In the present study we analysed the effects of diets containing high levels of plant proteins and with complete replacement of FO by VO on the liver transcriptome of Atlantic salmon, which is the primary metabolic organ of fish, as well as the influence of genotype on these responses. Here we focus on the separate effects of diet and genotype given that interactions, indicating pathways that were differentially affected by diet depending on the genetic background of the fish, were discussed in detail previously .
A common methodological difficulty in this type of nutritional experiment is that effects are typically quite subtle although physiological and metabolic pathways can be impacted by even small fold changes in gene expression. This has been demonstrated by several studies [7, 9, 11] and by previously reported data from the present study showing that low fold changes in gene expression were associated with biochemical differences in tissue lipid class and apolipoprotein composition . Furthermore, low fold changes observed in this study were generally corroborated by RT-qPCR, even if the low expression ratios meant that differences were not always significant. It should also be noted that a total match between the microarray and the RT-qPCR results is not expected due to the approach taken to design RT-qPCR primers on better annotated reference sequences rather than on less well characterized microarray clones. In view of the whole genome duplication event that occurred in salmonid fishes , transcriptomic and gene expression studies are often more challenging due to the presence of duplicated and highly similar genes whose transcripts might be differentially regulated, as observed previously for lipoprotein lipase . Therefore, collectively, and in conjunction with previous studies, data obtained in the present microarray study enabled identification of pathways that may be differentially affected by both dietary oil composition and genetic background related to flesh adiposity.
Effects of diet on lipid metabolism
Within the list of genes affected by diet, those involved in fatty acyl desaturation were prominent, leading to the identification, through GO enrichment analysis, of several terms related to LC-PUFA biosynthetic and metabolic processes. The up-regulation of Δ5 fad and Δ6 fad in both family groups when dietary FO was replaced by VO was confirmed by RT-qPCR. Several studies have previously demonstrated up-regulation of genes involved in LC-PUFA biosynthesis in salmon when FO is replaced by VO [10, 14, 15]. RT-qPCR also confirmed previous work showing that elovl2 is responsive to dietary n-3 LC-PUFA levels , being the only elongase whose expression was up-regulated when FO was replaced by VO. However, a significant effect was only observed in the Lean family group. In addition, both microarray and RT-qPCR analyses indicated that the up-regulation of Δ5 fad and Δ6 fad showed a considerably higher fold-change in the Lean fish, due mainly to lower basal expression of fads in Lean salmon, compared to Fat, when fed FO. These results indicate that the activity of this biosynthetic pathway may be dependent on the genetics of the fish, with different family groups showing differences in the magnitude of response. The liver fatty acid composition revealed that differences in EPA and DHA levels between fish fed either diet were smaller in the Lean fish, due to higher n-3 LC-PUFA in fish fed VO and lower n-3 LC-PUFA in fish fed FO, compared to the equivalent treatments in the Fat group. In addition, intermediates in the biosynthetic pathway, such as 20:4n-3 and 22:5n-3, tended to be present at higher levels in the Lean family group, suggesting that differences observed in the levels of mRNA of LC-PUFA biosynthesis genes, which have been shown to correlate with the enzymatic activity of this pathway in salmon [16, 17], were reflected in biochemical composition.
Another lipid metabolism gene significantly affected by diet was FAS, which was up-regulated in both family groups when fed VO. A well demonstrated effect of dietary FO supplementation in mammals is hypotriglyceridemia, resulting from a coordinated effect of n-3 LC-PUFA in suppressing hepatic lipogenesis and enhancing fatty acid oxidation in liver and muscle . Furthermore, this gene also appears to be regulated at a pre-translational level and hence changes in FAS transcription are likely to result in important effects in terms of enzyme activity . Similar mechanisms are believed to operate in fish but, although reduced hepatic lipogenic activity modulated by LC-PUFA has been demonstrated in vitro , a direct relationship with dietary FO and VO has not always been clear in vivo [21, 22]. The regulation of FAS in response to FO replacement by VO did not show an interaction with the flesh leanness/fatness phenotype in this study, as might have been expected. This was because genotype also had a significant effect, with the Lean group having lower levels of FAS expression than the Fat fish, with a similar fold-change in both diets.
Regulation of lipid metabolism is complex and controlled by several transcription factors and nuclear receptors, including PPARs and SREBPs. SREBP-1c is a major regulator of lipogenesis in mammals . Here we measured the expression of SREBP-1 as there is no evidence for the existence of alternatively spliced isoforms in salmon, and primers corresponded to an identical region in mammalian SREBP-1a and SREBP-1c . Our results agree with Minghetti et al. , who showed SREBP-1 was increased by cholesterol and decreased by EPA and DHA supplementation in a salmon cell line, denoting a similar nutritional regulation to mammals . However, there was a clear genetic effect as expression of SREBP-1 was 3-fold higher in Fat salmon fed VO, containing lower EPA, DHA and cholesterol, than in fish fed FO, whereas no regulation was observed in the Lean group.
