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1.
BMC Complement Altern Med ; 19(1): 255, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519174

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is characterized by excessive hepatic lipid accumulation. Many studies have suggested that lipid overload is the key initial factor that contributes to hepatic steatosis. Our previous study indicated that diosgenin (DSG) has a beneficial effect on energy metabolism, but the underlying mechanism remains unclear. METHODS: Human normal hepatocytes (LO2 cells) were incubated with palmitic acid to establish the cell model of nonalcoholic fatty liver. The effects of DSG on lipid metabolism, glucose uptake and mitochondrial function were evaluated. Furthermore, the mechanism of DSG on oxidative stress, lipid consumption and lipid synthesis in LO2 cells was investigated. RESULTS: The results indicated that palmitic acid induced obvious lipid accumulation in LO2 cells and that DSG treatment significantly reduced the intracellular lipid content. DSG treatment upregulated expression of lipolysis proteins, including phospho-AMP activated protein kinase (p-AMPK), phospho-acetyl-coA carboxylase (p-ACC) and carnitine acyl transferase 1A (CPT-1A), and inhibited expression of lipid synthesis-related proteins, including sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS). Additionally, DSG-treated cells displayed a marked improvement in mitochondrial function, with less production of reactive oxygen species and a higher mitochondrial membrane potential compared with the model group. CONCLUSION: This study suggests that DSG can reduce intracellular lipid accumulation in LO2 cells and that the underlying mechanism may be related to the improving oxidative stress, increasing fatty acid ß-oxidation and decreasing lipid synthesis. The above changes might be mediated by the activation of the AMPK/ACC/CPT-1A pathway and inhibition of the SREBP-1c/FAS pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Diosgenina/farmacologia , Ácido Graxo Sintases/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácido Palmítico/efeitos adversos , Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Linhagem Celular , Ácido Graxo Sintases/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
2.
Diabetes ; 68(4): 819-831, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30728184

RESUMO

Proximal tubular epithelial cells are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. Whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage has been suggested but is not entirely clear. Here we modeled mitochondrial overload by creating mice lacking the enzyme carnitine acetyltransferase (CrAT) in the proximal tubules, thus limiting a primary mechanism to export carbons under conditions of substrate excess. Mice developed tubular disease and, interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis regulator and fibrosis markers, increased oxidative stress, and abnormal profiles of acylcarnitines and organic acids suggesting profound impairments in all major forms of nutrient metabolism. When mice with CrAT deletion were fed a high-fat diet, kidney disease was more severe and developed faster. Primary proximal tubular cells isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology, suggesting mitochondrial respiratory abnormalities as a potential underlying mechanism. Our findings support the hypothesis that derailments of mitochondrial energy metabolism may be causative to chronic kidney disease. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell mitochondrial function and energy balance could be an important preventative strategy.


Assuntos
Carnitina O-Acetiltransferase/metabolismo , Nefropatias/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Animais , Apoptose/fisiologia , Carnitina O-Acetiltransferase/genética , Dieta Hiperlipídica , Complexo I de Transporte de Elétrons/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais Proximais/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia
3.
Nat Chem Biol ; 14(11): 1021-1031, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30327559

RESUMO

Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.


Assuntos
Tecido Adiposo/enzimologia , Aminoácidos de Cadeia Ramificada/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Obesidade/enzimologia , Células 3T3 , Adipócitos/citologia , Animais , Sistemas CRISPR-Cas , Carnitina O-Acetiltransferase/metabolismo , Citosol/metabolismo , Feminino , Hipóxia , Lentivirus/genética , Lipogênese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , RNA Interferente Pequeno/metabolismo
4.
J Ind Microbiol Biotechnol ; 45(11): 971-981, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30187242

RESUMO

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the ß-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH-cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.


