Antimicrobial Peptides from Photosynthetic Marine Organisms with Potential Application in Aquaculture.

Aquaculture production is at a record level and is estimated to increase in the coming years. However, this production can be negatively affected by infectious diseases produced by viruses, bacteria, and parasites, causing fish mortality and economic losses. Antimicrobial peptides (AMPs) are small peptides that may be promising candidates to replace antibiotics because they are the first line of defense in animals against a wide variety of pathogens and have no negative effects; they also show additional activities such as antioxidant or immunoregulatory functions, which makes them powerful alternatives for use in aquaculture. Moreover, AMPs are highly available in natural sources and have already been used in the livestock farming and food industries. Photosynthetic marine organisms can survive under all kinds of environmental conditions and under extremely competitive environments thanks to their flexible metabolism. For this reason, these organisms represent a powerful source of bioactive molecules as nutraceuticals and pharmaceuticals, including AMPs. Therefore, in this study we reviewed the present knowledge about AMPs from photosynthetic marine organism sources and analyzed whether they could be suitable for use in aquaculture.

Nature-identical compounds as feed additives in aquaculture.

Aquaculture sustainable development is necessary since it is categorized as the most important source of aquatic products for human consumption and it is expected to keep growing shortly. For this reason, the addition of natural immunostimulants to fish diet to improve fish health and to preserve the environment have great importance in aquaculture. In this sense, new biotechnological tools as nature-identical compounds are now being used as feed additives to strengthen and stimulate the fish immune system to prevent and/or control diseases due to their lesser cost and higher availability than plant compounds. This review aims to present the most recent studies in which nature-identical compounds have been used in the fish diet to establish their possible use in aquaculture. Nature-identical compounds can be considered a promising alternative to be added to fish diets to promote growth performance, manipulate the gut microbiota, and improve the immune and oxidative status of fish as wells as control bacterial infections in this important aquatic industry.

Immunomodulation and skin microbiota perturbations during an episode of chronic stress in gilthead seabream.

Fish stress is a major concern in the aquaculture industry. Many stressors coming from routine practices can predispose fish to compromised growth, immunity and overall health. This study focuses on the characterization of the skin microbiota using next generation sequencing (NGS) platform by targeting a genomic marker 16S and to determine growth performance and immune status of gilthead seabream (Sparus aurata) during an episode of chronic stress. Two groups were established: control group and chronically stressed group. Stressed fish were subjected to 1 min air exposure twice a week for 4 weeks. Results showed that stress negatively affected fish growth performance. Cellular and humoral systemic immunity remained unaffected while local immunity in skin was positively stimulated (total IgM and peroxidase). Skin mucus microbial composition showed significant differences especially after 14 days. Stressed fish showed a decrease in the abundance of the genera Acinetobacter, NS3a_marine_group and Pseudomonas, while Pseudoalteromonas and Marinagarivorans increased significantly. In conclusion, air exposure stress was associated with alterations in skin mucosal immunity and microbial composition that may have been beneficial to the host favoring adaptation to stress.

Implication of mucus-secreting cells, acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ-carrageenin to gilthead seabream (Sparus aurata) specimens.

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1ß, il-1ß) were up-regulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1ß and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Alteration of the Immune Response and the Microbiota of the Skin during a Natural Infection by Vibrio harveyi in European Seabass (Dicentrarchus labrax).

Disease outbreaks continue to represent one of the main bottlenecks for the sustainable development of the aquaculture industry. In marine aquaculture, many species from the Vibrio genus are serious opportunistic pathogens responsible for significant losses to producers. In this study, the effects on the immune response and the skin microbiota of European sea bass (Dicentrarchus labrax) were studied after a natural disease outbreak caused by V. harveyi. Data obtained from infected and non-infected fish were studied and compared. Regarding the local immune response (skin mucus) a decrease in the protease activity was observed in infected fish. Meanwhile, at a systemic level, a decrease in protease and lysozyme activity was reported while peroxidase activity showed a significant increase in serum from infected fish. A clear dysbiosis was observed in the skin mucus microbiota of infected fish in comparison with non-infected fish. Moreover, V. harveyi, was identified as a biomarker for the infected group and Rubritalea for healthy fish. This study highlights the importance of characterizing the mucosal surfaces and microbial composition of the skin mucus (as a non-invasive technique) to detect potential disease outbreaks in fish farms.

Antimicrobial Power of Organic Acids and Nature-Identical Compounds against Two Vibrio spp.: An In Vitro Study.

Vibrosis is one of the major threats in aquaculture farming, and due to the increasing antimicrobial resistance of different Vibrio species, there is an urgent need to replace conventional treatments with more sustainable solutions. Antimicrobial molecules such as organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in the infection management of terrestrial livestock, but little is known about their usage in aquaculture. The aim of this study was to perform a preliminary screening of the in vitro antimicrobial activity of a wide panel of OA and NIC against 2 Vibrio species characteristic of the Mediterranean area, Vibrio harveyi and Vibrio anguillarum, through minimal inhibitory/bactericidal concentration tests. The active principles that showed the best effective pathogen control were medium-chain fatty acids, sorbic and benzoic acid among OA and phenolic monoterpenoids (thymol, carvacrol and eugenol) and aromatic monoterpene aldehydes (vanillin and cinnamaldehyde) among NIC. These results showed how the usage of OA and NIC can open promising perspectives in terms of Vibrio spp. load control in aquaculture. Natural or nature-identical feed additives can make aquaculture production not only more efficient by reducing the need for medicated treatments, but also more sustainable.

Immunity and inflammatory responses in gilthead sea bream (Sparus aurata L.) exposed to sub-lethal mixture of carbamazepine, cadmium chloride and polybrominated diphenyl ether.

Chemical contaminants such as industrial and urban by-products, pharmaceuticals, drugs metabolites and, plastics, are continuously found in the oceans, affecting its quality and organism's welfare. Although these compounds are found at concentrations ranged ng L-1, there is an increasing concern about the potential adverse effects of the interactions among those substances present, simultaneously, in a mixture. In the present study, specimens of sea bream (Sparus aurata) were exposed, by food, to rising concentrations of a mixture of carbamazepine, polybrominated diphenyl ether-47 and cadmium chloride, for 15 days and then, maintained, with the same control diet, without contaminants, for other 15 days. Samples of skin mucus, serum, head-kidney, liver and intestine were sampled at 0, 15 and 30 days. Cellular immune parameters were evaluated on head-kidney, as well as humoral parameters were determined on skin mucus and serum. In addition, the expression of some genes, related to immunity, was analysed on liver and intestine. Both cellular and humoral response were affected at 15 days, showing slightly signs of recovery at 30 days. Besides, the expression of immune-related genes was highly affected, suggesting the development of inflammatory processes, as well as a reduction of immune parameters. Overall, the mixture of compounds severally affected the immune system of sea bream, suggesting a lower degree of recovery. The prolonged exposure to a mixture of these compounds could entail serious change on population immunity and, eventually, promote changes on marine biota.

Effects of aqueous and ethanolic leaf extracts from drumstick tree (Moringa oleifera) on gilthead seabream (Sparus aurata L.) leucocytes, and their cytotoxic, antitumor, bactericidal and antioxidant activities.

Aqueous and ethanolic extracts of drumstick, Moringa oleifera, leaves were evaluated in vitro to ascertain their principal active components and determine their immunostimulant, cytotoxic, antitumoral, bactericidal and antioxidant activities. Phytochemical screening of M. oleifera leaf extracts showed a greater abundance of phenolic and cyanogenic glycosides in aqueous than in ethanolic extracts, characterized by several flavonoids, condensed tannins and saponins. No significant effects on gilthead seabream (Sparus aurata) head-kidney leucocyte activities (phagocytic ability and capacity, respiratory burst and peroxidase) were detected after incubation for 24 h with different concentrations (0.001/1 mg mL-1) of either extract. In addition, the aqueous extract showed a marked cytotoxic effect on both SAF-1 (at doses above 0.01 mg mL-1) and PLHC-1 (at doses above 0.25 mg mL-1) cell lines. The ethanolic extract improved the viability of SAF-1 cells and decreased the viability of PLHC-1 cells when used at higher concentrations. Both the ethanolic and, particularly, the aqueous extracts showed significant bactericidal activity on pathogenic Vibrio anguillarum and Photobacterium damselae strains. The antiradical activity of M. oleifera, as determined by the ABTS assay, increased in a linear dose-response with increasing extract concentrations. The results as a whole for the cytotoxic, bactericidal and antioxidant activities of M. oleifera leaf extracts point to their possible use as additives in functional diets for farmed fish.

