Short-term induced swimming activity enhanced innate immune parameters and antioxidant status of European eel (Anguilla anguilla).

The swimming activity, although an essential trait in the life cycle of fish, is still poorly understood in farmed fish. The current study aimed to investigate the impact of short-term induced swimming on the immune and antioxidant defence systems in European eel (Anguilla anguilla). Sixteen male yellow European eels (total length: 39.9 ± 0.7 cm; body weight: 108.8 ± 6.1 g) were individually placed in swimming flumes and divided into two groups: i) no swimming (n = 8); and ii) induced-swimming (n = 8) at 0.3 body lengths (BL)·s-1 for 7 h. Swimming resulted in a 2-fold lower cortisol concentration in plasma, whereas plasma glucose, lactate, and several immune-related parameters did not present variations between groups. Interestingly, swimming led to higher lysozyme, peroxidase, and protease activities in skin mucus, whereas bactericidal activity did not show differences among groups. Additionally, the gene expression of interleukin 1 beta showed an up-regulation in the skin of fish with induced swimming, while no differences were observed in the head-kidney or gills. Furthermore, modulation of the antioxidant status was observed in the liver and posterior skeletal muscle after induced swimming. Fish subjected to swimming showed lower lipid peroxidation and higher reduced glutathione levels, increasing the reduced/oxidized glutathione ratio. However, no variations in the antioxidant status were observed between groups in the anterior skeletal muscle. This study showed modulation of immune and oxidative stress markers in European eels upon short-term induced swimming compared to non-swimming fish.

Morphology and metabolic traits related to swimming performance in Australasian snapper (Chrysophrys auratus) selected for fast growth.

Changes in body shape are linked to swimming performance and become relevant for selective breeding programmes in cultured finfish. We studied how the selection for fast growth could affect phenotypes by investigating the relationship between swimming performance and body shape. We also investigated how swimming might affect plasma metabolite concentrations. Critical swimming speed (UCrit), body traits (e.g., BW, body weight; BL, body length; K, condition factor), and plasma lactate and glucose concentrations were evaluated in two cohorts of Australasian snapper (Chrysophrys auratus): one derived from wild broodstock (F1), and the other selected for fast growth (F4). UCrit tests (n = 8) were applied in groups of 10 snapper of similar BW (71.7 g) and BL (14.6 cm). The absolute or relative UCrit values of both cohorts were similar (0.702 m⋅s-1 and 4.795 BL⋅s-1, respectively), despite the F4 cohort displaying a higher K. A positive correlation between K and absolute UCrit (Pearson's r = 0.414) was detected in the F4 cohort, but not in the F1 cohort, which may be linked to differences in body shape. A negative correlation between relative UCrit and body size (Pearson's r between -0.682 and -0.501), but no correlation between absolute UCrit and body size, was displayed in both cohorts. Plasma lactate and glucose concentrations were higher in the F4 cohort at UCrit. Whether a longer selective breeding programme could result in more changes in body shape, potentially affecting swimming performance, should be explored, along with the potential outcomes of the differences in metabolic traits detected.

Short-term swimming up-regulates pro-inflammatory mediators and cytokines in gilthead seabream (Sparus aurata).

Aerobic swimming exercise in fish has been shown to improve robustness of some species. However, the optimal conditions to be applied and the mechanisms underlying remain unknown. We investigated the effects of 6 h of induced swimming on the immune response of gilthead seabream (Sparus aurata), by analysing markers related to immune status in plasma, skin mucus, gills, heart and head-kidney. Forty fish were individually exercised in swim tunnels by applying different water currents: steady low (SL, 0.8 body lengths (BL) s-1), steady high (SH, 2.3 BL s-1), oscillating low (OL, 0.2/0.8 BL s-1) and oscillating high (OH, 0.8/2.3 BL s-1) velocities, including a non-exercised group with minimal water flow (MF, <0.1 BL s-1). Swimming conditions did not trigger a stress response or anaerobic metabolism, suggested by similar levels of cortisol, lactate, and glucose in plasma among groups. Blood haemoglobin and innate immune parameters in plasma and skin mucus also remained unaltered. However, decreased blood haematocrit was observed in fish swimming on the OL condition. Interestingly, gene expression analysis revealed that the OL condition led to the up-regulation of pro-inflammatory mediators (nfκb1 and mapk3) and cytokines (tnfα, il1ß and il6) in gills. A similar response occurred in heart, with an up-regulation of nfκb1, tnfα, il6 and cox2 in the OL condition. Gene expression of these cytokines was unaltered in the head-kidney. The inflammatory response in gills and heart of gilthead seabream triggered by the OL condition highlights the importance of establishing suitable rearing conditions to improve welfare of cultured fish.

