Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture

Ontogeny of the digestive enzyme activity of the pikeperch (Sander lucioperca) under culture condition

The pikeperch (Sander lucioperca) is a species with a high potential for aquaculture and a valuable food with high market acceptance. The aim of the study was to evaluate the functional ontogeny of digestive enzyme of pikeperch from hatching to 45 days-post fertilization, 777 degree-day (DPF, dd) under culture condition. The average total length (TL) of larvae measured at hatching was 3.6 ± 0.4 mm (5 DPF; 67 dd) and at the end of experiment (45 DPF, 777 dd) was 27.1 ± 1.1 mm. The survival rate was 80-90% during the experiment period. Inhibition zimography reveals the presence of nine bands with proteolytic activity in the digestive tract of juvenile pikeperch. Zimography results during the ontogeny revealed that in larvae at 8 DPF (108 dd) and 13 DPF (189 dd), three bands were presented. The variations observed in the enzymatic activity reflected a high amount of total protease activity at 10 DPF (133.5 dd). Regarding pepsin, its activity was observed for the first time at 26 DPF (378.9 dd). Lipase activity remained constant from hatching to 26 DPF (378.9 dd). The highest amount of α-amylase activity was detected at 15 DPF (211.5 dd) and 45 DPF (777 dd). The low lipase enzyme activity suggested that live feeds with low lipid were more suitable than diets containing high lipid levels; larvae had also early capability to digest nutrient-dense diet that was high in protein. According to results the pikeperch larvae possess after the exogenous feeding, a functional digestive system with high activities that indicated the gradual development of the digestive system

Characterisation and expression analysis of cathepsins and ubiquitin-proteasome genes in gilthead sea bream (Sparus aurata) skeletal muscle

The proteolytic enzymes involved in normal protein turnover in fish muscle are also responsible for post-mortem softening of the flesh and are therefore potential determinants of product quality. The main enzyme systems involved are calpains, cathepsins, and the ubiquitin-proteasome (UbP). In this study on Sparus aurata (Sa), the coding sequences of cathepsins (SaCTSB and SaCTSDb) and UbP family members (SaN3 and SaUb) were cloned from fast skeletal muscle, and their expression patterns were examined during ontogeny and in a fasting/re-feeding experiment. The amino acid sequences identified shared 66-100% overall identity with their orthologues in other vertebrates, with well conserved characteristic functional domains and catalytic residues. SaCTSDb showed phylogenetic, sequence and tissue distribution differences with respect to its paralogue SaCTSDa, previously identified in the ovary. Expression of gilthead sea bream cathepsins (B, L, Da, Db) and UbP members (N3, Ub, MuRF1 and MAFbx) in fast skeletal muscle was determined at three different life-history stages and in response to fasting and re-feeding in juveniles. Most of the proteolytic genes analysed were significantly up-regulated during fasting, and down-regulated with re-feeding and, between the fingerling (15 g) and juvenile/adult stages (~50/500 g), consistent with a decrease in muscle proteolysis in both later contexts. In contrast, SaCTSDa and SaMuRF1 expression was relatively stable with ontogeny and SaUb had higher expression in fingerlings and adults than juveniles. The data obtained in the present study suggest that cathepsins and UbP genes in gilthead sea bream are co-ordinately regulated during ontogeny to control muscle growth, and indicate that feeding regimes can modulate their expression, providing a potential dietary method of influencing post-mortem fillet tenderisation, and hence, product quality

Caffeic acid and hydroxytyrosol have anti-obesogenic properties in zebrafish and rainbow trout models

Some natural products, known sources of bioactive compounds with a wide range of properties, may have therapeutic values in human health and diseases, as well as agronomic applications. The effect of three compounds of plant origin with well-known dietary antioxidant properties, astaxanthin (ATX), caffeic acid (CA) and hydroxytyrosol (HT), on zebrafish (Danio rerio) larval adiposity and rainbow trout (Onchorynchus mykiss) adipocytes was assessed. The zebrafish obesogenic test (ZOT) demonstrated the anti-obesogenic activity of CA and HT. These compounds were able to counteract the obesogenic effect produced by the peroxisome proliferator-activated receptor gamma (PPAR.) agonist, rosiglitazone (RGZ). CA and HT suppressed RGZ-increased PPAR. protein expression and lipid accumulation in primary-cultured rainbow trout adipocytes. HT also significantly reduced plasma triacylglycerol concentrations, as well as mRNA levels of the fasn adipogenic gene in the adipose tissue of HT-injected rainbow trout. In conclusion, in vitro and in vivo approaches demonstrated the anti-obesogenic potential of CA and HT on teleost fish models that may be relevant for studying their molecular mode of action. Further studies are required to evaluate the effect of these bioactive components as food supplements for modulating adiposity in farmed fish

