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Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD(-/-)) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD(-/-) mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD(-/-) mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD(-/-) mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD(-/-) mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

Recent genome-wide association studies of the adult human metabolome have identified genetic variants associated with relative levels of several acylcarnitines, which are important clinical correlates for chronic conditions such as type 2 diabetes and obesity. We have previously shown that these same metabolite levels are highly heritable at birth; however, no studies to our knowledge have examined genetic associations with these metabolites measured at birth. Here, we examine, in 743 newborns, 58 single nucleotide polymorphisms (SNPs) in 11 candidate genes previously associated with differing relative levels of short-chain acylcarnitines in adults. Six SNPs (rs2066938, rs3916, rs3794215, rs555404, rs558314, rs1799958) in the short-chain acyl-CoA dehydrogenase gene ( ACADS ) were associated with neonatal C4 levels. Most significant was the G allele of rs2066938, which was associated with significantly higher levels of C4 ( P  = 1.5 × 10 −29 ). This SNP explains 25 % of the variation in neonatal C4 levels, which is similar to the variation previously reported in adult C4 levels. There were also significant ( P  < 1 × 10 −4 ) associations between neonatal levels of C5-OH and SNPs in the solute carrier family 22 genes ( SLC22A4 and SLC22A5 ) and the 3-methylcrotonyl-CoA carboxylase 1 gene ( MCCC1 ). We have replicated, in newborns, SNP associations between metabolic traits and the ACADS and SLC22A4 genes observed in adults. This research has important implications not only for the identification of rare inborn errors of metabolism but also for personalized medicine and early detection of later life risks for chronic conditions.

The small heterodimer partner (SHP) regulates fatty acid oxidation and lipogenesis in the liver by regulating peroxisome proliferator-activated receptor (PPAR) γ expression. SHP is also abundantly expressed in the myocardium. We investigated the effect of SHP expression on myocardia assessing not only heart structure and function but also lipid metabolism and related gene expression in a SHP deletion animal model. Transcriptional profiling with a microarray revealed that genes participating in cell growth, cytokine signalling, phospholipid metabolism, and extracellular matrix are up-regulated in the myocardia of SHP knockout (KO) mice compared to those of wild-type (WT) mice (nominal p value < 0.05). Consistent with these gene expression changes, the left ventricular masses of SHP KO mice were significantly higher than WT mice (76.8 ± 20.5 mg vs. 52.8 ± 6.8 mg, P = 0.0093). After 12 weeks of high fat diet (HFD), SHP KO mice gained less weight and exhibited less elevation in serum-free fatty acid and less ectopic lipid accumulation in the myocardium than WT mice. According to microarray analysis, genes regulated by PPARγ1 and PPARα were down-regulated in myocardia of SHP KO mice compared to their expression in WT mice after HFD, suggesting that the reduction in lipid accumulation in the myocardium resulted from a decrease in lipogenesis regulated by PPARγ. We confirmed the reduced expression of PPARγ1 and PPARα target genes such as CD36, medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase by SHP KO after HFD.

Filamentous fungi can use a variety of fatty acids (FA) as sole carbon and energy sources. Aspergillus nidulans has been shown to possess both peroxisomal and mitochondrial β-oxidation pathways. In these studies, the major peroxisomal long chain fatty acyl coenzyme A oxidase AoxA was identified. AoxA was shown to be localised to peroxisomes and deletion of the aoxA gene leads to reduced growth on long chain FA, but not on short chain FA. AoxA is predicted to be part of the same peroxisomal β-oxidation pathway as the bifunctional protein FoxA. In addition, an aoxA(p)lacZ reporter gene construct is induced by short and long chain FA and the induction is dependent on the transcriptional regulators FarA, FarB and ScfA with FarA being required for the induction by short chain as well as long chain FA and FarB and ScfA being required for induction of aoxA by short chain FA. It is proposed that there are additional peroxisomal β-oxidation pathways in A. nidulans, which include fatty acyl-CoA dehydrogenases with a partially overlapping substrate range and include a pathway for short chain FA.

