White Muscle Of Rainbow Trout Research Articles

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.

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.

Dietary methionine availability affects the main factors involved in muscle protein turnover in rainbow trout (Oncorhynchus mykiss).

Methionine is a limiting essential amino acid in most plant-based ingredients of fish feed. In the present study, we aimed to determine the effect of dietary methionine concentrations on several main factors involved in the regulation of mRNA translation and the two major proteolytic pathways (ubiquitin-proteasome and autophagy-lysosomal) in the white muscle of rainbow trout (Oncorhynchus mykiss). The fish were fed for 6 weeks one of the three isonitrogenous diets providing three different methionine concentrations (deficient (DEF), adequate (ADQ) and excess (EXC)). At the end of the experiment, the fish fed the DEF diet had a significantly lower body weight and feed efficiency compared with those fed the EXC and ADQ diets. This reduction in the growth of fish fed the DEF diet was accompanied by a decrease in the activation of the translation initiation factors ribosomal protein S6 and eIF2α. The levels of the main autophagy-related markers (LC3-II and beclin 1) as well as the expression of several autophagy genes (atg4b, atg12 l, Uvrag, SQSTM1, Mul1 and Bnip3) were higher in the white muscle of fish fed the DEF diet. Similarly, the mRNA levels of several proteasome-related genes (Fbx32, MuRF2, MuRF3, ZNF216 and Trim32) were significantly up-regulated by methionine limitation. Together, these results extend our understanding of mechanisms regulating the reduction of muscle growth induced by dietary methionine deficiency, providing valuable information on the biomarkers of the effects of low-fishmeal diets.

Differential effects of temperature and glucose on glycogenolytic enzymes in tissues of rainbow trout ( Oncorhynchus mykiss )

The pathways and regulatory mechanisms of glycogenolysis remain relatively unexplored in non-mammalian vertebrates, especially poikilotherms. We studied the temperature sensitivity and inhibition of glycogenolytic enzymes in liver, ventricle, and white muscle of rainbow trout acclimated to 14 °C. Glycogen phosphorylase (GP) and acid α-glucosidase (GAA) activities were measured in homogenates of tissues at physiological temperatures (4, 14, and 24 °C), and in the presence of allosteric inhibitor, glucose. Higher GP versus GAA activity in all three tissues suggested a predominance of phosphorolytic glycogenolysis over the lysosomal glucosidic pathway. GP activities at 14 °C were ~2-fold higher in the ventricle and white muscle versus the liver and selectively increased by AMP in striated muscle. Conversely, the activities of GAA and lysosomal marker acid phosphatase were 8- to 10-fold higher in the liver compared with the ventricle and white muscle. Thermal sensitivity (Q10) was increased for GP in all tissues below 14 °C and decreased in striated muscle in the absence of AMP above 14 °C. GAA had lower Q10 values than GP below 14 °C, and, unlike GP, Q10s for GAA were not different between tissues or affected by temperature. Both GP (in the absence of AMP) and GAA were inhibited by glucose in a dose-dependent manner, with the lowest IC50 values observed in the white muscle (1.4 and 6.3 mM, respectively). In conclusion, despite comparatively low kinetic potential, lysosomal GAA might facilitate glycogenolysis at colder body temperatures in striated muscle and intracellular glucose could limit phosphorolytic and glucosidic glycogenolysis in multiple tissues of the rainbow trout.

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.

Biochemical and physiological responses in liver and muscle of rainbow trout after long-term exposure to propiconazole

