RESUMO
Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Metabolismo dos Lipídeos/genética , Mitocôndrias/efeitos dos fármacos , Oxilipinas/metabolismo , Peritonite/genética , Sepse/genética , Acil-CoA Desidrogenase de Cadeia Longa/sangue , Acil-CoA Desidrogenase de Cadeia Longa/genética , Animais , Carnitina O-Palmitoiltransferase/sangue , Carnitina O-Palmitoiltransferase/genética , Coenzima A Ligases/sangue , Coenzima A Ligases/genética , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Interferon gama/farmacologia , Lipidômica/métodos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/sangue , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Oxirredução , Peritonite/sangue , Peritonite/induzido quimicamente , Peritonite/patologia , Células RAW 264.7 , Sepse/sangue , Sepse/patologiaRESUMO
Mutations in the HADHB gene lead to Mitochondrial Trifunctional Protein (MTP) deficiency. MTP deficiency is a rare autosomal recessive disorder affecting long-chain fatty acid oxidation. Patients affected by MTP deficiency are unable to metabolize long-chain fatty-acids and suffer a variety of symptoms exacerbated during fasting. The three phenotypes associated with complete MTP deficiency are an early-onset cardiomyopathy and early death, an intermediate form with recurrent hypoketotic hypoglycemia and a sensorimotor neuropathy with episodic rhabdomyolysis with small amount of residual enzyme activities. This review aims to discuss the pathophysiological mechanisms and clinical manifestations of each phenotype, which appears different and linked to HADHB expression levels. Notably, the pathophysiology of the sensorimotor neuropathy is relatively unknown and we provide a hypothesis on the qualitative aspect of the role of acylcarnitine buildup in Schwann cells in MTP deficiency patients. We propose that acylcarnitine may exit the Schwann cell and alter membrane properties of nearby axons leading to axonal degeneration based on recent findings in different metabolic disorders.
Assuntos
Cardiomiopatias/genética , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Proteína Mitocondrial Trifuncional/deficiência , Proteína Mitocondrial Trifuncional/genética , Doenças do Sistema Nervoso/genética , Rabdomiólise/genética , Cardiomiopatias/patologia , Humanos , Erros Inatos do Metabolismo Lipídico/patologia , Miopatias Mitocondriais/patologia , Mutação/genética , Doenças do Sistema Nervoso/patologia , Fenótipo , Rabdomiólise/patologiaRESUMO
Adequate protein nutrition is essential for good health. Effects of protein malnutrition in animals have been widely studied at the mRNA level with the development of DNA microarray technology. Although microRNAs (miRNAs) have attracted attention for their function in regulating gene expression and have been studied in several disciplines, fewer studies have clarified the effects of protein malnutrition on miRNA alterations. The present study aimed to elucidate the relationship between protein malnutrition and miRNAs. Six-week old Wistar male rats were fed a control diet (20% casein) or a low-protein diet (5% casein) for two weeks, and their livers were subjected to both DNA microarray and miRNA array analysis. miR-203 was downregulated and its putative target Hadhb (hydroxyacyl-CoA dehydrogenase ß subunit), known to regulate ß-oxidation of fatty acids, was upregulated by the low-protein diet. In an in vitro experiment, miR-203 or its inhibitor were transfected in HepG2 cells, and the pattern of Hadhb expression was opposite to that of miR-203 expression. In addition, to clarifying the hepatic miRNA profile in response to protein malnutrition, these results showed that a low-protein diet increased Hadhb expression through downregulation of miR-203 and induced ß-oxidation of fatty acids.
Assuntos
Dieta com Restrição de Proteínas , Regulação da Expressão Gênica , Desnutrição/patologia , MicroRNAs/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Animais , Células Hep G2 , Humanos , Masculino , Desnutrição/genética , Desnutrição/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Ratos , Ratos WistarRESUMO
Proper resolution of inflammation is vital for repair and restoration of homeostasis after tissue damage, and its dysregulation underlies various noncommunicable diseases, such as cardiovascular and metabolic diseases. Macrophages play diverse roles throughout initial inflammation, its resolution, and tissue repair. Differential metabolic reprogramming is reportedly required for induction and support of the various macrophage activation states. Here we show that a long noncoding RNA (lncRNA), lncFAO, contributes to inflammation resolution and tissue repair in mice by promoting fatty acid oxidation (FAO) in macrophages. lncFAO is induced late after lipopolysaccharide (LPS) stimulation of cultured macrophages and in Ly6Chi monocyte-derived macrophages in damaged tissue during the resolution and reparative phases. We found that lncFAO directly interacts with the HADHB subunit of mitochondrial trifunctional protein and activates FAO. lncFAO deletion impairs resolution of inflammation related to endotoxic shock and delays resolution of inflammation and tissue repair in a skin wound. These results demonstrate that by tuning mitochondrial metabolism, lncFAO acts as a node of immunometabolic control in macrophages during the resolution and repair phases of inflammation.
