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.

Background & Aims Ethanol-inducible cytochrome P450 2E1 (CYP2E1) activity contributes to oxidative stress. However, CYP2E1 may have an important role in the pathogenesis of high-fat mediated nonalcoholic steatohepatitis (NASH). Thus, the role of CYP2E1 in high-fat mediated NASH development was evaluated. Methods Male wild-type (WT) and Cyp2e1-null mice were fed a low-fat diet (LFD, 10% energy-derived) or high-fat diet (HFD, 60% energy-derived) for 10 weeks. Liver histology and tissue homogenates were examined for various parameters of oxidative stress and inflammation. Results Liver histology showed that only WT mice fed a HFD developed NASH despite increased steatosis in both WT and Cyp2e1-null mice fed HFD. Markers of oxidative stress such as elevated CYP2E1 activity and protein amounts, lipid peroxidation, protein carbonylation, nitration, and glycation with increased phospho-JNK were all markedly elevated only in the livers of HFD-fed WT mice. Furthermore, while the levels of inflammation markers osteopontin and F4/80 were higher in HFD-fed WT mice, TNFα and MCP-1 contents were lower compared to the corresponding LFD-fed WT. Finally, only HFD-fed WT mice exhibited increased insulin resistance and impaired glucose tolerance. Conclusions These data suggest that CYP2E1 is critically important in NASH development by promoting oxidative/nitrosative stress, protein modifications, inflammation and insulin resistance.

SummaryBackground & Aims-Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively-modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively-modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin MnTMPyP on oxidatively-modified proteins and their functions.

Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.

It is well-established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400 mg/kg) for 4 and 24 h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.

Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40~50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2−/− mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2−/− mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic IL-6 expression but surprisingly lower levels of steatosis and serum ALT. Higher IL-6 levels were also detected in ethanol-treated precision-cut-liver-slices from ALDH2−/− mice and in Kupffer cells isolated from ethanol-fed ALDH2−/− mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the LPS-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2−/− mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2−/− mice. Finally, ethanol-fed ALDH2−/− mice were more prone to CCl4-induced liver inflammation and fibrosis than ethanol-fed wild-type mice. Conclusions: ALDH2−/− mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis via MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, via acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are prone to liver inflammation and fibrosis following alcohol consumption.

Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury.