Antiviral activity of palmitic acid via autophagic flux inhibition in zebrafish (Danio rerio)

11 pages, 7 figures, 1 table.-- This is an open access article under the CC BY-NC-ND licenseFatty acids (FAs) are key elements that affect not only growth but also different immune functions, and therefore, nutrition is important for growing healthy fish. Zebrafish (Danio rerio) is a good model for assessing the beneficial effects of immunostimulants, including FAs, before applying them in aquaculture. Accordingly, this study evaluated the effects of palmitic acid (PA) treatment on different immune parameters of zebrafish and on the mortality caused by the spring viremia of carp virus (SVCV). The results suggest that PA modulates the infection outcome in vivo, which benefits zebrafish and results in reduced mortality and viral titres. The antiviral protection elicited by this FA seems to be associated with the inhibition of autophagy and is independent of other immune processes, such as neutrophil proliferation or type I interferon (IFN) activity. The use of PA as an immunostimulant at low concentrations showed great potential in the prevention of SVCV infections; therefore, this FA could help to prevent the mortality and morbidity caused by viral agents in aquacultured fish. Nevertheless, the potentially detrimental effects of suppressing autophagy in the organism should be taken into accountThis work was funded by the BIO2017-82851-C3-1R project of the Spanish Ministerio de Economía y Competitividad and IN607B 2019/01 from Consellería de Economía, Emprego e Industria (GAIN), Xunta de Galicia. Marta Librán-Pérez was the recipient of postdoctoral fellowships from the Ministerio de Ciencia, Innovación y Universidades (IJCI- 2017-31426). Patricia Pereiro wishes to thank the Axencia Galega de Innovación (GAIN, Xunta de Galicia) for her postdoctoral contract (IN606B-2018/010).Peer reviewe

Transcriptome Analysis of Turbot (Scophthalmus maximus) Infected With Aeromonas salmonicida Reveals a Direct Effect on Leptin Synthesis as a Neuroendocrine Mediator of Inflammation and Metabolism Regulation

15 pages, 8 figures, 1 table.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a disease affecting numerous fish species worldwide. It is a highly pathogenic bacterium for turbot, whose farming production represents an important economic activity in several European countries and China. To better understand the response of this organism to A. salmonicida, we conducted RNA-Seq analysis of the head kidney from experimentally infected and uninfected turbot juveniles at 24 hours post-infection (hpi). As expected, among the differentially expressed genes (DEGs) between infected and uninfected fish, we observed the modulation of a multitude of immune-related genes but also a high representation of genes linked to metabolism. Interestingly, one of the most upregulated genes was that encoding the hormone leptin. Leptin is a multifunctional hormone/cytokine that has been shown to play roles in the immune system, stress response, food intake, metabolism and energy balance. We used recombinant human leptin to elucidate its role during infection with A. salmonicida in turbot (anorexigenic activity, ability to modulate metabolism and the immune response, and its effect on survival and bacterial load during infection). We found that the intraperitoneal administration of leptin was able to alter the response to the bacteria at the immune level, but especially at the metabolic level, which resulted in a higher survival rate without affecting the bacterial load. Based on this, we hypothesized that leptin could offer great potential as a therapeutic treatment during furunculosis outbreaks by reducing the impact of sepsis. Our results reveal the complex interplay between bacterial activity and the regulation of food intake, metabolism and inflammationOur laboratory is funded by projects PID2020-119532RB-I00 from Ministerio de Ciencia e Innovación, MetDisFish from Ministerio de Agricultura, Pesca y Alimentación (MAPA) and European Maritime, Fisheries and Aquaculture Fund (EMFAF), 0474_BLUEBIOLAB from EU FEDER Programa Interreg España-Portugal and IN607B 2019/01 from Consellerıa de Economıa, Emprego e Industria (GAIN), Xunta de Galicia. ML-P and PP wish to thank the Ministerio de Ciencia e Innovación and Axencia Galega de Innovación (GAIN, Xunta de Galicia), respectively, for their postdoctoral contracts (IN606B-2018/010; IN606B-2018/010). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Conserved function of zebrafish (Danio rerio) Gdf15 as a sepsis tolerance mediator

