Table 1. Primer used for quantitative realtime PCR Gene HO1 PPARα MTP αSMA TGF β GAPDH Forward acatcgacagccccaccaagttcaa tgcaaacttggacttgaacg catgtcagccatcctgtttg tcctccctggagaagagctac tgcgcttgcagagattaaaa agaacatcatccctgcatcc Reverse ctgacgaagtgacgccatctgtgag tgatgtcacagaacggcttc ctcgcgataccacagactga tataggtggtttcgtggatgc ctgccgtacaactccagtga ttgtcattgagagcaatgcc HO1, heme oxygenase1; PPAR α, peroxisome proliferatoractivated receptor alpha; MTP, microsomal triglyceride transfer protein; αSMA, alpha smooth muscle actin; TGF β, trans forming growth factor beta; GAPDH, glyceraldehyde3phosphate dehydrogenase. Fig. 1. Hepatic HO1 expression and its activity in the absence of Bach1. (A) Wild type mice (open bar) and Bach1 −/− mice (closed bar) were fed either regular chow or MCD diet for 8 w prior to quantification of hepatic expression of HO1 mRNA by realtime PCR ( n = 5/each group). (B) HO1 activity in the liver from wild type (WT) or Bach1−/− (KO) mice were determined with standardlized of protein concentration (n = 5/each group). ZnPP was utilized as a HO1 inhibitor for negative control. * p<0.05, wild type vs Bach1−/− mice. were obtained from Charles River Laboratories (Yokohama, Japan). The generation of Bach1−/− mice on C57BL/6 background was described previously.(18) These mice at 10 weeks of age (5 mice per each group) were fed either methionine-choline deficient (MCD) diet (Oriental Yeast Co., Tokyo, Japan) or standard chow for 4 or 8 weeks with free access to drinking water. All animal protocols and studies were performed according to the guidelines of Institute of Laboratory Animal Science, Hiroshima University. Histological studies. Liver samples were fixed in 10% formalin, embedded in paraffin, and sliced into 4 μm thick. These were subjected to standard procedure of hematoxylin and eosin (HE) staining or Azan-Mallory staining. Biochemical assays. Serum alanine aminotransferase (ALT) levels were measured enzymatically. Hepatic triglyceride content was determined using Wako E test triglyceride kit (Wako chemical, Osaka, Japan) following lipid extraction.(20) Hepatic MDA levels were quantified using Lipid Peroxidation Assay Kit (Calbiochem, Gibbstown, NJ) with standardization of tissue protein concentra- tions. HO activity. HO activity in the liver was determined as pre- viously described.(21,22) In brief, microsome fraction isolated by ultracentrifugation was reacted with hemin as substrate, and bilirubin formation was measured using Beckman DU640 spectro- photometer (450 nm; Beckman Coulter, Fullerton, CA). The activity was expressed as nmol bilirubin formed per hour per mg of protein. To inhibit HO activity for a negative control, zinc protoporphyrin (ZnPP) in phosphate buffer was injected intra- peritoneally at a dose of 7.5 mg/kg once a week for 8 weeks. Quantitative realtime PCR. RNA was extracted from frozen liver tissues using RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized from 1 μg of total RNA 162 with GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA). Specific primers (Table 1) were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/) on the basis of nucleotide sequences from Genbank. Quantitative real-time PCR was per- formed on Light Cycler system using Light Cycler FastStart DNA Master Plus SYBR Green I (Roche Applied Science, Basel, Switzerland). The mRNA expressions were normalized to the housekeeping gene, GAPDH as an internal control. Statistical analysis. The results are expressed as the means ± SE. The statistical analysis was performed using Student’s t test and differences were considered statistically significant when p was less than 0.05. Results Hepatic HO1 expression and its activity in the absence of Bach1. Fig. 1 demonstrates the regulation of HO-1 expression and its activity by Bach1. As shown in Fig. 1A, real-time PCR quantified a 8-fold up-regulation of HO-1 mRNA in Bach1−/− mice on chow diet. Despite not being statistically significant, there was 2-fold up-regulation of mRNA by MCD diet in wild type mice. Consistent with these changes in mRNA expressions, Fig. 1B demonstrates 5-fold increase in hepatic HO-1 activity in the absence of Bach1, which was entirely repressed by intraperitoneal injection of ZnPP, an HO-1 inhibitor. Influence of Bach1 ablation on hepatic lipid metabolism. Eight weeks feeding of MCD diet induced moderate infiltration of inflammatory cells with macrovesicular steatosis in the liver of wild type mice as depicted in Fig. 2A (A, B). In contrast to wild type mice, Bach1−/− mice appeared devoid of hepatic steatosis (C, D). In keeping with these morphological changes, hepatic doi: 10.3164/jcbn.09122GFR ©2011 JCBN