Jean-Charles Gabillard

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.

An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss).

In mammals, feeding promotes protein accretion in skeletal muscle through a stimulation of the insulin- and amino acid- sensitive mammalian target of rapamycin (mTOR) signaling pathway, leading to the induction of mRNA translation. The purpose of the present study was to characterize both in vivo and in vitro the activation of several major kinases involved in the mTOR pathway in the muscle of the carnivorous rainbow trout. Our results showed that meal feeding enhanced the phosphorylation of the target of rapamycin (TOR), PKB, p70 S6 kinase, and eIF4E-binding protein-1, suggesting that the mechanisms involved in the regulation of mRNA translation are well conserved between lower and higher vertebrates. Our in vitro studies on primary culture of trout muscle cells indicate that insulin and amino acids regulate TOR signaling and thus may be involved in meal feeding effect in this species as in mammals. In conclusion, we report here for the first time in a fish species, the existence and the nutritional regulation of several major kinases involved in the TOR pathway, opening a new area of research on the molecular bases of amino acid utilization in teleosts.

Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss).

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P<0.05) to the rearing temperature without any relationship with nutritional status, suggesting a direct effect of temperature on its turnover. After 2 weeks of treatment, the levels of IGF1 mRNA in muscle at 8 degrees C were 2-fold higher (P<0.05) than at 16 degrees C in both ad libitum and restricted feed fish, whereas after 6 weeks, this difference was no longer observed. In both experiments, the levels of plasma IGF2 were 10-fold higher than the levels of plasma IGF1 (mean 105+/-3.0 versus 13.5+/-0.6 ng/ml), and plasma levels were correlated with their respective mRNA liver concentrations (r2=0.14 and 0.25, respectively; P<0.01). In the ad libitum feeding experiment, plasma and mRNA levels of IGF1 were related to the rearing temperature (P<0.05), while for IGF2 no effect was seen. In contrast, in the restricted feeding experiment, plasma and IGF2 mRNA levels were inversely proportional to the rearing temperature (P<0.0001) while plasma IGF1 was unaltered. Levels of plasma IGF1 were related to the growth rate in both experiments, while levels of plasma IGF2 appeared to be associated with the nutritional status of the fish. Our results suggest that the autocrine/paracrine expression of IGF1 and IGF2 in muscle is not a key regulator of the growth promoting effect of temperature. Conversely, temperature seems to promote growth through IGF1 secretion by the liver following GH stimulation, and impairment of nutritional status would prevent the IGF1 stimulation by temperature. In addition, the growth-promoting effect of temperature did not affect plasma IGF2, which appeared to be more related to the metabolic status of the fish.

Myostatin and the skeletal muscle atrophy and hypertrophy signaling pathways

Myostatin, a member of the transforming growth factor-β superfamily, is a potent negative regulator of skeletal muscle growth and is conserved in many species, from rodents to humans. Myostatin inactivation can induce skeletal muscle hypertrophy, while its overexpression or systemic administration causes muscle atrophy. As it represents a potential target for stimulating muscle growth and/or preventing muscle wasting, myostatin regulation and functions in the control of muscle mass have been extensively studied. A wealth of data strongly suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression. Moreover, myostatin plays a central role in integrating/mediating anabolic and catabolic responses. Myostatin negatively regulates the activity of the Akt pathway, which promotes protein synthesis, and increases the activity of the ubiquitin–proteasome system to induce atrophy. Several new studies have brought new information on how myostatin may affect both ribosomal biogenesis and translation efficiency of specific mRNA subclasses. In addition, although myostatin has been identified as a modulator of the major catabolic pathways, including the ubiquitin–proteasome and the autophagy–lysosome systems, the underlying mechanisms are only partially understood. The goal of this review is to highlight outstanding questions about myostatin-mediated regulation of the anabolic and catabolic signaling pathways in skeletal muscle. Particular emphasis has been placed on (1) the cross-regulation between myostatin, the growth-promoting pathways and the proteolytic systems; (2) how myostatin inhibition leads to muscle hypertrophy; and (3) the regulation of translation by myostatin.

Environmental temperature increases plasma GH levels independently of nutritional status in rainbow trout (Oncorhynchus mykiss)

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P<0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P<0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P<0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P<0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P<0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.

Leucine limitation regulates myf5 and myoD expression and inhibits myoblast differentiation

Satellite cells are the major pool of muscle stem cells after birth; they represent an important component required to maintain muscle mass and functionality during life. The molecular mechanisms involved in myogenic differentiation are relatively well-known. However, the role of extracellular stimulus in the control of differentiation remains largely unresolved. Notably little is known about the impact of nutrients on this process. Here we have studied the role of leucine, an essential amino acid, in the control of myogenic differentiation. Leucine is a well-known regulator of muscle protein synthesis. It acts not only as a substrate for translation but also as a regulator of gene expression and signaling pathways such as those involving mTOR and GCN2. In this study we demonstrated that the lack of leucine abolishes the differentiation of both C2C12 myoblasts and primary satellite cells. This effect is associated with a modification of the pattern of expression of the myogenic regulatory factors (MRF) myf5 and myoD. We report an up-regulation of myf5 mRNA and a decrease of myoD protein level during leucine starvation. This study demonstrates the importance of a nutrient, leucine, in the control of the myogenic differentiation program.

Myomaker mediates fusion of fast myocytes in zebrafish embryos.

Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

Myostatin inhibits proliferation but not differentiation of trout myoblasts

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100 nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity. In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN.

Revisiting the paradigm of myostatin in vertebrates: insights from fishes.

In the last decade, myostatin (MSTN), a member of the TGFβ superfamily, has emerged as a strong inhibitor of muscle growth in mammals. In fish many studies reveal a strong conservation of mstn gene organization, sequence, and protein structures. Because of ancient genome duplication, teleostei may have retained two copies of mstn genes and even up to four copies in salmonids due to additional genome duplication event. In sharp contrast to mammals, the different fish mstn orthologs are widely expressed with a tissue-specific expression pattern. Quantification of mstn mRNA in fish under different physiological conditions, demonstrates that endogenous expression of mstn paralogs is rarely related to fish muscle growth rate. In addition, attempts to inhibit MSTN activity did not consistently enhance muscle growth as in mammals. In vitro, MSTN stimulates myotube atrophy and inhibits proliferation but not differentiation of myogenic cells as in mammals. In conclusion, given the strong mstn expression non-muscle tissues of fish, we propose a new hypothesis stating that fish MSTN functions as a general inhibitors of cell proliferation and cell growth to control tissue mass but is not specialized into a strong muscle regulator.

Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts

Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing.