The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

Acetyl-CoA synthetase (ACSS2) does not generate butyryl- and crotonyl-CoA.

Acetyl and other acyl groups from different short-chain fatty acids (SCFA) competitively modify histones at various lysine sites. To fully understand the functional significance of such histone acylation, a key epigenetic mechanism, it is crucial to characterize the cellular sources of the corresponding acyl-CoA molecules required for the lysine modification. Like acetate, SCFAs such as propionate, butyrate and crotonate are thought to be the substrates used to generate the corresponding acyl-CoAs by enzymes known as acyl-CoA synthetases. The acetyl-CoA synthetase, ACSS2, which produces acetyl-CoA from acetate in the nucleocytoplasmic compartment, has been proposed to also mediate the synthesis of acyl-CoAs such as butyryl- and crotonyl-CoA from the corresponding SCFAs. This idea is now widely accepted and is sparking new research projects. However, based on our direct in vitro experiments with purified or recombinant enzymes and structural considerations, we demonstrate that ACSS2 is unable to mediate the generation of non-acetyl acyl-CoAs like butyryl- and crotonyl-CoA. It is therefore essential to re-examine published data and corresponding discussions in the light of this new finding.

Direct effects of adipocyte lipolysis on AMPK through intracellular long-chain acyl-CoA signaling.

Long-chain acyl-CoAs (LC-acyl-CoAs) are important intermediary metabolites and are also thought to function as intracellular signaling molecules; however, the direct effects of LC-acyl-CoAs have been difficult to determine in real-time and dissociate from Protein Kinase A (PKA) signaling. Here, we examined the direct role of lipolysis in generating intracellular LC-acyl-CoAs and activating AMPK in white adipocytes by pharmacological activation of ABHD5 (also known as CGI-58), a lipase co-activator. Activation of lipolysis in 3T3-L1 adipocytes independent of PKA with synthetic ABHD5 ligands, resulted in greater activation of AMPK compared to receptor-mediated activation with isoproterenol, a ß-adrenergic receptor agonist. Importantly, the effect of pharmacological activation of ABHD5 on AMPK activation was blocked by inhibiting ATGL, the rate-limiting enzyme for triacylglycerol hydrolysis. Utilizing a novel FRET sensor to detect intracellular LC-acyl-CoAs, we demonstrate that stimulation of lipolysis in 3T3-L1 adipocytes increased the production of LC-acyl-CoAs, an effect which was blocked by inhibition of ATGL. Moreover, ATGL inhibition blocked AMPKß1 S108 phosphorylation, a site required for allosteric regulation. Increasing intracellular LC-acyl-CoAs by removal of BSA in the media and pharmacological inhibition of DGAT1 and 2 resulted in greater activation of AMPK. Finally, inhibiting LC-acyl-CoA generation reduced activation of AMPK; however, did not lower energy charge. Overall, results demonstrate that lipolysis in white adipocytes directly results in allosteric activation of AMPK through the generation of LC-acyl-CoAs.

A comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene.

Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant (ETFDH-RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588 C > T p.Arg530Cys and c.1589 G > C p.Arg530Pro, FLAD1-RRMADD). And we compared our patient together with 9 FLAD1-RRMADD cases from literature to 106 ETFDH-RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1-RRMADD and ETFDH-RRMADD were carried out. On muscle pathology, both FLAD1-RRMADD and ETFDH-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH-RRMADD, while fibers with faint COX staining were more common in FLAD1-RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1-RRMADD and ETFDH-RRMADD groups. Our data revealed that FLAD1-RRMADD (p.Arg530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH-RRMADD except for muscle pathological features.

Coenzyme A Thioester Intermediates as Platform Molecules in Cell-Free Chemical Biomanufacturing.

The in vitro synthesis of Coenzyme A (CoA)-thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell-free biosynthetic cascades. In this review, we have systematically cataloged known CoA-dependent enzyme reactions that have been successfully implemented in vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl-CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA-dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA-dependent in vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.

Expanding Pseudomonas taiwanensis VLB120's acyl-CoA portfolio: Propionate production in mineral salt medium.

