Arno van Cruchten

The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl-CoA esters

Peroxisomes play a major role in human cellular lipid metabolism, including the β‐oxidation of fatty acids. The most frequent peroxisomal disorder is X‐linked adrenoleukodystrophy (X‐ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP‐binding‐cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X‐ALD is the accumulation of very long‐chain fatty acids (VLCFAs), due to an im paired peroxisomal β‐oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid β‐oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl‐CoA esters or as long‐chain acyl‐CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxalp and Pxa2p that form a heterodimeric complex in the perox isomal membrane. Using different strategies, including the analysis of intracellular acyl‐CoA esters by tandem‐MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl‐CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Δ mutant can be rescued, at least par tially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X‐linked adrenoleukodystrophy. Our data indicate that ALDP can function as a ho modimer and is involved in the transport of acyl‐CoA esters across the peroxisomal membrane.— van Roer mund, C. W. T., Visser, W. F., IJlst, L., van Cruchten, A., Boek, M., Kulik, W., Waterham, H. R., Wanders, R. J. A. The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl–CoA esters. FASEB J. 22, 4201–4208 (2008)

The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy.

X‐linked adrenoleukodystrophy (X‐ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X‐ALD is characterized by the accumulation of very long‐chain fatty acids (VLCFA; ≥C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X‐ALD. ALDP transports VLCFacyl‐CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal β‐oxidation of VLCFA but also raises cytosolic levels of VLCFacyl‐CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very‐long‐chain‐fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono‐unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X‐ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X‐ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X‐ALD.

New insights on the mechanisms of valproate-induced hyperammonemia: inhibition of hepatic N-acetylglutamate synthase activity by valproyl-CoA

BACKGROUND & AIMS Hyperammonemia is a frequent side-effect of valproic acid (VPA) therapy, which points to an imbalance between ammoniagenesis and ammonia disposal via the urea cycle. The impairment of this liver-specific metabolic pathway induced either by primary genetic defects or by secondary causes, namely associated with drugs administration, may result in accumulation of ammonia. To elucidate the mechanisms which underlie VPA-induced hyperammonemia, the aim of this study was to evaluate the effect of both VPA and its reactive intermediate, valproyl-CoA (VP-CoA), on the synthesis of N-acetylglutamate (NAG), a prime metabolite activator of the urea cycle. METHODS The amount of NAG in livers of rats treated with VPA was quantified by HPLC-MS/MS. The NAG synthase (NAGS) activity was evaluated in vitro in rat liver mitochondria, and the effect of both VPA and VP-CoA was characterized. RESULTS The present results clearly show that VP-CoA is a stronger inhibitor of NAGS activity in vitro than the parent drug VPA. The hepatic levels of NAG were significantly reduced in VPA-treated rats as compared with control tissues. CONCLUSIONS These data strongly suggest that the hyperammonemia observed in patients under VPA treatment may result from a direct inhibition of the NAGS activity by VP-CoA. The subsequent reduced availability of NAG will impair the flux through the urea cycle and compromise the major role of this pathway in ammonia detoxification.

Mitochondrial protein acetylation is driven by acetyl-CoA from fatty acid oxidation

Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC-MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.

Molecular Characterization of Iodotyrosine Dehalogenase Deficiency in Patients with Hypothyroidism

CONTEXT The recent cloning of the human iodotyrosine deiodinase (IYD) gene enables the investigation of iodotyrosine dehalogenase deficiency, a form a primary hypothyroidism resulting from iodine wasting, at the molecular level. OBJECTIVE In the current study, we identify the genetic basis of dehalogenase deficiency in a consanguineous family. RESULTS Using HPLC tandem mass spectrometry, we developed a rapid, selective, and sensitive assay to detect 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine in urine and cell culture medium. Two subjects from a presumed dehalogenase-deficient family showed elevated urinary 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine levels compared with 57 normal subjects without thyroid disease. Subsequent analysis of IYD revealed a homozygous missense mutation in exon 4 (c.658G>A p.Ala220Thr) that co-segregates with the clinical phenotype in the family. Functional characterization of the mutant iodotyrosine dehalogenase protein showed that the mutation completely abolishes dehalogenase enzymatic activity. One of the heterozygous carriers for the inactivating mutation recently presented with overt hypothyroidism indicating dominant inheritance with incomplete penetration. Screening of 100 control alleles identified one allele positive for this mutation, suggesting that the c.658G>A nucleotide substitution might be a functional single nucleotide polymorphism. CONCLUSIONS This study describes a functional mutation within IYD, demonstrating the molecular basis of the iodine wasting form of congenital hypothyroidism. This familial genetic defect shows a dominant pattern of inheritance with incomplete penetration.

