{"gene":"LEAP2","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2017,"finding":"LEAP2 (liver-expressed antimicrobial peptide 2) is an endogenous antagonist of the ghrelin receptor (GHSR). LEAP2 is produced in the liver and small intestine, its secretion is suppressed by fasting, and it fully inhibits GHSR activation by ghrelin in vitro. In vivo, LEAP2 blocks ghrelin-induced food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. Neutralizing antibodies that block endogenous LEAP2 enhance ghrelin action in vivo.","method":"In vitro receptor activation assays, in vivo pharmacology (LEAP2 administration and anti-LEAP2 neutralizing antibodies in mice), secretion/expression profiling","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods in a single rigorous study, replicated direction confirmed by neutralizing antibody experiments; foundational discovery paper","pmids":["29233536"],"is_preprint":false},{"year":2003,"finding":"LEAP2 is synthesized as a 77-residue precursor predominantly expressed in the liver, processed by a furin-like endoprotease to generate the largest native 40-amino-acid form; the mature peptide contains two disulfide bonds (cysteines in 1-3 and 2-4 positions) and exhibits dose-dependent antimicrobial activity against selected microbial model organisms, whereas smaller variants do not.","method":"Purification from human blood ultrafiltrate, molecular cloning, structural characterization (disulfide bond mapping), in vitro antimicrobial activity assay","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Strong — native peptide isolated and biochemically characterized, disulfide architecture defined, antimicrobial activity demonstrated in vitro with dose-response, replicated across subsequent studies","pmids":["12493837"],"is_preprint":false},{"year":2018,"finding":"The N-terminal region of LEAP2 alone is sufficient for binding to GHSR and confers receptor activity. Both full-length LEAP2 and its N-terminal fragment act as inverse agonists of GHSR (reducing constitutive activity) and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. The N-terminal LEAP2 fragment inhibits ghrelin-induced food intake in mice.","method":"Receptor binding assays, IP1 accumulation assay, calcium mobilization assay, in vivo food intake assay in mice (N-terminal LEAP2 fragment vs. full-length)","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal pharmacological assays (binding, IP1, Ca2+ mobilization) plus in vivo validation; inverse agonism and competitive antagonism established with fragment truncation","pmids":["30543423"],"is_preprint":false},{"year":2019,"finding":"LEAP2 and ghrelin compete for the same binding site on GHSR1a. When added simultaneously with ghrelin, LEAP2 behaves as a competitive antagonist; when added before ghrelin, it behaves as a non-competitive antagonist, attributable to slow dissociation from the receptor. The N-terminal fragment of LEAP2 is critical for receptor binding.","method":"Radioligand binding assays, GHSR activation assays with sequential vs. simultaneous ligand addition protocols","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding and activation assays with systematic addition-order experiments; single lab, two complementary methods","pmids":["30666806"],"is_preprint":false},{"year":2020,"finding":"Alanine-scanning mutagenesis of the LEAP2 N-terminal fragment identified Arg6 and Phe4 as essential residues for GHSR1a binding. Site-directed mutagenesis of GHSR1a revealed that Asp99 (extracellular) likely interacts with LEAP2 Arg6, while Phe279 and Phe312 (in the ligand-binding pocket) likely interact with LEAP2 Phe4, and Phe119 interacts with LEAP2 Trp5.","method":"Alanine-scanning mutagenesis of LEAP2, extensive site-directed mutagenesis of GHSR1a, receptor binding assays, structural modeling","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis with binding assays is tier 1, but structural assignment is partly modeled and from a single lab; key residues validated experimentally","pmids":["32803260"],"is_preprint":false},{"year":2021,"finding":"LEAP2 acts as both an antagonist of ghrelin-evoked GHSR activity and an inverse agonist of constitutive GHSR activity on CaV2.2 currents in neurons. LEAP2 also prevents GHSR from modulating D2R-dependent Gi signaling on CaV2.2. Using purified receptors in lipid nanodiscs and FRET, the N-terminal region of LEAP2 was shown to stabilize an inactive conformation of GHSR dissociated from Gq protein, thereby reversing the effect of GHSR on D2R-dependent Gi activation.","method":"Patch-clamp recordings (CaV2.2 currents), FRET with purified labeled receptors assembled into lipid nanodiscs, heterologous expression system","journal":"Frontiers in pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted receptor in nanodiscs with FRET plus electrophysiology; multiple orthogonal methods in a single study providing molecular mechanism of GHSR conformational stabilization by LEAP2","pmids":["34447311"],"is_preprint":false},{"year":2021,"finding":"Genetic deletion of LEAP2 in mice sensitizes them to acute ghrelin-induced food intake and GH secretion. Female LEAP2-KO mice on chronic high-fat diet exhibit increased body weight, food intake, energy expenditure reduction, hepatic fat accumulation, and greater c-Fos activation in arcuate nucleus and olfactory bulb following ghrelin administration, establishing LEAP2 as a physiologically relevant modulator of GHSR signaling in vivo.","method":"LEAP2-KO mouse generation, s.c. ghrelin administration, food intake measurement, GH secretion assay, c-Fos immunostaining, metabolic cage measurements, histology","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with multiple phenotypic readouts (food intake, GH, c-Fos, metabolic parameters, histology); comprehensive loss-of-function study","pmids":["34428557"],"is_preprint":false},{"year":2022,"finding":"Food deprivation-induced activation of CRF neurons in the hypothalamic paraventricular nucleus requires a decrease in plasma LEAP2 levels. Preventing the fasting-induced fall of LEAP2 (via continuous systemic LEAP2(1-12) infusion) reverses the activation of PVH-CRF neurons in food-deprived mice. This effect is ghrelin-independent but requires GHSR signaling at the hypothalamic level.","method":"Genetic mouse models (GHSR-KO, ghrelin-KO), pharmacological manipulation (anti-ghrelin antibody, GHSR ligands, LEAP2 infusion), c-Fos/CRF immunostaining, arcuate nucleus ablation","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic and pharmacological models tested in parallel, epistasis established between LEAP2 level decline and CRF neuron activation","pmids":["35504998"],"is_preprint":false},{"year":2022,"finding":"LEAP2 infusion reduces postprandial plasma glucose and GH concentrations and decreases ad libitum food intake in healthy men. In wild-type mice, similar effects are observed, but not in GHSR-null mice, establishing GHSR as the mediator of LEAP2's glucoregulatory and appetite-suppressing effects.","method":"Randomized double-blind placebo-controlled crossover trial (humans), GHSR-null mouse experiments with LEAP2 dosing","journal":"Cell reports. Medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — human RCT plus genetic null mouse rescue experiment; GHSR-dependency definitively established by null mouse","pmids":["35492241"],"is_preprint":false},{"year":2022,"finding":"Overexpression of LEAP2 in the arcuate nucleus via AAV reduces food intake and body weight in mice and increases POMC neuronal expression. Chemogenetic inhibition of POMC neurons abolishes the anorexigenic effect of centrally administered LEAP2, placing POMC neurons downstream of LEAP2 signaling in appetite suppression.","method":"AAV-mediated overexpression in arcuate nucleus, intracerebroventricular LEAP2 administration, chemogenetic POMC neuron inhibition (DREADD), food intake and body weight measurement","journal":"Frontiers in endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — viral overexpression combined with chemogenetics to place POMC