ACOT13

ACOT13 (Them2) is a mitochondria-associated long-chain fatty acyl-CoA thioesterase that channels fatty acid metabolites to regulate hepatic lipid homeostasis, gluconeogenesis, and adaptive thermogenesis. It forms a homotetramer via a hotdog-fold domain and preferentially hydrolyzes myristoyl-CoA and palmitoyl-CoA, with its catalytic efficiency enhanced by physical interaction with the lipid transfer protein PC-TP (StarD2) (PMID:19405909). Genetic ablation in mice reduces hepatic fatty acid oxidation and gluconeogenesis, confers resistance to diet-induced steatosis, and enhances brown adipose tissue thermogenesis by increasing intracellular fatty acid availability for mitochondrial uncoupling (PMID:22345407, PMID:24072708, PMID:24732803). In skeletal muscle, ACOT13 catalytic activity and PC-TP interaction drive secretion of extracellular vesicles that promote hepatic lipid accumulation and insulin resistance, establishing an inter-organ signaling axis dependent on its thioesterase function (PMID:39369989).

1. 2009 High

   Establishing the biochemical identity of ACOT13 resolved what enzymatic activity this gene encodes and how it is regulated: it is a homotetrameric hotdog-fold thioesterase with preference for C14–C16 acyl-CoAs, stimulated by PC-TP and associated with mitochondria.

   Evidence In vitro steady-state kinetics, size-exclusion chromatography, thermal denaturation, subcellular fractionation, and co-incubation with StarD2/PC-TP using recombinant protein

   PMID:19405909

   Open questions at the time

   • No crystal structure of the mammalian protein–PC-TP complex to define the activation mechanism
   • In vivo relevance of substrate inhibition near CMC not addressed
   • Regulation by post-translational modifications unknown
2. 2012 High

   Knockout studies in mice demonstrated that ACOT13 is required in vivo for hepatic fatty acid–CoA hydrolysis, PPARα activation, and gluconeogenesis, and that its loss protects against diet-induced steatosis—linking the enzyme to whole-body metabolic regulation.

   Evidence Them2−/− mouse model with mitochondrial thioesterase assay, acyl-CoA/FFA quantification, hepatic glucose production measurement, and high-fat diet challenge

   PMID:22345407

   Open questions at the time

   • Whether hepatic phenotypes are cell-autonomous versus secondary to systemic metabolic changes
   • Contribution of individual acyl-CoA species to PPARα activation not dissected
   • Tissue-specific conditional knockouts not performed
3. 2012 Medium

   Crystallization of zebrafish THEM2 at 1.80 Å confirmed the hotdog-fold architecture and homotetrameric assembly, providing a structural framework for the thioesterase superfamily placement.

   Evidence Recombinant zebrafish THEM2 expression, Ni-affinity and gel-filtration chromatography, synchrotron X-ray crystallography

   PMID:23192039

   Open questions at the time

   • No mammalian structure solved
   • Substrate-bound or PC-TP co-crystal structure unavailable
   • Active-site catalytic mechanism not defined from this structure alone
4. 2013 High

   Demonstrating that ACOT13 suppresses brown adipose tissue thermogenesis expanded its role beyond the liver: Them2−/− BAT showed enhanced mitochondrial fatty acid utilization and thermogenic gene expression, establishing ACOT13 as a gatekeeper of intracellular fatty acid channeling toward heat production.

   Evidence Them2−/− mice under cold challenge, electron microscopy of BAT mitochondria, primary brown adipocyte O₂ consumption and triglyceride hydrolysis assays

   PMID:24072708

   Open questions at the time

   • Direct lipid substrates mediating thermogenic suppression not identified
   • Whether PC-TP interaction is required for the BAT phenotype not tested
   • Signaling intermediates between ACOT13 activity and UCP1 induction unknown
5. 2014 High

   Epistasis experiments in primary hepatocytes showed that PC-TP acts through ACOT13 to promote fatty acid oxidation and gluconeogenesis, while ACOT13 has an independent role in glucose oxidation—defining the functional hierarchy between these two partners.

   Evidence Primary hepatocytes from Them2−/− and Pctp−/− mice, pharmacological PC-TP inhibition, fatty acid oxidation, gluconeogenesis, and glucose oxidation assays

   PMID:24732803

   Open questions at the time

   • Mechanism by which PC-TP delivers substrate to ACOT13 at the molecular level unresolved
   • Whether the Them2-specific glucose oxidation role requires catalytic activity not tested
   • In vivo validation of epistatic relationship not performed with tissue-specific double knockouts
6. 2024 High

   Active-site mutagenesis and skeletal muscle-specific reconstitution revealed that ACOT13 catalytic activity (not merely PC-TP scaffolding) is essential for driving hepatic steatosis through extracellular vesicle–mediated inter-organ communication, establishing a non-cell-autonomous metabolic signaling axis.

   Evidence N50A/D65A catalytic-dead mutant, AAV-mediated muscle reconstitution in Them2−/− mice on HFD, conditioned medium and EV isolation from myotubes, hepatocyte lipid accumulation assay

   PMID:39369989

   Open questions at the time

   • Cargo composition of ACOT13-dependent extracellular vesicles not characterized
   • Hepatocyte receptor or uptake mechanism for these EVs unknown
   • Whether this inter-organ axis operates under physiological (non-HFD) conditions not determined

• Key unresolved questions include the identity of EV cargo mediating ACOT13-dependent inter-organ signaling, the structural basis of the ACOT13–PC-TP complex in mammals, and whether ACOT13 catalytic versus scaffolding functions are separable in brown adipose tissue thermogenesis.
• No mammalian co-crystal structure of ACOT13–PC-TP
• EV cargo and hepatocyte receptor undefined
• Tissue-specific conditional knockout studies across all relevant tissues lacking