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 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

Oxidative Status of the Pyloric Caeca and Proximal Intestine in Gilthead Sea Bream Fed Diets Including Different Vegetable Oil Blends from Palm, Rapeseed and Linseed

Nowadays, including vegetable ingredients in fish diets without growth effects is common; however, their intestinal oxidative status under these conditions is less known. Five isonitrogenous and isolipidic diets with 75% vegetable oil (VO) inclusion were formulated for juvenile gilthead sea bream (Sparus aurata). As VO, one diet contained palm oil (diet P), another rapeseed oil (diet R), and the other three included linseed oil (L) combined with the above-mentioned VOs (named PL, RL and RPL diets). After 18 weeks, pyloric caeca (PC) and proximal intestine (PI) were analyzed for oxidative stress biomarkers, lipid peroxidation (LPO), and gene expression. Dietary linseed oil diminished the superoxide dismutase activity in both intestinal regions, catalase in PC and glutathione reductase in PI; rapeseed oil reduced the glutathione peroxidase (GPx) and glutathione-S-transferase activities in PC, and palm oil upregulated GPx activity in PI. The PL diet triggered LPO levels in the PI, and RPL-fed fish showed the highest levels of LPO in the PC due to lower antioxidant activities, while RL-fed fish presented the best oxidative status. The results suggest that the dietary amount of n-6 and the unsaturated/saturated fatty acids ratio are factors to be considered in aquafeed formulation, including VOs, to improve the intestinal oxidative status in fish

Effects of dietary vegetable oil mixtures including soybean oil on intestinal oxidative stress in gilthead sea bream (Sparus aurata)

Fish oil is commonly replaced by vegetable oils in sea bream diets, but little is known about their effects on intestinal health regarding oxidative stress biomarkers. The negative effects of lipid peroxidation on digestive mucosa could have consequences in animal nutrition and welfare. In this study, five isonitrogenous (46%) and isolipidic (22%) diets with 75% of vegetable oils inclusion were evaluated: soybean oil (S) alone or different mixtures containing soybean oil with linseed (SL), linseed and rapeseed (SLR), linseed and palm (SLP), and linseed, rapeseed, and palm (SLRP). Gilthead sea bream juveniles were fed twice a day for 18 weeks. Pyloric caeca and proximal intestine samples were collected 24 h post feeding for lipid peroxidation (LPO), antioxidant enzyme activities (SOD, CAT, GPx, GST, and GR) and gene expression analyses. Pyloric caeca presented larger unhealthy changes in oxidative status than proximal intestine. Although SL-fed fish showed the highest antioxidant activities, they were unable to cope with LPO that in pyloric caeca was 31.4 times higher than in the other groups. Instead, SLP fish presented the best oxidative status, with low LPO levels, antioxidant enzyme activities, and gene expression. In summary, between the vegetable oils dietary mixtures tested, SPL would maintain better intestinal health

Temperature affects musculoskeletal development and muscle lipid metabolism of gilthead sea bream (Sparus aurata)

World population is expected to increase to approximately 9 thousand million people by 2050 with a consequent food security decline. Besides, climate change is a major challenge that humanity is facing, with a predicted rise in mean sea surface temperature of more than 2°C during this century. This study aims to determine whether a rearing temperature of 19, 24, or 28°C may influence musculoskeletal development and muscle lipid metabolism in gilthead sea bream juveniles. The expression of growth hormone (GH)/insulin-like growth factors (IGFs) system-, osteogenic-, myogenic-, and lipid metabolism-related genes in bone and/or white muscle of treated fish, and the in vitro viability, mineralization, and osteogenic genes expression in primary cultured cells derived from bone of the same fish were analyzed. The highest temperature significantly down-regulated igf-1, igf-2, the receptor igf-1ra, and the binding proteins igfbp-4 and igfbp-5b in bone, and in muscle, igf-1 and igf-1ra, suggesting impaired musculoskeletal development. Concerning myogenic factors expression, contrary responses were observed, since the increase to 24°C significantly down-regulated myod1 and mrf4, while at 28°C myod2 and myogenin were significantly up-regulated. Moreover, in the muscle tissue, the expression of the fatty acid transporters cd36 and fabp11, and the lipases lipa and lpl-lk resulted significantly increased at elevated temperatures, whereas β-oxidation markers cpt1a and cpt1b were significantly reduced. Regarding the primary cultured bone-derived cells, a significant up-regulation of the extracellular matrix proteins on, op, and ocn expression was found with increased temperatures, together with a gradual decrease in mineralization along with fish rearing temperature. Overall, these results suggest that increasing water temperature in this species appears to induce unfavorable growth and development of bone and muscle, through modulating the expression of different members of the GH/IGFs axis, myogenic and osteogenic genes, while accelerating the utilization of lipids as an energy source, although less efficiently than at optimal temperatures

