RESUMEN
BACKGROUND: Acquisition and distribution of zinc supports a number of biological processes. Various molecular factors are involved in zinc metabolism but not fully explored. BASIC PROCEDURES: Spontaneous mutants were generated in yeast with excess zinc culture followed by whole genome DNA sequencing to discover zinc metabolism related genes by bioinformatics. An identified mutant was characterized through metallomic and molecular biology methods. MAIN FINDINGS: Here we reported that MTM1 knockout cells displayed much stronger zinc tolerance than wild type cells on SC medium when exposed to excess zinc. Zn accumulation of mtm1Δ cells was dramatically decreased compared to wild type cells under excessive zinc condition due to MTM1 deletion reduced zinc uptake. ZRC1 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain leading to increased vacuolar zinc accumulations in mtm1Δ cells. The mRNA levels of ZRT1 and ZAP1 decreased in mtm1Δ cells contributing to less Zn uptake. The zrc1Δmtm1Δ double knockout strain exhibited Zn sensitivity. MTM1 knockout did not afford resistance to excess zinc through an effect mediated through an influence on levels of ROS. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely impaired and not restored through Zn supplementation. Meanwhile, additional Zn showed no significant effect on the localization and expression of Mtm1p. PRINCIPAL CONCLUSIONS: Our study reveals the MTM1 gene plays an important role in the regulation of zinc homeostasis in yeast cells via changing zinc uptake and distribution. This discovery provides new insights for better understanding biochemical communication between vacuole and mitochondrial in relation to zinc-metabolism.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Zinc/metabolismo , Proteínas Portadoras/genética , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
TRMU is a nuclear gene crucial for mitochondrial DNA translation by encoding tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, which thiolates mitochondrial tRNA. Biallelic pathogenic variants in TRMU are associated with transient infantile liver failure. Other less common presentations such as Leigh syndrome, myopathy, and cardiomyopathy have been reported. Recent studies suggested that provision of exogenous L-cysteine or N-acetylcysteine may ameliorate the effects of disease-causing variants and improve the natural history of the disease. Here, we report six infants with biallelic TRMU variants, including four previously unpublished patients, all treated with exogenous cysteine. We highlight the first report of an affected patient undergoing orthotopic liver transplantation, the long-term effects of cysteine supplementation, and the ability of the initial presentation to mimic multiple inborn errors of metabolism. We propose that TRMU deficiency should be suspected in all children presenting with persistent lactic acidosis and hypoglycemia, and that combined N-acetylcysteine and L-cysteine supplementation should be considered prior to molecular diagnosis, as this is a low-risk approach that may increase survival and mitigate the severity of the disease course.
Asunto(s)
Enfermedad de Leigh/terapia , Fallo Hepático/terapia , Proteínas Mitocondriales/genética , Biosíntesis de Proteínas , ARNt Metiltransferasas/genética , Acetilcisteína/administración & dosificación , Acetilcisteína/metabolismo , Acidosis/genética , Acidosis/metabolismo , Cisteína/administración & dosificación , Cisteína/metabolismo , ADN Mitocondrial/genética , Femenino , Humanos , Lactante , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Enfermedad de Leigh/patología , Fallo Hepático/genética , Fallo Hepático/metabolismo , Fallo Hepático/patología , Trasplante de Hígado/métodos , Masculino , Mitocondrias/enzimología , Proteínas Mitocondriales/deficiencia , ARN de Transferencia/genética , ARNt Metiltransferasas/deficienciaRESUMEN
Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.
