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1.
Cell Mol Life Sci ; 78(14): 5631-5646, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34110423

RESUMO

Peroxisomes play an essential role in the ß-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA ß-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA ß-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA ß-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA ß-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA ß-oxidation is a regulator of hepatic cholesterol biosynthesis.


Assuntos
Colesterol/metabolismo , Ácidos Dicarboxílicos/urina , Erros Inatos do Metabolismo Lipídico/patologia , Hepatopatias/patologia , Mitocôndrias/patologia , Enzima Bifuncional do Peroxissomo/fisiologia , Animais , Feminino , Células HEK293 , Homeostase , Humanos , Erros Inatos do Metabolismo Lipídico/etiologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo
2.
Cell Rep ; 35(11): 109264, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34133930

RESUMO

MYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

3.
Cell Rep ; 35(9): 109210, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34077737

RESUMO

Natural killer (NK) cells are cytotoxic lymphocytes capable of rapid cytotoxicity, cytokine secretion, and clonal expansion. To sustain such energetically demanding processes, NK cells must increase their metabolic capacity upon activation. However, little is known about the metabolic requirements specific to NK cells in vivo. To gain greater insight, we investigated the role of aerobic glycolysis in NK cell function and demonstrate that their glycolytic rate increases rapidly following viral infection and inflammation, prior to that of CD8+ T cells. NK cell-specific deletion of lactate dehydrogenase A (LDHA) reveals that activated NK cells rely on this enzyme for both effector function and clonal proliferation, with the latter being shared with T cells. As a result, LDHA-deficient NK cells are defective in their anti-viral and anti-tumor protection. These findings suggest that aerobic glycolysis is a hallmark of NK cell activation that is key to their function.

4.
Elife ; 92020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33289483

RESUMO

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.


Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , RNA de Transferência de Glutamina/metabolismo , Aminoacilação de RNA de Transferência , Animais , Linhagem Celular Tumoral , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Glutamina/metabolismo , Humanos , Camundongos , Peptídeos/metabolismo
5.
Nature ; 581(7809): 475-479, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461639

RESUMO

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1)2-4. Products of microbial fermentation including butyrate facilitate the generation of peripherally induced Treg (pTreg) cells5-7, indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids8 with a range of physiological functions9. Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pTreg cells. We found that the secondary bile acid 3ß-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of Treg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing Treg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation.


Assuntos
Bactérias/metabolismo , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Bacteroides/metabolismo , Colo/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Fermentação , Microbioma Gastrointestinal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Consórcios Microbianos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo
6.
EMBO J ; 39(8): e103334, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32134147

RESUMO

The production and secretion of matrix proteins upon stimulation of fibroblasts by transforming growth factor-beta (TGFß) play a critical role in wound healing. How TGFß supports the bioenergetic cost of matrix protein synthesis is not fully understood. Here, we show that TGFß promotes protein translation at least in part by increasing the mitochondrial oxidation of glucose and glutamine carbons to support the bioenergetic demand of translation. Surprisingly, we found that in addition to stimulating the entry of glucose and glutamine carbon into the TCA cycle, TGFß induced the biosynthesis of proline from glutamine in a Smad4-dependent fashion. Metabolic manipulations that increased mitochondrial redox generation promoted proline biosynthesis, while reducing mitochondrial redox potential and/or ATP synthesis impaired proline biosynthesis. Thus, proline biosynthesis acts as a redox vent, preventing the TGFß-induced increase in mitochondrial glucose and glutamine catabolism from generating damaging reactive oxygen species (ROS) when TCA cycle activity exceeds the ability of oxidative phosphorylation to convert mitochondrial redox potential into ATP. In turn, the enhanced synthesis of proline supports TGFß-induced production of matrix proteins.


Assuntos
Fibrose/metabolismo , Glucose/metabolismo , Glutamina/metabolismo , Mitocôndrias/metabolismo , Prolina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Ciclo do Ácido Cítrico , Colágeno/metabolismo , Metabolismo Energético , Humanos , Camundongos , Células NIH 3T3 , Oxirredução , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo
7.
Cancer Cell ; 37(1): 71-84.e7, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31935373

RESUMO

Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.


