Members of my lab have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on promoting tissue injury by alcohol and other potentially toxic substances. Alcohol-induced oxidative and nitrative (nitroxidative) stress inactivated the ALDH2 activity, resulting in elevated amounts of acetaldehyde and lipid peroxides. In addition, CYP2E1-mediated nitroxidative stress can stimulate many different types of post-translational modification (PTM) of cellular proteins, contributing to mitochondrial dysfunction, ER stress and organ damage. In the past, we demonstrated the role of CYP2E1 in stimulating protein oxidation and nitration in rodent tissues. Therefore, we developed sensitive methods to specifically identify oxidatively-modified and nitrated proteins to establish their functional roles in causing tissue injury. In continuation, we have studied the role of stress-activated c-Jun N-terminal kinase (JNK) in promoting mitochondrial dysfunction and acute liver injury by carbon tetrachloride (CCl4) or acetaminophen. To achieve this goal, we established a sensitive method to identify the mitochondrial proteins that are phosphorylated by activated JNK following exposure to a hepatotoxic dose of CCl4 (50 mg CCl4/kg ip). JNK was rapidly activated and translocated to mitochondria within 1 h while markedly increased phosphorylation of many mitochondrial proteins observed between 1 and 8 h following CCl4 exposure. Pre-treatments with a highly-specific JNK inhibitor SU3327 or specific inhibitor of CYP2E1 chlormethiazole (CMZ) significantly reduced phosphorylation of mitochondrial proteins at 2 h and liver damage observed at 16 or 24 h post-CCl4 injection. Mass-spectrometry and differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in antioxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. ALDH2, NADH-ubiquinone oxidoreductase, and alpha-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but not phosphorylated in SU3327-pretreated mouse livers. Consistently, the suppressed activities of these enzymes in CCl4-exposed mice were restored by SU3327 pretreatment. These data provide a novel mechanism by which JNK, rapidly activated by CCl4 (or other toxic compounds), can promote mitochondrial dysfunction and acute hepatotoxicity through phosphorylation of numerous mitochondrial proteins (Jang et al., 2015). Based on these results, we plan to study the JNK-target proteins in liver and brain tissues of our animal models. In collaboration with Dr. Bonghee Lee at Gachon University, Korea, we recently reported a damaging role of another PTM i.e., advanced glycation end adduct with albumin (AGE-albumin) in alcohol-induced brain damage in rats. In this case, daily oral administrations of alcohol (5 g/kg/day for 10 consecutive days) activated microglial cells to produce large amounts of AGE-albumin, which eventually caused neuronal cell death in rat hippocampus via activated JNK and p38-kinase (Byun et al., 2014). In continuation, we also recently reported that the levels of AGE-albumin in the brain tissues of a mouse model of Parkinsons disease (PD) and autopsied brain tissues of PD patients were significantly elevated than the corresponding controls. Treatment with a soluble receptor for AGE (sRAGE) or AGE inhibitors significantly reduced neuronal damage in the rotenone-treated mice (Bayarsaikhan et al., 2016). These results suggest that AGE-albumin can be a neurotoxic agent and theranostic target in many neurodegenerative diseases, including alcohol-induced neurodegeneration. We recently reported the critical role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and advanced inflammatory liver disease in mice (Abdelmegeed et al, 2013). In continuation, we investigated the mechanisms of alcohol-induced gut leakiness and inflammatory liver disease in HIV-transgenic rats used as a surrogate model for HIV-infected people. Binge alcohol (3.5 g/kg oral gavage for 3 times at 12-h intervals) caused gut leakiness with elevated serum endotoxin and cytokines/chemokines, contributing to inflammatory liver disease. Based on these results (Banerjee et al., 2015), we have studied the mechanisms of alcohol-induced gut leakiness in rats. Specifically, we have studied the role of different PTMs of tight junction (TJ) proteins in binge alcohol-mediated gut leakiness and endotoxemia. Our unpublished results showed that the levels of intestinal TJ proteins were significantly decreased in rats following alcohol exposure. Our data also revealed that some TJ proteins were nitrated and then degraded by proteasomes, as reported previously for the nitrated proteins. To further characterize other types of PTM, we have purified that intestinal TJ proteins from wild-type rats before and after ethanol exposure. The identities of the purified intestinal TJ proteins and associated proteins involved in alcohol-induced gut leakiness are being determined by mass spectral analysis. By analyzing the mouse tissues in different ages, we also observed that CYP2E1 is involved in aging-related liver inflammation, apoptosis and fibrosis. Elevated levels of hepatosteatosis, ballooning degeneration, inflammatory cell infiltration and fibrosis were observed in aged WT mice compared to the young mice. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage, as evidenced by the levels of 8-oxo-2'-deoxyguanosine, were observed in aged WT. However, these aging-related oxidative changes were absent or very low in aged Cyp2e1-null mice. These data suggest that CYP2E1 is important in causing aging-related hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress (Abdelmegeed et al., 2016). Furthermore, our unpublished results showed that CYP2E1 is also very important in liver fibrosis caused by western fast food containing high fat and cholesterol contents, since Cyp2e1-null mice were significantly protected from the western diet-induced hepatic fibrosis. These results suggest that CYP2E1 could be a potential target for translational research in preventing advanced liver disease including fibrosis caused by both aging or high fat-containing western fast food. Based on our mechanistic studies on alcoholic fatty liver disease (AFLD) and NAFLD/NASH in our animal models, we performed translational research by evaluating the beneficial effects of walnuts as a dietary supplement against NAFLD/NASH in mouse models. Our results showed that supplementation with walnuts at physiologically relevant levels (20% energy-derived) for 20 weeks did not alter body weight or visceral fat mass but significantly prevented HFD (45% energy-derived)-induced fatty liver possibly by activating sirtuin 1 and AMP-kinase, a key metabolic regulator. These hepatic improvements by walnuts also coincided with reduction of HFD-induced inflammation of adipose tissues and macrophage infiltration. However, the dietary walnuts did not significantly alter HFD-induced peripheral glucose intolerance or insulin resistance despite a trend of improvement (Choi et al., 2016a). Despite the lack of insulin resistance and glucose intolerance, walnut supplementation improved the status of liver cells with decreased fat contents by suppressing the levels of CYP2E1, p-JNK and p-38K, and PARP, leading to decreased hepatocellular apoptosis (Choi et al., in press, 2016b). In addition, we plan to collaborate with members of Dr. Joe Hibbelns Section at LMBB to study the beneficial effects of n-3 fatty acids on liver steatosis and obesity in rodent models.
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