{"gene":"ACOT13","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2009,"finding":"ACOT13 (Them2) forms a stable homotetramer via a single hotdog fold domain and functions as a long-chain fatty acyl-CoA thioesterase, with lowest Km and highest kcat/Km for myristoyl-CoA and palmitoyl-CoA; substrate inhibition occurs near critical micellar concentrations. Interaction with StarD2/PC-TP increases the kcat of Them2, while phosphatidic acid/phosphatidylcholine vesicles decrease activity. Expression is mitochondria-associated and induced by PPARα activation.","method":"In vitro enzymatic assay (steady-state kinetics), size-exclusion chromatography (homotetramer determination), thermal denaturation, subcellular fractionation, co-incubation with StarD2/PC-TP","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro enzymatic assay with mutagenesis-equivalent substrate profiling, multiple orthogonal methods (kinetics, biophysics, fractionation)","pmids":["19405909"],"is_preprint":false},{"year":2012,"finding":"In Them2-/- mouse livers, mitochondrial thioesterase activity shows increased Km, fatty acyl-CoA concentrations rise by 28%, and free fatty acid concentrations fall by 23%, leading to reduced PPARα activation. Hepatic glucose production is decreased by 45% with reduced HNF4α expression. Them2-/- mice are resistant to high-fat diet-induced hepatic steatosis and increased glucose production, implicating Them2 in limiting β-oxidation and supporting gluconeogenesis via PC-TP interactions.","method":"Them2-/- mouse model, mitochondrial thioesterase activity assay, fatty acyl-CoA and free fatty acid quantification, hepatic glucose production measurement, PPARα and HNF4α expression analysis, high-fat diet challenge","journal":"FASEB Journal","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple defined metabolic readouts and mechanistic pathway placement","pmids":["22345407"],"is_preprint":false},{"year":2013,"finding":"In brown adipose tissue, Them2 suppresses adaptive thermogenesis: Them2-/- mice show reduced lipid droplets, altered mitochondrial ultrastructure, and increased thermogenic gene expression. Primary brown adipocytes from Them2-/- mice exhibit increased norepinephrine-mediated triglyceride hydrolysis, increased O2 consumption, and elevated thermogenic gene expression, indicating Them2 directly regulates intracellular fatty acid channeling to suppress heat production.","method":"Them2-/- mouse model, ambient temperature challenge (4–30°C), electron microscopy of BAT mitochondria, primary brown adipocyte culture, O2 consumption assay, triglyceride hydrolysis assay, thermogenic gene expression","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal cellular and molecular readouts in primary cells and in vivo","pmids":["24072708"],"is_preprint":false},{"year":2014,"finding":"Them2 and PC-TP interact to promote fatty acid oxidation and gluconeogenesis in hepatocytes under fasting-like conditions. Them2-/- and Pctp-/- primary hepatocytes each show decreased rates of fatty acid oxidation and gluconeogenesis. Chemical inhibition of PC-TP fails to reproduce these changes in Them2-/- hepatocytes, indicating PC-TP acts upstream of or through Them2. Additionally, glucose oxidation and lipogenesis under high glucose are decreased only in Them2-/- hepatocytes, revealing a Them2-specific role in glucose oxidation.","method":"Primary hepatocyte culture from Them2-/- and Pctp-/- mice, fatty acid oxidation assays, gluconeogenesis assays, glucose oxidation assays, pharmacological PC-TP inhibition","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO combined with pharmacological epistasis in primary cells with multiple metabolic readouts","pmids":["24732803"],"is_preprint":false},{"year":2012,"finding":"Zebrafish THEM2 (ortholog of ACOT13) was crystallized and X-ray diffraction data collected to 1.80 Å resolution, confirming the hotdog-fold thioesterase domain architecture and homotetrameric assembly consistent with the mammalian protein.","method":"Recombinant protein expression, Ni-affinity and gel-filtration chromatography, X-ray crystallography (synchrotron, 1.80 Å)","journal":"Acta Crystallographica Section F","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure determination, but from zebrafish ortholog; functional validation limited to crystallographic data","pmids":["23192039"],"is_preprint":false},{"year":2024,"finding":"Skeletal