RESUMO
Recently, we have identified a recessive mutation, an abnormal coat appearance in the BXH6 strain, a member of the HXB/BXH set of recombinant inbred (RI) strains. The RI strains were derived from the spontaneously hypertensive rat (SHR) and Brown Norway rat (BN-Lx) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the tuftelin 1 (Tuft1) gene on chromosome 2, which resulted in a premature stop codon. Compared with wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum monocyte chemoattractant protein-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and ß-oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for the important role of the Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.NEW & NOTEWORTHY A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a nonfunctional tuftelin 1 (Tuft1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene.
Assuntos
Códon sem Sentido , Glucose , Ratos , Animais , Glucose/metabolismo , Códon sem Sentido/genética , Metabolismo dos Lipídeos/genética , Proteoma/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos BN , Insulina/metabolismo , InflamaçãoRESUMO
Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293â¯cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15-80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10-30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/deficiência , Sobrevivência Celular , Células Clonais , Técnicas de Silenciamento de Genes , Glicólise , Células HEK293 , Humanos , Concentração Inibidora 50 , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Estresse Oxidativo , TermodinâmicaRESUMO
BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Vias Biossintéticas/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos , Feminino , Glicólise/efeitos dos fármacos , Humanos , Lactente , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Resultado do Tratamento , Adulto JovemRESUMO
Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.
Assuntos
Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genótipo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Proteolipídeos/metabolismo , Tamoxifeno/farmacologiaRESUMO
The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150-400â¯kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras , ATPases Translocadoras de Prótons/genéticaRESUMO
TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at â¼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.
Assuntos
Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Trifosfato de Adenosina/metabolismo , Animais , Cardiomiopatias/metabolismo , Feminino , Homozigoto , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Erros Inatos do Metabolismo/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/biossíntese , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Fosforilação Oxidativa , GravidezRESUMO
The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.
Assuntos
Complexo I de Transporte de Elétrons/genética , Síndrome de Fanconi/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Adulto , Alelos , Canadá , Mapeamento Cromossômico , Exoma/genética , Síndrome de Fanconi/patologia , Feminino , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Rim/metabolismo , Rim/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , MutaçãoRESUMO
Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Doença de Leigh/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Fibroblastos/patologia , Humanos , Doença de Leigh/genética , Doença de Leigh/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Especificidade de Órgãos , Especificidade da EspécieRESUMO
Resistin has been originally identified as an adipokine that links obesity to insulin resistance in mice. In our previous studies in spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin (Retn) transgene specifically in adipose tissue (SHR-Retn), we have observed an increased lipolysis and serum free fatty acids, ectopic fat accumulation in muscles, and insulin resistance. Recently, brown adipose tissue (BAT) has been suggested to play an important role in the pathogenesis of metabolic disturbances. In the current study, we have analyzed autocrine effects of transgenic resistin on BAT glucose and lipid metabolism and mitochondrial function in the SHR-Retn vs. nontransgenic SHR controls. We observed that interscapular BAT isolated from SHR-Retn transgenic rats compared with SHR controls showed a lower relative weight (0.71 ± 0.05 vs. 0.91 ± 0.08 g/100 g body wt, P < 0.05), significantly reduced both basal and insulin stimulated incorporation of palmitate into BAT lipids (658 ± 50 vs. 856 ± 45 and 864 ± 47 vs. 1,086 ± 35 nmol/g/2 h, P ≤ 0.01, respectively), and significantly decreased palmitate oxidation (37.6 ± 4.5 vs. 57 ± 4.1 nmol/g/2 h, P = 0.007) and glucose oxidation (277 ± 34 vs. 458 ± 38 nmol/g/2 h, P = 0.001). In addition, in vivo microPET imaging revealed significantly reduced (18)F-FDG uptake in BAT induced by exposure to cold in SHR-Retn vs. control SHR (232 ± 19 vs. 334 ± 22 kBq/ml, P < 0.05). Gene expression profiles in BAT identified differentially expressed genes involved in skeletal muscle and connective tissue development, inflammation and MAPK and insulin signaling. These results provide evidence that autocrine effects of resistin attenuate differentiation and activity of BAT and thus may play a role in the pathogenesis of insulin resistance in the rat.
