Rat liver: Dexamethasone treatment vs. Control

Sixteen male Sprague-Dawley rats were randomly allocated into 2 groups (8 rats per group) as follows: the control group (CON) and the dexamethasone-treated group (DEXA). Dexamethasone-treated rats received a daily intraperitoneal injection of 1.5 mg/kg of dexamethasone for 5 days. All rats were fasted during the night following the fifth day. On the sixth day, the animals were killed by decapitation. In order to focus our investigation on metabolism-related genes, we developed a metabolism dedicated microarray tool: the Mitoligo. Using this microarray tool, we were able to determine that energy metabolism was deeply modified by dexamethasone treatment. Dexamethasone treatment to rats induces a complete switch of the metabolism toward a maximal rate of ATP synthesis. In this study, we show that substrate supplying for oxidative phosphorylation is greatly enhanced. We also confirm that oxidative phosphorylation capacity is increased by dexamethasone treatment. Keywords: hormonal treatment The present investigation was performed according to the guiding principles of the French department of Animal and Environmental protection for the care and use of animals and in accordance with the American Physiological Society “Guiding Principles for Research Involving Animals and Human Beings”. Sixteen male Sprague-Dawley rats, born and bred in our animal facilities, were housed in individual cages from the age of 13 weeks (330-400g). Animals were provided with water ad libitum and a standard diet (U-A-R A04) composed of (% total weight) 16% protein, 3% fat, 60% carbohydrate and 21% water, fiber, vitamins and minerals. The metabolisable energy content was 2.9 kcal/day. Rats were randomly allocated into 2 groups (8 rats per group) as follows: the control group (CON) and the dexamethasone-treated group (DEXA). Dexamethasone-treated rats received a daily intraperitoneal injection of 1.5 mg/kg of dexamethasone for 5 days. All rats were fasted during the night following the fifth day. On the sixth day, the animals were killed by decapitation. Total RNA was isolated from tissue samples using a standard guanidium isothiocyanate protocol (TRIzol reagent, Invitrogen). RNA integrity was determined using a BioAnalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Cy3- and Cy5-labelled (amplified) aRNA was prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753). The reference sample consisted of a pool of an equal amount of aRNA obtained after the amplification of tRNA from each non-restricted control rat and was labelled with Cy5 (Amersham). For each of different rat within each group (including control group), two replicate tRNA extractions were prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753) and labeled individually with Cy3 (Amersham). Each Cy3-labeled sample was mixed with an equal amount of the Cy5- labeled reference sample, pre-incubated with yeast tRNA and polyA RNA, and hybridized to the microarrays. Microarrays were prepared in-house (Plateforme Puces à ADN-Ouest Génopole, Nantes, France) using 50-mer oligonucléotides probes. The probes were diluted in spotting buffer A (25 μM in buffer A 1x, Ocimum) and spotted onto epoxysilane-coated glass slides (Slide E, Schott nexterion) using the Lucidea Array Spotter from Amersham. Immediately after spotting, the slides were incubated at 42°C and 55% humidity for 12 h. Slides were washed once in 0.2% SDS and twice in H2O and then incubated for 20 minutes in H2O at 50°C, dried by centrifugation (1000 rpm, 3 minutes, 20°C), and stored at 20°C. The 982 genes that were represented on the microarray were selected for involvement in mitochondrial metabolism. 350 genes are specific for rat while 600 genes are specific of human mRNA. Each gene was spotted in triplicate on the microarrays. Hybridized arrays were scanned at 10 μm/pixel resolution by fluorescence confocal microscopy (Scanarray 3000, GSI-Lumonics). Signal intensities were extracted with Genepix Pro 5.1.0.19 image analysis software (Axon Instruments, USA). Data were retained based on concordance between the measurements of different types of replicates: biological replicates, technical replicates and combined biological and technical replicates. Microarray raw data were analyzed using Genespring GX 7.0 (Agilent technologies). Data were normalized using Lowess normalization. Normalized data were compared using ANOVA followed by tukey or student t-test.