183 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc acyl-CoA or acyl-carnitine levels are not surprising, since they are downstream of the deficient MCAD enzyme. C14, however, is upstream of MCAD, and at first sight it might therefore be expected to be elevated or unchanged. In the computational model the reduced C14-acyl-CoA could be attributed to a limitation of free CoA availability. Free CoA was sequestered into mediumchain acyl-CoAs, which strongly accumulated in MCADD. The decrease of free CoA, in turn limited the entry of new long-chain fatty acids into the mFAO pathway28,37 and thereby led to a reduced C14-acyl-CoA. Thus, our data provides experimental validation of this non-intuitive computational prediction. Together these data suggest that the maturation step into Mat-EHO organoids, accompanied by peroxisome and mitochondrial enrichment and a representative acylcarnitine profile, is a relevant step to study the pathophysiological mechanisms of MCADD. Adaptations of mitochondrial and peroxisomal β-oxidation and CoA metabolism in MCADD While peroxisomes have been reported to oxidize MCFAs29, we did not observe any regulation of peroxisomal enzymes involved in the import and oxidation of fatty acids in MCADD organoids, which suggests this pathway was not further activated. In addition to the oxidation of fatty acids, peroxisomes play a central role in the oxidation of dicarboxylic fatty acids (DCA)43. Under high fatty acid (FA) supply, such as fasting and mFAO disorders, excess FAs are channeled to DCA via ω-oxidation44,45. DCA accumulation in the liver can be toxic, causing inflammation, fibrosis and death46. In MCADD, patients in metabolic crisis have been reported to exhibit accumulation of medium-chain dicarboxylic fatty acids (MC-DCA) and high excretion in urine47,48. Excess DCAs can be metabolized by the peroxisomal β-oxidation43,49 or via the activity of nudix hydrolase 7 (NUDT7)41,42. The expression of peroxisomal ABCD3 transporter and LBP, both playing a major roles in DCA oxidation 50, was not regulated in the MCADD organoids. Recent animal studies suggest that Nudt7 contributes to the regulation of dicarboxylic fatty acid metabolism in the liver41,42. Male mice lacking Nudt7 (Nudt7 -/-) exposed to high fat diet showed accumulation of MC-DCA41. The authors proposed a major role of NUDT7 in the regulation of the levels of MC-DCA. The upregulation NUDT7 expression in the MCADD organoids may therefore play a role in preventing excessive accumulation of MC-DCA that otherwise could be toxic46.
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