neurons in the pathway; single lab, two orthogonal approaches","pmids":["36387867"],"is_preprint":false},{"year":2021,"finding":"A gut-derived LEAP2 fragment (LEAP2 38-47) stimulates insulin release in human pancreatic islets comparably to GLP-1, and this insulinotropic action is linked to attenuation of tonic GHSR activity. Small intestinal LEAP2 expression is upregulated after Roux-en-Y gastric bypass surgery.","method":"Genome-wide expression analysis of human EECs, in vitro human pancreatic islet secretion assay, GHSR activity assay, human infusion study","journal":"The Journal of clinical endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro islet assay with mechanistic link to GHSR activity; human infusion was negative for glucoregulatory effect in vivo","pmids":["33135737"],"is_preprint":false},{"year":2019,"finding":"A fluorescent LEAP2-based probe (F-LEAP2) labels GHSR specifically on the cell surface of GHSR-expressing cells, in contrast to fluorescent ghrelin which internalizes. F-LEAP2 acts as an inverse agonist of GHSR in vitro and reduces ghrelin-induced food intake in mice following central injection, consistent with LEAP2's inverse agonist mechanism.","method":"Fluorescent ligand design, receptor binding assay, cell surface labeling vs. internalization imaging, in vivo food intake assay","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct visualization of surface vs. internalized receptor labeling, mechanistic contrast with ghrelin established; single lab","pmids":["31499133"],"is_preprint":false},{"year":2022,"finding":"Beta-hydroxybutyrate (BHB) directly downregulates LEAP2 expression in isolated murine hepatocytes and reduces circulating LEAP2 levels in mice after oral BHB administration, identifying BHB as a cell-autonomous suppressor of hepatic LEAP2 production during fasting/ketosis.","method":"BHB treatment of isolated murine hepatocytes (in vitro), oral BHB administration in mice, hepatic/intestinal Leap2 expression measurement, plasma LEAP2 quantification","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro hepatocyte experiment combined with in vivo BHB administration; single lab, two complementary methods","pmids":["35352108"],"is_preprint":false},{"year":2025,"finding":"Glucagon infusion during somatostatin clamps significantly decreases plasma LEAP2 levels in humans. Insulin receptor antagonism offsets postprandial LEAP2 upregulation in mice. Insulin and glucagon receptor-expressing hepatocytes are the primary source of hepatic LEAP2 expression, coinciding with a putative enhancer-like signature bound by insulin- and glucagon-regulated transcription factors at the LEAP2 locus.","method":"Somatostatin clamp with glucagon infusion in humans, insulin receptor antagonist treatment in mice, hepatocyte-specific transcription factor binding analysis, plasma LEAP2 quantification","journal":"Cell reports. Medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human clamp study combined with mouse genetic/pharmacological intervention and genomic enhancer analysis; single lab, multiple orthogonal approaches","pmids":["40056903"],"is_preprint":false},{"year":2021,"finding":"LEAP2 has antagonized GHSR1a since at least the emergence of coelacanth fish. Coelacanth LEAP2 and ghrelin both bind coelacanth GHSR1a with IC50 values in the nanomolar range, and coelacanth LEAP2 efficiently antagonizes coelacanth ghrelin-induced GHSR1a activation, demonstrating evolutionary conservation of the LEAP2-GHSR1a antagonism system.","method":"Binding assays and activation assays using coelacanth GHSR1a expressed in cell system, competitive inhibition analysis","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and functional assays with fish ortholog; single lab, two complementary methods","pmids":["33966114"],"is_preprint":false},{"year":2020,"finding":"In teleost mudskipper, MOSPD2 (motile sperm domain-containing protein 2) was identified as a receptor mediating LEAP2's effects on monocytes/macrophages. BpMOSPD2 directly interacts with BpLEAP-2 (confirmed by Co-IP). Knockdown of BpMOSPD2 inhibited BpLEAP-2-induced chemotaxis, bacterial killing activity, and modulation of cytokine expression in MO/MΦ.","method":"Yeast two-hybrid cDNA library screening, co-immunoprecipitation, RNAi knockdown in primary mudskipper MO/MΦ, functional assays (chemotaxis, bacterial killing, cytokine qRT-PCR)","journal":"Zoological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus RNAi loss-of-function with multiple functional readouts; fish ortholog, single lab","pmids":["33124217"],"is_preprint":false},{"year":2025,"finding":"In teleost mudskipper, BpLEAP2 stimulation triggers retromer-dependent intracellular trafficking of BpMOSPD2 from the ER to early endosomes and then to the plasma membrane. Knockdown of retromer subunits (VPS35, VPS26, VPS29) abolishes BpMOSPD2 membrane localization and BpLEAP2-induced MO/MΦ migration. Co-IP with mass spectrometry confirmed direct interaction between BpMOSPD2 and BpVPS35.","method":"Subcellular fractionation, immunofluorescence, Co-IP combined with mass spectrometry, retromer subunit knockdown (RNAi), migration assay","journal":"Zoological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS plus subcellular fractionation and RNAi epistasis; mechanistic trafficking pathway established; fish ortholog, single lab","pmids":["41017400"],"is_preprint":false},{"year":2016,"finding":"LEAP2 overexpression in Xenopus embryos impairs normal embryonic development. In pluripotent embryonic cells, LEAP2 stimulates FGF signaling while reducing the activin response. LEAP2 also blocks FGF-induced migration of human vascular endothelial cells (HUVEC), suggesting an extracellular modulatory role for LEAP2 on FGF and activin signals.","method":"Xenopus gain-of-function overexpression, animal cap assays, HUVEC migration assay","journal":"Peptides","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, amphibian model; functional assays without mechanistic receptor identification; relevance to mammalian LEAP2 unclear","pmids":["27335344"],"is_preprint":false},{"year":2024,"finding":"Central (i.c.v.) administration of LEAP2 in mice reduces feeding and intake of palatable foods, attenuates accumbal dopamine release associated with palatable food exposure, and reduces the rewarding memory of high-preference foods. LEAP2 is expressed in reward-related brain areas including the laterodorsal tegmental area (LDTg), and infusion of LEAP2 into LDTg transiently reduces acute palatable food intake.","method":"i.c.v. and intra-LDTg LEAP2 administration in mice, microdialysis (accumbal dopamine), in situ expression profiling, behavioral food intake tests","journal":"Progress in neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct dopamine measurement by microdialysis plus regional brain injection to localize effect; single lab, multiple methods","pmids":["38641041"],"is_preprint":false},{"year":2024,"finding":"LEAP2 enhances insulin secretion in isolated islets from male but not female mice, and reverses acyl-ghrelin-stimulated somatostatin release in males but not females. Estradiol (E2) pre-treatment of male islets abolished both AG-induced insulinostatic effects and their reversal by LEAP2, demonstrating sex- and hormone-dependent modulation of islet function by LEAP2 acting via GHSR1a.","method":"Isolated mouse islet secretion experiments (radioimmunoassay), E2 pre-treatment, SSTR3 antagonist experiments, qPCR of islet gene expression","journal":"The Journal of endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct islet secretion assays with pharmacological and hormonal manipulation; sex-specific mechanistic effect established; single lab","pmids":["39292603"],"is_preprint":false},{"year":2023,"finding":"LEAP2 dietary regulation depends on meal composition: all meal challenges except fish oil increased jejunal Leap2 expression, while only a mixed meal increased liver Leap2 expression. Leap2 expression correlated with hepatic glycogen and jejunal lipid levels. Oleic acid (but not