Trout White Muscle Research Articles

Gene expression profiling of trout muscle during flesh quality recovery following spawning

BackgroundSexual maturation causes loss of fish muscle mass and deterioration of fillet quality attributes that prevent market success. We recently showed that fillet yield and flesh quality recover in female trout after spawning. To gain insight into the molecular mechanisms regulating flesh quality recovery, we used an Agilent-based microarray platform to conduct a large-scale time course analysis of gene expression in female trout white muscle from spawning to 33 weeks post-spawning.ResultsIn sharp contrast to the situation at spawning, muscle transcriptome of female trout at 33 weeks after spawning was highly similar to that of female trout of the same cohort that did not spawn, which is consistent with the post-spawning flesh quality recovery. Large-scale time course analysis of gene expression in trout muscle during flesh quality recovery following spawning led to the identification of approximately 3340 unique differentially expressed genes that segregated into four major clusters with distinct temporal expression profiles and functional categories. The first cluster contained approximately 1350 genes with high expression at spawning and downregulation after spawning and was enriched with genes linked to mitochondrial ATP synthesis, fatty acid catabolism and proteolysis. A second cluster of approximately 540 genes with transient upregulation 2 to 8 weeks after spawning was enriched with genes involved in transcription, RNA processing, translation, ribosome biogenesis and protein folding. A third cluster containing approximately 300 genes upregulated 4 to 13 weeks after spawning was enriched with genes encoding ribosomal subunits or regulating protein folding. Finally, a fourth cluster that contained approximately 940 genes with upregulation 8 to 24 weeks after spawning, was dominated by genes encoding myofibrillar proteins and extracellular matrix components and genes involved in glycolysis.ConclusionOverall, our study indicates that white muscle tissue restoration and flesh quality recovery after spawning are associated with transcriptional changes promoting anaerobic ATP production, muscle fibre hypertrophic growth and extracellular matrix remodelling. The generation of the first database of genes associated with post-spawning muscle recovery may provide insights into the molecular and cellular mechanisms controlling muscle yield and fillet quality in fish and provide a useful list of potential genetic markers for these traits.

Dietary Selenium Promotes Somatic Growth of Rainbow Trout (Oncorhynchus mykiss) by Accelerating the Hypertrophic Growth of White Muscle.

As a nutritionally essential trace element, selenium (Se) is crucial for fish growth. However, the underlying mechanisms remain unclear. Fish somatic growth relies on the white muscle growth. This study aimed to explore the effects and underlying mechanisms of Se on fish white muscle growth using a juvenile rainbow trout (Oncorhynchus mykiss) model. Fish were fed a basal diet unsupplemented or supplemented with selenium yeast at nutritional dietary Se levels (2 and 4mg/kg Se, respectively) for 30days. Results showed that dietary Se supplementation significantly enhanced trout somatic growth. Histological and molecular analysis of trout white muscle tissues at the vent level showed that dietary Se supplementation elevated the total cross-sectional area of white muscle, mean diameter of white muscle fibers, protein content, nuclei number, and DNA content of individual muscle fiber, and suppressed the activities of calpain system and ubiquitin-proteasome pathway. Overall, this study demonstrated that dietary Se within the nutritional range inhibits calpain- and ubiquitin-mediated protein degradation and promotes the fusion of myoblasts into the existed muscle fibers to promote the hypertrophic growth of white muscle, thereby accelerating the somatic growth of rainbow trout. Our results provide a mechanistic insight into the regulatory role of Se in fish growth.

