Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD?/?) Mouse to Compensate a Defective Fatty Acid β-Oxidation

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD^−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD^−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD^−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD^−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD^−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.     

Strategies for Correcting Very Long Chain Acyl-CoA Dehydrogenase Deficiency

Very long acyl-CoA dehydrogenase (VLCAD) deficiency is a genetic pediatric disorder presenting with a spectrum of phenotypes that remains for the most part untreatable. Here, we present a novel strategy for the correction of VLCAD deficiency by increasing mutant VLCAD enzymatic activity. Treatment of VLCAD-deficient fibroblasts, which express distinct mutant VLCAD protein and exhibit deficient fatty acid β-oxidation, with S-nitroso-N-acetylcysteine induced site-specific S-nitrosylation of VLCAD mutants at cysteine residue 237. Cysteine 237 S-nitrosylation was associated with an 8–17-fold increase in VLCAD-specific activity and concomitant correction of acylcarnitine profile and β-oxidation capacity, two hallmarks of the disorder. Overall, this study provides biochemical evidence for a potential therapeutic modality to correct β-oxidation deficiencies.     

Skeletal muscle carnitine metabolism in patients with unilateral peripheral arterial disease.

Patients with peripheral arterial disease (PAD) have abnormalities of carnitine metabolism that may contribute to their functional impairment. To test the hypothesis that muscle acylcarnitine generation (intermediates in oxidative metabolism) in patients with PAD provides a marker of the muscle dysfunction, 10 patients with unilateral PAD and 6 age-matched control subjects were studied at rest, and the patients were studied during exercise. At rest, biopsies of the gastrocnemius muscle in the patients' nonsymptomatic leg revealed a normal carnitine pool and lactate content compared with control subjects. In contrast, the patients' diseased leg had higher contents of lactate and long-chain acylcarnitines than controls. The muscle short-chain acylcarnitine content in the patients' diseased leg at rest was inversely correlated with peak exercise performance (r = -0.75, P less than 0.05). With graded treadmill exercise, only patients who exceeded their individual lactate threshold had an increase in muscle short-chain acylcarnitine content in the nonsymptomatic leg, which was identical to the muscle carnitine response in normal subjects. In the patients' diseased leg, muscle short-chain acylcarnitine content increased with exercise from 440 +/- 130 to 900 +/- 200 (SE) nmol/g (P less than 0.05). In contrast to the nonsymptomatic leg, there was no increase in muscle lactate content in the diseased leg with exercise, and the change in muscle carnitine metabolism was correlated with exercise duration (r = 0.82, P less than 0.01) and not with the lactate threshold. We conclude that energy metabolism in ischemic muscle of patients with PAD is characterized by the accumulation of acylcarnitines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice

Patients with mutations in the mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) gene are at risk for cardiomyopathy, myocardial dysfunction, ventricular tachycardia (VT), and sudden cardiac death. The mechanism is not known. Here we report a novel mechanism of VT in mice lacking VLCAD (VLCAD(-/-)). These mice exhibited polymorphic VT and increased incidence of VT after isoproterenol infusion. Polymorphic VT was induced in 10 out of 12 VLCAD(-/-) mice (83%) when isoproterenol was used. One out of 10 VLCAD(-/-) mice with polymorphic VT had VT with the typical bidirectional morphology. At the molecular level, VLCAD(-/-) cardiomyocytes showed increased levels of cardiac ryanodine receptor 2, phospholamban, and calsequestrin with increased [(3)H]ryanodine binding in heart microsomes. At the single cardiomyocyte level, VLCAD(-/-) cardiomyocytes showed significant increase in diastolic indo 1 and fura 2 fluorescence, with increased Ca(2+) transient amplitude. These changes were associated with altered Ca(2+) dynamics, to include: faster sarcomere contraction, larger time derivative of the upstroke, and shorter time-to-minimum sarcomere length compared with VLCAD(+/+) control cells. The L-type Ca(2+) current characteristics were not different under voltage-clamp conditions in the two VLCAD genotypes. Sarcoplasmic reticulum Ca(2+) load measured as normalized integrated Na(+)/Ca(2+) exchange current after rapid caffeine application was increased by 48% in VLCAD(-/-) cells. We conclude that intracellular Ca(2+) handling represents a possible molecular mechanism of arrhythmias in mice and perhaps in VLCAD-deficient humans.     

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-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 μM). 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.     

