{"gene":"TBC1D1","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2007,"finding":"TBC1D1 possesses Rab-GTPase-activating protein (GAP) activity; its GAP domain shares identical Rab substrate specificity with AS160 (TBC1D4). Ectopic expression of TBC1D1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect, demonstrating that this inhibition is GAP-activity-dependent. Insulin treatment leads to phosphorylation of TBC1D1 on a conserved Akt site.","method":"In vitro GAP activity assay, ectopic overexpression in 3T3-L1 adipocytes, point mutagenesis of catalytic residue, GLUT4 translocation assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay combined with active-site mutagenesis and cell-based GLUT4 translocation functional readout in a single study","pmids":["17274760"],"is_preprint":false},{"year":2008,"finding":"TBC1D1 is identified as the dominant PAS-immunoreactive protein at ~160 kDa in fast-twitch skeletal muscle (tibialis anterior, EDL) but not slow-twitch (soleus). In vivo stimulation by insulin, muscle contraction, and the AMPK activator AICAR each increased TBC1D1 PAS phosphorylation. Mass spectrometry identified multiple novel phosphorylation sites on TBC1D1, the majority being consensus or near-consensus AMPK sites. Purified Akt and AMPK both phosphorylate TBC1D1 in vitro, but AMPK (not Akt) caused a detectable shift in TBC1D1 electrophoretic mobility.","method":"Immunoprecipitation, mass spectrometry, in vitro kinase assay with purified Akt and AMPK, in vivo stimulation (insulin, contraction, AICAR) with PAS immunoblotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay combined with in vivo phosphorylation mapping by MS and multiple stimulation conditions","pmids":["18276596"],"is_preprint":false},{"year":2008,"finding":"AMPK activation in HEK-293 cells promotes 14-3-3 binding primarily to phospho-Ser237 of TBC1D1, while IGF-1/EGF/PMA promote 14-3-3 binding primarily via phospho-Thr596. In rat L6 myotubes, AMPK activators (AICAR, phenformin, A-769662) strongly phosphorylate Ser237 and promote 14-3-3 binding, whereas insulin promotes Thr596 phosphorylation but not 14-3-3 binding. In vitro phosphorylation experiments showed regulatory cross-talk among sites (e.g., phospho-Ser235 prevents subsequent phospho-Ser237).","method":"Site-specific phosphorylation analysis in HEK-293 and L6 cells, 14-3-3 binding assays, in vitro phosphorylation with purified kinases, pharmacological inhibition (LY294002)","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro kinase assay, cell-based phosphorylation, 14-3-3 binding) with site-specific resolution across multiple cell types","pmids":["17995453"],"is_preprint":false},{"year":2008,"finding":"Overexpressed TBC1D1 in 3T3-L1 adipocytes inhibits GLUT4 translocation even in response to activated Akt, and endogenous TBC1D1 (which is ~20-fold less abundant than AS160 in adipocytes) does not regulate insulin-stimulated GLUT4 translocation. AMPK activator AICAR partially reversed the inhibition of GLUT4 translocation caused by overexpressed TBC1D1. TBC1D1 is much more highly expressed in skeletal muscle than fat.","method":"RNAi knockdown of TBC1D1 in 3T3-L1 adipocytes, overexpression with constitutively active Akt, AICAR treatment, GLUT4 translocation assay, quantitative western blotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain- and loss-of-function experiments in adipocytes with defined functional readout, replicated across multiple experimental conditions","pmids":["18258599"],"is_preprint":false},{"year":2008,"finding":"TBC1D1 is an Akt substrate phosphorylated at Thr590 in 3T3-L1 adipocytes. RNAi silencing of TBC1D1 elevated basal 2-deoxyglucose uptake (~61%) and strongly increased GLUT1 (but not GLUT4) expression. Loss of TBC1D1 activated the mTOR–p70 S6K pathway, and the GLUT1 upregulation was blocked by rapamycin. Overexpression of the phosphorylation-defective T590A mutant inhibited insulin-stimulated p70 S6K phosphorylation.","method":"Mass spectrometry-based phosphoproteomic identification of Akt substrates, RNAi knockdown, rapamycin treatment, overexpression of T590A mutant, glucose uptake assay, western blotting","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — MS identification of phosphosite combined with RNAi, pharmacological rescue (rapamycin), and phosphomutant overexpression in a single study","pmids":["18215134"],"is_preprint":false},{"year":2008,"finding":"In vivo gene electroporation into mouse tibialis anterior showed that the obesity-associated R125W mutant TBC1D1 significantly decreased insulin-stimulated glucose transport through a mechanism requiring the GAP domain (disrupting GAP activity in the R125W background rescued glucose transport). A TBC1D1 quadruple phosphorylation-site mutant (4P, Ala substitutions at four AMPK/Akt sites) had no effect on insulin-stimulated transport but decreased contraction-stimulated glucose transport via the GAP domain, establishing that insulin- and contraction-regulated glucose transport occur via distinct mechanisms.","method":"In vivo gene injection/electroporation into mouse tibialis anterior, in vivo glucose transport assay, mutagenesis of phosphorylation sites and GAP domain","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mutagenesis study with defined functional readout, mechanistic dissection of insulin vs contraction pathways using multiple mutants","pmids":["20299473"],"is_preprint":false},{"year":2009,"finding":"Insulin-stimulated phosphorylation of multiple Akt sites on TBC1D1 is required for GLUT4 translocation: a TBC1D1 mutant with several Akt sites converted to alanine was considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1 in C2C12 myotubes. AMPK activation partially relieved TBC1D1-mediated inhibition of GLUT4 translocation. Akt sites on TBC1D1 were identified by mass spectrometry from C2C12 myotubes.","method":"Mass spectrometry identification of Akt phosphorylation sites in C2C12 myotubes, alanine-substitution mutagenesis, GLUT4 translocation assay, AMPK activator treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — MS phosphosite identification combined with mutagenesis and functional GLUT4 translocation assay in muscle cells","pmids":["19740738"],"is_preprint":false},{"year":2009,"finding":"In mouse EDL muscle, both contraction- and insulin-stimulated TBC1D1 Ser237 and Thr596 phosphorylation, and 14-3-3 protein binding to TBC1D1, are abolished in AMPK kinase-dead transgenic mice. AICAR and contraction induced comparable phosphorylation patterns; insulin increased Thr596 but not Ser237 phosphorylation, and insulin-stimulated Thr596 phosphorylation was fully abolished in AMPK KD mice, revealing an unexpected AMPK requirement for insulin-stimulated TBC1D1 Thr596 phosphorylation.","method":"Genetic epistasis using AMPK kinase-dead transgenic mice, site-specific phospho-antibodies for Ser237 and Thr596, 14-3-3 binding assay, ex vivo muscle contraction/AICAR/insulin stimulation","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function (AMPK KD transgenic) with site-specific phosphorylation readouts across multiple stimuli, providing definitive upstream kinase assignment","pmids":["19531644"],"is_preprint":false},{"year":2009,"finding":"In rat epitrochlearis muscle, contraction-stimulated PAS-TBC1D1 phosphorylation and glucose transport are abolished by the AMPK inhibitor compound C, but are unaffected by Wortmannin (PI3-kinase/Akt inhibitor). Conversely, insulin-stimulated phosphorylation of both AS160 and TBC1D1 requires PI3-kinase/Akt. This establishes that contraction regulates TBC1D1 and glucose transport through an AMPK-dependent, Akt-independent mechanism.","method":"Pharmacological inhibition (compound C for AMPK, Wortmannin for PI3K) in isolated rat skeletal muscle, PAS immunoblotting, 3-O-methylglucose transport assay","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — dual pharmacological epistasis with orthogonal kinase inhibitors, clear separation of insulin vs contraction pathway mechanisms","pmids":["19208911"],"is_preprint":false},{"year":2010,"finding":"In mouse skeletal muscle, contraction increases TBC1D1 phosphorylation on Ser231 and Ser660 (AMPK sites) but not Thr590 (Akt site). AICAR phosphorylates Ser231, Ser660, and Ser700 but not Thr590; insulin selectively increases Thr590. Contraction-stimulated Ser231, Ser660, and Ser700 phosphorylation is greatly reduced in AMPKα2-inactive transgenic mice, and Akt2-KO mice show blunted insulin-stimulated Thr590 phosphorylation. In vivo overexpression of a TBC1D1 mutated on four AMPK sites decreased glucose uptake in tibialis anterior.","method":"Site-specific phospho-antibodies, AMPKα2-inactive transgenic mice, Akt2-KO mice, in vivo electroporation/overexpression of phosphosite mutants, glucose uptake assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic models (AMPKα2i TG, Akt2 KO) combined with site-specific phosphorylation analysis and in vivo functional readout","pmids":["20701589"],"is_preprint":false},{"year":2010,"finding":"Exercise in human skeletal muscle increases TBC1D1 Ser237 phosphorylation (70–230%) and 14-3-3 binding capacity (60–250%) in an exercise-duration-dependent manner. Recombinant AMPK directly phosphorylates Ser237 and induces 14-3-3 binding in vitro. In AMPKα2-knockout (but not α1-knockout) mouse EDL, basal TBC1D1 protein levels and contraction-stimulated Ser237 phosphorylation are reduced, identifying AMPKα2 as the principal isoform regulating TBC1D1 Ser237 in EDL.","method":"Human exercise biopsy study (30 s, 2 min, 20 min cycling), in vitro AMPK phosphorylation assay, AMPKα1/α2 whole-body knockout mice, site-specific phospho-antibody, 14-3-3 binding overlay assay","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay combined with human exercise data and genetic knockout confirmation of isoform specificity","pmids":["20837646"],"is_preprint":false},{"year":2011,"finding":"Crystal structures of the RabGAP domains of human TBC1D1 (2.2 Å) and TBC1D4/AS160 (3.5 Å) were solved. Both have 16 α-helices and no β-sheet elements. Ala-scanning mutagenesis of inferred Rab-binding interface residues showed only one of five substitutions significantly perturbed catalytic efficiency; substitution of TBC1D1 Met930 outside the canonical yeast interface substantially reduced catalytic activity, and the M930A mutant also failed to promote GLUT4 translocation. Additional residue Leu1019 was predicted to contact Rab; mutants with lowest RabGAP activity confirmed these contacts are required for biological activity.","method":"X-ray crystallography (2.2 Å for TBC1D1 GAP domain), Ala-scanning mutagenesis, in vitro RabGAP activity assay, GLUT4 translocation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination combined with mutagenesis-validated enzymatic activity and cell-based functional assay in a