RESUMEN
MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.
Asunto(s)
Proteínas Represoras/genética , Animales , Autofagia/genética , Ingestión de Alimentos/genética , Metabolismo Energético/genética , Metabolismo de los Lípidos/genética , Longevidad/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/genética , ARN de Transferencia/metabolismo , Espermidina/metabolismoRESUMEN
OBJECTIVE: Asthma is a chronic inflammatory disease associated with increased cardiovascular events. This study assesses the presence of inflammation and the vascular reactivity of pulmonary arteries in patients with acute asthma. METHODS: Rings of human pulmonary arteries obtained from non-asthmatic and asthmatic patients were set up in organ bath for vascular tone monitoring. Reactivity was induced by vasoconstrictor and vasodilator agents. Protein expression of inflammatory markers was detected by western blot. Prostanoid releases and cyclic adenosine monophosphate (cAMP) levels were quantified using specific enzymatic kits. RESULTS: Protein expression of cluster of differentiation 68, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and cyclooxygenase-2 was significantly increased in arteries obtained from asthmatic patients. These effects were accompanied by an alteration of vasodilatation induced by iloprost and treprostinil, a decrease in cAMP levels and an increase in prostaglandin (PG) E2 and PGI2 synthesis. The use of forskolin (50 µmol/L) has restored the vasodilatation and cAMP release. No difference was observed between the two groups in reactivity induced by norepinephrine, angiotensin II, PGE2, KCl, sodium nitroprusside, and acetylcholine. CONCLUSION: Acute asthma causes inflammation of pulmonary arteries and decreases vasodilation induced by PGI2 analogs through the impairment of cAMP pathway.
Asunto(s)
Asma/fisiopatología , Prostaglandinas I/farmacología , Prostaglandinas Sintéticas/farmacología , Arteria Pulmonar/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Adulto , Asma/inmunología , Biomarcadores , Colforsina/farmacología , AMP Cíclico/antagonistas & inhibidores , Ciclooxigenasa 2/biosíntesis , Dinoprostona/biosíntesis , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Femenino , Humanos , Iloprost/farmacología , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/biosíntesis , Masculino , Persona de Mediana Edad , Prostaglandinas/inmunología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Vasoconstricción/efectos de los fármacosRESUMEN
CONTEXT: Extracellular matrix (ECM) synthesis regulation by sympathetic nervous system (SNS) or angiotensin II (ANG II) was widely reported, but interaction between the two systems on ECM synthesis needs further investigation. OBJECTIVE: We tested implication of SNS and ANG II on ECM synthesis in juvenile rat aorta. MATERIALS AND METHODS: Sympathectomy with guanethidine (50 mg/kg, subcutaneous) and blockade of the ANG II AT1 receptors (AT1R) blocker with losartan (20 mg/kg/day in drinking water) were performed alone or in combination in rats. mRNA and protein synthesis of collagen and elastin were examined by Q-RT-PCR and immunoblotting. RESULTS: Collagen type I and III mRNA were increased respectively by 62 and 43% after sympathectomy and decreased respectively by 31 and 60% after AT1R blockade. Combined treatment increased collagen type III by 36% but not collagen type I. The same tendency of collagen expression was observed at mRNA and protein levels after the three treatments. mRNA and protein level of elastin was decreased respectively by 63 and 39% and increased by 158 and 15% after losartan treatment. Combined treatment abrogates changes induced by single treatments. DISCUSSION AND CONCLUSION: The two systems act as antagonists on ECM expression in the aorta and combined inhibition of the two systems prevents imbalance of mRNA and protein level of collagen I and elastin induced by single treatment. Combined inhibition of the two systems prevents deposit or excessive reduction of ECM and can more prevent cardiovascular disorders.
