Effect of intermittent hypoxia on lipid metabolism in liver cells and the underlying mechanism

Abstract

To determine the effect of intermittent hypoxia on lipid metabolism in liver cells and to explore the possible molecular pathways involved in this process. An intermittent hypoxia cell model system was established by incubating the human hepatic cell lines L02 and HepG2 in an atmosphere of 2% O₂, 5% CO₂ and 93% N₂ for 8 hours per day over a period of 1, 2, 3, 4 or 5 days. Cells cultured in normoxia conditions (21% O₂) served as controls. Changes in intracellular lipid droplets and triglyceride (TG) levels were assessed by biochemical assays and oil red staining. Changes in intracellular reactive oxygen species (ROS) were assessed by inverted fluorescence microscopy and flow cytometry. Changes in expression of hypoxia-inducible factor (HIF)-1a and HIF-2a proteins, and the downstream ADFP, SREBP-1c and FAS proteins, were detected by western blotting. For both L02 and HepG2 cell lines, the cells grown under hypoxic conditions showed significantly higher lipid droplet accumulation and TG content than the cells grown under normoxic conditions (F(L02) =61.83, FHepG2 =104.19, P less than 0.01). Both oxygen concentration and time appeared to be correlated with these lipid-related changes (F(L02) =39.60, FHepG2 =76.39, P less than 0.01). The ROS fluorescence index was significantly increased after 2 days of intermittent hypoxia L02: 0.703 ± 0.129 vs. 3.310 ± 0.198, t =22.0637 and HepG2:0.617 ± 0.156 vs. 2.33 ± 0.42, t =7.2003, P < 0.05); in addition, increasing trends were observed in the ROS content and intensity of green fluorescence in conjunction with increased time of exposure to intermittent hypoxia (F(L02) =1021.84, FHepG2 =49.89, P less than 0.01). Compared with their respective control groups, the L02 and HepG2 cells both showed significantly upregulated expression of HIF-1a ADFP, SREBP-1c and FAS (L02:FHIF-1a =371.19, FsREBP-1c =204.49, FFAS =38.20, FADFP =154.31, P less than 0.05 and HepG2:FHF-1a =150.84, FSRERBP-1c =107.35, FFAs =279.71, FADFP =352.06, P less than 0.01). In contrast, the HIF-2a level was markedly decreased in the cells after 4 and 5 days of exposure to intermittent hypoxia (F(L02) =125.29, FHcpG2 =10.68, P less than 0.05). Under intermittent hypoxic conditions, ROS may regulate the expression of hypoxia-inducible factors and the adipose differentiation-related protein,as well as influence fatty acid metabolism via a HIF-1 a-SREBP-1 c-FAS signal and upregulation of the ADFP protein, in liver cells.