Proteolytic systems' expression during myogenesis and transcriptional regulation by amino acids in gilthead sea bream cultured muscle cells

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

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

Lysine and leucine deficiencies affect myocytes development and IGF signaling in gilthead sea bream (Sparus aurata)

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

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

Aplicability of sustained swimming for gilthead seabream (Sparus aurata) fingerlings farming

Trabajo presentado en Aquaculture Europe 2014, celebrado en San Sebastián (España) del 4 al 17 de octubre de 2014Fish growth is strongly influenced by environmental and nutritional factors. So, the optimization of culture conditions can help maximize fish growth, particularly with regards to their skeletal muscle. Moderate and sustained activity has been used to improve growth rate and feed conversion efficiencies in fish. In many fish species, swimming activity changes the amount and proportion of nutrients used as energy source and the aerobic capacity of the swimming muscles. Both aspects, diet composition and dynamic muscle metabolism, were determinants of the growth improvement observed in gilthead sea bream submitted to moderate activity (Martin-Pérez et al., 2012; Felip et al., 2013). The aim of this study was to analyse the effects of five weeks of sustained activity on growth parameters, muscle metabolism and regulatory factors in the early period of gilthead sea bream farming.Peer reviewe

Sustained exercise effects on GH/IGFs axis and growth in gilthead sea bream (Sparus aurata)

Trabajo presentado en la 27th Conference of European Comparative Endocrinologists, celebrada en Rennes (Francia), del 25 al 29 de agosto de 2014In vertebrates, the endocrine system regulates growth mainly through the growth hormone (GH)/insulin-like growth factors (IGFs) axis and, although exercise modulates growth, little is known about the response on these endocrine factors. The aim of this work was to characterize the effects of five weeks of moderate sustained swimming over the GH/IGFs axis and growth in gilthead sea bream juveniles. We analyzed the plasma GH and IGF-I levels and, the gene expression in liver and anterior and caudal muscles of: GH receptors (GHR1 and GHR2), three IGF-I splice variants (IGF-Ia/b/c), IGF-II, as well as for the first time in this species, that of the two IGF-I receptors (IGF-IRa and IGF-IRb). Fish under exercise (EX) grew significantly more than control fish (CT) without differences in feed intake. Moreover, plasma GH levels significantly decreased in EX fish in comparison with CT fish, together with reduced hepatic expression of both GHRs. GHR2 expression was also down-regulated in the anterior muscle, whereas GHR1 expression significantly increased in both muscle regions. These results indicated tissue-dependent differences of GHRs expression in response to exercise and suggest that GHR1 seems to be more important regulating muscle growth. On the other hand, plasma IGF-I levels in EX fish were significantly higher than those of CT fish, according to the increased IGF-I expression (mainly IGF-Ia) in the liver of the former. In both muscle regions, total IGF-I mRNA levels were similar; however, the expression of IGF-Ic increased significantly with exercise, suggesting a potential role for this splice variant mediating the swimming activity effects on muscle growth. Additionally, we found a significant up-regulation of IGFII expression in the caudal muscle region of EX fish, which may be related with the well-described local effects of this factor enhancing muscle growth. Furthermore, exercise decreased the hepatic IGF-IRa expression levels, while IGF-IRb was not detectable, showing a similar regulation as that observed for the GHRs. Contrary in muscle, both IGF-IRs were present, although their gene expression levels remained unchanged with exercise. Altogether, these data indicate that moderate sustained activity may be used to increase the IGF-I/GH ratio in plasma and, to modulate the muscle gene expression of different molecules of the GH/IGFs axis (i.e. GHR1 and IGF-II), to potentiate growth in gilthead sea bream.Peer reviewe

Characterization data of gilthead sea bream (Sparus aurata) IGF-I receptors (IGF-IRa/Rb)

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 brea

Lysine and leucine deficiencies affect myocytes development and IGF signaling in gilthead sea bream (Sparus aurata)

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

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.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”.Peer reviewe

Business Plan

The content of this bachelor thesis is a theoretical description and a specific elaboration of the business plan for the establishment of the BANH MI bar bageterie, which products and sells stuffed BANH MI baguettes. The theoretical part describes the legal forms of business in the Czech republic and provides a general description of the business plan. In the practical part, besides the specific plan, there is a comparison of the legal forms of business for the purpose of the selection the most suitable form for the future development of the company. The plan itself provides basic information about the company and its goals, market analysis , competitors, marketing strategy or financial plan. The final output consists of financial reports, which are processed in a realistic, optimistic and a pessimistic variant