single study","pmids":["21454505"],"is_preprint":false},{"year":2012,"finding":"The Rab-GTPase-activating proteins TBC1D1 and TBC1D4 together are essential for insulin-stimulated glucose uptake; double-knockout mice (D1/4KO) showed almost complete abolition of insulin-stimulated glucose uptake in skeletal muscle and adipocytes, whereas single knockouts showed only partial impairment. GLUT4 protein (but not mRNA) was substantially reduced in D1/4KO skeletal muscle and white adipose tissue. Cell surface labeling indicated that RabGAP deficiency impairs intracellular GLUT4 retention in the basal state.","method":"Generation of double-knockout (TBC1D1-/-/TBC1D4-/-) mice, hyperinsulinemic clamp, isolated skeletal muscle glucose uptake, cell surface GLUT4 labeling, GLUT4 protein/mRNA quantification","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic double-KO combined with multiple functional readouts (in vivo clamp, ex vivo glucose uptake, cell surface GLUT4 labeling)","pmids":["25249576"],"is_preprint":false},{"year":2013,"finding":"Conventional Tbc1d1-knockout mice show severely impaired insulin- and AICAR-stimulated glucose uptake in EDL (glycolytic) but not soleus muscle, and substantially increased fatty acid oxidation in oxidative soleus muscle. These mice had moderately reduced body weight, decreased respiratory quotient, and elevated resting metabolic rate, establishing that TBC1D1 plays a major role in glucose and lipid substrate preference in skeletal muscle.","method":"Conventional Tbc1d1-knockout mice, ex vivo glucose uptake assay (EDL, soleus), fatty acid oxidation assay, indirect calorimetry, body weight measurement","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conventional KO with multiple orthogonal metabolic readouts and muscle-type specificity established","pmids":["23892475"],"is_preprint":false},{"year":2013,"finding":"AICAR treatment prior to insulin stimulation enables TBC1D1 to acquire insulin responsiveness, triggering GLUT4 trafficking. This regulatory mode shift requires Ser237 phosphorylation and an intact phosphotyrosine-binding 1 (PTB1) domain of TBC1D1. Mutations in PTB1, including the obesity-associated R125W, abolish the acquisition of insulin responsiveness while leaving AICAR-responsive GLUT4-liberation activity intact.","method":"GLUT4 nanometry in cell-based reconstitution model, AICAR and insulin stimulation, site-directed mutagenesis of Ser237 and PTB1 domain (including R125W), temporal GLUT4 tracking","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — functional reconstitution with GLUT4 nanometry, multiple mutants, mechanistically resolved AICAR-to-insulin priming sequence","pmids":["23325788"],"is_preprint":false},{"year":2014,"finding":"TBC1D1 is phosphorylated by AKT and AMPK in response to insulin and muscle contraction, and both phosphorylation events increase 14-3-3 binding but do not alter intrinsic RabGAP activity on Rab8a, Rab10, and Rab14. Full-length TBC1D1 shows markedly higher catalytic activity toward Rab8a, Rab10, and Rab14 than the isolated GAP domain. TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic tail of insulin-regulated aminopeptidase (IRAP), a resident of GLUT4 storage vesicles, and this interaction is disrupted by AKT or AMPK phosphorylation, suggesting that TBC1D1 recruitment to GLUT4 vesicles (not GAP activity per se) is regulated by insulin/contraction signaling.","method":"Baculovirus/Sf9 expression of recombinant full-length TBC1D1, in vitro RabGAP assay, in vitro phosphorylation by AKT/AMPK, 14-3-3 binding assay, co-immunoprecipitation with IRAP cytoplasmic tail, PTB domain pulldown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified full-length protein, enzymatic assay, direct protein-protein interaction mapping, and phosphorylation-dependent disruption in a single rigorous study","pmids":["30275018"],"is_preprint":false},{"year":2015,"finding":"TBC1D1-knockout mice show impaired exercise-induced 2-deoxyglucose uptake specifically in white (non-oxidative) but not red skeletal muscle, and GLUT4 protein levels are reduced ~40% in white TBC1D1-/- muscle. TBC1D1-/- mice also display impaired exercise endurance. Normal body weight and glucose/insulin tolerance indicate TBC1D1 is dispensable for basal and insulin-stimulated glucose homeostasis under normal conditions.","method":"TBC1D1-knockout mouse generation, in vivo exercise-stimulated 2-deoxyglucose uptake, glucose/insulin tolerance tests, GLUT4 protein quantification, treadmill endurance test","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO model with in vivo exercise functional readout, muscle-type specificity demonstrated, multiple metabolic phenotypes characterized","pmids":["25576050"],"is_preprint":false},{"year":2015,"finding":"TBC1D1 deficiency in EDL but not soleus muscle impairs insulin-, AICAR-, and contraction-stimulated glucose transport. In TBC1D1-deficient (Nob1.10SJL) congenic mice, suppression of hepatic glucose production during hyperinsulinemic clamp was increased. A 50% reduction in GLUT4 protein in EDL from TBC1D1-deficient mice was identified as a mechanism for reduced glucose transport, with proximal AMPK/Akt signaling unaltered.","method":"TBC1D1-deficient congenic mouse model, euglycemic hyperinsulinemic clamp, isolated EDL/soleus glucose transport, GLUT4 protein quantification, proximal signaling analysis","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo clamp combined with ex vivo muscle-specific glucose transport and protein-level mechanistic analysis","pmids":["22693207"],"is_preprint":false},{"year":2018,"finding":"AMPK heterotrimers containing the α1 (but not α2) catalytic subunit form a stable, direct association with TBC1D1 but not its paralogue AS160. The interaction involves both PTB domains of TBC1D1 and is enhanced by AMPK activators (AICAR, A769662). This AMPKα1-TBC1D1 complex increases the efficiency of AMPK-mediated Ser237 phosphorylation. The obesity-associated R125W mutation in PTB1 reduces AMPKα1 binding and concomitantly reduces Ser237 phosphorylation.","method":"Co-immunoprecipitation, GST pulldown with purified proteins (direct interaction), pharmacological AMPK activation, site-directed mutagenesis (R125W, PTB domain mutants), in vitro kinase assay, quantitative phosphorylation analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct protein-protein interaction with purified components, isoform-specific genetic and pharmacological dissection, mutagenesis-validated mechanism in a single study","pmids":["30135087"],"is_preprint":false},{"year":2018,"finding":"In cells with both TBC1D1 and AS160, TBC1D1 functionally dominates AS160 in controlling GLUT4 release. AICAR and intracellular Ca2+ serve as proximal stimuli for TBC1D1-governed GLUT4 release. AS160 modulates sensitivity to external stimuli in TBC1D1-mediated GLUT4 release. The synergistic cooperative actions depend on TBC1D1 PTB1 domain, calmodulin-binding domain, and phosphorylation of AS160 Thr642 and TBC1D1 Ser237/Thr596.","method":"GLUT4 nanometry in cell-based reconstitution models with varying TBC1D1:AS160 expression ratios, site-directed mutagenesis (PTB1, calmodulin-binding domain, phosphosites), AICAR and Ca2+ stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — GLUT4 nanometry reconstitution combined with systematic mutagenesis of multiple regulatory domains and quantitative functional analysis","pmids":["30482843"],"is_preprint":false},{"year":2016,"finding":"A Tbc1d1 Ser231Ala knock-in mutation attenuates AICAR-induced glucose lowering and reduces AICAR-stimulated glucose uptake and GLUT4 cell surface content in isolated skeletal muscle. However, the Ser231Ala mutation does not impair exercise-induced muscle glucose uptake or exercise capacity, demonstrating that Ser231 phosphorylation is specifically required for AMPK-activator- but not exercise-stimulated glucose uptake.","method":"Tbc1d1 Ser231Ala knock-in mice, AICAR stimulation in vivo and ex vivo, exercise-stimulated glucose uptake, cell surface GLUT4 labeling, glucose tolerance test","journal":"Diabetologia","confidence":"High","confidence_rationale":"Tier 2 / Strong — precision knock-in mouse model with site-specific mechanistic dissection of AICAR vs. exercise stimuli, multiple functional readouts","pmids":["27826658"],"is_preprint":false},{"year":2014,"finding":"APPL2 interacts with TBC1D1 through its BAR domain. Insulin stimulates TBC1D1 phosphorylation on Ser235, which enhances APPL2 binding; this APPL2–TBC1D1 interaction suppresses insulin-evoked TBC1D1 Thr596 phosphorylation. Substitution of Ser235 with alanine diminishes APPL2-mediated inhibition of Thr596 phosphorylation and reverses the suppressive effects of TBC1D1 on insulin-induced GLUT4 translocation and glucose uptake in myotubes.","method":"Co-immunoprecipitation of APPL2 and TBC1D1, site-directed mutagenesis (S235A), overexpression and knockdown in C2C12 myotubes, GLUT4 translocation assay, glucose uptake assay, conditional muscle-specific APPL2 KO mice","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, phosphosite mutagenesis, and functional GLUT4/glucose uptake readouts in cells and conditional KO mice","pmids":["24879834"],"is_preprint":false},{"year":2016,"finding":"Rab28 is identified as an in vitro substrate for the GAP domains of both TBC1D1 and TBC1D4. Rab28 GTP-loading state is acutely regulated by insulin in skeletal muscle. siRNA-mediated knockdown of Rab28 in isolated mouse skeletal muscle decreases basal glucose uptake, and constitutively active Rab28-Q72L in adipocytes increases basal cell surface HA-GLUT4.","method":"In vitro GAP assay with purified Rab28 and TBC1D1/TBC1D4 GAP domains, Rab28-GTP loading assay in muscle, siRNA knockdown of Rab28 in skeletal muscle, overexpression of Rab28-Q72L in adipocytes, cell surface GLUT4 labeling","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay identifying substrate combined with in vivo Rab GTP-state measurement and bidirectional functional manipulation","pmids":["27929607"],"is_preprint":false},{"year":2019,"finding":"WNK1 kinase phosphorylates TBC1D1 on Ser565 (identified by mass spectrometry). Phosphomimetic and unphosphorylatable mutants of TBC1D1 at S565 both affected GLUT1 cell surface abundance, indicating a regulatory role for WNK1-mediated TBC1D1 phosphorylation in constitutive GLUT1 trafficking.","method":"WNK1 RNAi in HEK293 cells, mass spectrometry identification of Ser565 phosphosite, transfection of phosphomimetic/unphosphorylatable TBC1D1 mutants, cell surface GLUT1 quantification","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS phosphosite identification with functional mutagenesis in cells, single lab, single study","pmids":["31816312"],"is_preprint":false},{"year":2020,"finding":"TBC1D1 interacts with VPS13A and VPS13C, the Rab-binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1 