Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis

The study addresses how oxidative stress perturbs hepatic lipid metabolism and promotes steatosis and obesity by disrupting redox balance. While lipid droplets (LDs) are known to buffer reactive oxygen species (ROS) by sequestering vulnerable lipids, the sorting mechanisms that direct unsaturated fatty acid–triglycerides (UFA-TG) into LDs during oxidative stress remain poorly understood. The authors hypothesize that lipopolysaccharide-binding protein (LBP), an acute-phase protein, is upregulated by oxidative stress and modulates LD homeostasis by selectively capturing and depositing UFA-TG into LDs to prevent peroxidation and lipolysis. They further posit that redox signaling, via interaction with peroxiredoxin 4 (PRDX4), regulates LBP trafficking on and off LDs, thereby coupling redox sensing with lipid metabolism. This work aims to elucidate these mechanisms and their systemic metabolic consequences.

Background literature indicates oxidative stress as a key driver of hepatic steatosis and metabolic dysfunction, with LDs serving not only as lipid storage organelles but also as part of the cellular antioxidant system. Prior studies show ROS can stimulate LD biogenesis to protect polyunsaturated lipids from peroxidation, and numerous LD-associated proteins have been cataloged. LBP is known as an extracellular LPS ligand and has reported antioxidant and hepatoprotective roles; LBP knockout or downregulation reduces liver fat accumulation in diet-induced models, and circulating LBP correlates with insulin resistance and MAFLD. PRDX4, an ER-localized peroxidase, protects against hepatic steatosis and undergoes redox-state-dependent conformational changes that can modulate protein interactions. However, specific mechanisms by which proteins like LBP selectively sort UFA-TG into LDs under oxidative stress were unclear prior to this study.

- Multi-omics discovery: Hepatic transcriptomics and LD proteomics were performed in 8-week-old C57BL/6J mice before and after 24 h fasting to identify LD compositional changes under oxidative stress. - Cell assays: HepG2 cells treated with oxidative stressors (H2O2, starvation, heat shock) and lipids (Bodipy C12, palmitic acid) assessed for LBP expression and subcellular localization by confocal microscopy. - Animal models: Generated conventional LBP knockout (LBP−/−) and hepatocyte-specific LBP knock-in Alb-LBP-3"flag (LBPKIKI) mice; assessed steatosis on chow, high-fat diet (HFD), and ketogenic diet; measured hepatic triglycerides (TG), VLDL export (Poloxamer 407 assay), serum TG/FFA, glucose/insulin, GTT/ITT, and body fat. - Primary hepatocytes: Isolated from WT, LBP−/−, and LBPKIKI mice; LD formation evaluated after Bodipy C12. - Lipidomics: Non-targeted and targeted lipidomics on livers from WT and LBPKIKI mice (16-week HFD), and immunoprecipitated LBP-bound lipids before/after H2O2; chain length and saturation analyses. - Bioenergetics: Seahorse XF96 analyses of oxygen consumption rate (OCR) and fatty acid oxidation (FAO) during palmitate (PA) vs palmitoleate oxidation; inhibitor assays (BPTES, UK5099, Etomoxir); metabolic cage respiratory exchange ratio (RER). - Lipolysis assays: In vitro lipolysis in HepG2 under starvation, forskolin, and isoproterenol; in vivo fasting-induced lipolysis assessed by P-HSL translocation to LDs and biochemical readouts. - Protein–lipid interactions: In vitro lipid overlay binding assays testing fatty acids and triglycerides of varying chain length/unsaturation, with and without H2O2; microscale thermophoresis (MST) measured Kd for LBP–tridocosahexaenoin binding pre/post oxidation. - Structural biology and modeling: AlphaFold predictions, secondary structure prediction, de novo modeling of LBP; molecular docking/molecular dynamics simulations to map the C-segment (#4-helix) lipid-binding groove and effects of oxidation; mutagenesis (H294A/H294G; F436L) and functional localization assays. - Electron microscopy: APEX2-tagged LBP in HepG2 cells for TEM visualization of LBP within LDs. - Redox modulation: Antioxidant treatments (N-acetyl-L-cysteine, NAC) and polyene phosphatidylcholine (PC) to evaluate impacts on LBP localization, LD homeostasis, lipid composition; Bodipy FL C12-HPC used to track phosphatidylcholine interactions and LBP shuttling. - PRDX4 interaction: Database mining (IntAct), molecular docking of LBP–PRDX4, co-immunostaining/co-IP; PRDX4 knockdown (shRNA) and mutants (PRDX41–243, PRDX4C245A) to probe redox-state–dependent interactions and effects on LBP trafficking and LD dynamics. - Chronic stress models: Chronic jet lag and forced swimming tests evaluated for systemic LBP elevation and metabolic outcomes; NAC rescue experiments conducted in LBPKIKI mice under HFD and fasting. - Statistics: Appropriate ANOVA, t-tests, and enrichment analyses with defined replicates and significance thresholds (P < 0.05).