Essential amino acids exhibit variable effects on protein degradation in rainbow trout (Oncorhynchus mykiss) primary myocytes

The functional role of amino acids as regulators of protein degradation was investigated using primary myogenic precursor cell culture as in vitro model of rainbow trout white muscle. Seven-day old myocytes were starved of amino acids for two hours then exposed to media that contained amino acid treatments, during which protein degradation rates were analyzed over five hours by measuring cellular release of 3H-tyrosine. Increasing concentrations of essential amino acids (EAA) reduced protein degradation rates; this effect was dose-dependent within the physiological range found in plasma. Addition of leucine or phenylalanine at 5 mM and 2.5 mM, respectively, decreased rates of protein degradation compared to media without amino acid supplementation, suggesting that these amino acids directly regulate muscle proteolysis. Protein degradation rates were similar in cells exposed to media without EAA and media lacking only leucine, further supporting a role for leucine as a central regulator of protein turnover. Addition of 5 mM lysine or valine to media without amino acids increased protein degradation; this response was attenuated as EAA were added back into media, supporting that a lysine or valine imbalance is costly for muscle protein retention. In summary, there is evidence for amino acids as both positive and negative regulators of protein turnover in rainbow trout muscle. These findings suggest that there may be an optimal plasma amino acid profile that minimizes protein turnover and that this could be achieved through diet formulation.

A comparative evaluation of crowding stress on muscle HSP90 and myostatin expression in salmonids

Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin, and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48h crowding stress. Crowding stress resulted in a decrease of myostatin-1a in brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1a was upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.

Estimating in situ isotopic turnover in Rainbow Trout (Oncorhynchus mykiss) muscle and liver tissue

ABSTRACTStable isotope analysis (SIA) is a powerful tool for assessing food webs and diet. However, the underlying assumptions that stable isotope ratios in an organism's tissues reflect that of its present diet may be complicated by isotopic turnover and retention time. Quantification of in situ isotopic turnover of study organisms and different tissues via field studies is essential to inform scientifically sound food web and diet studies utilizing SIA. Rainbow Trout white muscle and liver tissue were collected monthly from North Twin Lake, Washington during the 2013 growing season. Using ANCOVAs, we estimated stable carbon retention time of 154 (95% CI: 106–224) and 292 (95% CI: 257–763) days for Rainbow Trout liver and white muscle, respectively. For nitrogen, we estimated a retention time of 186 (95% CI: 114–329) days for both white muscle and liver. Our analyses suggest longer isotopic retention times (and therefore slower isotope turnover rates) for adult fish in a natural system relative to those commonly reported in the literature. These findings can improve interpretation of SIA in future aquatic food web and diet studies involving adult coldwater fish in freshwater systems.

Is lipid correction necessary in the stable isotope analysis of fish tissues?

Stable isotope analysis (SIA) is a powerful tool for examining diet and food-web dynamics. SIA assumes "you are what you eat" relative to carbon (C) and nitrogen (N). However, fractionation of carbon during lipid synthesis violates this assumption; high-lipid tissues do not reflect δ(13) C values of diet and therefore have the potential to skew mixing model results and diet interpretations, making corrections necessary. Brook Trout (Salvelinus fontinalis) white muscle and liver samples from several fish species representing the temperate North American cold- and warm-water fish community were corrected for lipids via chemical lipid extraction and mathematical lipid normalization. To assess the accuracy of model-predicted lipid-free δ(13) C values calculated from four normalization models, we compared model-predicted values with those measured after lipid extraction. We found that chemical lipid extraction is unnecessary for Brook Trout white muscle tissue with low initial lipid content. However, in tissues with C:N ratios greater than 3.5, lipid extraction increased δ(13) C values in fish liver by more than 1.0‰, indicating that liver lipid content is sufficient to bias δ(13) C values. We also found that lipids were accurately accounted for with mathematical normalization and recommend that tissues with C:N ratios greater than 3.5 be corrected mathematically. Our findings indicate that mathematical normalization is sufficient to account for bias in δ(13) C values associated with lipid content in fish tissues when C:N ratios are above 3.5. C:N ratios below 3.5 indicate that tissues have insufficient levels of lipid to bias the δ(13) C values. Generally, these findings support the use of more timely and cost-effective processing and analysis methods in future aquatic food-web studies utilizing SIA. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro and in vivo effects of phytoestrogens on protein turnover in rainbow trout (Oncorhynchus mykiss) white muscle