docosahexaenoic acid) increased Leap2 expression in intestinal organoids, indicating nutrient-specific cell-autonomous regulation.","method":"Mouse meal challenge experiments, intestinal organoid culture with specific fatty acids, jejunal/hepatic gene expression analysis, portal vein plasma LEAP2 measurement","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — organoid cell-autonomous assay combined with in vivo portal/systemic sampling; single lab, multiple nutrient conditions","pmids":["37104087"],"is_preprint":false},{"year":2022,"finding":"Transcription factor CDX4 binds to the LEAP2 promoter region in the small intestine and positively regulates LEAP2 expression, as demonstrated by transcription factor prediction and dual luciferase assay.","method":"Dual luciferase reporter assay, transcription factor binding site analysis","journal":"Animals","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single luciferase assay in avian/chicken model without endogenous chromatin confirmation; single lab","pmids":["36552416"],"is_preprint":false},{"year":2024,"finding":"Central LEAP2 administration in mice prevents alcohol-induced locomotor stimulation, suppresses the memory of alcohol reward, attenuates dopamine release in the nucleus accumbens caused by alcohol, and reduces alcohol consumption in rats. These effects parallel LEAP2's modulation of the mesolimbic dopamine system.","method":"i.c.v. LEAP2 administration in mice/rats, locomotor activity measurement, conditioned place preference, in vivo microdialysis (nucleus accumbens dopamine), voluntary alcohol consumption paradigm","journal":"Translational psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct dopamine microdialysis combined with behavioral assays; mechanistic link to mesolimbic dopamine established; single lab","pmids":["39358354"],"is_preprint":false},{"year":2023,"finding":"In fish (Acrossocheilus fasciatus), the linear (disulfide-free) form of LEAP2 mature peptide shows potent antimicrobial activity and disrupts bacterial cell membrane integrity, while the oxidized (disulfide-bonded, cyclic) form has weak or no antibacterial activity. This establishes that disulfide bonds are not required for—and in fact impair—the antimicrobial function of LEAP2.","method":"Chemical synthesis of linear vs. oxidized LEAP2, circular dichroism spectroscopy, in vitro MIC assays, bacterial membrane integrity assay","journal":"BMC veterinary research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct structure-activity comparison with CD spectroscopy and functional assays; single lab, fish ortholog","pmids":["38835040"],"is_preprint":false}],"current_model":"LEAP2 is an endogenous antagonist and inverse agonist of the ghrelin receptor GHSR, produced primarily by hepatocytes and enterocytes; its N-terminal region (particularly Phe4 and Arg6) binds competitively to GHSR (interacting with receptor residues Asp99, Phe279, Phe312), stabilizes an inactive Gq-dissociated receptor conformation, blocks both ghrelin-induced and constitutive GHSR signaling (including D2R cross-talk and CaV2.2 modulation), and thereby suppresses food intake (partly via hypothalamic POMC neurons and mesolimbic dopamine), GH secretion, and blood glucose elevation; its hepatic expression is regulated by insulin, glucagon, and beta-hydroxybutyrate, and by CDX4 in the intestine, with circulating levels rising postprandially and in obesity and falling during fasting."},"narrative":{"mechanistic_narrative":"LEAP2 is a hepatocyte- and enterocyte-derived peptide hormone that functions as the endogenous competitive antagonist and inverse agonist of the ghrelin receptor GHSR, opposing ghrelin to suppress food intake, growth hormone release, and glucose elevation [PMID:29233536, PMID:30543423]. Originally isolated from human blood as a furin-processed 40-residue antimicrobial peptide bearing two disulfide bonds [PMID:12493837], its receptor-directed activity resides in the N-terminal region, which alone is sufficient to bind GHSR and to act as both an inverse agonist of constitutive receptor activity and an antagonist of ghrelin-evoked inositol-phosphate and calcium signaling [PMID:30543423, PMID:30666806]. Mutagenesis maps this interaction to LEAP2 residues Phe4 and Arg6 contacting GHSR residues Asp99, Phe279, and Phe312, and biophysical reconstitution shows the N-terminal region stabilizes an inactive, Gq-dissociated receptor conformation that also blocks GHSR modulation of D2R-dependent Gi signaling and CaV2.2 currents [PMID:32803260, PMID:34447311]. Physiologically, plasma LEAP2 falls with fasting and rises postprandially; genetic deletion sensitizes mice to ghrelin-driven feeding and GH secretion, and GHSR is required for LEAP2's anorexigenic and glucoregulatory actions, which engage hypothalamic POMC and CRF neurons and the mesolimbic dopamine system [PMID:34428557, PMID:35504998, PMID:35492241, PMID:36387867, PMID:38641041]. Hepatic and intestinal LEAP2 expression is nutrient- and hormone-regulated, suppressed by beta-hydroxybutyrate and glucagon and controlled by insulin- and glucagon-responsive transcription in hepatocytes [PMID:35352108, PMID:40056903, PMID:37104087]. The LEAP2–GHSR antagonism is evolutionarily ancient, conserved at least since coelacanth fish [PMID:33966114].","teleology":[{"year":2003,"claim":"Established LEAP2's molecular identity and biochemistry before any receptor role was known, defining the precursor, furin processing, disulfide architecture, and an antimicrobial activity.","evidence":"Purification from human blood ultrafiltrate, cloning, disulfide mapping, and in vitro antimicrobial assays","pmids":["12493837"],"confidence":"High","gaps":["No receptor or signaling function identified at this stage","Physiological role beyond antimicrobial activity unknown"]},{"year":2017,"claim":"Answered what LEAP2 does physiologically by identifying it as an endogenous GHSR antagonist that opposes ghrelin's effects on feeding, GH, and glucose.","evidence":"In vitro GHSR activation assays plus in vivo LEAP2 administration and anti-LEAP2 neutralizing antibodies in mice","pmids":["29233536"],"confidence":"High","gaps":["Binding site and structural basis of antagonism not defined","Whether activity resides in a peptide subregion unknown"]},{"year":2018,"claim":"Localized the receptor-active determinant to the LEAP2 N-terminus and distinguished inverse agonism from competitive antagonism.","evidence":"Binding, IP1, and calcium assays with full-length vs. N-terminal fragment plus in vivo food intake","pmids":["30543423"],"confidence":"High","gaps":["Specific contact residues not yet mapped","Kinetic basis of antagonism not addressed"]},{"year":2019,"claim":"Resolved the kinetic/competitive mechanism by showing LEAP2 and ghrelin share a binding site, with slow LEAP2 dissociation converting it to functional non-competitive blockade.","evidence":"Radioligand binding and sequential vs. simultaneous ligand addition activation assays","pmids":["30666806"],"confidence":"Medium","gaps":["Single lab, two methods","Residue-level contacts not defined"]},{"year":2020,"claim":"Mapped the LEAP2–GHSR interface, identifying Phe4/Arg6 on LEAP2 and Asp99/Phe279/Phe312/Phe119 on GHSR1a as the key contacts.","evidence":"Alanine scanning of LEAP2, site-directed mutagenesis of GHSR1a, binding assays, structural modeling","pmids":["32803260"],"confidence":"Medium","gaps":["Structural assignments partly modeled rather than solved","No experimental complex structure"]},{"year":2021,"claim":"Provided the conformational mechanism of antagonism, showing the LEAP2 N-terminus stabilizes an inactive Gq-dissociated GHSR state and blocks GHSR effects on D2R-Gi and CaV2.2 currents.","evidence":"FRET with purified receptors in lipid nanodiscs and patch-clamp electrophysiology","pmids":["34447311"],"confidence":"High","gaps":["No high-resolution structure of the LEAP2-bound receptor","Relevance of D2R cross-talk in vivo not established"]},{"year":2021,"claim":"Established LEAP2 as a physiologically required GHSR modulator