Asunto(s)
Angiotensina II/metabolismo , Aorta/metabolismo , Matriz Extracelular/metabolismo , Sistema Nervioso Simpático/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo III/metabolismo , Elastina/metabolismo , Guanetidina/farmacología , Losartán/farmacología , Masculino , ARN Mensajero , Ratas , Ratas Endogámicas WKY , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simpatectomía/métodosRESUMEN
The aim of our present study is to investigate the interaction between angiotensin II (ANG II) and sympathetic nervous system (SNS) on matrix metalloproteinase MMP-2 and MMP-9 expression and activity in juvenile rat aorta under normal conditions. Sympathectomy with guanethidine and blockade of the ANG II receptors (AT1R) by losartan were performed alone or in combination on new-born rats. mRNA, protein expression and activity of MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymography, respectively. MMP-2 mRNA and protein amount were decreased after sympathectomy or AT1R blockade and an additive effect was observed after combined treatment. However, MMP-9 expression was reduced to the same level in the three treated groups. There were some detectable gelatinolytic activity of the MMPs in both control and treated rats. We concluded that ANG II stimulates directly and indirectly (via sympathostimulator pathway) the MMP-2 expression but seems unable to affect MMP-9 expression through direct pathway. Combined inhibition of SNS and ANG II were more efficient than a single inhibition in reducing MMP amounts in rat vessels.
Asunto(s)
Aorta/enzimología , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Nervioso Simpático/metabolismo , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Aorta/patología , Modelos Biológicos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Nervioso Simpático/embriologíaRESUMEN
OBJECTIVE: Because inhibition of the renin-angiotensin system (RAS) reduces the onset of type 2 diabetes (T2D) and prevents atherosclerosis, we investigated the expression of RAS in the arterial wall of T2D and nondiabetic (CTR) patients. METHODS AND RESULTS: mRNA and protein levels of angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and AT1 receptor (AT1R) were determined in carotid atheroma plaque, nearby macroscopically intact tissue (MIT), and in vascular smooth muscle cells (VSMCs) before and after insulin stimulation from 21 T2D and 22 CTR patients. AGT and ACE mRNA and their protein levels were 2- to 3-fold higher in atheroma and in MIT of T2D patients. VSMCs from T2D patients had respectively 2.5- and 5-fold higher AGT and AT1R mRNA and protein contents. Insulin induced an increase in AGT and AT1R mRNA with similar ED50. These responses were blocked by PD98059, an inhibitor of MAP-kinase in the two groups whereas wortmannin, an inhibitor of PI3-kinase, partially prevented the response in CTR patients. Phosphorylated ERK1-2 was 4-fold higher in MIT from T2D than from CTR patients. CONCLUSIONS: The arterial RAS is upregulated in T2D patients, which can be partly explained by an hyperactivation of the ERK1-2 pathway by insulin.
Asunto(s)
Angiotensinógeno/efectos de los fármacos , Enfermedades de las Arterias Carótidas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Anciano , Análisis de Varianza , Angiotensinógeno/biosíntesis , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/metabolismo , Western Blotting , Enfermedades de las Arterias Carótidas/etiología , Enfermedades de las Arterias Carótidas/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Probabilidad , ARN/metabolismo , ARN Mensajero/análisis , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de Tejidos , Regulación hacia ArribaRESUMEN
Left ventricular hypertrophy (LVH) is commonly associated with hypertension and represents an independent cardiovascular risk factor. The aim of this study was to test the hypothesis that the cardiac overload related to hypertension is associated to a specific gene expression pattern independently of genetic background. Gene expression levels were obtained with microarrays for 15,866 transcripts from RNA of left ventricles from 12-wk-old rats of three hypertensive models [spontaneously hypertensive rat (SHR), Lyon hypertensive rat (LH), and heterozygous TGR(mRen2)27 rat] and their respective controls. More than 60% of the detected transcripts displayed significant changes between the three groups of normotensive rats, showing large interstrain variability. Expression data were analyzed with respect to hypertension, LVH, and chromosomal