Asunto(s)
Liasas de Carbono-Carbono/deficiencia , Retículo Endoplásmico/metabolismo , Metabolismo Energético , Fibroblastos/patología , Homeostasis , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/deficiencia , Proteínas de Transporte Nucleocitoplasmático/deficiencia , Adenosina Trifosfato/biosíntesis , Apoptosis , Liasas de Carbono-Carbono/metabolismo , Línea Celular , Respiración de la Célula , Análisis Mutacional de ADN , Fibroblastos/metabolismo , Humanos , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Superóxidos/metabolismoRESUMEN
Primary coenzyme Q10 (CoQ10 ; MIM# 607426) deficiencies are an emerging group of inherited mitochondrial disorders with heterogonous clinical phenotypes. Over a dozen genes are involved in the biosynthesis of CoQ10 , and mutations in several of these are associated with human disease. However, mutations in COQ5 (MIM# 616359), catalyzing the only C-methylation in the CoQ10 synthetic pathway, have not been implicated in human disease. Here, we report three female siblings of Iraqi-Jewish descent, who had varying degrees of cerebellar ataxia, encephalopathy, generalized tonic-clonic seizures, and cognitive disability. Whole-exome and subsequent whole-genome sequencing identified biallelic duplications in the COQ5 gene, leading to reduced levels of CoQ10 in peripheral white blood cells of all affected individuals and reduced CoQ10 levels in the only muscle tissue available from one affected proband. CoQ10 supplementation led to clinical improvement and increased the concentrations of CoQ10 in blood. This is the first report of primary CoQ10 deficiency caused by loss of function of COQ5, with delineation of the clinical, laboratory, histological, and molecular features, and insights regarding targeted treatment with CoQ10 supplementation.
Asunto(s)
Vías Biosintéticas/genética , Ataxia Cerebelosa/diagnóstico , Ataxia Cerebelosa/genética , Metiltransferasas/deficiencia , Encefalomiopatías Mitocondriales/diagnóstico , Encefalomiopatías Mitocondriales/genética , Proteínas Mitocondriales/deficiencia , Ubiquinona/análogos & derivados , Biopsia , Ataxia Cerebelosa/dietoterapia , Ataxia Cerebelosa/metabolismo , Variaciones en el Número de Copia de ADN , Suplementos Dietéticos , Transporte de Electrón , Femenino , Fibroblastos/metabolismo , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucocitos/metabolismo , Metiltransferasas/genética , Encefalomiopatías Mitocondriales/dietoterapia , Encefalomiopatías Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Músculos/patología , Consumo de Oxígeno , Linaje , Polimorfismo de Nucleótido Simple , Hermanos , Ubiquinona/biosíntesisRESUMEN
Coenzyme Q10 (CoQ10) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ10 is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ10 biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ10 biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ10 biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ10 deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ10 biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency.
Asunto(s)
Ataxia/metabolismo , Enfermedades Mitocondriales/metabolismo , Debilidad Muscular/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/deficiencia , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Hidroxibenzoatos/farmacología , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Espectrometría de Masas en Tándem , Ubiquinona/biosíntesis , Ubiquinona/metabolismo , Ácido Vanílico/farmacologíaRESUMEN
The mitochondrial citrate transport protein (CTP), encoded by SLC25A1, accommodates bidirectional trafficking of citrate between the mitochondria and cytosol, supporting lipid biosynthesis and redox homeostasis. Genetic CTP deficiency causes a fatal neurodevelopmental syndrome associated with the accumulation of L- and D-2-hydroxyglutaric acid, and elevated CTP expression is associated with poor prognosis in several types of cancer, emphasizing the importance of this transporter in multiple human pathologies. Here we describe the metabolic consequences of CTP deficiency in cancer cells. As expected from the phenotype of CTP-deficient humans, somatic CTP loss in cancer cells induces broad dysregulation of mitochondrial metabolism, resulting in accumulation of lactate and of the L- and D- enantiomers of 2-hydroxyglutarate (2HG) and depletion of TCA cycle intermediates. It also eliminates mitochondrial import of citrate from the cytosol. To quantify the impact of CTP deficiency on metabolic flux, cells were cultured with a set of 13C-glucose and 13C-glutamine tracers with resulting data integrated by metabolic flux analysis (MFA). CTP-deficient cells displayed a major restructuring of central carbon metabolism, including suppression of pyruvate dehydrogenase (PDH) and induction of glucose-dependent anaplerosis through pyruvate carboxylase (PC). We also observed an unusual lipogenic pathway in which carbon from glucose supplies mitochondrial production of alpha-ketoglutarate (AKG), which is then trafficked to the cytosol and used to supply reductive carboxylation by isocitrate dehydrogenase 1 (IDH1). The resulting citrate is cleaved to produce lipogenic acetyl-CoA, thereby completing a novel pathway of glucose-dependent reductive carboxylation. In CTP deficient cells, IDH1 inhibition suppresses lipogenesis from either glucose or glutamine, implicating IDH1 as a required component of fatty acid synthesis in states of CTP deficiency.