As one of the main precursors, acetyl-CoA leads to the predominant production of even-chain products. From an industrial biotechnology perspective, extending the acyl-CoA portfolio of a cell factory is vital to producing industrial relevant odd-chain alcohols, acids, ketones and polyketides. The bioproduction of odd-chain molecules can be facilitated by incorporating propionyl-CoA into the metabolic network. The shortest pathway for propionyl-CoA production, which relies on succinyl-CoA catabolism encoded by the sleeping beauty mutase operon, was evaluated in Pseudomonas taiwanensis VLB120. A single genomic copy of the sleeping beauty mutase genes scpA, argK and scpB combined with the deletion of the methylcitrate synthase PVLB_08385 was sufficient to observe propionyl-CoA accumulation in this Pseudomonas. The chassis' capability for odd-chain product synthesis was assessed by expressing an acyl-CoA hydrolase, which enabled propionate synthesis. Three fed-batch strategies during bioreactor fermentations were benchmarked for propionate production, in which a maximal propionate titre of 2.8 g L-1 was achieved. Considering that the fermentations were carried out in mineral salt medium under aerobic conditions and that a single genome copy drove propionyl-CoA production, this result highlights the potential of Pseudomonas to produce propionyl-CoA derived, odd-chain products.

An optimized fluorescent biosensor for monitoring long-chain fatty acyl-CoAs metabolism in vivo.

Long-chain fatty acyl-CoAs (LCACoAs) are intermediates in lipid metabolism that exert a wide range of cellular functions. However, our knowledge about the subcellular distribution and regulatory impacts of LCACoAs is limited by a lack of methods for detecting LCACoAs in living cells and tissues. Here, we report our development of LACSerHR, a genetically encoded fluorescent biosensor that enables precise measurement of subtle fluctuations in the levels of endogenous LCACoAs in vivo. LACSerHR significantly improve the fluorescent brightness and analyte affinity, in vitro and in vivo testing showcased LACSerHR's large dynamic range. We demonstrate LACSerHR's capacity for real-time evaluation of LCACoA levels in specific subcellular compartments, for example in response to disruption of ACSL enzyme function in HEK293T cells. Moreover, we show the application of LACSerHR for sensitive measurement of elevated LCACoA levels in the livers of mouse models for two common metabolic diseases (NAFLD and type 2 diabetes). Thus, our LACSerHR sensor is a powerful, broadly applicable tool for studying LCACoAs metabolism and disease.

Chemoselective Esterification of Natural and Prebiotic 1,2-Amino Alcohol Amphiphiles in Water.

In cells, a vast number of membrane lipids are formed by the enzymatic O-acylation of polar head groups with acylating agents such as fatty acyl-CoAs. Although such ester-containing lipids appear to be a requirement for life on earth, it is unclear if similar types of lipids could have spontaneously formed in the absence of enzymatic machinery at the origin of life. There are few examples of enzyme-free esterification of amphiphiles in water and none that can occur in water at physiological pH using biochemically relevant acylating agents. Here we report the unexpected chemoselective O-acylation of 1,2-amino alcohol amphiphiles in water directed by Cu(II) and several other transition metal ions. In buffers containing Cu(II) ions, mixing biological 1,2-amino alcohol amphiphiles such as sphingosylphosphorylcholine with biochemically relevant acylating agents, namely, acyl adenylates and acyl-CoAs, leads to the formation of the O-acylation product with high selectivity. The resulting O-acylated sphingolipids self-assemble into vesicles with markedly different biophysical properties than those formed from their N-acyl counterparts. We also demonstrate that Cu(II) can direct the O-acylation of alternative 1,2-amino alcohols, including prebiotically relevant 1,2-amino alcohol amphiphiles, suggesting that simple mechanisms for aqueous esterification may have been prevalent on earth before the evolution of enzymes.

An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling.

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

A distinct Acyl-CoA binding protein (ACBP6) shapes tissue plasticity during nutrient adaptation in Drosophila.

Nutrient availability is a major selective force in the evolution of metazoa, and thus plasticity in tissue function and morphology is shaped by adaptive responses to nutrient changes. Utilizing Drosophila, we reveal that distinct calibration of acyl-CoA metabolism, mediated by Acbp6 (Acyl-CoA binding-protein 6), is critical for nutrient-dependent tissue plasticity. Drosophila Acbp6, which arose by evolutionary duplication and binds acyl-CoA to tune acetyl-CoA metabolism, is required for intestinal resizing after nutrient deprivation through activating intestinal stem cell proliferation from quiescence. Disruption of acyl-CoA metabolism by Acbp6 attenuation drives aberrant 'switching' of metabolic networks in intestinal enterocytes during nutrient adaptation, impairing acetyl-CoA metabolism and acetylation amid intestinal resizing. We also identified STAT92e, whose function is influenced by acetyl-CoA levels, as a key regulator of acyl-CoA and nutrient-dependent changes in stem cell activation. These findings define a regulatory mechanism, shaped by acyl-CoA metabolism, that adjusts proliferative homeostasis to coordinately regulate tissue plasticity during nutrient adaptation.

