Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP ؉ -dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.Cell damage induced by oxidative stress and reactive oxygen species (ROS) 1 has been implicated in several human diseases including aging, alcohol-mediated organ damage, neurodegenerative diseases, many types of cancers, cardiovascular diseases, and UV-mediated skin disorders (1). As one of the major sources of ROS (2), mitochondria are highly susceptible to oxidative damage. ROS can damage mitochondrial enzymes directly (3), and they can cause mutation in mitochondrial DNAs (4). At the same time, ROS can change the mitochondrial transmembrane potential (⌬m), which is indicative of mitochondrial membrane integrity (5) and precedes cell death induced by various toxic compounds and cytokines (6). Recent reports indicate that mitochondrial ROS cause apoptosis (7, 8) by activating various apoptotic effectors such as cytochrome c release, procaspase-2, procaspase-9, procaspase-3, and latent apoptosis-inducing factor, which is released from the mitochondria during apoptosis (9 -11). Another report also suggested that mitochondrial ROS directly caused apoptosis of T cells (12). It was also reported that tumor necrosis factor ␣ causes a rapid production of mitochondrial ROS (13) and that ceramide, an apoptotic stimulus, also plays a crucial role in tumor necrosis factor ␣-induced mitochondrial ROS generation (14). Furthermore, several other investigators demonstrated that ROS are involved in the signaling pathway of certain growth factors (15) and cytokines (16). In addition, mitochondrial ROS, under hypoxic conditions, activate the transcription of the genes for glycolytic enzymes as well as erythropoietin and vascular endothelial growth factor by upregulating a transcriptional factor, hypoxia-inducible factor 1 (17), suggesting that mitochondrial ROS mediate cross-talk b...

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Emerging evidence suggests that the lack of PPARα enhances hepatic steatosis and inflammation in Ppara-null mice when fed a high-fat diet (HFD). Thus, the aim of this study was to determine whether Ppara-null mice are more susceptible to nonalcoholic steatohepatitis (NASH) than their wild-type (WT) counterparts following short-term feeding with a HFD. Age-matched male WT and Ppara-null mice were randomly assigned to consume ad libitum a standard Lieber-DeCarli liquid diet (STD) (35% energy from fat) or a HFD (71% energy from fat) for 3 wk. Liver histology, plasma transaminase levels, and indicators of oxidative/nitrosative stress and inflammatory cytokines were evaluated in all groups. Levels of lobular inflammation and the NASH activity score were greater in HFD-exposed Ppara-null mice than in the other 3 groups. Biochemical analysis revealed elevated levels of ethanol-inducible cytochrome P450 2E1 and TNFα accompanied by increased levels of malondialdehyde as well as oxidized and nitrated proteins in Ppara-null mice. Elevated oxidative stress and inflammation were associated with activation of c-Jun-N-terminal kinase and p38 kinase, resulting in increased hepatocyte apoptosis in Ppara-null mice fed a HFD. These results, with increased steatosis, oxidative stress, and inflammation observed in Ppara-null mice fed a HFD, demonstrate that inhibition of PPARα functions may increase susceptibility to high fat-induced NASH.

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) contributes to increased oxidative stress and steatosis in chronic alcohol-exposure models. However, its role in binge ethanol-induced gut leakiness and hepatic injury is unclear. This study was aimed to investigate the role of CYP2E1 in binge alcohol-induced gut leakiness and the mechanisms of steatohepatitis. Female wild-type (WT) and Cyp2e1-null mice were treated with three doses of binge ethanol (WT-EtOH or Cyp2e1-null-EtOH) (6 g/kg oral gavage at 12-h intervals) or dextrose (negative control). Intestinal histology of only WT-EtOH exhibited epithelial alteration and blebbing of lamina propria while liver histology obtained at 6 h after the last ethanol dose showed elevated steatosis with scattered inflammatory foci. These were accompanied by increased levels of serum endotoxin, hepatic enterobacteria and triglycerides. All these changes including the intestinal histology and hepatic apoptosis, determined by TUNEL assay, were significantly reversed when WT-EtOH mice were treated with the specific inhibitor of CYP2E1 chlormethiazole and the antioxidant N-acetyl-cysteine, both of which suppressed the oxidative markers including intestinal CYP2E1. WT-EtOH also exhibited elevated amounts of serum TNF-α, hepatic cytokines, CYP2E1 and lipid peroxidation with decreased levels of mitochondrial superoxide dismutase and suppressed aldehyde dehydrogenase 2 activity. Increased hepatocyte apoptosis with elevated levels of pro-apoptotic proteins and decreased levels of active (phosphorylated) p-AKT, p-AMPK and peroxisome proliferator-activated receptor-alpha (PPAR-α), all of which are involved in fat metabolism and inflammation, were observed in WT-EtOH. These changes were significantly attenuated in the corresponding Cyp2e1-null-EtOH mice. These data indicate that both intestinal and hepatic CYP2E1 induced by binge alcohol seem critical in the binge alcohol-mediated increased nitroxidative stress, gut leakage, endotoxemia, and altered fat metabolism, and inflammation, contributing to hepatic apoptosis and steatohepatitis.

Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S-nitrosylated mitochondrial proteins and to use a biotin-N-maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin-N-maleimide-labeled oxidized and/or S-nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S-nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial ␤-oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).