The effects of <i>CA</i>de on the early stage of adipogenesis in 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated into mature adipocytes for 8 days, as described under “Materials and Methods”, in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml) from D0 to D2 (D0-D2), from D2 to D8 (D2-D8) and from D0 to D8 (D0-D8). <b>A</b>) Total TG deposition of mature adipocytes. Data are mean ± SD of determinations from three independent experiments. Statistical analysis was performed using one-way ANOVA. ***<i>p</i><0.001, <i>vs</i>. Ctrl. Microscopic images (10X magnification) (<b>B</b>), and lipid quantization (<b>C</b>) of mature adipocytes stained with Oil-Red O. The results are means ± SD of the Oil-red O absorbance values measured at 490 nm from three independent experiments and are expressed as fold changes over control. Statistical analysis was performed using one-way ANOVA. **<i>p</i><0.01 <i>vs</i>. Ctrl. qPCR was performed to detect the mRNA expression of (<b>D</b>) <i>Pparγ</i> at D4, and (<b>E</b>) <i>Glut4</i> and (<b>F</b>) <i>Fapb4</i> at D8 of the adipogenesis. Results are means ± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using one-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 <i>vs</i>. Ctrl at D2, D4 or D8. <b>G</b>) The uptake of 2-DG was then evaluated in mature adipocytes upon stimulation with insulin (100 nmol/l; Ins) for 30 min. The results are means ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA. ***<i>p</i><0.001 <i>vs</i>. Ctrl—Ins; ^###<i>p</i><0.001, <i>CA</i>de D0-D8 + Ins <i>vs</i>. <i>CA</i>de D0-D8—Ins; ^{<i>¶¶¶</i>}<i>p</i><0.001, <i>CA</i>de D0-D2 + Ins <i>vs</i>. <i>CA</i>de D0-D2—Ins; ^†††<i>p</i><0.001, <i>CA</i>de D2-D8 + Ins <i>vs</i>. <i>CA</i>de D2-D8—Ins; ^§<i>p</i><0.05 <i>vs</i>. Ctrl + Ins.</p

The effects of <i>CA</i>de on gene expression during adipogenesis in 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated into mature adipocytes for 8 days, as described under “Materials and Methods”, in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml). At the indicated time points, cells were collected for the extraction of RNA. qPCR was performed to detect the mRNA expression of (<b>A</b>) <i>C/ebpβ</i> at D2, (<b>B</b>) <i>Pparγ</i> at D4, and (<b>C</b>) <i>Glut4</i> and (<b>D</b>) <i>Fapb4</i> at D8 of the adipogenesis. Results are means ± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using Student’s t-test. **<i>p</i><0.01, and ***<i>p</i><0.001 <i>vs</i>. Ctrl at D2, D4 or D8.</p

The effects of <i>CADE</i> on cell cycle progression during adipogenesis in 3T3-L1.

<p>MCE was induced in 3T3-L1 pre-adipocytes with differentiation medium, as described under “Materials and Methods”. Cells were then harvested at 12, 14, and 16 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml) and stained with PI solution for flow cytometer cell cycle analysis. <b>A</b>) Histograms of cell cycle distribution in G₀/G₁, S or G₂/M phases. <b>B</b>) Quantitative analysis of cell cycle distribution. The results are means ± SD of three independent experiments. Statistical analysis was performed using Student’s t-test *<i>p</i><0.05, and ***<i>p</i><0.001, <i>CA</i>de S Phase <i>vs</i>. Ctrl S Phase; ^#<i>p</i><0.05, <i>CA</i>de G₂/M Phase <i>vs</i>. Ctrl G₂/M Phase.</p

The effects of <i>CADE</i> on <i>C/ebpβ</i> gene expression and CREB activation during the early stage of adipogenesis in 3T3-L1.

