α-Naphthoflavone Increases Lipid Accumulation in Mature Adipocytes and Enhances Adipocyte-Stimulated Endothelial Tube Formation - PubMed (original) (raw)

α-Naphthoflavone Increases Lipid Accumulation in Mature Adipocytes and Enhances Adipocyte-Stimulated Endothelial Tube Formation

Mei-Lin Wang et al. Nutrients. 2015.

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated factor that regulates biological effects associated with obesity. The AhR agonists, such as environmental contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone (BNF), inhibit preadipocyte differentiation and interfere with the functions of adipose tissue, whereas the antagonist may have opposite or protective effects in obesity. This study investigated the effects of α-naphthoflavone (α-NF), an AhR antagonist, on adipogenesis- and angiogenesis-associated factors in mature adipocytes and on cross-talk of mature adipocytes with endothelial cells (ECs). Besides, the roles of the AhR on lipid accumulation and on secretion of vascular endothelial growth factor were also determined by introducing siRNA of AhR. Differentiated 3T3-L1 cells were treated with α-naphthoflavone (α-NF) (1-5 μM) for 16 h. Lipid accumulation and the expressions of AhR-associated factors in the cells were determined. The interaction between adipocytes and ECs was investigated by cultivating ECs with conditioned medium (CM) from α-NF-treated mature adipocytes, followed by the determination of endothelial tube formation. The results showed that α-NF significantly increased triglyceride (TG) accumulation in mature adipocytes, which was associated with increased expression of hormone-sensitive lipase (HSL), estrogen receptor (ER), as well as decreased expression of AhR, AhR nuclear translocator (ARNT), cytochrome P4501B1 (CYP1B1), and nuclear factor erythroid-2-related factor (NRF-2) proteins. In addition, CM stimulated formation of tube-like structures in ECs, and α-NF further enhanced such stimulation in association with modulated the secretions of various angiogenic mediators by mature adipocytes. Similarly, increased TG accumulation and vascular endothelial growth factor (VEGF) secretion were observed in AhR-knockout cells. In conclusion, α-NF increased TG accumulation in mature adipocytes and enhanced mature adipocyte-stimulated tube formation in ECs, suggesting that the AhR may suppress obesity-induced adverse effects, and α-NF abolished the protective effects of the AhR.

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Figures

Figure 1

Effect of α-naphthoflavone (α-NF) on cytotoxicity in differentiated adipocytes. Cells were treated with various concentrations of α-NF for 16 h, and cell viability was measured with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit. Values are the mean ± SD from three measurements. Data with different letters significantly differ (p < 0.05).

Figure 2

Effect of α-naphthoflavone (α-NF) on lipid accumulation in differentiated adipocytes. Cells were treated with various concentrations of α-NF for 16 h. Intracellular lipid accumulation was observed by oil-red-O staining (100× magnification) (A) and quantified (B) by the method described in “Materials and Methods”. Triglyceride (TG) contents (C) were quantified with a commercial kit. Pictures are representative of three independent experiments. Values are the mean ± SD from three measurements. Data with different letters significantly differ (p < 0.05).

Figure 2

Figure 3

Effects of α-naphthoflavone (α-NF) on vascular tube formation in endothelial cells activated with α-NF-treated conditioned medium (CM). After full differentiation, adipocytes were incubated with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% fetal bovine serum (FBS) in the presence of various concentrations of α-NF for 24 h, and CM was then used to cultivate endothelial EA hy926 cells, which were grown on Matrigel-coated plates for another 24 h. Formation of tube-like structures was observed and photographed under a phase-contrast microscope after staining with acetomethoxy derivate of calcein (calcein–AM) fluorescent dye (100× magnification). Pictures are representative of three independent experiments.

Figure 4

Effect of α-naphthoflavone (α-NF) on vascular endothelial growth factor (VEGF) (A), interleukin (IL)-6 (B), nitric oxide (NO) (C), and insulin growth factor (IGF)-1 (D) secretion in culture medium from differentiated adipocytes. After full differentiation, adipocytes were treated with various concentrations of α-NF in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% fetal bovine serum (FBS) for 16 h. The medium was collected to analyze nitrite using the Griess reagent, IGF-1, VEGF, and IL-6 by ELISA kits. Values are the mean ± SD from three measurements, and data with different letters significantly differ (p < 0.05).

Figure 4

Figure 5

Effects of α-naphthoflavone (α-NF) on the aryl hydrocarbon receptor (AhR), the AhR nuclear translocator (ARNT), cytochrome P4501B1 (CYP1B1), estrogen receptor (ER), hormone-sensitive lipase (HSL), glycerol phosphate dehydrogenase (GPDH), nuclear factor erythroid 2-related factor 2 (NRF2), and vascular endothelial growth factor receptor (VEGFR) protein expressions by differentiated adipocytes. After full differentiation, adipocytes were treated with various concentrations of α-NF in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% fetal bovine serum (FBS) for 16 h, and proteins were collected and then measured with a Western blot analysis. Levels of protein are expressed relative to the cells treated with no α-NF after being normalized by β-actin expression. Pictures are representatives of three independent experiments.

Figure 6

Effects of knocking out the aryl hydrocarbon receptor (AhR) on expression of the AhR (A), intracellular triglyceride accumulation (B), and vascular endothelial growth factor (VEGF) secretion by mature adipocytes. 3T3-L1 preadipocytes were transfected with siRNA for the AhR, and cells were cultivated in differentiating medium to obtain mature adipocytes. Expression of the AhR by mature adipocytes was determined by a Western blot analysis. Intracellular triglyceride (TG) contents and VEGF released into the medium were analyzed by commercial assay kits. Pictures are representative of three independent experiments. Values are the mean ± SD from three measurements. Data with different letters significantly differ (p < 0.05).

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α-Naphthoflavone Increases Lipid Accumulation in Mature Adipocytes and Enhances Adipocyte-Stimulated Endothelial Tube Formation - PubMed (original) (raw)