44 research outputs found

    Caffeic acid and hydroxytyrosol have anti-obesogenic properties in zebrafish and rainbow trout models

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    <div><p>Some natural products, known sources of bioactive compounds with a wide range of properties, may have therapeutic values in human health and diseases, as well as agronomic applications. The effect of three compounds of plant origin with well-known dietary antioxidant properties, astaxanthin (ATX), caffeic acid (CA) and hydroxytyrosol (HT), on zebrafish (<i>Danio rerio</i>) larval adiposity and rainbow trout (<i>Onchorynchus mykiss</i>) adipocytes was assessed. The zebrafish obesogenic test (ZOT) demonstrated the anti-obesogenic activity of CA and HT. These compounds were able to counteract the obesogenic effect produced by the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (RGZ). CA and HT suppressed RGZ-increased PPARγ protein expression and lipid accumulation in primary-cultured rainbow trout adipocytes. HT also significantly reduced plasma triacylglycerol concentrations, as well as mRNA levels of the <i>fasn</i> adipogenic gene in the adipose tissue of HT-injected rainbow trout. In conclusion, <i>in vitro</i> and <i>in vivo</i> approaches demonstrated the anti-obesogenic potential of CA and HT on teleost fish models that may be relevant for studying their molecular mode of action. Further studies are required to evaluate the effect of these bioactive components as food supplements for modulating adiposity in farmed fish.</p></div

    Identification of selected molecules of plant origin able to decrease adiposity <i>in vivo</i>.

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    <p>ZOT was conducted on larvae with a standard-length distribution from 7 to 9 mm and initially nourished with SD. Adiposity was quantified in fasting larvae in fish water with 0.1% DMSO as a vehicle control (CT) or 0.1% DMSO plus 50 μM CA (A), 100 μM HT (B), or 100 μM ATX (C). For each larva enrolled, WAT dynamics is expressed as a percentage of initial adiposity. Values are mean ± SEM, n = 4–6 independent experiments (10 animals per group). *P ≤ 0.05, **P ≤ 0.01 compared to CT, using two-tailed unpaired Student's t-test.</p

    qRT-PCR analysis of selected β-oxidation-related gene transcript levels in the dissected perivisceral WAT (A-C) and liver (D-F) of rainbow trout treated with CA and HT or untreated.

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    <p>After one-day fasting, juvenile rainbow trout received intraperitoneal injections of vehicle CT (DMSO in sesame oil 1:3, v/v), vehicle plus CA (10 μg/g body weight), or vehicle plus HT (20 μg/g body weight), and samples were taken after a 24 h exposure period in a fasting state. mRNA levels of <i>acsl1</i> (A, D), <i>hoad</i> (B, E) and <i>pparb</i> (C, F). All mRNA levels were normalized to the geometric mean of the two reference genes, <i>ef1a</i> and <i>actb</i>. Data are shown as mean ± SEM (n = 7–8 fish per condition). *<i>p</i> ≤ 0.05, compared to CT, using Student's t-test.</p

    CA and HT abolished the <i>in vivo</i> obesogenic effect of RGZ.

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    <p>ZOT was applied to larvae previously nourished with SD. Adiposity was quantified in the presence of 0.1% DMSO as a vehicle CT or 0.1% DMSO plus the indicated combination of compounds. Exposure to 1 μM RGZ induced a significantly smaller decrease in adiposity compared with CT. The effect of RGZ was abolished by CA 50 μM (A) and HT (100 μM) (B). Values are mean ± SEM, n = 4 independent experiments (10 animals per group). Significant differences are shown as different letters (<i>p</i> ≤ 0.05) using one-way ANOVA test followed by Tukey’s <i>post hoc</i> test.</p

    Differential effect of selected vegetal molecules of plant origin on WAT dynamics in different body parts of zebrafish larvae.

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    <p>Quantitative analysis of WAT dynamics was performed according to the ZOT protocol and the results are expressed as a percentage of initial adiposity relative to the amount of WAT fluorescence signal attached to each body region. Fasting larvae were exposed to 50 μM CA in 0.1% DMSO (A, C, E), 100 μM HT in 0.1% DMSO (F, H, J), 100 μM ATX in 0.1% DMSO (K, M, O), or 0.1% DMSO as a vehicle CT (A, B, D, F, G, I, K, L, N). Images of relevant regions in representative larvae are presented: head (B-E), viscera (G-J), and tail (L-O). Lateral views, anterior part on the left and dorsal part at the top, under fluorescence microscope after Nile red staining, recorded before (B, C, G, H, L, M) and after 24 h treatment (D, E, I, J, N, O) with (CA, HT, ATX) or without (CT) exposure to the compounds. Insets in each image are enlarged views of isolated adipocytes or groups of adipocytes from each panel, marked by a white rectangle. Values are mean + SEM, n = 4–6 independent experiments (10 animals per group). *<i>p</i> ≤ 0.05 compared to control for each region, using Student's t-test. Scale bar, 500 μm. Abbreviations: e, eye; pf, pectoral fin; pwat, perivisceral white adipose tissue; sb, swim bladder; ai, anterior intestine; swat, subcutaneous white adipose tissue.</p

    qRT-PCR analysis of selected lipid-metabolism-related gene transcript levels in the dissected perivisceral WAT (A-D) and liver (E-H) of rainbow trout treated with CA and HT or untreated.

