Geração de espécies reativas de oxigênio (ERO) mitocondriais: papel das Acil-CoA desidrogenases de cadeia muito longa / Generation of mitochondrial reactive oxygen species (ROS): role of very long chain acyl-coA dehydrogenases

Dietas hiperlipídicas e a esteatose hepática são condições extremamente prevalentes. Trabalhos anteriores mostraram que a esteatose está associada a um aumento na geração de espécies reativas de oxigênio (ERO), e que isso pode mediar danos no fígado. Neste trabalho nós investigamos os possíveis mecanismos que desencadeiam os aumentos nas taxas de geração de ERO por meio da administração de dietas hiperlipídicas. Nós descobrimos que mitocôndrias de animais sujeitos a dietas hiperlipídicas não apresentaram diferenças significativas quanto a capacidade respiratória máxima e acoplamento, mas eram capazes de gerar mais ERO especificamente quando usados substratos do metabolismo de ácidos graxos. Além disso, foi observado que muitas isoformas de acil-CoA desidrogenases estavam mais expressas nos fígados de animais alimentados pela dieta hiperlipídica. No entanto, quando realizados ensaios de atividade enzimática apenas a acil CoA desidrogenase de cadeia longa (VLCAD) foi mais ativa. Estudos conduzidos com mitocôndrias permeabilizadas e expostas a grupos acil-CoA de diferentes tamanhos sugerem que a VLCAD pode ser uma fonte da produção aumentada de ERO em animais submetidos a dietas hiperlipídicas. Esta produção foi estimulada pela ausência de NAD+. Concluindo, nossos estudos descobriram uma nova fonte importante na geração de ERO estimulada por dietas hiperlipídicas, a VLCAD

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis occurs concomitantly with enhanced reactive oxygen species (ROS) generation, which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is a source of enhanced ROS production in mitochondria from HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS

Short communication: high insulin concentrations inhibit fatty acid oxidation-related gene expression in calf hepatocytes cultured in vitro.

In dairy cows, ketosis is an important disease associated with negative energy balance, which leads to low blood glucose levels and high blood nonesterified fatty acid levels. The liver is the most active organ in cows for the metabolism of nonesterified fatty acids. Insulin is an anabolic hormone that plays numerous roles in the metabolism of carbohydrates, lipids, and proteins, as well as being a potent regulator of fatty acid oxidation. In this study, using fluorescent quantitative reverse-transcription PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of insulin (0, 5, 10, 20, 50, and 100 nmol/L) on fatty acid oxidation by monitoring mRNA and protein expression levels of key enzymes: long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase. The results showed that the mRNA and protein expression of long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase was markedly decreased when the concentration of insulin in the media was increased. These findings indicate that high levels of insulin significantly inhibit the expression of genes related to fatty acid oxidation and consequently results in a decreased level of fatty acid oxidation in calf hepatocytes.

Implementación diagnóstica de la deficiencia de acil-CoA deshidrogenasa de cadena muy larga / Diagnostic implementation of very long chain acyl-CoA dehydrogenase deficiency / Implementação diagnóstica da deficiência de acil-CoA desidrogenase de cadeia muito longa

La enzima acil-CoA deshidrogenasa de cadena muy larga (VLCAD) es un homodímero que cataliza la reacción inicial de la ß-oxidación mitocondrial de los ácidos grasos. El presente estudio tuvo como objetivo el análisis de los metabolitos producidos por fibroblastos incubados en presencia de sustratos tritiados vs. deuterados, como herramienta diagnóstica de la deficiencia de VLCAD. Fue encontrada severamente deprimida la oxidación de los sustratos tritiados en los fibroblastos de pacientes con esta enfermedad, asimismo la incubación con sustratos deuterados aportó un perfil característico en esta deficiencia. El método de valoración de agua tritiada, aunque inespecífico, debido a que la oxidación de sustratos tritiados también puede estar deprimida en otras deficiencias, es un buen método para sugerir una deficiencia de acil- CoA deshidrogenasa de cadena muy larga, si el análisis se compara con otros hallazgos propios de la deficiencia enzimática. Sin embargo, la determinación de los metabolitos deuterados es más específica, por encontrarse un perfil característico que muestra niveles elevados de los ácidos grasos octanoico, decanoico, dodecenoico, dodecanoico, tetradecenoico, tetradecanoico y hexadecenoico, lo que la hace diferente de otras deficiencias enzimáticas.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is a homodimer that catalyzes the initial reaction of fatty acid ß-oxidation. The objective of the present study was to analyse the metabolites produced by fibroblasts incubated with tritiated vs. deuterated substrates, as a diagnostic tool for the diagnosis of VLCAD deficiency. A severe depression for oxidizing the tritiated substrates was observed for these patients' fibroblasts, and a characteristic profile for this deficiency was found when incubating fibroblasts with deuterated substrates. The method which evaluates the production of tritiated water is nonspecific as the oxidation of tritiated substrates can be found depressed in other fatty acid ß-oxidation disorders; however this method can suggest VLCAD deficiency if the tritiated water measurement is compared with others findings related to this deficiency. On the other hand the measurement of deuterated metabolites is more specific as a characteristic profile was found for this deficiency showing increased levels of the following organic acids: octanoic, decanoic, dodecenoic, dodecanoic, tetradecenoic, tetradecanoic and hexadecanoic, which is different from other fatty acid ß-oxidation disorders.