Assuntos
Candida tropicalis/genética , Proteínas de Ligação a DNA/metabolismo , Ácidos Dicarboxílicos/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Alcanos/metabolismo , Candida tropicalis/metabolismo , Carnitina O-Acetiltransferase/genética , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácidos Graxos/metabolismo , Fermentação , Edição de Genes , Genoma Fúngico , Redes e Vias Metabólicas , Oxirredução , Plasmídeos , Regiões Promotoras Genéticas
5.
J Hum Genet ; 63(12): 1259-1267, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30266950

RESUMO

Essential hypersomnia (EHS) is a lifelong disorder characterized by excessive daytime sleepiness without cataplexy. EHS is associated with human leukocyte antigen (HLA)-DQB1*06:02, similar to narcolepsy with cataplexy (narcolepsy). Previous studies suggest that DQB1*06:02-positive and -negative EHS are different in terms of their clinical features and follow different pathological pathways. DQB1*06:02-positive EHS and narcolepsy share the same susceptibility genes. In the present study, we report a genome-wide association study with replication for DQB1*06:02-negative EHS (408 patients and 2247 healthy controls, all Japanese). One single-nucleotide polymorphism, rs10988217, which is located 15-kb upstream of carnitine O-acetyltransferase (CRAT), was significantly associated with DQB1*06:02-negative EHS (P = 7.5 × 10-9, odds ratio = 2.63). The risk allele of the disease-associated SNP was correlated with higher expression levels of CRAT in various tissues and cell types, including brain tissue. In addition, the risk allele was associated with levels of succinylcarnitine (P = 1.4 × 10-18) in human blood. The leading SNP in this region was the same in associations with both DQB1*06:02-negative EHS and succinylcarnitine levels. The results suggest that DQB1*06:02-negative EHS may be associated with an underlying dysfunction in energy metabolic pathways.


Assuntos
Carnitina O-Acetiltransferase/genética , Cromossomos Humanos Par 9/genética , Distúrbios do Sono por Sonolência Excessiva/genética , Cadeias beta de HLA-DQ/genética , Polimorfismo de Nucleotídeo Único , Distúrbios do Sono por Sonolência Excessiva/enzimologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino
6.
Biochem J ; 475(7): 1267-1269, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29632149

RESUMO

The movement of lipids across mitochondrial membranes represents a rate-limiting step in fatty acid oxidation within the heart. A key regulatory point in this process is flux through carnitine palmitoyltransferase-I (CPT-I), an enzyme located on the outer mitochondrial membrane. Malonyl-CoA (M-CoA) is a naturally occurring inhibitor of CPT-I; therefore, the abundance of M-CoA has long been considered a major regulator of fatty acid oxidation. A recent paper published in the Biochemical Journal by Altamimi et al. (Biochem. J. (2018) 475, 959-976) provides evidence for a novel mechanism to produce M-CoA. Specifically, these authors identified carnitine acetyltransferase within the cytosol and further show that flux in the reverse direction forms acetyl-CoA, which is the necessary substrate for the subsequent synthesis of M-CoA. The elegant study design and intriguing data presented by Altamimi et al. provide further insights into the reciprocal regulation of substrate selection within the heart, with implications for fuel utilization and the development of cardiac diseases.


Assuntos
Acetilcoenzima A , Carnitina O-Acetiltransferase , Carnitina O-Palmitoiltransferase , Citosol , Metabolismo Energético , Malonil Coenzima A
7.
Endocrinology ; 159(6): 2473-2483, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29697769

RESUMO

Behavioral adaptation to periods of varying food availability is crucial for survival, and agouti-related protein (AgRP) neurons have been associated with entrainment to temporal restricted feeding. We have shown that carnitine acetyltransferase (Crat) in AgRP neurons enables metabolic flexibility and appropriate nutrient partitioning. In this study, by restricting food availability to 3 h/d during the light phase, we examined whether Crat is a component of a food-entrainable oscillator (FEO) that helps link behavior to food availability. AgRP Crat knockout (KO) mice consumed less food and regained less body weight but maintained blood glucose levels during the 25-day restricted feeding protocol. Importantly, we observed no difference in meal latency, food anticipatory activity (FAA), or brown adipose tissue temperature during the first 13 days of restricted feeding. However, as the restricted feeding paradigm progressed, we noticed an increased FAA in AgRP Crat KO mice. The delayed increase in FAA, which developed during the last 12 days of restricted feeding, corresponded with elevated plasma levels of corticosterone and nonesterified fatty acids, indicating it resulted from greater energy debt incurred by KO mice over the course of the experiment. These experiments highlight the importance of Crat in AgRP neurons in regulating feeding behavior and body weight gain during restricted feeding but not in synchronizing behavior to food availability. Thus, Crat within AgRP neurons forms a component of the homeostatic response to restricted feeding but is not likely to be a molecular component of FEO.