in C2C12 myotubes. These interactions are mediated through the PTB domains of TBC1D1 and are not affected by AMPK activation, distinguishing them from the AMPK-regulated AMPKα1–TBC1D1 interaction. Depletion of VPS13A or VPS13C post-transcriptionally increases cellular GLUT4 protein and enhances cell surface GLUT4 in response to AMPK activation.","method":"Unbiased quantitative proteomics/co-immunoprecipitation to identify TBC1D1 interactors in C2C12 myotubes, siRNA depletion of VPS13A/VPS13C, cell surface GLUT4 assay, AMPK activation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — unbiased interactome MS combined with functional knockdown and GLUT4 readout, single lab","pmids":["33087848"],"is_preprint":false},{"year":2013,"finding":"TBC1D1 is expressed in pancreatic beta-cells and is phosphorylated in response to glucose. siRNA knockdown of TBC1D1 in beta-cells increased basal and glucose-stimulated insulin secretion and decreased beta-cell proliferation without affecting apoptosis.","method":"TBC1D1 expression analysis in human and rat beta-cells, phosphorylation assay (glucose stimulation), siRNA knockdown, insulin secretion assay, proliferation and apoptosis assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA knockdown with functional readouts in beta-cells, single lab, limited mechanistic pathway placement","pmids":["24239544"],"is_preprint":false},{"year":2022,"finding":"AMPK-TBC1D1 signaling regulates GTP-loading of Rab2A: nutrition repletion suppresses AMPK-TBC1D1 phosphorylation, increasing GTP-bound Rab2A, which stabilizes PPARγ protein and promotes hepatic lipid accumulation. In TBC1D1-S231A knock-in mice, increased GTP-Rab2A and fatty liver were observed. Inhibition of Rab2A expression alleviated hepatic lipid deposition in obese mice.","method":"TBC1D1-S231A knock-in mice, GTP-Rab2A pulldown assay, siRNA knockdown of Rab2A in hepatic cells and mice, western blotting for PPARγ stability, in vitro and in vivo lipid accumulation assays","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in mouse model combined with Rab2A GTP-state measurements and functional rescue, single lab","pmids":["35061665"],"is_preprint":false},{"year":2014,"finding":"Overexpression of TBC1D1 in mouse soleus muscle decreased basal and AICAR-stimulated palmitate oxidation by ~18–22% and increased glucose oxidation, without altering FAT/CD36, mitochondrial content, CPT1, AMPK, or ACC. TBC1D1-mediated reduction of fatty acid oxidation was associated with reduced β-hydroxyacyl-CoA dehydrogenase (β-HAD) enzyme activity.","method":"Electrotransfection of TBC1D1 cDNA into mouse soleus, palmitate oxidation assay, glucose oxidation assay, β-HAD enzyme activity measurement, protein expression analysis","journal":"American journal of physiology. Regulatory, integrative and comparative physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vivo overexpression with biochemical enzyme activity readout, single lab, single method for the β-HAD finding","pmids":["25163918"],"is_preprint":false},{"year":2017,"finding":"Ablation of TBC1D1 in rats impairs contraction-induced sarcolemmal GLUT4 redistribution and glucose uptake specifically in white gastrocnemius muscle but does not alter insulin-induced GLUT4 trafficking or whole-body insulin tolerance. TBC1D1-KO rats show increased skeletal muscle fatty acid oxidation and increased maximal ADP-stimulated mitochondrial respiration, but reduced exercise run time to exhaustion.","method":"Rat TBC1D1 KO model, sarcolemmal GLUT4 fractionation after contraction/insulin, in vivo insulin tolerance test, ex vivo fatty acid oxidation, permeabilized fiber respiration, treadmill exercise","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO animal model with direct GLUT4 membrane fractionation, multiple metabolic functional readouts, and contraction vs insulin pathway dissection","pmids":["28808062"],"is_preprint":false},{"year":2008,"finding":"The SJL mouse strain carries a naturally occurring mutation in Tbc1d1 resulting in a truncated protein lacking the TBC RabGAP domain. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, while overexpression had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation, and reduced glucose uptake in isolated skeletal muscle.","method":"Identification of naturally occurring Tbc1d1 truncation mutation in SJL strain, siRNA knockdown and overexpression in skeletal muscle cells, fatty acid uptake and oxidation assays, isolated muscle glucose uptake, indirect calorimetry in recombinant congenic mice","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — naturally occurring loss-of-function mutation combined with bidirectional manipulation in cells and in vivo metabolic phenotyping","pmids":["18931681"],"is_preprint":false}],"current_model":"TBC1D1 is a Rab GTPase-activating protein (RabGAP) predominantly expressed in fast-twitch skeletal muscle that functions as a key regulator of GLUT4 vesicle trafficking and glucose/lipid substrate utilization: its GAP activity toward Rab8a, Rab10, Rab14, and Rab28 retains GLUT4 in intracellular storage vesicles, and this activity is modulated by site-specific phosphorylation—AMPK phosphorylates Ser231/Ser237/Ser660/Ser700 in response to muscle contraction and AICAR, while Akt2 phosphorylates Thr590/Thr596 in response to insulin—leading to 14-3-3 protein binding and disruption of TBC1D1 interaction with the GLUT4 vesicle resident protein IRAP, thereby promoting GLUT4 translocation to the sarcolemma; the obesity-associated R125W mutation in the PTB1 domain impairs AMPKα1 association, reduces Ser237 phosphorylation efficiency, and abolishes exercise-priming acquisition of insulin responsiveness, while genetic ablation of TBC1D1 in mice and rats abolishes contraction-stimulated but not insulin-stimulated GLUT4 translocation in glycolytic muscle fibers and shifts substrate utilization toward enhanced fatty acid oxidation."},"narrative":{"mechanistic_narrative":"TBC1D1 is a Rab-GTPase-activating protein (RabGAP) that gates GLUT4 vesicle trafficking and substrate selection in skeletal muscle, acting as a convergence point for insulin and contraction/AMPK signaling [PMID:17274760, PMID:23892475]. Its GAP domain, whose crystal structure reveals an all-α-helical fold, hydrolyzes GTP on Rab8a, Rab10, Rab14, and Rab28, and full-length protein is markedly more active than the isolated domain; this catalytic activity is required to retain GLUT4 intracellularly, since overexpressed wild-type TBC1D1 blocks GLUT4 translocation while a GAP-dead mutant does not [PMID:17274760, PMID:21454505, PMID:30275018, PMID:27929607]. TBC1D1 is the dominant phospho-Akt-substrate species in fast-twitch (but not slow-twitch) muscle, where AMPK phosphorylates Ser231/Ser237/Ser660/Ser700 in response to contraction and AICAR, and Akt2 phosphorylates Thr590/Thr596 in response to insulin [PMID:18276596, PMID:20701589, PMID:20837646]. These phosphorylation events drive 14-3-3 binding without altering intrinsic RabGAP activity, instead disrupting the PTB-domain-mediated interaction with the GLUT4-vesicle protein IRAP, thereby releasing GLUT4 for translocation [PMID:17995453, PMID:30275018]. AMPKα1 (not α2) forms a stable direct complex with TBC1D1 via its PTB domains that enhances Ser237 phosphorylation efficiency [PMID:30135087]. Genetic ablation of TBC1D1 in mice and rats selectively abolishes contraction/AICAR-stimulated GLUT4 translocation and glucose uptake in glycolytic muscle, reduces GLUT4 protein, impairs exercise endurance, and shifts substrate use toward fatty acid oxidation, while TBC1D1/TBC1D4 double knockout almost completely abolishes insulin-stimulated glucose uptake [PMID:25249576, PMID:23892475, PMID:25576050, PMID:28808062]. The obesity-associated R125W mutation in the PTB1 domain impairs AMPKα1 association, reduces Ser237 phosphorylation, and abolishes the AICAR/exercise-priming acquisition of insulin responsiveness [PMID:23325788, PMID:30135087]. Beyond muscle, TBC1D1 also functions in pancreatic beta-cells and in hepatic lipid metabolism through AMPK-TBC1D1 control of Rab2A and PPARγ [PMID:24239544, PMID:35061665].","teleology":[{"year":2007,"claim":"Establishing that TBC1D1 is a catalytically active RabGAP whose enzymatic activity gates GLUT4 trafficking answered whether it is a functional regulator rather than an inert AS160 paralog.","evidence":"In vitro GAP assay, catalytic-residue mutagenesis, and GLUT4 translocation assay in 3T3-L1 adipocytes","pmids":["17274760"],"confidence":"High","gaps":["Did not identify the physiological Rab substrates in muscle","Performed in adipocytes where TBC1D1 is minimally expressed"]},{"year":2008,"claim":"Identifying TBC1D1 as the dominant fast-twitch-muscle PAS protein regulated by insulin, contraction, and AICAR, and mapping mostly AMPK-consensus phosphosites, placed it at the intersection of insulin and energy-sensing signaling.","evidence":"Immunoprecipitation, mass-spectrometry phosphosite mapping, in vitro Akt/AMPK kinase assays, and in vivo stimulation with PAS immunoblotting","pmids":["18276596"],"confidence":"High","gaps":["Functional consequence of individual sites not yet resolved","Did not establish which kinase acts in vivo"]},{"year":2008,"claim":"Resolving the stimulus-specific phosphorylation code (AMPK→Ser237/14-3-3 binding vs insulin→Thr596) and showing endogenous TBC1D1 does not govern insulin-stimulated GLUT4 in adipocytes refined where and how TBC1D1 acts.","evidence":"Site-specific phosphorylation and 14-3-3 binding assays in HEK293/L6 cells, plus RNAi and overexpression in 3T3-L1 adipocytes","pmids":["17995453","18258599","18215134"],"confidence":"High","gaps":["Did not link 14-3-3 binding to a downstream trafficking step","Tissue site of physiological action (muscle) not yet tested genetically"]},{"year":2008,"claim":"A naturally occurring SJL Tbc1d1 truncation lacking the GAP domain linking TBC1D1 to body weight and fatty acid oxidation provided the first in vivo evidence that it controls substrate utilization.","evidence":"Identification of SJL truncation, bidirectional manipulation in muscle cells, and metabolic phenotyping of congenic mice","pmids":["18931681"],"confidence":"High","gaps":["Mechanism linking GAP loss to fatty acid oxidation not defined","Confounded by other strain background loci"]},{"year":2009,"claim":"Genetic and pharmacological epistasis assigned contraction-stimulated TBC1D1 phosphorylation and glucose transport to an AMPK-dependent, Akt-independent route distinct from the insulin/PI3K route.","evidence":"AMPK kinase-dead transgenic mice, compound C and Wortmannin inhibition, and site-specific phospho-antibodies in isolated muscle; MS phosphosite mapping and mutagenesis in C2C12 myotubes","pmids":["19531644","19208911","19740738"],"confidence":"High","gaps":["Unexpected