Assuntos
Leucemia Mieloide Aguda/enzimologia , Fosfotransferases/metabolismo , Fosfato de Piridoxal/metabolismo , Vitamina B 6/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Regulação Leucêmica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfotransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Poliaminas/metabolismo , RNA Interferente Pequeno/metabolismo
8.
Methods Mol Biol ; 1978: 269-283, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119669

RESUMO

Stable isotope tracing allows a metabolic substrate to be followed through downstream biochemical reactions, thereby providing unparalleled insights into the metabolic wiring of cells. This approach stops short of modeling absolute fluxes but is relatively straightforward and has become increasingly accessible due to the widespread adoption of high-resolution mass spectrometers. Analysis of both dynamic and steady-state labeling patterns in downstream metabolites provides valuable qualitative information as to their origin and relative rates of production. Stable isotope tracing is, therefore, a powerful way to understand the impact of genetic alterations and defined perturbations on metabolism. In this chapter, we describe a liquid chromatography-mass spectrometry (LC-MS) protocol for stable isotope tracing using 13C-L-arginine in a macrophage cell line. A similar approach can be used to follow other stable isotope tracers, and notes are provided with advice on how this protocol can be generalized for use in other settings.


Assuntos
Cromatografia Líquida/métodos , Marcação por Isótopo/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/química , Redes e Vias Metabólicas/genética
9.
Mol Genet Metab ; 126(4): 388-396, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709776

RESUMO

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and α-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains.


Assuntos
Erros Inatos do Metabolismo/genética , Camundongos Endogâmicos/genética , Animais , Elementos de DNA Transponíveis/genética , Cetona Oxirredutases/genética , Masculino , Erros Inatos do Metabolismo/diagnóstico , Metabolômica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Análise de Sequência de RNA
10.
Brain ; 142(3): 542-559, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668673

RESUMO

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.


Assuntos
Epilepsia/etiologia , Proteínas/genética , Proteínas/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/fisiopatologia , Feminino , Células HEK293 , Humanos , Masculino , Fenótipo , Fosfato de Piridoxal/uso terapêutico , Piridoxina/deficiência , Vitamina B 6/metabolismo , Deficiência de Vitamina B 6/genética , Deficiência de Vitamina B 6/metabolismo , Peixe-Zebra
11.
FASEB J ; 33(3): 4355-4364, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30540494

RESUMO

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid ß-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ácidos Graxos/metabolismo , Proteína Multifuncional do Peroxissomo-2/fisiologia , Peroxissomos/enzimologia , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sistemas CRISPR-Cas , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/fisiologia , Células HEK293 , Humanos , Ácidos Láuricos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Oxirredução , Ácido Palmítico/metabolismo , Enzima Bifuncional do Peroxissomo/deficiência , Proteína Multifuncional do Peroxissomo-2/deficiência , Proteína Multifuncional do Peroxissomo-2/genética , Proteínas Recombinantes/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3277-3285, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28962896

RESUMO

OBJECTIVE: The transcription factor Krüppel-like factor 14 (KLF14) has been associated with type 2 diabetes and high-density lipoprotein-cholesterol (HDL-C) through genome-wide association studies. The mechanistic underpinnings of KLF14's control of metabolic processes remain largely unknown. We studied the physiological roles of KLF14 in a knockout (KO) mouse model. METHODS: Male whole body Klf14 KO mice were fed a chow or high fat diet (HFD) and diet induced phenotypes were analyzed. Additionally, tissue-specific expression of Klf14 was determined using RT-PCR, RNA sequencing, immunoblotting and whole mount lacZ staining. Finally, the consequences of KLF14 loss-of-function were studied using RNA sequencing in tissues with relatively high Klf14 expression levels. RESULTS: KLF14 loss-of-function did not affect HFD-induced weight gain or insulin resistance. Fasting plasma concentrations of glucose, insulin, cholesterol, HDL-C and ApoA-I were also comparable between Klf14+/+ and Klf14-/- mice on chow and HFD. We found that in mice expression of Klf14 was the highest in the anterior pituitary (adenohypophysis), lower but detectable in white adipose tissue and undetectable in liver. Loss of KLF14 function impacted on the pituitary transcriptome with extracellular matrix organization as the primary affected pathway and a predicted link to glucocorticoid receptor signaling. CONCLUSIONS: Whole body loss of KLF14 function in male mice does not result in metabolic abnormalities as assessed under chow and HFD conditions. Mostly likely there is redundancy for the role of KLF14 in the mouse and a diverging function in humans.


