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.
Highlights
Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases extending from pure fatty liver through nonalcoholic steatohepatitis (NASH) to cirrhosis and hepatocarcinoma that occurs in individuals who do not consume a significant amount of alcohol (Matteoni et al, 1999)
NADPH oxidase seems to play a crucial role in the pathogenesis of the nitro-oxidative stress and oxidative phosphorylation (OXPHOS) dysfunction, because the effects of saturated fatty acids are prevented in the absence of NADPH oxidase activity
In the present study, we show for the first time that saturated fatty acids, but not the monounsaturated oleic acid, decreased markedly the activity of all OXPHOS complexes in HepG2 cells (Fig. 1A)
Summary
Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver diseases extending from pure fatty liver through nonalcoholic steatohepatitis (NASH) to cirrhosis and hepatocarcinoma that occurs in individuals who do not consume a significant amount of alcohol (Matteoni et al, 1999). Mitochondrial dysfunction might play a crucial role in the induction of both ‘hits’, because mitochondria are involved in the β-oxidation of free fatty acids, and are the most important source of reactive oxygen species (ROS) (Fromenty et al, 2004). We have shown that oxidative phosphorylation (OXPHOS) is defective in individuals with NASH (Pérez-Carreras et al, 2003), in ob/ob mice with NAFLD (GarcíaRuiz et al, 2006) and in mice on a high-fat diet (García-Ruiz et al, 2014). We have shown that mice with diet-induced NASH have elevated NADPHox gene expression and activity (García-Ruiz et al, 2014), and other authors have found the same changes in mice fed a methionine-choline-deficient diet (Greene et al, 2014). Considering that fatty acids are increased in the liver of obese mice (García-Ruiz et al, 2014), it might be possible that these acids are responsible for the Disease Models & Mechanisms (2015) doi:10.1242/dmm.018234
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