Asunto(s)
Proteínas de Transporte de Anión/deficiencia , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas de Neoplasias , Neoplasias/metabolismo , Línea Celular Tumoral , Ácidos Grasos/genética , Humanos , Mitocondrias/genética , Mitocondrias/patología , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Transportadores de Anión OrgánicoRESUMEN
The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.
Asunto(s)
Conducta Animal , Ataxia Cerebelosa/enzimología , Cerebelo/enzimología , Proteínas Mitocondriales/deficiencia , Músculo Esquelético/enzimología , Ubiquinona/deficiencia , Animales , Células COS , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/fisiopatología , Ataxia Cerebelosa/psicología , Cerebelo/fisiopatología , Cerebelo/ultraestructura , Chlorocebus aethiops , Modelos Animales de Enfermedad , Tolerancia al Ejercicio , Femenino , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Metabolismo de los Lípidos , Masculino , Aprendizaje por Laberinto , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Modelos Moleculares , Actividad Motora , Fuerza Muscular , Músculo Esquelético/fisiopatología , Fenotipo , Unión Proteica , Conformación Proteica , Proteómica/métodos , Reconocimiento en Psicología , Prueba de Desempeño de Rotación con Aceleración Constante , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Convulsiones/enzimología , Convulsiones/genética , Convulsiones/fisiopatología , Relación Estructura-Actividad , Factores de Tiempo , Transfección , Ubiquinona/química , Ubiquinona/genéticaRESUMEN
Inherited ataxias are a group of heterogeneous disorders in children or adults but their genetic definition remains still undetermined in almost half of the patients. However, CoQ10 deficiency is a rare cause of cerebellar ataxia and ADCK3 is the most frequent gene associated with this defect. We herein report a 48 year old man, who presented with dysarthria and walking difficulties. Brain magnetic resonance imaging showed a marked cerebellar atrophy. Serum lactate was elevated. Tissues obtained by muscle and skin biopsies were studied for biochemical and genetic characterization. Skeletal muscle biochemistry revealed decreased activities of complexes I+III and II+III and a severe reduction of CoQ10 , while skin fibroblasts showed normal CoQ10 levels. A mild loss of maximal respiration capacity was also found by high-resolution respirometry. Molecular studies identified a novel homozygous deletion (c.504del_CT) in ADCK3, causing a premature stop codon. Western blot analysis revealed marked reduction of ADCK3 protein levels. Treatment with CoQ10 was started and, after 1 year follow-up, patient neurological condition slightly improved. This report suggests the importance of investigating mitochondrial function and, in particular, muscle CoQ10 levels, in patients with adult-onset cerebellar ataxia. Moreover, clinical stabilization by CoQ10 supplementation emphasizes the importance of an early diagnosis.