muscle Them2 promotes hepatic steatosis and insulin resistance through both its catalytic activity and interaction with PC-TP. Catalytic-dead mutant (N50A/D65A, maintaining homotetrameric structure and PC-TP binding) failed to promote high-fat diet-induced hepatic steatosis when restored in Them2-/- skeletal muscle via AAV. Conditioned medium and specifically secreted extracellular vesicles from WT myotubes (but not Them2-/- myotubes) promoted lipid accumulation in hepatocytes, dependent on Them2 catalytic activity and PC-TP interaction.","method":"Active-site mutagenesis (N50A/D65A), AAV-mediated skeletal muscle-specific reconstitution in Them2-/- mice, high-fat diet challenge, primary myotube-conditioned medium experiments, extracellular vesicle isolation, primary hepatocyte lipid accumulation assay, PC-TP pharmacological inhibition and genetic ablation","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — active-site mutagenesis combined with in vivo reconstitution and reductionist cell biology experiments","pmids":["39369989"],"is_preprint":false},{"year":2024,"finding":"ACOT13 overexpression in ADPKD cells (WT9-12) suppresses proliferation, induces cell cycle arrest, triggers apoptosis with increased cleaved caspase-3, reduces ATP production, and induces loss of mitochondrial membrane potential, indicating ACOT13 triggers mitochondria-mediated apoptosis in these cells.","method":"ACOT13 overexpression in WT9-12 cells, EdU staining, flow cytometry (cell cycle and apoptosis), cleaved caspase-3 Western blot, ATP production assay, mitochondrial membrane potential assay","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 — overexpression with multiple cellular readouts, but single lab, single cell line, no rescue/mutagenesis","pmids":["39172111"],"is_preprint":false},{"year":2026,"finding":"Under pathological conditions in nucleus pulposus cells, ACOT13 inhibits the AMPK/ACC signaling pathway, leading to disrupted fatty acid metabolism, mitochondrial dysfunction, and pyroptosis, thereby accelerating intervertebral disc degeneration.","method":"Single-cell sequencing and multi-omics analysis of clinical samples, GSEA pathway analysis, functional cell experiments with ACOT13 modulation","journal":"Journal of Nanobiotechnology","confidence":"Low","confidence_rationale":"Tier 3 — pathway placement inferred from omics and cell overexpression; limited mechanistic detail and mutagenesis absent","pmids":["41656235"],"is_preprint":false}],"current_model":"ACOT13 (Them2) is a mitochondria-associated homotetrameric hotdog-fold thioesterase that hydrolyzes long-chain fatty acyl-CoAs (preferring myristoyl-CoA and palmitoyl-CoA), and its catalytic activity together with its physical interaction with PC-TP (StarD2) regulates hepatic fatty acid oxidation, gluconeogenesis, and lipid homeostasis, while in skeletal muscle it promotes hepatic steatosis and insulin resistance via extracellular vesicle-mediated inter-organ communication, and in brown adipose tissue it suppresses adaptive thermogenesis by limiting intracellular fatty acid channeling."},"narrative":{"teleology":[{"year":2009,"claim":"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","pmids":["19405909"],"confidence":"High","gaps":["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"]},{"year":2012,"claim":"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","pmids":["22345407"],"confidence":"High","gaps":["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"]},{"year":2012,"claim":"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","pmids":["23192039"],"confidence":"Medium","gaps":["No mammalian structure solved","Substrate-bound or PC-TP co-crystal structure unavailable","Active-site catalytic mechanism not defined from this structure alone"]},{"year":2013,"claim":"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","pmids":["24072708"],"confidence":"High","gaps":["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"]},{"year":2014,"claim":"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","pmids":["24732803"],"confidence":"High","gaps":["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"]},{"year":2024,"claim":"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","pmids":["39369989"],"confidence":"High","gaps":["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"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Low","gaps":["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"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,5]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,2,3,5]}],"complexes":[],"partners":["PCTP"],"other_free_text":[]},"mechanistic_narrative":"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]."