Methionine deficiency does not increase polyamine turnover through depletion of hepatic S-adenosylmethionine in juvenile Atlantic salmon.

During the last few decades, plant protein ingredients such as soya proteins have replaced fishmeal in the diets of aquacultured species. This may affect the requirement and metabolism of methionine as soya contains less methionine compared with fishmeal. To assess whether methionine limitation affects decarboxylated S-adenosylmethionine availability and polyamine status, in the present study, juvenile Atlantic salmon were fed a methionine-deficient plant protein-based diet or the same diet supplemented with dl-methionine for 8 weeks. The test diets were compared with a fishmeal-based control diet to assess their effects on the growth performance of fish. Methionine limitation reduced growth and protein accretion, but when fish were fed the dl-methionine-supplemented diet their growth and protein accretion equalled those of fish fed the fishmeal-based control diet. Methionine limitation reduced free methionine concentrations in the plasma and muscle, while those in the liver were not affected. S-adenosylmethionine (SAM) concentrations were higher in the liver of fish fed the methionine-deficient diet, while S-adenosylhomocysteine concentrations were not affected. Putrescine concentrations were higher and spermine concentrations were lower in the liver of fish fed the methionine-deficient diet, while the gene expression of SAM decarboxylase (SAMdc) and the rate-limiting enzyme of polyamine synthesis ornithine decarboxylase (ODC) was not affected. Polyamine turnover, as assessed by spermine/spermidine acetyltransferase (SSAT) abundance, activity and gene expression, was not affected by treatment. However, the gene expression of the cytokine TNF-α increased in fish fed the methionine-deficient diet, indicative of stressful conditions in the liver. Even though taurine concentrations in the liver were not affected by treatment, methionine and taurine concentrations in muscle decreased due to methionine deficiency. Concomitantly, liver phospholipid and cholesterol concentrations were reduced, while NEFA concentrations were elevated. In conclusion, methionine deficiency did not increase polyamine turnover through depletion of hepatic SAM, as assessed by SSAT activity and abundance.

Aryl hydrocarbon receptor protein and Cyp1A1 gene induction by LPS and phenanthrene in Atlantic cod (Gadus morhua) head kidney cells.

The objective of this study was to evaluate interactions between environmental toxicants and cod immune cells during inflammation. Phenanthrene is abundant in plant oils (rapeseed, palm, and soya oil) as compared to fish oils, and consequently constitute an undesirable element in plant replacement diets in aquaculture. Phenanthrene was added to head kidney cell cultures, alone or together with LPS (lipopolysaccharide) or poly I: C (polyinosinic acid: polycytidylic acid), and the responses were evaluated in terms of protein and gene expression. The results showed that LPS, poly I: C or phenanthrene, added to the cultures separately, induced aryl hydrocarbon receptor (AhR) protein expression. Phenanthrene treatment in combination with LPS induced AhR protein expression and Cyp1A1 gene transcription, which not was observed combining poly I: C and phenanthrene. Phenanthrene exposure up regulated the transcription of common stress and detoxification enzymes like catalase, caspase 3 and glutathione S-transferase alfa 3 subunit B (GSTAB3), while LPS exposure alone or combined with phenanthrene down regulated GSTAB3 and catalase in cod leukocytes. It seems clear that immune regulation and phenanthrene induced signaling pathways interact; transcriptional down regulation of detoxification and antioxidant enzymes by LPS could indicate that combating bacterial infections is the number one priority in these cells, and that AhR and Cyp1A1 is somehow involved in this signaling cascade. LPS seems to affect the mitogen activated protein kinases (MAPKs) pathways (P-p38 and ERK1/2) thus modulating the AhR protein and Cyp1A1 gene transcription, while phenanthrene possibly activates AhR by ligand binding.

Arginine supplementation and exposure time affects polyamine and glucose metabolism in primary liver cells isolated from Atlantic salmon.