distribution. Only four genes had significantly modified expression in the three hypertensive models among which a single gene, coding for sialyltransferase 7A, was consistently overexpressed. Correlation analysis between expression data and left ventricular mass index (LVMI) over all rats identified a larger set of genes whose expression was continuously related with LVMI, including known genes associated with cardiac remodeling. Positioning the detected transcripts along the chromosomes pointed out high-density regions mostly located within blood pressure and cardiac mass quantitative trait loci. Although our study could not detect a unique reprogramming of cardiac cells involving specific genes at early stage of LVH, it allowed the identification of some genes associated with LVH regardless of genetic background. This study thus provides a set of potentially important genes contained within restricted chromosomal regions involved in cardiovascular diseases.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Hipertensión/genética , Hipertrofia Ventricular Izquierda/genética , Animales , Animales Modificados Genéticamente , Perfilación de la Expresión Génica , Ventrículos Cardíacos/patología , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Renina/genética , Sialiltransferasas/genética , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The interactions between the effects of the sympathetic nervous system (SNS) and angiotensin II (ANG II) on vascular extracellular matrix (ECM) synthesis were determined in rats. The mRNA and protein content of collagen I, collagen III and elastin in the abdominal aorta (AA) and femoral artery (FA) was investigated in Wistar-Kyoto rats treated for 5 weeks with guanethidine, a sympathoplegic, losartan, an ANG II AT1 receptor (AT1R) blocker, or both. The effects of noradrenaline (NE) and ANG II on collagen III and elastin mRNA, and the receptor involved, were tested in cultured vascular smooth muscle cells (VSMCs) in vitro. Guanethidine increased collagen types I and III and decreased elastin, while losartan had an opposite effect, although without effect on collagen III. The combination of treatments abrogated changes induced by simple treatment with collagen I and elastin, but increased collagen III mRNA in AA and not in FA. NE stimulated collagen III mRNA via ß receptors and elastin via α1 and α2 receptors. ANG II stimulated collagen III but inhibited elastin mRNA via AT1R. Overall, SNS and ANG II exert opposite and antagonistic effects on major components of ECM in the vascular wall. This may be of relevance for the choice of a therapeutic strategy in vascular diseases.
Asunto(s)
Angiotensina II/farmacología , Aorta Abdominal/fisiología , Colágeno/metabolismo , Elastina/metabolismo , Matriz Extracelular/metabolismo , Arteria Femoral/fisiología , Norepinefrina/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Western Blotting , Peso Corporal/efectos de los fármacos , Colágeno/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Elastina/genética , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/genética , Arteria Femoral/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Modelos Biológicos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The interactions between the sympathetic nervous system (SNS) and angiotensin II (ANG II), and their direct effects in vitro on the enzymes involved in vascular extracellular matrix (ECM) degradation, were examined. Rats were treated with guanethidine, losartan or the combined treatments. mRNA, protein and activity of matrix metalloproteinase (MMP)-2 and MMP-9 and mRNA of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) were quantified in abdominal aorta (AA) and femoral artery (FA). Norepinephrine (NE) or ANG II with adrenergic (ß, α1 and α2) or losartan antagonists was tested for MMP mRNA response in cultured vascular smooth muscle cells (VSMCs). Combined treatment enhances the inhibition of MMP-2 mRNA and protein level induced by simple treatment in AA. However MMP-9 in AA and MMP mRNA in FA were reduced in the same order by treatments. MMP activities were not affected by treatments. The t-PA/PAI-1 ratio, which reflects the fibrinolytic balance, remained higher after treatments. In cultured VSMCs, NE induced stimulation of MMP mRNA via α2 and ß adrenergic receptors and MMP-2 activity via ß adrenergic receptors, while ANG II-induced stimulation was abrogated by losartan. Overall, there is a synergic inhibition of both systems on the level of MMP-2 in AA.
