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

Oxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, significantly contributes to hepatic steatosis and obesity. Toxic lipid metabolites, such as diacylglycerols and lysophosphatidylcholine, generated during oxidative stress, cause cellular damage and lipotoxicity, a key factor in metabolic dysfunction progression. Even without overweight or acute hunger, chronic stress-induced oxidative stress disrupts lipid metabolism, increasing all-cause mortality risk. While antioxidants can alleviate metabolic dysfunction in animal models, long-term use poses safety concerns, highlighting the need to understand the molecular mechanisms driving oxidative stress-induced lipid metabolic dysfunction. Recent research indicates that lipid droplets (LDs), the primary organelles for neutral lipid accumulation, function not only in lipid storage and mobilization but also as crucial components of the cellular antioxidant system. LD homeostasis is critical for cell survival under stress. During oxidative stress, LD biogenesis is stimulated to protect vulnerable lipids, such as unsaturated fatty acids (UFAs), from ROS-induced peroxidation, maintaining lipid homeostasis. This process is precisely regulated by LD-associated proteins. Despite identifying numerous LD-associated proteins, the sorting mechanisms for UFA-triglycerides (TGs) in response to oxidative stress remain poorly understood. This study aimed to elucidate a fundamental mechanism regulating the oxidative stress response of LDs, focusing on the role of lipopolysaccharide-binding protein (LBP).

Previous studies have shown that LBP exhibits an antioxidant effect and that LBP knockout mice display reduced liver fat accumulation, indicating a potential role of LBP in lipid metabolism. Other research has characterized LBP's function as an extracellular lipopolysaccharide (LPS) ligand. Additionally, existing literature describes the various pathways involved in ROS-induced LD augmentation. However, the precise mechanism by which LBP regulates LD homeostasis under oxidative stress hasn't been fully explored. The study builds upon this existing knowledge by investigating the specific interactions of LBP with LDs and its impact on lipid metabolism and redox signaling in the context of oxidative stress.

The study employed a multi-faceted approach combining transcriptomic and proteomic analyses of liver tissue from 8-week-old C57BL/6J mice before and after a 24-h fast to identify changes in LD composition and regulation under oxidative stress. The integrative analysis of liver transcriptome and LD proteome data identified LBP as a key protein significantly altered under fasting-induced stress. Further experiments used HepG2 cells to investigate the effects of oxidative stress (induced by starvation, hydrogen peroxide, and heat shock) on LBP expression and localization within LDs. The study utilized conventional knockout (LBP⁻⁻) mice and Alb-LBP-3"flag (LBP^KIKI) mice to investigate the in vivo effects of LBP on steatosis. Lipidomics analysis was conducted on liver tissues from LBP^KIKI mice fed a high-fat diet to determine the impact of LBP on lipid composition. The study used Seahorse XF96 analyzer to assess the oxygen consumption rate (OCR) during fatty acid oxidation. Immunoprecipitation experiments analyzed LBP binding to lipids before and after oxidation treatment. A lipid-binding assay assessed LBP's binding to various fatty acids and triglycerides. Microscale thermophoresis (MST) measured the interaction dissociation constant (Kd) between LBP and tridocosahexaenoin. Proteomics and transcriptomics were used to assess the functional consequences of LBP-dependent deposition of LCPUFA-TG. N-acetyl-L-cysteine (NAC), a ROS scavenger, was administered to evaluate whether antioxidant therapy could alleviate LBP-induced steatosis. Molecular docking was used to explore the interaction between LBP and PRDX4. Fluorescence recovery after photobleaching (FRAP) experiments were performed to assess the mobility of LBP within LDs. The study also investigated the chronic effects of LBP upregulation on obesity by observing LBP^KIKI mice fed a high-fat diet and subjecting mice to chronic jet lag or forced swimming tests to induce chronic stress.

Fasting-induced oxidative stress upregulated hepatic LBP expression and its localization to LDs. Overexpression of LBP in HepG2 cells and in LBP^KIKI mice resulted in increased hepatic TG accumulation and steatosis. Lipidomics analysis revealed that LBP promotes the accumulation of long-chain polyunsaturated fatty acid-triglycerides (LCPUFA-TGs) in LDs, which are more prone to peroxidation. LBP overexpression enhanced the catabolism of saturated fatty acids (SCFAs) but not unsaturated fatty acids. LBP showed a stronger binding affinity to unsaturated fatty acids and triglycerides, particularly to cervonic acid, which was further enhanced by H2O2 treatment. LBP inhibits lipolysis by reducing phosphorylated hormone-sensitive lipase (P-HSL) levels, thus preventing the release of peroxidized lipids. NAC treatment significantly reduced LBP-mediated TG accumulation, increased phospholipid levels, and facilitated the removal of LBP from LDs by promoting its interaction with PRDX4. PRDX4, an ER-localized peroxidase, was identified as a redox state sensor for LBP, regulating its shuttle from LDs. The N-terminal segment of LBP showed high binding affinity for PRDX4, and this interaction is modulated by the redox state of PRDX4. The C-segment hydrophobic area and the #4-helix of LBP are crucial for TG capture and deposition. Chronic high expression of LBP induced obesity, insulin resistance, and glucose intolerance in mice. NAC treatment reversed LBP-induced TG accumulation in mice, and feeding a ketogenic diet prevented obesity in LBP^KIKI mice.

This study provides a novel mechanistic understanding of how LBP regulates hepatic lipid homeostasis under oxidative stress. The findings challenge the traditional view of LDs solely as inert lipid storage organelles, revealing their active role in antioxidant defense mediated by proteins like LBP. The study highlights LBP's dual roles in lipid metabolism and redox signaling and its importance in protecting against oxidative stress-induced cellular damage. The identification of PRDX4 as an oxidative sensor for LBP provides a new therapeutic target for MAFLD. The results suggest that therapeutic strategies targeting the LBP-PRDX4 axis, alongside antioxidant therapies, might be more effective than strategies focused solely on lipid metabolism. The lack of obesity in LBP^KIKI mice on a ketogenic diet suggests potential benefits of this dietary approach in mitigating LBP-induced metabolic dysfunction. Future research should explore the potential of targeting LBP for treating stress-related metabolic disorders.

This study demonstrates that LBP plays a crucial role in regulating lipid droplet homeostasis and protecting against oxidative stress-induced hepatic steatosis. LBP acts as an antioxidant by selectively sequestering unsaturated triglycerides into LDs, inhibiting lipolysis, and interacting with PRDX4, a redox state sensor. Chronic stress-induced LBP upregulation contributes to obesity and insulin resistance. Antioxidant therapy, such as NAC treatment, shows promise for alleviating LBP-induced metabolic dysfunction. Further research is needed to fully elucidate the role of LBP in various metabolic disorders and explore the therapeutic potential of targeting the LBP pathway.

The study primarily focuses on mouse models and HepG2 cells, which may not fully reflect the complexity of human metabolic processes. While the study identifies PRDX4 as a regulator of LBP trafficking, the detailed mechanisms of this interaction remain to be explored further. The long-term effects of manipulating LBP levels and the potential for off-target effects need additional investigation. The study predominantly uses high-fat diets, limiting the generalizability of findings to other forms of oxidative stress and metabolic dysfunction.