Matthew Picklo, University of North Dakota School of Medicine) complexes I (30 kD subunit), II

(30 kD subunit), III (core protein 1, Rieske iron sulfur), and IV (subunits I and IV) (Invitrogen, CA), phospho AMP-activated protein kinase (AMPK) and phospho ACC (Cell Signaling, MA), and detected using enhanced chemiluminescence (SuperSignal, West-Dura, Pierce, IL). All data represent the means ± standard error of the mean (SEM), n = 6 for control and ethanol groups and n = 5 for MitoQ-treated groups. Statistical significance was determined using Student’s t test, analysis of variance (ANOVA), and Newman-Keuls test as post-hoc test. P < 0.05 was taken as significantly different. Animals were pair-fed with either ethanol or control liquid diets containing MitoQ (0, 5, or 25 mg/kg/day) for 4 weeks. There was no CHIR-99021 cost significant difference in body weight gain; however, the ethanol group had increased liver weight as compared to pair-fed controls which was prevented by MitoQ, although no effect was seen on liver to body weight ratios (Table 1). Consumption of ethanol did Alisertib purchase not significantly increase serum alanine aminotransferase levels compared with controls. Ethanol consumption resulted in increased serum HDL levels as expected but was not changed by MitoQ.44 The serum

low-density lipoprotein (LDL) / very low-density lipoprotein (VLDL) also showed no significant changes with alcohol exposure or any treatment (Supporting Fig. 1). Chronic ethanol consumption is known to increase 4-HNE-protein adducts and iNOS-dependent protein nitration.7, 9, 20, 21 In agreement with these findings, liver tissues show intense staining for 4-HNE-protein adducts in chronic ethanol fed animals compared to pair-fed controls (Fig. 1A,B). HNE immunoreactivity was not uniform with hepatocytes around the central veins showing the most intense staining (zones 2, 3) and a gradual decline toward the periportal region (zone 1). MitoQ treatment completely abolished ethanol-induced 4-HNE staining

in all regions of the liver sections examined. Control experiments show no effect of MitoQ at either dose (Fig. 1A,B) oxyclozanide and omission of the primary antibody for HNE resulted in no detectable signal (result not shown). Consistent with previous studies, chronic ethanol feeding increased 3-NT and iNOS staining with highest in zone 3, with intermediate staining in zone 2 (Fig. 2A,B).9, 20, 45 MitoQ treatment significantly decreased 3-NT staining in the liver of ethanol fed rats; however, it did not have any effect on the induction of iNOS protein. Controls with excess free 3-NT or omission of the primary antibody for 3-NT resulted in no signal (result not shown). It has been shown that MitoQ inhibits mitochondrial ROS and the consequent activation of HIF1α.30, 31, 46 We first confirmed the activation of HIF1α in response to EtOH (Fig. 3A).