Author Contributions
Conceptualization, M.T., T.-K.K.Ø., and B.R.; methodology, X.W., M.T., and J.T.; formal analysis, M.B.; investigation, X.W., M.T., and J.T.; writing—original draft preparation, M.B.; writing—review and editing, M.B., T.-K.K.Ø., and B.R.; visualization, M.B.; supervision, B.R.; project administration, B.R.; and funding acquisition, M.B. and B.R. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Microscopic observation of lipid droplets (A–F) and total fatty acid content quantified by gas chromatography (G) in mature Atlantic salmon adipocytes. Differentiated cells at day 9 were incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h. Images were taken with a 20× magnification. All arrows, regardless of color, point to areas with a high presence of lipid droplets. Data are shown as mean + SEM (n = 3) and analyzed by one-way ANOVA (p = 0.0548). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 1.
Microscopic observation of lipid droplets (A–F) and total fatty acid content quantified by gas chromatography (G) in mature Atlantic salmon adipocytes. Differentiated cells at day 9 were incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h. Images were taken with a 20× magnification. All arrows, regardless of color, point to areas with a high presence of lipid droplets. Data are shown as mean + SEM (n = 3) and analyzed by one-way ANOVA (p = 0.0548). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 2.
Delta 9 desaturase activity (A) and relative changes in transcript levels (B) in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h. Data are shown as mean ± SEM (n = 3 for Δ9 desaturase activity, and n = 4 for the relative changes in transcript levels) and analyzed by one-way ANOVA followed by Tukey’s honestly significant difference test (p = 0.2197 and p < 0.0001, respectively). Different letters indicate significant differences between treatments. The activity was calculated based on the 16:0 and 16:1+18:1 produced from [1-14C] PA. OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid. Relative changes in transcript levels were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group was set to zero.
Figure 2.
Delta 9 desaturase activity (A) and relative changes in transcript levels (B) in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h. Data are shown as mean ± SEM (n = 3 for Δ9 desaturase activity, and n = 4 for the relative changes in transcript levels) and analyzed by one-way ANOVA followed by Tukey’s honestly significant difference test (p = 0.2197 and p < 0.0001, respectively). Different letters indicate significant differences between treatments. The activity was calculated based on the 16:0 and 16:1+18:1 produced from [1-14C] PA. OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid. Relative changes in transcript levels were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group was set to zero.
Figure 3.
(A) Relative changes in transcript levels of leptin in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18h GLU). Data are shown as mean ± SEM (n = 4). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18h GLU) as factors. (B) Changes in intracellular leptin concentrations in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3h GLU) and 18 h (18h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3h GLU, and 18h GLU) as factors. Lowercase letters indicate significant differences between fatty acids, and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). Relative changes in leptin transcript levels were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 3.
(A) Relative changes in transcript levels of leptin in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18h GLU). Data are shown as mean ± SEM (n = 4). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18h GLU) as factors. (B) Changes in intracellular leptin concentrations in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3h GLU) and 18 h (18h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3h GLU, and 18h GLU) as factors. Lowercase letters indicate significant differences between fatty acids, and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). Relative changes in leptin transcript levels were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.

Figure 4.
Changes in leptin secretion in the medium from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3 h GLU, and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids, and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 4.
Changes in leptin secretion in the medium from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3 h GLU, and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids, and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 5.
Relative changes in transcript levels of hormone-sensitive lipase (lipe) (A) and lipoprotein lipase (lpl) (B) in matured Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18 h GLU). Samples (n = 4) were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 5.
Relative changes in transcript levels of hormone-sensitive lipase (lipe) (A) and lipoprotein lipase (lpl) (B) in matured Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18 h GLU). Samples (n = 4) were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 6.
Total glycerol (A), non-esterified free fatty acids (NEFA) (B), and [1-14C] from radiolabelled PA recovered in secreted lipids (C) in the media from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 4 for glycerol and NEFA data, and n = 3 for radiolabeled lipids secreted data). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (3 h GLU and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 6.