Asunto(s)
Angiotensina II/farmacología , Aorta Abdominal/fisiología , Arteria Femoral/fisiología , Metaloproteinasas de la Matriz/metabolismo , Norepinefrina/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Aorta Abdominal/citología , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/enzimología , Presión Sanguínea/efectos de los fármacos , Densitometría , Arteria Femoral/efectos de los fármacos , Arteria Femoral/enzimología , Gelatina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Guanetidina/farmacología , Losartán/farmacología , Masculino , Metaloproteinasas de la Matriz/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Activador de Tejido Plasminógeno/genéticaRESUMEN
BACKGROUND: Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. CONCLUSIONS/SIGNIFICANCE: We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.
Asunto(s)
Dietilhexil Ftalato/análogos & derivados , Feto/metabolismo , Receptores Nucleares Huérfanos/genética , Ovario/citología , Ovario/metabolismo , Testículo/citología , Testículo/metabolismo , Dietilhexil Ftalato/toxicidad , Contaminantes Ambientales/toxicidad , Femenino , Feto/citología , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Lípidos/biosíntesis , Receptores X del Hígado , Masculino , Ovario/efectos de los fármacos , Óvulo/citología , Óvulo/efectos de los fármacos , Óvulo/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.
Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Humanos , Fosforilación , ARN Polimerasa III/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/metabolismo , Sirolimus/farmacología , TransfecciónRESUMEN
In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left ventricle (LV) in vivo. Sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA and protein synthesis of collagen types I and III were examined by Q-RT-PCR and immunoblotting in the LV. Collagen types I and III mRNA were decreased respectively by 53% and 22% after sympathectomy and only collagen type I mRNA was increased by 52% after AT1 receptor blockade. mRNA was not changed for collagen type I but was decreased by 25% for collagen type III after double treatment. Only collagen protein type III was decreased after sympathectomy by 12%, but collagen proteins were increased respectively for types I and III by 145% and 52% after AT1 receptor blockade and by 45% and 60% after double treatment. Deducted interpretations from our experimental approach suggest that Ang II stimulates indirectly (via SNS) and inhibits directly (via AT1 receptors) the collagen type I at transcriptional and protein levels. For collagen type III, it stimulates indirectly the transcription and inhibited directly the protein level. Therefore, the Ang II regulates collagen synthesis differently through indirect and direct pathways.
Asunto(s)
Angiotensina II/metabolismo , Colágeno/biosíntesis , Ventrículos Cardíacos/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina/fisiología , Sistema Nervioso Simpático/metabolismo , Angiotensina II/farmacología , Animales , Colágeno/efectos de los fármacos , Colágeno/genética , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo III/biosíntesis , Colágeno Tipo III/efectos de los fármacos , Colágeno Tipo III/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/inervación , Masculino , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Sistema Renina-Angiotensina/efectos de los fármacos , Simpatectomía , Sistema Nervioso Simpático/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
In the present study, we tested the hypothesis that angiotensin II (Ang II) has both direct (via AT1 receptors) and indirect (via sympathostimulator pathway) actions on the synthesis and activity of the enzymes involved in the extracellular matrix degradation in vivo. For this purpose, sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA of the plasminogen activator (t-PA) and its inhibitor (PAI-1), the mRNA, protein and activity of the matrix metalloproteinases MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymographic methods in the left ventricle. t-PA and PAI-1 mRNA were decreased after sympathectomy and remained unchanged after AT1 receptors blockade. mRNA was increased for t-PA and decreased by similar degree for PAI-1 after double treatment. MMPs mRNA and protein levels were decreased either after sympathectomy or AT1 receptors blockade and an additive effect was acquired after double treatment. MMPs activity was decreased by similar degree in the three treated groups. Deducted interpretations from our experimental approach suggest that Ang II inhibits directly (via AT1 receptors) and indirectly (via sympathostimulator pathway) t-PA mRNA synthesis. It seems unable to influence directly PAI-1 mRNA, but stimulates indirectly PAI-1 mRNA synthesis. Ang II stimulates directly (via AT1 receptors) and indirectly (via sympathostimulator pathway) MMPs synthesis at both transcriptional and protein levels. The enzymatic activity of MMPs does not seem to be influenced directly by Ang II but it could be stimulated indirectly (via sympathostimulator pathway).