via clean loss-of-function, linking deletion to ghrelin hypersensitivity and diet-induced metabolic phenotypes.","evidence":"LEAP2-KO mice with feeding, GH, c-Fos, metabolic, and histological readouts","pmids":["34428557"],"confidence":"High","gaps":["Sex-specific high-fat-diet effects not fully mechanistically explained","Tissue source contribution not dissected"]},{"year":2022,"claim":"Placed defined neuronal populations downstream of LEAP2: a fall in LEAP2 is required for fasting activation of PVH-CRF neurons, and POMC neurons mediate its anorexigenic effect.","evidence":"GHSR-KO/ghrelin-KO models, LEAP2(1-12) infusion, AAV overexpression in arcuate nucleus, and chemogenetic POMC inhibition","pmids":["35504998","36387867"],"confidence":"High","gaps":["Circuit connectivity between arcuate and PVH not resolved","POMC step shown in single lab"]},{"year":2022,"claim":"Demonstrated translational relevance and GHSR-dependence in humans, with LEAP2 infusion lowering postprandial glucose, GH, and food intake, absent in GHSR-null mice.","evidence":"Randomized placebo-controlled crossover trial in men plus GHSR-null mouse dosing","pmids":["35492241"],"confidence":"High","gaps":["Long-term metabolic effects not assessed","Female human data not reported"]},{"year":2022,"claim":"Defined nutritional and hormonal control of LEAP2 production, identifying beta-hydroxybutyrate as a cell-autonomous suppressor of hepatic expression during fasting/ketosis.","evidence":"BHB treatment of isolated hepatocytes and oral BHB in mice with plasma LEAP2 quantification","pmids":["35352108"],"confidence":"Medium","gaps":["Transcriptional mediators of BHB action not identified","Single lab"]},{"year":2023,"claim":"Extended regulation to meal composition and intestinal origin, showing nutrient-specific (oleic acid) induction of Leap2 in organoids and tissue-specific responses.","evidence":"Mouse meal challenges, intestinal organoid fatty-acid exposure, portal/systemic LEAP2 sampling","pmids":["37104087"],"confidence":"Medium","gaps":["Receptors/sensors for fatty acid induction not identified","Single lab"]},{"year":2024,"claim":"Connected LEAP2 to reward and addiction circuitry, showing central LEAP2 attenuates mesolimbic dopamine responses to palatable food and alcohol.","evidence":"i.c.v. and intra-LDTg LEAP2 administration, nucleus accumbens microdialysis, and behavioral reward/consumption assays in mice and rats","pmids":["38641041","39358354"],"confidence":"Medium","gaps":["Receptor identity in reward regions not directly proven","Single lab"]},{"year":2024,"claim":"Revealed sex- and hormone-dependent islet actions, with LEAP2 enhancing insulin secretion and reversing ghrelin-induced somatostatin release only in males, abolished by estradiol.","evidence":"Isolated mouse islet secretion assays with E2 and SSTR3 antagonist manipulation","pmids":["39292603"],"confidence":"Medium","gaps":["Mechanism of estradiol interference unknown","Human islet generalizability not established"]},{"year":2025,"claim":"Identified the transcriptional/endocrine wiring of hepatic LEAP2, implicating insulin- and glucagon-responsive transcription factors at an enhancer-like locus, with glucagon lowering and insulin raising plasma LEAP2.","evidence":"Human somatostatin-glucagon clamp, insulin receptor antagonism in mice, and hepatocyte transcription factor binding analysis","pmids":["40056903"],"confidence":"Medium","gaps":["Specific transcription factors not functionally validated","Single lab"]},{"year":null,"claim":"Whether LEAP2's ancestral antimicrobial activity and a non-GHSR receptor axis operate alongside GHSR antagonism in mammals remains unresolved.","evidence":"Fish-ortholog MOSPD2/retromer immune signaling and amphibian FGF/activin effects lack a defined mammalian receptor context","pmids":[],"confidence":"Low","gaps":["No mammalian receptor for LEAP2's antimicrobial/immune actions identified","Relationship between antimicrobial and GHSR-antagonist functions not integrated","MOSPD2 axis demonstrated only in teleost"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,3,5]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[2,5,11]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8,12,13]}],"complexes":[],"partners":["GHSR","MOSPD2","VPS35"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q969E1","full_name":"Liver-expressed antimicrobial peptide 2","aliases":[],"length_aa":77,"mass_kda":8.8,"function":"Has an antimicrobial activity","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q969E1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LEAP2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LEAP2","total_profiled":1310},"omim":[{"mim_id":"611373","title":"LIVER-EXPRESSED ANTIMICROBIAL PEPTIDE 2; LEAP2","url":"https://www.omim.org/entry/611373"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in 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study.","date":"2024","source":"Diabetes, obesity & metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/38888057","citation_count":4,"is_preprint":false},{"pmid":"38835040","id":"PMC_38835040","title":"Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.","date":"2024","source":"BMC veterinary research","url":"https://pubmed.ncbi.nlm.nih.gov/38835040","citation_count":3,"is_preprint":false},{"pmid":"40683494","id":"PMC_40683494","title":"Molecular characterization and functional prioritization of CD46, IL6R, KLRC1, LEAP2 and SMOX as candidate targets in acute kidney injury.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40683494","citation_count":3,"is_preprint":false},{"pmid":"40870497","id":"PMC_40870497","title":"Ghrelin and LEAP2: Their Interaction Effect on Appetite Regulation and the Alterations in Their Levels Following Bariatric Surgery.","date":"2025","source":"Medicina (Kaunas, Lithuania)","url":"https://pubmed.ncbi.nlm.nih.gov/40870497","citation_count":2,"is_preprint":false},{"pmid":"39347412","id":"PMC_39347412","title":"Deficiency of leap2 promotes somatic growth in zebrafish: Involvement of the growth hormone system.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/39347412","citation_count":2,"is_preprint":false},{"pmid":"40726500","id":"PMC_40726500","title":"Serum LEAP2 Levels Across the Spectrum of Metabolic Dysfunction-Associated Fatty Liver Disease: A Potential Noninvasive Biomarker for Severity Stratification.","date":"2025","source":"Diabetes, metabolic syndrome and obesity : targets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/40726500","citation_count":2,"is_preprint":false},{"pmid":"38636815","id":"PMC_38636815","title":"Quail GHRL and LEAP2 gene cloning, polymorphism detection, phylogenetic analysis, tissue expression profiling and its association analysis with feed intake.","date":"2024","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/38636815","citation_count":2,"is_preprint":false},{"pmid":"40121727","id":"PMC_40121727","title":"Molecular characterization of an antimicrobial peptide LEAP-2 in Onychostoma macrolepis: Expression pattern, antimicrobial ability and immunomodulation function.