In this study, the chronic toxic effects of PCZ, a triazole fungicide commonly present in surface and ground water, on morphological indices, ROS generation and RNA/DNA ratio in liver and white muscle of rainbow trout were investigated. Fish were exposed at sublethal concentrations of PCZ (0.2, 50 and 500μgL−1) for 7, 20 and 30d. Compared with the control, there were significant lower CF and HSI in fish exposed at the highest concentration of PCZ. ROS levels in both tissues increased significantly at higher PCZ concentrations (50 and 500μgL−1) after 20d and above, as well as in muscle of fish exposed at lowest PCZ concentration (0.2μgL−1) after 30d. The hepatic antioxidant enzymes (SOD and CAT) activities were induced significantly at higher concentrations (50, 500μgL−1) of PCZ after 20d and at 50μgL−1 after 30d. Additional, hepatic SOD activity was significantly induced at 0.2μgL−1 after 30d. Compared with the hepatic antioxidant enzymes activities in fish exposed to 50μgL−1 of PCZ, there was a decreasing trend in those exposed to 500μgL−1 after 30d exposure. However, both the antioxidant enzymes activities were significantly inhibited in muscle of fish exposed to 500μgL−1 PCZ after 30d. Moreover there was significant lower RNA/DNA ratio in both tissues after long-term exposure to higher concentration of PCZ. In short, environmental concentrations of PCZ could not induce obvious impacts on fish, but long-term exposure to higher concentrations of CBZ could affect seriously the health status of fish.

Dietary protein differentially regulates the kinetic behaviour of serine dehydratase and tyrosine aminotransferase of liver and white muscle of rainbow trout (Oncorhynchus mykiss)

We have determined the kinetic behaviour of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in the liver and white muscle of juvenile rainbow trout (Oncorhynchus mykiss) fed on a low-protein/high-fat (LP/HF) and control diets. The relationship between the kinetic parameters and tissue-DNA concentration has also been determined. SerDH and TyrAT showed hyperbolic kinetics in all cases. The partial replacement of protein with fat significantly increased hepatic SerDH-specific activity, maximum velocity (V(max)) and Michaelis constant (K(m)) while no significant changes were detected in the values of these parameters in white muscle. Nevertheless, hepatic and white-muscle TyrAT specific activity and V(max) decreased in trout fed on LP/HF diet with respect to control. The K(m) of hepatic TyrAT was also lower in trout fed on LP/HF diet than in control. The TyrAT kinetic parameters expressed per cell unit also changed in the same sense as previously described. These results show that, in trout, SerDH and TyrAT are two enzymes regulated by the partial replacement of dietary protein by fat.

Regulation of the kinetic behavior of serine dehydratase and tyrosine aminotransferase during the development of rainbow trout ( Oncorhynchus mykiss)

Serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) are regulatory enzymes of serine and tyrosine catabolism in trout. Here, we have studied the kinetic behavior of SerDH and TyrAT in liver and white muscle of rainbow trout ( Oncorhynchus mykiss) at different stages of development (fingerling: 14-week-old trout; juvenile: 24-, 28-, 40-week-old trout; adult: 96-week-old trout). SerDH and TyrAT showed hyperbolic kinetics in all developmental stages. A significant and progressive decrease in the specific activity, maximum velocity and catalytic efficiency of hepatic SerDH and hepatic and white-muscle TyrAT was observed during development. Nevertheless, the specific TyrAT activity per cell unit was similar in the fingerling and adult stages. No significant changes were found in the Michaelis constant of the different groups of age. This behavior is consistent with the existence of a regulation of the number of enzyme molecules. Total TyrAT activity was of similar magnitude in liver and in white muscle in the fingerling and adult stages. These results show that, in trout, SerDH and TyrAT are two inducible enzymes regulated by the development that showed their highest levels during the first stages, in which the highest whole-body growth rate was found. These results imply high serine and tyrosine requirements in fingerling and juvenile stages that should be considered in the formulation of trout diet for these stages.

Liver and muscle metabolic changes induced by dietary energy content and genetic selection in rainbow trout (Oncorhynchus mykiss)

