RESUMEN
White and brown adipocytes are believed to occupy different sites in the body. We studied the anatomical features and quantitative histology of the fat depots in obesity and type 2 diabetes-prone C57BL/6J mice acclimated to warm or cold temperatures. Most of the fat tissue was contained in depots with discrete anatomical features, and most depots contained both white and brown adipocytes. Quantitative analysis showed that cold acclimation induced an increase in brown adipocytes and an almost equal reduction in white adipocytes; however, there were no significant differences in total adipocyte count or any signs of apoptosis or mitosis, in line with the hypothesis of the direct transformation of white into brown adipocytes. The brown adipocyte increase was accompanied by enhanced density of noradrenergic parenchymal nerve fibers, with a significant correlation between the density of these fibers and the number of brown adipocytes. Comparison with data from obesity-resistant Sv129 mice disclosed a significantly different brown adipocyte content in C57BL/6J mice, suggesting that this feature could underpin the propensity of the latter strain to develop obesity. However, the greater C57BL/6J browning capacity can hopefully be harnessed to curb obesity and type 2 diabetes in patients with constitutively low amounts of brown adipose tissue.
Asunto(s)
Adipocitos Marrones/patología , Adipocitos Blancos/patología , Diabetes Mellitus Tipo 2/patología , Obesidad/patología , Sistema Nervioso Simpático/patología , Aclimatación , Animales , Recuento de Células , Transdiferenciación Celular , Frío , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Grasa Intraabdominal/patología , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas/patología , Grasa Subcutánea/patologíaRESUMEN
BACKGROUND: Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder resulting in a combined dopamine and serotonin deficiency. About 50% of the cases set in the neonatal period. Here, we report an atypical clinical presentation with moderate symptoms. PATIENT: At 10months old, the patient presented paroxysmal eye movements without seizures, and feeding difficulties which were attributed to gastroesophageal reflux. She was investigated at the age of 7years, because of orofacial dyspraxia, hypomimie, axial hypotonia and focal segmental dystonia, bilateral ptosis, without evidence for cognitive impairment. RESULTS: HVA [110nM; (reference value (rv): 202-596)] and HIAA (12nM; rv: 87-366) decreased, OMD (520nM; rv: 5-60) and 5-HTP (56nM; rv: 2-16) increased in CSF. We confirmed the diagnosis of AADC deficiency because the activity in plasma was low: 4pmol/min/ml; rv: 16-137. The kinetic analysis revealed a sixfold increase in the apparent affinity for L-dopa (4.26mM; control=0.71), but the V (max) was unchanged (37.5pmol dopamine/min/ml; control=39.1), suggesting a modification in the substrate binding-site. Molecular analysis revealed two heterozygous mutations in the DDC gene: c1040G > A; pR347Q already described, and a novel mutation c478C > T, pR160W. CONCLUSION: (1) CSF neurotransmitters metabolites suggested a moderate AADC deficiency; (2) The initial velocity saturation curve for L-dopa displayed a cooperative ligand binding behavior, in keeping with the modifications of the three-dimensional structure, induced by the amino acid substitutions (3) The treatment combination of L-dopa with pyridoxine dramatically improved the quality of life, the fatigability, and the paroxysmal eye movements.
RESUMEN
Brown adipose tissue is generally referred to as a specialized adipose tissue, as it presents many features of an adipose tissue. However, its specific morphology, innervation, vascularization and body location, as well as its unique physiological role in regulatable thermogenesis, highlight its peculiarity. Whereas the mechanism for energy dissipation by brown adipocytes as heat was elucidated several years ago, recent work has advanced our knowledge of these cells in terms of precursors, cell lineage and transcriptional regulators. The discovery of a proximity at the developmental level between brown adipocytes and myocytes will influence future research on human brown adipocytes with the aim of facilitating the burning of fatty acids in order to prevent or alleviate certain metabolic diseases.
