Values not sharing a common letter are statistically different,P< 0.05. == 3.2. another key molecular mechanism responsible for betaine hepatoprotection. == 1. Introduction == Chronic ethanol exposure has been shown to significantly alter liver mitochondrial structural and functional integrity. Ethanol consumption alters mitochondrial morphology, induces mitochondrial DNA damage, and impairs ribosomal activity and structure [14] resulting in depressed mitochondrial protein synthesis and associated loss of electron transport chain complexes levels and function. It has also been shown that alcohol exposure increases the sensitivity of liver mitochondria Cobalt phthalocyanine to induce mitochondrial permeability transition pore [5] that may be linked to higher cyclophilin D levels in liver mitochondria [6]. Together, these chronic ethanol-induced alterations result in depressed respiratory capacity and impaired oxidative phosphorylation, events critical to the development of alcoholic liver injury [710]. In recent years, advancements in proteomic technologies have facilitated the examination of alcohol-dependent alterations to the mitochondrial proteome [11]. Using both conventional and blue native (BN)-PAGE proteomics methods, Bailey et al. have reported that ethanol exposure results in the decrease of both nuclear and mitochondrial encoded gene products of the oxidative phosphorylation system [11]. Similar defects in the mitochondrial proteome such as reductions in cytochromecoxidase subunits and mitochondrial membrane potential, have also been reported in genetically altered mice exhibiting deficiency in liver levels of SAM [12], buttressing the concept that SAM plays a critical role in maintaining proper mitochondrial function. Several studies including ours have demonstrated that while the alcohol-induced decrease in hepatic SAM levels is detrimental, it is the decreased hepatocellular SAM : S-adenosylhomocysteine (SAH) ratio that adversely affects many crucial SAM-dependent methylation reactions and the ultimate generation of many hallmark features of alcoholic liver disease [1316]. We have further shown that the addition of SAM can normalize alcohol-induced SAM : SAH ratios [17] and preserve mitochondrial respiratory capacity by maintaining the mitochondrial genome and proteome while attenuating alcohol-dependent increases in mitochondrial superoxide production [3,18]. Betaine, a methyl donor and another key metabolite of the methionine cycle, has been shown to normalize hepatocellular SAM : SAH ratio, correct defective cellular methylation reactions, and prevent the alcohol-mediated steatosis, apoptosis, and accumulation of damaged proteins [14,17,1923]. Based on this, we investigated whether betaine prevents alcohol-induced changes to the mitochondrial oxidative phosphorylation system in a rat model of chronic alcohol exposure. This assessment was complemented by determinations of liver cytochrome P450 2E1 (CYP2E1) protein and activity, glutathione (GSH) levels, SAM : SAH ratios, NOS2 expression, NO generation, and triglyceride levels. == 2. Materials and Methods == == 2.1. Diet Formulation == Nutritionally adequate Lieber-DeCarli control and ethanol liquid diets [24] were purchased from Dyets, Inc. (Bethlehem, Pa, USA). The ethanol diet consisted of 18% of total energy as protein, 35% as fat, 11% as carbohydrate, and 36% as ethanol. In the control diet, ethanol was replaced isocalorically with carbohydrate such that both ethanol and control rats consumed identical amounts Cobalt phthalocyanine of all nutrients except carbohydrate. == 2.2. Ethanol and Betaine Feeding Procedure == Cobalt phthalocyanine Male Wistar rats (Charles River Laboratories, Wilmington, Mass, USA) weighing 180 to 200 g (approximately 4548 days old) were weight-matched and divided into four groups. Group 1 was fed the control diet. Group 2 was fed the same diet as Group 1 except 1% (w/v) betaine was added to the diet. Group 3 was fed the ethanol diet, and Group 4 was fed the ethanol diet containing 1% (w/v) betaine. Rats in groups 13 were Cobalt phthalocyanine fed the amount of diet consumed by rats in group 4. Overall, each group consisted of 8 rats fed the appropriate diet for 4-5 weeks. Twenty four hours before sacrifice, the total daily volume of the diet was divided with 1/4 given at 8:00 am, 1/4 at 12:00 noon, and 1/2 at 4:00 pm. In addition, animals were given 1/4 their respective Rabbit polyclonal to ANKRA2 diets 6090 minutes prior to death. This regimen was followed to minimize differences in feeding patterns that exists between the groups of rats. The care, use, and procedures performed on these rats were approved by the Institutional Cobalt phthalocyanine Animal Care and Use Committee at the Omaha Veterans Affairs Medical Center and complied with NIH guidelines. == 2.3. Liver Histology and Detection of Lipid Accumulation == Formalin fixed liver tissue was processed for hematoxylin-eosin staining and evaluated for steatosis and inflammation. In addition, fresh frozen liver sections were fixed in.