AMPK requirement for insulin-stimulated Thr596 left mechanistically unexplained","Did not resolve which Rab is targeted by each pathway"]},{"year":2010,"claim":"Site-resolved genetics and human exercise data assigned specific AMPK sites (Ser231/Ser660/Ser700) to contraction and Thr590 to insulin, and identified AMPKα2 as the principal isoform phosphorylating Ser237 in EDL.","evidence":"AMPKα2-inactive transgenic and Akt2-KO mice, AMPKα1/α2 knockouts, in vivo electroporation of phosphosite mutants, human exercise biopsies, and in vitro AMPK phosphorylation","pmids":["20701589","20837646"],"confidence":"High","gaps":["Reconciling α2 (EDL phosphorylation) vs α1 (stable complex) roles not addressed","Causal link from each site to GLUT4 movement not demonstrated"]},{"year":2010,"claim":"Dissecting the obesity-associated R125W and a quadruple phosphosite mutant in vivo demonstrated that insulin- and contraction-regulated glucose transport act through distinct mechanisms, both requiring the GAP domain.","evidence":"In vivo gene electroporation of mutants into mouse tibialis anterior with glucose transport assay","pmids":["20299473"],"confidence":"High","gaps":["Molecular basis of R125W effect not yet defined","Did not identify the disrupted binding partner"]},{"year":2011,"claim":"Crystal structures of the TBC1D1 and AS160 GAP domains plus mutagenesis pinpointed Rab-contact residues (Met930, Leu1019) required for both catalysis and GLUT4 translocation, linking structure to function.","evidence":"X-ray crystallography (2.2 Å), Ala-scanning mutagenesis, in vitro RabGAP and GLUT4 translocation assays","pmids":["21454505"],"confidence":"High","gaps":["No co-structure with a bound Rab","Did not include regulatory PTB/phospho regions"]},{"year":2012,"claim":"Double knockout of TBC1D1 and TBC1D4 nearly abolished insulin-stimulated glucose uptake, establishing the two RabGAPs as collectively essential and redundant for GLUT4-dependent uptake.","evidence":"TBC1D1/TBC1D4 double-knockout mice with hyperinsulinemic clamp, ex vivo glucose uptake, and cell-surface GLUT4 labeling","pmids":["25249576"],"confidence":"High","gaps":["Relative tissue-specific contribution of each paralog not quantified","Mechanism of GLUT4 protein loss not defined"]},{"year":2013,"claim":"Conventional knockout phenotyping fixed TBC1D1 as a determinant of glucose/lipid substrate preference, impairing glycolytic-muscle glucose uptake while raising oxidative-muscle fatty acid oxidation; reconstitution defined an AICAR-priming step conferring insulin responsiveness.","evidence":"Tbc1d1-KO mice with ex vivo glucose uptake, fatty acid oxidation, and calorimetry; GLUT4 nanometry reconstitution with Ser237 and PTB1 (R125W) mutants","pmids":["23892475","23325788"],"confidence":"High","gaps":["Molecular substrate effector of the lipid-oxidation shift not identified","Mechanism of PTB1-dependent priming not biochemically resolved"]},{"year":2014,"claim":"Defining APPL2 as a Ser235-phosphorylation-enhanced binding partner that suppresses Thr596 phosphorylation revealed an additional layer of negative regulation of insulin-evoked TBC1D1 inactivation.","evidence":"Reciprocal Co-IP, S235A mutagenesis, knockdown/overexpression in C2C12 myotubes, and muscle-specific APPL2-KO mice","pmids":["24879834"],"confidence":"High","gaps":["Structural basis of APPL2 BAR-domain binding not resolved","Physiological weight of this axis vs direct AMPK/Akt signaling unclear"]},{"year":2016,"claim":"A Ser231Ala knock-in separated AMPK-activator-stimulated from exercise-stimulated glucose uptake, and Rab28 was added as a physiological insulin-regulated GAP substrate.","evidence":"Tbc1d1 Ser231Ala knock-in mice with AICAR/exercise glucose-uptake assays; in vitro GAP assay, Rab28-GTP loading, and Rab28 manipulation in muscle/adipocytes","pmids":["27826658","27929607"],"confidence":"High","gaps":["Why exercise tolerates Ser231 loss but AICAR does not is unresolved","Rab28's vesicle-level mechanism not defined"]},{"year":2018,"claim":"Demonstrating a stable, direct AMPKα1–TBC1D1 complex via both PTB domains that boosts Ser237 phosphorylation, and that R125W weakens this binding, supplied the mechanistic basis for the obesity mutation's loss of signaling efficiency.","evidence":"Co-IP, GST pulldown with purified proteins, pharmacological AMPK activation, R125W/PTB mutagenesis, and in vitro kinase assays; plus GLUT4 nanometry showing TBC1D1 dominance over AS160","pmids":["30135087","30482843"],"confidence":"High","gaps":["Stoichiometry and structure of the AMPKα1–TBC1D1 complex unknown","How α1 anchoring relates to α2 catalytic phosphorylation not reconciled"]},{"year":2018,"claim":"In vitro reconstitution with full-length protein clarified that insulin/contraction phosphorylation does not change intrinsic RabGAP activity but instead disrupts the PTB-domain–IRAP interaction, reframing regulation as control of TBC1D1 recruitment to GLUT4 vesicles.","evidence":"Recombinant full-length TBC1D1 RabGAP and phosphorylation assays, 14-3-3 binding, and Co-IP/pulldown with the IRAP cytoplasmic tail","pmids":["30275018"],"confidence":"High","gaps":["In-cell validation of phosphorylation-dependent IRAP release not shown","Whether 14-3-3 directly competes with IRAP not resolved"]},{"year":2022,"claim":"Extending TBC1D1 beyond muscle, work in beta-cells and liver implicated it in insulin secretion and in AMPK-TBC1D1–Rab2A–PPARγ control of hepatic lipid accumulation, and additional kinases/interactors (WNK1, VPS13A/C) were tied to GLUT trafficking.","evidence":"Beta-cell siRNA and secretion assays; TBC1D1-S231A knock-in mice with Rab2A-GTP and PPARγ readouts; WNK1 RNAi/MS and Ser565 mutants; proteomic interactome with VPS13A/C knockdown","pmids":["24239544","35061665","31816312","33087848"],"confidence":"Medium","gaps":["Single-lab findings without independent replication","Mechanistic connection between Rab2A regulation and the canonical GLUT4 role unclear","VPS13/WNK1 interactions lack reciprocal in vivo validation"]},{"year":null,"claim":"How the distinct AMPKα1 anchoring versus AMPKα2 catalytic roles, the IRAP/14-3-3 recruitment switch, and the multiple Rab substrates are integrated into a single trafficking mechanism in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of TBC1D1 bound to its physiological Rab or to AMPK","Causal in vivo demonstration that phospho-dependent IRAP release liberates GLUT4 is lacking","Which Rab (Rab8a/10/14/28/2A) operates in each tissue and stimulus is undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,11,15,18,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,11,15,22]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,7,15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[15,24]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,12,13,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,7,8,18]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,15,28]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[13,27,28,29]}],"complexes":[],"partners":["IRAP","PRKAA1","YWHAZ","APPL2","VPS13A","VPS13C","WNK1","TBC1D4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86TI0","full_name":"TBC1 domain family member 1","aliases":[],"length_aa":1168,"mass_kda":133.1,"function":"May act as a GTPase-activating protein for Rab family protein(s). May play a role in the cell cycle and differentiation of various tissues. Involved in the trafficking and translocation of GLUT4-containing vesicles and insulin-stimulated glucose uptake into cells (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86TI0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TBC1D1","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":[{"gene":"CALM1","stoichiometry":0.2},{"gene":"CALM2","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TBC1D1","total_profiled":1310},"omim":[{"mim_id":"617939","title":"ZINC FINGER PROTEIN 69; ZFP69","url":"https://www.omim.org/entry/617939"},{"mim_id":"612465","title":"TBC1 DOMAIN FAMILY, MEMBER 4; TBC1D4","url":"https://www.omim.org/entry/612465"},{"mim_id":"609850","title":"TBC1 DOMAIN FAMILY, MEMBER 1; TBC1D1","url":"https://www.omim.org/entry/609850"},{"mim_id":"608410","title":"BODY MASS INDEX QUANTITATIVE TRAIT LOCUS 7; BMIQ7","url":"https://www.omim.org/entry/608410"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TBC1D1"},"hgnc":{"alias_symbol":["TBC","TBC1","KIAA1108"],"prev_symbol":[]},"alphafold":{"accession":"Q86TI0","domains":[{"cath_id":"2.30.29.30","chopping":"18-79_88-157","consensus_level":"high","plddt":77.7511,"start":18,"end":157},{"cath_id":"2.30.29.30","chopping":"170-203_276-379","consensus_level":"high","plddt":79.2023,"start":170,"end":379},{"cath_id":"-","chopping":"390-461","consensus_level":"medium","plddt":86.9682,"start":390,"end":461},{"cath_id":"1.10.8.270","chopping":"824-919","consensus_level":"medium","plddt":93.5304,"start":824,"end":919},{"cath_id":"1.10.472.80","chopping":"929-1071","consensus_level":"medium","plddt":94.8156,"start":929,"end":1071},{"cath_id":"1.10.287","chopping":"1083-1132","consensus_level":"medium","plddt":89.24,"start":1083,"end":1132}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86TI0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86TI0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86TI0-F1-predicted_aligned_error_v6.png","plddt_mean":70.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TBC1D1","jax_strain_url":"https://www.jax.org/strain/search?query=TBC1D1"},"sequence":{"accession":"Q86TI0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86TI0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86TI0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86TI0"}},"corpus_meta":[{"pmid":"18477703","id":"PMC_18477703","title":"Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic.","date":"2008","source":"American journal of physiology. 