Goldfish enter a hypometabolic state to survive chronic hypoxia. We recently described tissue-specific contributions of membrane lipid composition remodeling and mitochondrial function to metabolic suppression across different goldfish tissues. However, the molecular and especially epigenetic foundations of hypoxia tolerance in goldfish under metabolic suppression are not well understood. Here we show that components of the molecular oxygen-sensing machinery are robustly activated across tissues irrespective of hypoxia duration. Induction of gene expression of enzymes involved in DNA methylation turnover and microRNA biogenesis suggest a role for epigenetic transcriptional and post-transcriptional suppression of gene expression in the hypoxia-acclimated brain. Conversely, mechanistic target of rapamycin-dependent translational machinery activity is not reduced in liver and white muscle, suggesting this pathway does not contribute to lowering cellular energy expenditure. Finally, molecular evidence supports previously reported chronic hypoxia-dependent changes in membrane cholesterol, lipid metabolism and mitochondrial function via changes in transcripts involved in cholesterol biosynthesis, β-oxidation, and mitochondrial fusion in multiple tissues. Overall, this study shows that chronic hypoxia robustly induces expression of oxygen-sensing machinery across tissues, induces repressive transcriptional and post-transcriptional epigenetic marks especially in the chronic hypoxia-acclimated brain and supports a role for membrane remodeling and mitochondrial function and dynamics in promoting metabolic suppression.

In rainbow trout, dietary carbohydrates are poorly metabolized compared to other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose fluxes is unknown. The goal of this study was to determine the effects of 4h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose fluxes, glucose appearance (Ra), and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity and cell signaling molecules relevant to glucose metabolism were assessed in liver and muscle. Results show that alanine inhibits Ra glucose (from 13.2+/-2.5 to 7.3+/-1.6 micromol / kg min) and Rd glucose (from 13.2+/-2.5 to 7.4+/-1.5 micromol / kg min) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in Rd glucose can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, alanine-dependent glucose-lowering effects in trout did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion which could indirectly promote alanine oxidation to provide the needed energy.

Due to their reported ‘glucose‐intolerant’ phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue‐specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post‐translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post‐translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.