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

The probiotic Lactobacillus rhamnosus mimics the dark-driven regulation of appetite markers and melatonin receptors' expression in zebrafish (Danio rerio) larvae: Understanding the role of the gut microbiome.

The use of probiotics has been recently considered a novel therapeutic strategy to prevent pathologies such as obesity; however, the specific mechanisms of action by which probiotics exert their beneficial effects on metabolic health remain unclear. The aim of the present study was to investigate the short-term effects of a probiotic Lactobacillus rhamnosus supplementation (PROB) on appetite regulation, growth-related markers, and microbiota diversity in zebrafish (Danio rerio) larvae, compared to a group subjected to a constant darkness photoperiod (DARK), as well as to evaluate the effects of both treatments on melatonin receptors' expression. After a 24 h treatment, both PROB and DARK conditions caused a significant increase in leptin a expression. Moreover, mRNA abundances of leptin b and proopiomelanocortin a were elevated in the PROB group, and DARK showed a similar tendency, supporting a negative regulation of appetite markers by the treatments. Moreover, both PROB and DARK also enhanced the abundances of melatonin receptors transcript (melatonin receptor 1 ba and bb) and protein (melatonin receptor 1) suggesting a potential involvement of melatonin in mediating these effects. Nevertheless, treatments did not exhibit a significant effect on the expression of most of the growth hormone/insulin-like growth factor axis genes evaluated. Finally, only the DARK condition significantly modulated gut microbiota diversity at such short time, altogether highlighting the rapid effects of this probiotic on modulating appetite regulatory and melatonin receptors' expression, without a concomitant variation of gut microbiota

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

Photoperiod manipulation affects transcriptional profile of genes related to lipid metabolism and apoptosis in zebrafish (Danio rerio) larvae: potential roles of gut microbiota

Gut microbiota plays a fundamental role in maintaining host's health by controlling a wide range of physiological processes. Administration of probiotics and manipulation of photoperiod have been suggested as modulators of microbial composition and are currently undergoing an extensive research in aquaculture as a way to improve health and quality of harvested fish. However, our understanding regarding their effects on physiological processes is still limited. In the present study we investigated whether manipulation of photoperiod and/or probiotic administration was able to alter microbial composition in zebrafish larvae at hatching stage. Our findings show that probiotic does not elicit effects while photoperiod manipulation has a significant impact on microbiota composition. Moreover, we successfully predicted lipid biosynthesis and apoptosis to be modulated by microbial communities undergoing continuous darkness. Interestingly, expression levels of caspase 3 gene (casp3) and lipid-related genes (hnf4a, npc1l1, pparγ, srebf1, agpat4 and fitm2) were found to be significantly overexpressed in dark-exposed larvae, suggesting an increase in the occurrence of apoptotic processes and a lipid metabolism impairment, respectively (p < 0.05). Our results provide the evidence that microbial communities in zebrafish at early life stages are not modulated by a short administration of probiotics and highlight the significant effect that dark photoperiod elicits on zebrafish microbiota and potentially on health

Long-term feeding of Atlantic salmon with varying levels of dietary EPA+DHA alters the mineral status, but does not affect the stress responses after mechanical delousing stress

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Nutritional value of Ethyl Ester oils in fish diets: A review on their potential impact on growth and health

The Norwegian Pelagic sector plays a significant role in producing pelagic fish products for human consumption, including concentrated marine omega-3 fatty acids (FA) used in dietary supplements. To fully utilize fish sources, the sector must explore to use valuable side stream products as ingredients in aquaculture feeds, such as ethyl ester (EE) oils derived from production of concentrated omega-3 FA for dietary supplement. Despite annual production of approximately 10,000 tons of EE oils in Norway, there is limited knowledge about their suitability in aquafeeds. This knowledge gap may stem from the absence of EE in aquatic animals' natural diets, unlike in human dietary supplements. Moreover, varying FA compositions of EE oils used in different fish trials present challenges in drawing definitive conclusions about their effects. Fish metabolism differs from mammals in digesting dietary lipids, lacking the necessary lipase enzymes for monoacylglycerol production during TAG digestion. This may affect the efficiency of lipid transport and metabolism in salmonids, potentially influencing intestinal health and overall lipid metabolism. Furthermore, studies suggest lower digestibility of EE oils compared to free FA or TAG forms, indicating potential limitations in their re-esterification process in enterocytes. Concerns also arise regarding ethanol release during EE hydrolysis in the gut and its potential impact on intestinal health. Additionally, EE oils' susceptibility to oxidation suggests variations in stability in feeds depending on composition and antioxidant content. In SalmoE2, we aim to assess the safety and suitability of EE oils in salmonid diets, addressing the challenge of locally sourcing sustainable ingredients with low CO2 footprints. Understanding the effects of EE oils on salmon and trout performance, health, and fillet quality is crucial for both the pelagic industry and feed producers, optimizing the exploitation of side stream products while promoting sustainable marine product usage. Quantitative data on safety, optimal inclusion, and potential benefits of EE oil use in salmonid diets are essential.Nutritional value of Ethyl Ester oils in fish diets: A review on their potential impact on growth and healthpublishedVersio