},"prefetch_data":{"uniprot":{"accession":"Q9NPJ3","full_name":"Acyl-coenzyme A thioesterase 13","aliases":["Hotdog-fold thioesterase superfamily member 2","Palmitoyl-CoA hydrolase","Thioesterase superfamily member 2","THEM2"],"length_aa":140,"mass_kda":15.0,"function":"Catalyzes the hydrolysis of acyl-CoAs into free fatty acids and coenzyme A (CoASH), regulating their respective intracellular levels (PubMed:16934754, PubMed:19170545). Has acyl-CoA thioesterase activity towards medium (C12) and long-chain (C18) fatty acyl-CoA substrates (By similarity) (PubMed:16934754, PubMed:19170545). Can also hydrolyze 3-hydroxyphenylacetyl-CoA and 3,4-dihydroxyphenylacetyl-CoA (in vitro) (By similarity) (PubMed:16934754, PubMed:19170545). May play a role in controlling adaptive thermogenesis (By similarity)","subcellular_location":"Cytoplasm, cytosol; Mitochondrion; Nucleus; Cytoplasm, cytoskeleton, spindle","url":"https://www.uniprot.org/uniprotkb/Q9NPJ3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACOT13","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACOT13","total_profiled":1310},"omim":[{"mim_id":"615652","title":"ACYL-CoA THIOESTERASE 13; ACOT13","url":"https://www.omim.org/entry/615652"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cell Junctions","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":28.6}],"url":"https://www.proteinatlas.org/search/ACOT13"},"hgnc":{"alias_symbol":["HT012"],"prev_symbol":["THEM2"]},"alphafold":{"accession":"Q9NPJ3","domains":[{"cath_id":"3.10.129.10","chopping":"2-136","consensus_level":"high","plddt":96.7116,"start":2,"end":136}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NPJ3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NPJ3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NPJ3-F1-predicted_aligned_error_v6.png","plddt_mean":96.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACOT13","jax_strain_url":"https://www.jax.org/strain/search?query=ACOT13"},"sequence":{"accession":"Q9NPJ3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NPJ3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NPJ3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NPJ3"}},"corpus_meta":[{"pmid":"22262880","id":"PMC_22262880","title":"Genetic variants of FOXP2 and KIAA0319/TTRAP/THEM2 locus are associated with altered brain activation in distinct language-related regions.","date":"2012","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/22262880","citation_count":126,"is_preprint":false},{"pmid":"22345407","id":"PMC_22345407","title":"Thioesterase superfamily member 2/acyl-CoA thioesterase 13 (Them2/Acot13) regulates hepatic lipid and glucose metabolism.","date":"2012","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/22345407","citation_count":61,"is_preprint":false},{"pmid":"31181634","id":"PMC_31181634","title":"gga-miRNA-18b-3p Inhibits Intramuscular Adipocytes Differentiation in Chicken by Targeting the ACOT13 Gene.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31181634","citation_count":46,"is_preprint":false},{"pmid":"19405909","id":"PMC_19405909","title":"Thioesterase superfamily member 2 (Them2)/acyl-CoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs.","date":"2009","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/19405909","citation_count":46,"is_preprint":false},{"pmid":"24072708","id":"PMC_24072708","title":"Thioesterase superfamily member 2/Acyl-CoA thioesterase 13 (Them2/Acot13) regulates adaptive thermogenesis in mice.