Hepatic Glycerol Metabolism-Related Genes in Carnivorous Rainbow Trout (Oncorhynchus mykiss): Insights Into Molecular Characteristics, Ontogenesis, and Nutritional Regulation.

Glycerol metabolism in rainbow trout is poorly studied even though it is at the interface between lipid and glucose metabolism. Moreover, glycerol can be an important ingredient in new aquafeed formulation to decrease the catabolism of dietary amino acids. Thus, the present study aimed to characterize for the first time the different genes coding for key enzymes and proteins involved in hepatic glycerol metabolism. From the trout genomes, all the paralogous genes coding for glycerol transport (aqp9b), glycerol kinase (gk2a and gk5), glycerol-3-phosphate phosphatase (pgp), and glycerol-3-phosphate dehydrogenase (gpd1a, gpd1b, and gpd1c) were identified. The ontogenesis determined that the capacity to metabolize glycerol begins with the apparition of the liver during the development (stage 22) and are more expressed at the endogenous-exogenous feeding period (stage 35). The postprandial regulation of the expression of these genes in juvenile trout showed that the postprandial peak of expression is between 4 and 24 h after the last meal for many of the genes, demonstrating that glycerol metabolism could be nutritionally regulated at a molecular level. However, surprisingly, no regulation of the mRNA abundance for the glycerol metabolism-related genes by different levels of dietary glycerol (0, 2.5, and 5%) have been detected, showing that hepatic glycerol metabolism is poorly regulated at a molecular level by dietary glycerol in rainbow trout juveniles.

Protective effects of seaweed supplemented diet on antioxidant and immune responses in European seabass (Dicentrarchus labrax) subjected to bacterial infection.

European seabass (Dicentrarchus labrax) production is often hampered by bacterial infections such as photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp). Since diet can impact fish immunity, this work investigated the effect of dietary supplementation of 5% Gracilaria sp. aqueous extract (GRA) on seabass antioxidant capacity and resistance against Phdp. After infection, mortality was delayed in fish fed GRA, which also revealed increased lysozyme activity levels, as well as decreased lipid peroxidation, suggesting higher antioxidant capacity than in fish fed a control diet. Dietary GRA induced a down-regulation of hepatic stress-responsive heat shock proteins (grp-78, grp-170, grp-94, grp-75), while bacterial infection caused a down-regulation in antioxidant genes (prdx4 and mn-sod). Diet and infection interaction down-regulated the transcription levels of genes associated with oxidative stress response (prdx5 and gpx4) in liver. In head-kidney, GRA led to an up-regulation of genes associated with inflammation (il34, ccr9, cd33) and a down-regulation of genes related to cytokine signalling (mif, il1b, defb, a2m, myd88). Additionally, bacterial infection up-regulated immunoglobulins production (IgMs) and down-regulated the transcription of the antimicrobial peptide leap2 in head kidney. Overall, we found that GRA supplementation modulated seabass resistance to Phdp infection.

Metabolic Effects of Dietary Glycerol Supplementation in Muscle and Liver of European Seabass and Rainbow Trout by 1H NMR Metabolomics.

The sustainable growth of fish aquaculture will require the procurement of non-marine feed sources. Glycerol is a potential feed supplement whose metabolism may spare the catabolism of dietary amino acids, thereby extending the use of the feed protein to other physiological functions such as growth. In the present study, the effects of dietary glycerol supplementation on the muscle and liver metabolomes of rainbow trout (Oncorhynchus mykiss) and European seabass (Dicentrarchus labrax) were evaluated. Fish juveniles were fed diets with 0%, 2.5%, and 5% glycerol. Muscle and liver aqueous fractions were extracted and 1H NMR spectra were acquired. Metabolite profiles derived from the 1H NMR signals were assessed using univariate and multivariate statistical analyses. The adenylate energy charge was determined in the muscle. For both species, the muscle metabolite profile showed more variability compared to that of the liver and was most perturbed by the 5.0% glycerol diet. For the liver metabolite profile, rainbow trout showed fewer differences compared to European seabass. No differences were observed in energy charge between experimental groups for either species. Thus, rainbow trout appeared to be less susceptible to tissue metabolite perturbations, compared to seabass, when the diet was supplemented with up to 5% glycerol.

Fatty Acid Profile of Pacific Oyster, Crassostrea gigas, Fed Different Ratios of Dietary Seaweed and Microalgae during Broodstock Conditioning.