A enzima acil-CoA desidrogenase de cadeia muito longa (VLCAD) é um homodímero que catalisa a reação inicial da ß-oxidação mitocondrial dos ácidos graxos. O presente estudo teve como objetivo a análise dos metabólitos produzidos por Ibroblastos incubados em presença de substratos tritiados vs. deuterados, como ferramenta de diagnóstico da deIciência de VLCAD. Foi encontrada severamente deprimida a oxidação dos substratos tritiados nos Ibroblastos de pacientes com esta doença, do mesmo modo a incubação com substratos deuterados deu um perIl característico nesta deIciência. O método de avaliação de água tritiada, embora não especíIco, devido a que a oxidação de substratos tritiados também pode estar deprimida em outras deIciências, é um bom método para sugerir uma deIciência de acil-CoA desidrogenase de cadeia muito longa, se a análise é comparada com outros achados próprios da deIciência enzimática. Entretanto, a determinação dos metabólitos deuterados é mais especiIca, devido a que se encontra um perIl característico que mostra níveis elevados dos ácidos graxos octanoico, decanoico, dodecenoico, dodecanoico, tetradecenoico, tetradecanoico e hexadecenoico, o que a torna diferente de outras deIciências enzimáticas.

A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency.

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.

Influence of valproic acid on the expression of various acyl-CoA dehydrogenases in rats.

BACKGROUND: Valproic acid (2-propyl-N-pentanoic acid, VPA) causes severe hepatic dysfunction, similar to Reye's syndrome, in a small number of patients. An enhanced excretion of dicarboxylic acids by patients indicates an interference with mitochondrial beta-oxidation. We investigated the expression of various acyl-coenzyme A (acyl-CoA) dehydrogenases (ACD), which catalyze the first step of beta-oxidation in VPA-treated rats. METHODS: The control group received normal saline and the experimental group received VPA (500 mg/kg per day) by intraperitoneal injections for 7 days. Various clinical chemistry parameters in rat blood and free and total carnitine levels in plasma and tissue were determined. Mitochondria were isolated from rat liver and heart and the relative amount of each ACD protein was determined by immunoblot analysis. Total RNA was prepared from various tissues and the mRNA levels for various ACD were measured by slot-blot hybridization analysis using respective cDNA probes. RESULTS: Administration of VPA to rats caused various metabolic effects including hypoglycemia, hyperammonemia and decreased beta-hydroxybutyrate concentration. Free carnitine levels in plasma and heart were also decreased. Enzyme activities of various acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, decreased moderately in heart (57-79%), and slightly in liver (78-95%). The most prominent effects were observed in mRNA levels involved in fatty acid oxidation (short-, medium- and long-chain acyl-CoA dehydrogenase). Each mRNA increased in the liver, kidney, skeletal muscle and heart to varying degrees when rats were fed ad libitum. The increase of short- and medium- chain acyl-CoA dehydrogenase mRNA in the heart were particularly large. However, 3 day starvation strongly inhibited expression of ACD in VPA-treated rats. There was an apparent decrease in the amount of ACD mRNA and proteins in VPA-treated liver. CONCLUSIONS: Valproic acid causes enhanced expression of fatty ACD mRNA, especially in the heart, by a feedback mechanism related to inhibition of beta-oxidation in rats fed ad libitum. However, it impairs the expression of ACD in the liver when there is a drastic change in nutritional state.