Assuntos
Ácidos Graxos/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , RNA Longo não Codificante/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Camundongos , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Oxirredução , Cultura Primária de Células , RNA Longo não Codificante/genética , Pele/imunologia , Pele/lesões , Cicatrização/imunologiaRESUMO
1. Limiting the growth of adipose tissue in chickens is a major issue in the poultry industry. In chickens, de novo synthesis of lipids occurs primarily in the liver. Thus, it is necessary to understand how fatty acid accumulation in the liver is controlled. The miR-33 is an intronic microRNA (miRNA) of the chicken sterol regulatory element binding transcription factor 2 (SREBF2), which is a master switch in activating many genes involved in the uptake and synthesis of cholesterol, triglycerides, fatty acids and phospholipids. 2. In the current study, the genes CROT and HADHB known to encode enzymes critical for fatty acid oxidation were predicted to be potential targets of miR-33 in chickens via the miRNA target prediction programs 'miRanda' and 'TargetScan'. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3'-untranslated region (3'UTR) of the chicken CROT and HADHB mRNA was down-regulated by overexpression of the chicken miR-33 (P < 0.05). This down-regulation was completely abolished when the predicted miR-33 target sites in the CROT and HADHB 3'UTR were mutated. 3. Transfecting miR-33 mimics into the LMH cells led to a decrease in the mRNA expression of CROT and HADHB (P < 0.01), and this transfection had a similar effect on the proteins (P < 0.05). In contrast, the expression of CROT in primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33 (P < 0.05). 4. Using quantitative RT-PCR, it was shown that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas the CROT and HADHB mRNA levels decreased during the same period. 5. These findings support the conclusion that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the CROT and HADHB genes, which encode enzymes critical for lipid oxidation.
Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Animais , Proteínas Aviárias/metabolismo , Sequência de Bases , Galinhas/metabolismo , Íntrons , Fígado/metabolismo , MicroRNAs/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Plasmídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
The heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and ß-subunits harbours three enzymes that each perform a different function in mitochondrial fatty acid ß-oxidation. Pathogenic variants in the MTP genes (HADHA and HADHB) cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by phenotypic heterogeneity ranging from severe, early-onset, cardiac disease to milder, later-onset, myopathy and neuropathy. Since metabolic myopathies and neuropathies are a group of rare genetic disorders and their associated muscle symptoms may be subtle, the diagnosis is often delayed. Here we evaluated data of 161 patients with myopathy and 242 patients with neuropathy via next generation sequencing (NGS) and report the diagnostic yield in three patients of this cohort by the detection of disease-causing variants in the HADHA or HADHB gene. The mitigated phenotypes of this treatable disease were missed by the newborn screening, highlighting the importance of phenotype-based NGS analysis in patients with rare and clinically very variable disorders such as MTP deficiency.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Mutação/genética , Adolescente , Cardiomiopatias/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Erros Inatos do Metabolismo Lipídico/genética , Masculino , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/deficiência , Proteína Mitocondrial Trifuncional/genética , Doenças do Sistema Nervoso/genética , Fenótipo , Rabdomiólise/genética , SíndromeRESUMO
Mitochondrial trifunctional protein deficiency (MTPD) is a rare disorder caused by mutations in the HADHA and HADHB genes. Here, we report on two Han Chinese patients with HADHB mutation-associated infantile axonal Charcot-Marie-Tooth disease (IACMT). Both patients were unrelated. Case 1 was a 19-year-old man, and case 2 was a 5-year-old boy. Both had delayed motor development and slowly-progressing distal muscle weakness with areflexia and foot deformities. The electrophysiology findings were compatible with axonal polyneuropathy in both patients. Blood tandem mass spectrometry showed increased concentrations of multiple acylcarnitines. Nerve biopsies showed axonal neuropathy with a moderate loss of myelinated fibers. Gene analysis identified two compound heterozygous mutations (c.184A>G/c.340A>G and c.488G>A/c.1175C>T, respectively) in the HADHB gene. The c.488G>A mutation was novel. This study broadens the phenotype of MTPD and suggests that the genetic testing of patients suffering from IACMT should include the HADHB gene.â©.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Sequência de Aminoácidos , Povo Asiático , Axônios/patologia , Biópsia , Carnitina/análogos & derivados , Carnitina/metabolismo , Pré-Escolar , Análise Mutacional de DNA , Humanos , Masculino , Proteína Mitocondrial Trifuncional/deficiência , Mutação/genética , Polineuropatias/patologia , Nervo Sural/patologia , Adulto JovemRESUMO