Asunto(s)
Ataxia/genética , Ataxia Cerebelosa/genética , Codón sin Sentido , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Debilidad Muscular/genética , Ubiquinona/análogos & derivados , Ubiquinona/deficiencia , Ataxia/complicaciones , Ataxia/diagnóstico , Ataxia/fisiopatología , Ataxia Cerebelosa/complicaciones , Ataxia Cerebelosa/diagnóstico , Ataxia Cerebelosa/fisiopatología , Diagnóstico Tardío , Proteínas del Complejo de Cadena de Transporte de Electrón/deficiencia , Fibroblastos/metabolismo , Expresión Génica , Homocigoto , Humanos , Ácido Láctico/sangre , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/fisiopatología , Proteínas Mitocondriales/deficiencia , Debilidad Muscular/complicaciones , Debilidad Muscular/diagnóstico , Debilidad Muscular/fisiopatología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Piel/metabolismo , Ubiquinona/genéticaRESUMEN
The hypothalamus has been implicated in skeletal metabolism. Whether hunger-promoting neurons of the arcuate nucleus impact the bone is not known. We generated multiple lines of mice to affect AgRP neuronal circuit integrity. We found that mice with Ucp2 gene deletion, in which AgRP neuronal function was impaired, were osteopenic. This phenotype was rescued by cell-selective reactivation of Ucp2 in AgRP neurons. When the AgRP circuitry was impaired by early postnatal deletion of AgRP neurons or by cell autonomous deletion of Sirt1 (AgRP-Sirt1(-/-)), mice also developed reduced bone mass. No impact of leptin receptor deletion in AgRP neurons was found on bone homeostasis. Suppression of sympathetic tone in AgRP-Sirt1(-/-) mice reversed osteopenia in transgenic animals. Taken together, these observations establish a significant regulatory role for AgRP neurons in skeletal bone metabolism independent of leptin action.
Asunto(s)
Proteína Relacionada con Agouti/genética , Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/metabolismo , Fémur/metabolismo , Propranolol/farmacología , Tibia/metabolismo , Proteína Relacionada con Agouti/deficiencia , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/patología , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/patología , Fémur/efectos de los fármacos , Fémur/patología , Regulación de la Expresión Génica , Homeostasis , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/patología , Canales Iónicos/deficiencia , Canales Iónicos/genética , Leptina/genética , Leptina/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Norepinefrina/metabolismo , Fenotipo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Transducción de Señal , Sirtuina 1/deficiencia , Sirtuina 1/genética , Tibia/efectos de los fármacos , Tibia/patología , Proteína Desacopladora 2RESUMEN
Type 2 diabetes is characterized by insulin resistance and mitochondrial dysfunction in classical target tissues such as muscle, fat, and liver. Using a murine model of type 2 diabetes, we show that there is hypothalamic insulin resistance and mitochondrial dysfunction due to downregulation of the mitochondrial chaperone HSP60. HSP60 reduction in obese, diabetic mice was due to a lack of proper leptin signaling and was restored by leptin treatment. Knockdown of Hsp60 in a mouse hypothalamic cell line mimicked the mitochondrial dysfunction observed in diabetic mice and resulted in increased ROS production and insulin resistance, a phenotype that was reversed with antioxidant treatment. Mice with a heterozygous deletion of Hsp60 exhibited mitochondrial dysfunction and hypothalamic insulin resistance. Targeted acute downregulation of Hsp60 in the hypothalamus also induced insulin resistance, indicating that mitochondrial dysfunction can cause insulin resistance in the hypothalamus. Importantly, type 2 diabetic patients exhibited decreased expression of HSP60 in the brain, indicating that this mechanism is relevant to human disease. These data indicate that leptin plays an important role in mitochondrial function and insulin sensitivity in the hypothalamus by regulating HSP60. Moreover, leptin/insulin crosstalk in the hypothalamus impacts energy homeostasis in obesity and insulin-resistant states.
Asunto(s)
Chaperonina 60/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Leptina/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Línea Celular , Chaperonina 60/deficiencia , Chaperonina 60/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Obesidad/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
OBJECTIVE: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. APPROACH AND RESULTS: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. CONCLUSIONS: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.