14 pages, 9 figures, 1 tableGDF15 is frequently detected in patients suffering from various diseases, especially those associated with pro-inflammatory processes and/or metabolic disorders. Accordingly, sepsis, whose major complications are related to metabolic alterations and systemic inflammation, significantly increases the secretion of GDF15. Indeed, this cytokine could be considered a marker of sepsis severity. However, until the last several years, the involvement of GDF15 in these disorders had not been widely characterized. In mice, GDF15 was recently described as a pivotal inducer of sepsis tolerance by mediating metabolic alterations that reduce tissue damage. In this work we describe a zebrafish gdf15 gene. We found that gdf15 follows an expression pattern similar to that observed in mammals, being highly expressed in the liver and kidney and induced after pro-inflammatory stimuli. Moreover, larvae overexpressing gdf15 were more resistant to bacterial and viral challenges without affecting the pathogen load. Consequently, Gdf15 also protected zebrafish larvae against LPS-induced mortality. As in mice, zebrafish Gdf15 seems to induce sepsis tolerance by altering the metabolic parameters of the individualsThis work was funded by the BIO 2017-82851-C3-1R project of the Spanish Ministerio de Economía y Competitividad. Patricia Pereiro and Marta Librán-Pérez wish to thank the Axencia Galega de Innovación (GAIN, Xunta de Galicia) and Ministerio de Ciencia, Innovación y Universidades, respectively, for their postdoctoral contracts (IN606B-2018/010; IJCI-2017-31426). Our laboratory is funded by EU Feder Programa Interreg España-Portugal OPE01833 and IN607B 2019/01 from Consellería de Economía, Emprego e Industria (GAIN), Xunta de GaliciaPeer reviewe

β-glucan administration induces metabolic changes and differential survival rates after bacterial or viral infection in turbot (Scophthalmus maximus)

10 pages, 6 figuresThe innate immune response is able to ward off pathogens and remember previous infections using different mechanisms; this kind of immune reaction has been called “trained immunity”. Changes in cellular metabolism (aerobic glycolysis) have been observed during training with some immunostimulants like β-glucans or during viral and bacterial infections. We hypothesize that β-glucans can induce metabolic changes used by the host to fight pathogens. Accordingly, we evaluated changes in metabolic parameters in turbot that could affect their survival after a previous intraperitoneal treatment with β-glucans and subsequent administration of Viral Hemorrhagic Septicemia Virus (VHSV) or bacteria (Aeromonas salmonicida subsp. salmonicida). The results obtained support that β-glucans, VHSV and A. salmonicida induce changes in lactate, glucose and ATP levels in plasma, head kidney and liver and in the mRNA expression of enzymes related to glucose and fatty acid metabolism in head kidney. Additionally, the metabolic changes induced by β-glucans are beneficial for VHSV replication, but they are harmful to A. salmonicida, resulting in reduced mortality. β-glucans appear to have great therapeutic potential and can induce trained immunity against bacterial disease but not against viral disease, which seems to take advantage of β-glucan metabolic alterationsThis work was funded by the projects AGL2014-51773-C3 and BIO2017-82851-C3-1-R from the Spanish Ministerio de Economía y Competitividad, Proyecto Intramural Especial, PIE 201230E057 from Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC) and IN607B 2016/12 from Consellería de Economía, Emprego e Industria (GAIN), Xunta de Galicia. M.L-P was the recipient of postdoctoral fellowships from the Ministerio de Economía, Industria y Competitividad (FJCI-2015-24106)Peer reviewe

Conserved function of zebrafish (Danio rerio) Gdf15 as a sepsis tolerance mediator

GDF15 is frequently detected in patients suffering from various diseases, especially those associated with pro-inflammatory processes and/or metabolic disorders. Accordingly, sepsis, whose major complications are related to metabolic alterations and systemic inflammation, significantly increases the secretion of GDFt 5. Indeed, this cytokine could be considered a marker of sepsis severity. However, until the last several years, the involvement of GDF15 in these disorders had not been widely characterized. In mice, GDF15 was recently described as a pivotal inducer of sepsis tolerance by mediating metabolic alterations that reduce tissue damage. In this work we describe a zebra fish gdf15 gene. We found that gdf15 follows an expression pattern like that observed in mammals, being highly expressed in the liver and kidney and induced after pro-inflammatory stimuli. Moreover, larvae overexpressing gdf15 were more resistant to bacterial and viral challenges without affecting the pathogen load. Consequently, Gdft 5 also protected zebrafish larvae against LPS-induced mortality. As in mice, zebrafish Gdf15 seems to induce sepsis tolerance by altering the metabolic parameters of the individualsThis work was funded by the BIO 2017-82851-0-1 R project of the Spanish Ministerio de Economía y Competitividad. Patricia Pereiro and Marta Libran-Perez wish to thank the Axencia Galega de lnnovación (GAIN, Xunta de Galicia) and Ministerio de Ciencia, lnnovación y Universidades, respectively, for their postdoctoral contracts (IN606B-2018/010; IJCl-2017-31426). Our laboratory is funded by 0474_BLUEBIOLAB from EU FEDER Programa lnterreg Espana-Portugal and IN607B 2019/01 from Consellerfa de Economfa, Emprego e lndustria (GAIN), Xunta de GaliciaN

Oleic acid and octanoic acid sensing capacity in rainbow trout Oncorhynchus mykiss is direct in hypothalamus and Brockmann bodies.