Background: DNA methylation is an important epigenetic modification, associated with gene expression. 5-Methylcytosine and 5-hydroxymethylcytosine are two epigenetic hallmarks that maintain the equilibrium of epigenetic reprogramming. Disequilibrium in genomic methylation leads to carcinogenesis. The purpose of this study was to elucidate the epigenetic mechanisms of DNA methylation and hydroxymethylation in the carcinogenesis of colorectal cancer. Methods: Genome-wide patterns of DNA methylation and hydroxymethylation in six paired colorectal tumor tissues and corresponding normal tissues were determined using immunoprecipitation and sequencing. Transcriptional expression was determined by RNA sequencing (RNA-Seq). Groupwise differential methylation regions (DMR), differential hydroxymethylation regions (DhMR), and differentially expressed gene (DEG) regions were identified. Epigenetic biomarkers were screened by integrating DMR, DhMR, and DEGs and confirmed using functional analysis. Results: We identified a genome-wide distinct hydroxymethylation pattern that could be used as an epigenetic biomarker for clearly differentiating colorectal tumor tissues from normal tissues. We identified 59,249 DMRs, 187,172 DhMRs, and 948 DEGs by comparing between tumors and normal tissues. After cross-matching genes containing DMRs or DhMRs with DEGs, we screened seven genes that were aberrantly regulated by DNA methylation in tumors. Furthermore, hypermethylation of the HADHB gene was persistently found to be correlated with downregulation of its transcription in colorectal cancer (CRC). These findings were confirmed in other patients of colorectal cancer. Tumor functional analysis indicated that HADHB reduced cancer cell migration and invasiveness. These findings suggested its possible role as a tumor suppressor gene (TSG). Conclusion: This study reveals the global patterns of methylation and hydroxymethylation in CRC. Several CRC-associated genes were screened with multi-omic analysis. Aberrant methylation and hydroxymethylation were found to be in the carcinogenesis of CRC.
Assuntos
Neoplasias Colorretais/genética , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/metabolismo , Metilação de DNA , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Masculino , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder of mitochondrial fatty-acid oxidation. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is often reported in Caucasian countries due to a common mutation. However, the molecular and clinical basis of complete TFP deficiency has not been extensively reported. In this study, 14 Japanese cases (13 families) with complete TFP deficiency, including 9 previously reported cases, were analyzed to clarify the clinical and molecular characteristics of TFP deficiency. The clinical types of the 14 patients were as follows: 12 cases of neonatal (n=7) or myopathic (n=5) types and 2 cases of intermediate type. Peripheral neuropathy was found in four cases and hypocalcemia due to hypoparathyroidism, which is rarely reported in Caucasian patients, had developed in four cases. Maternal hemolysis, elevated liver enzymes and low platelet count syndrome and acute fatty liver of pregnancy were noted in two and one mothers, respectively. Fourteen mutations were identified in 26 alleles in Japanese patients, including two novel mutations (HADHA: c.361C>T, and HADHA-HADHB: g.26233880_ 26248855del), although no common mutations were found. This study suggests that the molecular and clinical aspects of Japanese patients with TFP deficiencies differ from those of Caucasian patients.
Assuntos
Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/diagnóstico , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Rabdomiólise/diagnóstico , Rabdomiólise/genética , Adolescente , Povo Asiático/genética , Criança , Pré-Escolar , Ativação Enzimática , Família , Feminino , Testes Genéticos , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Proteína Mitocondrial Trifuncional/genética , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Mutação , População Branca/genéticaRESUMO
INTRODUCTION: Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four ß-subunits that possess LCKT (long-chain 3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in ß-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. MATERIALS AND METHODS: A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. RESULTS: Here, we report a novel mutation in a patient with MTP deï¬ciency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. CONCLUSIONS: Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.