Arginine has been demonstrated to enhance glucose and lipid oxidation in mammals through activation of polyamine turnover. We aimed to investigate how arginine affects energy utilization through polyamine metabolism and whether this effect is time dependent. Primary liver cells were isolated from Atlantic salmon (2.2 kg body weight) fed diets containing 25.5 (low arginine, LA) or 36.1 (high arginine, HA) g arginine/kg dry matter for 12 weeks, to investigate the effect of long-term arginine supplementation. The cells were cultured for 24 h in L-15 medium to which either alpha-difluoromethylornithine (DFMO) or N (1),N (11)-diethylnorspermine (DENSPM) was added. Analysis of the medium by nuclear magnetic resonance revealed significant differences between the two dietary groups as well as between cells exposed to DFMO and DENSPM, with decreased glucose, fumarate and lactate concentrations in media of the HA cells. Liver cells from fish fed the HA diet had higher spermidine/spermine-N1-acetyltransferase protein abundance and lower adenosine triphosphate concentration as compared to the LA-fed fish, while gene expression was not affected by either diet or treatment. Primary liver cells isolated from salmon fed a commercial diet and cultured in L-15 media with or without arginine supplementation (1.82 or 3.63 mM) for 48 h, representing short-term effect of arginine supplementation, showed differential expression of genes for apoptosis and polyamine synthesis due to arginine supplementation or inhibition by DFMO. Overall, arginine concentration and exposure time affected energy metabolism and gene regulation more than inhibition or activation of key enzymes of polyamine metabolism, suggesting a polyamine-independent influence of arginine on cellular energy metabolism and survival.

A co culture approach show that polyamine turnover is affected during inflammation in Atlantic salmon immune and liver cells and that arginine and LPS exerts opposite effects on p38MAPK signaling.

This study assess which pathways and molecular processes are affected by exposing salmon head kidney cells or liver cells to arginine supplementation above the established requirements for growth support. In addition to the conventional mono cultures of liver and head kidney cells, co cultures of the two cell types were included in the experimental set up. Responses due to elevated levels of arginine were measured during inflammatory (lipopolysaccharide/LPS) and non -inflammatory conditions. LPS up regulated the genes involved in polyamine turnover; ODC (ornithine decarboxylase), SSAT (spermidine/spermine-N1-acetyltransferase) and SAMdc (S-adenosyl methionine decarboxylase) in head kidney cells when co cultured with liver cells. Regardless of treatment, liver cells in co culture up regulated ODC and down regulated SSAT when compared to liver mono cultures. This suggests that polyamines have anti-inflammatory properties and that both salmon liver cells and immune cells seem to be involved in this process. The transcription of C/EBP ß/CCAAT, increased during inflammation in all cultures except for liver mono cultures. The observed up regulation of this gene may be linked to glucose transport due to the highly variable glucose concentrations found in the cell media. PPARα transcription was also increased in liver cells when receiving signals from head kidney cells. Gene transcription of Interleukin 1ß (IL-1ß), Interleukin-8 (IL-8), cyclooxygenase 2 (COX2) and CD83 were elevated during LPS treatment in all the head kidney cell cultures while arginine supplementation reduced IL-1ß and IL-8 transcription in liver cells co cultured with head kidney cells. This is probably connected to p38MAPK signaling as arginine seem to affect p38MAPK signaling contrary to the LPS induced p38MAPK signaling, suggesting anti-inflammatory effects of arginine/arginine metabolites. This paper shows that co culturing these two cell types reveals the connection between metabolism and inflammation, suggesting different pathways and candidate biomarkers to be further explored.

Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for ß-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and ß-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

Cytokine gene expression and prostaglandin production in head kidney leukocytes isolated from Atlantic cod (Gadus morhua) added different levels of arachidonic acid and eicosapentaenoic acid.

Primary head kidney leukocytes from Atlantic cod were isolated to evaluate the use of arachidonic acid and eicosapentaenoic acid by cyclooxygenases and the production of prostaglandins E2 and E3. The expression of cyclooxygenase genes and selected interleukin genes like Interleukin 1ß, Interleukin 6, interleukin 8 and interleukin 10 were monitored. Increasing concentrations of eicosapentaenoic acid and arachidonic acid in equal amounts increased cyclooxygenase2 transcription as well as cell secretion of prostaglandin E2. Even though the ratio of the two fatty acids was 1:1, the ratio between prostaglandin E2 and E3 was 50:1. The addition of arachidonic acid alone increased prostaglandin E2 secretion but did not induce cyclooxygenase2 transcription. However, when the concentration of eicosapentaenoic acid was increased, maintaining arachidonic acid constant, both prostaglandin E3 and prostaglandin E2 production was induced and the prostaglandin E2 production was higher than in cell cultures only added arachidonic acid. An up-regulation of cyclooxygenase2 transcription was also observed. The addition of the two fatty acids also affected the immune response by alteration of leukocytic cytokines gene expression. According to our results the Cyclooxygenase in cod seem to prefer arachidonic acid as substrate. Therefore, we suggest that the shift from marine oils (rich in n-3 fatty acids) to plant oils (higher in n-6 fatty acids) in the diet of commercially reared Atlantic cod could have negative effects on the whole organism through the increase in the production of prostaglandins belonging to those derived from n-6 fatty acids.