Medium-chain specific acyl-CoA dehydrogenase (MCAD) is a mitochondrial homotetrameric flavoprotein that catalyzes the first step in fatty acid beta-oxidation. MCAD deficiency arises from variants that either impair enzymatic activity or destabilize interactions between subunits, leading to protein aggregation. Standard enzymatic assays measure the overall MCAD activity but cannot differentiate between tetramers and other protein forms—critical for understanding the impact of pathogenic variants on structure destabilization. In this study, we adapted a native gel colorimetric assay to quantify the activity of MCAD tetramers separately from other protein forms, providing novel insights into how pathogenic variants affect MCAD structure and function. The assay showed a linear correlation between protein amount and enzymatic activity for octanoyl-CoA, a physiological MCAD substrate. Applying this method to clinically relevant MCAD variants allowed us to distinguish subtle differences in protein shape, enzymatic activity, and FAD content, offering profound implications for understanding the molecular basis of MCADD. This methodology can be extended to analyze variants in other acyl-CoA dehydrogenase family members—such as glutaryl-CoA, isovaleryl-CoA or short-chain fatty acyl-CoA dehydrogenases—that are implicated in disorders of fatty acid and amino acid metabolism.

Tumor-associated macrophages are increasingly viewed as a target of great relevance in the tumor microenvironment, because of their important role in cancer progression and metastasis. However, the endogenous regulatory mechanisms underlying tumor-associated macrophage differentiation remain largely unknown. Here, we report that caspase-1 promotes tumor-associated macrophage differentiation by cleaving peroxisome proliferator-activated receptor gamma (PPARγ) at Asp64, thus generating a 41 kDa fragment. This truncated PPARγ translocates to mitochondria, where it directly interacts with medium-chain acyl-CoA dehydrogenase (MCAD). This binding event attenuates MCAD activity and inhibits fatty acid oxidation, thereby leading to the accumulation of lipid droplets and promoting tumor-associated macrophage differentiation. Furthermore, the administration of caspase-1 inhibitors or the infusion of bone marrow-derived macrophages genetically engineered to overexpress murine MCAD markedly suppresses tumor growth. Therefore, targeting the caspase-1/PPARγ/MCAD pathway might be a promising therapeutic approach to prevent tumor progression. Tumor associated macrophages (TAMs) promote cancer progression. Here, the author show that caspase-1 promotes TAMs differentiation by attenuating medium-chain acyl-CoA dehydrogenase activity and that inhibition of this axis results in suppression of tumour growth in a transgenic mouse model of breast cancer.

Fatty acids are a primary source of carbon for Pseudomonas aeruginosa (PA) in the airways of people with cystic fibrosis (CF). Here, we use tandem mass-tag proteomics to analyse the protein expression profile of a CF clinical isolate grown on different fatty acids. Two fatty acyl-CoA dehydrogenases (designated FadE1 and FadE2) are strongly induced during growth on fatty acids. FadE1 displays a strong preference for long-chain acyl-CoAs, whereas FadE2 exclusively utilizes medium-chain acyl-CoAs. Structural analysis of the enzymes enables us to identify residues comprising the substrate selectivity filter in each. Engineering these residues enables us to invert the substrate specificity of each enzyme. Mutants in fadE1 displayed impaired virulence in an infection model, and decreased growth on long chain fatty acids. The unique features of the substrate binding pocket enable us to identify an inhibitor that is differentially active against FadE1 and FadE2. Wang et al show that the pathogen Pseudomonas aeruginosa employs two distinct acyl-CoA dehydrogenases for β-oxidation. They identify the substrate selectivity filter in each enzyme, and show that the enzymes are required for optimal virulence.

A combination of selenium (Se) with other trace element is associated with partially modulate fatty acid distribution as well as reduction of the body weight and feed efficiency. To investigate whether or not Se treatment has an impact on lipid metabolism, we examined the levels of lipid metabolism-related factors, including abdominal fat, adiponectin, cholesterol, very long chain dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) in 20-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats following sodium selenite treatment for 2 weeks. Herein, we observed that (a) Se treatment induced insulin-like effects by lowering the serum glucose level in rats; (b) Se-treated rats showed significance values decreases in abdominal fat mass, adipocyte size, and adiponectin, which are associated with lipid metabolism; (c) Se treatment led to reduced levels of cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; (d) fat tissue in Se-treated rats displayed significantly lower expression of adipocyte marker genes along with increased expression of VLCAD and MCAD; and (e) fatty liver formation and β-oxidation gene expression were both significantly reduced in liver tissue of Se-treated rats. Therefore, our results suggest that Se may induce inhibition of adipocyte hypertrophy and abdominal fat accumulation along with suppression of fatty liver formation by the differential regulation of the gene expression for fatty acid β-oxidation in the OLETF model.

Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal β-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid β-oxidation via two enzymes with an overlapping substrate repertoire.