Genistein induces adipogenic and autophagic effects in rainbow trout (Oncorhynchus mykiss) adipose tissue: in vivo and in vitro 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 17β-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 trou

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

Omega-3 canola oil effectively replaces fish oil as a new safe dietary source of docosahexaenoic acid (DHA) in feed for juvenile Atlantic salmon

Limited availability of fish oils (FO), rich in n-3 long-chain (≥C20) PUFA, is a major constraint for further growth of the aquaculture industry. Long-chain n-3 rich oils from crops GM with algal genes are promising new sources for the industry. This project studied the use of a newly developed n-3 canola oil (DHA-CA) in diets of Atlantic salmon fingerlings in freshwater. The DHA-CA oil has high proportions of the n-3 fatty acids (FA) 18 : 3n-3 and DHA and lower proportions of n-6 FA than conventional plant oils. Levels of phytosterols, vitamin E and minerals in the DHA-CA were within the natural variation of commercial canola oils. Pesticides, mycotoxins, polyaromatic hydrocarbons and heavy metals were below lowest qualifiable concentration. Two feeding trials were conducted to evaluate effects of two dietary levels of DHA-CA compared with two dietary levels of FO at two water temperatures. Fish increased their weight approximately 20-fold at 16°C and 12-fold at 12°C during the experimental periods, with equal growth in salmon fed the FO diets compared with DHA-CA diets. Salmon fed DHA-CA diets had approximately the same EPA+DHA content in whole body as salmon fed FO diets. Gene expression, lipid composition and oxidative stress-related enzyme activities showed only minor differences between the dietary groups, and the effects were mostly a result of dietary oil level, rather than the oil source. The results demonstrated that DHA-CA is a safe and effective replacement for FO in diets of Atlantic salmon during the sensitive fingerling life-stage.acceptedVersio

The autophagy response during adipogenesis of primary cultured rainbow trout (Oncorhynchus mykiss) adipocytes

Adipogenesis is a tightly regulated process, and the involvement of autophagy has been recently proposed in mammalian models. In rainbow trout, two well-defined phases describe the development of primary cultured adipocyte cells: proliferation and differentiation. Nevertheless, information on the transcriptional profile at the onset of differentiation and the potential role of autophagy in this process is scarce. In the present study, the cells showed an early and transient induction of several adipogenic transcription factors genes' expression (i.e., cebpa and cebpb) along with the morphological changes (round shape filled with small lipid droplets) typical of the onset of adipogenesis. Then, the expression of various lipid metabolism-related genes involving the synthesis (fas), uptake (fatp1 and cd36), accumulation (plin2) and mobilization (hsl) of lipids, characteristic of the mature adipocyte, increased. In parallel, several autophagy markers (i.e., atg4b, gabarapl1 and lc3b) mirrored the expression of those adipogenic-related genes, suggesting a role of autophagy during in vitro fish adipogenesis. In this regard, the incubation of preadipocytes with lysosomal inhibitors (Bafilomycin A1 or Chloroquine), described to prevent autophagy flux, delayed the process of adipogenesis (i.e., cell remodelling), thus suggesting a possible relationship between autophagy and adipocyte differentiation in trout. Moreover, the disruption of the autophagic flux altered the expression of some key adipogenic genes such as cebpa and pparg. Overall, this study contributes to improve our knowledge on the regulation of rainbow trout adipocyte differentiation, and highlights for the first time in fish the involvement of autophagy on adipogenesis, suggesting a close-fitting connection between both processes