Impact of Hydroxytyrosol-Rich Extract Supplementation in a High-Fat Diet on Gilthead Sea Bream (Sparus aurata) Lipid Metabolism

Abstract

High-fat diets (HFDs) enhance fish growth by optimizing nutrient utilization (i.e., protein-sparing effect); however, their potential negative effects have also encouraged the search for feed additives. This work has investigated the effects of an extract rich in a polyphenolic antioxidant, hydroxytyrosol (HT), supplemented (0.52 g HT/kg feed) in a HFD (24% lipid) in gilthead sea bream (Sparus aurata). Fish received the diet at two ration levels, standard (3% of total fish weight) or restricted (40% reduction) for 8 weeks. Animals fed the supplemented diet at a standard ration had the lowest levels of plasma free fatty acids (4.28 ± 0.23 mg/dL versus 6.42 ± 0.47 in the non-supplemented group) and downregulated hepatic mRNA levels of lipid metabolism markers (ppara, pparb, lpl, fatp1, fabp1, acox1, lipe and lipa), supporting potential fat-lowering properties of this compound in the liver. Moreover, the same animals showed increased muscle lipid content and peroxidation (1.58- and 1.22-fold, respectively, compared to the fish without HT), suggesting the modulation of body adiposity distribution and an enhanced lipid oxidation rate in that tissue. Our findings emphasize the importance of considering this phytocompound as an optimal additive in HFDs for gilthead sea bream to improve overall fish health and condition.

Keywords: Sparus aurata; aquafeeds; feed additive; high-fat diet; lipid metabolism; olive polyphenols.

Figure 1

(A) Representative images of liver sections stained with hematoxylin and eosin (H&E) and (B) percentage area occupied by lipid droplets within hepatocytes. (C) Representative images of H&E stained adipose tissue sections, (D) area and (E) number of adipocytes/mm². Tissue samples are from gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with (HF+HT) hydroxytyrosol (0.52 g HT per kg feed) at standard (ST) (3% total fish weight/tank) or restricted ration (RE) (40% reduction) for 9 weeks. Magnification 10×. Data are shown as mean + SEM (n = 6 fish). Statistical differences are indicated in three components: diet (D), ration (R) and interaction (D*R), using two-way ANOVA (p < 0.05, shown in bold).

Figure 2

Relative gene expression of (A) transcription factors, (B) fatty acid uptake and transport markers, (C) peroxisomal and mitochondrial β-oxidation markers, and (D) key lipolytic and lipogenic enzymes in liver of gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with hydroxytyrosol (HF+HT) (0.52 g HT per kg feed) at standard (ST) (3% total fish weight/tank) or restricted ration (RE) (40% reduction) for 8 weeks. Data are shown as mean + SEM (n = 10 fish). Statistical differences are indicated in three components: diet (D), ration (R) and interaction (D*R), using two-way ANOVA (p < 0.05, shown in bold). Comparisons among groups were analyzed by a Tukey’s post hoc test when the interaction between the two factors was significant, and significant differences are indicated by asterisks (p < 0.05 shown as *; p < 0.01 **; p < 0.001 ***).

Figure 3

Relative gene expression of (A) transcription factors, (B) fatty acid uptake and transport markers, (C) peroxisomal and mitochondrial β-oxidation markers, and (D) key lipolytic and lipogenic enzymes in adipose tissue of gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with hydroxytyrosol (HF+HT) (0.52 g HT per kg feed) at standard (ST) (3% total fish weight/tank) or restricted ration (RE) (40% reduction) for 8 weeks. Data are shown as mean + SEM (n = 10 fish). Statistical differences are indicated in three components: diet (D), ration (R) and interaction (D*R), using two-way ANOVA (p < 0.05, shown in bold). Comparisons among groups were analyzed by a Tukey’s post hoc test when the interaction between the two factors was significant, and significant differences are indicated by asterisks (p < 0.01 shown as **; p < 0.001 ***).

Figure 4

Relative gene expression of (A) fatty acid uptake and transport markers and (B) peroxisomal and mitochondrial β-oxidation markers in white muscle of gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with hydroxytyrosol (HF+HT) (0.52 g HT per kg feed) at standard (ST) (3% total fish weight/tank) or restricted ration (RE) (40% reduction) for 8 weeks. Data are shown as mean + SEM (n = 10 fish). Statistical differences are indicated in three components: diet (D), ration (R) and interaction (D*R), using two-way ANOVA (p < 0.05, shown in bold). Comparisons among groups were analyzed by a Tukey’s post hoc test when the interaction between the two factors was significant, and significant differences are indicated by asterisks (p < 0.05 shown as *).

Figure 5

Representative Western blots and quantification of CD36 protein levels normalized to total protein (Revert^TM) in liver samples of gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with hydroxytyrosol (HF+HT) (0.52 g HT per kg feed) at standard (ST) (3% total fish weight/tank) or restricted ration (RE) (40% reduction) for 8 weeks. Data are shown as mean + SEM (n = 4 fish). Statistical differences are indicated in three components: diet (D), ration (R) and interaction (D*R), using two-way ANOVA (p < 0.05, shown in bold).

Figure 6

(A) Viability and (B) quantification of lipid content by Oil Red O staining in adipocytes incubated at day 8 of culture for 72 h with growth media (GM) (0.1% ethanol), GM 5 µL/mL of lipid mixture (LM), or hydroxytyrosol (HT) at different concentrations (10 and 100 µM) + 5 µL/mL of LM. Cells were extracted from gilthead sea bream juveniles fed with the experimental diets. A high-fat diet alone (HF) or supplemented with (HF+HT) HT (0.52 g HT per kg feed), at a standard ration (3% total fish weight/tank) for 9 weeks. Data are shown as mean + SEM (n = 4 independent cultures). Statistical differences are indicated in three components: diet (D), treatment (T) and interaction (D*T), using two-way ANOVA (p < 0.05, shown in bold). Comparisons among groups were analyzed by a Tukey’s post hoc test when treatment factor was significant, and significant differences are indicated by letters.