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Deuterated water ((2)H(2)O) is widely used for measuring de novo lipogenesis (DNL). (2)H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid (2)H enrichment requires correcting. In mice, we measured the (2)H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-(2)H(7)]glucose to determine (2)H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid (2)H enrichments were compared with (13)C enrichment of the same sites from an equivalent amount of [U-(13)C(6)]glucose provided alongside the [U-(2)H(7)]glucose tracer. Transfer of glucose (2)H to fatty acid position 2 and methyl sites was low ((2)H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to (13)C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose (2)H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to (13)C, P < 10(−5) vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-(13)C(6)]glucose and (2)H(2)O had equivalent (2)H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water (2)H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive (2)H enrichment from (2)H(2)O prior to DNL. Under such conditions, (2)H enrichment of fatty acids from (2)H(2)O does not need correction.

Huntington&rsquo, s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the HTT gene. HD usually manifests in mid-life with loss of GABAergic projection neurons from the striatum accompanied by progressive atrophy of the putamen followed by other brain regions, but linkages between the genetics and neurodegeneration are not understood. We measured metabolic perturbations in HD-human brain in a case-control study, identifying pervasive lowering of vitamin B5, the obligatory precursor of coenzyme A (CoA) that is essential for normal intermediary metabolism. Cerebral pantothenate deficiency is a newly-identified metabolic defect in human HD that could potentially: (i) impair neuronal CoA biosynthesis, (ii) stimulate polyol-pathway activity, (iii) impair glycolysis and tricarboxylic acid cycle activity, and (iv) modify brain-urea metabolism. Pantothenate deficiency could lead to neurodegeneration/dementia in HD that might be preventable by treatment with vitamin B5.

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human per-oxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Inter-subunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Discovery Grant 9600 from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair Program (to P. C. L.) and by Grant BFU2012-36827 from the Ministerio de Ciencia e Innovación (MICINN) and Grant SGR2009-00327 from the Generalitat de Catalunya (to I. F.)

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00203-012-0829-7) contains supplementary material, which is available to authorized users.

Obesity has dramatically increased in prevalence, making it essential to understand its accompanying metabolic changes. Modeling diet-induced obesity in Drosophila melanogaster (fruit flies), we elucidated transcriptional and metabolic changes in w1118 flies on a high-fat diet (HFD). Mass spectrometry-based metabolomics revealed altered fatty acid, amino acid, and carbohydrate metabolism with HFD. Microarray analysis uncovered transcriptional changes in nitrogen metabolism, including CG9510, homolog of human argininosuccinate lyase (ASL). CG9510 knockdown in flies phenocopied traits observed with HFD, namely increased triglyceride levels and decreased cold tolerance. Restoration of CG9510 expression ameliorated observed negative consequences of HFD. Metabolomic analysis of CG9510 knockdown flies confirmed functional similarity to ASL, regulating the balance of carbon and nitrogen metabolism. In summary, we found that HFD suppresses CG9510 expression, a gene required for proper triglyceride storage and stress tolerance. These results draw an important link between regulation of amino acid metabolism and the response to diet-induced obesity. © 2013 The Authors.

Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism. © 2013 Society for Industrial Microbiology and Biotechnology., This work has been funded in part by the Chalmers Foundation, the Knut and Alice Wallenberg Foundation, and the European Research Council. C.S.A. is the recipient of an FPU predoctoral fellowship from the Spanish Ministerio de Educación. A. K. is a recipient of an Angpanneföreningens Forskningsstiftelse project grant.