The fatty acid (FA) profile of oysters generally reflects the dietary FA composition. Moreover, incorporation of FA into tissues is modulated by various metabolic factors, and final composition will depend upon the dietary sources, cumulative intake, and oysters' development stage. Thus, the aim of this study was to assess the impact of dietary incorporation of seaweed (SW) Ulva rigida, in replacement of traditional microalgae diet, on the FA composition of Pacific oysters Crassostrea gigas, during broodstock conditioning. The dietary conditioning consisted of direct replacement of microalgae (33% Tisochrysis lutea, 50.25% Skeletonema costatum, and 16.75% Chaetoceros calcitrans) by SW at four different substitution levels (0%, 25%, 50%, and 100% diet). The dietary docosahexaenoic acid (DHA) (22:6n-3) and eicosapentaenoic acid (EPA) (20:5n-3) contents showed a positive correlation with the dietary microalgae level. During the trial, oysters fed with higher percentages of microalgae revealed a depletion of DHA and accumulation of EPA. The 100% SW caused a significant reduction in oxygen consumption and, consequently, in the standard metabolic rate. Based on these results, a partial substitution of up to 25% of dietary microalgae seems to be a suitable alternative, because it elicited similar results to the commercial 100% microalgae diet.

Acute Stress and an Electrolyte- Imbalanced Diet, but Not Chronic Hypoxia, Increase Oxidative Stress and Hamper Innate Immune Status in a Rainbow Trout (Oncorhynchus mykiss) Isogenic Line.

In aquaculture, fish may be exposed to sub-optimal rearing conditions, which generate a stress response if full adaptation is not displayed. However, our current knowledge of several coexisting factors that may give rise to a stress response is limited, in particular when both chronic and acute stressors are involved. This study investigated changes in metabolic parameters, oxidative stress and innate immune markers in a rainbow trout (Oncorhynchus mykiss) isogenic line exposed to a combination of dietary (electrolyte-imbalanced diet, DEB 700 mEq Kg-1) and environmental (hypoxia, 4.5 mg O2 L-1) challenges and their respective controls (electrolyte-balanced diet, DEB 200 mEq Kg-1 and normoxia, 7.9 or mg O2 L-1) for 49 days. At the end of this period, fish were sampled or subjected to an acute stressor (2 min of handling/confinement) and then sampled. Feeding trout an electrolyte-imbalanced diet produced a reduction in blood pH, as well as increases in cortisol levels, hepato-somatic index (HSI) and total energy content in the liver. The ratio between the lactate dehydrogenase (LDH) and isocitrate dehydrogenase (IDH) activities decreased in the liver of trout fed the DEB 700 diet, but increased in the heart, suggesting a different modulation of metabolic capacity by the dietary challenge. Several markers of oxidative stress in the liver of trout, mainly related to the glutathione antioxidant system, were altered when fed the electrolyte-imbalanced diet. The dietary challenge was also associated with a decrease in the alternative complement pathway activity (ACH50) in plasma, suggesting an impaired innate immune status in that group. Trout subjected to the acute stressor displayed reduced blood pH values, higher plasma cortisol levels as well as increased levels of metabolic markers associated with oxidative stress in the liver. An interaction between diet and acute stressor was detected for oxidative stress markers in the liver of trout, showing that the chronic electrolyte-imbalance impairs the response of rainbow trout to handling/confinement. However, trout reared under chronic hypoxia only displayed changes in parameters related to energy use in both liver and heart. Taken together, these results suggest that trout displays an adaptative response to chronic hypoxia. Conversely, the dietary challenge profoundly affected fish homeostasis, resulting in an impaired physiological response leading to stress, which then placed constraints on a subsequent acute challenge.

Viability of dietary substitution of live microalgae with dry Ulva rigida in broodstock conditioning of the Pacific oyster (Crassostrea gigas).

The current study evaluated the microalgae replacement by dry macroalgae (Ulva rigida) in the reproductive success and biochemical composition of the Pacific oyster (Crassostrea gigas) during broodstock conditioning. Five nutritional regimes were tested: 100% macroalgae (diet 1), 50% macroalgae+50% microalgae (diet 2), 25% macroalgae+75% microalgae (diet 3) and 100% microalgae (diet 4). An unfed group was used as a negative control. The microalgae blend was composed of 33% Isochrysis galbana and 67% diatoms (75% Skeletonema costatum+25% Chaetoceros calcitrans). Gonadal maturation was reflected in the physiological condition of the individuals. All treatments, except diet 1, showed an increase in condition index and were fully matured at the end of the trial, with the best physiological condition observed in oysters fed diet 3 and diet 4. Protein and total lipid content increased during the conditioning period, whereas glycogen content decreased. Oysters conditioned with diet 3 had higher protein and total lipid content and lower glycogen content than the other treatments. In addition, diet 3 showed the highest percentage of viable veliger larvae. The current study demonstrated that it is possible to replace 25% of microalgae with macroalgae in the broodstock conditioning, minimizing the operative cost in bivalve hatcheries.This article has an associated First Person interview with the first author of the paper.

