Metabolomics unveiled the impact of alterations in nucleotide glucose metabolism on palmitic acid-induced TLR4 activation in THP-1 macrophages

ABSTRACT OBJECTIVE The association of palmitic acid with macrophage inflammation and its promotion of the expression of inflammatory factors through TLR4 have been demonstrated. It has been observed that immature TLR4 localizes to the endoplasmic reticulum and Golgi apparatus, necessitating glycosylation for migration to the cell membrane. The objective of this study was to identify potential biomarkers associated with N-glycosylation subsequent to the activation of TLR4 inflammatory signaling in human macrophages by palmitic acid. APPROACH AND RESULTS The co-cultivation of palmitic acid with THP-1 macrophages was conducted for a duration of 24 hours, followed by the collection of cell extracts for subsequent metabolomic and lipidomic analyses using high performance liquid chromatography-tandem mass spectrometry. Multivariate and univariate statistical analyses were conducted to identify potential biomarkers, in accordance with established scientific protocols. The impact of palmitic acid on the TLR4 signaling pathway and macrophage N-glycosylation was assessed at various time points using Western blot analysis, immunofluorescence staining, Elisa assays, and chemical labeling techniques. The TLR4 inflammatory signaling pathway was examined in macrophages at different time points, revealing that PA induced the upregulation of MyD88 and TRAF6 expression as well as NF-κB phosphorylation, indicating the activation of classical NF-κB signaling. After 24 hours, TLR4 translocated from the cell membrane to the cytoplasm and initiated internalization, accompanied by significant colocalization with GalAz in the cytosol. In addition, the metabolites in the cell extract were found to be altered in both the control and model groups. Significantly alterations in two N-glycosylation related metabolites were observed in the model group, including guanosine diphosphate-L-fucose and uridine diphosphate-N-acetylglucosamine/uridine diphosphate-N-acetylgalactosamine. CONCLUSIONS THP-1 macrophages incubated with palmitic acid exhibited a distinct metabolomic profile compared to the control group. Our findings suggest that metabolomics analysis holds promise in identifying disease-specific biomarkers for diagnosing fatty acid-induced inflammatory responses in macrophages.