Cholesterol Metabolism Regulation by Native and Oxidized Low-Density Lipoprotein in a Liver Cell Line

Metabolic dysfunction-associated steatohepatitis (MASLD) is a highly prevalent chronic liver disease. Oxidized low-density lipoprotein (oxLDL) is implicated in MASLD progression; however, its effects on the hepatic cholesterol metabolism remain unclear. Therefore, we explored the effects of native LDL (nLDL) and oxLDL on the cholesterol metabolism and lipid composition in the human liver-derived C3A cell line.

Orbitrap liquid chromatography-tandem mass spectrometry was used to measure free cholesterol (FC), cholesteryl ester (CE), and CE hydroperoxide (CEOOH) levels in LDL particles and LDL-treated C3A cells. The expression of cholesterol metabolic genes was examined using real-time PCR, and proprotein convertase subtilisin kexin type 9 (PCSK9) protein expression was quantified using Western blotting and an enzyme-linked immunosorbent assay.

Both LDL types, particularly nLDL, increased intracellular CE levels. nLDL preferentially increased CE species containing unsaturated fatty acids, whereas oxLDL promoted the accumulation of saturated fatty acid (SFAs)-containing CEs and CEOOH, while FC levels remained unchanged. Both LDL types downregulated SREBP2 and downstream cholesterol synthesis genes and reduced LDLR expression. The PCSK9 protein levels also decreased. SOAT1 expression was reduced, indicating that CE accumulation was primarily driven by exogenous LDL-derived lipids rather than enhanced esterification. In addition, the genes involved in cholesterol efflux (ABCA1), xenobiotic metabolism, and scavenger receptor-related pathways were downregulated in C3A cells.

Both nLDL and oxLDL particles drive CE accumulation through impaired cholesterol release rather than increased cholesterol synthesis. Furthermore, LDL oxidation increases CEOOH and saturated CEs, thus potentially enhancing hepatocellular lipotoxicity.