title: In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis.
abstract: 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.
authors.affiliation: Research Center, Laboratory of Gastroenterology and Hepatology, University Hospital "12 de Octubre", Complutense University, 28041-Madrid, Spain. inmagr86@hotmail.com.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: García-Ruiz
authors.affiliation: Anglo-American Medical Unit, 28001-Madrid, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Solís-Muñoz
authors.affiliation: Department of Bromatology and Food Hygiene, Military Center of Veterinary of Defense, 28024-Madrid, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Fernández-Moreira
authors.affiliation: Research Center, Laboratory of Gastroenterology and Hepatology, University Hospital "12 de Octubre", Complutense University, 28041-Madrid, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Muñoz-Yagüe
authors.affiliation: Research Center, Laboratory of Gastroenterology and Hepatology, University Hospital "12 de Octubre", Complutense University, 28041-Madrid, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Solís-Herruzo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D005227:Fatty Acids / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D019255:NADPH Oxidases / Q000378:metabolism; D065626:Non-alcoholic Fatty Liver Disease / Q000378:metabolism* / Q000473:pathology*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D019308:Palmitic Acid / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D005227:Fatty Acids; D021122:Protein Subunits; D017392:Thiobarbituric Acid Reactive Substances; D019308:Palmitic Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases
keywords: Mitochondrial respiratory chain; NADPH oxidase; Nitro-oxidative stress; Nonalcoholic steatohepatitis; OXPHOS; Oxidative phosphorylation; Proteomic