Myomaker and Myomixer Characterization in Gilthead Sea Bream under Different Myogenesis Conditions.

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish.

Recombinant Bovine Growth Hormone-Induced Metabolic Remodelling Enhances Growth of Gilthead Sea-Bream (Sparus aurata): Insights from Stable Isotopes Composition and Proteomics.

Growth hormone and insulin-like growth factors (GH/IGF axis) regulate somatic growth in mammals and fish, although their action on metabolism is not fully understood in the latter. An intraperitoneal injection of extended-release recombinant bovine growth hormone (rbGH, Posilac®) was used in gilthead sea bream fingerlings and juveniles to analyse the metabolic response of liver and red and white muscles by enzymatic, isotopic and proteomic analyses. GH-induced lipolysis and glycogenolysis were reflected in liver composition, and metabolic and redox enzymes reported higher lipid use and lower protein oxidation. In white and red muscle reserves, rBGH increased glycogen while reducing lipid. The isotopic analysis of muscles showed a decrease in the recycling of proteins and a greater recycling of lipids and glycogen in the rBGH groups, which favoured a protein sparing effect. The protein synthesis capacity (RNA/protein) of white muscle increased, while cytochrome-c-oxidase (COX) protein expression decreased in rBGH group. Proteomic analysis of white muscle revealed only downregulation of 8 proteins, related to carbohydrate metabolic processes. The global results corroborated that GH acted by saving dietary proteins for muscle growth mainly by promoting the use of lipids as energy in the muscles of the gilthead sea bream. There was a fuel switch from carbohydrates to lipids with compensatory changes in antioxidant pathways that overall resulted in enhanced somatic growth.

Short-Term Responses to Fatty Acids on Lipid Metabolism and Adipogenesis in Rainbow Trout (Oncorhynchus mykiss).

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 ß-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.

Genistein Induces Adipogenic and Autophagic Effects in Rainbow Trout (Oncorhynchus mykiss) Adipose Tissue: In Vitro and In Vivo Models.

Soybeans are one of the most used alternative dietary ingredients in aquafeeds. However, they contain phytoestrogens like genistein (GE), which can have an impact on fish metabolism and health. This study aimed to investigate the in vitro and in vivo effects of GE on lipid metabolism, apoptosis, and autophagy in rainbow trout (Oncorhynchus mykiss). Primary cultured preadipocytes were incubated with GE at different concentrations, 10 or 100 µM, and 1 µM 17ß-estradiol (E2). Furthermore, juveniles received an intraperitoneal injection of GE at 5 or 50 µg/g body weight, or E2 at 5 µg/g. In vitro, GE 100 µM increased lipid accumulation and reduced cell viability, apparently involving an autophagic process, indicated by the higher LC3-II protein levels, and higher lc3b and cathepsin d transcript levels achieved after GE 10 µM. In vivo, GE 50 µg/g upregulated the gene expression of fatty acid synthase (fas) and glyceraldehyde-3-phosphate dehydrogenase in adipose tissue, suggesting enhanced lipogenesis, whereas it increased hormone-sensitive lipase in liver, indicating a lipolytic response. Besides, autophagy-related genes increased in the tissues analyzed mainly after GE 50 µg/g treatment. Overall, these findings suggest that an elevated GE administration could lead to impaired adipocyte viability and lipid metabolism dysregulation in rainbow trout.

Gene expression analyses in malformed skeletal structures of gilthead sea bream (Sparus aurata).

The incidence of skeletal anomalies in reared fish has been translated for years in important economic losses for the aquaculture industry. In the present study, we have analysed the gene expression of extracellular matrix components and transcription factors involved in bone development in gilthead sea bream presenting different skeletal anomalies: lordosis (LD), lordosis-scoliosis-kyphosis (LSK) or opercular, dental or jaw malformations in comparison with control (CT) specimens. Results showed a possible link between the presence of LD and LSK and the significant downregulation of genes involved in osteoblasts' maturation and matrix mineralization (collagen type 1-alpha, osteopontin, osteocalcin, matrix Gla protein and tissue non-specific alkaline phosphatase), as well as in bone resorption (cathepsin K and matrix metalloproteinase 9) compared to CT animals. Contrarily, the key osteogenic transcription factor runx2 was upregulated in the malformed vertebra suggesting impaired determination of mesenchymal stem cells towards the osteoblastic lineage. Despite the gene expression patterns of the other malformed structures were not affected in comparison with CT fish, the results of the present study may contribute in the long term to identify potential candidate gene profiles associated with column deformities that may help reducing the incidence of appearance of skeletal anomalies in this important aquaculture species.