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24072708","citation_count":31,"is_preprint":false},{"pmid":"24732803","id":"PMC_24732803","title":"Thioesterase superfamily member 2 (Them2) and phosphatidylcholine transfer protein (PC-TP) interact to promote fatty acid oxidation and control glucose utilization.","date":"2014","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24732803","citation_count":30,"is_preprint":false},{"pmid":"34886911","id":"PMC_34886911","title":"Identification of ACOT13 and PTGER2 as novel candidate genes of autosomal dominant polycystic kidney disease through whole exome sequencing.","date":"2021","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/34886911","citation_count":6,"is_preprint":false},{"pmid":"39172111","id":"PMC_39172111","title":"Acyl-CoA thioesterase 13 (ACOT13) attenuates the progression of autosomal dominant polycystic kidney disease in vitro via triggering mitochondrial-related cell apoptosis.","date":"2024","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/39172111","citation_count":1,"is_preprint":false},{"pmid":"23192039","id":"PMC_23192039","title":"Molecular cloning, expression, purification and crystallographic analysis of zebrafish THEM2.","date":"2012","source":"Acta crystallographica. Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/23192039","citation_count":0,"is_preprint":false},{"pmid":"39369989","id":"PMC_39369989","title":"Activity and phosphatidylcholine transfer protein interactions of skeletal muscle thioesterase Them2 enable hepatic steatosis and insulin resistance.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39369989","citation_count":0,"is_preprint":false},{"pmid":"41656235","id":"PMC_41656235","title":"A novel nanotherapeutic strategy: rescuing nucleus pulposus cells from fatty acid metabolic disorder and pyroptosis through ACOT13 by Chinese herbal formula nanoparticles.","date":"2026","source":"Journal of nanobiotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/41656235","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7212,"output_tokens":2347,"usd":0.028421},"stage2":{"model":"claude-opus-4-6","input_tokens":5663,"output_tokens":1997,"usd":0.11736},"total_usd":0.145781,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"ACOT13 (Them2) forms a stable homotetramer via a single hotdog fold domain and functions as a long-chain fatty acyl-CoA thioesterase, with lowest Km and highest kcat/Km for myristoyl-CoA and palmitoyl-CoA; substrate inhibition occurs near critical micellar concentrations. Interaction with StarD2/PC-TP increases the kcat of Them2, while phosphatidic acid/phosphatidylcholine vesicles decrease activity. Expression is mitochondria-associated and induced by PPARα activation.\",\n      \"method\": \"In vitro enzymatic assay (steady-state kinetics), size-exclusion chromatography (homotetramer determination), thermal denaturation, subcellular fractionation, co-incubation with StarD2/PC-TP\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro enzymatic assay with mutagenesis-equivalent substrate profiling, multiple orthogonal methods (kinetics, biophysics, fractionation)\",\n      \"pmids\": [\"19405909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Them2-/- mouse livers, mitochondrial thioesterase activity shows increased Km, fatty acyl-CoA concentrations rise by 28%, and free fatty acid concentrations fall by 23%, leading to reduced PPARα activation. Hepatic glucose production is decreased by 45% with reduced HNF4α expression. Them2-/- mice are resistant to high-fat diet-induced hepatic steatosis and increased glucose production, implicating Them2 in limiting β-oxidation and supporting gluconeogenesis via PC-TP interactions.\",\n      \"method\": \"Them2-/- mouse model, mitochondrial thioesterase activity assay, fatty acyl-CoA and free fatty acid quantification, hepatic glucose production measurement, PPARα and HNF4α expression analysis, high-fat diet challenge\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple defined metabolic readouts and mechanistic pathway placement\",\n      \"pmids\": [\"22345407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In brown adipose tissue, Them2 suppresses adaptive thermogenesis: Them2-/- mice show reduced lipid droplets, altered mitochondrial ultrastructure, and increased thermogenic gene expression. Primary brown adipocytes from Them2-/- mice exhibit increased norepinephrine-mediated triglyceride hydrolysis, increased O2 consumption, and elevated thermogenic gene expression, indicating Them2 directly regulates intracellular fatty acid channeling to suppress heat production.