Voluntary exercise, rat, colon mucosa. Rattus norvegicus

Colon cancer is the second most cancer type in Europe. Its development is highly influenced by life style factors such as nutrition and physical inactivity. Detailed biological mechanisms are so far unclear. The purpose of this study was to investigate the effects of chronic wheel running on gene expression in rat colon mucosa. Therefore six-week-old male Wistar rats (exercise (EX) group, n=20) completed a stress free voluntary running exercise period of 12 weeks. Sedentary rats served as a control (CO, n=9) group. In the colon mucosa, steady-state mRNA expression levels of approximately 10,000 genes were compared between both groups by micro-array analysis (MWG rat 10k array). 8,846 mRNAs were detected above background level. Chronic exercise led to a decreased expression of 47 genes at a threshold-factor of 2.0. 3 genes were found to be up-regulated in the EX group. The identified genes encode proteins involved in signal transduction (n=11), transport (n=8), immune system (n=7), cytoskeleton (n=6), protein targeting (n=6) and metabolism (n=5). Among the genes regulated by chronic exercise, the betaine-homocysteine methyltransferase 2 (BHMT2) seems to be of particular interest. Physical activity may protect against aberrant methylation by repressing the BHMT2 gene and thus contribute to a decreased risk of developing colon cancer. In summary, our experiment presents the first gene expression pattern in rat colon mucosa following chronic wheel running and therefore represents an important step in understanding the molecular mechanisms responsible for the preventive effect of physical activity on the development of colon cancer. Keywords: voluntary running exercise, animal model Overall design: Pooled and high-concentrated total RNA-samples of the EX and CO group were obtained. Reverse transcription, labelling and hybridization were performed according to the manufacturer’s manual (MWG Biotech AG; Ebersberg, Germany). For the fluorescence detection Cy3- and Cy5-labeled dCTP (Amersham Bioscience Europe, Freiburg, Germany) were used to produce fluorescence labelled first-strand cDNAs. Arrays were scanned (Affimetrix 428 Array Scanner, Santa Clara, CA) under dried conditions. Data were normalized and analyzed via ImaGene 4.2 software (BioDiscovery, Los Angeles, CA). Three independent hybridizations were carried out. Genes were considered as regulated if: a) the expression ratio was more than 2-fold in at least two hybridizations and b) signal intensity values were at least 2-fold above overall background signal intensity in every group. Criteria for the inclusion into the EX pool sample were an adequate RNA-amount, RNA purity and the running distance per night of the respective rat: rats with extremely low or extremely high running distances were excluded to standardize the influence of physical activity on the colon mucosa. Ten rats from the EX group ran between 5747±2888 m and 8446±3434 m per night. Out of these specimen eight samples (animal number 1, 2, 3, 6, 8, 17, 19 and 20) had the right amount (more than 60 µg total RNA) and purity (checked by optical density (OD) 260 280-1 nm absorption) of total RNA. Eight samples were randomly taken for the CO pool.