Hypoxia, but not an electrolyte-imbalanced diet, reduces feed intake, growth and oxygen consumption in rainbow trout (Oncorhynchus mykiss).

Oxygen limitation and dietary imbalances are key aspects influencing feed intake (FI) and growth performance in cultured fish. This study investigated the combined effects of hypoxia and dietary electrolyte balance on the growth performance, body composition and nutrient utilization in a rainbow trout (Oncorhynchus mykiss) isogenic line. Fish were fed ad libitum two experimental diets: electrolyte-balanced or -imbalanced diets (DEB 200 or 700 mEq kg-1, respectively) and exposed to normoxia or hypoxia (7.9 or 4.5 mg O2 l-1, respectively) for 42 days. DEB did not affect FI, growth performance or body composition. Nevertheless, hypoxia had a negative impact, reducing FI (6%), growth rate (8%), oxygen consumption (19%), energy (5%) and lipid (42%) contents. Digestible energy intake and heat production were higher in normoxic fish (40% and 23%, respectively), retaining 64% more energy in lipid or protein. Hypoxia reduced the apparent digestibility of dry matter, ash, protein, lipid, carbohydrates and energy. Trout fed DEB 700 diet were energetically less efficient, reflected in higher heat production and energy requirements for maintenance. FI was inhibited by low dissolved oxygen levels, but not by electrolyte-imbalanced diet, in spite of the higher energy requirements for maintenance. This study highlights the importance that dietary-electrolyte content and DO levels have on energy balance and growth performance when fish are fed to satiation.

Dietary supplementation of heat-treated Gracilaria and Ulva seaweeds enhanced acute hypoxia tolerance in gilthead sea bream (Sparus aurata).

Intensive aquaculture practices involve rearing fish at high densities. In these conditions, fish may be exposed to suboptimal dissolved O2 levels with an increased formation of reactive O2 species (ROS) in tissues. Seaweeds (SW) contain biologically active substances with efficient antioxidant capacities. This study evaluated the effects of dietary supplementation of heat-treated SW (5% Gracilaria vermiculophylla or 5% Ulva lactuca) on stress bioindicators in sea bream subjected to a hypoxic challenge. 168 fish (104.5 g average weight) were distributed in 24 tanks, in which eight tanks were fed one of three experimental diets for 34 days: (i) a control diet without SW supplementation, (ii) a control diet supplemented with Ulva, or (iii) a control diet with Gracilaria Thereafter, fish from 12 tanks (n=4 tanks/dietary treatment) were subjected to 24 h hypoxia (1.3 mg O2 l-1) and subsequent recovery normoxia (8.6 mg O2 l-1). Hypoxic fish showed an increase in hematocrit values regardless of dietary treatment. Dietary modulation of the O2-carrying capacity was conspicuous during recovery, as fish fed SW supplemented diets displayed significantly higher haemoglobin concentration than fish fed the control diet. After the challenge, survival rates in both groups of fish fed SW were higher, which was consistent with a decrease in hepatic lipid peroxidation in these groups. Furthermore, the hepatic antioxidant enzyme activities were modulated differently by changes in environmental O2 condition, particularly in sea bream fed the Gracilaria diet. After being subjected to hypoxia, the gene expression of antioxidant enzymes and molecular chaperones in liver and heart were down regulated in sea bream fed SW diets. This study suggests that the antioxidant properties of heat-treated SW may have a protective role against oxidative stress. The nature of these compounds and possible mechanisms implied are currently being investigated.

Dietary electrolyte balance affects growth performance, amylase activity and metabolic response in the meagre (Argyrosomus regius).