Total glycerol (A), non-esterified free fatty acids (NEFA) (B), and [1-14C] from radiolabelled PA recovered in secreted lipids (C) in the media from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 4 for glycerol and NEFA data, and n = 3 for radiolabeled lipids secreted data). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (3 h GLU and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 7.
Distribution between free fatty acids (FFA) (A), phospholipids (PL) (B), mono and diacylglycerol (MDG) and triglycerides (TAG) (C), and cholesteryl esters and wax esters (CE + WE) (D) produced from [1-14C] PA in the media from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (3 h GLU and 18 h GLU) as factors. Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 7.
Distribution between free fatty acids (FFA) (A), phospholipids (PL) (B), mono and diacylglycerol (MDG) and triglycerides (TAG) (C), and cholesteryl esters and wax esters (CE + WE) (D) produced from [1-14C] PA in the media from mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18 h GLU). Data are shown as mean ± SEM (n = 3). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (3 h GLU and 18 h GLU) as factors. Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 8.
(A) Mitochondria area from immunostaining images in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18h GLU). Data are shown as mean ± SEM (n = 4–6). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3 h GLU, and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). (B) Relative changes in transcript levels of mfn1 and fis1 (C) in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18 h GLU). Samples (n = 4) were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.
Figure 8.
(A) Mitochondria area from immunostaining images in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 3 (3 h GLU) and 18 h (18h GLU). Data are shown as mean ± SEM (n = 4–6). Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control, 3 h GLU, and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids and capital letters indicate significant differences between conditions (p < 0.05; Tukey’s post hoc test). (B) Relative changes in transcript levels of mfn1 and fis1 (C) in mature Atlantic salmon adipocytes incubated with oleic, palmitic, or eicosapentaenoic acid for 72 h (Control) and serum-deprived in the presence of glucagon thereafter for 18 h (18 h GLU). Samples (n = 4) were analyzed with real-time qPCR; data are presented as −ΔΔCt ± SEM, and the OA group from the Control condition was set to zero. Results are compared by two-way ANOVA using the fatty acid tested (OA, PA, and EPA) and the experimental condition (Control and 18 h GLU) as factors. Lowercase letters indicate significant differences between fatty acids (p < 0.05; Tukey’s post hoc test). Asterisks (*) indicate significant differences between conditions (p < 0.05; Sidak’s test). OA = oleic acid, PA = palmitic acid, and EPA = eicosapentaenoic acid.

Table 1.
Fatty acid composition (% of total) in mature Atlantic salmon adipocytes. Differentiated cells at day 9 were incubated with oleic (OA), palmitic (PA), or eicosapentaenoic acid (EPA) for 72 h (mean ± SEM; n = 3).
Table 1.
Fatty acid composition (% of total) in mature Atlantic salmon adipocytes. Differentiated cells at day 9 were incubated with oleic (OA), palmitic (PA), or eicosapentaenoic acid (EPA) for 72 h (mean ± SEM; n = 3).
Fatty Acid | OA | PA | EPA | p |
---|
16:0 | 10.0 ± 4.5b | 36.4 ± 4.3a | 19.0 ± 0.8b | 0.0058 |
Other SFA§ | 15.5 ± 0.7 | 17.5 ± 1.8 | 16.3 ± 3.1 | 0.8031 |
16:1 n−7 | nd | 3.8 ± 3.8 | 1.0 ± 0.4 | 0.4910 |
18:1 n−7 | 1.9 ± 0.3 | 5.0 ± 2.6 | 2.0 ± 0.1 | 0.3218 |
18:1 n−9 | 47.4 ± 6.7a | 15.8 ± 1.0b | 17.5 ± 0.2b | 0.0020 |
Other MUFA† | 8.5 ± 3.4 | 9.9 ± 5.9 | 8.4 ± 1.2 | 0.9593 |
18:2 n−6 | 3.7 ± 1.0 | 2.2 ± 0.6 | 3.8 ± 0.1 | 0.2617 |
20:4 n−6 | 4.6 ± 0.6 | 4.0 ± 0.9 | 3.3 ± 0.5 | 0.4239 |
20:5 n−3 | ND | 2.9 ± 1.3b | 18.6 ± 3.1a | 0.0010 |
22:5 n−3 | 2.1 ± 0.3 | 4.3 ± 2.7 | 3.5 ± 0.4 | 0.6279 |
22:6 n-−3 | 4.0 ± 0.4 | 2.6 ± 0.7 | 3.3 ± 0.4 | 0.2751 |
Other FA‡ | 5.1 ± 0.0 | 0.7 ± 0.0 | 3.2 ± 0.7 | 0.2252 |
Sum identified | 97.5 ± 0.8 | 96.0 ± 1.9 | 96.8 ± 0.6 | 0.6994 |
Sum EPA/DHA | 4.0 ± 0.4b | 5.6 ± 0.6b | 21.9 ± 3.3a | 0.0011 |
Sum N−3 | 7.8 ± 1.2b | 10.1 ± 2.2b | 26.4 ± 3.9a | 0.0053 |
Sum N−6 | 8.2 ± 1.6 | 6.2 ± 1.2 | 9.1 ± 0.6 | 0.2900 |
Sum N−0 | 25.5 ± 4.8b | 53.9 ± 5.9a | 35.3 ± 3.9ab | 0.0172 |
Table 2.
