The increase in oxidized DCF levels induced by MG was similar with that induced by glucose

n adult rat offspring exposed to ethanol in utero, and whether the anti-oxidant and anti-ER stress TUDCA can reverse these anomalies. Experimental Design Animal experiments were performed as described before, except that ethanol was given for one gestation week instead of throughout pregnancy. Pregnant Sprague-Dawley rats were randomly assigned: a) to be gavaged with ethanol during the first, second or third gestation week; b) to be gavaged with water instead of ethanol and be pair-fed the amount of chow consumed by the ethanol dams; c) to be gavaged with water and be given free access to chow. After weaning onto normal chow, subgroups of male offspring from ethanol dams were given tauroursodeoxycholic acid sodium salt by daily IP injection for 3 weeks as of 13 weeks of age, whereas the others were given normal saline. At 16 weeks, male rat offspring from each group, representing individual litters, were fasted for 15 h and the procedures described below were performed. Materials and Methods Ethics Statement All studies were approved by the Committee for Animal Use in Research and Teaching of the University of Manitoba prior to commencement of the studies, in full compliance with the Canadian Council on Animal Care who has certified that the animal care and use program at the University of Manitoba is in accordance with the standards of Good Animal Practice. Glucose Tolerance 7531648 Test Rat offspring were fasted overnight and then submitted to IP glucose tolerance test by 9:00 a.m. the next morning. 2 Reversing Early Ethanol-Enhanced Gluconeogenesis Glucose, 2 g/kg body weight, was IP injected and saphenous vein blood was sequentially collected for the determination of glucose and insulin. The rats were killed by exsanguination, and liver and plasma were stored, respectively, at 280uC and 220uC until used. Duplicate volumes of samples and DCF standard were added to a 96 well plate followed by 50 ml of a catalyst that helps accelerate the oxidative reaction. After MRT-67307 site incubation for 5 min at room temperature, 100 ml of DCFH solution were added to samples and the incubation continued for 30 min in the dark. DCF fluorescence was read in a Fluostar Optima fluorescence microplate reader at 480 nm excitation/530 nm emission. The free radical content of liver samples was determined in comparison with the DCF standard curve. HDAC Activity Assay HDAC activity was measured colorimetrically using a kit from BioVision following the manufacturer’s 22582137 instructions, as described. Briefly, 100 mg of nuclear proteins were added to each well of a 96-well plate. A standard curve was prepared using a deacetylated standard included in the kit, with HeLa nuclear extract and trichostatin A as positive and negative controls, respectively. Absorbance was read in an ELISA plate reader at 405 nm and HDAC activity was expressed as mm/mg protein. Four month-old male rat offspring were exposed to ethanol or water only during early, mid, or late pregnancy. Control and pair-fed offspring were from dams with free access to chow and dams fed the amount of chow consumed by EtOH dams, respectively. Briefly, rats were IP injected with sodium pyruvate dissolved in saline, and blood glucose was determined with Ascensia Elite XL in saphenous vein blood every 30 min for 2 h. Western Blotting Antibodies against GADD 153 or C/EBP homologous protein, HDAC3, HDAC4, HDAC5, HDAC7, HDAC4/5/7, forkhead transcription factor or foxo1, acetylated-FKHR, SIRT2, PEPCK, glucose6-phosphatase and b-actin,