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The growth hormone (GH)/insulin-like growth factors (IGFs) endocrine axis is the main growth-regulator system in vertebrates. Some authors have demonstrated the positive effects on growth of a sustained-release formulation of a recombinant bovine GH (rBGH) in different fish species. The aim of this work was to characterize the effects of a single injection of rBGH in fingerlings of gilthead sea bream on growth, GH-IGF axis, and both myogenic and osteogenic processes. Thus, body weight and specific growth rate were significantly increased in rBGH-treated fish respect to control fish at 6 weeks post-injection, whereas the hepatosomatic index was decreased and the condition factor and mesenteric fat index were unchanged, altogether indicating enhanced somatic growth. Moreover, rBGH injection increased the plasma IGF-I levels in parallel with a rise of hepatic mRNA from total IGF-I, IGF-Ic and IGF-II, the binding proteins IGFBP-1a and IGFBP-2b, and also the receptors IGF-IRb, GHR-I and GHR-II. In skeletal muscle, the expression of IGF-Ib and GHR-I was significantly increased but that of IGF-IRb was reduced; the mRNA levels of myogenic regulatory factors, proliferation and differentiation markers (PCNA and MHC, respectively), or that of different molecules of the signaling pathway (TOR/AKT) were unaltered. Besides, the growth inhibitor myostatin (MSTN1 and MSTN2) and the hypertrophic marker (MLC2B) expression resulted significantly enhanced, suggesting altogether that the muscle is in a non-proliferative stage of development. Contrarily in bone, although the expression of most molecules of the GH/IGF axis was decreased, the mRNA levels of several osteogenic genes were increased. The histology analysis showed a GH induced lipolytic effect with a clear decrease in the subcutaneous fat layer. Overall, these results reveal that a better growth potential can be achieved on this species and supports the possibility to improve growth and quality through the optimization of its culture conditions., E.J.V. and E.L. are supported by a predoctoral fellowship from the “Ministerio de Ciencia e Innovación” (MICINN), and A.M. by the University of Barcelona (APIF-2012). This study was supported by the project AGL2012-39768 (MICINN) to J.G., and the “Xarxa de Refèrencia d’R+D+I en Aqüicultura” and the 2014SGR-01371 from the “Generalitat de Catalunya”.

Research on the regulation of fish muscle physiology and growth was addressed originally by classical in vivo approaches; however, systemic interactions resulted in many questions that could be better considered through in vitro myocyte studies. The first paper published by our group in this field was with Tom Moon on brown trout cardiomyocytes, where the insulin and IGF-I receptors were characterized and the down-regulatory effects of an excess of peptides demonstrated. We followed the research on cultured skeletal muscle cells through the collaboration with INRA focused on the characterization of IGF-I receptors and its signaling pathways through in vitro development. Later on, we showed the important metabolic role of IGFs, although these studies were only the first stage of a prolific area of work that has offered a useful tool to advance in our knowledge of the endocrine and nutritional regulation of fish growth and metabolism. Obviously, the findings obtained in vitro serve the purpose to propose the scenario that will need confirmation in vivo, but this technique has made possible many different, easy, fast and better controlled studies. In this review, we have summarized the main advances that the use of cultured muscle cells has permitted, focusing mainly in the role of IGFs regulating fish metabolism and growth. Although many articles have already appeared using this model system in salmonids, gilthead sea bream or zebrafish, it is reasonable to expect new studies with cultured cells using innovative approaches that will help to understand fish physiology and its regulation.

The enhancement of the endocrine growth hormone (GH)/insulin-like growth factor I (IGF-I) system by the treatment with a sustained release formulation of a recombinant bovine GH (rBGH), is a good strategy to investigate growth optimization in aquaculture fish species. To further deepen into the knowledge of rBGH effects in fish and to estimate the growth potential of juveniles of gilthead sea bream, the present work evaluated rBGH injection on growth, GH/IGF-I axis and lipid metabolism modulation, and explored the conservation of GH effects provoked by the in vivo treatment using in vitro models of different tissues. The rBGH treatment increased body weight and specific growth rate (SGR) in juveniles and potentiated hyperplastic muscle growth while reducing circulating triglyceride levels. Moreover, the results demonstrated that the in vivo treatment enhanced also lipolysis in both isolated hepatocytes and adipocytes, as well as in day 4 cultured myocytes. Furthermore, these cultured myocytes extracted from rBGH-injected fish presented higher gene expression of GH/IGF-I axis-related molecules and myogenic regulatory factors, as well as stimulated myogenesis (i.e. increased protein expression of a proliferation and a differentiation marker) compared to Control fish-derived cells. These data, suggested that cells in vitro can retain some of the pathways activated by in vivo treatments in fish, what can be considered an interesting line of applied research. Overall, the results showed that rBGH stimulates somatic growth, including specifically muscle hyperplasia, as well as lipolytic activity in gilthead sea bream juveniles., E.J.V., E.L. and M.P.-A. were supported by predoctoral fellowships from the “Ministerio de Economía y Competitividad” (MINECO). This study was supported by the projects from the MINECO AGL2012-39768 and AGL2015-70679-R to J.G., and AGL2014-57974-R to I.N. and E.C., and the “Xarxa de Refèrencia d'R+D+I en Aqüicultura” and the 2014SGR-01371 from the “Generalitat de Catalunya”.