Skeletal muscle lipid metabolism with obesity

The objectives of this study were to 1). examine skeletal muscle fatty acid oxidation in individuals with varying degrees of adiposity and 2). determine the relationship between skeletal muscle fatty acid oxidation and the accumulation of long-chain fatty acyl-CoAs. Muscle was obtained from normal-weight [n = 8; body mass index (BMI) 23.8 +/- 0.58 kg/m(2)], overweight/obese (n = 8; BMI 30.2 +/- 0.81 kg/m(2)), and extremely obese (n = 8; BMI 53.8 +/- 3.5 kg/m(2)) females undergoing abdominal surgery. Skeletal muscle fatty acid oxidation was assessed in intact muscle strips. Long-chain fatty acyl-CoA concentrations were measured in a separate portion of the same muscle tissue in which fatty acid oxidation was determined. Palmitate oxidation was 58 and 83% lower in skeletal muscle from extremely obese (44.9 +/- 5.2 nmol x g(-1) x h(-1)) patients compared with normal-weight (71.0 +/- 5.0 nmol x g(-1) x h(-1)) and overweight/obese (82.2 +/- 8.7 nmol x g(-1) x h(-1)) patients, respectively. Palmitate oxidation was negatively (R = -0.44, P = 0.003) associated with BMI. Long-chain fatty acyl-CoA content was higher in both the overweight/obese and extremely obese patients compared with normal-weight patients, despite significantly lower fatty acid oxidation only in the extremely obese. No associations were observed between long-chain fatty acyl-CoA content and palmitate oxidation. These data suggest that there is a defect in skeletal muscle fatty acid oxidation with extreme obesity but not overweight/obesity and that the accumulation of intramyocellular long-chain fatty acyl-CoAs is not solely a result of reduced fatty acid oxidation.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

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 β-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.     

Plasma acylcarnitines inadequately reflect tissue acylcarnitine metabolism

Acylcarnitines have been linked to obesity-induced insulin resistance. However the majority of these studies have focused on acylcarnitines in plasma. It is currently unclear to what extent plasma levels of acylcarnitines reflect tissue acylcarnitine metabolism. We investigated the correlation of plasma acylcarnitine levels with selected tissue acylcarnitines as measured with tandem mass spectrometry, in both fed and fasted BALB/cJ (BALB) and C57BL/6N (Bl6) mice. Fasting affected acylcarnitine levels in all tissues. These changes varied substantially between the different tissue compartments. No significant correlations were found between plasma acylcarnitine species and their tissue counterparts in both mouse strains, with the exception of plasma C4OH-carnitine in BALB mice. We suggest that this lack of correlation is due to differences in acylcarnitine turnover rates between plasma and tissue compartments and the fact that the plasma acylcarnitine profile is a composition of acylcarnitines derived from different compartments. Therefore, plasma acylcarnitine levels do not reflect tissue levels and should be interpreted with caution. A focus on tissue acylcarnitine levels is warranted in metabolic studies.     

Effect of endurance training on lipid metabolism in women: a potential role for PPARalpha in the metabolic response to training

Endurance training increases fatty acid oxidation (FAO) and skeletal muscle oxidative capacity. However, the source of the additional fat and the mechanisms for increasing FAO capacity in muscle are not clear. We measured whole body and regional lipolytic activity and whole body and plasma FAO in six lean women during 90 min of bicycling exercise (50% pretraining peak O(2) consumption) before and after 12 wk of endurance training. We also assessed skeletal muscle content of peroxisome proliferator-activated receptor-alpha (PPARalpha) and its target proteins that regulate FAO [medium-chain and very long chain acyl-CoA dehydrogenase (MCAD and VLCAD)]. Despite a 25% increase in whole body FAO during exercise after training (P < 0.05), training did not alter regional adipose tissue lipolysis (abdominal: 0.56 +/- 0.26 and 0.57 +/- 0.10 micromol x 100 g(-1) x min(-1); femoral: 0.13 +/- 0.07 and 0.09 +/- 0.02 micromol x 100 g(-1) x min(-1)), whole body palmitate rate of appearance in plasma (168 +/- 18 and 150 +/- 25 micromol/min), and plasma FAO (554 +/- 61 and 601 +/- 45 micromol/min). However, training doubled the levels of muscle PPARalpha, MCAD, and VLCAD. We conclude that training increases the use of nonplasma fatty acids and may enhance skeletal muscle oxidative capacity by PPARalpha regulation of gene expression.     

