22 research outputs found

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

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    <p>MCE was induced in 3T3-L1 pre-adipocytes with differentiation medium, as described under ā€œMaterials and Methodsā€. 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 Ī¼g/ml) and stained with PI solution for flow cytometer cell cycle analysis. <b>A</b>) Histograms of cell cycle distribution in G<sub>0</sub>/G<sub>1</sub>, S or G<sub>2</sub>/M phases. <b>B</b>) Quantitative analysis of cell cycle distribution. The results are means Ā± SD of three independent experiments. Statistical analysis was performed using Studentā€™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; <sup>#</sup><i>p</i><0.05, <i>CA</i>de G<sub>2</sub>/M Phase <i>vs</i>. Ctrl G<sub>2</sub>/M Phase.</p

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

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    <p>3T3-L1 pre-adipocytes were differentiated into mature adipocytes for 8 days, as described under ā€œMaterials and Methodsā€, in absence (Ctrl) or presence of <i>CA</i>de (100 Ī¼g/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/ebpĪ²</i> at D2, (<b>B</b>) <i>PparĪ³</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 Ā± SD of three independent experiments and are expressed as relative changes over control. Statistical analysis was performed using Studentā€™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>CA</i>de on the early stage of adipogenesis in 3T3-L1 cells.

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    <p>3T3-L1 pre-adipocytes were differentiated into mature adipocytes for 8 days, as described under ā€œMaterials and Methodsā€, in absence (Ctrl) or presence of <i>CA</i>de (100 Ī¼g/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 Ā± 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 Ā± 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>PparĪ³</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 Ā± 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 Ā± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA. ***<i>p</i><0.001 <i>vs</i>. Ctrlā€”Ins; <sup>###</sup><i>p</i><0.001, <i>CA</i>de D0-D8 + Ins <i>vs</i>. <i>CA</i>de D0-D8ā€”Ins; <sup><i>Ā¶Ā¶Ā¶</i></sup><i>p</i><0.001, <i>CA</i>de D0-D2 + Ins <i>vs</i>. <i>CA</i>de D0-D2ā€”Ins; <sup>ā€ ā€ ā€ </sup><i>p</i><0.001, <i>CA</i>de D2-D8 + Ins <i>vs</i>. <i>CA</i>de D2-D8ā€”Ins; <sup>Ā§</sup><i>p</i><0.05 <i>vs</i>. Ctrl + Ins.</p

    <i>Citrus aurantium</i> L. dry extracts promote <i>C/ebpĪ²</i> expression and improve adipocyte differentiation in 3T3-L1 cells

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    <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/EbpĪ²</i>), <i>peroxisome proliferator activated receptor gamma</i> (<i>PparĪ³</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/ebpĪ²</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

    The effects of single flavonoids on <i>C/ebpĪ²</i> gene expression during the early stage of adipogenesis in 3T3-L1.

    No full text
    <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 Ī¼g/ml narirutin (N) or 3.9 Ī¼g/ml hesperidin (H) or 5.5 Ī¼g/ml vicenin-2 (V). Cells treated with <i>CA</i>de (100 Ī¼g/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/ebpĪ²</i> at 2h (<b>A</b>), 4h (<b>B</b>), and 8h (<b>C</b>) upon adipogenesis. Results are means Ā± 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

    The effects of <i>CADE</i> on <i>C/ebpĪ²</i> gene expression and CREB activation during the early stage of adipogenesis in 3T3-L1.

    No full text
    <p>Adipogenesis was induced in 3T3-L1 pre-adipocytes with the differentiation medium (MDI), as described under ā€œMaterials and Methodsā€. 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 Ī¼g/ml) and processed for qPCR and western blot analysis. <b>A</b>) qPCR of <i>C/ebpĪ²</i> mRNA expression. Results are means Ā± 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; <sup>###</sup><i>p</i><0.001, <i>CA</i>de 2 h <i>vs</i>. Ctrl 2 h; <sup><i>Ā¶</i></sup><i>p</i><0.05, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h; <sup>ā€ ā€ ā€ </sup><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 Ser<sup>133</sup> phosphorylated form of the cAMP response element-binding protein (CREB) and of the Ī²-Actin protein. <b>C)</b> CREB protein binding on <i>C/ebpĪ²</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 Ī¼g/ml). ChIP enrichment is relative to input chromatin. Data are expressed as mean Ā± SD of values from at least three independent experiments. Statistical analysis was performed using Studentā€™s t-test. **<i>p</i><0.01, <i>CA</i>de 4 h <i>vs</i>. Ctrl 4 h.</p

    Identification of a Potent Tryptophan-Based TRPM8 Antagonist With in Vivo Analgesic Activity

    No full text
    TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of <i>N</i>,<i>N</i>ā€²-dibenzyl tryptamine <b>1</b>, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative <b>14</b> was identified as a potent (IC<sub>50</sub> 0.2 Ā± 0.2 nM) and selective TRPM8 antagonist. In vivo, <b>14</b> showed significant target coverage in both an icilin-induced WDS (at 1ā€“30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1ā€“1 Ī¼g s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1ā€“4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain

    Identification of a Potent Tryptophan-Based TRPM8 Antagonist With in Vivo Analgesic Activity

    No full text
    TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of <i>N</i>,<i>N</i>ā€²-dibenzyl tryptamine <b>1</b>, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative <b>14</b> was identified as a potent (IC<sub>50</sub> 0.2 Ā± 0.2 nM) and selective TRPM8 antagonist. In vivo, <b>14</b> showed significant target coverage in both an icilin-induced WDS (at 1ā€“30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1ā€“1 Ī¼g s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1ā€“4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain

    Identification of a Potent Tryptophan-Based TRPM8 Antagonist With in Vivo Analgesic Activity

    No full text
    TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of <i>N</i>,<i>N</i>ā€²-dibenzyl tryptamine <b>1</b>, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative <b>14</b> was identified as a potent (IC<sub>50</sub> 0.2 Ā± 0.2 nM) and selective TRPM8 antagonist. In vivo, <b>14</b> showed significant target coverage in both an icilin-induced WDS (at 1ā€“30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1ā€“1 Ī¼g s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1ā€“4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain
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