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Atlantic salmon were fed fish meal-based diets supplemented with either 100% fish oil (FO) or 100% rapeseed oil (RO) from an initial weight of 85 g to a final average weight of 280 g. The effects of these diets on the capacity of Atlantic salmon hepatocytes to elogate, desaturate, and esterify [1-14C]18∶1n−9 and the immediate substrates for the Δ5 desaturase, [1-14C]20∶3 n−6 and [1-14C]20∶4n−3, were investigated. Radiolabeled 18∶1n−9 was mainly esterified into cellular TAG, whereas the more polyunsaturated FA, [1-14C]20∶3n−6 and [1-14C]20∶4n−3, were primarily esterified into cellular PL. More of the elongation product, [1-14C]20∶1n−9, was produced from 18∶1n−9 and more of the desaturation and elongation products, 22∶5n−6 and 22∶6n−3, were produced from [1-14C]20∶3n−6 and [1-14C]20∶4n−3, respectively, in RO hepatocytes than in FO hepatocytes. Further, we studied whether increased addition of [1-14C]18∶1n−9 to the hepatocyte culture media would affect the capacity of hepatocytes to oxidize 18∶1n−9 to acid-soluble products and CO2. An increase in exogenous concentration of 18∶1n−9 from 7 to 100 μM resulted in a nearly twofold increase in the amount of 18∶1n−9 that was oxidized. The conversion of 20∶4n−3 and 20∶3n−6 to the longer-chain 22∶6n−3 and 22∶5n−6 was enhanced by RO feeding in Atlantic salmon hepatocytes. The increased capacity of RO hepatocytes to produce 22∶6n−3 was, however, not enought to achieve the levels found in FO hepatocytes. Our data further showed that there were no differences in the hepatocyte FA oxidation capacity and the lipid deposition of carcass and liver between the two groups. [ABSTRACT FROM AUTHOR]

Adequate dietary supply of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) is required to maintain health and growth of Atlantic salmon (Salmo salar). However, salmon can also convert α-linolenic acid (18:3n-3) into eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) by sequential desaturation and elongation reactions, which can be modified by 20:5n-3 and 22:6n-3 intake. In mammals, dietary 20:5n-3 + 22:6n-3 intake can modify Fads2 expression (Δ6 desaturase) via altered DNA methylation of its promoter. Decreasing dietary fish oil (FO) has been shown to increase Δ5fad expression in salmon liver. However, it is not known whether this is associated with changes in the DNA methylation of genes involved in polyunsaturated fatty acid synthesis. To address this, we investigated whether changing the proportions of dietary FO and vegetable oil altered the DNA methylation of Δ6fad_b, Δ5fad, Elovl2, and Elovl5_b promoters in liver and muscle from Atlantic salmon and whether any changes were associated with mRNA expression. Higher dietary FO content increased the proportions of 20:5n-3 and 22:6n-3 and decreased Δ6fad_b mRNA expression in liver, but there was no effect on Δ5fad, Elovl2, and Elovl5_b expression. There were significant differences between liver and skeletal muscle in the methylation of individual CpG loci in all four genes studied. Methylation of individual Δ6fad_b CpG loci was negatively related to its expression and to proportions of 20:5n-3 and 22:6n-3 in the liver. These findings suggest variations in dietary FO can induce gene-, CpG locus-, and tissue-related changes in DNA methylation in salmon., (© 2019 AOCS.)

The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

Atlantic salmon (Salmo salar) (90 g) were fed four different diets for 21 weeks (final weight 344 g). The levels of n-3 highly unsaturated fatty acids (HUFA) ranged from 11% of the total fatty acids (FA) in the low n-3 diet to 21% in the intermediate n-3 diet, to 55 and 58% in the high n-3 diets. The high n-3 diets were enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). Increasing dietary levels of n-3 HUFA led to increasing percentages (from 31 to 52%) of these FA in liver lipids. The group fed the highest level of DHA had higher expressions of peroxisome proliferator-activated receptor (PPAR) beta and the FA beta-oxidation genes acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase (CPT)-II, compared to the low n-3 groups. The high n-3 groups had reduced activity of mitochondrial cytochrome c oxidase and beta-oxidation capacity, together with increased activities of superoxide dismutase (SOD) and caspase-3 activities. In the group fed the highest level of n-3 HUFA, decreased percentages of major phospholipids (PL) in the mitochondrial and microsomal membranes of the liver were also apparent. The percentage of mitochondrial cardiolipin (Ptd(2)Gro) was 3.1 in the highest n-3 group compared to 6.6 in the intermediate group. These data clearly show an increased incidence of oxidative stress in the liver of fish fed the high n-3 diets.

The white muscle of Atlantic salmon metabolizes FA with different chain lengths and different saturations at different rates, but few details are available on the processes involved or the products formed. We have investigated how multinucleated muscle cells (myotubes) in culture metabolize [1-(14)C]8:0, [1-(14)C]18:1n-9, and [1-(14)C]20:5n-3. The myotubes were formed by the differentiation of isolated myosatellite cells from the white skeletal muscle of salmon fry. Almost all (98%) of the [1-(14)C]8:0 substrate was oxidized to acid-soluble products (ASP) and (14)CO2 after 48 h of incubation, whereas only approximately 50% of the [1-(14)C]18:1n-9 and [1-(14)C]20:5n-3 substrates were oxidized. However, only one cycle of beta-oxidation was measured by the method used. For all three substrates, the main ASP were acetate and a combined fraction of oxaloacetate and malate. Nearly twice as much radioactivity from the [1-(14)C]20:5n-3 substrate was found in the cellular lipids as radioactivity from [1-(14)C]18:1n-9, indicating that [1-(14)C]20:5n-3 was taken up into muscle cells more rapidly than [1-(14)C]18:1n-9. Approximately 10% of the added [1-(14)C]20:5n-3 substrate and 5% of the added [1-(14)C]18:1n-9 substrate was secreted from the muscle cells into the culture media as esterified lipids. Immunocytochemical staining showed that the cells synthesized apolipoprotein A-I. Differentiated muscle cells also expressed peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta, two transcription factors that are involved in regulating beta-oxidation.

The aim of the present study was to develop a cell culture system for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon adipose tissue. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker for cell proliferation. The cells started to proliferate within 48 h after seeding and continued to proliferate throughout the culture period of 2 wk. Undifferentiated preadipocytes showed a fibroblast-like morphology with a homogeneous cytoplasm devoid of lipid droplets. At confluence, an exogenous lipid mixture was added to the cell cultures. The preadipocytes became larger and rounder during the subsequent days, and the cytoplasm gradually filled with lipid-rich droplets. These droplets were revealed by oil red O staining. Immunocytochemical staining showed that differentiated adipocytes expressed detectable levels of the three regulatory proteins associated with adipocyte differentiation: peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and leptin. The cells also showed activity of glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), a biochemical marker of adipocyte differentiation. The morphological and biochemical data presented here show that fish preadipocytes have properties that are similar to those of preadipocytes in mammals. We conclude therefore that salmon adipose tissue contains a sizable population of preadipocytes. Exogenous lipids promote the activation of adipose-related genes and induce the differentiation of fish preadipocytes in vitro.