Asunto(s)
Aorta/metabolismo , Aneurisma de la Aorta/prevención & control , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aorta/fisiopatología , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/patología , Aneurisma de la Aorta/fisiopatología , Presión Sanguínea , Células Cultivadas , Colágeno Tipo I/metabolismo , Grupo Citocromo b/metabolismo , Dilatación Patológica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Tejido Elástico/metabolismo , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Genotipo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Miocitos del Músculo Liso/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Oxidantes/farmacología , Fenotipo , ARN Mensajero/metabolismo , Rigidez Vascular , Vasoconstrictores/farmacología , VasodilataciónRESUMEN
Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1(+/-) mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1(+/-) mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ(10) was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3(+/-) mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno/genética , Ubiquinona/metabolismo , Animales , Respiración de la Célula/genética , Masculino , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Mitocondrias/enzimología , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/deficiencia , Oxigenasas de Función Mixta , Partículas Submitocóndricas/metabolismo , Ubiquinona/administración & dosificación , Ubiquinona/genéticaRESUMEN
Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.
Asunto(s)
Hipoxia de la Célula/fisiología , Hígado Graso , Hepatocitos/patología , Canales Iónicos/deficiencia , Proteínas Mitocondriales/deficiencia , Oxígeno/farmacología , Adenosina Trifosfato/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Emulsiones/farmacología , Ácidos Grasos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Obesos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosfolípidos/farmacología , Aceite de Soja/farmacología , Proteína Desacopladora 2RESUMEN
The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.
Asunto(s)
Metabolismo Energético/fisiología , Técnicas de Inactivación de Genes , Interferencia de ARN/fisiología , Adenosina Trifosfato/metabolismo , Animales , Dióxido de Carbono/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Ciclo del Ácido Cítrico/fisiología , Embrión de Mamíferos/citología , Metabolismo Energético/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Fibroblastos/metabolismo , Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/genética , Eliminación de Gen , Glucólisis/fisiología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ácido Láctico/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas de Transporte de Nucleótidos/genética , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , ARN Interferente Pequeño/genéticaRESUMEN
Steatotic livers represent a growing proportion of marginal organs available for transplantation. These livers are highly prone to primary nonfunction following transplantation and are therefore routinely turned down for surgery. Given the elevated levels and sensitivity for reactive oxygen species (ROS) in these livers, we evaluated whether pretreatment with a targeted ROS scavenger, vitamin E succinate, increased survival and decreased injury after ischemia/reperfusion (I/R). For this study, ob/ob mice received 50 IU/d vitamin E succinate in supplemented vs control chow for 7 days, and were subjected to 15 minutes of total hepatic ischemia and 24 hours of reperfusion. Treatment resulted in a 5-fold decrease in serum alanine aminotransferase (ALT) levels after reperfusion, mirrored by significant decreases in hepatocellular necrosis. These results suggested that targeted antioxidants such as vitamin E succinate may prove to be highly applicable for the pretreatment of steatotic donor livers, increasing their tolerance for I/R and the transplantation process.
Asunto(s)
Hígado/patología , Daño por Reperfusión/prevención & control , Tocoferoles/uso terapéutico , Animales , Canales Iónicos/deficiencia , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Proteínas Mitocondriales/deficiencia , Necrosis , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Proteína Desacopladora 2RESUMEN
Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.
Asunto(s)
Fármacos Antiobesidad/uso terapéutico , Aceites de Pescado/uso terapéutico , Canales Iónicos/fisiología , Proteínas Mitocondriales/fisiología , Obesidad/terapia , Animales , Grasas de la Dieta/administración & dosificación , Femenino , Expresión Génica , Canales Iónicos/deficiencia , Canales Iónicos/genética , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Obesidad/etiología , ARN Mensajero/análisis , Aceite de Cártamo/administración & dosificación , Caracteres Sexuales , Proteína Desacopladora 2RESUMEN
Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced beta cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes K(ATP) channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced beta cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating beta cell dysfunction.