We combined genetic selection and dietary treatment to produce a model to study metabolic pathways involved in genetic and nutritional control of fat deposition in fish muscle. Two experimental lines of rainbow trout, selected for a lean (L) or fat (F) muscle, were fed with diets containing either 10 or 23% lipids from the first feeding, up to 6 mo. At the end of the feeding trial, trout were distinguished by very different muscle fat content (from 4.2 to 10% wet weight), and line x diet interactions were observed for parameters related to fat storage. We analyzed the activity and gene expression of key enzymes involved in lipid metabolism (fatty acid synthase, hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase 1 isoforms, and peroxisome proliferator-activated receptor alpha) and glycolysis (hexokinase 1 and pyruvate kinase) as well as energy production (isocitrate dehydrogenase, citrate synthase, and cytochrome oxidase) in the liver and the white muscle of rainbow trout. The lipid-rich diet repressed the activity of the lipogenic enzymes and stimulated enzymes involved in fatty acid oxidation and glycolysis in liver but had little effect on muscle enzymes assessed in this study. Regarding the selection effect, enzyme activity and expression suggest that compared with the L line, the F line presented reduced hepatic fatty acid oxidation as well as reduced mitochondrial oxidative capacities and enhanced glucose utilization in both liver and muscle. Very few line x diet interactions were found, suggesting that the two factors (i.e., dietary energy content and selection) used in this study to modify muscle lipid content exerted some additive but mostly independent effects on these metabolic actors.

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.

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.

ISOLATION AND CHARACTERIZATION OF CALPAINS FROM RAINBOW TROUT MUSCLE AND THEIR ROLE IN TEXTURE DEVELOPMENT

ABSTRACT A single anion‐exchange column was used to resolve calpain I and II and their endogenous inhibitor, calpastatin, from the white muscle of rainbow trout (Oncorhynchus mykiss). Calcium requirement assays showed that calpain I and II required approximately 0.2 mM and 2.5 mM, respectively, for half maximal activities. Isolation of calpain I and calpastatin were confirmed by Western blot. Incubation of trout muscle for 12 h postmortem, in a buffer containing CaCl2, decreased (P &lt; 0.0001) shear force compared to samples incubated in a buffer containing EDTA. This effect indicates that softening of fish muscle can be accelerated by activation of calpains with exogenous calcium.

Palmitate movement across red and white muscle membranes of rainbow trout.

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.

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.

Utilization of (3S ,3′S )-astaxanthin acyl esters in pigmentation of rainbow trout (Oncorhynchus mykiss )

The deposition of natural, optically active, astaxanthin fatty acid esters in rainbow trout (Oncorhynchus mykiss) was studied. Mono-esterified and di-esterified (3S,3¢S) astaxanthin were purified from the green microalga Haematococcus pluvialis and incorporated into extruded diets and compared with diets containing synthetic racemic astaxanthin (Carophyll Pink) and a total carotenoid extract from the alga. All sources of astaxanthin achieved >4 mg kg )1 in the white muscle after 6 weeks feeding. No significant diAerence (P > 0.05) between the deposition of astaxanthin or total carotenoid for the diAerent diets was observed. Other xanthophylls, namely lutein, zeaxanthin and idoxanthin were found in the white muscle of rainbow trout fed all diets and together accounted for 10‐14% of total carotenoid. Astaxanthin was deposited in the white muscle in the stereochemical form administered in the diet, i.e. racemic astaxanthin for Carophyll Pink and 100% (3S,3¢S)-astaxanthin for the algal sources. In contrast, epimerization of (3S,3¢S) astaxanthin from the alga was observed for the astaxanthin esters deposited in the skin of rainbow trout, with a ratio close to 1.0:2.0:1.0 (3S,3¢S:3R,3¢S:3R,3¢R).

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.

Lipid oxidation fuels recovery from exhaustive exercise in white muscle of rainbow trout.

The oxidative utilization of lipid and carbohydrate was examined in white muscle of rainbow trout (Oncorhynchus mykiss) at rest, immediately after exhaustive exercise, and for 32-h recovery. In addition to creatine phosphate and glycolysis fueling exhaustive exercise, near maximal activation of pyruvate dehydrogenase (PDH) at the end of exercise points to oxidative phosphorylation of carbohydrate as an additional source of ATP during exercise. Within 15 min postexercise, PDH activation returned to resting values, thus sparing accumulated lactate from oxidation. Glycogen synthase activity matched the rate of glycogen resynthesis and represented near maximal activation. Decreases in white muscle free carnitine, increases in long-chain fatty acyl carnitine, and sustained elevations of acetyl-CoA and acetyl carnitine indicate a rapid utilization of lipid to supply ATP for recovery. Increases in malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palmitoyltransferase-1 in trout muscle during recovery, but instead it may act to elongate short-chain fatty acids for mitochondrial oxidation. In addition, decreases in intramuscular triacylglycerol and in plasma nonesterified fatty acids indicate that both endogenous and exogenous lipid fuels may be oxidized during recovery.

