Variable impacts of L-arginine or L-NAME during early life on molecular and cellular markers of muscle growth mechanisms in rainbow trout.

Two experiments were conducted to test if manipulations of the Arginine-Nitric oxide pathway during the early life of rainbow trout would act on its early myogenic process. In experiment 1, trout embryos were immersed at 72° days post-fertilization (°dpf) or 104°dpf in water alone (control treatment, C) or containing 2 mM/L L-Arg (treatment A) or 1 mM/L of L-NAME, a NOS inhibitor (treatment N). We observed the beginning of expression of myf5 and fmhc genes at 72°dpf and 96°dpf, respectively. "A" treatment doubled the free Arg content of eggs but did not affect either the pattern of expression of myf5 and fmhc, nor white muscle cross-sectional area and number of white muscle fibres at hatching, nor embryo survival and fry growth. "N" treatment also did not affect these markers. In experiment 2, trout fry were fed from first feeding onwards and during 20 days either a control diet (C) or the same diet supplemented with L-NAME (0.1 g/100 g diet, N-diet). In C-fed fry, distribution of a single meal after overnight fasting induced changes in pcna, myod1, myog, fmhc, inos, nnos and ctsd gene expressions. N-feeding decreased fry growth but did not change their growth trajectory or survival. Twenty days of N-feeding led, compared to C-feeding, to changes in kinetics of transcription of pcna, myod1, myog, fmhc, inos, nnos, ctsd genes and to decreased white muscle cross-sectional area, total number of white muscle fibres, and number of large muscle fibres. L-NAME feeding thus decreased fry muscle growth by altering both hyperplasia and hypertrophy.

Higher glycolytic capacities in muscle of carnivorous rainbow trout juveniles after high dietary carbohydrate stimulus at first feeding.

BACKGROUND: Rainbow trout is a "glucose-intolerant" carnivorous species. Using the metabolic programming strategy, we used early nutritional stimuli in order to modify carbohydrate utilization in trout juveniles. METHOD: Fish were fed two diets during the first feeding, namely HP (no carbohydrate / high protein) diet and LP (high carbohydrate / low protein) diet. HP diet was used as the control diet and LP diet as an early stimulus diet. We also used another early stimulus with fish fed HP diet every other day during the first feeding (HP restriction feeding - HPR). After the first-feeding stage (4 weeks), all fish were subsequently subjected to a growth trial with a commercial diet followed by a challenge test with the LP diet (11 weeks). At the end of the first feeding stimulus and of the challenge test, we investigated growth performance, glucose metabolism-related parameters and global DNA CmCGG methylation in trout. RESULTS: LP and HPR dietary stimuli have been a success as shown by the direct modifications of growth performance and mRNA levels for glucose metabolism-related genes at the end of first feeding compared to alevins fed the HP diet. At the end of the challenge trial, no variation in growth performance and hepatic metabolism of LP-history and HPR-history in trout juveniles were observed. However, in muscle of trout juvenile subjected to LP diet at the first feeding, we found an up-regulation of mRNA levels of some glucose metabolism (glucose transport and glycolysis)-related genes and an increase of activities of important glycolysis-related enzymes (hexokinase, phosphofructokinase and pyruvate kinase). These observations are associated with a decrease in the content of glycogen compared to fish fed the HP diet. Moreover, global CmCGG DNA methylation in the muscle of fish with LP history was significantly lower than those fed the HP diet. CONCLUSION: Dietary LP stimulus at first feeding could permanently modify glucose metabolism and global CmCGG DNA methylation level in muscle of trout juveniles, showing that the first feeding stage is efficient for programming the glucose metabolism in fish.

Muscle growth mechanisms in response to isoenergetic changes in dietary non-protein energy source at low and high protein levels in juvenile rainbow trout.