Assuntos
Adaptação Fisiológica/genética , Proteína Relacionada com Agouti/metabolismo , Restrição Calórica , Carnitina O-Acetiltransferase/fisiologia , Comportamento Alimentar/fisiologia , Homeostase/genética , Neurônios/metabolismo , Animais , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Condicionamento Físico Animal/fisiologia
8.
Cancer Res ; 78(10): 2490-2502, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29535221

RESUMO

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the ß-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle ß-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to ß-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.


Assuntos
Acetilcoenzima A/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Carcinogênese/genética , Movimento Celular/genética , Sobrevivência Celular/genética , Neoplasias da Próstata/patologia , Acil-CoA Oxidase/genética , Animais , Sistemas CRISPR-Cas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Carnitina O-Acetiltransferase/genética , Linhagem Celular Tumoral , Ácidos Graxos/metabolismo , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Oxirredução , Oxirredutases/genética , Células PC-3
9.
Biochem J ; 475(5): 959-976, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29438065

RESUMO

The role of carnitine acetyltransferase (CrAT) in regulating cardiac energy metabolism is poorly understood. CrAT modulates mitochondrial acetyl-CoA/CoA (coenzyme A) ratios, thus regulating pyruvate dehydrogenase activity and glucose oxidation. Here, we propose that cardiac CrAT also provides cytosolic acetyl-CoA for the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation. We show that in the murine cardiomyocyte cytosol, reverse CrAT activity (RCrAT, producing acetyl-CoA) is higher compared with the liver, which primarily uses ATP-citrate lyase to produce cytosolic acetyl-CoA for lipogenesis. The heart displayed a lower RCrAT Km for CoA compared with the liver. Furthermore, cytosolic RCrAT accounted for 4.6 ± 0.7% of total activity in heart tissue and 12.7 ± 0.2% in H9C2 cells, while highly purified heart cytosolic fractions showed significant CrAT protein levels. To investigate the relationship between CrAT and acetyl-CoA carboxylase (ACC), the cytosolic enzyme catalyzing malonyl-CoA production from acetyl-CoA, we studied ACC2-knockout mouse hearts which showed decreased CrAT protein levels and activity, associated with increased palmitate oxidation and acetyl-CoA/CoA ratio compared with controls. Conversely, feeding mice a high-fat diet for 10 weeks increased cardiac CrAT protein levels and activity, associated with a reduced acetyl-CoA/CoA ratio and glucose oxidation. These data support the presence of a cytosolic CrAT with a low Km for CoA, favoring the formation of cytosolic acetyl-CoA, providing an additional source to the classical ATP-citrate lyase pathway, and that there is an inverse relation between CrAT and the ratio of acetyl-CoA/CoA as evident in conditions affecting the regulation of cardiac energy metabolism.


Assuntos
Acetilcoenzima A/metabolismo , Carnitina O-Acetiltransferase/fisiologia , Citosol/metabolismo , Metabolismo Energético/genética , Miocárdio/metabolismo , Animais , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxirredução
10.
Cell Rep ; 22(7): 1745-1759, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29444428