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    <p>After one-day fasting, juvenile rainbow trout received intraperitoneal injections of vehicle CT (DMSO in sesame oil 1:3, v/v), vehicle plus CA (10 μg/g body weight), or vehicle plus HT (20 μg/g body weight), and samples were taken after a 24 h exposure period in a fasting state. mRNA levels of <i>fasn</i> (A, E), <i>lpl</i> (B, F), <i>lipe1</i> (C, G) and <i>pnpla2</i> (D, H). All mRNA levels were normalized to the geometric mean of the two reference genes, <i>ef1a</i> and <i>actb</i>. Data are shown as mean ± SEM (n = 7–8 fish per condition). *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.0001, compared to CT, using Student's t-test.</p

    Characterization of potential PPARγ signaling pathway antagonism and inhibition of adipogenesis produced by CA and HT in primary-cultured rainbow trout adipocytes.

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    <p>Representative PPARγ immunofluorescence images (A), quantification of PPARγ immunofluorescence protein signal (B), anti-PPARγ immunoreactive band and quantification of PPARγ protein expression by Western blot (C, D), specific lipid content (E) and mRNA levels of adipogenic genes <i>pparg</i> (F) and <i>cebpa</i> (G). Immunofluorescence images show Hoechst nuclei staining (left panels), PPARγ (medium panels) and overlay (right panels). Scale bar, 100 μm. For both protein expression analyses (immunofluorescence, A and B; Western blot, C and D) cells were incubated with vehicle (DM) plus CA (50 μM), HT (100 μM), RGZ (1 μM), or the indicated combination of CA or HT with RGZ, or vehicle CT alone, for 24 h (day 7 of culture). RGZ was used as a potential rainbow trout PPARγ agonist. Representative Western blot images of anti-PPARγ immunoreactive band (top) and the same membrane labelled with Ponceau S (bottom) (D). Lipid content expressed spectrophotometrically as the ratio of absorbance value between ORO and Coomassie blue staining (E). For lipid content analysis cells were incubated with vehicle (DM) plus CA (50 μM), HT (100 μM), LIP (10 μl/ml), or the indicated combination of CA or HT with LIP, or vehicle CT alone, for 72 h (day 7 of culture). mRNA levels of <i>pparg</i> (F) and <i>cebpa</i> (G) were normalized to the geometric mean of the two reference genes, <i>ef1a</i> and <i>ubiquitin</i>. For gene expression analyses cells were incubated with vehicle (DM) plus CA (50 μM), HT (100 μM), RGZ (1 μM), LIP (10 μl/ml), or the indicated combination of CA or HT with RGZ or LIP, or vehicle CT alone for 24 h (day 7 of culture). Data are shown as mean ± SEM (n = 3–7 cell cultures). Significant differences (<i>p</i> ≤ 0.05) are indicated by different letters, using one-way ANOVA followed by Tukey’s <i>post hoc</i> test (B, C, E, F) or the non-parametric Kruskal-Wallis test followed by paired U-Mann Whitney test (G).</p

    The effect of <i>smyhc1</i> knockdown, BTS treatment or <i>hsp90α1</i> mutation on M-line organization in skeletal muscles of zebrafish embryos.

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    <p>Anti-myomesin antibody staining shows the M-line organization in control (A), <i>smyhc1</i> knockdown (B), BTS-treated (C), or <i>slo<sup>tu44c</sup></i> mutant (D) embryos at 72 hpf. Scale bar = 25 µm in A.</p

    Effects of <i>smyhc1</i> knockdown on muscle development in zebrafish embryos.

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    <p>A and B. <i>In situ</i> hybridization shows <i>myod</i> expression in control (A) or <i>smyhc1</i>-ATG-MO (B) injected embryos at 14 hpf. Adaxial cells that give rise to slow muscles are indicated by arrows. C–F. <i>In situ</i> hybridization shows slow-specific <i>troponin C</i> expression in control (C, D) or <i>smyhc1</i>-ATG-MO (E, F) injected embryos at 24 hpf. D and F are cross sections of C and E, respectively. Arrows in D and F indicate slow muscles.</p
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