This study investigates muscle growth mechanisms in juvenile rainbow trout in response to isoenergetic changes in dietary non-protein energy (NPE) source (F, fat vs. C, carbohydrates) at two levels of digestible protein to digestible energy (DP/DE) ratio. Fish (initial weight 32.4 g) were fed four diets having similar DE levels (~18 kJ g-1) with a high (HP/E~26 mg kJ-1) vs. low (LP/E~14 mg kJ-1) DP/DE ratio using F or C as major NPE source (7 week-experiment). The lowering of dietary DP/DE ratio increased myoblast determination protein 1a (myod1a) and decreased myostatin 1b (mstn1b) and cathepsin D (ctsd) muscle mRNA levels. The isoenergetic change in dietary NPE from F to C decreased myod1a and proliferative cell nuclear antigen (pcna) muscle mRNA levels. An interaction between DP/DE ratio and NPE source was observed in muscle transcript levels of myogenic factor 6 (mrf4/myf6), fast myosin heavy chain (fmhc) and fast myosin light chain 2 (fmlc2). White muscle total cross-sectional area decreased at low dietary DP/DE ratio and also when NPE source changed from F to C, linked i) to a decreased total number of white muscle fibres, indicating that low dietary DP/DE restricted muscle hyperplasia and that dietary carbohydrate were less efficiently used than fat to sustain muscle hyperplasia, and ii) to decreased percentage of large muscle fibres, indicating limited fibre hypertrophy. Not only the DP level or the DP/DE ratio, but also the isoenergetic change in dietary NPE source (fat vs carbohydrates) thus appears as a potent regulator of muscle hyperplasia and hypertrophy.

Transient up- and down-regulation of expression of myosin light chain 2 and myostatin mRNA mark the changes from stratified hyperplasia to muscle fiber hypertrophy in larvae of gilthead sea bream (Sparus aurata L.).

Hyperplasia and hypertrophy are the two mechanisms by which muscle develops and grows. We study these two mechanisms, during the early development of white muscle in Sparus aurata, by means of histology and the expression of structural and regulatory genes. A clear stage of stratified hyperplasia was identified early in the development of gilthead sea bream but ceased by 35 dph when hypertrophy took over. Mosaic recruitment of new white fibers began as soon as 60 dph. The genes mlc2a and mlc2b were expressed at various levels during the main phases of hyperplasia and hypertrophy. The genes myog and mlc2a were significantly up-regulated during the intensive stratified formation of new fibers and their expression was significantly correlated. Expression of mstn1 and igf1 increased at 35 dph, appeared to regulate the hyperplasia-to-hypertrophy transition, and may have stimulated the expression of mlc2a, mlc2b and col1a1 at the onset of mosaic hyperplasia. The up-regulation of mstn1 at transitional phases in muscle development indicates a dual regulatory role of myostatin in fish larval muscle growth.

Early decrease in dietary protein:energy ratio by fat addition and ontogenetic changes in muscle growth mechanisms of rainbow trout: short- and long-term effects.

As the understanding of the nutritional regulation of muscle growth mechanisms in fish is fragmentary, the present study aimed to (1) characterise ontogenetic changes in muscle growth-related genes in parallel to changes in muscle cellularity; (2) determine whether an early decrease in dietary protein:energy ratio by fat addition affects the muscle growth mechanisms of rainbow trout (Oncorhynchus mykiss) alevins; and (3) determine whether this early feeding of a high-fat (HF) diet to alevins had a long-term effect on muscle growth processes in juveniles fed a commercial diet. Developmental regulation of hyperplasia and hypertrophy was evidenced at the molecular (expression of myogenic regulatory factors, proliferating cell nuclear antigen and myosin heavy chains (MHC)) and cellular (number and diameter of white muscle fibres) levels. An early decrease in dietary protein:energy ratio by fat addition stimulated the body growth of alevins but led to a fatty phenotype, with accumulation of lipids in the anterior part, and less caudal muscle when compared at similar body weights, due to a decrease in both the white muscle hyperplasia and maximum hypertrophy of white muscle fibres. These HF diet-induced cellular changes were preceded by a very rapid down-regulation of the expression of fast-MHC. The present study also demonstrated that early dietary composition had a long-term effect on the subsequent muscle growth processes of juveniles fed a commercial diet for 3 months. When compared at similar body weights, initially HF diet-fed juveniles indeed had a lower mean diameter of white muscle fibres, a smaller number of large white muscle fibres, and lower expression levels of MyoD1 and myogenin. These findings demonstrated the strong effect of early feed composition on the muscle growth mechanisms of trout alevins and juveniles.

Nutrient deposition partitioning and priorities between body compartments in two size classes of rainbow trout in response to feed restriction.

