Hepatic transcriptome response to glucocorticoid receptor activation in rainbow trout.

Stress induced elevation in plasma cortisol levels is shown to be involved in the metabolic re-adjustments to stress but the genetic basis of this response is still unclear. Cortisol signaling in teleosts is thought to be mediated predominantly by the glucocorticoid receptor (GR), a ligand-bound transcription factor and previous studies have shown that RU486, a GR antagonist offsets corticosteroid signaling in teleosts. In order to elucidate the genetic basis of GR-mediated metabolic re-adjustments, we exposed in vitro primary culture of trout hepatocytes to cortisol (100 ng/ml; stressed levels) or RU486 (1000 ng/ml) or a combination of both (RU486 + Cortisol), and analyzed the transcriptional responses using a low density custom-made targeted trout cDNA array. These results were further validated using quantitative real-time PCR. Cortisol treatment significantly increased glucose production in hepatocytes and this response is blocked by RU486 suggesting the activation of GR-mediated corticosteroid signaling. Cortisol treatment significantly modulated the transcript abundance of several genes, previously shown to be involved in molecular and biochemical responses to stress and most of these effects are abolished by RU486, suggesting GR-mediated effects. In addition, cortisol treatment affected key genes involved in reproduction supporting previous findings on inhibitory effects of cortisol on reproduction. Taken together, our results provide evidence on the transcriptional changes involved in cortisol signaling in response to stress. Keywords: cDNA microarray, RU486, Cortisol, GR signaling, rainbow trout, hepatocytes Microarray experiments were designed to comply with minimum information about microarray experiment (MIAME) guidelines. The experimental design include treatment of primary culture of trout hepatocytes with either control (0.01% ethanol; vehicle), Cortisol (100 ng/ml), RU486 (GR antagonist; 1000 ng/ml) or a combination of both RU486 (1000 ng/ml) and cortisol (100 ng/ml). Hepatocytes were sampled 24 h after treatment and used for microarray analyses. RNA isolated from these samples was co-hydridized with a common reference standard. A common reference standard was made by pooling equal amounts of RNA from untreated hepatocytes isolated from the experimental fish. A total of 16 hybridizations were performed (4 treatments X 4 biological replicates per treatment) in this experiment. Dye swapping was carried out so that each treatment had two samples labeled with either Cy3 or Cy5.