Microarray datasets were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) for hepatic gene expression analysis. GSE15653 was used to compare gene expression among lean individuals, obese individuals without T2D, and obese individuals with T2D. GSE13251 was analyzed to compare normal and NAFLD liver samples; in this study, NAFLD samples were referred to as MASLD to reflect updated disease terminology. Probe annotations were cross-referenced with GEO-provided annotation files, and LGALS3BP expression was examined among the extracted target genes.

All mice were maintained under standard conditions (12-hour light-dark cycle) with unrestricted access to food and water. LGALS3BP-knockout (KO) mice were generated using the CRISPR-Cas9 system (Macrogen, Seoul, Korea) by targeting exon 3 of LGALS3BP, as previously described [36]. Male wild-type (WT) and LGALS3BP KO mice were fed either chow diet (CD) or HFD (60% kcal from fat; Research Diet, New Brunswick, NJ, USA), with body weights recorded weekly for 12 or 24 weeks. At the study endpoints, mice were fasted for 6 hours prior to anesthesia.

Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Serum concentrations of total cholesterol (CHOL), triglycerides (TG), low-density lipoprotein cholesterol (LDL-CHOL), and high-density lipoprotein cholesterol (HDL-CHOL) were analyzed using an IDEXX Catalyst One chemistry analyzer (IDEXX Laboratories, Westbrook, ME, USA) according to the manufacturer’s instructions. Serum insulin levels were measured in 6-hour fasted mice using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (ab277390, Abcam, Cambridge, UK).

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were conducted in mice after a 6-hour fast. For GTT, mice were intraperitoneally injected with glucose (1 g/kg; Sigma, St. Louis, MO, USA), and for ITT, insulin (1 IU/kg; Sigma) was administered intraperitoneally. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes using ACCU-CHECK test strips (Roche, Basel, Switzerland). Areas under the curve (AUC) for GTT were calculated by subtracting basal glucose values, whereas ITT AUC values were derived directly from glucose measurements. Serum glucose concentrations were measured at baseline before GTT.

Total RNA was extracted from liver tissues and cells using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) and Hybrid R kit (GeneAll, Seoul, Korea), respectively, according to the manufacturers’ instructions. cDNA synthesis was performed, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with TATA-box binding protein (TBP) as an internal control. Cycling conditions comprised 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 20 seconds, for a total of 35 cycles. Relative gene expression was calculated using the 2−ΔΔCT method. Primer sequences are listed in Supplemental Table S1 .

Tissues and cells were lysed in tissue protein extraction reagent (T-PER) or radioimmunoprecipitation assay buffer (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Fisher, Waltham, MA, USA). Equal protein amounts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocking, membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were detected with the LAS-3000 (Fujifilm, Tokyo, Japan) and quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). Primary antibodies are detailed in Supplemental Table S2.

Alpha mouse liver 12 (AML12), Hepa-1c1c7, and HepG2 cells were cultured in their respective media with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco), and maintained at 37°C in a humidified atmosphere containing 5% CO₂. Primary hepatocytes were isolated from C57BL/6J mice using a two-step collagenase perfusion method, and maintained in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) containing 10% FBS and 1% penicillin-streptomycin. AML12 cells were cultured in DMEM/F12 (Welgene, Seoul, Korea) supplemented with 10% FBS, 1% penicillin-streptomycin, 1×insulin-transferrin-selenium-pyruvate (Sigma), and 40 nM dexamethasone (Sigma). Hepa-1c1c7 cells were maintained in Minimum Essential Medium Eagle, Alpha Modification (Alpha MEM, Welgene), whereas HepG2 cells were cultured in MEM (Welgene) supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Welgene).

Prior to treatments, cells were serum-starved overnight. Palmitate (Sigma) conjugated with bovine serum albumin (BSA; Sigma) at a molar ratio of 6:1 was applied at a final concentration of 300 μM for durations of 6 or 16 hours. Insulin (Sigma) treatments were performed at concentrations of 10 or 100 nM for 10 minutes. Recombinant LGALS3BP (R&D Systems, Minneapolis, MN, USA) was applied at concentrations of 2.5 or 5 μg/mL for 1 to 6 hours, depending on the experimental conditions. Lipoteichoic acid (LTA, Sigma) was applied at a final concentration of 1 μg/mL for 1 to 8 hours. Following treatments, cells were lysed for subsequent analyses by qRT-PCR or Western blotting.