Relative distribution of radioactivity from [1-14C PA] recovered in different cellular lipid classes in Atlantic salmon differentiated adipocytes incubated with oleic (OA), palmitic (PA), or eicosapentaenoic acid (EPA) for 72 h.
Table 2.
Relative distribution of radioactivity from [1-14C PA] recovered in different cellular lipid classes in Atlantic salmon differentiated adipocytes incubated with oleic (OA), palmitic (PA), or eicosapentaenoic acid (EPA) for 72 h.
(%) | OA | PA | EPA | p |
---|
PL | 46.17 ± 3.37 b | 47.26 ± 7.02 b | 82.33 ± 2.71 a | 0.0026 |
FFA | 2.63 ± 0.25 b | 12.00 ± 2.28 a | 3.35 ± 0.97 b | 0.0064 |
TAG | 5.84 ± 1.10 b | 11.28 ± 1.32 ab | 11.93 ± 1.55 a | 0.0338 |
DAG | 2.08 ± 0.43 | 1.9 ± 0.72 | 2.39 ± 0.67 | 0.8583 |
CE | 43.29 ± 4.67 a | 27.56 ± 4.56 b | ND | 0.0367 |
Total nmol 1 | 5.8 ± 0.35 a | 1.8 ± 0.07 b | 6.7 ± 0.46 a | 0.0001 |
Table 3.
Atlantic salmon primer sequences used for real-time PCR.
Table 3.
Atlantic salmon primer sequences used for real-time PCR.
Gene | Accession No. | Direction | Primer Sequence 5′→ 3′ |
---|
ef1α | AF321836 | Forward | CACCACCGGCCATCTGATCTACAA |
Reverse | TCAGCAGCCTCCTTCTGAACTTC |
etif3 | DW542195 | Forward | CAGGATGTTGTTGCTGGATGGG |
Reverse | ACCCAACTGGGCAGGTCAAGA |
β-actin | AF012125 | Forward | ACATCAAGGAGAAGCTGTGC |
Reverse | GACAACGGAACCTCTCGTTA |
scd | BT044999 | Forward | TGAAATAGTGCTGTCCCGGGCTC |
Reverse | TGGGGAAACCTCTTAGCCACTCCG |
leptin | FJ830677 | Forward | CCAGGCCGCCAGCAGAAACA |
Reverse | GCGCCACTGGACCCACACTC |
lipe | XM_014201599 | Forward | TCCCCAGACGTTTGTGTCAGATGC |
Reverse | GCTTTGGATCCCCCATTAGTTCCTG |
lpl | BI468076 | Forward | TGCTGGTAGCGGAGAAAGACAT |
Reverse | CTGACCACCAGGAAGACACCAT |
mfn1 | BT072406 | Forward | AGTGTGTCCAGTCTTCCGCACA |
Reverse | ACAGGCTACAGCACCCAACCTT |
fis1 | BT072691 | Forward | CCCCAGGGGGCATCCTGTCTTA |
Reverse | TTGCAGCTGGCCGATCTAGCG |