Asunto(s)
Adipocitos Marrones/fisiología , Tejido Adiposo Pardo/fisiología , Metabolismo Energético/fisiología , Termogénesis/fisiología , Tejido Adiposo Pardo/citología , Metabolismo Energético/genética , Humanos , Células Musculares/fisiología , Termogénesis/genéticaRESUMEN
Carnitine palmitoyltransferase 2 (CPT2) deficiency is a rare mitochondrial fatty acid oxidation (FAO) disorder characterized by myalgia, exercise intolerance, and rhabdomyolysis. We evaluate the efficacy of bezafibrate (BZ), a hypolipidemic drug, as a treatment for this form of CPT2 deficiency. A pilot trial was conducted with BZ in six patients for 6 months. There was a follow-up period of 3 years. The oxidation rates of the long-chain fatty acid derivative palmitoyl-CoA, measured in the mitochondria of the patients' muscles, were markedly lower than normal before treatment and increased significantly (+39 to +206%; P = 0.028) in all patients after BZ treatment. The evaluation of the therapeutic effects by the patients themselves (using the Short Form Health Survey (SF-36)), as well as by the physicians, indicated an improvement in the condition of the patients; there was an increase in physical activity and a decline in muscular pain. The results suggest that BZ has a therapeutic effect in the muscular form of CPT2 deficiency.
Asunto(s)
Bezafibrato/uso terapéutico , Carnitina O-Palmitoiltransferasa/biosíntesis , Carnitina O-Palmitoiltransferasa/deficiencia , Hipolipemiantes/uso terapéutico , Enfermedades Musculares/tratamiento farmacológico , Enfermedades Musculares/etiología , Actividades Cotidianas , Acil-CoA Deshidrogenasa de Cadena Larga/biosíntesis , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Adulto , Carnitina O-Palmitoiltransferasa/genética , Prueba de Esfuerzo , Femenino , Estudios de Seguimiento , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/enzimología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Enfermedades Musculares/genética , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Dolor/epidemiología , Dolor/etiología , Palmitoil Coenzima A/metabolismo , Proyectos Piloto , Rabdomiólisis/tratamiento farmacológico , Rabdomiólisis/enzimología , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Normative data for amniotic fluid (AF) levels of organic acids at different gestational ages are lacking. They can provide a useful framework to investigate the accuracy of prenatal diagnosis for organic acidemias. METHODS: We report on the concentration of 21 organic acids in AF obtained by gas chromatography/mass spectrometry between the 12th and 34th weeks of gestation from 92 pregnancies that were not at risk for organic acidurias. RESULTS: We infer normal reference values that can be compared with 134 pregnancies at risk for several metabolic conditions, that is, propionic acidemia, methylmalonic acidemia (methylmalonyl-CoA mutase deficiency or defects in cobalamin metabolism), 4-hydroxybutyric acidemia, glutaric acidemia and pyroglutamic acidemia. CONCLUSION: Most of the metabolites tested did not show conspicuous variations across gestational ages in normal fetuses, with ranges that were consistently similar to available reference values from pooled samples in previous reports. With rare exceptions, knowledge of pathological versus normal values for relevant metabolites leads to clear-cut differentiation of affected versus unaffected fetuses. Nevertheless, it is strongly recommended that mutational analysis and/or additional biochemical approaches complement organic acid analysis for an adequate diagnostic workup.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Líquido Amniótico/química , Ácidos Carboxílicos/análisis , Diagnóstico Prenatal/métodos , Adulto , Femenino , Edad Gestacional , Humanos , Embarazo , Diagnóstico Prenatal/estadística & datos numéricos , Valores de ReferenciaRESUMEN
BACKGROUND: Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This disorder can be treated specifically using high doses of cysteamine. Accurate measurement of intracellular cystine content is necessary for the diagnosis and monitoring of treatment with cysteamine. Here we describe a new method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry assay. We compare this novel method with the cystine-binding protein assay. METHOD: Cells were isolated and lysed in the presence of N-ethylmaleimide to avoid interference from cysteine. After deproteinization, addition of stable isotope d6 cystine and butylation, cystine was measured using an API 3000 MSMS. RESULTS: The cystine assay was linear to at least 50 micromol/L. Within-run and between-run coefficients of variation were 2.9% and 5.7% respectively. CONCLUSION: It is possible to measure very low concentrations of intracellular cystine with liquid chromatography-tandem mass spectrometry. The results obtained with this novel method correlate very well with those obtained using the cystine-binding protein assay.