Assuntos
Tecido Adiposo Marrom/metabolismo , Comunicação Autócrina/fisiologia , Glucose/metabolismo , Palmitatos/metabolismo , Resistina/genética , Tecido Adiposo Marrom/fisiologia , Animais , Comunicação Autócrina/genética , Ácidos Graxos não Esterificados/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/genética , Mitocôndrias/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Oxirredução , Ratos , Ratos Endogâmicos SHR , Ratos Transgênicos , Transcriptoma/genéticaRESUMO
Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another type is represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes-mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, â¼60 kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above â¼90% mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene-protein level.
Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Mutação/genética , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Deleção de Genes , Humanos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/deficiência , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.
Assuntos
Transporte de Elétrons , Glicerolfosfato Desidrogenase/química , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Ferricianetos/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Glicerofosfatos/metabolismo , Peróxido de Hidrogênio/metabolismo , Mamíferos , Mitocôndrias/enzimologia , Ratos , Succinato Desidrogenase/química , Succinato Desidrogenase/metabolismo , Ubiquinona/metabolismoRESUMO
Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).
Assuntos
Tecido Adiposo Marrom/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Temperatura Baixa , Espectroscopia de Ressonância de Spin Eletrônica , Glicerofosfatos/farmacologia , Guanosina Difosfato/farmacologia , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Canais Iônicos/genética , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Consumo de Oxigênio/efeitos dos fármacos , Ionóforos de Próton/farmacologia , Ácido Pirúvico/farmacologia , Ácido Succínico/farmacologia , Superóxidos/metabolismo , Proteína Desacopladora 1RESUMO
Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.
Assuntos
Trifosfato de Adenosina/biossíntese , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Mitocôndrias Cardíacas/metabolismo , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Fosfatos/química , Fosfatos/metabolismo , Ratos , Ratos WistarRESUMO
Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.
Assuntos
Glicerolfosfato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Regulação Alostérica , Animais , Ácidos Graxos/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicólise , Humanos , Especificidade de Órgãos , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismoRESUMO
Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.
Assuntos
Domínio Catalítico , Estrutura Secundária de ProteínaRESUMO
Renal nerves play a critical role in cardiorenal interactions. Renal denervation (RDN) improved survival in some experimental heart failure (HF) models. It is not known whether these favorable effects are indirect, explainable by a decrease in vascular afterload, or diminished neurohumoral response in the kidneys, or whether RDN procedure per se has direct myocardial effects in the failing heart. To elucidate mechanisms how RDN affects failing heart, we studied load-independent indexes of ventricular function, gene markers of myocardial remodeling, and cardiac sympathetic signaling in HF, induced by chronic volume overload (aorto-caval fistula, ACF) of Ren2 transgenic rats. Volume overload by ACF led to left ventricular (LV) hypertrophy and dysfunction, myocardial remodeling (upregulated Nppa, MYH 7/6 genes), increased renal and circulating norepinephrine (NE), reduced myocardial NE content, increased monoaminoxidase A (MAO-A), ROS production and decreased tyrosine hydroxylase (+) nerve staining. RDN in HF animals decreased congestion in the lungs and the liver, improved load-independent cardiac function (Ees, PRSW, Ees/Ea ratio), without affecting arterial elastance or LV pressure, reduced adverse myocardial remodeling (Myh 7/6, collagen I/III ratio), decreased myocardial MAO-A and inhibited renal neprilysin activity. RDN increased myocardial expression of acetylcholinesterase (Ache) and muscarinic receptors (Chrm2), decreased circulating and renal NE, but increased myocardial NE content, restoring so autonomic control of the heart. These changes likely explain improvements in survival after RDN in this model. The results suggest that RDN has remote, load-independent and favorable intrinsic myocardial effects in the failing heart. RDN therefore could be a useful therapeutic strategy in HF.
RESUMO
Recently, red beetroot has attracted attention as a health-promoting functional food. Studies have shown that beetroot administration can reduce blood pressure and ameliorate parameters of glucose and lipid metabolism; however, mechanisms underlying these beneficial effects of beetroot are not yet fully understood. In the current study, we analysed the effects of beetroot on parameters of glucose and lipid metabolism in two models of metabolic syndrome: (i) transgenic spontaneously hypertensive rats expressing human C-reactive protein (SHR-CRP rats), and (ii) hereditary hypertriglyceridemic (HHTg) rats. Treatment with beetroot juice for 4 weeks was, in both models, associated with amelioration of oxidative stress, reduced circulating lipids, smaller visceral fat depots, and lower ectopic fat accumulation in the liver compared to the respective untreated controls. On the other hand, beetroot treatment had no significant effects on the sensitivity of the muscle and adipose tissue to insulin action in either model. Analyses of hepatic proteome revealed significantly deregulated proteins involved in glycerophospholipid metabolism, mTOR signalling, inflammation, and cytoskeleton rearrangement.