LGALS3BP silencing was performed using Lipofectamine RNAiMax (Invitrogen, Waltham, MA, USA) and siRNA (Bioneer, Daejeon, Korea) (Supplemental Table S3) via reverse transfection. Cells were analyzed 24 hours after transfection.

HepG2 cells were serum-starved overnight, washed with phosphate-buffered saline, and incubated for 4 hours in glucose-free DMEM (Gibco) without phenol red, supplemented with 2 mM sodium pyruvate, 20 mM sodium lactate, 2 mM L-glutamine, and 15 mM HEPES, in the presence of recombinant LGALS3BP (5 μg/mL). Glucose concentration in the culture medium was measured using a glucose assay kit (Abcam) according to the manufacturer’s instructions.

We used RNA sequencing data from our previously published study on Hepa-1c1c7 cells treated with recombinant LGALS3BP or vehicle control [34]. Differentially expressed genes (DEGs) were identified using Cuffdiff (ver 2.2.1) with an adjusted P<0.05 and |log1.5-fold change| >1. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on upregulated DEGs using Enrichr (https://maayanlab.cloud/Enrichr/), and pathways with an adjusted P<0.05 were considered significantly enriched. Enriched pathways and genes associated with NF-κB signaling pathways were visualized using SRplot (https://www.bioinformatics.com.cn).

Structural modeling of the LGALS3BP-TLR2 complex was carried out using AlphaFold Multimer (DeepMind and Isomorphic Labs) with UniProt sequences (mouse LGALS3BP: ID Q07797; mouse TLR2: ID Q9QUN7). Default parameters were applied, and models with an interface predicted template modeling (iPTM) score ≥0.5 were selected for further analysis. Structural visualization and identification of key interaction sites were conducted using PyMOL v3.1.3 (Schrödinger, LLC, New York, NY, USA).

To validate computational predictions, immunoprecipitation assays were performed in Hepa-1c1c7 cells transfected with Flag- or Myc-tagged full-length LGALS3BP as well as Myc-tagged LGALS3BP domain constructs. Protein complexes were immunoprecipitated using anti-Flag, anti-Myc, or control immunoglobulin G (IgG) antibodies, followed by Western blot analysis to detect co-precipitated TLR2, TLR3, and TLR4.

The full-length protein sequences of both LGALS3BP and TLR2 were used for structural visualization. Key interaction regions were identified by focusing on binding between TLR2 (residues 25–784) and specific domains of LGALS3BP. Structural proximity and binding complementarity were analyzed to identify key interaction sites, integrating computational and experimental data to elucidate the mechanism underlying the LGALS3BP-TLR2 interaction.

Statistical analyses were conducted using GraphPad Prism version 10.0 (GraphPad Software Inc., San Diego, CA, USA). A two-tailed Student’s t test or Welch’s correction was applied for comparisons between two groups, while one-way or two-way analysis of variance (ANOVA) followed by appropriate post hoc tests (Tukey’s or Dunnett’s multiple comparisons) was used for multiple-group comparisons. Data are presented as mean± standard error of the mean or standard deviation. Statistical significance was defined as P<0.05.

All animal care procedures and experiments were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the IACUC of Chonnam National University Medical School (Approval No. CNU IACUC-H-2024-36).

To explore the role of LGALS3BP in T2D and liver metabolic disorders, we analyzed hepatic gene expression data from a publicly available transcriptome dataset. In GSE15653, which contains liver samples from five lean participants, four obese participants without T2D, and nine obese participants with T2D, LGALS3BP expression progressively increased among obese individuals, with significantly higher levels in those with T2D compared to lean controls (Fig. 1A). Similarly, in GSE135251, which includes liver samples from 10 control participants and 206 MASLD patients, LGALS3BP expression was significantly upregulated in MASLD patients compared to controls (Fig. 1B). Hepatic LGALS3BP mRNA and protein levels were nearly twice as high in mice fed a HFD for 24 weeks, a well-established MASLD model (Fig. 1C). Consistent with these findings, serum ELISA analysis indicated significantly elevated circulating LGALS3BP levels in HFD-fed mice compared to chow-fed controls (Fig. 1C). Palmitate-induced lipotoxicity significantly increased LGALS3BP protein levels in whole-cell lysates of AML12, Hepa-1c1c7, and HepG2 cells, with significant increases observed after 6 hours of treatment in Hepa-1c1c7 cells and after 16 hours in AML12 and HepG2 cells (Fig. 1D).