Asunto(s)
Cromatografía Liquida/métodos , Cistina/análisis , Granulocitos/química , Espectrometría de Masas en Tándem/métodos , Cistinosis/diagnóstico , Proteínas de Escherichia coli/metabolismo , HumanosRESUMEN
The term 'uncoupling protein' was originally used for the mitochondrial membrane protein UCP1, which is uniquely present in mitochondria of brown adipocytes, thermogenic cells that regulate body temperature in small rodents, hibernators and mammalian newborns. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP-synthase resulting in a futile proton cycling and dissipation of oxidation energy as heat. Recent identification of new homologues to UCP1 expressed in brown and white adipose tissue, muscle, brain and other tissues together with the hypothesis that these novel uncoupling proteins (UCPs) may regulate thermogenesis and/or fatty acid metabolism and furthermore may protect against free radical oxygen species production have generated considerable optimism for rapid advances in the identification of new targets for pharmacological management of complex pathological syndromes such as obesity, type 2 diabetes or chronic inflammatory diseases. However, since the physiological and biochemical roles of the novel UCPs are not yet clear, the main challenge today consists first of all in providing mechanistic explanation for their functions in cellular physiology. This lively awaited information may be the basis for potential pharmacological targeting of the UCPs in future.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Proteínas Mitocondriales/fisiología , Apoptosis/fisiología , Aterosclerosis/fisiopatología , Peso Corporal/fisiología , Respiración de la Célula/fisiología , Senescencia Celular/fisiología , Diabetes Mellitus Tipo 2/fisiopatología , Metabolismo Energético/fisiología , Humanos , Inflamación/fisiopatología , Canales Iónicos , Procesos Neoplásicos , Especies Reactivas de Oxígeno/metabolismo , Termogénesis/fisiología , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
BACKGROUND: Uncoupling protein 2 (UCP2) regulates the production of reactive oxygen species in macrophages. However, its role in atherosclerosis is unknown. METHODS AND RESULTS: Irradiated low-density lipoprotein receptor deficient mice (LDLR-/-) were transplanted with bone marrow from either UCP2 deficient mice (Ucp2-/-) or wild type mice (Ucp2+/+). Mice were fed an atherogenic diet for 7 weeks. Engraftment of bone marrow cells was confirmed by the presence of UCP2 protein expression in spleen cell mitochondria of Ucp2+/+ transplanted mice and its absence in Ucp2-/- transplanted mice. Leukocyte counts and plasma cholesterol levels were comparable in both groups. We found a marked increase in atherosclerotic lesion size in the thoracic aorta of Ucp2-/- transplanted mice compared with control Ucp2+/+ transplanted mice (8.3+/-0.9% versus 4.3+/-0.4%, respectively; P<0.005), as well as in the aortic sinus (150 066+/-12 388 microm2 versus 105 689+/-9 727 microm2, respectively; P<0.05). This was associated with increased nitrotyrosine staining, which suggests enhanced oxidative stress. Analysis of plaque composition revealed a significant increase in macrophage accumulation (P<0.05) and apoptosis (P<0.05), along with a decrease in collagen content (P<0.05), suggesting a potentially more vulnerable phenotype. CONCLUSION: These results suggest a protective role for UCP2 against atherosclerosis.
Asunto(s)
Arteriosclerosis/etiología , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/fisiología , Animales , Arteriosclerosis/sangre , Arteriosclerosis/patología , Trasplante de Médula Ósea , Cardiotónicos , Colesterol/sangre , Femenino , Canales Iónicos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Proteínas/genética , Receptores de LDL/genética , Proteína Desacopladora 2RESUMEN
Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipose tissue (BAT) and generates heat by uncoupling respiration from ATP synthesis. A defect in BAT thermogenesis has been described in different models of rodent obesity. In humans, the implication of BAT in energy expenditure is still under discussion. A BclI polymorphism associated with fat gain over time has been described in the upstream region of the human UCP1 (hUCP1) gene. In this study, a new polymorphic site linked to the BclI site is described which results in a C to A point mutation, 89 bp downstream of the BclI site, ie at position -3737 bp. This site is located in the recently analysed regulatory region of the hUCP1 gene. The mutation disrupts a consensus site for the binding of ATF/CREB transcription factor family and inhibits the factor binding in vitro. However, transient transfection of a rodent brown adipocyte cell line shows that the isoproterenol (ISO) stimulation of the hUCP1 gene transcription is not significantly affected by this mutation. However, we postulate that the C/A substitution, in human, may contribute to a minor defect in the regulation of hUCP1 transcription and that would explain fat gain over time.