In a previous study, we provided evidence for the presence in hypothalamus and Brockmann bodies (BB) of rainbow trout Oncorhynchus mykiss of sensing systems responding to changes in levels of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). Since those effects could be attributed to an indirect effect, in the present study, we evaluated in vitro if hypothalamus and BB respond to changes in FA in a way similar to that observed in vivo. In a first set of experiments, we evaluated in hypothalamus and BB exposed to increased oleic acic or octanoic acid concentrations changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, reactive oxygen species (ROS) effectors, components of the KATP channel, and (in hypothalamus) neuropeptides related to food intake. In a second set of experiments, we evaluated in hypothalamus the response of those parameters to oleic acid or octanoic acid in the presence of inhibitors of fatty acid sensing components. The responses observed in vitro in hypothalamus are comparable to those previously observed in vivo and specific inhibitors counteracted in many cases the effects of FA. These results support the capacity of rainbow trout hypothalamus to directly sense changes in MCFA or LCFA levels. In BB increased concentrations of oleic acid or octanoic acid induced changes that in general were comparable to those observed in hypothalamus supporting direct FA sensing in this tissue. However, those changes were not coincident with those observed in vivo allowing us to suggest that the FA sensing capacity of BB previously characterized in vivo is influenced by other neuroendocrine systems

Counter-regulatory response to a fall in circulating fatty acid levels in rainbow trout. Possible involvement of the hypothalamus-pituitary-interrenal axis.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma

mRNA levels in Brockmann bodies of rainbow trout incubated <i>in vitro</i> for 1 h at 15°C in modified Hanks’ medium containing 2 mM D-glucose alone (control) or containing 1, 10, or 100 µM oleic acid or octanoic acid.

<p>Each value is the mean of 5 independent experiments carried out with pools of Brockmann bodies from 3–4 different fish. Data is expressed as fold-induction (+, increase; −, decrease) with respect to the control group (expression results were normalized by β-actin mRNA levels, mRNA levels-no variation). *, significantly different (P<0.05) from control fish. Different letters indicate significant differences (P<0.05) among concentration within each fatty acid treatment.</p

Nucleotide sequences of the PCR primers used to evaluate mRNA abundance by RT-PCR (qPCR).

<p>ACC, Acetyl-CoA carboxylase; ACLY, ATP-citrate lyase; CART, cocaine- and amphetamine-related transcript; CPT1, carnitine palmitoyl transferase type 1; CS, citrate synthetase; FAS, fatty acid synthetase; FAT/CD36, fatty acid translocase; Kir6.x-like, inward rectifier K⁺ channel pore type 6.-like; LXRα, liver X receptor α; MCD, malonyl CoA dehydrogenase; NPY, neuropeptide Y; POMC, pro-opio melanocortin; PPARα, peroxisome proliferator-activated receptor type α; SREBP1c, sterol regulatory element-binding protein type 1c; SUR-like, sulfonylurea receptor-like; UCP2a, mitochondrial uncoupling protein 2a.</p

Response of metabolite levels, enzyme activities, and mRNA abundance of several parameters related to fatty acid sensing in hypothalamus of rainbow trout incubated <i>in vitro</i> for 1 h at 15°C in modified Hanks’ medium containing 100 µM oleic acid (Ol) or 100 µM octanoic acid (Oc) alone (controls) or 100 µM oleic acid or 100 µM octanoic acid and selected inhibitors related to fatty acid sensing capacity in mammals.

<p>These included: 40 µg.ml⁻¹ C75 (FAS inhibitor), 50 µM etomoxir (CPT1 inhibitor), 1 µM trolox (ROS scavenger), 20 µM genipin (UCP2 inhibitor), 500 µM diazoxide (sulfonyl urea receptor 1 antagonist), 5 µM triacsin C (ACS inhibitor), 50 nM SSO (FAT/CD36 inhibitor), and 40 µg.ml⁻¹ TOFA (ACC inhibitor). Only those parameters for which significant effects of oleic acid or octanoic acid treatment alone were noticed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059507#pone-0059507-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059507#pone-0059507-t002" target="_blank">Table 2</a>) were evaluated for inhibitor action. Values represent the mean of 5 independent experiments carried out with pools of hypothalami from 3–4 different fish. +, inhibitor significantly (P<0.05) counteracted the effect of oleic acid or octanoic acid alone.</p><p>C75, 4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid. Etomoxir, R(+)-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylic acid). Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Genipin, methyl (1<i>R</i>,2<i>R</i>,6<i>S</i>)-2-hydroxy-9-(hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate. Diazoxide, 7-chloro-3-methyl-4<i>H</i>-1,2,4-benzothiadiazine 1,1-dioxide. Triacsin C, <i>N</i>-(((2<i>E</i>,4<i>E</i>,7<i>E</i>)-undeca-2,4,7-trienylidene)amino)nitrous amide). SSO, sulfo-N-succinimidyl oleate. TOFA, 5-(tetradecyloxy)-2-furoic acid.</p