with NAC had no effect on intracellular glutathione level. In contrast, F1 maintained intracellular GSH at control levels at all four doses. Subsequent studies performed with 200 microg/ml of F1, F2, or NAC (optimal dose) revealed normalization of 4-HNE, whereas restoration of 13C from glucose to alanine or protein to control values was only noted in the F1 group. Spermine-induced alterations in VSMC ultrastructure were prevented in approximately 90% of cells treated with F1 but only approximately 50% of cells treated with either NAC or F2. In conclusion, F1 was more effective than NAC or F2 in ameliorating spermine-induced reduction in intracellular GSH levels and cellular alterations in VSMC. The cystine-based GSH precursor (F1) is a promising antioxidant, and further studies are needed to examine the effect of this compound in preventing CKD-associated vascular disease.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Cistina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Selenometionina/farmacología , Aldehídos/metabolismo , Glucosa/metabolismo , Glutatión/metabolismo , Humanos , Espacio Intracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Espermina/farmacologíaRESUMEN
CKD (chronic kidney disease) is a public health problem, mediated by haemodynamic and non-haemodynamic events including oxidative stress. We investigated the effect of two GSH (glutathione) precursors, NAC (N-acetylcysteine) and cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1) in preventing spermine (uraemic toxin)-induced apoptosis in cultured human aortic VSMC (vascular smooth muscle cells). VSMCs exposed to spermine (15 microM) with or without antioxidants (doses 50, 100, 200 and 500 microg/ml) were assessed for apoptosis, JNK (c-Jun-NH2-terminal kinase) activation and iNOS (inducible nitric oxide synthase) induction and activation of intrinsic pathway signalling. Spermine exposure resulted in activation of JNK and iNOS induction and apoptosis. NAC and F1 (dose range 50-500 microg/ml) attenuated spermine-induced acceleration of VSMC apoptosis but only F1 (at 200 and 500 microg/ml) maintained spermine-induced apoptosis at control levels. Spermine-induced JNK activation was prevented by 200 microg/ml of both NAC and F1, while iNOS induction was blocked only by F1. Notably, the adverse effects of spermine on BAX/BCL-2 ratio, cytochrome c release and caspase activation was fully attenuated by F1. In conclusion, F1 was more effective than NAC in preventing spermine-induced apoptosis and downstream changes in related signal transduction pathways in VSMCs. Further studies are needed to examine the effect of these compounds in preventing CKD-associated vascular disease.
Asunto(s)
Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Cistina/farmacología , Glutatión/análogos & derivados , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Espermina/farmacología , Acetilcisteína/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacología , Cistina/metabolismo , Activación Enzimática , Glutatión/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Aging in rodents and humans is characterized by loss of muscle mass (sarcopenia). Testosterone supplementation increases muscle mass in healthy older men. Here, using a mouse model, we investigated the molecular mechanisms by which testosterone prevents sarcopenia and promotes muscle growth in aging. Aged mice of 22 months of age received a single sc injection of GnRH antagonist every 2 wk to suppress endogenous testosterone production and were implanted subdermally under anesthesia with 0.5 or 1.0 cm testosterone-filled implants for 2 months (n = 15/group). Young and old mice (n = 15/group), of 2 and 22 months of age, respectively, received empty implants and were used as controls. Compared with young animals, a significant (P < 0.05) increase in muscle cell apoptosis coupled with a decrease in gastrocnemius muscles weight (by 16.7%) and muscle fiber cross-sectional area, of both fast and slow fiber types, was noted in old mice. Importantly, such age-related changes were fully reversed by higher dose (1 cm) of testosterone treatment. Testosterone treatment effectively suppressed age-specific increases in oxidative stress, processed myostatin levels, activation of c-Jun NH(2)-terminal kinase, and cyclin-dependent kinase inhibitor p21 in aged muscles. Furthermore, it restored age-related decreases in glucose-6-phosphate dehydrogenase levels, phospho-Akt, and Notch signaling. These alterations were associated with satellite cell proliferation and differentiation. Collectively these results suggest involvement of multiple signal transduction pathways in sarcopenia. Testosterone reverses sarcopenia through stimulation of cellular metabolism and survival pathway together with inhibition of death pathway.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Miostatina/fisiología , Sarcopenia/prevención & control , Testosterona/sangre , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Apoptosis/efectos de los fármacos , Activación Enzimática , Humanos , Hipertrofia , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miostatina/genética , Tamaño de los Órganos , Testosterona/farmacología , Testosterona/uso terapéuticoRESUMEN
PNRC2 was identified in our laboratory as a general cofactor for nuclear receptors. To better characterize the physiological function of PNRC2, we used gene-targeting technology to generate PNRC2-null mice (PNRC2(-/-) mice). These PNRC2(-/-) mice are viable and fertile. PNRC2-null mice, especially male mice, are lean and are resistant to high fat diet-induced obesity but without the induction of insulin resistance. Male mice devoid of PNRC2 protein have a higher metabolic rate than wild-type mice. They consume more oxygen and produce more heat. Consistent with reduced adipose mass, the levels of leptin are lower in PNRC2(-/-) mice. This study provides evidence that PNRC2 plays one or more important roles in controlling the energy balance between energy storage and energy expenditure. PNRC2 may be a new target in the treatment of obesity and related metabolic diseases.
Asunto(s)
Adiposidad/fisiología , Metabolismo Energético/fisiología , Transactivadores/fisiología , Adiposidad/genética , Animales , Northern Blotting , Southern Blotting , Índice de Masa Corporal , Temperatura Corporal , Peso Corporal/genética , Peso Corporal/fisiología , Calorimetría Indirecta , Grasas de la Dieta , Ingestión de Alimentos/genética , Ingestión de Alimentos/fisiología , Metabolismo Energético/genética , Femenino , Genotipo , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/fisiopatología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genéticaRESUMEN
To test the hypothesis that c-Jun NH2-terminal kinase (JNK) and nitric oxide (NO)-mediated signaling plays an important role in muscle cell apoptosis, we examined the contribution of these molecules in muscle cell apoptosis during cardiotoxin (ctx)-induced muscle injury in mice. Compared to controls, where no apoptosis was detected, the percent of muscle cell apoptosis rose significantly (P < 0.05) at 4 h (27%) after ctx-treatment and increased further progressively up to 16 h posttreatment (80%), before it fell again at 24 h posttreatment (38%). Initiation of apoptosis was preceded by JNK activation and elevated levels of B-cell lymphoma-2 (BCL-2) in the mitochondrial fractions (BAX levels remained unaffected). Ctx treatment also resulted in the inactivation of BCL-2 through phosphorylation at serine 70, thereby perturbing the BAX/BCL-2 rheostat, and the subsequent activation of the cytochrome c-mediated death pathway. Concomitant administration of SP600125, a selective JNK inhibitor, or aminoguanidine (AG), a selected inducible nitric oxide synthase (iNOS) inhibitor, effectively diminished BCL-2 phosphorylation, suppressed cytochrome c release from mitochondria and caspase activation, and significantly prevented ctx-induced muscle cell apoptosis. In additional studies, we examined the role of testosterone in preventing such ctx-induced muscle cell apoptosis. Collectively, the present study emphasizes the role of a new signal transduction pathway involving JNK and iNOS that promotes ctx-induced myocyte apoptosis by provoking BCL-2 phosphorylation, leading to its inactivation, and subsequent activation of the intrinsic pathway signaling. Testosterone therapy has no protective effect in acute muscle injury associated with increased muscle cell death after ctx-treatment.
Asunto(s)
Cardiotoxinas/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Mitocondrias/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Testosterona/fisiología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citologíaRESUMEN
Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.