Soybeans and other legumes investigated as fishmeal replacements in aquafeeds contain phytoestrogens capable of binding to and activating estrogen receptors. Estradiol has catabolic effects in salmonid white muscle, partially through increases in protein turnover. The current study determines whether phytoestrogens promote similar effects. In rainbow trout (Oncorhynchus mykiss) primary myocyte cultures, the phytoestrogens genistein, daidzein, glycitein, and R- and S-equol reduced rates of protein synthesis and genistein, the phytoestrogen of greatest abundance in soy, also increased rates of protein degradation. Increased expression of the ubiquitin ligase fbxo32 and autophagy-related genes was observed with high concentrations of genistein (100μM), and R- and S-equol (100μM) also up-regulated autophagy-related genes. In contrast, low genistein concentrations in vitro (0.01–0.10μM) and in vivo (5μg/g body mass) decreased fbxo32 expression, suggesting a potential metabolic benefit for low levels of genistein exposure. Phytoestrogens reduced cell proliferation, indicating that effects of phytoestrogens extend from metabolic to mitogenic processes. Co-incubation of genistein with the estrogen receptor (ER) antagonist, ICI 182,780, ameliorated effects of genistein on protein degradation, but not protein synthesis or cell proliferation, indicating that effects of genistein are mediated through ER-dependent and ER-independent mechanisms. Collectively, these data warrant additional studies to determine the extent to which dietary phytoestrogens, especially genistein, affect physiological processes that impact growth and nutrient retention.

Exhausting exercise and tissue-specific expression of monocarboxylate transporters in rainbow trout

Transmembrane lactate movements are mediated by monocarboxylate transporters (MCTs), but these proteins have never been characterized in rainbow trout. Our goals were to clone potential trout MCTs, determine tissue distribution, and quantify the effects of exhausting exercise on MCT expression. Such information could prove important to understand the mechanisms underlying the classic "lactate retention" seen in trout white muscle after intense exercise. Four isoforms were identified and partially characterized in rainbow trout: MCT1a, MCT1b, MCT2, and MCT4. MCT1b was the most abundant in heart and red muscle but poorly expressed in the gill and brain where MCT1a and MCT2 were prevalent. MCT expression was strongly stimulated by exhausting exercise in brain (MCT2: +260%) and heart (MCT1a: +90% and MCT1b: +50%), possibly to increase capacity for lactate uptake in these highly oxidative tissues. By contrast, the MCTs of gill, liver, and muscle remained unaffected by exercise. This study provides a possible functional explanation for postexercise "lactate retention" in trout white muscle. Rainbow trout may be unable to release large lactate loads rapidly during recovery because: 1) they only poorly express MCT4, the main lactate exporter found in mammalian glycolytic muscles; 2) the combined expression of all trout MCTs is much lower in white muscle than in any other tissue; and 3) exhausting exercise fails to upregulate white muscle MCT expression. In this tissue, carbohydrates act as an "energy spring" that alternates between explosive power release during intense swimming (glycogen to lactate) and recoil during protracted recovery (slow glycogen resynthesis from local lactate).

Effects of sex steroids on indices of protein turnover in rainbow trout ( Oncorhynchusmykiss) white muscle

Effects of 17β-estradiol (E2), testosterone, and 5α-dihydrotestosterone (DHT) on protein turnover and proteolytic gene expression were determined in rainbow trout ( Oncorhynchus mykiss) primary myocytes and white muscle tissue. E2 reduced rates of protein synthesis and increased rates of protein degradation in primary myocytes by 45% and 27%, respectively. DHT reduced rates of protein synthesis by 27%. Testosterone did not affect protein synthesis and neither testosterone nor DHT affected rates of protein degradation. Single injections of E2 increased expression of ubiquitin ligase genes fbxo32, fbxo25, and murf1, and the proteasome subunit psmd6 by 24 h after injection. Within the cathepsin-lysosome pathway, E2 increased expression of cathepsins ctsd and ctsl, as well as autophagy-related genes atg4b and lc3b. Additionally, E2 injection up-regulated the expression of casp3 and casp9 caspase genes. Incubation of primary myocytes with E2 also increased expression of ubiquitin ligase genes. Therefore, catabolic effects of E2 on protein turnover result in part from E2-induced increases in proteolytic gene expression directly in muscle. Injection of testosterone increased milli-calpain ( capn2) and casp3 expression, and DHT increased ctsd expression in vivo, whereas both androgens up-regulated fbxo32 expression in primary myocytes. These results suggest that effects of androgens on protein turnover in muscle are not driven primarily by direct effects of these hormones in this tissue.