Asunto(s)
Glucosa/antagonistas & inhibidores , Compuestos Heterocíclicos con 3 Anillos/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas Mitocondriales/efectos de los fármacos , Obesidad/metabolismo , Piranos/farmacología , Adenosina Trifosfato/metabolismo , Aldehídos/antagonistas & inhibidores , Aldehídos/metabolismo , Animales , Medicamentos Herbarios Chinos/farmacología , Glucosa/farmacología , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/química , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/fisiología , Canales Iónicos , Glicósidos Iridoides , Iridoides , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones Obesos , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/metabolismo , Conformación Molecular , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Protones , Piranos/química , Proteína Desacopladora 2RESUMEN
A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.
Asunto(s)
Colchicina/análisis , Colorantes Fluorescentes/síntesis química , Hepatopatías/diagnóstico , Hígado/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/farmacocinética , Pirroles/síntesis química , Pirroles/farmacocinética , Ácidos Sulfónicos/síntesis química , Ácidos Sulfónicos/farmacocinética , Animales , Bilis/química , Bilis/metabolismo , Bilirrubina/análogos & derivados , Bilirrubina/química , Colchicina/química , Evaluación Preclínica de Medicamentos , Estudios de Factibilidad , Colorantes Fluorescentes/química , Hígado/efectos de los fármacos , Hepatopatías/genética , Hepatopatías/metabolismo , Masculino , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Pirimidinonas/análisis , Pirroles/análisis , Pirroles/química , Ratas , Ratas Gunn , Ratas Sprague-Dawley , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Fluorescencia , Ácidos Sulfónicos/análisis , Orina/químicaRESUMEN
Fatty acids are a major fuel for the body and fatty acid oxidation is particularly important during fasting, sustained aerobic exercise & stress. The myocardium and resting skeletal muscle utilise long-chain fatty acids as a major source of energy. Inherited disorders of fatty acid oxidation seriously compromise the function of muscle and other highly energy-dependent tissues such as brain, nerve, heart, kidney & liver. Defects of fatty acid oxidation lead to a range of neuromyopathic disease in both adults and children. Such defects encompass a wide spectrum of clinical disease, presenting in the neonatal period or infancy with recurrent hypoketotic hypoglycaemic encephalopathy, liver dysfunction and hyperammonaemia with neurosensory deficits secondary to the acute onset. In addition, there may be cardiac arrhythmias and/or progressive cardiomyopathy, which may give rise to secondary hypoxic-ischaemic encephalopathy. In older children, adolescence or adults there is often exercise intolerance with episodic myalgia or rhabdomyolysis in association with prolonged aerobic exercise or other exacerbating factors. Some disorders are particularly associated with toxic metabolites that may contribute to encephalopathy, polyneuropathy, axonopathy and pigmentary retinopathy. Diagnosis is through clinical suspicion with appropriate investigations in blood and urine taken during crisis. Definitive diagnosis is usually by fibroblast assay. Treatment is generally through avoidance of fasting, frequent carbohydrate rich feeds and in long-chain defects MCT supplementation. Novel treatments include the use of D,L-3-hydroxybutyrate and the potential use of fibrates to increase mutant protein levels in mild disorders.
Asunto(s)
Encefalopatías Metabólicas/etiología , Ácidos Grasos/metabolismo , Acil-CoA Deshidrogenasa/deficiencia , Encefalopatías Metabólicas/terapia , Carnitina/biosíntesis , Carnitina/deficiencia , Dietoterapia , Humanos , Proteínas Mitocondriales/deficiencia , Oxidación-ReducciónRESUMEN
In an infant who suffered from prolonged icterus and hepatocellular dysfunction we detected an increase of citrulline and dibasic amino acids in plasma and urine. The amino acid levels along with all the abnormal liver tests normalized upon replacing breast-milk by formula feeding; there was no relapse after human milk was tentatively reintroduced. A novel mutation, a approximately 9.5-kb genomic duplication, was identified in the citrin gene (SLC25A13) resulting in the insertion of exon 15. No mutation was detected in the CAT2A specific exon of the SLC7A2 gene which encodes for the liver transporter of cationic amino acids. This is the first report of infantile citrin deficiency in non-Asian patients.