Carnitine and acylcarnitine profiles in dried blood spots of patients with acute myocardial infarction

Earlier studies have suggested an important role of carnitine pathway in cardiovascular pathology. However, the redistribution of carnitine and acylcarnitine pools, as a result of altered carnitine metabolism, is not clearly known in patients with acute myocardial infarction (AMI). We compared the carnitine and acylcarnitine profiles of 65 AMI patients, including 26 ST-elevated myocardial infarction (STEMI) and 39 non-ST-elevated myocardial infarction (NSTEMI), 28 patients with chest pain and 154 normal controls. The levels of carnitine and acylcarnitines in the blood spots were determined using LC-MS/MS. Total and free carnitine levels were significantly higher in all the patient groups in the following order: STEMI > NSTEMI > chest pain. The levels of short- and medium-chain acylcarnitines were significantly higher in patient groups. Among the long-chain acylcarnitines, C14:2 and C16:1 levels were significantly increased in STEMI and NSTEMI. The ratio of free carnitine to short-chain or medium-chain acylcarnitines was significantly decreased in STEMI, NSTEMI and chest pain patients however a significant increase was observed in the ratio of carnitine to long-chain acylcarnitines in all the patient groups as compared to normal controls. In conclusion, alterations in carnitine and acylcarnitine levels in the blood of AMI patients indicate the possibility of impaired carnitine homeostasis in ischemic myocardium. The clinical implications of these findings for the risk screening or diagnosis and prognosis of AMI require additional follow-up studies on large number of patients. We also suggest that a dual-marker strategy using carnitine (longer plasma half-life) in combination with troponin (shorter plasma half-life) could be a more promising biomarker strategy in risk stratification of patients.     

Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency

Recent trials have reported the ability of triheptanoin to improve clinical outcomes for the severe symptoms associated with long-chain fatty acid oxidation disorders, including very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency.However, the milder myopathic symptoms are still challenging to treat satisfactorily. Myopathic pathogenesis is multifactorial, but oxidative stress is an important component. We have previously shown that metabolic stress increases the oxidative burden in VLCAD-deficient cell lines and can deplete the antioxidant glutathione (GSH).We investigated whether medium-chain fatty acids provide protection against GSH depletion during metabolic stress in VLCAD-deficient fibroblasts. To investigate the effect of differences in anaplerotic capacity, we included both even-(octanoate) and odd-numbered (heptanoate) medium-chain fatty acids. Overall, we show that modulation of the concentration of medium-chain fatty acids in culture media affects levels of GSH retained during metabolic stress in VLCAD-deficient cell lines but not in controls.Lowered glutamine concentration in the culture media during metabolic stress led to GSH depletion and decreased viability in VLCAD deficient cells, which could be rescued by both heptanoate and octanoate in a dose-dependent manner. Unlike GSH levels, the levels of total thiols increased after metabolic stress exposure, the size of this increase was not affected by differences in cell culture medium concentrations of glutamine, heptanoate or octanoate.Addition of a PPAR agonist further exacerbated stress-related GSH-depletion and viability loss, requiring higher concentrations of fatty acids to restore GSH levels and cell viability.Both odd- and even-numbered medium-chain fatty acids efficiently protect VLCADdeficient cells against metabolic stress-induced antioxidant depletion.     

Carnitine metabolism during exercise in patients with peripheral vascular disease

The distribution between carnitine and the acyl derivatives of carnitine reflects changes in the metabolic state of a variety of tissues. Patients with peripheral vascular disease (PVD) develop skeletal muscle ischemia with exertion. This impairment in oxidative metabolism during exercise may result in the generation of acylcarnitines. To test this hypothesis, 11 patients with PVD and 7 age-matched control subjects were evaluated with graded treadmill exercise. Subjects with PVD walked to maximal claudication pain at a peak O2 consumption (VO2) of 19.9 +/- 1.3 ml X kg-1 X min-1 (mean +/- SE). Control subjects were taken to a near-maximal work load at a VO2 of 31.3 +/- 1.0 ml X kg-1 X min-1. In patients with PVD, the plasma concentration of total acid-soluble, long-chain acylcarnitine and total carnitine was increased at peak exercise compared with resting values. Four minutes postexercise, the plasma short-chain acylcarnitine concentration was also increased. In control subjects taken to the higher work load, only the long-chain acylcarnitine concentration was increased at peak exercise. In patients with PVD, plasma short-chain acylcarnitine concentration at rest was negatively correlated with subsequent maximal walking time (r = -0.51, P less than 0.05). In conclusion, acylcarnitines increased in patients with PVD who walked to maximal claudication pain, whereas control subjects did not show equivalent changes even when taken to a higher work load. The relationship between short-chain acylcarnitine concentration at rest and subsequent exercise performance suggests that repeated episodes of ischemia may cause chronic accumulation of short-chain acylcarnitine in plasma in proportion to the severity of disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carnitine metabolism in the vitamin B-12-deficient rat.

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.     

A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

Background

Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.

Methodology

LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.

Principal Findings

VLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.

Conclusions

Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.     

In vitro probe acylcarnitine profiling assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry predicts severity of patients with glutaric aciduria type 2

Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency, MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase. The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M). In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate their clinical severity or not. Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines; C14, C12, C10 or C8 as well as C2, were extremely low. In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S. The ratio of C16/C14, C16/C12, or C16/C10, in the culture medium was significantly higher in MAD-S compared with that in MAD-M. Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S, while their effects were less pronounced in MAD-M. In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates. This strategy may be applicable for other metabolic disorders.     