Assuntos
Cardiomiopatias/genética , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/genética , Rabdomiólise/genética , Análise Mutacional de DNA , Humanos , Recém-Nascido , Irã (Geográfico) , Masculino , Proteína Mitocondrial Trifuncional/genética , Mutação de Sentido Incorreto , Triagem Neonatal , Espectrometria de Massas em TandemRESUMO
A 45-year-old man presented to us due to slowly progressive muscle weakness and sensory disturbances in his lower limbs since his 40's. He reported multiple episodes of exercise-induced severe muscle fatigue and brown urine in his childhood, which disappeared by age 20. A nerve conduction study showed peripheral axonal neuropathy and then Charcot-Marie-Tooth disease (CMT) was considered as the most likely diagnosis; however, exome sequencing failed to identify a mutation in the known genes of CMTs. Since age 55, he recurrently developed severe rhabdomyolysis that required hospitalization. On suspicion of lipid metabolism disorders, we performed serum acylcarnitine analysis, and which revealed mildly elevated long-chain fatty acids. We re-examined variants obtained via exome sequencing and found a mutation in HADHB. Mitochondrial trifunctional protein (MTP) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid beta-oxidation caused by HADHA or HADHB mutation. It can be a life-threatening multiorgan disorder with early infantile onset, but it can also present in childhood or adolescence with peripheral neuropathy and recurrent rhabdomyolysis. This case of adult-diagnosed MTP deficiency was characterized by slowly progressive peripheral neuropathy masquerading CMT in addition to muscular symptoms. MTP deficiency should be considered in patients with the combination of peripheral neuropathy and recurrent rhabdomyolysis.
Assuntos
Cardiomiopatias/diagnóstico , Erros Inatos do Metabolismo Lipídico/diagnóstico , Miopatias Mitocondriais/diagnóstico , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/diagnóstico , Rabdomiólise/diagnóstico , Biomarcadores/sangue , Cardiomiopatias/complicações , Cardiomiopatias/genética , Carnitina/análogos & derivados , Carnitina/sangue , Doença de Charcot-Marie-Tooth , Diagnóstico Diferencial , Progressão da Doença , Testes Genéticos , Humanos , Erros Inatos do Metabolismo Lipídico/complicações , Erros Inatos do Metabolismo Lipídico/genética , Masculino , Pessoa de Meia-Idade , Miopatias Mitocondriais/complicações , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Mutação , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso Periférico/etiologia , Recidiva , Rabdomiólise/complicações , Rabdomiólise/etiologia , Rabdomiólise/genéticaAssuntos
Proteína Mitocondrial Trifuncional/genética , Doenças do Sistema Nervoso , Cardiomiopatias , Humanos , Erros Inatos do Metabolismo Lipídico , Miopatias Mitocondriais , Proteína Mitocondrial Trifuncional/deficiência , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Mutação , RabdomióliseRESUMO
Myocardial fatty acid ß-oxidation is critical for the maintenance of energy homeostasis and contractile function in the heart, but its regulation is still not fully understood. While thioredoxin-interacting protein (TXNIP) has recently been implicated in cardiac metabolism and mitochondrial function, its effects on ß-oxidation have remained unexplored. Using a new cardiomyocyte-specific TXNIP knockout mouse and working heart perfusion studies, as well as loss- and gain-of-function experiments in rat H9C2 and human AC16 cardiomyocytes, we discovered that TXNIP deficiency promotes myocardial ß-oxidation via signaling through a specific microRNA, miR-33a. TXNIP deficiency leads to increased binding of nuclear factor Y (NFYA) to the sterol regulatory element binding protein 2 (SREBP2) promoter, resulting in transcriptional inhibition of SREBP2 and its intronic miR-33a. This allows for increased translation of the miR-33a target genes and ß-oxidation-promoting enzymes, carnitine octanoyl transferase (CROT), carnitine palmitoyl transferase 1 (CPT1), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase-ß (HADHB), and AMPKα and is associated with an increase in phospho-AMPKα and phosphorylation/inactivation of acetyl-CoA-carboxylase. Thus, we have identified a novel TXNIP-NFYA-SREBP2/miR-33a-AMPKα/CROT/CPT1/HADHB pathway that is conserved in mouse, rat, and human cardiomyocytes and regulates myocardial ß-oxidation.
Assuntos
Proteínas de Transporte/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Tiorredoxinas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Carnitina Aciltransferases/genética , Carnitina Aciltransferases/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Genótipo , Humanos , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Miócitos Cardíacos/enzimologia , Oxirredução , Fenótipo , Interferência de RNA , Ratos , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Tiorredoxinas/genética , TransfecçãoRESUMO
Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 µM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.