- Oxidative stress upregulates LBP and targets it to lipid droplets: Fasting, H2O2, starvation, and heat shock increased hepatic/cellular LBP expression and its colocalization with LDs; fatty acid exposure alone did not induce LBP. - LBP drives LD enlargement and hepatic TG accumulation: LBP overexpression in HepG2 promoted large LDs; LBPKIKI mice showed severe steatosis on chow/HFD; LBP−/− hepatocytes had negligible LD formation; reintroduction of LBP restored hepatic TG and LD formation. LBP-APEX2 localized predominantly to large LDs by TEM. - Selective sequestration of long-chain polyunsaturated TG (LCPUFA-TG): Lipidomics showed enrichment of LCPUFA-TG in LBPKIKI livers; targeted lipidomics indicated increased PUFA and decreased short-chain saturated fatty acids (SCFAs). LBP did not colocalize with PA-derived LDs; LBP enhanced mitochondrial catabolism of PA and increased OCR during PA oxidation but not with palmitoleate; effect required intact LBP (abolished by F436L loss-of-function mutation). - Oxidation enhances LBP–TG binding: After H2O2, LBP bound more TGs, preferring long-chain unsaturated species. MST showed Kd for LBP–tridocosahexaenoin improved from 27.434 µM to 378.62 nM post-oxidation, indicating markedly increased affinity. - Antioxidant function via lipolysis suppression: Integrated omics showed LBP−/− upregulated TG transport/antioxidant genes and downregulated lipolysis genes. LBP overexpression reduced cellular ROS and lipid peroxides under starvation, promoted sequestration of peroxidized TGs in LDs, and inhibited lipolysis (reduced P-HSL and its LD translocation). In vivo fasting-induced lipolysis was attenuated by LBP. - NAC counters LBP-induced steatosis and modulates lipid balance: NAC reduced hepatic TG and LDs in LBPKIKI mice, increased phospholipids, and promoted LBP export from LDs. PC also promoted LBP exit to ER but less efficiently than NAC; excessive PC during ongoing oxidative stress was detrimental. - PRDX4 as redox-state chaperone for LBP: PRDX4 interacted with LBP (enhanced under starvation), co-localized on LDs, and regulated LBP export; PRDX4 knockdown prevented LBP export and increased LD size, while its overexpression promoted LBP exit and reduced LDs in the presence of PC. Oxidative stress (H2O2) disrupted the LBP–PRDX4 interaction. PRDX4 mutants mimicking oxidized/locally unfolded states showed increased binding to LBP’s N-terminus. - Structural basis in LBP C-segment: AlphaFold and de novo modeling identified a conserved #4-helix (residues 286–297) within the C-segment hydrophobic groove critical for TG capture; docking revealed more hydrogen bonds and lower binding energy with oxidized TGs. His294 was essential; H294A/H294G reduced LD translocation. F436L narrowed the groove and reduced TG entry and hepatic TG accumulation upon reintroduction. - Systemic metabolic consequences: LBPKIKI mice had higher hepatic TG export (Poloxamer 407), developed obesity on HFD with increased adiposity, higher fasting glucose/insulin, and impaired GTT/ITT. Bioenergetics favored lipid over glucose oxidation (lower RER; OCR responses to UK5099 and Etomoxir). Chronic stress (CJL, FST) elevated serum LBP, hepatic/serum TG, body fat ratio, and induced insulin resistance/glucose intolerance. NAC reduced body weight gain and fat mass; brief NAC after fasting raised serum FFA and lowered TG. Ketogenic diet (90% saturated fat) did not induce fatty liver or obesity in LBPKIKI or controls, consistent with LBP’s preference for PUFA-TG.

The findings establish LBP as a stress-inducible effector that couples redox signaling to lipid metabolism by selectively capturing LCPUFA-TG and depositing them into LDs, thereby limiting peroxidative damage and suppressing lipolysis during oxidative stress. This mechanism clarifies how LD homeostasis is tuned to protect vulnerable unsaturated lipids and aligns with observed reductions in steatosis when LBP is absent. The oxidation-enhanced affinity of LBP for PUFA-TG, mediated by a conserved #4-helix in the C-segment and key residues (e.g., His294), provides a structural rationale for selective lipid sequestration. PRDX4 functions as a redox-state sensor and chaperone that regulates LBP shuttling: under oxidative conditions, LBP accumulates on LDs to exert antioxidant protection; upon redox resolution and phospholipid rebalance, PRDX4 promotes LBP export, permitting lipolysis and LD remodeling. Systemically, chronic LBP elevation shifts fuel preference toward lipid oxidation, elevates hepatic TG export, and contributes to obesity and insulin resistance, particularly under chronic stress. Therapeutically, antioxidants like NAC, which both resolve oxidative stress and increase phospholipids, facilitate LBP export from LDs and reverse steatosis/obesity phenotypes more effectively than phosphatidylcholine alone during ongoing oxidative stress.

This study identifies LBP as a key regulator of LD homeostasis under oxidative stress, selectively sequestering LCPUFA-TG into LDs to protect against peroxidation and inhibit lipolysis. Oxidation enhances LBP–TG binding via a conserved C-segment #4-helix, while PRDX4 mediates redox-dependent LBP trafficking between LDs and ER. Elevated LBP drives hepatic steatosis and systemic metabolic disturbances, including obesity and insulin resistance, particularly under chronic stress. Antioxidant therapy (e.g., NAC) effectively counteracts LBP-driven lipid accumulation by restoring redox balance and phospholipid/TG homeostasis, suggesting a redox-based therapeutic avenue for MAFLD and stress-induced metabolic dysfunction. Future research should delineate the biophysical properties of LBP-enriched LDs, quantify LBP’s contribution to obesity and metabolic disease progression, and assess population-level links between stress, LBP, and cardiometabolic outcomes.

- The precise biophysical characterization of LDs containing LBP and how this affects lipase access and LD dynamics remains unresolved. - The extent to which LBP causally contributes to obesity and associated metabolic diseases (e.g., diabetes, cardiovascular disease) over chronic timescales is not fully defined. - Structural inferences (e.g., #4-helix role) are supported by modeling, docking, and mutagenesis but lack direct high-resolution structural validation with bound lipid ligands. - Some interventions (e.g., phosphatidylcholine) may cause harm under unresolved oxidative stress; contextual therapeutic windows require further study. - Translational generalizability to humans and the impact of genetic variation (e.g., F436L) warrant clinical investigation.