Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease

Abstract

BackgroundIn vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH.MethodsHuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured.ResultsFFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species.ConclusionsThe exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.

Up-regulated HOTAIR induced by fatty acids inhibits PTEN expression and increases triglycerides accumulation in HepG2 cells

“…The present study used FFAs to establish a cell model of human NAFLD. The results showed triglyceride accumulation in HepG2 cells after treatment with FFAs, consistent with previous studies [27][28][29][30].…”

“…Saturated palmitic (PA; C16:0) and monounsaturated oleic (OA; C18:1) acids are the most abundant FFAs in the liver triglycerides of patients with NAFLD [27,28]. Previous studies showed that in vitro NAFLD models induced by FFAs mixture (PA/OA, 1:2 ratio) could lead to triglyceride accumulation in hepatocytes similar to that of patients with NAFLD, with minor toxic and apoptotic effects [29,30]. The present study used FFAs to establish a cell model of human NAFLD.…”

Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro

“…Interestingly, phospo-IκBα and phospho-p65 levels were further enhanced by combined stimulation with both stimuli ( Figure 2D). Interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) are two pro-inflammatory genes which are regulated by NFκB and which correlate with hepatic inflammation in ALD and NAFLD [26,27]. Notably, expression levels of IL-8 and ICAM-1 corresponded with phospo-IκBα and phospho-p65 levels in PHH stimulated with alcohol, oleate or their combination ( Figure 2E).…”

The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH

“…Friedman (Mount Sinai School of Medicine, New York, NY). Cells were grown in DMEM high glucose medium (ECB7501L-50, Euroclon, Italy) supplemented with 10% v/v (HuH7) (Chavez-Tapia et al, 2012) and 1% v/v (LX-2) (Xu et al, 2005) of fetal bovine serum (FBS) (ECS0180L, Euroclon, Italy), 2 mM L-Glutamine (ECB3000D, Euroclon, Italy) and 10,000 U/mL penicillin and 10 mg/mL streptomycine (ECB3001D, Euroclon, Italy). Simultaneous co-culture experiments were performed in 1% v/v FBS medium, in order to avoid LX-2 auto activation.…”

“…Intracellular lipid droplets were determined by fluorescence microscopy based on Nile Red (N3013, Sigma Chemical USA) staining as described by Chavez-Tapia et al (2012). Briefly, cells were seeded in a coverslip and treated with FFA (1200 lM -24 h).…”