\",\n      \"method\": \"Them2-/- mouse model, ambient temperature challenge (4–30°C), electron microscopy of BAT mitochondria, primary brown adipocyte culture, O2 consumption assay, triglyceride hydrolysis assay, thermogenic gene expression\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal cellular and molecular readouts in primary cells and in vivo\",\n      \"pmids\": [\"24072708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Them2 and PC-TP interact to promote fatty acid oxidation and gluconeogenesis in hepatocytes under fasting-like conditions. Them2-/- and Pctp-/- primary hepatocytes each show decreased rates of fatty acid oxidation and gluconeogenesis. Chemical inhibition of PC-TP fails to reproduce these changes in Them2-/- hepatocytes, indicating PC-TP acts upstream of or through Them2. Additionally, glucose oxidation and lipogenesis under high glucose are decreased only in Them2-/- hepatocytes, revealing a Them2-specific role in glucose oxidation.\",\n      \"method\": \"Primary hepatocyte culture from Them2-/- and Pctp-/- mice, fatty acid oxidation assays, gluconeogenesis assays, glucose oxidation assays, pharmacological PC-TP inhibition\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO combined with pharmacological epistasis in primary cells with multiple metabolic readouts\",\n      \"pmids\": [\"24732803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Zebrafish THEM2 (ortholog of ACOT13) was crystallized and X-ray diffraction data collected to 1.80 Å resolution, confirming the hotdog-fold thioesterase domain architecture and homotetrameric assembly consistent with the mammalian protein.\",\n      \"method\": \"Recombinant protein expression, Ni-affinity and gel-filtration chromatography, X-ray crystallography (synchrotron, 1.80 Å)\",\n      \"journal\": \"Acta Crystallographica Section F\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure determination, but from zebrafish ortholog; functional validation limited to crystallographic data\",\n      \"pmids\": [\"23192039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Skeletal muscle Them2 promotes hepatic steatosis and insulin resistance through both its catalytic activity and interaction with PC-TP. Catalytic-dead mutant (N50A/D65A, maintaining homotetrameric structure and PC-TP binding) failed to promote high-fat diet-induced hepatic steatosis when restored in Them2-/- skeletal muscle via AAV. Conditioned medium and specifically secreted extracellular vesicles from WT myotubes (but not Them2-/- myotubes) promoted lipid accumulation in hepatocytes, dependent on Them2 catalytic activity and PC-TP interaction.\",\n      \"method\": \"Active-site mutagenesis (N50A/D65A), AAV-mediated skeletal muscle-specific reconstitution in Them2-/- mice, high-fat diet challenge, primary myotube-conditioned medium experiments, extracellular vesicle isolation, primary hepatocyte lipid accumulation assay, PC-TP pharmacological inhibition and genetic ablation\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — active-site mutagenesis combined with in vivo reconstitution and reductionist cell biology experiments\",\n      \"pmids\": [\"39369989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACOT13 overexpression in ADPKD cells (WT9-12) suppresses proliferation, induces cell cycle arrest, triggers apoptosis with increased cleaved caspase-3, reduces ATP production, and induces loss of mitochondrial membrane potential, indicating ACOT13 triggers mitochondria-mediated apoptosis in these cells.\",\n      \"method\": \"ACOT13 overexpression in WT9-12 cells, EdU staining, flow cytometry (cell cycle and apoptosis), cleaved caspase-3 Western blot, ATP production assay, mitochondrial membrane potential assay\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — overexpression with multiple cellular readouts, but single lab, single cell line, no rescue/mutagenesis\",\n      \"pmids\": [\"39172111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Under pathological conditions in nucleus pulposus cells, ACOT13 inhibits the AMPK/ACC signaling pathway, leading to disrupted fatty acid metabolism, mitochondrial dysfunction, and pyroptosis, thereby accelerating intervertebral disc degeneration.