Effects of ß2-adrenoceptor agonists on gilthead sea bream (Sparus aurata) cultured muscle cells.

ß2-adrenoceptors are a subtype of G-protein coupled receptors whose activation leads to increased protein synthesis and decreased degradation in mammalian skeletal muscle, causing hypertrophy. In this study, we compared the effects of the classical ß2-agonist noradrenaline (NA) with two representatives of a new generation of agonists (formoterol, FOR and salmeterol, SALM) on growth and metabolism of primary cultured muscle cells of gilthead sea bream. Activation of signaling pathways, cell development and expression of relevant genes were analyzed in day 4 myocytes. The three agonists increased either cAMP levels or PKA phosphorylation, plus TOR phosphorylation, and the proportion of proliferating cell nuclear antigen (PCNA)-positive cells, in parallel with pcna mRNA levels. Thus, demonstrating that these cells are ß2-agonists-responsive, and supporting enhanced cell proliferation. The expression of the myogenic factor myf5 was significantly down-regulated, suggesting that the cells were already destined to the muscular linage; while insulin-like growth factors (igf-1 and igf-2) transcript levels were up-regulated, proposing an additional anabolic effect through their local production. Furthermore, SALM treatment up-regulated expression of the lipases (hsl and lipa) and the ß-oxidation marker cpt1a, and all three agonists increased mitochondrial dehydrogenase hadh mRNA levels. These data correspond with a situation of enhanced lipolytic and ß-oxidation capacity, a fact supported by the higher glycerol released into the media induced by the agonists. Overall, these results suggest a hyperplastic growth condition and a favorable protein/fat ratio profile upon these treatments; consequently, ß2-agonists (especially SALM) may be considered good candidates to optimize the growth in this aquaculture species.

A long-term growth hormone treatment stimulates growth and lipolysis in gilthead sea bream juveniles.

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.

Recombinant bovine growth hormone (rBGH) enhances somatic growth by regulating the GH-IGF axis in fingerlings of gilthead sea bream (Sparus aurata).

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 6weeks 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.

Gene expression profile during proliferation and differentiation of rainbow trout adipocyte precursor cells.

BACKGROUND: Excessive accumulation of adipose tissue in cultured fish is an outstanding problem in aquaculture. To understand the development of adiposity, it is crucial to identify the genes which expression is associated with adipogenic differentiation. Therefore, the transcriptomic profile at different time points (days 3, 8, 15 and 21) along primary culture development of rainbow trout preadipocytes has been investigated using an Agilent trout oligo microarray. RESULTS: Our analysis identified 4026 genes differentially expressed (fold-change >3) that were divided into two major clusters corresponding to the main phases observed during the preadipocyte culture: proliferation and differentiation. Proliferation cluster comprised 1028 genes up-regulated from days 3 to 8 of culture meanwhile the differentiation cluster was characterized by 2140 induced genes from days 15 to 21. Proliferation was characterized by enrichment in genes involved in basic cellular and metabolic processes (transcription, ribosome biogenesis, translation and protein folding), cellular remodelling and autophagy. In addition, the implication of the eicosanoid signalling pathway was highlighted during this phase. On the other hand, the terminal differentiation phase was enriched with genes involved in energy production, lipid and carbohydrate metabolism. Moreover, during this phase an enrichment in genes involved in the formation of the lipid droplets was evidenced as well as the activation of the thyroid-receptor/retinoic X receptor (TR/RXR) and the peroxisome proliferator activated receptors (PPARs) signalling pathways. The whole adipogenic process was driven by a coordinated activation of transcription factors and epigenetic modulators. CONCLUSIONS: Overall, our study demonstrates the coordinated expression of functionally related genes during proliferation and differentiation of rainbow trout adipocyte cells. Furthermore, the information generated will allow future investigations of specific genes involved in particular stages of fish adipogenesis.

Moderate and sustained exercise modulates muscle proteolytic and myogenic markers in gilthead sea bream (Sparus aurata).

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.

Effects of sustained exercise on GH-IGFs axis in gilthead sea bream (Sparus aurata).