Specific assay of medium-chain acyl-CoA dehydrogenase based on the spectrophotometric measurement of product formation.

A spectrophotometric method for assaying medium-chain acyl-CoA dehydrogenase is described. The assay measures at 308 nm the formation of cinnamoyl-CoA from 3-phenylpropionyl-CoA in the presence of phenazine methosulfate as electron acceptor. Apparent kinetic constants (Km, Vmax) determined with 3-phenylpropionyl-CoA are similar to constants obtained with octanoyl-CoA, the preferred substrate of this enzyme. The assay is specific for medium-chain acyl-CoA dehydrogenase because long-chain and short-chain acyl-CoA dehydrogenases exhibit little or no activity with 3-phenylpropionyl-CoA as substrate. Since absorbance changes at 308 nm caused by other reactions are less than 5% of the absorbance change due to cinnamoyl-CoA formation catalyzed by medium-chain acyl-CoA dehydrogenase, the assay can be used to measure the activity of this enzyme in crude tissue homogenates. Specific activities of medium-chain acyl-CoA dehydrogenase determined by use of this assay in homogenates of rat liver, heart, and leukocytes were found to be 29, 68, and 2.1 mU/mg of protein, respectively.

A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS.

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.

Effects of diabetes mellitus on renal fatty acid activation and desaturation.

We report the first direct measurement of delta-6 desaturase and delta-9 desaturase (EC 1.3.99.3, acyl-CoA dehydrogenase) activities in the rat kidney. Crude renal cortical homogenates from alloxan-diabetic and from normal rats were assayed for delta-6 and delta-9 desaturase activities. The delta-6 desaturation pathway activity measured with 9,12-octadecadienoic acid (linoleic acid) as substrate was increased, while the delta-9 desaturation pathway measured with hexadecanoic acid (palmitic acid) as substrate was unchanged in diabetic renal cortex, suggesting that the two enzymes are regulated independently in this tissue. In contrast to the kidney, delta-6 desaturase pathway activity was unchanged and the delta-9 desaturase pathway activity was greatly depressed in diabetic liver. When exogenous long-chain acyl-CoA synthetase (EC 6.2.1.3; acid: CoA ligase, AMP-forming) was added to the delta-6 desaturase assay system, the rate of delta-6 desaturation in normal kidney increased to a rate similar to that found in diabetic kidney; rates in diabetic extracts were unchanged. These results suggest that the rate of fatty acid substrate activation to the coenzyme A ester limits the rate of delta-6 desaturation in normal renal cortex. These results also suggest that the rate of fatty acid activation by long-chain acyl-CoA synthetase activity is increased in diabetic renal cortex. Direct measurement of the activity of long-chain acyl-CoA synthetase demonstrated that its activity was indeed increased significantly in the renal cortex of diabetic rats.

Fluorometric assay of acyl-CoA dehydrogenases in normal and mutant human fibroblasts.

A fluorimetric, ETF-linked procedure to determine activities of acyl-CoA dehydrogenase in cultured human fibroblasts is described. The assay readily distinguishes between cell lines deficient in medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and controls, and may allow for the diagnosis of heterozygous carriers of these disorders. The method has been made feasible with the development of rapid and efficient procedures to isolate ETF, and offers several advantages over procedures that are currently employed.

Fatty acyl-CoA dehydrogenase deficiency: enzyme measurement and studies on alternative metabolism.

Fatty acyl-CoA dehydrogenase deficiencies are defined as disorders of the metabolism of straight chain acyl-CoA esters at the level of short chain acyl-CoA, general (medium chain) acyl-CoA and long chain acyl-CoA dehydrogenases. Patients with proven or indicated defects in either general (medium chain) or long chain acyl-CoA dehydrogenase have been reported. In recent years assays for the enzymatic diagnosis in cells, especially cultured skin fibroblasts, from such patients have been developed. The different methods are reviewed. The urinary excretion profile of organic acids from patients with fatty acyl-CoA dehydrogenase deficiencies are characterized by the presence of different compounds originating from the primary accumulated acyl-CoA ester(s). The most important biochemical processes involved in the formation of these compounds are glycine conjugation and omega/omega-1 oxidation. The biochemistry of these pathways is discussed and the knowledge gained from in vitro and in vivo studies is used to explain the excretion pattern in some of the patients with general (medium chain) acyl-CoA dehydrogenase deficiency.