Detection of nonsterol isoprenoids by HPLC–MS/MS

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C(18) HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0mumol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.

Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS.

The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis.

A sensitive mass spectrometry platform identifies metabolic changes of life history traits in C. elegans

Abnormal nutrient metabolism is a hallmark of aging, and the underlying genetic and nutritional framework is rapidly being uncovered, particularly using C. elegans as a model. However, the direct metabolic consequences of perturbations in life history of C. elegans remain to be clarified. Based on recent advances in the metabolomics field, we optimized and validated a sensitive mass spectrometry (MS) platform for identification of major metabolite classes in worms and applied it to study age and diet related changes. Using this platform that allowed detection of over 600 metabolites in a sample of 2500 worms, we observed marked changes in fatty acids, amino acids and phospholipids during worm life history, which were independent from the germ-line. Worms underwent a striking shift in lipid metabolism after early adulthood that was at least partly controlled by the metabolic regulator AAK-2/AMPK. Most amino acids peaked during development, except aspartic acid and glycine, which accumulated in aged worms. Dietary intervention also influenced worm metabolite profiles and the regulation was highly specific depending on the metabolite class. Altogether, these MS-based methods are powerful tools to perform worm metabolomics for aging and metabolism-oriented studies.

Comparative Study of Thymine and Uracil Metabolism in Healthy Persons and in a Patient with Dihydropyrimidine Dehydrogenase Deficiency

Dihydropyrimidine dehydrogenase (EC 1.3.1.2) deficiency has so far been reported in 8 patients, 7 of which were diagnosed in The Netherlands1–5. The patients did not exhibit a characteristic clinical picture, although a form of epilepsy was seen in half of the patients. The other 4 patients were below the age of 4 years at the time of diagnosis. Therefore, it cannot be excluded that epileptic symptoms will develop later on. All patients were-discovered by the increased excretion of thymine and uracil. In most of the cases also an elevated excretion of 5-hydroxymethyluracil (5-HMU) was present.

Cerebrospinal fluid in tuberculous meningitis exhibits only the L-enantiomer of lactic acid

BackgroundThe defining feature of the cerebrospinal fluid (CSF) collected from infants and children with tuberculous meningitis (TBM), derived from an earlier untargeted nuclear magnetic resonance (NMR) metabolomics study, was highly elevated lactic acid. Undetermined was the contribution from host response (L-lactic acid) or of microbial origin (D-lactic acid), which was set out to be determined in this study.MethodsIn this follow-up study, we used targeted ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) to determine the ratio of the L and D enantiomers of lactic acid in these CSF samples.ResultsHere we report for the first time that the lactic acid observed in the CSF of confirmed TBM cases was in the L-form and solely a response from the host to the infection, with no contribution from any bacteria. The significance of elevated lactic acid in TBM appears to be that it is a crucial energy substrate, used preferentially over glucose by microglia, and exhibits neuroprotective capabilities.ConclusionThese results provide experimental evidence to support our conceptual astrocyte–microglia lactate shuttle model formulated from our previous NMR-based metabolomics study — highlighting the fact that lactic acid plays an important role in neuroinflammatory diseases such as TBM. Furthermore, this study reinforces our belief that the determination of enantiomers of metabolites corresponding to infectious diseases is of critical importance in substantiating the clinical significance of disease markers.