PPARs have been less studied in fish than in mammals but present evidence suggests PPARα and PPARβ have similar ligands and functions to their mammalian homologues, while PPARγ may present some functional differences [24, 25]. LC-PUFA are well recognised enhancers of PPARα activity in fish, and while the response of PPARβ to LC-PUFA might be variable between fish species, an enhancement of activity in sea bass, plaice and sea bream [24–26] and of expression in Atlantic salmon  has been observed. In addition, and unlike rodents, PPARα and PPARβ have a similar pattern of expression in response to fasting and feeding in sea bream liver, indicating that they may be regulated similarly . In the present study, PPARα was down-regulated when VO replaced FO but only in the Lean family group and, although not statistically significant, PPARβ showed a similar trend, suggesting similar transcriptional regulation of these nuclear receptors by dietary fatty acid composition. These results thus indicate that the genetic background of the fish might affect PPAR transcriptional responses to LC-PUFA. In contrast, no nutritional regulation was observed for PPARγ transcription in liver, in accordance with previous studies in fish, including salmon, and its predominant role in adipocytes [24, 28].
The hypotriglyceridemic effects of n-3 LC-PUFA in mammals involve activation of PPARα, leading to up-regulation of β-oxidation genes (including carnitine palmitoyltransferase I - CPT1 and acyl-CoA oxidase - ACO) and suppression of SREBP-1c transcription that down-regulates lipogenic enzymes [29, 30]. As previously reported, FAS expression was up-regulated in both family groups fed the VO diet but neither CPT1 nor ACO expression, was affected . As elovl2 expression was only altered in the Lean fish and both Δ5 fad and Δ6 fad showed greater up-regulation in Lean salmon fed VO, we may speculate that PPARα (and potentially also PPARβ) expression may be involved in down-regulation of LC-PUFA biosynthesis. Paradoxically, fatty acyl desaturases are regulated by both SREBPs and PPARs in mammals . In addition, PPARα agonists regulate the transcriptional activity of elongases in rat, although only elovl5 and not elovl2 . However, in mammals, PPARα ligands induce the transcription of elongases and desaturases while we observed an up-regulation of elovl2 and a stronger stimulation of Δ5 fad and Δ6 fad transcription when PPARα expression was lower. In the rat and human Δ6 fad gene promoters, both PUFA and PPARα response regions have been identified which suppress and induce, respectively, Δ6 fad expression . The molecular mechanisms of transcriptional regulation of these genes are complex and will require further investigation in salmon . In contrast, target genes of SREBP-1 remain elusive and, although it may regulate FAS expression , this was only observed in Fat fish whereas, in the Lean group, another mechanism is required to explain up-regulation of FAS in VO-fed fish as expression of SREBP-1 was unaffected. Nonetheless, the action of SREBP-1 is under the regulation of liver X receptor (LXR) and these complex pathways have only recently started to be investigated in fish .
Another gene affected by diet was squalene epoxidase (SQLE), which was up-regulated by VO but only markedly in the Lean family group. This enzyme catalyses the first oxygenation step in sterol biosynthesis, a pathway identified earlier as presenting a diet × genotype interaction . In contrast, cytochrome P450 reductase (CPR) was down-regulated in salmon fed VO, particularly in Lean fish. This enzyme has multiple roles as the electron donor for several oxygenase enzymes, such as cytochrome P450 (involved in drug and xenobiotic metabolism, and sterol and bile acid synthesis), HOX and cytochrome b5 (which supports both sterol and LC-PUFA biosynthesis pathways). In addition, it has key roles in the biosynthesis of several signalling factors and the regulation of oxidative response genes [reviewed by ]. CPR is transcriptionally regulated by PPARα in mouse and, given the comparable PPARα and CPR expression in Lean salmon fed VO, similar regulation likely occurs in salmon. However, changes in CPR expression can be related to several processes that were affected by FO replacement. Thus, CPR expression could be linked to changes in both cholesterol and LC-PUFA biosynthesis, both more marked in Lean fish, although this is unlikely because VO induced up-regulation of these pathways. A more likely association is with cell oxidant metabolism, also suggested by the microarray results as being possibly down-regulated in VO-fed fish. In particular, down-regulation of HOX in salmon fed VO, more marked for Lean fish correlating with CPR expression, might be an indication of this.