Asunto(s)
Proteínas Portadoras/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , Proteínas de la Membrana/genética , Polimorfismo Genético , Tejido Adiposo Pardo/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Sitios de Unión , Proteínas Portadoras/fisiología , Línea Celular , AMP Cíclico/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Canales Iónicos , Isoproterenol/farmacología , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales , Mutagénesis , Transfección , Proteína Desacopladora 1RESUMEN
OBJECTIVE: It has been reported that an increased availability of free fatty acids (NEFA) not only interferes with glucose utilization in insulin-dependent tissues, but may also result in an uncoupling effect of heart metabolism. We aimed therefore to investigate the effect of an increased availability of NEFA on gene expression of proteins involved in transmembrane fatty acid (FAT/CD36) and glucose (GLUT4) transport and of the uncoupling proteins UCP2 and 3 at the heart and skeletal muscle level. STUDY DESIGN: Euglycemic hyperinsulinemic clamp was performed after 24 h Intralipid(R) plus heparin or saline infusion in lean Zucker rats. Skeletal and heart muscle glucose utilization was calculated by 2-deoxy-[1-(3)H]-D-glucose technique. Quantification of FAT/CD36, GLUT4, UCP2 and UCP3 mRNAs was obtained by Northern blot analysis or RT-PCR. RESULTS: In Intralipid(R) plus heparin infused animals a significant decrease in insulin-mediated glucose uptake was observed both in the heart (22.62+/-2.04 vs 10.37+/-2.33 ng/mg/min; P<0.01) and in soleus muscle (13.46+/-1.53 vs 6.84+/-2.58 ng/mg/min; P<0.05). FAT/CD36 mRNA was significantly increased in skeletal muscle tissue (+117.4+/-16.3%, P<0.05), while no differences were found at the heart level in respect to saline infused rats. A clear decrease of GLUT4 mRNA was observed in both tissues. The 24 h infusion of fat emulsion resulted in a clear enhancement of UCP2 and UCP3 mRNA levels in the heart (99.5+/-15.3 and 80+/-4%) and in the skeletal muscle (291.5+/-24.7 and 146.9+/-12.7%). CONCLUSIONS: As a result of the increased availability of NEFA, FAT/CD36 gene expression increases in skeletal muscle, but not at the heart level. The augmented lipid fuel supply is responsible for the depression of insulin-mediated glucose transport and for the increase of UCP2 and 3 gene expression in both skeletal and heart muscle.
Asunto(s)
Proteínas Portadoras/genética , Emulsiones Grasas Intravenosas/administración & dosificación , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Músculos/metabolismo , Transportadores de Anión Orgánico/genética , Proteínas/genética , Animales , Glucemia/metabolismo , Northern Blotting , Antígenos CD36 , Ácidos Grasos no Esterificados/sangre , Expresión Génica , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Transportador de Glucosa de Tipo 4 , Heparina/administración & dosificación , Insulina/sangre , Insulina/farmacología , Canales Iónicos , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculos/efectos de los fármacos , Miocardio/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Zucker , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
Energy exists as organic molecules and heat in living organisms. In adult mammals, body weight and fat content remain unchanged if energy intake is strictly equivalent to energy expenditure. In other words, regulation of body weight requires energy of foods to be entirely dissipated as heat. Imbalance between ingested energy and thermogenesis induces obesity or thinness. Alterations of food intake or energy expenditure represent the two causes of body weight disturbance. It is accepted that individuals differ in food efficiency i.e. ability to metabolize foods and store fat or totally burn nutrients. Mechanisms of food efficiency and futile cycles are presented. I started my research work analysing thermogenic mechanism in brown adipose tissue. Actually, in addition to white adipose tissue which is the major type of adipose tissue, mammals own another type of adipose tissue referred to as brown adipose tissue. This later tissue is an activatable thermogenic organ which oxidizes fatty acids and releases heat in blood stream. Brown fat is activated during exposure to the cold (in rodents), at birth, and during arousal in hibernators. My initial work helped to characterize a mitochondrial protein named uncoupling protein or UCP which is responsible for activation of fatty acid oxidation and heat production in brown adipocytes. Actually, in most cells, fifty per cent of oxidation energy is recovered as ATP in mitochondria through the process of coupling of respiration to ADP phosphorylation. In contrast to mitochondria of most tissues, brown adipocyte mitochondria can escape the obligatorily coupling of respiration and waste almost ninety per cent of respiration energy as thermogenesis. UCP characterization and its molecular cloning as well as antibodies obtention were used to better understand cellular thermogenesis. Brown adipocytes were identified in babies and adult patients with pheochromocytoma. More recently, research on the brown fat UCP helped us to identify UCP2, a UCP homolog present in most human and animal tissues. A family of UCPs exist in animals and plants. These UCPs may function as mitochondrial uncouplers. However, the ancient function of the UCPs may be rather associated to adaptation to oxygen and control of free radicals than to thermogenesis. Further studies of UCPs will improve the knowledge of mitochondrial metabolism and substrate oxidation. In other respects, analysis of molecular mechanisms controlling respiration uncoupling may contribute to new strategies of treatment of metabolic disorders such as obesity.