Adaptations in growth dynamics in fish, i.e. how fish prioritise tissue accretion between organs, remains poorly understood. In the present study, we investigated the effects of graded feed restriction levels on nutrient deposition in 1.3 g fingerlings and 70 g juveniles. At the whole-body level, highly restricted juveniles strove to maintain body protein while mobilising lipid reserves and compensating for mass loss by increasing water content. In contrast, fingerlings maintained body water and energy contents. Additionally, we investigated deposition patterns in four body compartments (red and white axial muscles, viscera and rest of the carcass) in juveniles and changes in the cellularity of the white and red muscles in fingerlings. We provide evidence of priorities in growth and nutrient deposition in body compartments in response to low feeding levels. In juveniles, feed intake (FI) primarily affected the white muscle, while the red muscle and the viscera appeared to be preserved. Specific proteins (45 and 173 kDa) were preferentially deposited in the white muscle, while others (22 and 32 kDa) were preferentially mobilised. In fingerlings' muscle anterior to the anus, the cross-sectional surface areas increased with increasing FI in a logarithmic fashion in the white muscle, and in linear fashion in the red muscle. The maximum diameter of white fibres decreased linearly with fish length, while that of red fibres remained stable. This suggests an adaptation mechanism by decreasing white muscle hyperplasia in favour of hypertrophy when feed is restricted. Overall, these results indicate some mechanisms by which fish cope with low food availability. Our findings also suggest different adaptation strategies employed by fish of different body weights.

Postprandial regulation of growth- and metabolism-related factors in zebrafish.

Zebrafish (Danio rerio) have been proposed as a possible model organism for nutritional physiology. However, this potential has not yet been realized and studies on the field remain scarce. In this work, we investigated in this species the effect of a single meal as well as that of an increase in the ratio of dietary carbohydrates/proteins on the postprandial expression of several hepatic and muscle metabolism-related genes and proteins. Fish were fed once either a commercial diet (experiment 1) or one of two experimental diets (experiment 2) containing different protein and carbohydrate levels after 72 h of starvation. Refeeding induced the postprandial expression of genes of glycolysis (GK, HK1) and lipogenesis (FAS, G6PDH, ACCa) and inhibited those of gluconeogenesis (cPEPCK) and beta-oxidation (CPT1b) in the viscera. In the muscle, refeeding increased transcript levels of myogenesis (Myf5, Myogenin), inhibited those of Ub-proteasomal proteolytic system (Atrogin1, Murf1a, Murf1b), and induced the activation of key signaling factors of protein synthesis (Akt, 4EBP1, S6K1, S6). However, diet composition had a low impact on the studied factors. Together, these results highlight some specificity of the zebrafish metabolism and demonstrate the interest and the limits of this species as a model organism for nutritional physiology studies.

Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

Early thermal history significantly affects the seasonal hyperplastic process occurring in the myotomal white muscle of Dicentrarchus labrax juveniles.

The effect of early (embryonic and larval) thermal history on subsequent (juvenile) white muscle hyperplasia was studied in a teleost fish, the European sea bass (Dicentrarchus labrax L.). D. labrax, incubated and reared at constant temperatures of 13 degrees C, 15 degrees C or 20 degrees C from the embryonic stage of half epiboly up to 18-19 mm in total length, were transferred to ambient seawater temperature and reared for the subsequent 14 months on commercial feed. The somatic growth of juveniles was linked to annual variations of ambient seawater temperature and inversely related to early rearing temperature, so that, after 14 months, the juveniles originally reared at low temperatures had compensated for the growth retardation experienced during early life. The white muscle growth process of juveniles was quantified after two periods of growth opportunity at ambient seawater temperature (100 and 400 days post-transfer) as well as, in order to follow total-length-dependent effects of early temperature and to discriminate total-length-independent effects of early temperature, on juveniles from the three batches sampled at six successive equivalent total lengths (31-33, 84-88, 141-145, 166-172, 196-206 and 211-220 mm). Our data demonstrate the existence of a seasonal recruitment of new white muscle fibres when seawater temperature increases and of a shrinkage of the largest white muscle fibres during the winter months. The seasonal recruitment of new white muscle fibres occurring in juveniles is linked to their early rearing temperature. Juveniles originating from low temperatures have a higher and longer capacity to recruit new white muscle fibres when seawater temperature increases, supporting their better somatic growth. This finding is discussed in relation to the early (embryonic and larval) myogenic processes of the three populations and is related to their sex ratio.