\",\n      \"method\": \"Single-cell sequencing and multi-omics analysis of clinical samples, GSEA pathway analysis, functional cell experiments with ACOT13 modulation\",\n      \"journal\": \"Journal of Nanobiotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway placement inferred from omics and cell overexpression; limited mechanistic detail and mutagenesis absent\",\n      \"pmids\": [\"41656235\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACOT13 (Them2) is a mitochondria-associated homotetrameric hotdog-fold thioesterase that hydrolyzes long-chain fatty acyl-CoAs (preferring myristoyl-CoA and palmitoyl-CoA), and its catalytic activity together with its physical interaction with PC-TP (StarD2) regulates hepatic fatty acid oxidation, gluconeogenesis, and lipid homeostasis, while in skeletal muscle it promotes hepatic steatosis and insulin resistance via extracellular vesicle-mediated inter-organ communication, and in brown adipose tissue it suppresses adaptive thermogenesis by limiting intracellular fatty acid channeling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"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].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"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.\",\n      \"evidence\": \"In vitro steady-state kinetics, size-exclusion chromatography, thermal denaturation, subcellular fractionation, and co-incubation with StarD2/PC-TP using recombinant protein\",\n      \"pmids\": [\"19405909\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal structure of the mammalian protein–PC-TP complex to define the activation mechanism\",\n        \"In vivo relevance of substrate inhibition near CMC not addressed\",\n        \"Regulation by post-translational modifications unknown\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"Them2−/− mouse model with mitochondrial thioesterase assay, acyl-CoA/FFA quantification, hepatic glucose production measurement, and high-fat diet challenge\",\n      \"pmids\": [\"22345407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether hepatic phenotypes are cell-autonomous versus secondary to systemic metabolic changes\",\n        \"Contribution of individual acyl-CoA species to PPARα activation not dissected\",\n        \"Tissue-specific conditional knockouts not performed\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"Recombinant zebrafish THEM2 expression, Ni-affinity and gel-filtration chromatography, synchrotron X-ray crystallography\",\n      \"pmids\": [\"23192039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No mammalian structure solved\",\n        \"Substrate-bound or PC-TP co-crystal structure unavailable\",\n        \"Active-site catalytic mechanism not defined from this structure alone\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"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.\",\n      \"evidence\": \"Them2−/− mice under cold challenge, electron microscopy of BAT mitochondria, primary brown adipocyte O₂ consumption and triglyceride hydrolysis assays\",\n      \"pmids\": [\"24072708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct lipid substrates mediating thermogenic suppression not identified\",\n        \"Whether PC-TP interaction is required for the BAT phenotype not tested\",\n        \"Signaling intermediates between ACOT13 activity and UCP1 induction unknown\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"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.\",\n      \"evidence\": \"Primary hepatocytes from Them2−/− and Pctp−/− mice, pharmacological PC-TP inhibition, fatty acid oxidation, gluconeogenesis, and glucose oxidation assays\",\n      \"pmids\": [\"24732803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which PC-TP delivers substrate to ACOT13 at the molecular level unresolved\",\n        \"Whether the Them2-specific glucose oxidation role requires catalytic activity not tested\",\n        \"In vivo validation of epistatic relationship not performed with tissue-specific double knockouts\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"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\",\n      \"pmids\": [\"39369989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Cargo composition of ACOT13-dependent extracellular vesicles not characterized\",\n        \"Hepatocyte receptor or uptake mechanism for these EVs unknown\",\n        \"Whether this inter-organ axis operates under physiological (non-HFD) conditions not determined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"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.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No mammalian co-crystal structure of ACOT13–PC-TP\",\n        \"EV cargo and hepatocyte receptor undefined\",\n        \"Tissue-specific conditional knockout studies across all relevant tissues lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PCTP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}