Asunto(s)
Metabolismo Energético , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Composición Corporal , Peso Corporal , Proteínas Portadoras/metabolismo , Ingestión de Energía , Humanos , Canales Iónicos , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Termogénesis , Proteína Desacopladora 1RESUMEN
Uncoupling protein 2 (UCP2) mRNA is expressed in a panoply of tissues, including the brain, where it is widely distributed. In the mouse brain, it is expressed in the hypothalamus (suprachiasmatic, paraventricular, dorsomedial, ventromedial and arcuate nuclei), the thalamus (submedius nucleus) and the brain-stem (dorsal motor nucleus of the vagus nerve). In the rat brain, it is also expressed in the hippocampus. The presence of UCP2 mRNA in neurons expressing corticotropin-releasing factor and arginine-vasopressin suggests a role for UCP2 in the control of neuroendocrine and behavioural functions. We have recently demonstrated that UCP2-deficient mice can resist the lethal effect of toxoplasmosis through an enhanced production of reactive oxygen species (ROS) from the macrophages. This finding provides evidence that UCP2 can be part of a mechanism preventing ROS production. UCP2 could therefore be involved in protecting the brain against oxidative stress. The involvement of UCP2 in neuroprotection is also consistent with the recent observation that kainic acid, which promotes Ca(2+) uptake in the glutamate-activated neurons in the hippocampal CA1 field, can induce the UCP2 gene in the activated CA1 cells. The role of UCP2 in neuroprotection warrants further investigation.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Biosíntesis de Proteínas , Proteínas/fisiología , Animales , Canales Iónicos , Modelos Biológicos , Oxidación-Reducción , Estrés Oxidativo , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno , Distribución Tisular , Proteína Desacopladora 2Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/fisiología , Desacopladores , Tejido Adiposo Pardo/ultraestructura , Animales , Proteínas Portadoras/genética , Humanos , Canales Iónicos , Mitocondrias/fisiología , Proteínas/genética , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
We observed a 17-month-old girl with profound and initially isolated episodes of hypothermia. Thereafter, she developed growth delay, repetitive corneal and bone lesions. Persistent hyperlactataemia in plasma and in CSF prompted us to investigate respiratory chain enzymes. A deficit in respiratory chain complexes III and IV was demonstrated in isolated skeletal muscle mitochondria, circulating lymphocytes and fibroblasts by spectrophotometric and polarographic studies. Moreover, UCP3 mRNA expression in muscle was decreased.