RESUMO

AgRP neurons control peripheral substrate utilization and nutrient partitioning during conditions of energy deficit and nutrient replenishment, although the molecular mechanism is unknown. We examined whether carnitine acetyltransferase (Crat) in AgRP neurons affects peripheral nutrient partitioning. Crat deletion in AgRP neurons reduced food intake and feeding behavior and increased glycerol supply to the liver during fasting, as a gluconeogenic substrate, which was mediated by changes to sympathetic output and peripheral fatty acid metabolism in the liver. Crat deletion in AgRP neurons increased peripheral fatty acid substrate utilization and attenuated the switch to glucose utilization after refeeding, indicating altered nutrient partitioning. Proteomic analysis in AgRP neurons shows that Crat regulates protein acetylation and metabolic processing. Collectively, our studies highlight that AgRP neurons require Crat to provide the metabolic flexibility to optimize nutrient partitioning and regulate peripheral substrate utilization, particularly during fasting and refeeding.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Carnitina O-Acetiltransferase/metabolismo , Ácidos Graxos/metabolismo , Animais , Colecistocinina/administração & dosagem , Ingestão de Alimentos , Jejum , Comportamento Alimentar , Deleção de Genes , Glucose/metabolismo , Teste de Tolerância a Glucose , Injeções Intraperitoneais , Injeções Intraventriculares , Insulina/administração & dosagem , Integrases/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Knockout , Proteômica , Reprodutibilidade dos Testes
11.
Theriogenology ; 105: 150-157, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28965027

RESUMO

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.


Assuntos
Carnitina O-Acetiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/farmacologia , Técnicas de Cultura Embrionária/veterinária , Peroxirredoxinas/metabolismo , Ovinos/embriologia , Animais , Carnitina O-Acetiltransferase/genética , Carnitina O-Palmitoiltransferase/genética , Criopreservação/métodos , Criopreservação/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Congelamento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oócitos , Peroxirredoxinas/genética , Ovinos/fisiologia , Vitrificação
12.
Mol Metab ; 6(2): 219-225, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28180063

RESUMO

OBJECTIVE: Fatty acid oxidation in macrophages is thought to regulate inflammatory status and insulin-sensitivity. An important unanswered question in this field is whether carnitine acetyl-transferase (CrAT) that regulates fatty acid oxidation and mitochondrial acetyl-CoA balance is required to integrate nutrient stress sensing to inflammatory response in macrophages. METHODS: Mice with myeloid lineage-specific Crat deletion were subjected to several metabolic stressors, including high-fat diet-induced obesity, fasting, and LPS-induced endotoxemia. Their metabolic homeostasis was compared to that of Crat-sufficient littermate controls. Inflammatory potential of Crat-deficient and Crat-sufficient macrophages were measured both in vitro and in vivo. RESULTS: Our studies revealed that ablation of CrAT in myeloid lineage cells did not impact glucose homeostasis, insulin-action, adipose tissue leukocytosis, and inflammation when animals were confronted with a variety of metabolic stressors, including high-fat diet, fasting, or LPS-induced acute endotoxemia. CONCLUSIONS: These findings demonstrate that unlike muscle cells, substrate switch mechanisms that control macrophage energy metabolism and mitochondrial short-chain acyl-CoA pools during nutrient stress are controlled by pathways that are not solely reliant on CrAT.


Assuntos
Carnitina O-Acetiltransferase/biossíntese , Macrófagos/enzimologia , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Tecido Adiposo/metabolismo , Animais , Carnitina/metabolismo , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Dieta Hiperlipídica , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Homeostase/fisiologia , Inflamação/enzimologia , Inflamação/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Obesidade/enzimologia , Obesidade/metabolismo , Oxirredução
13.
Cell Rep ; 14(2): 243-54, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26748706

RESUMO

Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.


Assuntos
Carnitina O-Acetiltransferase/metabolismo , Lisina/metabolismo , Proteínas Mitocondriais/metabolismo , Acetilação , Animais , Humanos , Camundongos
14.
Br J Nutr ; 115(6): 937-50, 2016 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-26819073

RESUMO

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.


Assuntos
Arginina/administração & dosagem , Betaína/administração & dosagem , Dieta com Restrição de Proteínas/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Metabolismo dos Lipídeos , Músculo Liso/metabolismo , Gordura Subcutânea Abdominal/metabolismo , Adiposidade , Animais , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Cruzamentos Genéticos , Dieta com Restrição de Proteínas/efeitos adversos , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Qualidade dos Alimentos , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Carne/análise , Músculo Liso/enzimologia , Músculo Liso/crescimento & desenvolvimento , Especificidade de Órgãos , Portugal , RNA Mensageiro/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Gordura Subcutânea Abdominal/enzimologia , Gordura Subcutânea Abdominal/crescimento & desenvolvimento , Sus scrofa
15.
Cell Metab ; 22(1): 65-76, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26154055

RESUMO

Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.