IGF and GH mRNA levels are suppressed upon exposure to micromolar concentrations of cobalt and zinc in rainbow trout white muscle

The objective of this study was to assess the effects of cobalt and zinc exposure of rainbow trout ( Oncorhynchus mykiss) on insulin like growth factors (IGF) and growth hormone (GH). Mature rainbow trouts were exposed to 0.42, 2.1, 4.2, 21 and 42 μmol/L Co 2+ (added as CoCl 2·6H 2O) and 0.34, 1.7, 3.4, 17 and 34 μmol/L Zn 2+ (added as ZnSO 4i·7H 2O). After 6, 12, 24 and 48 h of treatment, expressions of white muscle IGF-I, IGF-II and GH mRNAs were measured by means of quantitative Real Time PCR. During the exposure experiments, no mortalities occurred. The most effective metal concentrations, which caused significant alterations, were determined to be 42 μmol/L Co 2+ (10 mg CoCl 2·6H 2O/L) and 3.4 μmol/L Zn + 2 (1 mg ZnSO 4·7H 2O/L). The following results were obtained for these concentrations. Expression of IGF-I did not change at 6 h in zinc treatment while the decrease (p < 0.05) was observed at 12 h and 24 h, and this decrease became stronger at 48 h. Cobalt exposure caused a decrease in IGF-I mRNA level at 6 h, 12 h, 24 h and 48 h (p < 0.05). Both zinc and cobalt exposure resulted in significant decreases in GH expression at 6 h. Exposure of trout to Zn resulted in a decrease in expression of IGF-II starting from 6 h whereas the significant decrease started at 6 h in cobalt exposure and this decrease elevated at 24 h. The results indicate that micromolar cobalt and zinc exposure causes significant attenuation in the expressions of these three genes' time dependently. Our findings show that IGF-I is the most resistant and GH is the most sensitive component against cobalt and zinc exposure. We conclude that IGF/GH axis might be strongly affected by the short term exposure to low micromolar concentrations of zinc and cobalt due to alterations of these genes.

Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish

Juvenile salmonid fish readily form dominance hierarchies when faced with limited resources. While these social interactions may result in profound behavioural and physiological stress, it is unknown if this social stress is evident at the level of the cellular stress response--specifically, the induction of stress or heat shock proteins (Hsps). Thus, the goal of our study was to determine if Hsps are induced during hierarchy formation in juvenile rainbow trout (Oncorhynchus mykiss). To this end, we measured levels of three Hsps, Hsp70, Hsc (heat shock cognate)70 and Hsp90 in the white muscle, liver and brain of trout that had been interacting for 36 h, 72 h or 6 days. Our data indicate that Hsps are induced in both dominant and subordinate fish in a time- and tissue-specific manner. In further mechanistic experiments on fasted and cortisol-treated fish, we demonstrated that high plasma cortisol does not affect Hsp induction in trout white muscle or liver, but both conditions may be part of the mechanism for Hsp induction with social stress in the brain. We conclude that the behavioural and physiological stress experienced by juvenile rainbow trout in dominance hierarchies can be extended to the induction of Hsps.