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Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25163918","citation_count":15,"is_preprint":false},{"pmid":"30135087","id":"PMC_30135087","title":"Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity.","date":"2018","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/30135087","citation_count":15,"is_preprint":false},{"pmid":"23374713","id":"PMC_23374713","title":"The TBC1D1 gene: structure, function, and association with obesity and related traits.","date":"2013","source":"Vitamins and hormones","url":"https://pubmed.ncbi.nlm.nih.gov/23374713","citation_count":14,"is_preprint":false},{"pmid":"34342853","id":"PMC_34342853","title":"Tris (2,3-Dibromopropyl) Isocyanurate (TDBP-TAZTO or TBC) Shows Different Toxicity Depending on the Degree of Differentiation of the Human Neuroblastoma (SH-SY5Y) Cell Line.","date":"2021","source":"Neurotoxicity research","url":"https://pubmed.ncbi.nlm.nih.gov/34342853","citation_count":14,"is_preprint":false},{"pmid":"22538644","id":"PMC_22538644","title":"TBC: a clustering algorithm based on prokaryotic taxonomy.","date":"2012","source":"Journal of microbiology (Seoul, Korea)","url":"https://pubmed.ncbi.nlm.nih.gov/22538644","citation_count":14,"is_preprint":false},{"pmid":"29296513","id":"PMC_29296513","title":"Vps34 and the Armus/TBC-2 Rab GAPs: Putting the brakes on the endosomal Rab5 and Rab7 GTPases.","date":"2017","source":"Cellular logistics","url":"https://pubmed.ncbi.nlm.nih.gov/29296513","citation_count":13,"is_preprint":false},{"pmid":"23364847","id":"PMC_23364847","title":"Exploratory study on association of genetic variation in TBC1D1 with antipsychotic-induced weight gain.","date":"2013","source":"Human psychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/23364847","citation_count":13,"is_preprint":false},{"pmid":"33023658","id":"PMC_33023658","title":"Increased glucose metabolism in Arid5b-/- skeletal muscle is associated with the down-regulation of TBC1 domain family member 1 (TBC1D1).","date":"2020","source":"Biological research","url":"https://pubmed.ncbi.nlm.nih.gov/33023658","citation_count":12,"is_preprint":false},{"pmid":"27089993","id":"PMC_27089993","title":"Tbc1d1 deletion suppresses obesity in leptin-deficient mice.","date":"2016","source":"International journal of obesity (2005)","url":"https://pubmed.ncbi.nlm.nih.gov/27089993","citation_count":11,"is_preprint":false},{"pmid":"17376030","id":"PMC_17376030","title":"Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways.","date":"2007","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/17376030","citation_count":11,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53402,"output_tokens":9687,"usd":0.152755,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":20049,"output_tokens":5822,"usd":0.122898,"stage2_stop_reason":"end_turn"},"total_usd":0.275653,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"TBC1D1 possesses Rab-GTPase-activating protein (GAP) activity; its GAP domain shares identical Rab substrate specificity with AS160 (TBC1D4). Ectopic expression of TBC1D1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect, demonstrating that this inhibition is GAP-activity-dependent. Insulin treatment leads to phosphorylation of TBC1D1 on a conserved Akt site.\",\n      \"method\": \"In vitro GAP activity assay, ectopic overexpression in 3T3-L1 adipocytes, point mutagenesis of catalytic residue, GLUT4 translocation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay combined with active-site mutagenesis and cell-based GLUT4 translocation functional readout in a single study\",\n      \"pmids\": [\"17274760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TBC1D1 is identified as the dominant PAS-immunoreactive protein at ~160 kDa in fast-twitch skeletal muscle (tibialis anterior, EDL) but not slow-twitch (soleus). In vivo stimulation by insulin, muscle contraction, and the AMPK activator AICAR each increased TBC1D1 PAS phosphorylation. Mass spectrometry identified multiple novel phosphorylation sites on TBC1D1, the majority being consensus or near-consensus AMPK sites. Purified Akt and AMPK both phosphorylate TBC1D1 in vitro, but AMPK (not Akt) caused a detectable shift in TBC1D1 electrophoretic mobility.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, in vitro kinase assay with purified Akt and AMPK, in vivo stimulation (insulin, contraction, AICAR) with PAS immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay combined with in vivo phosphorylation mapping by MS and multiple stimulation conditions\",\n      \"pmids\": [\"18276596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AMPK activation in HEK-293 cells promotes 14-3-3 binding primarily to phospho-Ser237 of TBC1D1, while IGF-1/EGF/PMA promote 14-3-3 binding primarily via phospho-Thr596. In rat L6 myotubes, AMPK activators (AICAR, phenformin, A-769662) strongly phosphorylate Ser237 and promote 14-3-3 binding, whereas insulin promotes Thr596 phosphorylation but not 14-3-3 binding. In vitro phosphorylation experiments showed regulatory cross-talk among sites (e.g., phospho-Ser235 prevents subsequent phospho-Ser237).\",\n      \"method\": \"Site-specific phosphorylation analysis in HEK-293 and L6 cells, 14-3-3 binding assays, in vitro phosphorylation with purified kinases, pharmacological inhibition (LY294002)\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro kinase assay, cell-based phosphorylation, 14-3-3 binding) with site-specific resolution across multiple cell types\",\n      \"pmids\": [\"17995453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Overexpressed TBC1D1 in 3T3-L1 adipocytes inhibits GLUT4 translocation even in response to activated Akt, and endogenous TBC1D1 (which is ~20-fold less abundant than AS160 in adipocytes) does not regulate insulin-stimulated GLUT4 translocation. AMPK activator AICAR partially reversed the inhibition of GLUT4 translocation caused by overexpressed TBC1D1. TBC1D1 is much more highly expressed in skeletal muscle than fat.\",\n      \"method\": \"RNAi knockdown of TBC1D1 in 3T3-L1 adipocytes, overexpression with constitutively active Akt, AICAR treatment, GLUT4 translocation assay, quantitative western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain- and loss-of-function experiments in adipocytes with defined functional readout, replicated across multiple experimental conditions\",\n      \"pmids\": [\"18258599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TBC1D1 is an Akt substrate phosphorylated at Thr590 in 3T3-L1 adipocytes. RNAi silencing of TBC1D1 elevated basal 2-deoxyglucose uptake (~61%) and strongly increased GLUT1 (but not GLUT4) expression. Loss of TBC1D1 activated the mTOR–p70 S6K pathway, and the GLUT1 upregulation was blocked by rapamycin. Overexpression of the phosphorylation-defective T590A mutant inhibited insulin-stimulated p70 S6K phosphorylation.\",\n      \"method\": \"Mass spectrometry-based phosphoproteomic identification of Akt substrates, RNAi knockdown, rapamycin treatment, overexpression of T590A mutant, glucose uptake assay, western blotting\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — MS identification of phosphosite combined with RNAi, pharmacological rescue (rapamycin), and phosphomutant overexpression in a single study\",\n      \"pmids\": [\"18215134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In vivo gene electroporation into mouse tibialis anterior showed that the obesity-associated R125W mutant TBC1D1 significantly decreased insulin-stimulated glucose transport through a mechanism requiring the GAP domain (disrupting GAP activity in the R125W background rescued glucose transport). A TBC1D1 quadruple phosphorylation-site mutant (4P, Ala substitutions at four AMPK/Akt sites) had no effect on insulin-stimulated transport but decreased contraction-stimulated glucose transport via the GAP domain, establishing that insulin- and contraction-regulated glucose transport occur via distinct mechanisms.\",\n      \"method\": \"In vivo gene injection/electroporation into mouse tibialis anterior, in vivo glucose transport assay, mutagenesis of phosphorylation sites and GAP domain\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mutagenesis study with defined functional readout, mechanistic dissection of insulin vs contraction pathways using multiple mutants\",\n      \"pmids\": [\"20299473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Insulin-stimulated phosphorylation of multiple Akt sites on TBC1D1 is required for GLUT4 translocation: a TBC1D1 mutant with several Akt sites converted to alanine was considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1 in C2C12 myotubes. AMPK activation partially relieved TBC1D1-mediated inhibition of GLUT4 translocation. Akt sites on TBC1D1 were identified by mass spectrometry from C2C12 myotubes.\",\n      \"method\": \"Mass spectrometry identification of Akt phosphorylation sites in C2C12 myotubes, alanine-substitution mutagenesis, GLUT4 translocation assay, AMPK activator treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — MS phosphosite identification combined with mutagenesis and functional GLUT4 translocation assay in muscle cells\",\n      \"pmids\": [\"19740738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In mouse EDL muscle, both contraction- and insulin-stimulated TBC1D1 Ser237 and Thr596 phosphorylation, and 14-3-3 protein binding to TBC1D1, are abolished in AMPK kinase-dead transgenic mice. AICAR and contraction induced comparable phosphorylation patterns; insulin increased Thr596 but not Ser237 phosphorylation, and insulin-stimulated Thr596 phosphorylation was fully abolished in AMPK KD mice, revealing an unexpected AMPK requirement for insulin-stimulated TBC1D1 Thr596 phosphorylation.\",\n      \"method\": \"Genetic epistasis using AMPK kinase-dead transgenic mice, site-specific phospho-antibodies for Ser237 and Thr596, 14-3-3 binding assay, ex vivo muscle contraction/AICAR/insulin stimulation\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function (AMPK KD transgenic) with site-specific phosphorylation readouts across multiple stimuli, providing definitive upstream kinase assignment\",\n      \"pmids\": [\"19531644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In rat epitrochlearis muscle, contraction-stimulated PAS-TBC1D1 phosphorylation and glucose transport are abolished by the AMPK inhibitor compound C, but are unaffected by Wortmannin (PI3-kinase/Akt inhibitor). Conversely, insulin-stimulated phosphorylation of both AS160 and TBC1D1 requires PI3-kinase/Akt. This establishes that contraction regulates TBC1D1 and glucose transport through an AMPK-dependent, Akt-independent mechanism.\",\n      \"method\": \"Pharmacological inhibition (compound C for AMPK, Wortmannin for PI3K) in isolated rat skeletal muscle, PAS immunoblotting, 3-O-methylglucose transport assay\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dual pharmacological epistasis with orthogonal kinase inhibitors, clear separation of insulin vs contraction pathway mechanisms\",\n      \"pmids\": [\"19208911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In mouse skeletal muscle, contraction increases TBC1D1 phosphorylation on Ser231 and Ser660 (AMPK sites) but not Thr590 (Akt site). AICAR phosphorylates Ser231, Ser660, and Ser700 but not Thr590; insulin selectively increases Thr590. Contraction-stimulated Ser231, Ser660, and Ser700 phosphorylation is greatly reduced in AMPKα2-inactive transgenic mice, and Akt2-KO mice show blunted insulin-stimulated Thr590 phosphorylation. In vivo overexpression of a TBC1D1 mutated on four AMPK sites decreased glucose uptake in tibialis anterior.\",\n      \"method\": \"Site-specific phospho-antibodies, AMPKα2-inactive transgenic mice, Akt2-KO mice, in vivo electroporation/overexpression of phosphosite mutants, glucose uptake assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic models (AMPKα2i TG, Akt2 KO) combined with site-specific phosphorylation analysis and in vivo functional readout\",\n      \"pmids\": [\"20701589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Exercise in human skeletal muscle increases TBC1D1 Ser237 phosphorylation (70–230%) and 14-3-3 binding capacity (60–250%) in an exercise-duration-dependent manner. Recombinant AMPK directly phosphorylates Ser237 and induces 14-3-3 binding in vitro. In AMPKα2-knockout (but not α1-knockout) mouse EDL, basal TBC1D1 protein levels and contraction-stimulated Ser237 phosphorylation are reduced, identifying AMPKα2 as the principal isoform regulating TBC1D1 Ser237 in EDL.\",\n      \"method\": \"Human exercise biopsy study (30 s, 2 min, 20 min cycling), in vitro AMPK phosphorylation assay, AMPKα1/α2 whole-body knockout mice, site-specific phospho-antibody, 14-3-3 binding overlay assay\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay combined with human exercise data and genetic knockout confirmation of isoform specificity\",\n      \"pmids\": [\"20837646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of the RabGAP domains of human TBC1D1 (2.2 Å) and TBC1D4/AS160 (3.5 Å) were solved. Both have 16 α-helices and no β-sheet elements. Ala-scanning mutagenesis of inferred Rab-binding interface residues showed only one of five substitutions significantly perturbed catalytic efficiency; substitution of TBC1D1 Met930 outside the canonical yeast interface substantially reduced catalytic activity, and the M930A mutant also failed to promote GLUT4 translocation. Additional residue Leu1019 was predicted to contact Rab; mutants with lowest RabGAP activity confirmed these contacts are required for biological activity.\",\n      \"method\": \"X-ray crystallography (2.2 Å for TBC1D1 GAP domain), Ala-scanning mutagenesis, in vitro RabGAP activity assay, GLUT4 translocation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determination combined with mutagenesis-validated enzymatic activity and cell-based functional assay in a single study\",\n      \"pmids\": [\"21454505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The Rab-GTPase-activating proteins TBC1D1 and TBC1D4 together are essential for insulin-stimulated glucose uptake; double-knockout mice (D1/4KO) showed almost complete abolition of insulin-stimulated glucose uptake in skeletal muscle and adipocytes, whereas single knockouts showed only partial impairment. GLUT4 protein (but not mRNA) was substantially reduced in D1/4KO skeletal muscle and white adipose tissue. Cell surface labeling indicated that RabGAP deficiency impairs intracellular GLUT4 retention in the basal state.\",\n      \"method\": \"Generation of double-knockout (TBC1D1-/-/TBC1D4-/-) mice, hyperinsulinemic clamp, isolated skeletal muscle glucose uptake, cell surface GLUT4 labeling, GLUT4 protein/mRNA quantification\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic double-KO combined with multiple functional readouts (in vivo clamp, ex vivo glucose uptake, cell surface GLUT4 labeling)\",\n      \"pmids\": [\"25249576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Conventional Tbc1d1-knockout mice show severely impaired insulin- and AICAR-stimulated glucose uptake in EDL (glycolytic) but not soleus muscle, and substantially increased fatty acid oxidation in oxidative soleus muscle. These mice had moderately reduced body weight, decreased respiratory quotient, and elevated resting metabolic rate, establishing that TBC1D1 plays a major role in glucose and lipid substrate preference in skeletal muscle.\",\n      \"method\": \"Conventional Tbc1d1-knockout mice, ex vivo glucose uptake assay (EDL, soleus), fatty acid oxidation assay, indirect calorimetry, body weight measurement\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conventional KO with multiple orthogonal metabolic readouts and muscle-type specificity established\",\n      \"pmids\": [\"23892475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AICAR treatment prior to insulin stimulation enables TBC1D1 to acquire insulin responsiveness, triggering GLUT4 trafficking. This regulatory mode shift requires Ser237 phosphorylation and an intact phosphotyrosine-binding 1 (PTB1) domain of TBC1D1. Mutations in PTB1, including the obesity-associated R125W, abolish the acquisition of insulin responsiveness while leaving AICAR-responsive GLUT4-liberation activity intact.\",\n      \"method\": \"GLUT4 nanometry in cell-based reconstitution model, AICAR and insulin stimulation, site-directed mutagenesis of Ser237 and PTB1 domain (including R125W), temporal GLUT4 tracking\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — functional reconstitution with GLUT4 nanometry, multiple mutants, mechanistically resolved AICAR-to-insulin priming sequence\",\n      \"pmids\": [\"23325788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TBC1D1 is phosphorylated by AKT and AMPK in response to insulin and muscle contraction, and both phosphorylation events increase 14-3-3 binding but do not alter intrinsic RabGAP activity on Rab8a, Rab10, and Rab14. Full-length TBC1D1 shows markedly higher catalytic activity toward Rab8a, Rab10, and Rab14 than the isolated GAP domain. TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic tail of insulin-regulated aminopeptidase (IRAP), a resident of GLUT4 storage vesicles, and this interaction is disrupted by AKT or AMPK phosphorylation, suggesting that TBC1D1 recruitment to GLUT4 vesicles (not GAP activity per se) is regulated by insulin/contraction signaling.\",\n      \"method\": \"Baculovirus/Sf9 expression of recombinant full-length TBC1D1, in vitro RabGAP assay, in vitro phosphorylation by AKT/AMPK, 14-3-3 binding assay, co-immunoprecipitation with IRAP cytoplasmic tail, PTB domain pulldown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified full-length protein, enzymatic assay, direct protein-protein interaction mapping, and phosphorylation-dependent disruption in a single rigorous study\",\n      \"pmids\": [\"30275018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TBC1D1-knockout mice show impaired exercise-induced 2-deoxyglucose uptake specifically in white (non-oxidative) but not red skeletal muscle, and GLUT4 protein levels are reduced ~40% in white TBC1D1-/- muscle. TBC1D1-/- mice also display impaired exercise endurance. Normal body weight and glucose/insulin tolerance indicate TBC1D1 is dispensable for basal and insulin-stimulated glucose homeostasis under normal conditions.\",\n      \"method\": \"TBC1D1-knockout mouse generation, in vivo exercise-stimulated 2-deoxyglucose uptake, glucose/insulin tolerance tests, GLUT4 protein quantification, treadmill endurance test\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO model with in vivo exercise functional readout, muscle-type specificity demonstrated, multiple metabolic phenotypes characterized\",\n      \"pmids\": [\"25576050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TBC1D1 deficiency in EDL but not soleus muscle impairs insulin-, AICAR-, and contraction-stimulated glucose transport. In TBC1D1-deficient (Nob1.10SJL) congenic mice, suppression of hepatic glucose production during hyperinsulinemic clamp was increased. A 50% reduction in GLUT4 protein in EDL from TBC1D1-deficient mice was identified as a mechanism for reduced glucose transport, with proximal AMPK/Akt signaling unaltered.\",\n      \"method\": \"TBC1D1-deficient congenic mouse model, euglycemic hyperinsulinemic clamp, isolated EDL/soleus glucose transport, GLUT4 protein quantification, proximal signaling analysis\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo clamp combined with ex vivo muscle-specific glucose transport and protein-level mechanistic analysis\",\n      \"pmids\": [\"22693207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMPK heterotrimers containing the α1 (but not α2) catalytic subunit form a stable, direct association with TBC1D1 but not its paralogue AS160. The interaction involves both PTB domains of TBC1D1 and is enhanced by AMPK activators (AICAR, A769662). This AMPKα1-TBC1D1 complex increases the efficiency of AMPK-mediated Ser237 phosphorylation. The obesity-associated R125W mutation in PTB1 reduces AMPKα1 binding and concomitantly reduces Ser237 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown with purified proteins (direct interaction), pharmacological AMPK activation, site-directed mutagenesis (R125W, PTB domain mutants), in vitro kinase assay, quantitative phosphorylation analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct protein-protein interaction with purified components, isoform-specific genetic and pharmacological dissection, mutagenesis-validated mechanism in a single study\",\n      \"pmids\": [\"30135087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In cells with both TBC1D1 and AS160, TBC1D1 functionally dominates AS160 in controlling GLUT4 release. AICAR and intracellular Ca2+ serve as proximal stimuli for TBC1D1-governed GLUT4 release. AS160 modulates sensitivity to external stimuli in TBC1D1-mediated GLUT4 release. The synergistic cooperative actions depend on TBC1D1 PTB1 domain, calmodulin-binding domain, and phosphorylation of AS160 Thr642 and TBC1D1 Ser237/Thr596.\",\n      \"method\": \"GLUT4 nanometry in cell-based reconstitution models with varying TBC1D1:AS160 expression ratios, site-directed mutagenesis (PTB1, calmodulin-binding domain, phosphosites), AICAR and Ca2+ stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — GLUT4 nanometry reconstitution combined with systematic mutagenesis of multiple regulatory domains and quantitative functional analysis\",\n      \"pmids\": [\"30482843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A Tbc1d1 Ser231Ala knock-in mutation attenuates AICAR-induced glucose lowering and reduces AICAR-stimulated glucose uptake and GLUT4 cell surface content in isolated skeletal muscle. However, the Ser231Ala mutation does not impair exercise-induced muscle glucose uptake or exercise capacity, demonstrating that Ser231 phosphorylation is specifically required for AMPK-activator- but not exercise-stimulated glucose uptake.\",\n      \"method\": \"Tbc1d1 Ser231Ala knock-in mice, AICAR stimulation in vivo and ex vivo, exercise-stimulated glucose uptake, cell surface GLUT4 labeling, glucose tolerance test\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — precision knock-in mouse model with site-specific mechanistic dissection of AICAR vs. exercise stimuli, multiple functional readouts\",\n      \"pmids\": [\"27826658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"APPL2 interacts with TBC1D1 through its BAR domain. Insulin stimulates TBC1D1 phosphorylation on Ser235, which enhances APPL2 binding; this APPL2–TBC1D1 interaction suppresses insulin-evoked TBC1D1 Thr596 phosphorylation. Substitution of Ser235 with alanine diminishes APPL2-mediated inhibition of Thr596 phosphorylation and reverses the suppressive effects of TBC1D1 on insulin-induced GLUT4 translocation and glucose uptake in myotubes.