The endocrine system regulates growth mainly through the growth hormone (GH)/insulin-like growth factors (IGFs) axis and, although exercise promotes growth, little is known about its modulation of these factors. The aim of this work was to characterize the effects of 5 wk of moderate sustained swimming on the GH-IGFs axis in gilthead sea bream fingerlings. Plasma IGF-I/GH ratio and tissue gene expression of total IGF-I and three splice variants, IGF-II, three IGF binding proteins, two GH receptors, two IGF-I receptors, and the downstream molecules were analyzed. Fish under exercise (EX) grew more than control fish (CT), had a higher plasma IGF-I/GH ratio, and showed increased hepatic IGF-I expression (mainly IGF-Ia). Total IGF-I expression levels were similar in the anterior and caudal muscles; however, IGF-Ic expression increased with exercise, suggesting that this splice variant may be the most sensitive to mechanical action. Moreover, IGFBP-5b and IGF-II increased in the anterior and caudal muscles, respectively, supporting enhanced muscle growth. Furthermore, in EX fish, hepatic IGF-IRb was reduced together with both GHRs; GHR-II was also reduced in anterior muscle, while GHR-I showed higher expression in the two muscle regions, indicating tissue-dependent differences and responses to exercise. Exercise also increased gene and protein expression of target of rapamycin (TOR), suggesting enhanced muscle protein synthesis. Altogether, these data demonstrate that moderate sustained activity may be used to increase the plasma IGF-I/GH ratio and to potentiate growth in farmed gilthead sea bream, modulating the gene expression of different members of the GH-IGFs axis (i.e., IGF-Ic, IGF-II, IGFBP-5b, GHR-I, and TOR).

IGF-I and IGF-II effects on local IGF system and signaling pathways in gilthead sea bream (Sparus aurata) cultured myocytes.

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.

Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout.

As leptin has a key role on appetite, knowledge about leptin regulation is important in order to understand the control of energy balance. We aimed to explore the modulatory effects of adiposity on plasma leptin levels in vivo and the role of potential regulators on leptin expression and secretion in rainbow trout adipocytes in vitro. Fish were fed a regular diet twice daily ad libitum or a high-energy diet once daily at two ration levels; satiation (SA group) or restricted (RE group) to 25% of satiation, for 8weeks. RE fish had significantly reduced growth (p<0.001) and adipose tissue weight (p<0.001), and higher plasma leptin levels (p=0.022) compared with SA fish. Moreover, plasma leptin levels negatively correlated with mesenteric fat index (p=0.009). Adipocytes isolated from the different fish were treated with insulin, ghrelin, leucine, eicosapentaenoic acid or left untreated (control). In adipocytes from fish fed regular diet, insulin and ghrelin increased leptin secretion dose-dependently (p=0.002; p=0.033, respectively). Leptin secretion in control adipocytes was significantly higher in RE than in SA fish (p=0.022) in agreement with the in vivo findings, indicating that adipose tissue may contribute to the circulating leptin levels. No treatment effects were observed in adipocytes from the high-energy diet groups, neither in leptin expression nor secretion, except that leptin secretion was significantly reduced by leucine in RE fish adipocytes (p=0.025). Overall, these data show that the regulation of leptin in rainbow trout adipocytes by hormones and nutrients seems to be on secretion, rather than at the transcriptional level.

Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro.

Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes, ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or ß-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, fatty acid synthase and peroxisome proliferator-activated receptor-ß, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

Interplay of adiponectin, TNFα and insulin on gene expression, glucose uptake and PPARγ, AKT and TOR pathways in rainbow trout cultured adipocytes.

Adipose tissue is being increasingly recognized as an important endocrine organ that produces and releases a variety of factors. In the present study we have evaluated in primary cultures of rainbow trout adipocytes, obtained from visceral adipose tissue, the interplay of the adiponectin system, TNFα and insulin at a transcriptional level and, their effects on the adipogenic transcription factor PPARγ, as well as on the activation of main insulin signaling pathways. Likewise, the implication of these adipokines in the regulation of glucose uptake in the adipocyte and their interactions with insulin or IGF-I were also evaluated. Similarly to the mammalian model, insulin enhanced adiponectin gene expression, while it exerted a negative modulation on adiponectin receptors. TNFα increased the mRNA levels of adiponectin receptor 1, but neither adiponectin nor TNFα modulated each other expression. Therefore, the reciprocal suppressive effect of both adipokines previously reported in mammals was not present in this model. Furthermore, the anti-adipogenic effect of TNFα was revealed by the down-regulation of PPARγ at a protein level, meanwhile adiponectin increased PPARγ expression in insulin-stimulated adipocytes, supporting its insulin-sensitizing role. Both adipokines stimulated glucose uptake without modifying AKT or TOR phosphorylation; however, glucose uptake in insulin-treated adipocytes was enhanced by TNFα but not by adiponectin. All in all, these results contribute to gain knowledge on the role of adipokines in rainbow trout adipose tissue and, to better understand the mechanisms that regulate glucose metabolism in this species.

