In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis

• Published in: Disease models & mechanisms Vol. 8; no. 2; pp. 183 - 191
• Main Authors: García-Ruiz, Inmaculada, Solís-Muñoz, Pablo, Fernández-Moreira, Daniel, Muñoz-Yagüe, Teresa, Solís-Herruzo, José A
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 01.02.2015; The Company of Biologists Limited; The Company of Biologists
• Subjects: Adenosine Triphosphate - metabolism; Cytochrome; DNA, Mitochondrial - metabolism; Enzymes; Fatty acids; Fatty Acids - pharmacology; Gene expression; Gene Expression Regulation, Neoplastic - drug effects; Gene Silencing - drug effects; Hep G2 Cells; Humans; Liver diseases; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - pathology; Mitochondrial DNA; Mitochondrial respiratory chain; NADPH oxidase; NADPH Oxidases - metabolism; Nitro-oxidative stress; Non-alcoholic Fatty Liver Disease - metabolism; Non-alcoholic Fatty Liver Disease - pathology; Nonalcoholic steatohepatitis; Oxidation; Oxidative phosphorylation; Oxidative Phosphorylation - drug effects; Oxidative stress; Oxidative Stress - drug effects; OXPHOS; Palmitic Acid - pharmacology; Pathogenesis; Phosphorylation; Protein Subunits - genetics; Protein Subunits - metabolism; Proteins; Proteomic; Thiobarbituric Acid Reactive Substances - metabolism; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Nitro-oxidative stress; Nonalcoholic steatohepatitis; Oxidative phosphorylation; NADPH oxidase; Proteomic; OXPHOS; Mitochondrial respiratory chain
• ISSN: 1754-8403; 1754-8411
• DOI: 10.1242/dmm.018234

Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.