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40121727","citation_count":1,"is_preprint":false},{"pmid":"40618862","id":"PMC_40618862","title":"LEAP2: from feeding regulation to its implications in eating disorders.","date":"2025","source":"Physiology & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/40618862","citation_count":0,"is_preprint":false},{"pmid":"41069857","id":"PMC_41069857","title":"Ghrelin-GHSR-LEAP2 system in the pathophysiology of type 2 diabetes.","date":"2025","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/41069857","citation_count":0,"is_preprint":false},{"pmid":"41520761","id":"PMC_41520761","title":"Additive effect of leptin and palm-LEAP2(1-14) ameliorates obesity-induced metabolic stress in ob/ob mice.","date":"2026","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41520761","citation_count":0,"is_preprint":false},{"pmid":"41503208","id":"PMC_41503208","title":"Exercise maintains LEAP2 levels after weight loss in females with obesity.","date":"2025","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/41503208","citation_count":0,"is_preprint":false},{"pmid":"41299349","id":"PMC_41299349","title":"Does fetal or maternal leap-2 level affect infant birth weight?","date":"2025","source":"BMC pregnancy and childbirth","url":"https://pubmed.ncbi.nlm.nih.gov/41299349","citation_count":0,"is_preprint":false},{"pmid":"41571146","id":"PMC_41571146","title":"LEAP2 acts in hepatocytes and at central level, alleviates steatosis and inflammation but resistance in obese and aging.","date":"2026","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41571146","citation_count":0,"is_preprint":false},{"pmid":"41619965","id":"PMC_41619965","title":"LEAP2 modulates β-adrenergic triggered cardiac responses and provokes antihypertensive effects.","date":"2026","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41619965","citation_count":0,"is_preprint":false},{"pmid":"40082911","id":"PMC_40082911","title":"Identification and characterisation of LEAP2 from Chinese spiny frogs (Quasipaa spinosa) with antimicrobial and macrophage activation properties.","date":"2025","source":"BMC veterinary research","url":"https://pubmed.ncbi.nlm.nih.gov/40082911","citation_count":0,"is_preprint":false},{"pmid":"41406822","id":"PMC_41406822","title":"Effect of intracerebroventricular (ICV) injection of antimicrobial peptide expressed in the body-2 (LEAP-2) and its interaction with cannabinoid and ghrelin systems on food intake in broiler chickens.","date":"2025","source":"Poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/41406822","citation_count":0,"is_preprint":false},{"pmid":"41077091","id":"PMC_41077091","title":"LEAP2 deficiency does not impair essential iron-dependent functions in zebrafish.","date":"2025","source":"Fish & shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41077091","citation_count":0,"is_preprint":false},{"pmid":"41911360","id":"PMC_41911360","title":"LEAP2 Reduces Ad Libitum Food Intake and Attenuates Postprandial Glucose Excursions in Men With Obesity.","date":"2026","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/41911360","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":54317,"output_tokens":6322,"usd":0.12889,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15388,"output_tokens":4104,"usd":0.08977,"stage2_stop_reason":"end_turn"},"total_usd":0.21866,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"LEAP2 (liver-expressed antimicrobial peptide 2) is an endogenous antagonist of the ghrelin receptor (GHSR). LEAP2 is produced in the liver and small intestine, its secretion is suppressed by fasting, and it fully inhibits GHSR activation by ghrelin in vitro. In vivo, LEAP2 blocks ghrelin-induced food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. Neutralizing antibodies that block endogenous LEAP2 enhance ghrelin action in vivo.\",\n      \"method\": \"In vitro receptor activation assays, in vivo pharmacology (LEAP2 administration and anti-LEAP2 neutralizing antibodies in mice), secretion/expression profiling\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods in a single rigorous study, replicated direction confirmed by neutralizing antibody experiments; foundational discovery paper\",\n      \"pmids\": [\"29233536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"LEAP2 is synthesized as a 77-residue precursor predominantly expressed in the liver, processed by a furin-like endoprotease to generate the largest native 40-amino-acid form; the mature peptide contains two disulfide bonds (cysteines in 1-3 and 2-4 positions) and exhibits dose-dependent antimicrobial activity against selected microbial model organisms, whereas smaller variants do not.\",\n      \"method\": \"Purification from human blood ultrafiltrate, molecular cloning, structural characterization (disulfide bond mapping), in vitro antimicrobial activity assay\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — native peptide isolated and biochemically characterized, disulfide architecture defined, antimicrobial activity demonstrated in vitro with dose-response, replicated across subsequent studies\",\n      \"pmids\": [\"12493837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The N-terminal region of LEAP2 alone is sufficient for binding to GHSR and confers receptor activity. Both full-length LEAP2 and its N-terminal fragment act as inverse agonists of GHSR (reducing constitutive activity) and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. The N-terminal LEAP2 fragment inhibits ghrelin-induced food intake in mice.\",\n      \"method\": \"Receptor binding assays, IP1 accumulation assay, calcium mobilization assay, in vivo food intake assay in mice (N-terminal LEAP2 fragment vs. full-length)\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal pharmacological assays (binding, IP1, Ca2+ mobilization) plus in vivo validation; inverse agonism and competitive antagonism established with fragment truncation\",\n      \"pmids\": [\"30543423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LEAP2 and ghrelin compete for the same binding site on GHSR1a. When added simultaneously with ghrelin, LEAP2 behaves as a competitive antagonist; when added before ghrelin, it behaves as a non-competitive antagonist, attributable to slow dissociation from the receptor. The N-terminal fragment of LEAP2 is critical for receptor binding.\",\n      \"method\": \"Radioligand binding assays, GHSR activation assays with sequential vs. simultaneous ligand addition protocols\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding and activation assays with systematic addition-order experiments; single lab, two complementary methods\",\n      \"pmids\": [\"30666806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Alanine-scanning mutagenesis of the LEAP2 N-terminal fragment identified Arg6 and Phe4 as essential residues for GHSR1a binding. Site-directed mutagenesis of GHSR1a revealed that Asp99 (extracellular) likely interacts with LEAP2 Arg6, while Phe279 and Phe312 (in the ligand-binding pocket) likely interact with LEAP2 Phe4, and Phe119 interacts with LEAP2 Trp5.\",\n      \"method\": \"Alanine-scanning mutagenesis of LEAP2, extensive site-directed mutagenesis of GHSR1a, receptor binding assays, structural modeling\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis with binding assays is tier 1, but structural assignment is partly modeled and from a single lab; key residues validated experimentally\",\n      \"pmids\": [\"32803260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LEAP2 acts as both an antagonist of ghrelin-evoked GHSR activity and an inverse agonist of constitutive GHSR activity on CaV2.2 currents in neurons. LEAP2 also prevents GHSR from modulating D2R-dependent Gi signaling on CaV2.2. Using purified receptors in lipid nanodiscs and FRET, the N-terminal region of LEAP2 was shown to stabilize an inactive conformation of GHSR dissociated from Gq protein, thereby reversing the effect of GHSR on D2R-dependent Gi activation.\",\n      \"method\": \"Patch-clamp recordings (CaV2.2 currents), FRET with purified labeled receptors assembled into lipid nanodiscs, heterologous expression system\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted receptor in nanodiscs with FRET plus electrophysiology; multiple orthogonal methods in a single study providing molecular mechanism of GHSR conformational stabilization by LEAP2\",\n      \"pmids\": [\"34447311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Genetic deletion of LEAP2 in mice sensitizes them to acute ghrelin-induced food intake and GH secretion. Female LEAP2-KO mice on chronic high-fat diet exhibit increased body weight, food intake, energy expenditure reduction, hepatic fat accumulation, and greater c-Fos activation in arcuate nucleus and olfactory bulb following ghrelin administration, establishing LEAP2 as a physiologically relevant modulator of GHSR signaling in vivo.