Growth, protein-turnover rates and nucleic-acid concentrations in the white muscle of rainbow trout during development

We have studied the growth rate, nucleic-acid concentration, protein-accumulation rate ( K G), and several other parameters relating to protein turnover, such as the protein-synthesis ( K S), and protein-degradation rates ( K D), protein-synthesis capacity ( C S), protein-synthesis efficiency ( K RNA), protein-synthesis rate per DNA unit ( K DNA) and protein-retention efficiency (PRE), in the white muscle of rainbow trout during development. Both growth rate and relative food intake decreased significantly with age and weight, as did the food-efficiency ratio (FER) and protein-efficiency ratio (PER). Although absolute RNA and DNA contents increased with age, their relative concentrations decreased. The RNA/DNA ratio increased sharply from 14 to 28 weeks but afterwards decreased towards initial values. Hypertrophy increased rapidly to the 28-week stage but thenceforth increased much more slowly. Hyperplasia, on the other hand, continued to increase linearly, resulting in a significant four- to fivefold predominance in this type of growth at the end of the 96-week experimental period. K G decreased significantly with age, as did K S, and C S, whereas at the 14-week stage, K D was significantly lower than at other ages. K RNA increased until 28 weeks. K DNA increased significantly in juvenile fish compared to both fingerlings and adults, where it showed similar lower values. PRE remained high at all ages.

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.

Lactate transport by rainbow trout white muscle: kinetic characteristics and sensitivity to inhibitors

This study used an isolated-perfused tail-trunk preparation of rainbow trout to examine the uptake and release of lactate (Lac) and metabolic protons (delta H+M) in resting and exercised fish white muscle. In exercised muscle, L(+)-Lac efflux was inhibited (approximately 40%) by 5 mM alpha-cyano-4-hydroxycinnamate (CIN), but not by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) or 0.1 mM amiloride. These results suggest that Lac release occurs through a Lac(-)-H- symport and the free diffusion of lactic acid (HLac) or Lac-, but not via the Lac-/HCO3(-)-Cl- antiporter. Lac efflux was accompanied by delta H+m influx in all treatments, and increased delta H+m influx occurred after SITS treatment. In resting muscle, Lac uptake rates were greater than Lac efflux rates in the postexercise preparation. L-Lac influx exhibited partial saturation kinetics, whereas D(-)-Lac influx was linearly related to its extracellular concentration (0-32 mM). At 16 mM extracellular L-Lac, with a negligible transmembrane L-HLac gradient and an outwardly directed not driving force on L-Lac-, CIN, and SITS reduced net L-Lac uptake by 75 and 45%, respectively. At 16 mM extracellular concentration, D-Lac influx was 64% of the net L-Lac influx. These results suggest that in trout muscle at 16 mM extracellular L-Lac, the Lac -H+ symport accounts for 30-36%, the Lac-/HCO3(-)-Cl- antiport for 39-45%, and diffusion for 19-25% of uptake, although the latter is probably overestimated and the former underestimated for methodological reasons. Net L-Lac efflux was not affected by extracellular D-Lac concentration and/or D-Lac influx, implying the existence of a concurrent L-Lac efflux during L-Lac influx. The D-Lac influx kinetics data indicated that the Lac-/HCO3 antiport was not saturable in the extracellular D-Lac concentration range of 0-32 mM. This study clearly demonstrates the involvement of carrier-mediated transport in transmembrane Lac movement in fish muscle and supports the "active lactate retention" mechanism proposed by Turner and Wood (J. Exp. Biol. 105: 895-401, 1983).