Eight-week-old male C57BL/6N mice were obtained from Harlan (Indianapolis, IN). All mice were housed in a pathogen-free, temperature-controlled animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care with 12 hour light/12 hour dark cycles. All experiments were carried out according to the criteria outlined in the Guide for Care and Use of Laboratory Animals and with approval of the University of Louisville Animal Care and Use Committee.
Animals were fed a modified Lieber-DeCarli liquid diet containing EtOH (35% of total calories) and enriched in unsaturated fat (USF, corn oil, 55-60% of LA) or saturated fat (SF, medium-chain triglyceride:beef tallow, 82:18 ratio, Research Diet, New Brunswick, NJ,Fig. 1 ). Soybean oil was used in both diets to provide essential free fatty acids. Control liquid maltose-dextrin diets provided 40% of energy from fat, 43% - from carbohydrate, 17% - from protein. Initially, all mice were given the control liquid maltose dextrin diets (SF or USF, no EtOH) ad libitum for one week. Afterwards, mice were fed either the liquid ethanol diet or the control liquid maltose-dextrin diet. Ethanol was gradually increased every 3-4 days from 11.2% to 35% of total calories (5.0% [vol/vol]). The mice were fed with the EtOH diet (5% EtOH vol/vol) ad libitum for 8 weeks. The control mice were pair-fed with SF or USF maltose-dextrin diets on an isocaloric basis.
At the end of the feeding experiment, mice were fasted overnight, anesthetized with sodium-pentobarbital (nembutal, 80 mg/kg, intraperitonially), and blood, liver, and intestinal samples were collected for assays. The killing sequence was randomized in order to eliminate any time dependent variation due to length of fasting. Blood samples were collected from the inferior vena cava using heparinized syringes and were then centrifuged at 300 g for 15 minutes at 4°C. Whole livers and intestines were removed and their weights were measured. Part of the liver from left lobe was harvested and fixed in 10% neutral-buffered formalin, while the remaining liver tissue was snap frozen in liquid N2 and stored at −80°C. Freshly isolated intestinal segments (duodenum, jejunum, ileum) were used for ex vivo intestinal permeability assay.
Animals were fed a modified Lieber-DeCarli liquid diet containing EtOH (35% of total calories) and enriched in unsaturated fat (USF, corn oil, 55-60% of LA) or saturated fat (SF, medium-chain triglyceride:beef tallow, 82:18 ratio, Research Diet, New Brunswick, NJ,
At the end of the feeding experiment, mice were fasted overnight, anesthetized with sodium-pentobarbital (nembutal, 80 mg/kg, intraperitonially), and blood, liver, and intestinal samples were collected for assays. The killing sequence was randomized in order to eliminate any time dependent variation due to length of fasting. Blood samples were collected from the inferior vena cava using heparinized syringes and were then centrifuged at 300 g for 15 minutes at 4°C. Whole livers and intestines were removed and their weights were measured. Part of the liver from left lobe was harvested and fixed in 10% neutral-buffered formalin, while the remaining liver tissue was snap frozen in liquid N2 and stored at −80°C. Freshly isolated intestinal segments (duodenum, jejunum, ileum) were used for ex vivo intestinal permeability assay.