Maslinic acid added to the diet increases growth and protein-turnover rates in the white muscle of rainbow trout ( Oncorhynchus mykiss)

Maslinic acid (2-α, 3-β-dihydroxiolean-12-en-28-oic acid) is a triterpenoid compound present in fruit and leaves of Olea europaea that can be used as an additive in the diet of trout. The present work investigates the effects of maslinic acid on growth, protein-turnover rates and nucleic acid concentration in trout white muscle. Five groups of 180 trout of a mean body mass of 20 g were fed for 225 days with diets containing 0, 1, 5, 25 and 250 mg of maslinic acid per kg of diet. At the end of the experiment, white-muscle weight and protein-accumulation rate of trout fed with maslinic acid were higher than in control. The total content of DNA, RNA, and protein in trout fed with 25 and 250 mg of maslinic acid kg − 1 were significantly higher than in control. The protein:DNA ratio was also slightly higher than control. In the same groups of trout, fractional ( K S) and absolute ( A S) protein-synthesis rates increased to more than 80% over the control values while no differences were found in the fractional protein-degradation rate ( K D). These results, similar to previous findings in liver, show that maslinic acid can act as a growth factor when added to a standard trout diet.

Parvalbumin expression in trout swimming muscle correlates with relaxation rate

Rainbow trout (Oncorhynchus mykiss) display longitudinal and developmental shifts in muscle relaxation rate. This study aimed to determine the role of variations in parvalbumin content in modulating muscle relaxation. Parvalbumin is a low molecular weight protein that buffers myoplasmic Ca2+ and enhances muscle relaxation. In some fish, longitudinal variations in muscle relaxation have been linked to variations in the total amount of parvalbumin present in muscle and in the relative expression of two parvalbumin isoforms. We have demonstrated previously that anterior slow-twitch or red myotomal muscle relaxes more rapidly than that from the posterior for both rainbow and brook trout. Further, younger rainbow trout parr have faster red muscle relaxation rates than older smolts. Here we report similar results for fast-twitch or white muscle. We quantified the parvalbumin expression in red and white muscle from different body positions of rainbow trout parr and smolts and for brook trout (Salvelinus fontinalis) adults. There was a significant shift in total parvalbumin content of muscle: the faster muscle from the anterior myotome contained greater amounts of parvalbumin. For brook trout, longitudinal variation in relaxation rate was also associated with shifts in the relative expression of the two parvalbumin isoforms. The faster muscle of parr contained more parvalbumin. Lastly, trout white muscle tended to have higher levels of parvalbumin and greater levels of the Parv2 (relative to Parv1) isoform as compared to red muscle. Parvalbumin expression correlated with muscle relaxation rate in trout, although there were species-specific differences in the importance of altering total parvalbumin content versus shifts in relative parvalbumin isoform expression.

Fuel use during glycogenesis in rainbow trout (Oncorhynchus mykissWalbaum) white muscle studiedin vitro

The purpose of this study was to examine fuel used during muscle glycogenesis in rainbow trout Oncorhynchus mykiss using an in vitro muscle slice preparation to test the hypothesis that intracellular lactate is the major glycogenic substrate and the muscle relies upon extracellular substrates for oxidation. Fish were exhaustively exercised to reduce muscle glycogen content, muscle slices were taken from exhausted fish and incubated for 1 h in medium containing various substrates at physiological concentrations. 14C-labeled lactate, glycerol or palmitate was added and 14C incorporation into muscle glycogen and/or CO2 was measured. Lactate clearance in the absence of net glycogenesis suggests that when suitable oxidizable extracellular substrates were lacking, intracellular lactate was oxidized. Only muscle incubated in lactate, glycerol or palmitate synthesized glycogen, with the greatest synthesis in muscle incubated in lactate plus glycerol. The major fate of these extracellular substrates was oxidative, with lactate oxidized at rates 10 times that of palmitate and 100 times that of glycerol. Neither extracellular lactate nor glycerol contributed significantly to glycogenesis, with lactate carbon contributing less than 0.1% of the total glycogen synthesized, and glycerol less than 0.01%. There was 100 times more extracellular lactate-carbon incorporated into CO2 than into glycogen. In the presence of extracellular lactate, palmitate or glycerol, intracellular lactate was spared an oxidative fate, allowing it to serve as the primary substrate for in situ glycogenesis, with oxidation of extracellular substrates driving ATP synthesis. The primary fate of extracellular lactate is clearly oxidative, while that of intracellular, glycolytically derived lactate is glycogenic, which suggests intracellular compartmentation of lactate metabolism.

