RESUMO
Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid ß-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.
Assuntos
Acil-CoA Oxidase/antagonistas & inibidores , Acil-CoA Oxidase/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo , Sirtuínas/metabolismo , Acil-CoA Oxidase/genética , Animais , Dano ao DNA , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Peróxido de Hidrogênio , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxirredução , Peroxissomos/química , Prognóstico , Sirtuínas/genéticaRESUMO
A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, acyl-CoA oxidase-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat diet- or obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism.
Assuntos
Acil-CoA Oxidase/metabolismo , Ácidos Graxos Insaturados/farmacologia , Lipídeos/química , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acil-CoA Oxidase/antagonistas & inibidores , Animais , Peso Corporal , Dieta Hiperlipídica , Insulina/metabolismo , Fígado/metabolismo , Masculino , Mitocôndrias/metabolismo , Oxigênio/química , Peroxissomos/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Cold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's "stress receptors" and "nutrient receptors". Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. METHODS: To investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. RESULTS: In C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.
Assuntos
Ácidos Graxos , Metabolismo dos Lipídeos , Ubiquitinação , Animais , Masculino , Camundongos , Ácidos Graxos/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/metabolismo , Proteólise , Acil-CoA Oxidase/antagonistas & inibidores , Acil-CoA Oxidase/metabolismo , Acetilglucosamina/metabolismoRESUMO
N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.
Assuntos
Acil-CoA Oxidase/genética , Acil-CoA Oxidase/isolamento & purificação , Arthrobacter/enzimologia , Etilmaleimida/farmacologia , Acil-CoA Oxidase/antagonistas & inibidores , Sequência de Aminoácidos , Clonagem Molecular , Colorimetria , Resistência a Medicamentos , Estabilidade Enzimática , Escherichia coli/enzimologia , Ácidos Graxos não Esterificados/análise , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Alinhamento de SequênciaRESUMO
Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.
Assuntos
Gorduras Insaturadas na Dieta/uso terapêutico , Suplementos Nutricionais , Dioxóis/uso terapêutico , Hiperlipidemias/prevenção & controle , Hipolipemiantes/uso terapêutico , Lignanas/uso terapêutico , Fígado/metabolismo , Ácido gama-Linolênico/uso terapêutico , Acil-CoA Oxidase/antagonistas & inibidores , Acil-CoA Oxidase/química , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Animais , Gorduras Insaturadas na Dieta/efeitos adversos , Sacarose Alimentar/efeitos adversos , Enoil-CoA Hidratase/antagonistas & inibidores , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/sangue , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Hiperlipidemias/metabolismo , Ácidos Linoleicos/uso terapêutico , Lipídeos/sangue , Fígado/enzimologia , Masculino , Oenothera biennis , Oxirredução , Óleo de Palmeira/efeitos adversos , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Óleos de Plantas/uso terapêutico , Ratos Sprague-Dawley , Óleo de Cártamo/efeitos adversos , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/química , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
Oct-2-en-4-ynoyl-CoA was found to be a specific inhibitor of acyl-CoA oxidase in fatty acid oxidation in peroxisomes that has no inhibitory effect on acyl-CoA dehydrogenase in mitochondria. The inhibition reaction involves a nucleophilic attack of Glu421 to the delta carbon of the inhibitor. The result indicates that acyl-CoA oxidase and acyl-CoA dehydrogenase have certain differences in active-site structure, which makes it possible to control fatty acid oxidation selectively in either mitochondria or peroxisomes with different enzyme inhibitors.
Assuntos
Acil Coenzima A/farmacologia , Acil-CoA Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Acil Coenzima A/síntese química , Acil Coenzima A/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Beauveria bassiana produces acyl-Co oxidase (ACO) in the P(20000 g) fraction of glucose and alkane-grown cultures that catalyze the oxidation of acyl-CoAs of different chain length. The activity was measured indirectly over the formation of H2O2 via the oxidative-coupled assay system. ACO activity was assessed spectrophotometrically in the P(20000 g) fraction of glucose-grown (FS0) and n-alkane grown cultures (FS(alk)), employing acyl-CoAs of 16 to 24 carbons as substrates. A significant increment in the activity was observed in FS(alk) as compared to that of controls (FS0) in all conditions tested. Tetracosane-grown cultures showed the highest activity with lignoceroyl-CoA. The reaction conditions were optimized employing lignoceroyl-CoA as substrate. A variable lag phase was observed when the activity was measured as a function of time. In the presence of 3-amino-1,2,4-triazole (AT) to prevent H2O2 consumption by endogenous catalase, the lag phase became shorter and disappeared when AT concentrations were raised from 40 to 200 mM, thus enhancing acyl-CoA oxidation. Enzyme activity reached its maximal value in the presence of 240 microg peroxidase, 0.08% Triton X-100 and 36 microM bovine serum albumin. The apparent Km using lignoceroyl as substrate was estimated 2.5 microM. ACO showed high activity and stability between 30 and 40 degrees C, as well as between 7.0 and 9.0 pH, for 120 min, being 7.0 the optimum pH.