Assuntos
Fatores de Transcrição Kruppel-Like/deficiência , Síndrome Metabólica/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucocorticoides/metabolismo , Análise de Sequência de RNA
13.
Clin Chem ; 63(4): 842-851, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28196920

RESUMO

BACKGROUND: Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS: We incubated 10 µL leukocyte lysate and 25 µL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid-liquid extraction. The extracts were evaporated and reconstituted in 200 µL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS: A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%-1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44-1.75 nmol · h-1 · mg-1 and 2.0-6.5 nmol · h-1 · mg-1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0-28.1 nmol · h-1 · mg-1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS: This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.


Assuntos
Cromatografia Líquida , Doença de Depósito de Glicogênio Tipo II/sangue , Leucócitos/enzimologia , Triagem Neonatal , Espectrometria de Massas em Tandem , alfa-Glucosidases/sangue , Adulto , Alelos , Criança , Pré-Escolar , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Lactente , Recém-Nascido , Leucócitos/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismo
14.
Annu Rev Physiol ; 78: 23-44, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26474213

RESUMO

Mitochondrial fatty acid ß-oxidation (FAO) is the major pathway for the degradation of fatty acids and is essential for maintaining energy homeostasis in the human body. Fatty acids are a crucial energy source in the postabsorptive and fasted states when glucose supply is limiting. But even when glucose is abundantly available, FAO is a main energy source for the heart, skeletal muscle, and kidney. A series of enzymes, transporters, and other facilitating proteins are involved in FAO. Recessively inherited defects are known for most of the genes encoding these proteins. The clinical presentation of these disorders may include hypoketotic hypoglycemia, (cardio)myopathy, arrhythmia, and rhabdomyolysis and illustrates the importance of FAO during fasting and in hepatic and (cardio)muscular function. In this review, we present the current state of knowledge on the biochemistry and physiological functions of FAO and discuss the pathophysiological processes associated with FAO disorders.


Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/genética , Mitocôndrias/fisiologia , Animais , Glucose/metabolismo , Homeostase/genética , Homeostase/fisiologia , Humanos , Mitocôndrias/metabolismo , Oxirredução
15.
Biochim Biophys Acta ; 1831(9): 1467-74, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23850792

RESUMO

Fatty acid ß-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal ß-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid ß-oxidation deficiency or overload.


Assuntos
Carnitina Aciltransferases/fisiologia , Carnitina O-Palmitoiltransferase/fisiologia , Carnitina/análogos & derivados , Carnitina/metabolismo , Fibroblastos/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Peroxissomos/metabolismo , Pele/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina Aciltransferases/metabolismo , Células Cultivadas , Fibroblastos/citologia , Imunofluorescência , Humanos , Ácidos Láuricos/química , Erros Inatos do Metabolismo Lipídico/patologia , Oxirredução , Pele/citologia
16.
Biochim Biophys Acta ; 1832(6): 773-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23485643

RESUMO

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid ß-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.