Asunto(s)
Proteínas Portadoras/genética , Deficiencia de Citocromo-c Oxidasa , Complejo III de Transporte de Electrones/deficiencia , Hipotermia/etiología , Miopatías Mitocondriales/diagnóstico , ARN Mensajero/genética , Biopsia , Preescolar , Femenino , Fracturas Espontáneas/diagnóstico , Fracturas Espontáneas/enzimología , Fracturas Espontáneas/genética , Humanos , Hipotermia/enzimología , Hipotermia/genética , Lactante , Recién Nacido , Canales Iónicos , Miopatías Mitocondriales/enzimología , Miopatías Mitocondriales/genética , Proteínas Mitocondriales , Músculo Esquelético/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Proteína Desacopladora 3RESUMEN
The metabolic utilization of substrates results in ATP synthesis and energy loss as heat. In tissues and cells the mitochondria reoxidize reduced coenzymes and phosphorylate ADP. A significant proportion of the energy is released through thermogenesis by mitochondria. This is due to a less than perfect coupling of cellular respiration to ATP synthesis. Previous studies of brown adipocytes, which are cells specialized in regulatory thermogenesis, have shown that heat production is due to the regulated activity and synthesis of a particular proton transporter in the inner membrane of brown adipocyte mitochondria--uncoupling protein (UCP) 1. UCP homologues have recently been identified. UCP2 is widely expressed in human tissues, whereas UCP3 is expressed predominantly in human skeletal muscles. These novel UCPs represent genes which are potentially important for regulation of metabolic pathways and energy expenditure in humans. Biochemical and genetic studies support a role for these novel UCPs in metabolic regulations in humans. However, several physiological studies question such a role. Importantly, UCP2 and UCP3 seem to be able to control the activity of mitochondria in response to oxidants.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas Mitocondriales , Proteínas/metabolismo , Termogénesis/fisiología , Metabolismo Energético/fisiología , Homeostasis/fisiología , Humanos , Canales Iónicos , Investigadores , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Desacopladores/metabolismo , Secuencia de Aminoácidos , Animales , Metabolismo Basal/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Canales Iónicos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Proteínas Desacopladoras Mitocondriales , Modelos Moleculares , Plantas , Proteínas/química , Proteínas/metabolismo , Retinoides/farmacología , Homología de Secuencia , Desacopladores/química , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3 , Levaduras/genética , Levaduras/metabolismoRESUMEN
The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.
Asunto(s)
Proteínas Aviares , Proteínas Portadoras/fisiología , Proteínas Mitocondriales , Músculo Esquelético/fisiología , Termogénesis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Pollos , Cartilla de ADN , ADN Complementario , Proteínas Desacopladoras Mitocondriales , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Uncoupling protein-2 (UCP2) is present in many tissues with relevance to fuel metabolism, and its expression is increased in fat and muscle in response to elevated circulating free fatty acids resulting from fasting and high fat feeding. We proposed a role for peroxisome proliferator-activated receptor-gamma (PPARgamma) as a mediator of these physiological changes in UCP2, because thiazolidinediones also increase expression of UCP2 in these cell types (). To determine the molecular basis for this regulation, we isolated the 7.3-kilobase promoter region of the mouse UCP2 gene. The -7.3-kilobase/+12-base pair fragment activates transcription of a reporter gene by 50-100-fold. Deletion and point mutation analysis, coupled with gel shift assays, indicate the presence of a 43-base pair enhancer (-86/-44) that is responsible for the majority of both basal and PPARgamma-dependent transcriptional activity. The distal (-86/-76) part of the enhancer specifically binds Sp1, Sp2, and Sp3 and is indistinguishable from a consensus Sp1 element in competition experiments. Point mutation in this sequence reduces basal activity by 75%. A second region (-74/-66) is identical to the sterol response element consensus and specifically binds ADD1/SREBP1. However, deletion of this sequence does not affect basal transcriptional activity or the response to PPARgamma. The proximal portion of the enhancer contains a direct repeat of two E-Box motifs, which contributes most strongly to basal and PPARgamma-dependent transcription of the UCP2 promoter. Deletion of this region results in a 10-20-fold reduction of transcriptional activity and complete loss of PPARgamma responsiveness. Point mutations in either E-Box, but not in the spacer region between them, eliminate the stimulatory response to PPARgamma. However, gel shift assays show that PPARgamma does not bind to this region. Taken together, these data indicate that PPARgamma activates the UCP2 gene indirectly by altering the activity or expression of other transcription factors that bind to the UCP2 promoter.
Asunto(s)
Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/genética , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Canales Iónicos , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Regiones Promotoras Genéticas , Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína Desacopladora 2RESUMEN
Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria. We generated a highly sensitive polyclonal antibody against human UCP2. Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(-/-) mice, leading to non-ambiguous identification of UCP2. We detected UCP2 in spleen, lung, stomach, and white adipose tissue. No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue. The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria. Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations. Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels. This is explained by translational regulation of the UCP2 mRNA. We have shown that an upstream open reading frame located in exon two of the ucp2 gene strongly inhibits the expression of the protein. This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions.
Asunto(s)
Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Estrés Oxidativo , Biosíntesis de Proteínas , Proteínas/metabolismo , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Exones , Humanos , Canales Iónicos , Ratones , Ratones Noqueados , Sistemas de Lectura Abierta , Proteínas/genética , ARN Mensajero/genética , Ratas , Proteína Desacopladora 2RESUMEN
The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.