Hydroxytyrosol-rich extract from olive juice as an additive in gilthead sea bream juveniles fed a high-fat diet: Regulation of somatic growth.

The dietary inclusion of plant-based products in fish feeds formulation is required for the sustainable development of aquaculture. Moreover, considering functional diets, hydroxytyrosol, one of the major phenolic compounds found in olives (Olea europaea), has been identified as a potential candidate to be used in the aquafeeds industry due to its health promoting abilities. The aim of this study was to evaluate the effects of the inclusion of an olive juice extract rich in hydroxytyrosol as an additive (0.52 g HT/kg feed) in a high-fat (24% lipids) diet in gilthead sea bream (Sparus aurata) juveniles. Moreover, the experimental diets, with or without the extract, were administered daily at a standard (3% of total biomass in the tank) or restricted ration (40% reduction) for 8-9 weeks. Growth and biometric parameters, insulin-like growth factor 1 (IGF-1) plasma levels and growth hormone/IGF axis-, myogenic- and osteogenic-related genes expression in liver, white muscle and/or bone were analyzed. Moreover, in vitro cultures of vertebra bone-derived cells from fish fed the diets at a standard ration were performed at weeks 3 and 9 to explore the effects of hydroxytyrosol on osteoblasts development. Although neither body weight or any other biometric parameter were affected by diet composition after 4 or 8 weeks, the addition of the hydroxytyrosol-rich extract to the diet increased IGF-1 plasma levels, regardless of the ration regime, suggesting an anabolic condition. In muscle, the higher mRNA levels of the binding protein igfbp-5b and the myoblast fusion marker dock5 in fish fed with the hydroxytyrosol-rich diet suggested that this compound may have a role in muscle, inducing development and a better muscular condition. Furthermore in bone, increased osteogenic potential while delayed matrix mineralization after addition to the diet of the olive juice extract was supported by the upregulated expression of igf-1 and bmp4 and reduced transcript levels of osteopontin. Overall, this study provides new insights into the beneficial use of hydroxytyrosol as a dietary additive in gilthead sea bream functional diets to improve muscle-skeletal condition and, the aquaculture industry

Musculoskeletal growth modulation in gilthead sea bream juveniles reared at high water temperature and fed with palm and rapeseed oils-based diets

The upward trend of seawater temperature has encouraged improving the knowledge of its consequences on fish, considering also the development of diets including vegetable ingredients as an approach to achieve a more sustainable aquaculture. This study aims to determine the effects on musculoskeletal growth of: (1) a high-water temperature of 28 °C (versus 21 °C) in gilthead sea bream juveniles (Sparus aurata) fed with a diet rich in palm oil and, (2) feeding the fish reared at 28 °C with two other diets containing rapeseed oil or an equilibrated combination of both vegetable oils. Somatic parameters and mRNA levels of growth hormone-insulin-like growth factors (GH-IGFs) axis-, osteogenic-, myogenic-, lipid metabolism- and oxidative stress-related genes in vertebra bone and/or white muscle are analyzed. Overall, the data indicate that high-water rearing temperature in this species leads to different adjustments through modulating the gene expression of members of the GH-IGFs axis (down-regulating igf-1, its receptors, and binding proteins) and also, to bone turnover (reducing the resorption-activity genes cathepsin K (ctsk) and matrix metalloproteinase-9 (mmp9)) to achieve harmonic musculoskeletal growth. Moreover, the combination of palm and rapeseed oils seems to be the most beneficial at high-water rearing temperature for both balanced somatic growth and muscular fatty acid uptake and oxidation