\",\n      \"method\": \"LEAP2-KO mouse generation, s.c. ghrelin administration, food intake measurement, GH secretion assay, c-Fos immunostaining, metabolic cage measurements, histology\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with multiple phenotypic readouts (food intake, GH, c-Fos, metabolic parameters, histology); comprehensive loss-of-function study\",\n      \"pmids\": [\"34428557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Food deprivation-induced activation of CRF neurons in the hypothalamic paraventricular nucleus requires a decrease in plasma LEAP2 levels. Preventing the fasting-induced fall of LEAP2 (via continuous systemic LEAP2(1-12) infusion) reverses the activation of PVH-CRF neurons in food-deprived mice. This effect is ghrelin-independent but requires GHSR signaling at the hypothalamic level.\",\n      \"method\": \"Genetic mouse models (GHSR-KO, ghrelin-KO), pharmacological manipulation (anti-ghrelin antibody, GHSR ligands, LEAP2 infusion), c-Fos/CRF immunostaining, arcuate nucleus ablation\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic and pharmacological models tested in parallel, epistasis established between LEAP2 level decline and CRF neuron activation\",\n      \"pmids\": [\"35504998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LEAP2 infusion reduces postprandial plasma glucose and GH concentrations and decreases ad libitum food intake in healthy men. In wild-type mice, similar effects are observed, but not in GHSR-null mice, establishing GHSR as the mediator of LEAP2's glucoregulatory and appetite-suppressing effects.\",\n      \"method\": \"Randomized double-blind placebo-controlled crossover trial (humans), GHSR-null mouse experiments with LEAP2 dosing\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human RCT plus genetic null mouse rescue experiment; GHSR-dependency definitively established by null mouse\",\n      \"pmids\": [\"35492241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Overexpression of LEAP2 in the arcuate nucleus via AAV reduces food intake and body weight in mice and increases POMC neuronal expression. Chemogenetic inhibition of POMC neurons abolishes the anorexigenic effect of centrally administered LEAP2, placing POMC neurons downstream of LEAP2 signaling in appetite suppression.\",\n      \"method\": \"AAV-mediated overexpression in arcuate nucleus, intracerebroventricular LEAP2 administration, chemogenetic POMC neuron inhibition (DREADD), food intake and body weight measurement\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — viral overexpression combined with chemogenetics to place POMC neurons in the pathway; single lab, two orthogonal approaches\",\n      \"pmids\": [\"36387867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A gut-derived LEAP2 fragment (LEAP2 38-47) stimulates insulin release in human pancreatic islets comparably to GLP-1, and this insulinotropic action is linked to attenuation of tonic GHSR activity. Small intestinal LEAP2 expression is upregulated after Roux-en-Y gastric bypass surgery.\",\n      \"method\": \"Genome-wide expression analysis of human EECs, in vitro human pancreatic islet secretion assay, GHSR activity assay, human infusion study\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro islet assay with mechanistic link to GHSR activity; human infusion was negative for glucoregulatory effect in vivo\",\n      \"pmids\": [\"33135737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A fluorescent LEAP2-based probe (F-LEAP2) labels GHSR specifically on the cell surface of GHSR-expressing cells, in contrast to fluorescent ghrelin which internalizes. F-LEAP2 acts as an inverse agonist of GHSR in vitro and reduces ghrelin-induced food intake in mice following central injection, consistent with LEAP2's inverse agonist mechanism.\",\n      \"method\": \"Fluorescent ligand design, receptor binding assay, cell surface labeling vs. internalization imaging, in vivo food intake assay\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct visualization of surface vs. internalized receptor labeling, mechanistic contrast with ghrelin established; single lab\",\n      \"pmids\": [\"31499133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Beta-hydroxybutyrate (BHB) directly downregulates LEAP2 expression in isolated murine hepatocytes and reduces circulating LEAP2 levels in mice after oral BHB administration, identifying BHB as a cell-autonomous suppressor of hepatic LEAP2 production during fasting/ketosis.\",\n      \"method\": \"BHB treatment of isolated murine hepatocytes (in vitro), oral BHB administration in mice, hepatic/intestinal Leap2 expression measurement, plasma LEAP2 quantification\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro hepatocyte experiment combined with in vivo BHB administration; single lab, two complementary methods\",\n      \"pmids\": [\"35352108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Glucagon infusion during somatostatin clamps significantly decreases plasma LEAP2 levels in humans. Insulin receptor antagonism offsets postprandial LEAP2 upregulation in mice. Insulin and glucagon receptor-expressing hepatocytes are the primary source of hepatic LEAP2 expression, coinciding with a putative enhancer-like signature bound by insulin- and glucagon-regulated transcription factors at the LEAP2 locus.\",\n      \"method\": \"Somatostatin clamp with glucagon infusion in humans, insulin receptor antagonist treatment in mice, hepatocyte-specific transcription factor binding analysis, plasma LEAP2 quantification\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human clamp study combined with mouse genetic/pharmacological intervention and genomic enhancer analysis; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"40056903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LEAP2 has antagonized GHSR1a since at least the emergence of coelacanth fish. Coelacanth LEAP2 and ghrelin both bind coelacanth GHSR1a with IC50 values in the nanomolar range, and coelacanth LEAP2 efficiently antagonizes coelacanth ghrelin-induced GHSR1a activation, demonstrating evolutionary conservation of the LEAP2-GHSR1a antagonism system.\",\n      \"method\": \"Binding assays and activation assays using coelacanth GHSR1a expressed in cell system, competitive inhibition analysis\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and functional assays with fish ortholog; single lab, two complementary methods\",\n      \"pmids\": [\"33966114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In teleost mudskipper, MOSPD2 (motile sperm domain-containing protein 2) was identified as a receptor mediating LEAP2's effects on monocytes/macrophages. BpMOSPD2 directly interacts with BpLEAP-2 (confirmed by Co-IP). Knockdown of BpMOSPD2 inhibited BpLEAP-2-induced chemotaxis, bacterial killing activity, and modulation of cytokine expression in MO/MΦ.\",\n      \"method\": \"Yeast two-hybrid cDNA library screening, co-immunoprecipitation, RNAi knockdown in primary mudskipper MO/MΦ, functional assays (chemotaxis, bacterial killing, cytokine qRT-PCR)\",\n      \"journal\": \"Zoological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus RNAi loss-of-function with multiple functional readouts; fish ortholog, single lab\",\n      \"pmids\": [\"33124217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In teleost mudskipper, BpLEAP2 stimulation triggers retromer-dependent intracellular trafficking of BpMOSPD2 from the ER to early endosomes and then to the plasma membrane. Knockdown of retromer subunits (VPS35, VPS26, VPS29) abolishes BpMOSPD2 membrane localization and BpLEAP2-induced MO/MΦ migration. Co-IP with mass spectrometry confirmed direct interaction between BpMOSPD2 and BpVPS35.