<p>Adipogenesis was induced in 3T3-L1 pre-adipocytes with the differentiation medium (MDI), as described under “Materials and Methods”. Cells were then harvested at 1, 2, 4 and 8 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml) and processed for qPCR and western blot analysis. <b>A</b>) qPCR of <i>C/ebpβ</i> mRNA expression. Results are means ± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using one-way ANOVA. ***<i>p</i><0.01, <i>vs</i>. 3T3-L1 cells at 0 h; ^###<i>p</i><0.001, <i>CA</i>de 2 h <i>vs</i>. Ctrl 2 h; ^<i>¶</i><i>p</i><0.05, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h; ^†††<i>p</i><0.001, <i>CA</i>de 8 h <i>vs</i>. Ctrl 8 h. <b>B</b>) The representative western blot show levels of the total and Ser¹³³ phosphorylated form of the cAMP response element-binding protein (CREB) and of the β-Actin protein. <b>C)</b> CREB protein binding on <i>C/ebpβ</i> promoter was evaluated by ChIP analysis on 3T3-L1 cells harvested at 4 h after the initiation of differentiation in absence (Ctrl) or presence of <i>CA</i>de (100 μg/ml). ChIP enrichment is relative to input chromatin. Data are expressed as mean ± SD of values from at least three independent experiments. Statistical analysis was performed using Student’s t-test. **<i>p</i><0.01, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h.</p

Effects of <i>CA</i>de on 3T3-L1 pre-adipocytes cell viability.

<p>Effects of <i>CA</i>de on 3T3-L1 pre-adipocytes cell viability.</p

The effects of single flavonoids on <i>C/ebpβ</i> gene expression during the early stage of adipogenesis in 3T3-L1.

<p>Adipogenesis was induced in 3T3-L1 pre-adipocytes with the differentiation medium (MDI). Cells were then harvested at 2, 4 and 8 h after the initiation of differentiation in the absence (Ctrl) or presence of 6.7 μg/ml narirutin (N) or 3.9 μg/ml hesperidin (H) or 5.5 μg/ml vicenin-2 (V). Cells treated with <i>CA</i>de (100 μg/ml) were also used. At the indicated time points, cells were collected for the extraction of RNA. qPCR was performed to detect the mRNA expression of <i>C/ebpβ</i> at 2h (<b>A</b>), 4h (<b>B</b>), and 8h (<b>C</b>) upon adipogenesis. Results are means ± SD of three independent experiments and are expressed as relative changes over control cells at time 0. Statistical analysis was performed using one-way ANOVA. **<i>p</i><0.01, and ***<i>p</i><0.001 <i>vs</i>. Ctrl at 2, 4 or 8 h.</p

<i>Citrus aurantium</i> L. dry extracts promote <i>C/ebpβ</i> expression and improve adipocyte differentiation in 3T3-L1 cells

<div><p>Metabolic and/or endocrine dysfunction of the white adipose tissue (WAT) contribute to the development of metabolic disorders, such as Type 2 Diabetes (T2D). Therefore, the identification of products able to improve adipose tissue function represents a valuable strategy for the prevention and/or treatment of T2D. In the current study, we investigated the potential effects of dry extracts obtained from <i>Citrus aurantium</i> L. fruit juice (<i>CA</i>de) on the regulation of 3T3-L1 cells adipocyte differentiation and function <i>in vitro</i>. We found that <i>CA</i>de enhances terminal adipocyte differentiation of 3T3-L1 cells raising the expression of <i>CCAAT/enhancer binding protein beta</i> (<i>C/Ebpβ</i>), <i>peroxisome proliferator activated receptor gamma</i> (<i>Pparγ</i>), <i>glucose transporter type 4</i> (<i>Glut4</i>) and <i>fatty acid binding protein 4</i> (<i>Fabp4</i>). <i>CA</i>de improves insulin-induced glucose uptake of 3T3-L1 adipocytes, as well. A focused analysis of the phases occurring in the pre-adipocytes differentiation to mature adipocytes furthermore revealed that <i>CA</i>de promotes the early differentiation stage by up-regulating <i>C/ebpβ</i> expression at 2, 4 and 8 h post the adipogenic induction and anticipating the 3T3-L1 cell cycle entry and progression during mitotic clonal expansion (MCE). These findings provide evidence that the exposure to <i>CA</i>de enhances <i>in vitro</i> fat cell differentiation of pre-adipocytes and functional capacity of mature adipocytes, and pave the way to the development of products derived from <i>Citrus aurantium</i> L. fruit juice, which may improve WAT functional capacity and may be effective for the prevention and/or treatment of T2D.</p></div