RESUMO
OBJECTIVE: Non-shivering thermogenesis (NST) mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) can be activated via the adrenergic system in response to cold or diet, contributing to both thermal and energy homeostasis. Other mechanisms, including metabolism of skeletal muscle, may also be involved in NST. However, relative contribution of these energy dissipating pathways and their adaptability remain a matter of long-standing controversy. METHODS: We used warm-acclimated (30 °C) mice to characterize the effect of an up to 7-day cold acclimation (6 °C; CA) on thermoregulatory thermogenesis, comparing inbred mice with a genetic background conferring resistance (A/J) or susceptibility (C57BL/6 J) to obesity. RESULTS: Both warm-acclimated C57BL/6 J and A/J mice exhibited similar cold endurance, assessed as a capability to maintain core body temperature during acute exposure to cold, which improved in response to CA, resulting in comparable cold endurance and similar induction of UCP1 protein in BAT of mice of both genotypes. Despite this, adrenergic NST in BAT was induced only in C57BL/6 J, not in A/J mice subjected to CA. Cold tolerance phenotype of A/J mice subjected to CA was not based on increased shivering, improved insulation, or changes in physical activity. On the contrary, lipidomic, proteomic and gene expression analyses along with palmitoyl carnitine oxidation and cytochrome c oxidase activity revealed induction of lipid oxidation exclusively in skeletal muscle of A/J mice subjected to CA. These changes appear to be related to skeletal muscle NST, mediated by sarcolipin-induced uncoupling of sarco(endo)plasmic reticulum calcium ATPase pump activity and accentuated by changes in mitochondrial respiratory chain supercomplexes assembly. CONCLUSIONS: Our results suggest that NST in skeletal muscle could be adaptively augmented in the face of insufficient adrenergic NST in BAT, depending on the genetic background of the mice. It may provide both protection from cold and resistance to obesity, more effectively than BAT.
Assuntos
Tecido Adiposo Marrom , Proteômica , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , Camundongos Endogâmicos C57BL , Termogênese/fisiologia , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Camundongos Endogâmicos , Adrenérgicos/metabolismoRESUMO
Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.
Assuntos
Medula Óssea , Ácidos Graxos Ômega-3 , Humanos , Camundongos , Animais , Medula Óssea/metabolismo , Adiposidade , Osso e Ossos/metabolismo , Obesidade/complicações , Obesidade/prevenção & controle , Obesidade/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: The general self-rated health (SRH) question is the most common health measure employed in large population surveys. This study contributes to research on the concurrent validity of SRH using representative data with biomarkers from the Czech Republic, a population not previously used to assess the SRH measure. This work determines the relative contribution of biomedical and social characteristics to an individual's SRH assessment. Studies have already explored the associations between SRH and markers of physical health. However, according to a PubMed systematic literature search, the issue of the relative importance of physiological and psychosocial factors that affect individuals' assessments of their SRH has generally been neglected. METHODOLOGY/PRINCIPAL FINDINGS: Using data from a specialized epidemiological survey of the Czech population (N = 1021), this study adopted ordinary least squares regression to analyze the extent to which variance in SRH is explained by biomedical measures, mental health, health behavior, and socioeconomic characteristics. This analysis showed that SRH variance can be largely attributed to biomedical and psychological measures. Socioeconomic characteristics (i.e. marital status, education, economic activity, and household income) contributed to around 5% of the total variance. After controlling for age, sex, location, and socioeconomic status, biomarkers (i.e. C-reactive protein, blood glucose, triglyceride, low-density lipoprotein, and high-density lipoprotein), number of medical conditions, and current medications explained 11% of the total SRH variance. Mental health indicators contributed to an additional 9% of the variance. Body mass index and health behaviors (i.e. smoking and alcohol consumption) explained less than 2% of the variance. CONCLUSIONS/SIGNIFICANCE: The results suggested that SRH was a valid measure of physiological and mental health in the Czech sample, and the observed differences were likely to have reflected inequalities in bodily and mental functions between social groups.