Increase in mitochondrial content of long-chain acyl-CoA in brown adipose tissue during cold-acclimation

The mitochondrial content of long-chain acyl-CoA esters in the brown adipose tissue of guinea pigs increased 3.5-fold from a level of 92 +/- 17 pmol per mg protein (+/- S.E.; n = 7) in the control animals adapted at 22 degrees C to a new steady-state level of 328 +/- 20 pmol per mg protein (+/- S.E.; n = 46) after 10 days of cold-acclimation (5 degrees C). These low values of long-chain acyl-CoA species and the slow adaptive response for their increase do not support the proposal (Cannon, B., Sindin, U. and Romert, L. (1977) FEBS Lett. 4, 43-46) that the fatty acid CoA-esters have a physiological function in the regulation of the H+ (or OH-) permeability of the mitochondrial inner membrane. Experimental evidence is presented supporting the proposal that the long-chain acyl-CoA species are largely confined to the cytosolic side of the inner membrane. The activity of the adenine nucleotide translocase, as estimated at 25 degrees C in the reverse direction, was found to increase 5-fold upon depletion of the mitochondria of fatty acids (free and esterified) by preincubation with bovine serum albumin. The presence of potent inhibitors, i.e., long-chain acyl-CoA species, of adenine nucleotide translocation in brown adipose tissue of thermogenically active animals further supports the conclusion that ATP hydrolyzing mechanisms contribute insignificantly to long-term thermogenesis. The low values of long-chain acyl-CoA hydrolase (EC 3.1.2.1) activity, as measured in intact mitochondria and on a mitochondrial matrix fraction (i.e., 1.6 nmol X min-1 per mg protein), do not support the proposal that the hydrolase activity plays a significant role in the loose-coupling of brown adipose tissue mitochondria, either by a futile cycle mechanism or promoted by free fatty acid-induced uncoupling.     

Abnormal mitochondrial bioenergetics and heart rate dysfunction in mice lacking very-long-chain acyl-CoA dehydrogenase

Mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is associated with severe hypoglycemia, cardiac dysfunction, and sudden death in neonates and children. Sudden death is common, but the underlying mechanisms are not fully understood. We report on a mouse model of VLCAD deficiency with a phenotype induced by the stresses of fasting and cold, which includes hypoglycemia, hypothermia, and severe bradycardia. The administration of glucose did not rescue the mice under stress conditions, but rewarming alone consistently led to heart rate recovery. Brown adipose tissue (BAT) from the VLCAD-/- mice showed elevated levels of the uncoupling protein isoforms and peroxisome proliferator-activated receptor-alpha. Biochemical assessment of the VLCAD(/- mice BAT showed increased oxygen consumption, attributed to uncoupled respiration in the absence of stress. ADP-stimulated respiration was 23.05 (SD 4.17) and 68.24 (SD 6.3) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.001), and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated respiration was 35.9 (SD 3.6) and 49.3 (SD 9) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.20), but these rates were insufficient to protect them in the cold. We conclude that disturbed mitochondrial bioenergetics in BAT is a critical contributing factor for the cold sensitivity in VLCAD deficiency. Our observations provide insights into the possible mechanisms of stress-induced death in human newborns with abnormal fat metabolism and elucidate targeting of specific substrates for particular metabolic needs.     

Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-A resolution. The overall fold of the N-terminal approximately 400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an alpha-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12A and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial beta-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.     

Comparative biochemistry of beta-oxidation. An investigation into the abilities of isolated heart mitochondria of various animal species to oxidize long-chain fatty acids, including the C22:1 monoenes.

Rates of acylcarnitine oxidation by isolated heart mitochondria from various animal species were measured polarographically, and by using a spectrophotometric assay [see Osmundsen & Bremer (1977) Biochem. J. 164, 621-633]. Polarographic measurements do not give a correct guide to abilities to beta-oxidize very-long-chain acylcarnitines, in particular C22:1 fatty acylcarnitines. 2. No significant species differences were detected in the abilities to beta-oxidize various C22:1 fatty acylcarnitines. Significant species differences were, however, detected when rates of beta-oxidation were correlated with rates of respiration brought about by very-long-chain acylcarnitines. We concluded that some aspects of oxidative metabolism (possibly the oxidation of tricarboxylic acid-cycle intermediates) are inhibited by very-long-chain fatty acids in some species (e.g. the rat and the cat but not in others (e.g. the pig and the rabbit). 3. It is proposed that the pattern of variation of rates of oxidation of various acylcarnitines (as measured spectrophotometrically) of various chain lengths can be used as a guide to the chain-length specificities of the acyl-CoA dehydrogenases of beta-oxidation (EC 1.3.99.3).