\",\n      \"method\": \"Co-immunoprecipitation of APPL2 and TBC1D1, site-directed mutagenesis (S235A), overexpression and knockdown in C2C12 myotubes, GLUT4 translocation assay, glucose uptake assay, conditional muscle-specific APPL2 KO mice\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, phosphosite mutagenesis, and functional GLUT4/glucose uptake readouts in cells and conditional KO mice\",\n      \"pmids\": [\"24879834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rab28 is identified as an in vitro substrate for the GAP domains of both TBC1D1 and TBC1D4. Rab28 GTP-loading state is acutely regulated by insulin in skeletal muscle. siRNA-mediated knockdown of Rab28 in isolated mouse skeletal muscle decreases basal glucose uptake, and constitutively active Rab28-Q72L in adipocytes increases basal cell surface HA-GLUT4.\",\n      \"method\": \"In vitro GAP assay with purified Rab28 and TBC1D1/TBC1D4 GAP domains, Rab28-GTP loading assay in muscle, siRNA knockdown of Rab28 in skeletal muscle, overexpression of Rab28-Q72L in adipocytes, cell surface GLUT4 labeling\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay identifying substrate combined with in vivo Rab GTP-state measurement and bidirectional functional manipulation\",\n      \"pmids\": [\"27929607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WNK1 kinase phosphorylates TBC1D1 on Ser565 (identified by mass spectrometry). Phosphomimetic and unphosphorylatable mutants of TBC1D1 at S565 both affected GLUT1 cell surface abundance, indicating a regulatory role for WNK1-mediated TBC1D1 phosphorylation in constitutive GLUT1 trafficking.\",\n      \"method\": \"WNK1 RNAi in HEK293 cells, mass spectrometry identification of Ser565 phosphosite, transfection of phosphomimetic/unphosphorylatable TBC1D1 mutants, cell surface GLUT1 quantification\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS phosphosite identification with functional mutagenesis in cells, single lab, single study\",\n      \"pmids\": [\"31816312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TBC1D1 interacts with VPS13A and VPS13C, the Rab-binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1 in C2C12 myotubes. These interactions are mediated through the PTB domains of TBC1D1 and are not affected by AMPK activation, distinguishing them from the AMPK-regulated AMPKα1–TBC1D1 interaction. Depletion of VPS13A or VPS13C post-transcriptionally increases cellular GLUT4 protein and enhances cell surface GLUT4 in response to AMPK activation.\",\n      \"method\": \"Unbiased quantitative proteomics/co-immunoprecipitation to identify TBC1D1 interactors in C2C12 myotubes, siRNA depletion of VPS13A/VPS13C, cell surface GLUT4 assay, AMPK activation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — unbiased interactome MS combined with functional knockdown and GLUT4 readout, single lab\",\n      \"pmids\": [\"33087848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TBC1D1 is expressed in pancreatic beta-cells and is phosphorylated in response to glucose. siRNA knockdown of TBC1D1 in beta-cells increased basal and glucose-stimulated insulin secretion and decreased beta-cell proliferation without affecting apoptosis.\",\n      \"method\": \"TBC1D1 expression analysis in human and rat beta-cells, phosphorylation assay (glucose stimulation), siRNA knockdown, insulin secretion assay, proliferation and apoptosis assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA knockdown with functional readouts in beta-cells, single lab, limited mechanistic pathway placement\",\n      \"pmids\": [\"24239544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AMPK-TBC1D1 signaling regulates GTP-loading of Rab2A: nutrition repletion suppresses AMPK-TBC1D1 phosphorylation, increasing GTP-bound Rab2A, which stabilizes PPARγ protein and promotes hepatic lipid accumulation. In TBC1D1-S231A knock-in mice, increased GTP-Rab2A and fatty liver were observed. Inhibition of Rab2A expression alleviated hepatic lipid deposition in obese mice.\",\n      \"method\": \"TBC1D1-S231A knock-in mice, GTP-Rab2A pulldown assay, siRNA knockdown of Rab2A in hepatic cells and mice, western blotting for PPARγ stability, in vitro and in vivo lipid accumulation assays\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mouse model combined with Rab2A GTP-state measurements and functional rescue, single lab\",\n      \"pmids\": [\"35061665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Overexpression of TBC1D1 in mouse soleus muscle decreased basal and AICAR-stimulated palmitate oxidation by ~18–22% and increased glucose oxidation, without altering FAT/CD36, mitochondrial content, CPT1, AMPK, or ACC. TBC1D1-mediated reduction of fatty acid oxidation was associated with reduced β-hydroxyacyl-CoA dehydrogenase (β-HAD) enzyme activity.\",\n      \"method\": \"Electrotransfection of TBC1D1 cDNA into mouse soleus, palmitate oxidation assay, glucose oxidation assay, β-HAD enzyme activity measurement, protein expression analysis\",\n      \"journal\": \"American journal of physiology. Regulatory, integrative and comparative physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vivo overexpression with biochemical enzyme activity readout, single lab, single method for the β-HAD finding\",\n      \"pmids\": [\"25163918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ablation of TBC1D1 in rats impairs contraction-induced sarcolemmal GLUT4 redistribution and glucose uptake specifically in white gastrocnemius muscle but does not alter insulin-induced GLUT4 trafficking or whole-body insulin tolerance. TBC1D1-KO rats show increased skeletal muscle fatty acid oxidation and increased maximal ADP-stimulated mitochondrial respiration, but reduced exercise run time to exhaustion.\",\n      \"method\": \"Rat TBC1D1 KO model, sarcolemmal GLUT4 fractionation after contraction/insulin, in vivo insulin tolerance test, ex vivo fatty acid oxidation, permeabilized fiber respiration, treadmill exercise\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO animal model with direct GLUT4 membrane fractionation, multiple metabolic functional readouts, and contraction vs insulin pathway dissection\",\n      \"pmids\": [\"28808062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The SJL mouse strain carries a naturally occurring mutation in Tbc1d1 resulting in a truncated protein lacking the TBC RabGAP domain. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, while overexpression had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation, and reduced glucose uptake in isolated skeletal muscle.\",\n      \"method\": \"Identification of naturally occurring Tbc1d1 truncation mutation in SJL strain, siRNA knockdown and overexpression in skeletal muscle cells, fatty acid uptake and oxidation assays, isolated muscle glucose uptake, indirect calorimetry in recombinant congenic mice\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — naturally occurring loss-of-function mutation combined with bidirectional manipulation in cells and in vivo metabolic phenotyping\",\n      \"pmids\": [\"18931681\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TBC1D1 is a Rab GTPase-activating protein (RabGAP) predominantly expressed in fast-twitch skeletal muscle that functions as a key regulator of GLUT4 vesicle trafficking and glucose/lipid substrate utilization: its GAP activity toward Rab8a, Rab10, Rab14, and Rab28 retains GLUT4 in intracellular storage vesicles, and this activity is modulated by site-specific phosphorylation—AMPK phosphorylates Ser231/Ser237/Ser660/Ser700 in response to muscle contraction and AICAR, while Akt2 phosphorylates Thr590/Thr596 in response to insulin—leading to 14-3-3 protein binding and disruption of TBC1D1 interaction with the GLUT4 vesicle resident protein IRAP, thereby promoting GLUT4 translocation to the sarcolemma; the obesity-associated R125W mutation in the PTB1 domain impairs AMPKα1 association, reduces Ser237 phosphorylation efficiency, and abolishes exercise-priming acquisition of insulin responsiveness, while genetic ablation of TBC1D1 in mice and rats abolishes contraction-stimulated but not insulin-stimulated GLUT4 translocation in glycolytic muscle fibers and shifts substrate utilization toward enhanced fatty acid oxidation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TBC1D1 is a Rab-GTPase-activating protein (RabGAP) that gates GLUT4 vesicle trafficking and substrate selection in skeletal muscle, acting as a convergence point for insulin and contraction/AMPK signaling [#0, #13]. Its GAP domain, whose crystal structure reveals an all-\\u03b1-helical fold, hydrolyzes GTP on Rab8a, Rab10, Rab14, and Rab28, and full-length protein is markedly more active than the isolated domain; this catalytic activity is required to retain GLUT4 intracellularly, since overexpressed wild-type TBC1D1 blocks GLUT4 translocation while a GAP-dead mutant does not [#0, #11, #15, #22]. TBC1D1 is the dominant phospho-Akt-substrate species in fast-twitch (but not slow-twitch) muscle, where AMPK phosphorylates Ser231/Ser237/Ser660/Ser700 in response to contraction and AICAR, and Akt2 phosphorylates Thr590/Thr596 in response to insulin [#1, #9, #10]. These phosphorylation events drive 14-3-3 binding without altering intrinsic RabGAP activity, instead disrupting the PTB-domain-mediated interaction with the GLUT4-vesicle protein IRAP, thereby releasing GLUT4 for translocation [#2, #15]. AMPK\\u03b11 (not \\u03b12) forms a stable direct complex with TBC1D1 via its PTB domains that enhances Ser237 phosphorylation efficiency [#18]. Genetic ablation of TBC1D1 in mice and rats selectively abolishes contraction/AICAR-stimulated GLUT4 translocation and glucose uptake in glycolytic muscle, reduces GLUT4 protein, impairs exercise endurance, and shifts substrate use toward fatty acid oxidation, while TBC1D1/TBC1D4 double knockout almost completely abolishes insulin-stimulated glucose uptake [#12, #13, #16, #28]. The obesity-associated R125W mutation in the PTB1 domain impairs AMPK\\u03b11 association, reduces Ser237 phosphorylation, and abolishes the AICAR/exercise-priming acquisition of insulin responsiveness [#14, #18]. Beyond muscle, TBC1D1 also functions in pancreatic beta-cells and in hepatic lipid metabolism through AMPK-TBC1D1 control of Rab2A and PPAR\\u03b3 [#25, #26].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that TBC1D1 is a catalytically active RabGAP whose enzymatic activity gates GLUT4 trafficking answered whether it is a functional regulator rather than an inert AS160 paralog.