Quantification and mechanisms of oleic acid-induced steatosis in HepG2 cells

Abstract

Developing a quantifiable in vitro model of steatosis is critical in understanding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and searchingfor effective therapies. Using an ORO-based colorimetric measurement, we developed a convenient assay to qualify the degree of OA-induced steatosis in HepG2 cells. We demonstrated that in the absence of exogenous inflammatory mediators, OA-induced steatosis was associated with increased production and secretion of tumor necrosis factor alpha and decreased expression of peroxisome proliferators-activated receptor alpha in HepG2 cells. OA-induced steatosis was also associated with increased lipid peroxidation, apoptosis, but decreased proliferation in these cells. The increased lipid peroxidation was related to decreased SOD-1, a free radical scavenger enzyme; while increased apoptosis was related to increased active caspase-9. The decreased proliferation mediated by OA-induced steatosis was associated with increased production of p27 with unchanged alanine transaminase (ALT) level in the culture medium, indicating OA-induced steatosis alters cell cycle progression without direct toxicity to these cells. In conclusion, the present study developed a colorimetric assay that accurately quantifies OA-induced steatosis in HepG2 cells. In the absence of exogenous inflammatory mediators, OA-induced steatosis results in a series of pathophysilogical changes in HepG2 cells, indicating direct pathogenic roles of hepatocytes in NAFLD.

Keywords: Steatosis; apoptosis; cell proliferation; lipid peroxidation; peroxisome proliferators-activated receptor α; tumor necrosis factor α.

Figure 1

OA-induced steatosis in HepG2 cells determined by ORO staining and ORO-based colorimetric assay. (A) Non-treated HepG2 cells did not show ORO staining. (B) Positive ORO staining as shown in red in cytoplasm of HepG2 cells treated with 1 mM OA for 24 hours. (C) OA (0.1-2.0 mM) treatment of HepG2 cells for 24 hours induced steatosis in a dose-dependent manner (r² = 0.97, p=0.001), as determined by ORO-based colorimetric assay.

Figure 2

OA-induced Steatosis and Its Effects on TNF-α and PPARα Expression. OA-induced steatosis in HepG2 cells resulted in (A) increased production of TNF-α; (B) increased secretion of TNF-α from HepG2 cells to the culture medium; (C) inhibited PPARα expression. (D) β-actin was used as an internal control. “#” means p value < 0.05 compared to the untreated cells.

Figure 3

OA-induced steatosis and its effects on lipid peroxidation, cell proliferation and apoptosis. OA-induced steatosis in HepG2 cells resulted in (A) increased lipid peroxidation; (B) decreased cell proliferation, as determined by MTT assay; (C) no changes in ALT level in these cells and the culture medium, compared to untreated HepG2 cells and medium; and (D) increased apoptosis of HepG2 cells. “#” means p value < 0.05 compared to the untreated cells.

Figure 4

OA-induced Steatosis and Its Effects on the Modulators Related to Lipid Peroxidation, Cell Proliferation and Apoptosis. OA-induced steatosis in HepG2 cells resulted in (A) increased SOD-1 expression; (B) increased p27; (C) no significant change in p21 expression; (D) increased production of activated cleaved caspase-9; and no significant change in of Bcl-2 (E) and Bax (F) expression. (G) β-actin was used as an internal control. “#” means p value < 0.05 compared to the untreated cells.