Dietary ion content is known to alter the acid-base balance in freshwater fish. The current study investigated the metabolic impact of acid-base disturbances produced by differences in dietary electrolyte balance (DEB) in the meagre (Argyrosomus regius), an euryhaline species. Changes in fish performance, gastric chyme characteristics, pH and ion concentrations in the bloodstream, digestive enzyme activities and metabolic rates were analyzed in meagre fed ad libitum two experimental diets (DEB 200 or DEB 700mEq/kg) differing in the Na2CO3 content for 69days. Fish fed the DEB 200 diet had 60-66% better growth performance than the DEB 700 group. Meagre consuming the DEB 200 diet were 90-96% more efficient than fish fed the DEB 700 diet at allocating energy from feed into somatic growth. The pH values in blood were significantly lower in the DEB 700 group 2h after feeding when compared to DEB 200, indicating that acid-base balance in meagre was affected by electrolyte balance in diet. Osmolality, and Na+ and K+ concentrations in plasma did not vary with the dietary treatment. Gastric chyme in the DEB 700 group had higher pH values, dry matter, protein and energy contents, but lower lipid content than in the DEB 200 group. Twenty-four hours after feeding, amylase activity was higher in the gastrointestinal tract of DEB 700 group when compared to the DEB 200 group. DEB 700 group had lower routine metabolic (RMR) and standard metabolic (SMR) rates, indicating a decrease in maintenance energy expenditure 48h after feeding the alkaline diet. The current study demonstrates that feeding meagre with an alkaline diet not only causes acid-base imbalance, but also negatively affects digestion and possibly nutrient assimilation, resulting in decreased growth performance.

Dietary Oil Source and Selenium Supplementation Modulate Fads2 and Elovl5 Transcriptional Levels in Liver and Brain of Meagre (Argyrosomus regius).

The meagre (Argyrosomus regius) is taking on increasing importance in the aquaculture industry. In view of the limited supply of fish oil (FO) as a feed ingredient, the study of the capacity to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from alternative dietary oil sources is important. We analyzed changes in fatty acid (FA) desaturase 2 (fads2) and FA elongase 5 (elovl5) mRNA levels in livers and brains in response to FO replacement with a blend of vegetable oils (VO) and selenium (Se) supplementation. Fish were fed for 60 days with either a diet containing FO or a diet including VO, each supplemented or not with organic Se. Results showed that fads2 and elovl5 transcription was higher in liver when fish were fed VO diets. The brain mRNA levels of both genes were not affected by the dietary replacement of FO by VO. FA composition in the liver and skeletal muscle was altered by FO replacement, particularly by decreasing eicosapentaenoic acid and docosahexaenoic acid contents. The α-linolenic, linoleic, and arachidonic acid contents increased in both liver and brain of fish fed VO diets. The effect of Se supplementation on lipid metabolism was evident only in fish fed FO, showing a decrease in the transcription of hepatic fads2. Results indicate that the total replacement of FO by VO in diets modulates the expression of genes involved in LC-PUFA biosynthesis in meagre, affecting the FA profile of the fish flesh.

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss).

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout.

Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming.

AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has not been investigated in fish. We found that sustained swimming (40 days at 0.75 body lengths s(-1)) increased AMPK activity in red and white trout skeletal muscle (3.9- and 2.2-fold, respectively) as well as the expression of AMPK target genes involved in energy use: lipoprotein lipase and citrate synthase in red and white muscle and CPT1ß1b and PGC-1α in red muscle. Furthermore, electrical pulse stimulation of cultured trout myotubes increased AMPK activity and glucose uptake (1.9- and 1.2-fold, respectively) in an AMPK-dependent manner. These results suggest that AMPK may play an important mediatory role in the metabolic adaptation to swimming in fish skeletal muscle.

Effects of sustained swimming on the red and white muscle transcriptome of rainbow trout (Oncorhynchus mykiss) fed a carbohydrate-rich diet.

Training at sustainable swimming speeds can produce changes in fish skeletal muscle that are important for aquaculture due to their growth-potentiating effects. Such changes may be even more relevant when fish are fed diets containing an increasing proportion of carbohydrates as an energy source. We evaluated the effects of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet using microarray and qPCR. Analysis of the red and white muscle transcriptome in resting or swimming (1.3 body lengths/s) fish for 30days revealed significant changes in the expression of a large number of genes (395 and 597, respectively), with a total of 218 differentially expressed genes (DEGs) common for both muscles. A large number of the genes involved in glucose use and energy generation, contraction, development, synthesis and catabolism of proteins were up-regulated in red and white muscle. Additionally, DEGs in both muscles were involved in processes of defense response and apoptosis. Skeletal muscle contraction activates a transcriptional program required for the successful adaptation of both muscles to the changing demands imposed by swimming conditions. Future studies should further clarify the mechanisms involved in the adaptation of both tissues to exercise and assess possible benefits of such conditions for cultured fish.

Deep RNA sequencing of the skeletal muscle transcriptome in swimming fish.

Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (n = 10) or swum (n = 10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes) was sequenced and resulted in 15-17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (>500 nucleotides), a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an up-regulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells.

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.