Assuntos
Bezafibrato/farmacologia , Cardiomiopatias/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Hipolipemiantes/farmacologia , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Miopatias Mitocondriais/tratamento farmacológico , Subunidade alfa da Proteína Mitocondrial Trifuncional/deficiência , Subunidade beta da Proteína Mitocondrial Trifuncional/deficiência , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/tratamento farmacológico , Rabdomiólise/tratamento farmacológico , Cardiomiopatias/genética , Linhagem Celular , Genótipo , Humanos , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/genética , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Mutação/genética , Doenças do Sistema Nervoso/genética , Rabdomiólise/genéticaRESUMO
OBJECTIVE: miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. APPROACH AND RESULTS: High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33-treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33-treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33-treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. CONCLUSIONS: We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.
Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Obesidade/terapia , Oligonucleotídeos Antissenso/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Colesterol/sangue , Modelos Animais de Doenças , Insulina/sangue , Resistência à Insulina , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Obesidade/genética , Obesidade/metabolismo , Oligonucleotídeos Antissenso/genética , Oxirredução , Fenótipo , Fatores de Tempo , Triglicerídeos/sangue , Aumento de PesoRESUMO
BACKGROUND: miRNAs are single-stranded, small RNA molecules with a length of 18-25 nucleotides. They bind to the 3' untranslated regions of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation. The roles of these molecules differ in biological processes, such as cell differentiation, proliferation, apoptosis and tumor genesis. miRNA-33 is encoded by the gene introns of proteins that bind sterol-regulatory elements. This molecule cooperates with these proteins to control cholesterol homeostasis, fatty acid levels and the genes that are related to the expression of fat metabolism. The examination of miR-33 expression and its target genes can promote the in-depth study of the miRNA regulation mechanism in the formation process of goose fatty liver and can lay a foundation for research into human fatty liver. METHODOLOGY/PRINCIPAL FINDINGS: (1) Through real-time fluorescent quantitative polymerase chain reaction (TaqMan MicroRNA Assay), we detected the expression of miR-33 during the feeding of Landes geese. The expression level of miR-33 increases significantly in the liver after 19 days in comparison with the control group; (2) By using the bioinformatics software programs TargetScan, miRDB and miRCosm to predict the target genes of miR-33 according to laboratory prophase transcriptome results and references, we screen nine target genes: adenosine triphosphate binding cassette transporters A1, adenosine triphosphate binding cassette transporters G1, Neimann Pick C, carnitine O-octanoyltransferase (CROT), cyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), AMP-activated protein kinase, alpha subunit 1 (AMPKα1), insulin receptor substrate 2, glutamic pyruvate transaminase and adipose differentiation-related protein. The dual luciferase reporter gene system in the CHO cell line verifies that CROT, HADHB and NPC1 are the target genes of miR-33 in geese. The inhibition rate of CROT is highest and reaches 70%; (3) The seed sequence (5' 2-8 bases) is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33. CONCLUSIONS/SIGNIFICANCE: (1) After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2) CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.
Assuntos
Fígado Gorduroso/metabolismo , MicroRNAs/genética , Transcriptoma , Animais , Carnitina Aciltransferases/genética , Carnitina Aciltransferases/metabolismo , Gansos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismoRESUMO
We report a fetal case with fatal outcome having a novel mutation in the HADHB gene, coding the beta-subunit of the mitochondrial trifunctional protein. Parents had a previous pregnancy loss due to fetal heart failure and hydrops. The next pregnancy led to left ventricular noncompaction and increasing pleural effusions after 29 gestational weeks. The fetus was small for gestational age, and long bones were abnormally short. The baby was born severely asphyxiated at 32 gestational weeks by cesarean section. Intensive care was withdrawn due to failure to thrive and suspicion of a severe mitochondrial disorder. Postmortem brain MRI suggested microcephaly with a simplified gyral pattern. The lateral cerebral ventricles were normal. Chromosome analysis was normal (46, XX). Fibroblasts cultured from a skin biopsy of the baby revealed the large homozygous deletion c.1109+243_1438-703del in the HADHB gene, and heterozygous mutations were detected in both parents. The deletion has not been reported earlier. CONCLUSION: It is important to differentiate systemic metabolic diseases from disorders that affect only the cardiac muscle. Trifunctional protein deficiency is a relatively rare disorder of the fatty acid ß-oxidation cycle. The mutation in the HADHB gene causes a systemic disease with early-onset cardiomyopathy. Understanding the molecular genetic defect of the patient allows appropriate genetic counseling of the family. WHAT IS KNOWN: ⢠Mitochondrial disorders as a group are an important etiology for fetal cardiomyopathies including human trifunctional protein (TFP) disorders and several other mitochondrial diseases. WHAT IS NEW: ⢠We report a fetal case with fatal outcome having a novel mitochondrial trifunctional protein mutation (c.1109+243_1438-703del in the HADHB gene).