Alterations in expression of genes associated with muscle metabolism and growth during nutritional restriction and refeeding in rainbow trout

Rainbow trout, as well as many other species of fish, demonstrate the ability to survive starvation for long periods of time. During starvation, growth rate is decreased and muscle exhibits signs of wasting. However, upon resumption of feeding, accelerated growth is often observed. Alterations in muscle metabolism occur during feed restriction and refeeding, although the ways in which these alterations affect the molecular pathways that control muscle growth have not been fully determined. To analyze changes in muscle metabolism and growth during starvation and refeeding, real-time PCR was used to test the expression of six metabolic-related genes and eight muscle-specific genes in rainbow trout white muscle prior to and after 30 days of starvation, and after 4 and 14 days of refeeding. The six metabolic-related genes chosen are indicative of specific metabolic pathways: glycolysis, glycogenesis, gluconeogenesis, the pentose phosphate pathway, and fatty acid formation. The eight muscle specific genes chosen are key components in muscle growth and structural integrity, i.e., MRFs, MEFs, myostatins, and myosin. Alterations in expression of the tested metabolic-related genes and muscle-specific genes suggest that during both starvation and refeeding, changes in specific metabolic pathways initiate shifts in muscle that result mainly in the modification of myotube hypertrophy. The expression levels of many of the metabolic-related genes were altered during the refeeding period compared to those observed before the starvation period began. However, the accelerated growth often observed during refeeding is likely driven by changes in normal muscle metabolism, and the altered expression observed here may be a demonstration of those changes.

Effect of refeeding on IGFI, IGFII, IGF receptors, FGF2, FGF6, and myostatin mRNA expression in rainbow trout myotomal muscle

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic growth and especially in muscle growth is poorly understood. In this study we examined the expression profile of major muscle growth regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like growth factor I (IGFI) mRNA level in muscle increased dramatically 8- and 15-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like growth factor II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean fibroblast growth factor 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas fibroblast growth factor 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential growth factors we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory growth induced by refeeding.

Lactate efflux from sarcolemmal vesicles isolated from rainbow trout Oncorhynchus mykiss white muscle is via simple diffusion.

Lactic acid is produced as an end product of glycolysis in rainbow trout white muscle following exhaustive exercise. The metabolically produced lactic acid causes an intramuscular acidosis that must be cleared, either via net transport out of the muscle or by conversion to glycogen, thereby replenishing the muscle energy store. Trout muscle has been shown to retain lactate and utilise it as a substrate for in situ glycogen resynthesis. The giant sarcolemmal vesicle preparation was used to characterise the potential for lactate loss from white muscle of rainbow trout. Minimal lactate loss was expected due to the requirement within the intramuscular compartment of lactate for glycogen resynthesis. The sarcolemma was found to be highly resistant to lactate loss, with efflux rates approximately 500-fold lower than influx rates [0.049+/-0.006 nmol mg(-1) min(-1) (N=21) versus 26.4+/-6.3 nmol mg(-1) min(-1) (N=5), respectively, at 25 mmol l(-1) lactate concentration]. Lactate efflux was linear over the range 10-250 mmol l(-1) lactate, and greatest under conditions when intravesicular pH was lower than extravesicular pH, but was unaffected by alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. These results suggest that lactate is relatively impermeant to the trout white muscle membrane and any lactate loss occurs via passive diffusion. This resistance to lactate diffusion can explain why trout muscle retains lactate post-exercise, despite transmembrane gradients that should favour net efflux.

Glycogen phosphorylase and pyruvate dehydrogenase transformation in white muscle of trout during high-intensity exercise.