Effect of diet on carbohydrate and intermediate metabolism
Within the metabolism genes that were identified by the microarray analysis as being significantly affected by dietary oil substitution, a few relate to carbohydrate metabolism, particularly glucose and intermediary metabolism. Given that similar effects were observed in previous salmonid studies, and that a few signal transduction genes present in the list of diet significant effects are also potentially implicated in these pathways, these results warrant further discussion, even if the observed fold changes were low. An association between lipid and carbohydrate metabolism in salmon is not surprising given that the pathways of lipogenesis, lipolysis, glycolysis, gluconeogenesis and pentose phosphate shunt are all interrelated in the regulation of body energy homeostasis. In mammals, the role of LC-PUFA as "fuel partitioners" involves both directing fatty acids away from anabolic and towards catabolic routes as well as enhancing glucose flux to glycogen, mediated by effects on SREBP-1 and transcription factors that regulate key genes of lipid metabolism and glycolysis . Similar mechanisms may operate in fish but differences are likely given that carnivorous fish like salmon have low capacity to use carbohydrate and appear to show features of glucose intolerance [36, 37]. Nonetheless, dietary n-3/n-6 ratio has been shown to influence mRNA levels of the glucose transporter GLUT4 in Atlantic salmon muscle, with some reflection in plasma glucose . In addition to a decreased hexokinase and phosphoenolpyruvate carboxykinase expression, complete replacement of FM and FO by vegetable alternatives in rainbow trout resulted in a slightly increased expression of glycerol kinase, as observed here . This enzyme is at the intersection of lipid-carbohydrate metabolism and over-expression of this gene in human muscle and rat hepatoma cells resulted in higher TAG synthesis and up-regulation of the pentose phosphate pathway providing reducing power for lipogenesis . Panserat et al.  hypothesised that the up-regulation of glycerol kinase may be related to higher lipid biosynthesis in liver when trout were fed plant-based diets. Similarly, our results, associated with the observed changes in FAS mRNA when VO replaced FO, suggest a possible relationship with lipogenesis. Also possibly related with this was the up-regulation of two different biotinidase clones with the potential to increase availability of substrates for FAS and/or gluconeogenesis in VO-fed fish. This gene, besides being involved in the regulation of gene expression, including genes of glucose metabolism, codes for an enzyme that recycles biotin, which is a co-factor for several carboxylases responsible for production of substrates for lipogenesis and gluconeogenesis .
Another gene affected by diet was alpha-enolase, which was slightly down regulated in Lean fish fed VO. A similar effect was observed in liver of salmon fed rapeseed oil in comparison to FO . This glycolytic enzyme participates in the conversion of glucose to pyruvate, a key intermediate at the intersection of multiple metabolic pathways, including lipogenesis. Thus, this might result in lower levels of pyruvate for conversion to acetyl-CoA in VO-fed fish. This result does not necessarily conflict with an increase in lipogenesis given that, in fish, carbon skeletons for de novo fatty acid production are mainly derived from amino acid catabolism rather than from carbohydrates, whose main contribution towards lipogenesis is to supply NADPH via the pentose-phosphate pathway .
Finally, a few signalling genes that were significantly affected by diet might also have an effect on glucose metabolism, assuming that similar cascades exist in fish. One of these is phosphoinositide 3-protein kinase (PI3K), which mediates insulin's effects on glucose, lipid and protein metabolism, and that was significantly down regulated in VO-fed fish. Among other roles, it regulates glucose cellular uptake in mammals, recruiting GLUT4 transporters to the cell surface . In addition, it is found upstream of a signal transduction cascade regulating glycogen synthesis through glycogen synthase, by inactivating glycogen synthase kinase-3 (GSK3) [41, 42]. In our study, expression of GSK3-binding protein (GBP) was significantly increased in VO-fed Lean fish. GBP is a protein that blocks GSK3, which in turn inactivates glycogen synthase . Hence, it is possible that the oil composition of the diet might also affect glucose metabolism and glycogen storage.