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, Co-IP combined with mass spectrometry, retromer subunit knockdown (RNAi), migration assay\",\n      \"journal\": \"Zoological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS plus subcellular fractionation and RNAi epistasis; mechanistic trafficking pathway established; fish ortholog, single lab\",\n      \"pmids\": [\"41017400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LEAP2 overexpression in Xenopus embryos impairs normal embryonic development. In pluripotent embryonic cells, LEAP2 stimulates FGF signaling while reducing the activin response. LEAP2 also blocks FGF-induced migration of human vascular endothelial cells (HUVEC), suggesting an extracellular modulatory role for LEAP2 on FGF and activin signals.\",\n      \"method\": \"Xenopus gain-of-function overexpression, animal cap assays, HUVEC migration assay\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, amphibian model; functional assays without mechanistic receptor identification; relevance to mammalian LEAP2 unclear\",\n      \"pmids\": [\"27335344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Central (i.c.v.) administration of LEAP2 in mice reduces feeding and intake of palatable foods, attenuates accumbal dopamine release associated with palatable food exposure, and reduces the rewarding memory of high-preference foods. LEAP2 is expressed in reward-related brain areas including the laterodorsal tegmental area (LDTg), and infusion of LEAP2 into LDTg transiently reduces acute palatable food intake.\",\n      \"method\": \"i.c.v. and intra-LDTg LEAP2 administration in mice, microdialysis (accumbal dopamine), in situ expression profiling, behavioral food intake tests\",\n      \"journal\": \"Progress in neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct dopamine measurement by microdialysis plus regional brain injection to localize effect; single lab, multiple methods\",\n      \"pmids\": [\"38641041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LEAP2 enhances insulin secretion in isolated islets from male but not female mice, and reverses acyl-ghrelin-stimulated somatostatin release in males but not females. Estradiol (E2) pre-treatment of male islets abolished both AG-induced insulinostatic effects and their reversal by LEAP2, demonstrating sex- and hormone-dependent modulation of islet function by LEAP2 acting via GHSR1a.\",\n      \"method\": \"Isolated mouse islet secretion experiments (radioimmunoassay), E2 pre-treatment, SSTR3 antagonist experiments, qPCR of islet gene expression\",\n      \"journal\": \"The Journal of endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct islet secretion assays with pharmacological and hormonal manipulation; sex-specific mechanistic effect established; single lab\",\n      \"pmids\": [\"39292603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LEAP2 dietary regulation depends on meal composition: all meal challenges except fish oil increased jejunal Leap2 expression, while only a mixed meal increased liver Leap2 expression. Leap2 expression correlated with hepatic glycogen and jejunal lipid levels. Oleic acid (but not docosahexaenoic acid) increased Leap2 expression in intestinal organoids, indicating nutrient-specific cell-autonomous regulation.\",\n      \"method\": \"Mouse meal challenge experiments, intestinal organoid culture with specific fatty acids, jejunal/hepatic gene expression analysis, portal vein plasma LEAP2 measurement\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — organoid cell-autonomous assay combined with in vivo portal/systemic sampling; single lab, multiple nutrient conditions\",\n      \"pmids\": [\"37104087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Transcription factor CDX4 binds to the LEAP2 promoter region in the small intestine and positively regulates LEAP2 expression, as demonstrated by transcription factor prediction and dual luciferase assay.\",\n      \"method\": \"Dual luciferase reporter assay, transcription factor binding site analysis\",\n      \"journal\": \"Animals\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single luciferase assay in avian/chicken model without endogenous chromatin confirmation; single lab\",\n      \"pmids\": [\"36552416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Central LEAP2 administration in mice prevents alcohol-induced locomotor stimulation, suppresses the memory of alcohol reward, attenuates dopamine release in the nucleus accumbens caused by alcohol, and reduces alcohol consumption in rats. These effects parallel LEAP2's modulation of the mesolimbic dopamine system.\",\n      \"method\": \"i.c.v. LEAP2 administration in mice/rats, locomotor activity measurement, conditioned place preference, in vivo microdialysis (nucleus accumbens dopamine), voluntary alcohol consumption paradigm\",\n      \"journal\": \"Translational psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct dopamine microdialysis combined with behavioral assays; mechanistic link to mesolimbic dopamine established; single lab\",\n      \"pmids\": [\"39358354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In fish (Acrossocheilus fasciatus), the linear (disulfide-free) form of LEAP2 mature peptide shows potent antimicrobial activity and disrupts bacterial cell membrane integrity, while the oxidized (disulfide-bonded, cyclic) form has weak or no antibacterial activity. This establishes that disulfide bonds are not required for—and in fact impair—the antimicrobial function of LEAP2.\",\n      \"method\": \"Chemical synthesis of linear vs. oxidized LEAP2, circular dichroism spectroscopy, in vitro MIC assays, bacterial membrane integrity assay\",\n      \"journal\": \"BMC veterinary research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct structure-activity comparison with CD spectroscopy and functional assays; single lab, fish ortholog\",\n      \"pmids\": [\"38835040\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LEAP2 is an endogenous antagonist and inverse agonist of the ghrelin receptor GHSR, produced primarily by hepatocytes and enterocytes; its N-terminal region (particularly Phe4 and Arg6) binds competitively to GHSR (interacting with receptor residues Asp99, Phe279, Phe312), stabilizes an inactive Gq-dissociated receptor conformation, blocks both ghrelin-induced and constitutive GHSR signaling (including D2R cross-talk and CaV2.2 modulation), and thereby suppresses food intake (partly via hypothalamic POMC neurons and mesolimbic dopamine), GH secretion, and blood glucose elevation; its hepatic expression is regulated by insulin, glucagon, and beta-hydroxybutyrate, and by CDX4 in the intestine, with circulating levels rising postprandially and in obesity and falling during fasting.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LEAP2 is a hepatocyte- and enterocyte-derived peptide hormone that functions as the endogenous competitive antagonist and inverse agonist of the ghrelin receptor GHSR, opposing ghrelin to suppress food intake, growth hormone release, and glucose elevation [#0, #2]. Originally isolated from human blood as a furin-processed 40-residue antimicrobial peptide bearing two disulfide bonds [#1], its receptor-directed activity resides in the N-terminal region, which alone is sufficient to bind GHSR and to act as both an inverse agonist of constitutive receptor activity and an antagonist of ghrelin-evoked inositol-phosphate and calcium signaling [#2, #3]. Mutagenesis maps this interaction to LEAP2 residues Phe4 and Arg6 contacting GHSR residues Asp99, Phe279, and Phe312, and biophysical reconstitution shows the N-terminal region stabilizes an inactive, Gq-dissociated receptor conformation that also blocks GHSR modulation of D2R-dependent Gi signaling and CaV2.2 currents [#4, #5]. Physiologically, plasma LEAP2 falls with fasting and rises postprandially; genetic deletion sensitizes mice to ghrelin-driven feeding and GH secretion, and GHSR is required for LEAP2's anorexigenic and glucoregulatory actions, which engage hypothalamic POMC and CRF neurons and the mesolimbic dopamine system [#6, #7, #8, #9, #18]. Hepatic and intestinal LEAP2 expression is nutrient- and hormone-regulated, suppressed by beta-hydroxybutyrate and glucagon and controlled by insulin- and glucagon-responsive transcription in hepatocytes [#12, #13, #20]. The LEAP2–GHSR antagonism is evolutionarily ancient, conserved at least since coelacanth fish [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established LEAP2's molecular identity and biochemistry before any receptor role was known, defining the precursor, furin processing, disulfide architecture, and an antimicrobial activity.