We examined the regulation of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) in white muscle of rainbow trout during a continuous bout of high-intensity exercise that led to exhaustion in 52 s. The first 10 s of exercise were supported by creatine phosphate hydrolysis and glycolytic flux from an elevated glycogenolytic flux and yielded a total ATP turnover of 3.7 micromol x g wet tissue(-1) x s(-1). The high glycolytic flux was achieved by a large transformation of Phos into its active form. Exercise performed from 10 s to exhaustion was at a lower ATP turnover rate (0.5 to 1.2 micromol x g wet tissue(-1) x s(-1)) and therefore at a lower power output. The lower ATP turnover was supported primarily by glycolysis and was reduced because of posttransformational inhibition of Phos by glucose 6-phosphate accumulation. During exercise, there was a gradual activation of PDH, which was fully transformed into its active form by 30 s of exercise. Oxidative phosphorylation, from PDH activation, only contributed 2% to the total ATP turnover, and there was no significant activation of lipid oxidation. The time course of PDH activation was closely associated with an increase in estimated mitochondrial redox (NAD(+)-to-NADH concentration ratio), suggesting that O2 was not limiting during high-intensity exercise. Thus anaerobiosis may not be responsible for lactate production in trout white muscle during high-intensity exercise.

Myosin heavy chain expression in the red, white, and ventricular muscle of juvenile stages of rainbow trout.

Juvenile stages of rainbow trout, smaller parr and older juveniles, termed smolts, show differences in red muscle contractile properties: parr red muscle has faster kinetics and a faster maximum shortening velocity than smolt red muscle. A developmental reduction in the number of MHC isoforms as detected by SDS-PAGE between parr and smolt has also been observed. To investigate whether this shift in contractile kinetics results from differential gene expression, three different MHC cDNA fragments, one each from red, white, and ventricular muscle, were identified. The red muscle and ventricular forms are novel MHCs, and the white muscle form is identical to a published MHC from adult trout white muscle. Tissue and developmental stage-specific expression patterns of these MHC isoforms were examined using isoform-specific RT-PCR. Ventricular muscle typically showed only the ventricular form; 60% parr and 80% smolts expressed the ventricular form only. Approximately half of the white muscle samples of either parr or smolts, 58% and 50%, respectively, expressed only white muscle MHC. Red muscle samples were the most heterogeneous, with red muscle MHC found in combination with either the white or ventricular form or both. Combining samples from the anterior and posterior, 8% of parr red muscle samples expressed solely the red muscle MHC form, and 30% of smolt red muscle samples expressed the red muscle form alone. Variations in the relative contribution of each MHC to the red muscle of parr and smolt may explain observed differences in protein composition and contractile properties. J. Exp. Zool. 290:751-758, 2001.

Lactate transport across sarcolemmal vesicles isolated from rainbow trout white muscle.

Rainbow trout (Oncorhynchus mykiss) retain the majority of lactate produced during exhaustive exercise within white muscle. Previous studies have suggested that this retention is partially via a re-uptake of released lactate. The purpose of this work was to study lactate uptake using trout white muscle sarcolemmal vesicles. Lactate uptake by trout white muscle is partially through a low-affinity, high-capacity carrier (apparent K(m)=55.6 mmol l(-)(1) and V(max)=44.5 nmol mg(-)(1 )protein min(-)(1)). At high concentrations (20 and 50 mmol l(-)(1)), pyruvate partially (up to 39 %) inhibited lactate uptake, suggesting the involvement of a monocarboxylate carrier. The anion transport inhibitor 4-acetoamido-4'-isothiocyanstilbene-2,2'-disulphonic acid (SITS) and the monocarboxylate transport inhibitor (&agr;)-cyano-4-hydroxycinnamate (CHC) stimulated apparent lactate uptake. The model developed suggests that lactate is taken up by the vesicles, at least in part by a pyruvate-sensitive monocarboxylate carrier, and that its subsequent efflux is inhibited by SITS and CHC, suggesting that lactate export from trout white muscle is also carrier-mediated.