IGF-I and amino acids effects through TOR signaling on proliferation and differentiation of gilthead sea bream cultured myocytes.

Skeletal muscle growth and development is controlled by nutritional (amino acids, AA) as well as hormonal factors (insulin-like growth factor, IGF-I); however, how its interaction modulates muscle mass in fish is not clearly elucidated. The purpose of this study was to analyze the development of gilthead sea bream cultured myocytes to describe the effects of AA and IGF-I on proliferating cell nuclear antigen (PCNA) and myogenic regulatory factors (MRFs) expression, as well as on the transduction pathways involved in its signaling (TOR/AKT). Our results showed that AA and IGF-I separately increased the number of PCNA-positive cells and, together produced a synergistic effect. Furthermore, AA and IGF-I, combined or separately, increased significantly Myogenin protein expression, whereas MyoD was not affected. These results indicate a role for these factors in myocyte proliferation and differentiation. At the mRNA level, AA significantly enhanced PCNA expression, but no effects were observed on the expression of the MRFs or AKT2 and FOXO3 upon treatment. Nonetheless, we demonstrated for the first time in gilthead sea bream that AA significantly increased the gene expression of TOR and its downstream effectors 4EBP1 and 70S6K, with IGF-I having a supporting role on 4EBP1 up-regulation. Moreover, AA and IGF-I also activated TOR and AKT by phosphorylation, respectively, being this activation decreased by specific inhibitors. In summary, the present study demonstrates the importance of TOR signaling on the stimulatory role of AA and IGF-I in gilthead sea bream myogenesis and contributes to better understand the potential regulation of muscle growth and development in fish.

Adipose tissue and liver metabolic responses to different levels of dietary carbohydrates in gilthead sea bream (Sparus aurata).

This study analyzes the effects of replacing dietary lipids by carbohydrates and carbohydrates by fiber on gilthead sea bream growth, as well as lipid and glucose metabolism in adipose tissue and liver over the course of a 15-week feeding trial. Six different diets were formulated and fish were classified into two experimental groups sharing one diet. In the first group (LS), fish were fed four diets where lipids were reduced (23%-17%) by increasing carbohydrates (12%-28%) and, the second group (SF) consisted on three diets where the amount of carbohydrates (28%-11%) was exchanged at expenses of fiber (1%-18%). Differences in growth were not observed; nevertheless, the hepatosomatic index was positively related to dietary starch levels, apparently not due to enhanced hepatic lipogenesis, partly supported by unchanged G6PDH expression. In the LS group, lipogenic activity of adipose tissue was stimulated with low-lipid/high-carbohydrate diets by up-regulating G6PDH expression and a tendency to increase FAS, and promoted carbohydrate utilization versus fatty acid oxidation by modulating the transcription factors LXRα, PPARα and PPARß expression. In the SF group, PPARs and LXRα increased parallel to fiber levels in adipose tissue. Furthermore, an adaptation of hepatic GK to dietary starch inclusion was observed in both groups; however, the lack of effects on G6Pase expression indicated that gluconeogenesis was not nutritionally regulated under the conditions examined. Overall, metabolic adaptations directed to an efficient use of dietary carbohydrates are present in gilthead sea bream, supporting the possibility of increasing carbohydrate or fiber content in diets for aquaculture sustainability.

Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata).

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of the gilthead sea bream. Gene expression profiles during culture maturation were compared with those obtained from a fasting-refeeding model which is widely used to modulate myogenesis in vivo. Myogenesis is controlled by numerous extracellular signals together with intracellular transcriptional factors whose coordinated expression is critical for the appropriate development of muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated during the proliferating phase of the myogenic cultures coincident with the highest expression of proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance was highest during the differentiation phase of the culture when myotubes were present, and was correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30days of fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction of myogenic cell activity with fasting. The primary cell cultures and fasting-feeding experiments described provide a foundation for the future investigations on the regulation of muscle growth in gilthead sea bream.