To investigate the unexplored role of LGALS3BP in modulating insulin signaling pathways, LGALS3BP KO and WT littermates were fed either a CD or HFD for 12 weeks (Fig. 2A). While LGALS3BP deficiency slightly reduced body weight, no statistically significant differences were observed between KO and WT mice on either diet (Fig. 2B). Despite similar weight gain, HFD-fed LGALS3BP KO mice exhibited significantly lower serum ALT and AST levels, suggesting that LGALS3BP deficiency confers protection against HFD-induced liver injury (Fig. 2C). HFD-fed LGALS3BP KO mice demonstrated improved glucose tolerance and insulin sensitivity compared to WT mice, as assessed by GTT and ITT, respectively (Fig. 2D, E). Consistent with these findings, LGALS3BP KO livers exhibited significantly reduced expression of key gluconeogenic genes, including glucose-6-phosphatase 1 (G6pase1), phosphoenolpyruvate carboxykinase 1 (Pck1), glycogen phosphorylase (Pygl), and pyruvate dehydrogenase kinase 4 (Pdk4), all of which regulate hepatic glucose metabolism (Fig. 2F). Furthermore, fasting serum glucose and insulin levels were significantly lower in HFD-fed LGALS3BP KO mice than in WT mice (Fig. 2G, H). At the molecular level, the phosphorylation levels of protein kinase B (AKT) (Ser473) and forkhead box protein O1 (FoxO1) (Ser256) were significantly increased in LGALS3BP KO livers, whereas phosphorylation levels of glycogen synthase kinase 3 beta (GSK3β) (Ser9) and mammalian target of rapamycin complex 1 (mTOR) (Ser2448) remained comparable between groups (Fig. 2I). Despite these metabolic improvements, hepatic lipid accumulation and TG content were not significantly different between WT and LGALS3BP KO mice in liver tissues and serum (Supplemental Fig. S1A-C). Additionally, LGALS3BP KO mice exhibited significantly lower serum LDL-CHOL levels, while total CHOL and HDL-CHOL levels remained comparable between WT and KO mice (Supplemental Fig. S1D-F). Interestingly, further analysis revealed that hepatic expression levels of genes involved in both lipid accumulation and fatty acid oxidation pathways were significantly downregulated in LGALS3BP KO mice compared with WT controls (Supplemental Fig. S1G, H).

To further investigate the impact of LGALS3BP deficiency on insulin sensitivity, we used mouse primary hepatocytes, AML12, Hepa-1c1c7, and HepG2 cells. LGALS3BP knockdown or KO significantly enhanced insulin signaling, evidenced by increased phosphorylation of AKT (Ser473) and FoxO1 (Ser256) following insulin stimulation, whereas insulin receptor beta (Ins Rβ) (Tyr1361) phosphorylation remained comparable between insulin-treated groups (Fig. 3A-D). In addition, to further assess the restoration of insulin signaling, we examined the phosphorylation of key downstream effectors, GSK3β (Ser9) and mammalian target of rapamycin complex 1 (mTORC1) (Ser2448). In primary hepatocytes and AML12 cells, LGALS3BP depletion significantly increased both p-GSK3β and p-mTORC1 levels, supporting enhanced insulin responsiveness (Fig. 3A, B). However, these effects were not consistently observed in Hepa-1c1c7 and HepG2 hepatic cancer cell lines (Fig. 3C, D), suggesting that LGALS3BP’s regulation of these downstream insulin targets may vary depending on cellular context. Accompanying this enhanced insulin signaling was significant downregulation of gluconeogenesis-related genes (G6pase and Pck1) in LGALS3BP KO hepatocytes and knockdown cells under basal conditions (Fig. 3E-G). Under lipotoxic conditions induced by palmitate, LGALS3BP KO hepatocytes displayed significantly increased phosphorylation of AKT (Ser473) and FoxO1 (Ser256) compared to WT cells (Supplemental Fig. S2A). Furthermore, palmitate-induced upregulation of gluconeogenic genes (G6pase and Pck1) as well as Pdk4 was significantly attenuated in LGALS3BP KO hepatocytes, along with reduced expression of the lipogenic transcription factor sterol regulatory element-binding protein 1c (Srebp1c) (Fig. 3H). Interestingly, inflammatory markers associated with IR, such as interleukin 1 beta (Il1β) and Tnfα, were also significantly downregulated in LGALS3BP KO hepatocytes (Supplemental Fig. S2B), suggesting a role for LGALS3BP in regulating insulin sensitivity and inflammation under IR conditions. Dose-dependent insulin stimulation under BSA conditions resulted in significantly higher AKT (Ser473) phosphorylation in LGALS3BP KO hepatocytes compared to WT controls. Under palmitate-induced lipotoxicity, which typically suppresses insulin-dependent AKT phosphorylation, LGALS3BP KO hepatocytes maintained higher AKT (Ser473) phosphorylation following insulin stimulation compared to WT cells (Fig. 3I).