Assuntos
Aminoácidos de Cadeia Ramificada/química , Aminoácidos de Cadeia Ramificada/metabolismo , Carnitina O-Acetiltransferase/química , Carnitina O-Acetiltransferase/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Aminoácidos de Cadeia Ramificada/genética , Carnitina O-Acetiltransferase/genética , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/genética , Humanos , Especificidade por Substrato/fisiologia
17.
FASEB J ; 27(5): 2039-44, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23322164

RESUMO

Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid ß-oxidation disorders (mFAODs). It is generally assumed that this plasma acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/acylcarnitine translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P<0.01). OCTN2-deficient cell lines also showed extracellular accumulation of octanoylcarnitine (2.8-fold, P<0.01), suggesting that the cellular export of acylcarnitines does not depend on OCTN2. In contrast, in CPT2- and CACT-deficient cells, the accumulation of octanoylcarnitine in the medium did not significantly increase in the MCAD knockdown. Similar results were obtained using pharmacological inhibition of CPT2 in fibroblasts from MCAD-deficient individuals. This shows that CPT2 and CACT are crucial for mitochondrial acylcarnitine formation and export to the extracellular fluids in mFAOD.


Assuntos
Carnitina Aciltransferases/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/análogos & derivados , Doenças Mitocondriais/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Carnitina/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina O-Palmitoiltransferase/deficiência , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto
18.
Proc Natl Acad Sci U S A ; 109(21): 7974-81, 2012 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-22566635

RESUMO

We recently reported a deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene in a proband with autism. TMLHE maps to the X chromosome and encodes the first enzyme in carnitine biosynthesis, 6-N-trimethyllysine dioxygenase. Deletion of exon 2 of TMLHE causes enzyme deficiency, resulting in increased substrate concentration (6-N-trimethyllysine) and decreased product levels (3-hydroxy-6-N-trimethyllysine and γ-butyrobetaine) in plasma and urine. TMLHE deficiency is common in control males (24 in 8,787 or 1 in 366) and was not significantly increased in frequency in probands from simplex autism families (9 in 2,904 or 1 in 323). However, it was 2.82-fold more frequent in probands from male-male multiplex autism families compared with controls (7 in 909 or 1 in 130; P = 0.023). Additionally, six of seven autistic male siblings of probands in male-male multiplex families had the deletion, suggesting that TMLHE deficiency is a risk factor for autism (metaanalysis Z-score = 2.90 and P = 0.0037), although with low penetrance (2-4%). These data suggest that dysregulation of carnitine metabolism may be important in nondysmorphic autism; that abnormalities of carnitine intake, loss, transport, or synthesis may be important in a larger fraction of nondysmorphic autism cases; and that the carnitine pathway may provide a novel target for therapy or prevention of autism.


Assuntos
Transtorno Autístico , Carnitina/deficiência , Cromossomos Humanos X/genética , Genes Ligados ao Cromossomo X/genética , Erros Inatos do Metabolismo , Oxigenases de Função Mista/genética , Transtorno Autístico/epidemiologia , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Carnitina/biossíntese , Cognição/fisiologia , Éxons/genética , Deleção de Genes , Humanos , Masculino , Erros Inatos do Metabolismo/epidemiologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Oxigenases de Função Mista/sangue , Oxigenases de Função Mista/urina , Penetrância , Fatores de Risco , Irmãos
19.
PLoS One ; 6(10): e25443, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22003392

RESUMO

miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinson's disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of α-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46≤OR≤0.63) and highly significant in the meta-dataset (3.36×10⁻4


Assuntos
Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica , MicroRNAs/genética , Doença de Parkinson/genética , Integração de Sistemas , alfa-Sinucleína/metabolismo , Idoso , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Doença de Parkinson/metabolismo
20.
Biochim Biophys Acta ; 1802(9): 728-32, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20538056

RESUMO

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8 microM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.


Assuntos
Carnitina O-Palmitoiltransferase/fisiologia , Carnitina/análogos & derivados , Metaboloma , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Carnitina/análise , Carnitina/metabolismo , Carnitina/farmacocinética , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , Catálise , Humanos , Cinética , Metaboloma/fisiologia , Organismos Geneticamente Modificados , Concentração Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
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