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>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 <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

<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

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

Table_1_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.xlsx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p

Table_4_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.xlsx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p

DataSheet_1_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.docx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p