Effect of Jasonia glutinosa on immune and oxidative status of gilthead seabream (Sparus aurata L.).

Jasonia glutinosa (rock-tea, RT) has numerous biological activities. In the present work, the beneficial effects of dietary RT on gilthead seabream (Sparus aurata L.) were studied. Fish fed experimental diets containing 0 (control), 10 or 30% of RT for 15 and 30 days. Samples from skin mucus, blood, head kidney, liver and gut were obtained at 15 and 30 days. The antioxidant properties from RT were analysed such as the antioxidant capacity and phenolic content. The heat shock protein 70 level (HSP70) and the total oxidized proteins were evaluated on skin mucus as stress markers. Immune parameters, both humoral (peroxidase activity, immunoglobulin M levels and complement activity) and cellular (phagocytic, respiratory burst and peroxidase activities) were determined in skin mucus, serum or head-kidney leucocytes, respectively. Concomitantly, the expression of different genes related to inflammation and oxidative stress was studied both in liver and gut. Skin mucus peroxidase was significantly increased on fish fed 10% RT for 15 days with respect to the control group. In addition, Serum IgM levels were significantly increased while HSP70 levels and oxidized proteins were significantly decreased on skin mucus from fish fed 30% RT for 30 days, respectively. Besides, cellular immune parameters (phagocytosis, respiratory burst and peroxidase activity) were significantly higher in leucocytes from fish fed the RT diets for 15 days, but not for 30 days. Finally, the gene expression of antioxidant enzymes was up-regulated in liver at 15 and in liver and gut at 30 days. However, the expression of il1b and hsp70 was down-regulated in the liver of fish fed 30% RT for 30 days with respect to the values of control fish. The possible inclusion of RT in fish diets as an additive with antioxidant and/or immunostimulant activities is discussed.

Effects of dietary Origanum vulgare on gilthead seabream (Sparus aurata L.) immune and antioxidant status.

Origanum sp. is a very common genus of aromatic plants worldwide distributed around the Mediterranean area and O. vulgare (oregano) is the most important species of this genus throughout the world. Due the known medicinal properties of oregano, the effect of diets enriched with 0% (control), 0.5% and 1% oregano leaves powder was studied on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.). Fish fed with oregano 0.5% and 1% enriched diets improved both humoral (IgM and bactericidal activity in skin mucus and protease activity in serum) and cellular (head kidney leucocytes phagocytic ability) immunity at 15 and 30 days. Furthermore, the addition of oregano did not provoke any significant effect neither in the growth promotion nor in the liver antioxidant enzymes activity studied in the serum and skin mucus. The possibility of using O. vulgare as a functional additive to fish diet is discussed.

An in vitro study of the effect of carob (Ceratonia siliqua L.) leaf extracts on gilthead seabream (Sparus aurata L.) leucocyte activities. Antioxidant, cytotoxic and bactericidal properties.

Carob leaves, the main residues of the carob tree, were investigated as a renewable and abundant source of bioactive compounds for fish aquaculture. Aqueous and ethanolic extracts obtained from carob leaves were characterized in terms of biochemical composition, antiradical and cytotoxic effects and immunostimulant and antibacterial activities. The ethanolic extract showed higher levels of total phenolics, flavonoids and condensed tannins and higher antioxidant activity than the aqueous extract. No significant immunostimulant effects were observed on gilthead seabream (Sparus aurata) head kidney leucocytes (viability, phagocytosis and respiratory burst activities and peroxidase content) after incubation for 24 h with different extracts. Furthermore, the ethanolic extracts used at 0.5, 0.75 and 1 mg mL-1 and aqueous extracts at 1 g mL-1 had a cytotoxic effect on PLHC-1 cells. When the bactericidal activity was tested against three fish pathogenic bacteria (Vibrio harveyi, Vibrio anguillarum and Photobacterium damselae) notable activity of the different extracts was detected against P. damselae at all three concentrations. A similar effect was demonstrated against V. haryeri when ethanolic extracts were used in the same range of concentrations. This work demonstrates interesting in vitro effects of carob leaf extracts and suggests it could be used as an alternative to chemical compounds with farmed fish. The concentration and nature of the extracts were very important in terms of any positive results.

Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-ß), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Coping with exposure to hypoxia: modifications in stress parameters in gilthead seabream (Sparus aurata) fed spirulina (Arthrospira platensis) and brewer's yeast (Saccharomyces cerevisiae).

This study aimed to investigate the stress response of Sparus aurata specimens fed with nutraceutical aquafeed brewer's yeast (Saccharomyces cerevisiae) and spirulina (Arthrospira platensis). For that purpose, 96 (169.0 ± 2.8 g) animals were distributed randomly in 12 tanks (8 fish per tank, 4 replicates) and divided in 3 groups (D1, casein/gelatin, control; D2, brewer's yeast; D3, spirulina) and fed for 30 days. At the end of this period, fish from two replicates of each experimental diet were submitted to air exposure for 60 s while the fish from the other two replicates were maintained undisturbed (control). Afterwards, samples of blood, skin mucus, and head kidney were collected. The results revealed that after air exposure, cortisol, and glucose levels increased in the groups fed D1 (18.5 ± 2.6 mg/mL; 7.3 ± 0.6 mmol/L, respectively) and D2 (20.0 ± 6.2 mg/mL; 7.7 ± 0.6 mmol/L), but glucose not increased in fish fed D3 (13.7 ± 2.6 mg/mL; 5.5 ± 0.3 mmol/L). Lactate levels increased in all stressed groups, but in D1, its levels were significantly higher. After stress procedure, immunoglobulin M (IgM) levels in mucus increased only in fish fed D3 (0.1901 ± 0.0126 U/mL). Furthermore, there was a reduction in the expression of some genes involved in stress response (coxIV, prdx3, csfl-r, ucp1, and sod in fish fed D2 and D3). csf1 decreased only in stressed fish fed D2. However, cat increased in fish fed with D3. In summary, these findings points to the beneficial effects of spirulina and brewer's yeast to improve stress resistance in aquaculture practices of gilthead seabream.

Effects of dietary inclusion of Moringa oleifera leaves on growth and some systemic and mucosal immune parameters of seabream.

The effect of the dietary incorporation of drumstick, Moringa oleifera, leaf meal (MOL; 0, 5, 10 and 15%) on the growth, feed utilization, some skin mucus and systemic immune parameters and intestinal immune-related gene expression in gilthead seabream (Sparus aurata) specimens. The experiment lasted 4 weeks. The results revealed that MOL can be incorporated in S. aurata diet up to 10% with no significant negative effect on growth and feed utilization. However, there was a significant decrease with MOL at a level of 15% after 2 weeks of feeding. The systemic immune status of fish fed with the different levels of MOL showed an improvement in head kidney leucocyte phagocytosis, respiratory burst and peroxidase activities. Also, serum humoral components, including protease, ACH50 and lysozyme activities and IgM level, increased with MOL inclusion especially at the 5% level. MOL at 5% improved skin-mucosal immunity such as protease, antiprotease, peroxidase and lysozyme activities. Moreover, the feeding of MOL revealed an upregulation of the intestinal mucosal immunity genes (lyso and c3), tight junction proteins (occludin and zo-1) and anti-inflammatory cytokines (tgf-ß) with a downregulation of pro-inflammatory cytokine (tnf-α). Therefore, it is recommended to incorporate MOL in S. aurata diets at a level of 5% for the best immune status or 10% for the high growth performance and acceptable immune surveillance. Graphical abstract ᅟ.

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities.

Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.

