Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis

Abstract

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Peroxisomal L-bifunctional Protein Deficiency Causes Male-specific Kidney Hypertrophy and Proximal Tubular Injury in Mice

“…In the first model (A), such a toxic metabolite is produced by one of the CYP enzymes whose expression is known to be sexually dimorphic 21,22,34 . Dicarboxylic acids and eicosanoids are likely candidate metabolites as both undergo several cycles of peroxisomal β-oxidation after initial ω-oxidation by CYP enzymes 10,12,[40][41][42][43][44][45] . Unfortunately, our metabolomics analysis did not reveal a clear candidate toxic molecule, but the detection may have been obscured by the ongoing disease process.…”

“…A third cohort of mice was used for the orchiectomy experiment and consisted of 5 WT (average age before orchiectomy = 3.2 months) and 5 Ehhadh KO mice (average age before orchiectomy = 3.4 months). The metabolomics dataset was obtained from a fourth cohort that was generated for a previous study 10 , consisting of 7 WT (males, average age = 8.6 months) and 5 Ehhadh KO (males, average age = 8.6 month) mice.…”

“…Only genes with at least one count per million in at least 2 samples were considered. Differential gene expression analysis was conducted with the R package limma, as previously described 10 Differentially expressed genes (DEGs) were defined using an adjusted p value of < 0.05 with no logFC cut-off. The raw data and the count matrix for all genes can be accessed on the National Center for the Biotechnology Information database (GSE169676).…”

“…EHHADH is mainly expressed in liver and kidney. We and others have characterized a specific role of EHHADH in the hepatic metabolism of long-chain dicarboxylic acids (DCAs) [10][11][12] , but the role of EHHADH in the metabolic homeostasis of the kidney is currently unknown.…”

The peroxisomal transporter ABCD3 plays a major role in dicarboxylic fatty acid metabolism

“…Studies in fibroblasts from the ABCD3-deficient patient as well as phytol loading in an Abcd3 KO mouse model, have confirmed a role for ABCD3 in the transport of branched-chain fatty acids (Ferdinandusse et al 2015). Previous studies from our laboratory using CRISPR-Cas9 genome editing in HEK-293 cells confirmed an essential role for ABCD3 in the peroxisomal β-oxidation (FAO) of lauric, palmitic and hexadecanedioic acid (Violante et al 2019; Ranea-Robles et al 2021b). Even though an important role for ABCD3 in DCA metabolism has been hypothesized based on the available in vitro evidence, this has not yet been addressed in vivo .…”