Elevated Pentraxin 3 in Obese Adipose Tissue Promotes Adipogenic Differentiation by Activating Neuropeptide Y Signaling

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  Figure 1. Elevated pentraxin 3 (PTX3) in obese adipose tissue induces adipocyte differentiation. (A) PTX3 immunohistochemical (IHC) signals in adipose tissue from wild-type (WT) and db/db mice. This IHC staining pattern demonstrates a marked increase of adipocyte cells in the db/db mouse. Scale bar: 200 µm. (B) The size of adipocytes of WT (n = 20) and db/db (n = 20) mice was determined from (A). (C) Circulating PTX3 protein levels in plasma from WT (n = 5) and db/db (n = 5) mice determined by ELISA. (D) PTX3 promotes lipid accumulation in 3T3-L1 cells. 3T3-L1 preadipocytes were induced to differentiate into adipocytes by culturing in the presence of a differentiation cocktail for 2 days. Cells continuously treated with 5 µg/ml insulin for an additional 4 days were used as positive controls. In the PTX3-treated cells, the differential cocktail was replaced with PTX3 (200 ng/ml) and the cells were cultured for an additional 4 days without insulin. The cells were then fixed, stained with Oil-Red-O, and imaged under a microscope. Scale bar: 100 microm (µM). (E) Quantification of the total number of lipid droplets in (D) (n = 5). (F) PTX3 upregulates the expression of adipocyte differentiation…

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  Figure 2. Pentraxin 3 (PTX3) induces neuropeptide Y (NPY) and neuropeptide Y receptor (NPYR) expression in murine preadipocytes. (A,B) 3T3-L1 cells (A) and bone marrow-derived macrophages (B) were treated with the tumor necrosis factor-α, interleukin-1β, IL-8 cytokines, and PTX3 (10 ng/ml) for 24 h. The mRNA expression level of NPY was determined using quantitative real-time PCR. GAPDH was used as an internal control (mean ± SD). Data values are the fold-changes of the mean vehicle control value (n = 4). (C) Effects of PTX3 on NPY/NPYR expression in 3T3-L1 cells. 3T3-L1 cells were incubated with the indicated concentration of PTX3 for 24 h and the mRNA expression of NPY, NPY1R, NPY2R, and NPY5R were determined using RT-PCR. GAPDH was used as an internal control. (D) PTX3 upregulates the expression of NPY, NPY2R, and NPY5R. The NPY, NPY1R, NPY2R, and NPY5R mRNA levels in 3T3-L1 cells treated with the indicated concentrations of PTX3 for 24 h were quantified using quantitative real-time PCR (n = 4). Data are representative of three independent experiments. Data are the fold-changes in the mean vehicle control value and represent the mean ± SD. *p < 0.05 compared with the vehicle control. p…

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  Figure 3. Neuropeptide Y (NPY) induces adipogenesis via reactive oxygen species (ROS) production in murine preadipocytes. (A) ROS levels were increased in NPY-treated 3T3-L1 cells. The cells were incubated with 2′,7′-dichlorofluorescin diacetate (10 µM) for 30 min at 37°C and subsequently treated with the indicated concentrations of NPY with or without N-acetylcysteine (NAC, 10 mM) for 4 h. ROS levels were assessed using a FACS Canto flow cytometry system (n = 3). (B) The pharmacological inhibition of NPYR blocks NPY-induced ROS production. 3T3-L1 cells were treated with NPY (200 nM) in the presence or absence of NPY1R antagonist (BIBO3304), NPY2R antagonist (BIIE0246), or NPY5R antagonist (CGP71683 hydrochloride) and the ROS levels were subsequently analyzed. *p < 0.05 compared with the vehicle control (n = 3). (C) 3T3-L1 cells treated with NPY with or without NAC (10 mM) were stained with Oil-Red-O and imaged under a microscope. Scale bar: 100 microm (µM). (D) The total number of lipid droplets in (C) was quantified (n = 5). (E) Relative mRNA levels of adipocyte differentiation markers were analyzed using quantitative real-time PCR (qPCR). qPCR data are the fold-changes in the mean…