Taurine attenuates apoptosis in primary liver cells isolated from Atlantic salmon (Salmo salar).

One of the many functions of taurine is to protect cells against oxidation, by protecting mitochondrial integrity and respiration. Taurine metabolism has attracted much attention in fish nutrition due to the fact that as plant ingredients replace fishmeal, dietary taurine has declined. As the endogenous synthesis of taurine might be too low to protect cells against oxidative stress and apoptosis, the present study aimed to test whether taurine may protect liver cells from apoptosis. Liver cells isolated from Atlantic salmon (Salmo salar) were grown in media supplemented with a physiological concentration of taurine (25 (se 0·5) mm) or without any taurine supplementation (14 (se 3) µm) for 3 d. To increase oxidation in the mitochondria and maximise any cellular response of taurine supplementation, 100 µm-CdCl2 was added or not added to the cells at day 3. At day 4, cells were harvested and assessed for viability. As expected, the addition of CdCl2 decreased cell viability without showing any interaction with taurine supplementation. Cells grown in the taurine-supplemented media had lower protein abundance of active caspase-3. In addition, the protein abundance of phosphorylated mitogen-activating phosphokinase (P-p63, P-p42/44 and P-p38) as well as cytochrome P450 were reduced when taurine was added to the media. Cells grown without taurine supplementation had a more condensed chromatin and more smeared DNA, also pointing to a higher apoptosis in these cells. In conclusion, taurine attenuated apoptosis in primary liver cells isolated from Atlantic salmon, and as such, taurine may be conditionally indispensable in Atlantic salmon.

Transcriptional evidence for low contribution of oil droplets to acute toxicity from dispersed oil in first feeding Atlantic cod (Gadus morhua) larvae.

We evaluated the potential contribution of oil droplets to the toxicity of dispersed oil to first feeding fish larvae. Atlantic cod larvae were exposed to five concentrations of either artificially weathered (200°C residue) dispersed oil (D1-D5) containing oil droplets [medium size 11-13 µm based on volume] and water-soluble fraction [WSF] or the filtered dispersions containing only the corresponding equilibrium WSFs only (W1-W5). The larvae were exposed for 4 days and harvested for transcriptional analysis at 13 days post hatching. The most significant differently expressed genes were observed in cod larvae exposed to the highest concentration of the dispersed oil (containing 10.41 ± 0.46 µg ∑PAH/L), with CYP1A showing the strongest response. Functional analysis further showed that the top scored network as analyzed with Ingenuity Pathway Analysis was "Drug Metabolism, Endocrine System Development and Function, Lipid Metabolism". Oil exposure also increased the expression of genes involved in bone resorption and decreased the expression of genes related to bone formation. In conclusion, oil exposure affects drug metabolism, endocrine regulation, cell differentiation and proliferation, apoptosis, fatty acid biosynthesis and tissue development in Atlantic cod larvae. The altered gene transcription was dominated by the WSF and the corresponding oil droplet fraction only had a moderate contribution to the observed changes.

Ontogenetic expression of maternal and zygotic genes in Atlantic cod embryos under ambient and thermally stressed conditions.

The embryonic stages of Atlantic cod (Gadus morhua) are especially sensitive to incubation temperature. The purpose of the present study was to follow the ontogenetic expression of selected genes of maternal (pou2 and nanog) and zygotic origin (hsp70, hsp90α and stip1), in Atlantic cod embryos under ambient and thermally stressed conditions. The study also investigated how reference genes can be applied to studies on embryonic development, when maternal genes are degraded and the zygotic transcription stabilizes. Three batches of eggs were reared and gene expression profiles from the reference and target genes were determined. The embryos were reared at ambient 6 °C, and 10 °C for continuous long-term and acute short-term heat exposure. Both pou2 and nanog showed reduced expression whereas the zygotic and reference genes showed increased expression until stabilizing at gastrulation, when a normalized ontogenetic expression profile of target genes could be generated. pou2 and nanog were not affected by thermal stress. In contrast, hsp70 and hsp90α were upregulated after short-term heat exposure at the early blastula (hsp70 only), late blastula, 50% epiboly and 90% epiboly stages (hsp90α only). Long-term heat exposure of Atlantic cod embryos upregulated both hsp70 (90% epiboly) and hsp90α (90% epiboly and 20-somites). The results suggest that a cellular defense mechanism is activated even in the earliest stages of embryonic development, a period critical to developmental temperature.