\",\n      \"evidence\": \"In vitro GAP assay, catalytic-residue mutagenesis, and GLUT4 translocation assay in 3T3-L1 adipocytes\",\n      \"pmids\": [\"17274760\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological Rab substrates in muscle\", \"Performed in adipocytes where TBC1D1 is minimally expressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying TBC1D1 as the dominant fast-twitch-muscle PAS protein regulated by insulin, contraction, and AICAR, and mapping mostly AMPK-consensus phosphosites, placed it at the intersection of insulin and energy-sensing signaling.\",\n      \"evidence\": \"Immunoprecipitation, mass-spectrometry phosphosite mapping, in vitro Akt/AMPK kinase assays, and in vivo stimulation with PAS immunoblotting\",\n      \"pmids\": [\"18276596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of individual sites not yet resolved\", \"Did not establish which kinase acts in vivo\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolving the stimulus-specific phosphorylation code (AMPK\\u2192Ser237/14-3-3 binding vs insulin\\u2192Thr596) and showing endogenous TBC1D1 does not govern insulin-stimulated GLUT4 in adipocytes refined where and how TBC1D1 acts.\",\n      \"evidence\": \"Site-specific phosphorylation and 14-3-3 binding assays in HEK293/L6 cells, plus RNAi and overexpression in 3T3-L1 adipocytes\",\n      \"pmids\": [\"17995453\", \"18258599\", \"18215134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not link 14-3-3 binding to a downstream trafficking step\", \"Tissue site of physiological action (muscle) not yet tested genetically\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"A naturally occurring SJL Tbc1d1 truncation lacking the GAP domain linking TBC1D1 to body weight and fatty acid oxidation provided the first in vivo evidence that it controls substrate utilization.\",\n      \"evidence\": \"Identification of SJL truncation, bidirectional manipulation in muscle cells, and metabolic phenotyping of congenic mice\",\n      \"pmids\": [\"18931681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking GAP loss to fatty acid oxidation not defined\", \"Confounded by other strain background loci\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Genetic and pharmacological epistasis assigned contraction-stimulated TBC1D1 phosphorylation and glucose transport to an AMPK-dependent, Akt-independent route distinct from the insulin/PI3K route.\",\n      \"evidence\": \"AMPK kinase-dead transgenic mice, compound C and Wortmannin inhibition, and site-specific phospho-antibodies in isolated muscle; MS phosphosite mapping and mutagenesis in C2C12 myotubes\",\n      \"pmids\": [\"19531644\", \"19208911\", \"19740738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Unexpected AMPK requirement for insulin-stimulated Thr596 left mechanistically unexplained\", \"Did not resolve which Rab is targeted by each pathway\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Site-resolved genetics and human exercise data assigned specific AMPK sites (Ser231/Ser660/Ser700) to contraction and Thr590 to insulin, and identified AMPK\\u03b12 as the principal isoform phosphorylating Ser237 in EDL.\",\n      \"evidence\": \"AMPK\\u03b12-inactive transgenic and Akt2-KO mice, AMPK\\u03b11/\\u03b12 knockouts, in vivo electroporation of phosphosite mutants, human exercise biopsies, and in vitro AMPK phosphorylation\",\n      \"pmids\": [\"20701589\", \"20837646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciling \\u03b12 (EDL phosphorylation) vs \\u03b11 (stable complex) roles not addressed\", \"Causal link from each site to GLUT4 movement not demonstrated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Dissecting the obesity-associated R125W and a quadruple phosphosite mutant in vivo demonstrated that insulin- and contraction-regulated glucose transport act through distinct mechanisms, both requiring the GAP domain.\",\n      \"evidence\": \"In vivo gene electroporation of mutants into mouse tibialis anterior with glucose transport assay\",\n      \"pmids\": [\"20299473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of R125W effect not yet defined\", \"Did not identify the disrupted binding partner\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystal structures of the TBC1D1 and AS160 GAP domains plus mutagenesis pinpointed Rab-contact residues (Met930, Leu1019) required for both catalysis and GLUT4 translocation, linking structure to function.\",\n      \"evidence\": \"X-ray crystallography (2.2 \\u00c5), Ala-scanning mutagenesis, in vitro RabGAP and GLUT4 translocation assays\",\n      \"pmids\": [\"21454505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-structure with a bound Rab\", \"Did not include regulatory PTB/phospho regions\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Double knockout of TBC1D1 and TBC1D4 nearly abolished insulin-stimulated glucose uptake, establishing the two RabGAPs as collectively essential and redundant for GLUT4-dependent uptake.\",\n      \"evidence\": \"TBC1D1/TBC1D4 double-knockout mice with hyperinsulinemic clamp, ex vivo glucose uptake, and cell-surface GLUT4 labeling\",\n      \"pmids\": [\"25249576\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative tissue-specific contribution of each paralog not quantified\", \"Mechanism of GLUT4 protein loss not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Conventional knockout phenotyping fixed TBC1D1 as a determinant of glucose/lipid substrate preference, impairing glycolytic-muscle glucose uptake while raising oxidative-muscle fatty acid oxidation; reconstitution defined an AICAR-priming step conferring insulin responsiveness.\",\n      \"evidence\": \"Tbc1d1-KO mice with ex vivo glucose uptake, fatty acid oxidation, and calorimetry; GLUT4 nanometry reconstitution with Ser237 and PTB1 (R125W) mutants\",\n      \"pmids\": [\"23892475\", \"23325788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular substrate effector of the lipid-oxidation shift not identified\", \"Mechanism of PTB1-dependent priming not biochemically resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defining APPL2 as a Ser235-phosphorylation-enhanced binding partner that suppresses Thr596 phosphorylation revealed an additional layer of negative regulation of insulin-evoked TBC1D1 inactivation.\",\n      \"evidence\": \"Reciprocal Co-IP, S235A mutagenesis, knockdown/overexpression in C2C12 myotubes, and muscle-specific APPL2-KO mice\",\n      \"pmids\": [\"24879834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of APPL2 BAR-domain binding not resolved\", \"Physiological weight of this axis vs direct AMPK/Akt signaling unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A Ser231Ala knock-in separated AMPK-activator-stimulated from exercise-stimulated glucose uptake, and Rab28 was added as a physiological insulin-regulated GAP substrate.\",\n      \"evidence\": \"Tbc1d1 Ser231Ala knock-in mice with AICAR/exercise glucose-uptake assays; in vitro GAP assay, Rab28-GTP loading, and Rab28 manipulation in muscle/adipocytes\",\n      \"pmids\": [\"27826658\", \"27929607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why exercise tolerates Ser231 loss but AICAR does not is unresolved\", \"Rab28's vesicle-level mechanism not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating a stable, direct AMPK\\u03b11\\u2013TBC1D1 complex via both PTB domains that boosts Ser237 phosphorylation, and that R125W weakens this binding, supplied the mechanistic basis for the obesity mutation's loss of signaling efficiency.\",\n      \"evidence\": \"Co-IP, GST pulldown with purified proteins, pharmacological AMPK activation, R125W/PTB mutagenesis, and in vitro kinase assays; plus GLUT4 nanometry showing TBC1D1 dominance over AS160\",\n      \"pmids\": [\"30135087\", \"30482843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structure of the AMPK\\u03b11\\u2013TBC1D1 complex unknown\", \"How \\u03b11 anchoring relates to \\u03b12 catalytic phosphorylation not reconciled\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"In vitro reconstitution with full-length protein clarified that insulin/contraction phosphorylation does not change intrinsic RabGAP activity but instead disrupts the PTB-domain\\u2013IRAP interaction, reframing regulation as control of TBC1D1 recruitment to GLUT4 vesicles.\",\n      \"evidence\": \"Recombinant full-length TBC1D1 RabGAP and phosphorylation assays, 14-3-3 binding, and Co-IP/pulldown with the IRAP cytoplasmic tail\",\n      \"pmids\": [\"30275018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell validation of phosphorylation-dependent IRAP release not shown\", \"Whether 14-3-3 directly competes with IRAP not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extending TBC1D1 beyond muscle, work in beta-cells and liver implicated it in insulin secretion and in AMPK-TBC1D1\\u2013Rab2A\\u2013PPAR\\u03b3 control of hepatic lipid accumulation, and additional kinases/interactors (WNK1, VPS13A/C) were tied to GLUT trafficking.\",\n      \"evidence\": \"Beta-cell siRNA and secretion assays; TBC1D1-S231A knock-in mice with Rab2A-GTP and PPAR\\u03b3 readouts; WNK1 RNAi/MS and Ser565 mutants; proteomic interactome with VPS13A/C knockdown\",\n      \"pmids\": [\"24239544\", \"35061665\", \"31816312\", \"33087848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab findings without independent replication\", \"Mechanistic connection between Rab2A regulation and the canonical GLUT4 role unclear\", \"VPS13/WNK1 interactions lack reciprocal in vivo validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct AMPK\\u03b11 anchoring versus AMPK\\u03b12 catalytic roles, the IRAP/14-3-3 recruitment switch, and the multiple Rab substrates are integrated into a single trafficking mechanism in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of TBC1D1 bound to its physiological Rab or to AMPK\", \"Causal in vivo demonstration that phospho-dependent IRAP release liberates GLUT4 is lacking\", \"Which Rab (Rab8a/10/14/28/2A) operates in each tissue and stimulus is undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 11, 15, 18, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 11, 15, 22]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 7, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [15, 24]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 12, 13, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 7, 8, 18]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 15, 28]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [13, 27, 28, 29]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"IRAP\", \"PRKAA1\", \"YWHAZ\", \"APPL2\", \"VPS13A\", \"VPS13C\", \"WNK1\", \"TBC1D4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}