Activation of oxygen-responsive pathways is associated with altered protein metabolism in Arctic char exposed to hypoxia.

Fish exposed to fluctuating oxygen concentrations often alter their metabolism and/or behaviour to survive. Hypoxia tolerance is typically associated with the ability to reduce energy demand by supressing metabolic processes such as protein synthesis. Arctic char is amongst the most sensitive salmonid to hypoxia, and typically engage in avoidance behaviour when faced with lack of oxygen. We hypothesized that a sensitive species will still have the ability (albeit reduced) to regulate molecular mechanisms during hypoxia. We investigated the tissue-specific response of protein metabolism during hypoxia. Little is known about protein degradation pathways during hypoxia in fish and we predict that protein degradation pathways are differentially regulated and play a role in the hypoxia response. We also studied the regulation of oxygen-responsive cellular signalling pathways [hypoxia inducible factor (HIF), unfolded protein response (UPR) and mTOR pathways] since most of what we know comes from studies on cancerous mammalian cell lines. Arctic char were exposed to cumulative graded hypoxia trials for 3 h at four air saturation levels (100%, 50%, 30% and 15%). The rate of protein synthesis was measured using a flooding dose technique, whereas protein degradation and signalling pathways were assessed by measuring transcripts and phosphorylation of target proteins. Protein synthesis decreased in all tissues measured (liver, muscle, gill, digestive system) except for the heart. Salmonid hearts have preferential access to oxygen through a well-developed coronary artery, therefore the heart is likely to be the last tissue to become hypoxic. Autophagy markers were upregulated in the liver, whereas protein degradation markers were downregulated in the heart during hypoxia. Further work is needed to determine the effects of a decrease in protein degradation on a hypoxic salmonid heart. Our study showed that protein metabolism in Arctic char is altered in a tissue-specific fashion during graded hypoxia, which is in accordance with the responses of the three major hypoxia-sensitive pathways (HIF, UPR and mTOR). The activation pattern of these pathways and the cellular processes that are under their control varies greatly among tissues, sometimes even going in the opposite direction. This study provides new insights on the effects of hypoxia on protein metabolism. Adjustment of these cellular processes is likely to contribute to shifting the fish phenotype into a more hypoxia-tolerant one, if more than one hypoxia event were to occur. Our results warrant studying these adjustments in fish exposed to long-term and diel cycling hypoxia.

Diel cycling hypoxia enhances hypoxia tolerance in rainbow trout (Oncorhynchus mykiss): evidence of physiological and metabolic plasticity.

Many fish naturally encounter a daily cycle of hypoxia, but it is unclear whether this exposure hardens hypoxia-intolerant fish to future hypoxia or leads to accumulated stress and death. The rainbow trout (Oncorhynchus mykiss) is a putatively hypoxia-sensitive species found in rivers and estuaries that may routinely experience hypoxic events. Trout were exposed to one of four 135 h treatments in a swim-tunnel respirometer: (1) air-saturated control (20.7 kPa PO2 ); (2) diel cycling O2 (20.7-4.2 kPa PO2  over 24 h); (3) acute hypoxia (130 h at 20.7 kPa PO2  followed by 5 h at 4.2 kPa PO2 ); and (4) the mean oxygen tension (12.4 kPa PO2 ) experienced by the diel cycled fish. Some responses were similar in diel O2 cycled and mean PO2 -treated fish, but overall, exposure to ecologically representative diel hypoxia cycles improved hypoxia tolerance. Diel hypoxia-induced protective responses included increased inducible HSP70 concentration and mean corpuscular hemoglobin concentration, as well as reduced plasma cortisol. Acclimation to diel hypoxia allowed metabolic rates to decline during hypoxia, reduced oxygen debt following subsequent exposures, and allowed fish to return to an anabolic phenotype. The data demonstrate that acute diel cycling hypoxia improves hypoxia tolerance in previously intolerant fish through the activation of cellular protective mechanisms and a reduction in metabolic O2 requirements.

Effects of fasting and refeeding on protein and glucose metabolism in Arctic charr.

Refeeding, following a period of food deprivation will often lead to compensatory growth. Although many studies have focused on molecular mechanisms behind this accelerated growth response in fish, little is known on the roles of protein and metabolism. We also assessed, for the first time, the potential roles of miRNAs in regulating compensatory growth. Artcic charr, Salvelinus alpinus, a northern freshwater species, was deprived of food for 101 days and then fed to satiety for 126 days. The refeeding period resulted in compensatory growth, with a partial compensation of body mass. The feed deprivation period lead to a decrease in hepatosomatic index (HSI) and intestinal somatic index (ISI). HSI and ISI were then gradually replenished during early refeeding, following a lag phase prior to the compensatory growth response. mRNA transcripts regulating protein degradation via the autophagy pathway (Cathepsin D and Cathepsin L) in muscle were upregulated during feed restriction and downregulated after refeeding, which could allow for greater protein accretion in muscle, facilitating compensatory growth. Transcript levels from the ubiquitin proteasome pathway (Mafbx and Murf1) and the calpain system (Calpain 7 and Calpastatin) suggested that these pathways were not involved in regulating compensatory growth. Furthermore, we've shown that miRNAs (miR-29a and miR-223) could be involved in fish glycogen homeostasis during the early stages of refeeding. These findings provide a deeper understanding of the molecular mechanisms regulating growth in fish.

Adjustments of Protein Metabolism in Fasting Arctic Charr, Salvelinus alpinus.

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (KS) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both KS and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). KS was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.