\",\n      \"evidence\": \"Purification from human blood ultrafiltrate, cloning, disulfide mapping, and in vitro antimicrobial assays\",\n      \"pmids\": [\"12493837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No receptor or signaling function identified at this stage\", \"Physiological role beyond antimicrobial activity unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Answered what LEAP2 does physiologically by identifying it as an endogenous GHSR antagonist that opposes ghrelin's effects on feeding, GH, and glucose.\",\n      \"evidence\": \"In vitro GHSR activation assays plus in vivo LEAP2 administration and anti-LEAP2 neutralizing antibodies in mice\",\n      \"pmids\": [\"29233536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding site and structural basis of antagonism not defined\", \"Whether activity resides in a peptide subregion unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Localized the receptor-active determinant to the LEAP2 N-terminus and distinguished inverse agonism from competitive antagonism.\",\n      \"evidence\": \"Binding, IP1, and calcium assays with full-length vs. N-terminal fragment plus in vivo food intake\",\n      \"pmids\": [\"30543423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific contact residues not yet mapped\", \"Kinetic basis of antagonism not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the kinetic/competitive mechanism by showing LEAP2 and ghrelin share a binding site, with slow LEAP2 dissociation converting it to functional non-competitive blockade.\",\n      \"evidence\": \"Radioligand binding and sequential vs. simultaneous ligand addition activation assays\",\n      \"pmids\": [\"30666806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, two methods\", \"Residue-level contacts not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapped the LEAP2–GHSR interface, identifying Phe4/Arg6 on LEAP2 and Asp99/Phe279/Phe312/Phe119 on GHSR1a as the key contacts.\",\n      \"evidence\": \"Alanine scanning of LEAP2, site-directed mutagenesis of GHSR1a, binding assays, structural modeling\",\n      \"pmids\": [\"32803260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural assignments partly modeled rather than solved\", \"No experimental complex structure\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the conformational mechanism of antagonism, showing the LEAP2 N-terminus stabilizes an inactive Gq-dissociated GHSR state and blocks GHSR effects on D2R-Gi and CaV2.2 currents.\",\n      \"evidence\": \"FRET with purified receptors in lipid nanodiscs and patch-clamp electrophysiology\",\n      \"pmids\": [\"34447311\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of the LEAP2-bound receptor\", \"Relevance of D2R cross-talk in vivo not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established LEAP2 as a physiologically required GHSR modulator via clean loss-of-function, linking deletion to ghrelin hypersensitivity and diet-induced metabolic phenotypes.\",\n      \"evidence\": \"LEAP2-KO mice with feeding, GH, c-Fos, metabolic, and histological readouts\",\n      \"pmids\": [\"34428557\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sex-specific high-fat-diet effects not fully mechanistically explained\", \"Tissue source contribution not dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed defined neuronal populations downstream of LEAP2: a fall in LEAP2 is required for fasting activation of PVH-CRF neurons, and POMC neurons mediate its anorexigenic effect.\",\n      \"evidence\": \"GHSR-KO/ghrelin-KO models, LEAP2(1-12) infusion, AAV overexpression in arcuate nucleus, and chemogenetic POMC inhibition\",\n      \"pmids\": [\"35504998\", \"36387867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Circuit connectivity between arcuate and PVH not resolved\", \"POMC step shown in single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated translational relevance and GHSR-dependence in humans, with LEAP2 infusion lowering postprandial glucose, GH, and food intake, absent in GHSR-null mice.\",\n      \"evidence\": \"Randomized placebo-controlled crossover trial in men plus GHSR-null mouse dosing\",\n      \"pmids\": [\"35492241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Long-term metabolic effects not assessed\", \"Female human data not reported\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined nutritional and hormonal control of LEAP2 production, identifying beta-hydroxybutyrate as a cell-autonomous suppressor of hepatic expression during fasting/ketosis.\",\n      \"evidence\": \"BHB treatment of isolated hepatocytes and oral BHB in mice with plasma LEAP2 quantification\",\n      \"pmids\": [\"35352108\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional mediators of BHB action not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended regulation to meal composition and intestinal origin, showing nutrient-specific (oleic acid) induction of Leap2 in organoids and tissue-specific responses.\",\n      \"evidence\": \"Mouse meal challenges, intestinal organoid fatty-acid exposure, portal/systemic LEAP2 sampling\",\n      \"pmids\": [\"37104087\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptors/sensors for fatty acid induction not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected LEAP2 to reward and addiction circuitry, showing central LEAP2 attenuates mesolimbic dopamine responses to palatable food and alcohol.\",\n      \"evidence\": \"i.c.v. and intra-LDTg LEAP2 administration, nucleus accumbens microdialysis, and behavioral reward/consumption assays in mice and rats\",\n      \"pmids\": [\"38641041\", \"39358354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor identity in reward regions not directly proven\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed sex- and hormone-dependent islet actions, with LEAP2 enhancing insulin secretion and reversing ghrelin-induced somatostatin release only in males, abolished by estradiol.\",\n      \"evidence\": \"Isolated mouse islet secretion assays with E2 and SSTR3 antagonist manipulation\",\n      \"pmids\": [\"39292603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of estradiol interference unknown\", \"Human islet generalizability not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified the transcriptional/endocrine wiring of hepatic LEAP2, implicating insulin- and glucagon-responsive transcription factors at an enhancer-like locus, with glucagon lowering and insulin raising plasma LEAP2.\",\n      \"evidence\": \"Human somatostatin-glucagon clamp, insulin receptor antagonism in mice, and hepatocyte transcription factor binding analysis\",\n      \"pmids\": [\"40056903\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific transcription factors not functionally validated\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether LEAP2's ancestral antimicrobial activity and a non-GHSR receptor axis operate alongside GHSR antagonism in mammals remains unresolved.\",\n      \"evidence\": \"Fish-ortholog MOSPD2/retromer immune signaling and amphibian FGF/activin effects lack a defined mammalian receptor context\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mammalian receptor for LEAP2's antimicrobial/immune actions identified\", \"Relationship between antimicrobial and GHSR-antagonist functions not integrated\", \"MOSPD2 axis demonstrated only in teleost\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 3, 5]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [2, 5, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8, 12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GHSR\", \"MOSPD2\", \"VPS35\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":6,"faith_pct":100.0}}