Caffeic acid and hydroxytyrosol have anti-obesogenic properties in zebrafish and rainbow trout models
Fig 4
Characterization of potential PPARγ signaling pathway antagonism and inhibition of adipogenesis produced by CA and HT in primary-cultured rainbow trout adipocytes.
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 pparg (F) and cebpa (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 pparg (F) and cebpa (G) were normalized to the geometric mean of the two reference genes, ef1a and ubiquitin. 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 (p ≤ 0.05) are indicated by different letters, using one-way ANOVA followed by Tukey’s post hoc test (B, C, E, F) or the non-parametric Kruskal-Wallis test followed by paired U-Mann Whitney test (G).