Effect of diet on oxidative stress and immune response
Increased oxidative stress associated with the consumption of FO has been typically reported in fish and mammals [27, 44, 45]. Accordingly, genes related to oxidant metabolism were found in the significant list for diet. A thioredoxin domain-containing protein, possessing an antioxidant role , and GST, which detoxifies peroxidised lipids and xenobiotics , were down-regulated in salmon fed VO, consistent with the higher auto-oxidative potential of LC-PUFA in FO. However, quantification of GST by RT-qPCR was not consistent with the microarray result, although the possibility exists that different GST genes with differential regulation exist in salmon and this requires clarification. In addition, the observed down-regulation of HOX in VO-fed fish, validated by RT-qPCR, might be related to a decrease in oxidative stress in these fish. This enzyme catalyses the degradation of heme and can be induced by oxidative stress  and may be increased during pro-inflammatory states, being thought to increase resistance to oxidative injury and ameliorate inflammation . The n-3 LC-PUFA in FO have important anti-inflammatory actions in mammals , which does not correlate with the expression of HOX and its putative role in inflammation in this case. Inflammation is an important mechanism in immune defence but, in fish, the demonstrated effects of LC-PUFA on immune and inflammatory mechanisms have been inconsistent . However, a recent study has clearly shown an effect of dietary oil composition on the progression of a myxosporean parasite infection in Gilthead sea bream, with fish fed the VO diet showing higher signs of the disease and faster course of infection in comparison with those on a FO diet . On the other hand, the synthesis of pro-inflammatory eicosanoids was increased in the intestine of salmon fed vegetable-based diets in response to acute stress . In the present study immune response was the second highest category of genes affected by diet, after metabolism. Whether this is due to the potential anti-inflammatory role of dietary FO or whether VO diets can have detrimental health effects is not clear as the fold-changes were subtle, as expected in unchallenged animals. Nonetheless, the majority of genes related to processes of both innate and adaptive immunity were up-regulated in fish fed VO. Only T-cell and leukotriene B4 (LTB4) receptors, that are reduced after antigen and LTB4 exposure, respectively, and, in the case of LTB4 receptor, increased after EPA administration [53–55], were down-regulated in salmon fed VO.
Differences in gene expression between Lean and Fat genotypes
Muscle adiposity is a trait of great importance in animal production, aquaculture included, and hence physiological changes induced by genetic selection for this phenotype have been examined in various animals, including rainbow trout [7, 8]. In the present study the main differences between family groups were associated with signal transduction pathways, followed by metabolism. Only a small number of lipid metabolism genes varied in relation to muscle adiposity, as reported previously in rainbow trout, where the main differences were related to lipogenesis and mitochondrial oxidative metabolism [7, 8]. In our study glycerophospholipid metabolism may have been down-regulated in the Lean family group through AGPAT and LPP2, two enzymes acting consecutively on de novo TAG and phospholipid biosynthesis [56, 57]. Quantification of AGPAT and LPP2 expression by RT-qPCR confirmed this down-regulation but fold-changes were too subtle to be significant. AGPAT converts lysophosphatidic acid into phosphatidic acid (PA), while LPP2 then catalyzes the conversion of PA to diacylglycerol. All these molecules can function as second messengers and are involved in the regulation of multiple signalling pathways. Therefore, down-regulation of this pathway in the Lean group has the potential to lower lipid biosynthesis, at least partly explaining the flesh lipid phenotype, but may also alter levels of lipid signalling molecules. On the other hand, differences in muscle adiposity might also be caused by higher hepatic "de novo" fatty acid synthesis in the Fat family group, as indicated by the expression of FAS. In a previous study, no differences were found in the expression of ACO and CPT1, which suggested that the phenotypes could not be explained by differences in β-oxidation . By contrast, in rainbow trout Fat and Lean families, β-oxidation and mitochondrial oxidative metabolism, but not lipogenesis, were affected by genetic selection , although another study using the same trout lines suggested differences related to lipogenesis rather than fatty acid oxidation . Thus, both metabolic processes are likely involved and discrepancies in the data are likely due to lack of methodological sensitivity to detect the small fold-changes that are possibly characteristic of these biological processes and typical in this type of experiment.
PPARα, PPARβ and SREBP-1 were also regulated in response to genotype, being down-regulated in Lean fish, but only when fed the VO diet. In cobia, Rachycentron canadum, a negative correlation was found between PPARα mRNA levels in liver and body lipid deposition . Furthermore, PPARβ appears to play a similar role in fish to that in mammals, as a ubiquitous regulator of fat burning and with a role in energy mobilisation during early development [24, 25]. Therefore, both PPARα and PPARβ might have a role in the control of adipogenesis in fish and it may be the case that, similarly to chickens , Fat salmon might have higher lipid turnover than their Lean counterparts when fed a diet that predisposes for hepatic fat deposition, even though the end result is higher lipid accumulation in liver . To explain this, Collin et al.  suggested that a fat chicken family is better "equipped" to deal with higher circulating levels of TAG when fed a high fat diet, compared to lean chicken. On the other hand, we observed a direct relationship between SREBP-1 and FAS expression in the Fat family group in response to diet, as well as in VO-fed fish in response to genotype. It thus appears that SREBP-1 may be partly responsible for higher lipogenesis in Fat fish, compared to Lean, when fed VO.