The tolerance of fish to fasting offers a model to study the regulatory mechanisms and changes produced when feeding is restored. Gilthead sea bream juveniles were exposed to a 21-days fasting period followed by 2 h to 7-days refeeding. Fasting provoked a decrease in body weight, somatic indexes, and muscle gene expression of members of the Gh/Igf system, signaling molecules (akt, tor and downstream effectors), proliferation marker pcna, myogenic regulatory factors, myostatin, and proteolytic molecules such as cathepsins or calpains, while most ubiquitin-proteasome system members increased or remained stable. In bone, downregulated expression of Gh/Igf members and osteogenic factors was observed, whereas expression of the osteoclastic marker ctsk was increased. Refeeding recovered the expression of Gh/Igf system, myogenic and osteogenic factors in a sequence similar to that of development. Akt and Tor phosphorylation raised at 2 and 5 h post-refeeding, much faster than its gene expression increased, which occurred at day 7. The expression in bone and muscle of the inhibitor myostatin (mstn2) showed an inverse profile suggesting an inter-organ coordination that needs to be further explored in fish. Overall, this study provides new information on the molecules involved in the musculoskeletal system remodeling during the early stages of refeeding in fish.

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.

Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.

The combination of physical exercise and a balanced diet presents substantial health benefits and could improve fish production. However, the redox balance can be affected by training regimen, dietary macronutrient ratio and their interaction. In this study, we conjointly evaluated the effects of physical activity (by voluntary swimming (VS) or sustained swimming as exercise (Ex)) and diet composition (by high-protein (HP) or high-lipid (HE) commercial diets) after 6 weeks on oxidative stress status in liver, white muscle and red muscle of gilthead sea bream juveniles. The HE diet increased the biochemical redox markers’ thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPP) and reduced thiols (-SH) in the different tissues. Exercise increased AOPP and -SH levels in liver but reduced TBARS levels in white muscle. Regarding the expression of oxidative stress, chaperones and apoptosis-related genes, the VSHE group showed the highest values and the VSHP the lowest, whereas the application of sustained swimming partially equalized those differences. Diet composition modulated the enzyme activity, prioritizing the superoxide dismutase and catalase in the HE-fed groups and the glutathione-related enzymes in the HP groups. Exercise also altered enzyme activity, but in a tissue-dependent manner. Overall, the redox balance in gilthead sea bream juveniles can be affected by diet composition and sustained swimming. However, the response will partly depend on the interaction between these factors and the tissue studied. Therefore, the combination of an adequate diet and sustained exercise could be used in fish production to improve the physiological redox status.

Fish are rich in n-3 long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Due to the increasing use of vegetable oils (VO), their proportion in diets has lowered, affecting lipid metabolism and fillet composition. Rainbow trout cultured preadipocytes were treated with representative FA found in fish oils (EPA and DHA) or VO (linoleic, LA and alpha-linolenic, ALA acids), while EPA and LA were also orally administered, to evaluate their effects on adipogenesis and lipid metabolism. In vitro, all FA increased lipid internalization, with ALA producing the highest effect, together with upregulating the FA transporter fatp1. In vivo, EPA or LA increased peroxisome proliferator-activated receptors ppara and pparb transcripts abundance in adipose tissue, suggesting elevated β-oxidation, contrary to the results obtained in liver. Furthermore, the increased expression of FA synthase (fas) and the FA translocase/cluster of differentiation (cd36) in adipose tissue indicated an enhanced uptake of lipids and lipogenesis de novo, whereas stable or low hepatic expression of genes involved in lipid transport and turnover was found. Thus, fish showed a similar tissue metabolic response to the short-term availability of EPA or LA in vivo, while in vitro VO-derived FA demonstrated greater potential inducing fat accumulation.