GLUT1-mediated HMGB1 O-GlcNAcylation drives hyperglycemia-Induced neutrophil extracellular trap networks formation via TLR4 signaling and exacerbates fibroblast inflammation.

Neutrophil extracellular traps (NETs) exacerbate fibroblast inflammatory injury in hyperglycemic conditions, yet the role of glucose metabolism and O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in this process remains unclear. Here, we investigate how glucose transporter protein 1 (GLUT1)-dependent glucose uptake regulates O-GlcNAcylation of high-mobility group box 1 (HMGB1) to drive NET formation and fibroblast inflammation. Mouse peripheral blood neutrophils (MPBN) were treated with high glucose (25 mM) and phorbol ester (PMA) to induce NETs. Co-culture of NETs with mouse fibroblasts (L929) reduced fibroblast viability by 1.1 fold and migration by 1.2 fold within 24 h, while upregulating pro-inflammatory cytokines (Tumor Necrosis Factor-α (TNF-α): +1.3-fold; Interleukin-1β (IL-1β): +1.1-fold; Interleukin-6 (IL-6): +1.1-fold) and suppressing collagen synthesis (Collagen I (COL-I): - 1.7-fold; Collagen III (COL-III): -2.5-fold). Critically, high glucose elevated GLUT1 expression in MPBN (+ 1.2-fold), further amplified under co-culture conditions(+ 1.2-fold). Functional assays using GLUT1 knockdown confirmed that GLUT1 activity was essential for glucose uptake and subsequent O-GlcNAc modification of HMGB1, stabilizing its expression. Enhanced O-GlcNAcylation of high-mobility group box 1 (HMGB1) directly promoted NET formation, evidenced by elevated markers (Citrullinated histone H3 (Cit-H3): +1.6-fold; Myeloperoxidase (MPO): +1.2-fold; Circulating free DNA (cfDNA): +2-fold) and activation of c-Jun N-terminal kinase (JNK)/p38 phosphorylation. These effects were abolished by toll-like receptor 4 (TLR4) inhibition, linking HMGB1-TLR4 signaling to NET-driven inflammation. Mechanistically, GLUT1 knockdown reduced HMGB1 O-GlcNAcylation and reversed NET-induced fibroblast dysfunction. Our findings provide direct evidence that hyperglycemia enhances GLUT1 expression and activity, driving HMGB1 O-GlcNAcylation to maintain NETs formation through TLR4, which promotes fibroblast inflammatory injury. This pathway highlights a metabolic-inflammation axis relevant to diabetic complications.