Assuntos
Cardiomiopatias/genética , Ventrículos do Coração/anormalidades , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/genética , Rabdomiólise/genética , Adulto , Cardiomiopatias/diagnóstico , Ecocardiografia , Evolução Fatal , Feminino , Doenças Fetais , Feto , Humanos , Erros Inatos do Metabolismo Lipídico/diagnóstico , Miopatias Mitocondriais/diagnóstico , Proteína Mitocondrial Trifuncional/genética , Mutação , Doenças do Sistema Nervoso/diagnóstico , Gravidez , Rabdomiólise/diagnósticoRESUMO
Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. ß-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired ß-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid ß-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA ß-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and ß subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA ß-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA ß-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA ß-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA ß-oxidation and inducing cytokine expression by association with MTP.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/metabolismo , Ácidos Graxos/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/genética , Ácidos Graxos/genética , Células HEK293 , Humanos , Camundongos , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Oxirredução , Mutação Puntual , Proteínas não Estruturais Virais/genéticaRESUMO
Acute fatty liver of pregnancy (AFLP) is a devastating disorder of the maternal liver in the third trimester. Recent studies have demonstrated an association between AFLP and fetal fatty acid oxidation disorders. Here, we report a case of AFLP caused by fetal mitochondrial trifunctional protein (TFP) deficiency. A 21-year-old parous woman presented with nausea, genital bleeding and abdominal pain at 33 weeks of gestation. Laboratory data revealed hepatic failure and disseminated intravascular coagulopathy. The patient underwent emergency cesarean section and was diagnosed with AFLP from the clinical characteristics. She was successfully treated with frequent plasma exchange. The newborn presented severe heart failure and died on the 39th day after birth. Tandem mass spectrometry indicated long-chain fatty acid oxidation disorder. Gene analysis demonstrated homozygous mutation in exon 13 of HADHB, the gene responsible for mitochondrial TFP deficiency. The parents carried a heterozygous mutation at the same location in HADHB.
Assuntos
Cardiomiopatias/complicações , Fígado Gorduroso/etiologia , Insuficiência Cardíaca/complicações , Erros Inatos do Metabolismo Lipídico/complicações , Miopatias Mitocondriais/complicações , Proteína Mitocondrial Trifuncional/deficiência , Doenças do Sistema Nervoso/complicações , Complicações na Gravidez/etiologia , Rabdomiólise/complicações , Cardiomiopatias/genética , Evolução Fatal , Fígado Gorduroso/terapia , Feminino , Insuficiência Cardíaca/genética , Humanos , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/genética , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Mutação , Doenças do Sistema Nervoso/genética , Troca Plasmática , Gravidez , Complicações na Gravidez/terapia , Rabdomiólise/genética , Adulto JovemRESUMO
Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of four α- and four ß-subunits that catalyzes the final three steps of mitochondrial ß-oxidation of long chain fatty acids. HADHA and HADHB encode the α-subunit and the ß-subunit of MTP, respectively. To date, only two cases with MTP deficiency have been reported to be associated with hypoparathyroidism and peripheral polyneuropathy. Here, we report on two siblings with autosomal recessive infantile onset hypoparathyroidism, peripheral polyneuropathy, and rhabdomyolysis. Sequence analysis of HADHA and HADHB in both siblings shows that they were homozygous for a mutation in exon 14 of HADHB (c.1175C>T, [p.A392V]) and the parents were heterozygous for the mutation. Biochemical analysis revealed that the patients had MTP deficiency. Structural analysis indicated that the A392V mutation identified in this study and the N389D mutation previously reported to be associated with hypoparathyroidism are both located near the active site of MTP and affect the conformation of the ß-subunit. Thus, the present patients are the second and third cases of MTP deficiency associated with missense HADHB mutation and infantile onset hypoparathyroidism. Since MTP deficiency is a treatable disease, MTP deficiency should be considered when patients have hypoparathyroidism as the initial presenting feature in infancy.