Swimming activity primarily accelerates growth in fish by increasing protein synthesis and energy efficiency. The role of muscle in this process is remarkable and especially important in teleosts, where muscle represents a high percentage of body weight and because many fish species present continuous growth. The aim of this work was to characterize the effects of 5 wk of moderate and sustained swimming in gene and protein expression of myogenic regulatory factors, proliferation markers, and proteolytic molecules in two muscle regions (anterior and caudal) of gilthead sea bream fingerlings. Western blot results showed an increase in the proliferation marker proliferating cell nuclear antigen (PCNA), proteolytic system members calpain 1 and cathepsin D, as well as vascular endothelial growth factor protein expression. Moreover, quantitative real-time PCR data showed that exercise increased the gene expression of proteases (calpains, cathepsins, and members of the ubiquitin-proteasome system in the anterior muscle region) and the gene expression of the proliferation marker PCNA and the myogenic factor MyoD in the caudal area compared with control fish. Overall, these data suggest a differential response of the two muscle regions during swimming adaptation, with tissue remodeling and new vessel formation occurring in the anterior muscle and enhanced cell proliferation and differentiation occurring in the caudal area. In summary, the present study contributes to improving the knowledge of the role of proteolytic molecules and other myogenic factors in the adaptation of muscle to moderate sustained swimming in gilthead sea bream.

In this data article we describe the coding sequence of two IGF-IR paralogues (IGF-IRa and IGF-IRb) obtained from gilthead sea bream embryos. The putative protein architecture (domains and other important motifs) was determined and, amino acid sequences alignment and phylogenetic analysis of both receptors together with IGF-IR orthologues from different vertebrates was performed. Additionally, a semi-quantitative conventional PCR was done to analyze the mRNA expression of both receptors in different tissues of gilthead sea bream. These data will assist in further physiological studies in this species. In this sense, the expression of both receptors during ontogeny in muscle as well as the differential effects of IGF-I and IGF-II on their regulation during in vitro myogenesis has been recently studied (doi: 10.1016/j.ygcen.2015.11.011; [1]). Keywords: IGF-I receptors, Gilthead sea bream

The insulin-like growth factors (IGFs) have a fundamental role in a vast range of functions acting through a tyrosine-kinase receptor (IGF-IR). IGFs in muscle can affect the expression of components of the local IGF system, myogenic regulatory factors (MRFs), proliferating (proliferating cell nuclear antigen, PCNA) or differentiating molecules (myosin heavy chain, MHC) and, lead to the activation of different signaling pathways. The response of all these genes to IGFs incubation at two different times in day 4 cultured myocytes of gilthead sea bream was analyzed. Both IGFs increased the expression of IGF-I and IGFBP-5, but showed different effects on the receptors, with IGF-I suppressing the expression of both isoforms (IGF-IRa and IGF-IRb) and IGF-II up-regulating only IGF-IRb. Moreover, the protein levels of PCNA and target of rapamycin (TOR) increased after IGF-II incubation, although a decline in Myf5 and a rise in MHC gene expression was caused by IGF-I. Taken together, these results provide evidence for the importance of IGFs on controlling muscle development and growth in gilthead sea bream and suggest that each IGF may be preferentially acting through a specific IGF-IR. Moreover, the data support the hypothesis that IGF-II has a more important role during proliferation, whereas IGF-I seems to be relevant for the differentiation phase of myogenesis.

Proteolytic systems exert an important role in vertebrate muscle controlling protein turnover, recycling of amino acids (AA) or its use for energy production, as well as other functions like myogenesis. In fish, proteolytic systems are crucial for the relatively high muscle somatic index they possess, and because protein is the most important dietary component. Thus in this study, the molecular profile of proteolytic markers (calpains, cathepsins and ubiquitin-proteasome system (UbP) members) were analyzed during gilthead sea bream (Sparus aurata) myogenesis in vitro and under different AA treatments. The gene expression of calpains (capn1, capn3 and capns1b) decreased progressively during myogenesis together with the proteasome member n3; whereas capn2, capns1a, capns1b and ubiquitin (ub) remained stable. Contrarily, the cathepsin D (ctsd) paralogs and E3 ubiquitin ligases mafbx and murf1, showed a significant peak in gene expression at day 8 of culture that slightly decreased afterwards. Moreover, the protein expression analyzed for selected molecules presented in general the same profile of the mRNA levels, which was confirmed by correlation analysis. These data suggest that calpains seem to be more important during proliferation, while cathepsins and the UbP system appear to be required for myogenic differentiation. Concerning the transcriptional regulation by AA, the recovery of their levels after a short starvation period did not show effects on cathepsins expression, whereas it down-regulated the expression of capn3, capns1b, mafbx, murf1 and up-regulated n3. With regards to AA deficiencies, the major changes occurred at day 2, when leucine limitation suppressed ctsb and ctsl expression. Besides at the same time, both leucine and lysine deficiencies increased the expression of mafbx and murf1 and decreased that of n3. Overall, the opposite nutritional regulation observed, especially for the UbP members, points out an efficient and complementary role of these factors that could be useful in gilthead sea bream diets optimization.

Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of an adequate level of Lysine in fishmeal diet formulation for optimum growth.