Assuntos
Acetilcoenzima A/metabolismo , Carnitina O-Acetiltransferase/metabolismo , Carnitina/análogos & derivados , Fadiga Muscular , Músculos/enzimologia , Animais , Carnitina/sangue , Carnitina/metabolismo , Exercício Físico , Humanos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Condicionamento Físico Animal
16.
J Biol Chem ; 290(13): 8121-32, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25645937

RESUMO

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.


Assuntos
Acetilcoenzima A/metabolismo , Carnitina O-Acetiltransferase/metabolismo , Miocárdio/enzimologia , Complexo Piruvato Desidrogenase/metabolismo , Animais , Isótopos de Carbono/metabolismo , Deutério/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Técnicas In Vitro , Corpos Cetônicos/metabolismo , Masculino , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
17.
Biochem Soc Trans ; 42(4): 1043-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25110000

RESUMO

CoA (coenzyme A) and its derivatives have a critical role in regulating cardiac energy metabolism. This includes a key role as a substrate and product in the energy metabolic pathways, as well as serving as an allosteric regulator of cardiac energy metabolism. In addition, the CoA ester malonyl-CoA has an important role in regulating fatty acid oxidation, secondary to inhibiting CPT (carnitine palmitoyltransferase) 1, a key enzyme involved in mitochondrial fatty acid uptake. Alterations in malonyl-CoA synthesis by ACC (acetyl-CoA carboxylase) and degradation by MCD (malonyl-CoA decarboxylase) are important contributors to the high cardiac fatty acid oxidation rates seen in ischaemic heart disease, heart failure, obesity and diabetes. Additional control of fatty acid oxidation may also occur at the level of acetyl-CoA involvement in acetylation of mitochondrial fatty acid ß-oxidative enzymes. We find that acetylation of the fatty acid ß-oxidative enzymes, LCAD (long-chain acyl-CoA dehydrogenase) and ß-HAD (ß-hydroxyacyl-CoA dehydrogenase) is associated with an increase in activity and fatty acid oxidation in heart from obese mice with heart failure. This is associated with decreased SIRT3 (sirtuin 3) activity, an important mitochondrial deacetylase. In support of this, cardiac SIRT3 deletion increases acetylation of LCAD and ß-HAD, and increases cardiac fatty acid oxidation. Acetylation of MCD is also associated with increased activity, decreases malonyl-CoA levels and an increase in fatty acid oxidation. Combined, these data suggest that malonyl-CoA and acetyl-CoA have an important role in mediating the alterations in fatty acid oxidation seen in heart failure.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Glucose/metabolismo , Animais , Carnitina O-Acetiltransferase/metabolismo , Ácidos Graxos/metabolismo , Humanos , Lisina/metabolismo , Oxirredução , Sirtuína 3/metabolismo
18.
BMC Genomics ; 15: 514, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24962334

RESUMO

BACKGROUND: The carnitine acetyltransferase (CrAT) is a mitochondrial matrix protein that directly influences intramitochondrial acetyl-CoA pools. Murine CrAT is encoded by a single gene located in the opposite orientation head to head to the PPP2R4 gene, sharing a very condensed bi-directional promoter. Since decreased CrAT expression is correlated with metabolic inflexibility and subsequent pathological consequences, our aim was to reveal and define possible activators of CrAT transcription in the normal embryonic murine liver cell line BNL CL. 2 and via which nuclear factors based on key metabolites mainly regulate hepatic expression of CrAT. Here we describe a functional characterization of the CrAT promoter region under conditions of L-carnitine deficiency and supplementation as well as fenofibrate induction in cell culture cells. RESULTS: The murine CrAT promoter displays some characteristics of a housekeeping gene: it lacks a TATA-box, is very GC-rich and harbors two Sp1 binding sites. Analysis of the promoter activity of CrAT by luciferase assays uncovered a L-carnitine sensitive region within -342 bp of the transcription start. Electrophoretic mobility shift and supershift assays proved the sequence element (-228/-222) to be an L-carnitine sensitive RXRα binding site, which also showed sensitivity to application of anti-PPARα and anti-PPARbp antibodies. In addition we analysed this specific RXRα/PPARα site by Southwestern Blotting technique and could pin down three protein factors binding to this promoter element. By qPCR we could quantify the nutrigenomic effect of L-carnitine itself and fenofibrate. CONCLUSIONS: Our results indicate a cooperative interplay of L-carnitine and PPARα in transcriptional regulation of murine CrAT, which is of nutrigenomical relevance. We created experimental proof that the muCrAT gene clearly is a PPARα target. Both L-carnitine and fenofibrate are inducers of CrAT transcripts, but the important hyperlipidemic drug fenofibrate being a more potent one, as a consequence of its pharmacological interaction.


Assuntos
Carnitina O-Acetiltransferase/genética , Carnitina/antagonistas & inibidores , Fenofibrato/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , PPAR alfa/antagonistas & inibidores , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Carnitina/metabolismo , Carnitina/farmacologia , Núcleo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Camundongos , Dados de Sequência Molecular , PPAR alfa/metabolismo , PPAR alfa/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética
19.
J Org Chem ; 79(10): 4644-9, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24742295

RESUMO

N-Bromosuccinimide-induced electrophilic multicomponent reaction has been applied to the synthesis of Reboxetine intermediate, a highly potent selective norepinephrine reuptake inhibitor. By simply changing the olefinic partner, the synthesis of a carnitine acetyltransferase inhibitor, which contains a 2,6,6-trisubstituted morpholine system, can be accomplished.


Assuntos
Inibidores da Captação Adrenérgica/química , Inibidores da Captação Adrenérgica/farmacologia , Bromosuccinimida/química , Carnitina O-Acetiltransferase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Morfolinas/síntese química , Norepinefrina/química , Norepinefrina/farmacologia , Carnitina O-Acetiltransferase/química , Inibidores Enzimáticos/química , Morfolinas/química , Reboxetina
20.
J Surg Res ; 188(2): 480-8, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24565505

RESUMO

BACKGROUND: During the course of sepsis, often myocardial depression with hemodynamic impairment occurs. Acetylcholine, the main transmitter of the parasympathetic Nervus vagus, has been shown to be of importance for the transmission of signals within the immune system and also for a variety of other functions throughout the organism. Hypothesizing a potential correlation between this dysfunction and hemodynamic impairment, we wanted to assess the impact of vagal stimulation on myocardial inflammation and function in a rat model of lipopolysaccharide (LPS)-induced septic shock. As the myocardial tissue is (sparsely) innervated by the N. vagus, there might be an important anti-inflammatory effect in the heart, inhibiting proinflammatory gene expression in cardiomyocytes and improving cardiac function. MATERIALS AND METHODS: We performed stimulation of the right cervical branch of the N. vagus in vagotomized, endotoxemic (1 mg/kg body weight LPS, intravenously) rats. Hemodynamic parameters were assessed over time using a left ventricular pressure-volume catheter. After the experiments, hearts and blood plasma were collected, and the expression of proinflammatory cytokines was measured using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: After vagotomy, the inflammatory response was aggravated, measurable by elevated cytokine levels in plasma and ventricular tissue. In concordance, cardiac impairment during septic shock was pronounced in these animals. To reverse both hemodynamic and immunologic effects of diminished vagal tone, even a brief stimulation of the N. vagus was enough during initial LPS infusion. CONCLUSIONS: Overall, the N. vagus might play a major role in maintaining hemodynamic stability and cardiac immune homeostasis during septic shock.


Assuntos
Regulação para Baixo/fisiologia , Endotoxemia/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Choque Séptico/fisiopatologia , Nervo Vago/patologia , Anestesia , Animais , Carnitina O-Acetiltransferase/metabolismo , Colina O-Acetiltransferase/metabolismo , Endotoxemia/patologia , Hemodinâmica , Inflamação/patologia , Inflamação/fisiopatologia , Interleucina-1beta/sangue , Masculino , Ratos , Ratos Endogâmicos Lew , Choque Séptico/patologia , Fator de Necrose Tumoral alfa/sangue , Vagotomia
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