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.

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.

The GH/IGF system is a complex regulation network strongly dependent on nutrient availability. While the effect of starvation on the GH/IGF system has been extensively studied, the time course of events leading to the restoration of GH/IGF system activity after starvation is largely unknown. We, therefore, measured the plasma levels of GH, IGF-I and IGF-II and the expression of the GH/IGF system in liver and muscle. Starvation increased the plasma GH level and 1 day of refeeding completely restored it (1$10G0$27 vs 1$12G0$28 ng/ml). Thereafter, plasma GH continued to decrease until day 7 and returned to control values from day 15. Starvation decreased plasma IGF-I and IGF-II and refeeding raised plasma IGF-I only from day 4. In contrast, the plasma IGF-II level doubled after 1 day's refeeding (26$5G1$9 vs 44$0G3$4 ng/ml; P!0$01). Starved fish exhibited higher GH receptor (GHR)1 mRNA abundance in liver and muscle than in controls, whereas GHR2 mRNA abundance was increased only in muscle. In liver, 1 day of refeeding, decreased GHR1 (twofold), but increased GHR2 mRNA abundance (twofold). Thereafter, a progressive return to normal values was observed. Liver IGFBP-4 mRNA abundance was lowered in starved fish followed by a progressive restoration during refeeding. Starvation had no effect on liver IGFBP-2 and IGFBP-6 mRNA abundance, whereas refeeding provoked a peak of IGFBP-2 and IGFBP-6 expression at day 7. In muscle, starvation led to a decrease of the IGFBP-2 mRNA level, which was restored only from day 7. IGFBP-4 mRNA abundance in starved fish was lower than in the controls and refeeding led to a transient upregulation (sevenfold) of IGFBP-4 gene at day 1. IGF-I, IGFBP-5, and IGFBP-related protein 1 (rP1) expression profiles were similar, showing a decrease of expression after starvation, a first peak of expression at day 2, a second peak at day 7, and a return to normal value from day 15. Moreover, IGF-I, IGFBP-5, and IGFBP-rP1 mRNA abundance were positively correlated (rZ0$6-0$8; P!0$0001). In conclusion, plasma IGF-I was restored later than plasma GH level, which suggests that plasma IGF-I levels cannot account for plasma GH changes. The coordinated regulation of IGF-I, IGFBP-5, and IGFBP-rP1 expression would be a signature for the resumption of myogenic activity.

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Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named "reserve" cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.

In the present study we report the full coding sequence of rainbow trout IGF-binding protein-1 (IGFBP1), -2, -3, -5, and -6 and IGFBP-related protein-1 (IGFBP-rP1) mRNAs as well as the partial coding sequence of IGFBP-4 mRNA. To our knowledge, this is the first report of IGFBP4, IGFBP6, and IGFBP-rP1 in a nonmammalian species. The tissue distribution of all mRNAs was studied, and the ovarian expression profiles of IGFBP2 to -6 and IGFBP-rP1 between late vitellogenesis and oocyte maturation were characterized. In addition, in vitro hormonal regulation by the maturation-inducing steroid 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP), gonadotropin, and estradiol were studied. We observed that besides IGFBP1, which was only found in liver, IGFBP2 to -6 and IGFPB-rP1 were expressed in the preovulatory ovary. IGFBP3 was also detected in liver, trunk, kidney, skin, and gills, whereas IGFBP2 to -6 and IGFBP-rP1 exhibited a wider tissue distribution. In the preovulatory ovary, IGFBP3 was strongly down-regulated during the postvitellogenesis period, whereas IGFBP5 exhibited a limited up-regulation. In addition, IGFBP6 and IGFBP-rP1 were up-regulated during oocyte maturation. Hormonal regulation data indicated that all ovarian IGFBPs and IGFBP-rP1 transcripts are regulated under gonadotropic stimulation at a concentration that induced 100% oocyte maturation. In addition, IGFBP2 to -5 transcripts are regulated by 17,20betaP and estradiol. Together, our observations strongly suggest that during final oocyte maturation, a down-regulation of IGFBP3, -4, and -5 occurs in the oocyte in response to gonadotropic and 17,20betaP (IGFBP3 and -5) stimulation, whereas an up-regulation of IGFBP2 and -6 occurs in follicular layers or extrafollicular tissue in response to gonadotropic stimulation.