Quantitative Analysis of Acetyl-CoA, Malonyl-CoA, and Succinyl-CoA in Myocytes.

Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.

Conformational Tuning Shapes the Balance between Functional Promiscuity and Specialization in Paralogous Plasmodium Acyl-CoA Binding Proteins.

Paralogous proteins confer enhanced fitness to organisms via complex sequence-conformation codes that shape functional divergence, specialization, or promiscuity. Here, we dissect the underlying mechanism of promiscuous binding versus partial subfunctionalization in paralogues by studying structurally identical acyl-CoA binding proteins (ACBPs) from Plasmodium falciparum that serve as promising drug targets due to their high expression during the protozoan proliferative phase. Combining spectroscopic measurements, solution NMR, SPR, and simulations on two of the paralogues, A16 and A749, we show that minor sequence differences shape nearly every local and global conformational feature. A749 displays a broader and heterogeneous native ensemble, weaker thermodynamic coupling and cooperativity, enhanced fluctuations, and a larger binding pocket volume compared to A16. Site-specific tryptophan probes signal a graded reduction in the sampling of substates in the holo form, which is particularly apparent in A749. The paralogues exhibit a spectrum of binding affinities to different acyl-CoAs with A749, the more promiscuous and hence the likely ancestor, binding 1000-fold stronger to lauroyl-CoA under physiological conditions. We thus demonstrate how minor sequence changes modulate the extent of long-range interactions and dynamics, effectively contributing to the molecular evolution of contrasting functional repertoires in paralogues.

The Physiological and Pathological Role of Acyl-CoA Oxidation.

Fatty acid metabolism, including ß-oxidation (ßOX), plays an important role in human physiology and pathology. ßOX is an essential process in the energy metabolism of most human cells. Moreover, ßOX is also the source of acetyl-CoA, the substrate for (a) ketone bodies synthesis, (b) cholesterol synthesis, (c) phase II detoxication, (d) protein acetylation, and (d) the synthesis of many other compounds, including N-acetylglutamate-an important regulator of urea synthesis. This review describes the current knowledge on the importance of the mitochondrial and peroxisomal ßOX in various organs, including the liver, heart, kidney, lung, gastrointestinal tract, peripheral white blood cells, and other cells. In addition, the diseases associated with a disturbance of fatty acid oxidation (FAO) in the liver, heart, kidney, lung, alimentary tract, and other organs or cells are presented. Special attention was paid to abnormalities of FAO in cancer cells and the diseases caused by mutations in gene-encoding enzymes involved in FAO. Finally, issues related to α- and ω- fatty acid oxidation are discussed.

Multi-Method Quantification of Acetyl-Coenzyme A and Further Acyl-Coenzyme A Species in Normal and Ischemic Rat Liver.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.

Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism.

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

The Role of Acyl-CoA ß-Oxidation in Brain Metabolism and Neurodegenerative Diseases.

This review highlights the complex role of fatty acid ß-oxidation in brain metabolism. It demonstrates the fundamental importance of fatty acid degradation as a fuel in energy balance and as an essential component in lipid homeostasis, brain aging, and neurodegenerative disorders.

Enzymes of the crotonase superfamily: Diverse assembly and diverse function.

The crotonase fold is generated by a framework of four repeats of a ßßα-unit, extended by two helical regions. The active site of crotonase superfamily (CS) enzymes is located at the N-terminal end of the helix of the third repeat, typically being covered by a C-terminal helix. A major subset of CS-enzymes catalyzes acyl-CoA-dependent reactions, allowing for a diverse range of acyl-tail modifications. Most of these enzymes occur as trimers or hexamers (dimers of trimers), but monomeric forms are also observed. A common feature of the active sites of CS-enzymes is an oxyanion hole, formed by two peptide-NH hydrogen bond donors, which stabilises the negatively charged thioester oxygen atom of the reaction intermediate. Structural properties and possible use of these enzymes for biotechnological applications are discussed.

Inhibitory Investigations of Acyl-CoA Derivatives against Human Lipoxygenase Isozymes.

Lipid metabolism is a complex process crucial for energy production resulting in high levels of acyl-coenzyme A (acyl-CoA) molecules in the cell. Acyl-CoAs have also been implicated in inflammation, which could be possibly linked to lipoxygenase (LOX) biochemistry by the observation that an acyl-CoA was bound to human platelet 12-lipoxygenase via cryo-EM. Given that LOX isozymes play a pivotal role in inflammation, a more thorough investigation of the inhibitory effects of acyl-CoAs on lipoxygenase isozymes was judged to be warranted. Subsequently, it was determined that C18 acyl-CoA derivatives were the most potent against h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs were more potent against human 5-LOX. Specifically, oleoyl-CoA (18:1) was most potent against h12-LOX (IC50 = 32 µM) and h15-LOX-2 (IC50 = 0.62 µM), stearoyl-CoA against h15-LOX-1 (IC50 = 4.2 µM), and palmitoleoyl-CoA against h5-LOX (IC50 = 2.0 µM). The inhibition of h15-LOX-2 by oleoyl-CoA was further determined to be allosteric inhibition with a Ki of 82 +/- 70 nM, an α of 3.2 +/- 1, a ß of 0.30 +/- 0.07, and a ß/α = 0.09. Interestingly, linoleoyl-CoA (18:2) was a weak inhibitor against h5-LOX, h12-LOX, and h15-LOX-1 but a rapid substrate for h15-LOX-1, with comparable kinetic rates to free linoleic acid (kcat = 7.5 +/- 0.4 s-1, kcat/KM = 0.62 +/- 0.1 µM-1s-1). Additionally, it was determined that methylated fatty acids were not substrates but rather weak inhibitors. These findings imply a greater role for acyl-CoAs in the regulation of LOX activity in the cell, either through inhibition of novel oxylipin species or as a novel source of oxylipin-CoAs.

Development of a fluorescent-labeled trapping reagent to evaluate the risk posed by acyl-CoA conjugates.

Although acyl-CoA conjugates are known to have higher reactivity than acyl glucuronides, few studies have been conducted to evaluate the risk of the conjugates. In the present study, we aimed to develop a trapping assay for acyl-CoA conjugates using trapping reagents we have developed previously. It was revealed that Cys-Dan, which has both a thiol and an amino group, was the most effective in forming stable adducts containing an amide bond after intramolecular acyl migration. Additionally, we also developed a hepatocyte-based trapping assay in the present study to overcome the shortcomings of liver microsomes. Although liver microsomes are commonly used as enzyme sources in trapping assays, they lack some of the enzymes required for drug metabolism and detoxification systems. In human hepatocytes, our three trapping reagents, CysGlu-Dan, Dap-Dan and Cys-Dan, captured CYP-dependent reactive metabolites, reactive acyl glucuronides, and reactive acyl-CoA conjugates, respectively. The work suggests that the trapping assay with the reagents in hepatocytes is useful to evaluate the risk of reactive metabolites in drug discovery.

Role of 2-hydroxy acyl-CoA lyase HACL2 in odd-chain fatty acid production via α-oxidation in vivo.

Although most fatty acids (FAs) are even chain, certain tissues, including brain, contain relatively large quantities of odd-chain FAs in their sphingolipids. One of the pathways producing odd-chain FAs is the α-oxidation of 2-hydroxy (2-OH) FAs, where 2-OH acyl-CoA lyases (HACL1 and HACL2) catalyze the key cleavage reaction. However, the contribution of each HACL to odd-chain FA production in vivo remains unknown. Here, we found that HACL2 and HACL1 play major roles in the α-oxidation of 2-OH FAs (especially very-long-chain types) and 3-methyl FAs (other α-oxidation substrates), respectively, using ectopic expression systems of human HACL2 and HACL1 in yeast and analyzing Hacl1 and/or Hacl2 knockout (KO) CHO-K1 cells. We then generated Hacl2 KO mice and measured the quantities of odd-chain and 2-OH lipids (free FAs and sphingolipids [ceramides, sphingomyelins, and monohexosylceramides]) in 17 tissues. We observed fewer odd-chain lipids and more 2-OH lipids in many tissues of Hacl2 KO mice than in wild-type mice, and of these differences the reductions were most prominent for odd-chain monohexosylceramides in the brain and ceramides in the stomach. These results indicate that HACL2-involved α-oxidation of 2-OH FAs is mainly responsible for odd-chain FA production in the brain and stomach.