Christoff Odendaal1$, Ligia Akemi Kiyuna1$, Madhulika Singh2$, Albert Gerding1,3, Miriam Langelaar-Makkinje1, Marianne van der Zwaag4, Asmara Drachman2, Vladimíra Cetkovská2, Gaby Liem Foeng Kioen2, Anne-Claire M.F. Martines1, Nicolette C. A. Huijkman1, Hein Schepers4, Bart van de Sluis1, Dirk-Jan Reijngoud1, Ody C.M. Sibon4, Amy C. Harms2, Thomas Hankemeier2, Barbara M. Bakker1

1Laboratory of Pediatrics, 3Departments of Laboratory Medicine and 4Biomedical Sciences of Cells and Systems, University of Groningen, UMCG, The Netherlands

2Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Leiden University, The Netherlands.

$ These authors contributed equally

Coenzyme A (CoA) is a vital cofactor that is involved in 8-10% of all metabolic reactions in human cells. In so-called ‘CoA Sequestration, Toxicity, and Redistribution’ (CASTOR) diseases, specific enzyme deficiencies lead to the accumulation of a corresponding CoA ester that is not efficiently metabolised. Common symptoms include acidosis, hypoglycaemia and hyperammonaemia. It has been proposed that a depletion of free, non-esterified CoA (CoASH) is underlying these symptoms, but there is limited direct evidence for this hypothesis. Here, we focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most prevalent fatty-acid oxidation (mFAO) disorder, in which patients accumulate medium-chain acylcarnitine esters. The aim of this study is to investigate if the loss of MCAD leads to the accumulation of medium-chain acyl-CoA esters, sequestration of CoASH, and remodelling of CoA metabolism.
In agreement with the CASTOR hypothesis, kinetic computational simulations of the mFAO pathway predicted elevated medium-chain C8-acyl-CoA levels and reduced CoASH and short-chain acyl-CoA esters in MCAD knockout (-KO) versus wild-type (WT) hepatocytes. Remarkably, the model predictions were replicated experimentally in MCAD-KO HepG2 cells. Moreover, long-chain acyl-CoA esters, upstream of the deficient enzyme, were also reduced in both the MCAD-KO computational model as well as in MCAD-KO HepG2 cells. According to the model, this may point to a limitation imposed by reduced CoASH, as the generation of new long-chain acyl-CoA esters and their entry into the mFAO pathway also require CoASH. Incorporation of 13C315N1- labelled pantothenate (vitamine B5, the precursor of CoA) showed no difference in the CoA biosynthesis rate between MCAD-KO and WT HepG2 cells. In MCAD-KO mice exposed to severe energetic stress (14h overnight fasting at room temperature followed by 4h fasting at 4°C), however, the total CoA concentration (free plus esterified) was increased. This was accompanied by the upregulation of genes involved in CoA biosynthesis (pantothenate kinases) and CoA release (carnitine acyltransferases and acyl-CoA thioesterases (ACOTs)). Computational simulations showed that the combined effect of elevated CoA and ACOT activity is an effective way to increase free CoASH, while relieving the excessive accumulation of C8-acyl-CoA.
To our knowledge, these results represent the first experimental evidence of the CASTOR mechanism in MCADD. Furthermore, using in vivo and computational models of MCADD, this study provides insights into a potential compensatory remodelling of CoA metabolism, activated under catabolic stress.