Given the elevated circulating LGALS3BP levels observed in the MASLD mouse model (Fig. 1B), we investigated whether direct exposure to LGALS3BP impairs hepatic insulin signaling. Although insulin-induced Ins Rβ (Tyr1361) phosphorylation was comparable between control and LGALS3BP-deficient cells (Fig. 3A-D), recombinant LGALS3BP significantly suppressed insulin-stimulated phosphorylation of Ins Rβ and its downstream signaling proteins, including PDK1 (Ser241) and AKT (Ser473), in hepatic cells (Fig. 4A-C). These differences may reflect the dual nature of LGALS3BP, which is constitutively expressed under physiological conditions but rapidly upregulated during acute inflammatory responses. Consistent with these findings, LGALS3BP treatment significantly upregulated gluconeogenic genes (G6pase and Pck1) in both Hepa-1c1c7 and HepG2 cells (Fig. 4D, E). Additionally, LGALS3BP treatment increased glucose production in HepG2 cells by approximately threefold under basal conditions compared to controls (Fig. 4F), further supporting its role in promoting hepatic IR by enhancing gluconeogenesis and glucose output.

To elucidate the molecular mechanisms through which LGALS3BP contributes to hepatic IR, RNA sequencing was performed on Hepa-1c1c7 cells treated with recombinant LGALS3BP or vehicle control. DEG analysis and subsequent KEGG pathway enrichment analysis identified significant upregulation of pathways associated with metabolic dysfunction and inflammation, including TNF signaling, NF-κB signaling, TLR signaling, NAFLD, and IR-related pathways (Fig. 5A). The NF-κB and TLR pathways, which are closely linked to inflammation-induced IR, were notably enriched. Additionally, enrichment of the phosphatidylinositol 3-kinase (PI3K)-Akt, FoxO, and advanced glycation end-products–receptor for advanced glycation end-products (AGE-RAGE) signaling pathways—known regulators of glucose metabolism, oxidative stress, and cell survival—suggests that LGALS3BP exerts multifaceted effects in metabolic dysfunction. Further analysis of DEGs within the NF-κB pathway revealed significant upregulation of key signaling molecules, including myeloid differentiation primary response 88 (Myd88), TNF receptor associated factor 2 (Traf2), Traf3, Rela, Relb, nuclear factor kappa B subunit 1 (Nfkb1), and Nfkb2, following LGALS3BP treatment (Fig. 5B). To validate these findings, we assessed the expression of NF-κB-dependent pro-inflammatory genes. In Hepa-1c1c7 cells, recombinant LGALS3BP treatment significantly increased expression of Tnfα, Il1β, Il6, and C-C motif chemokine ligand 2 (Ccl2). In HepG2 cells, recombinant LGALS3BP similarly increased the expression levels of TNFα, IL1β, and IL6, but CCL2 expression remained unchanged (Fig. 5C). Consistent with these results, immunoblot analysis revealed that LGALS3BP treatment activated key mediators of inflammatory signaling by increasing the phosphorylation of IKKα/β, a central regulator of NF-κB activation [11,18,19]. Additionally, IRS1 phosphorylation at Ser307, a modification known to impair insulin signaling [9,10,19], was significantly elevated following LGALS3BP treatment (Fig. 5D).

Since TLRs (including TLR2, TLR3, and TLR4) are critical regulators in activating inflammatory signaling [12,22,23], we investigated whether LGALS3BP interacts with TLRs to facilitate this activation. Immunoprecipitation assays confirmed specific interactions between Flag-tagged LGALS3BP and TLR2, with no corresponding bands in the IgG control (Fig. 5E). To further characterize this interaction, we employed AlphaFold3 structural modeling to predict the three-dimensional structure of the LGALS3BP-TLR2 complex. The model revealed that residues 153–221 of the Broad-complex, Tramtrack, and Bric-à-brac (BTB) domain on LGALS3BP and residues 320–398 of the leucine-rich repeat (LRR) domain on TLR2 were involved in this interaction, forming a broad and stable interface with multiple contact points (Fig. 5F, Supplemental Table S4).

To evaluate the functional relevance of this interaction, we examined IKKα/β activation following stimulation with LTA, a well-established TLR2 ligand. LTA treatment induced a time-dependent increase in phosphorylation of IKKα/β, NF-κB, and IRS1 (Ser307), progressively increasing over the time course (Fig. 5G). LGALS3BP knockdown significantly reduced LTA-induced phosphorylation of IKKα/β, NF-κB, and IRS1 (Ser307) (Fig. 5H), confirming that LGALS3BP enhances TLR2-mediated IKKα/β activation and IR. Furthermore, LTA stimulation increased Ccl2 and Il6 expression, while LGALS3BP knockdown attenuated this effect (Supplemental Fig. S3A). Recombinant LGALS3BP displayed a synergistic effect with LTA, further amplifying inflammatory gene expression compared to LTA alone (Supplemental Fig. S3B).

To investigate the role of LGALS3BP in hepatic inflammation and TLR2-mediated inflammatory responses in vivo, we employed a long-term (24-week) HFD feeding model using WT and LGALS3BP KO mice. In HFD-fed WT mice, hepatic Tlr2 mRNA expression was significantly upregulated compared to CD-fed WT mice. In contrast, HFD-fed LGALS3BP KO mice exhibited notably reduced Tlr2 mRNA levels compared to HFD-fed WT mice. Similarly, key pro-inflammatory markers, including Tnfα, Il1β, Ccl2, and F4/80, were markedly elevated in HFD-fed WT mice compared to CD-fed WT mice. However, these inflammatory markers were significantly attenuated in HFD-fed LGALS3BP KO mice compared to HFD-fed WT mice, whereas Il6 expression remained unchanged between groups (Fig. 6A).

Consistent with these transcriptional findings, immunoblot analyses revealed significantly reduced TLR2 protein levels in HFD-fed LGALS3BP KO mice compared to WT controls. Since TLR2 activation initiates downstream signaling cascades involving IKK, JNK, and extracellular signal-regulated kinase (ERK) [19,37,38], we assessed their phosphorylation status in liver tissue. HFD-fed LGALS3BP KO mice exhibited decreased phosphorylation of IKKα/β and NF-κB p65, correlating with reduced TLR2 expression (Fig. 6B). Additionally, ERK1/2 phosphorylation was significantly reduced in LGALS3BP KO mice, while total ERK1/2 protein levels remained unchanged (Fig. 6B). Moreover, HFD-fed KO mice exhibited significantly lower IRS1 Ser307 phosphorylation, further supporting its role in inflammation-driven IR (Fig. 6B). However, the expression levels of lipid-accumulation-related genes did not differ significantly between LGALS3BP KO and WT mice, whereas cluster of differentiation 36 (Cd36) expression was markedly reduced, indicating that LGALS3BP deficiency attenuates hepatic inflammation without substantially altering lipid metabolism under chronic metabolic stress (Supplemental Fig. S4). Given that chronic inflammation under prolonged HFD conditions contributes to hepatic fibrosis as part of the wound-healing response, we examined fibrosis markers in the liver. LGALS3BP KO mice exhibited significantly decreased expression of fibrosis markers, including transforming growth factor beta 1 (Tgfβ1), collagen type I alpha 1 (Col1a1), S100 calcium-binding protein A6 (S100a6), and platelet-derived growth factor subunit B (Pdgfb), suggesting that LGALS3BP deficiency mitigates fibrotic progression in the liver (Fig. 6C). These observations align well with our previous study demonstrating that LGALS3BP deficiency reduces hepatic fibrosis by limiting TGFB1 availability [35]. These findings collectively suggest that LGALS3BP may represent a promising therapeutic target for ameliorating hepatic fibrosis and IR in metabolic dysfunction-associated steatohepatitis.