In vitro effects of virgin microplastics on fish head-kidney leucocyte activities.

Microplastics are well-documented pollutants in the marine environment that result from production or fragmentation of larger plastic items. The knowledge about the direct effects of microplastics on immunity, including fish, is still very limited. We investigated the in vitro effects of microplastics [polyvinylchloride (PVC) and polyethylene (PE)] on gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) head-kidney leucocytes (HKLs). After 1 and 24 h of exposure of HKLs with 0 (control), 1, 10 and 100 mg mL-1 MPs in a rotatory system, cell viability, innate immune parameters (phagocytic, respiratory burst and peroxidase activities) and the expression of genes related to inflammation (il1b), oxidative stress (nrf2, prdx3), metabolism of xenobiotics (cyp1a1, mta) and cell apoptosis (casp3) were studied. Microplastics failed to affect the cell viability of HKLs. In addition, they provoke very few significant effects on the main cellular innate immune activities, as decrease on phagocytosis or increase in the respiratory burst of HKLs with the highest dose of microplastics tested. Furthermore, microplastics failed to affect the expression of the selected genes on sea bass or seabream, except the nrf2 which was up-regulated in seabream HKLs incubated with the highest doses. Present results seem to suggest that continue exposure of fish to PVC or PE microplastics could impair fish immune parameters probably due to the oxidative stress produced in the fish leucocytes.

Dietary dehydrated lemon peel improves the immune but not the antioxidant status of gilthead seabream (Sparus aurata L.).

Lemon (Citrus limon) is the third most important species of citrus in the world, while Spain is the major producer in Europe. Numerous beneficial effects of lemon are known, which explains their use in traditional medicine. The paper describes the effect of dietary dehydrated lemon peel (a sub-product of the lemon industry) on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.) over a period of 30 days. Fish fed diets enriched with dehydrated lemon peel (1.5% and 3%) for 15 days showed improved growth and both humoral (seric immunoglobulin M) and cellular (peroxidase activity and phagocytic ability of head kidney leucocytes) immunity, as well as the expression of some immune-related genes (nkefa, il1ß, igth and csfr1). However, decreases growth promotion was observed after thirty days of trial. Neither the anti-oxidant enzymes activity nor the expression of several anti-oxidants and anti-stress genes in liver was improved by the diet. The possible inclusion of dehydrated lemon peel in fish diets for its immunostimulant effects is discussed.

Liquid fructose in Western-diet-fed mice impairs liver insulin signaling and causes cholesterol and triglyceride loading without changing calorie intake and body weight.

BACKGROUND/OBJECTIVES: Liquid fructose associates with prevalence of type 2 diabetes mellitus and obesity. Intervention studies suggest that metabolically unfit individuals are more responsive than healthy individuals to liquid fructose. We determined whether mice consuming an obesogenic Western diet were more responsive than chow-fed mice to the alterations induced by liquid fructose supplementation (LFS). METHODS: C57BL/6N mice were fed chow or Western diet±ad libitum 15% fructose solution for 12 weeks. Food and liquid intake and body weight were monitored. Plasma analytes and liver lipids, histology and the expression of genes related to lipid handling, endoplasmic reticulum stress, inflammation and insulin signaling were analyzed. RESULTS: Western diet increased energy intake, visceral adipose tissue (vWAT), body weight, plasma and liver triglycerides and cholesterol, and inflammatory markers in vWAT vs. chow-fed mice. LFS did not change energy intake, vWAT or body weight. LFS significantly increased plasma and liver triglycerides and cholesterol levels only in Western-diet-fed mice. These changes associated with a potentiation of the increased liver expression of PPARγ and CD36 that was observed in Western-fed mice and related to the increased liver mTOR phosphorylation induced by LFS. Furthermore, LFS in Western-diet-fed mice induced the largest reduction in liver IRS2 protein and a significant decrease in whole-body insulin sensitivity. CONCLUSIONS: LFS in mice, in a background of an unhealthy diet that already induces fatty liver visceral fat accretion and obesity, increases liver lipid burden, hinders hepatic insulin signaling and diminishes whole-body insulin sensitivity without changing energy intake.