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  Figure 4. Pentraxin 3 (PTX3)-mediated adipogenesis is inhibited by antioxidants. (A) Antioxidants prevent PTX3-mediated increases in lipid accumulation in 3T3 cells. Oil-Red-O staining of 3T3 cells treated with PTX3 with or without the antioxidant N-acetylcysteine (NAC) (10 mM) is shown. Scale bar: 100 microm (µM). (B) Quantification of the total number of lipid droplets in (A) (n = 5). (C) NAC inhibits PTX3-mediated increases in fatty acid binding protein-4 (FABP-4) expression. FABP-4 expression was analyzed using quantitative real-time PCR (n = 4). (D) PTX3 has no effect on reactive oxygen species (ROS) production in 3T3-L1 cells. The cells were stained with 2′, 7′-dichlorofluorescin diacetate for 30 min at 37°C and subsequently treated with the indicated concentrations of PTX3 for 4 h. ROS levels were detected using a fluorescent plate reader (n = 3). Data are representative of three independent experiments. **p < 0.005 compared with the vehicle control. p values were calculated using the Kruskal–Wallis test. Data values represent the mean ± SD.

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  Figure 5. Pentraxin 3 (PTX3)-induced adipogenesis is mediated by neuropeptide Y/NPYR signaling in murine preadipocytes. (A) 3T3-L1 preadipocytes were cultured in medium supplemented with a differentiation cocktail for 2 days and then treated with PTX3 (200 ng/ml) with or without NPY1R antagonist (BIBO3304), NPY2R antagonist (BIIE0246), or NPY5R antagonist (CGP71683 hydrochloride) for an additional 4 days without insulin. The cells were fixed, stained with Oil-Red-O, and imaged under a microscope. Scale bar: 100 microm (µM). (B) Quantification of the total number of lipid droplets in (A) (n = 5). (C) PTX3 modulates the expression of adipogenesis-associated transcription factors in murine preadipocytes. Adipocytes were treated with PTX3 (200 ng/ml) with or without NPY1R antagonist (BIBO3304) (1 µM), NPY2R antagonist (BIIE0246) (1 µM), or NPY5R antagonist (CGP71683 hydrochloride) (1 µM). Total RNA was isolated from 3T3-L1 preadipocytes, and C/EBP-α and peroxisome proliferator-activated receptor γ (PPAR-γ) levels were determined by RT-PCR. (D) PTX3 upregulates the expression of adipocyte differentiation markers via NPYR signaling. C/EBP-α, PPAR-γ, and fatty acid binding protein-4 expression…

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  Figure 6. Pentraxin 3 (PTX3) and neuropeptide Y (NPY) induce adipogenesis in human mesenchymal stem cells (hMSCs). (A) Antioxidants prevent PTX3- or NPY-mediated increases in lipid accumulation in hMSCs. Oil-Red-O staining of hMSC cells treated with PTX3 or NPY with or without N-acetylcysteine (NAC) (10 mM) is shown. Scale bar: 100 microm (µM). (B) Quantification of the total number of lipid droplets in (A) (n = 5). (C) The antioxidant NAC inhibits PTX3- and NPY-mediated increases in fatty acid binding protein-4 expression, as determined by quantitative real-time PCR (qPCR) (n = 4). (D) hMSCs were transfected with either control or PTX3 targeting small interfering RNA (siRNA) and the silencing efficacy of PTX3 targeting siRNA was confirmed with qPCR (n = 4). (E) NPY mRNA expression level in the cells shown in (D) was determined using qPCR (n = 4). (F) Reactive oxygen species (ROS) levels were analyzed in hMSCs transfected with either control or PTX3 targeting siRNA (n = 4). (G) Oil-Red-O staining of hMSCs transfected with siRNAs is shown. Scale bar: 100 microm (µM). (H) The total number of lipid droplets in (G) was quantified (n = 5). Data are representative of more than two independent…