Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors

Abstract

Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/beta-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-alpha and -delta and peroxisome proliferator-activated receptor-gamma2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of beta-catenin and AR. Immunoprecipitation studies demonstrated association of beta-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-alpha in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of beta-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.

Fig. 1

AR expression in 3T3-L1 cells. A, Immunocytochemical detection: 3T3-L1 preadipocytes were plated on 8-well chamber slides, either in the presence or the absence of DHT (10 nm) or T (100 nm) for 24 h. The cells were fixed with 2% paraformaldehyde and AR protein was detected by immunofluorescence using a polyclonal anti-AR antibody and FITC-conjugated secondary antibody (green). The arrows show the location of nuclei. B, Western blot analysis: Cells were treated with various concentrations of DHT (0–30 nm) for 24 h, harvested, and analyzed by Western blot analysis using anti-AR antibody. The membrane was stripped and reprobed with anti-GAPDH antibody.

Fig. 2

Testosterone and DHT inhibit adipogenic differentiation of 3T3-L1 preadipocytes. Top panel, 3T3-L1 cells were induced to differentiate in 6-well plates for 12 d as described in Materials and Methods with various concentrations of testosterone (T) and DHT. In certain conditions, cells were simultaneously treated with flutamide (Fl is 200 nm flutamide). Cells were fixed with 2% paraformaldehyde and stained with 0.5% Oil Red O stain. Representative photomicrographs (200×) are shown for each treatment group. Bottom panel, 3T3-L1 (4 × 10⁵) preadipocytes were induced to differentiate for 12 d with or without various treatments (androgens and 200 nm flut-amide), fixed with 2% paraformaldehyde, and stained with 0.5% Oil Red O. Quantitative analysis of adipocyte differentiation was done by measuring the OD 520 nm of the Oil Red O-stained adipocytes eluted with isopropanol and Igepal CA 40. The data represent the mean ± sem of three independent experiments (significance: **, P < 0.01; ***, P < 0.001 compared with control group; and ##, P < 0.01; and ###, P < 0.001 compared with DHT (10) and T (100) groups, respectively).

Fig. 3

Time course of C/EBP-β and C/EBP-δ expression during adipogenic differentiation with and without androgens and dose-dependent inhibition of C/EBP-α and PPAR-γ expression in 3T3-L1 preadipocytes by androgens. A, Confluent 3T3-L1 cells were treated with 100 nm testosterone (T) and allowed to differentiate in AM for various time points. Cells were harvested, lysed, and 100 μg of total protein was electrophoresed on SDS-containing polyacrylamide gels, and Western blot analysis was performed using anti-C/EBP-β, anti-C/EBP-δ, or anti-GAPDH antibodies. Three independent experiments were conducted and data from one representative experiment are shown. B, Top panel, 3T3-L1 cells were treated with either testosterone (0–100 nm) or DHT (0–10 nm) for 12 d and allowed to differentiate in AM as described in Materials and Methods. Cells were harvested, lysed, and 100 μg of total protein was electrophoresed on SDS-containing polyacrylamide gels, and Western blot analysis was performed using anti-C/EBP-α, anti-PPAR-γ, or anti-GAPDH antibodies. Three independent experiments were conducted and data from one representative experiment are shown. C, Densitometric analysis of the PPAR-γ expression (B, top panel) in 3T3-L1 cells after various doses of androgens normalization with GAPDH. D, 3T3-L1 cells were treated with testosterone (0 –100 nm) or DHT (0 –10 nm) and allowed to differentiate in AM as in panel B for 12 d. Cells were harvested, lysed, and 100 μg of total protein was electrophoresed on SDS-containing polyacrylamide gels, and Western blot analysis was performed using anti-C/EBP-α or anti-GAPDH antibodies. Three independent experiments were conducted and data from one representative experiment are shown. E, Densitometric analysis of the expression of C/EBP-α protein (panel C) in 3T3-L1 cells with various doses of androgens after normalization with GAPDH. F, Total cellular RNA from 3T3-L1 cells was prepared using Trizol reagent after incubation with graded concentrations of testosterone (0–100 nm) for 12 d. Quantitative analysis of C/EBP-α and PPAR-γ2 mRNA levels was performed by real-time RT-PCR after normalizing with GAPDH using gene-specific primers. (Significance: *, P < 0.02; and **, P < 0.005 compared with the control group.)

Fig. 4

AR mediated nuclear translocation of β-catenin in 3T3-L1 preadipocyte cells after testosterone or DHT treatment. A, Cells were grown for 24 h on eight-well chamber slides at 40% confluence and fixed for 20 min with 2% paraformaldehyde after various treatments, as shown. Localization of β-catenin was detected by using anti-β-catenin antibody and Texas Red-conjugated secondary antibody (red). The cells were counter stained with a nuclear stain DAPI (blue). B, Top panel, Cells were treated with T (100 nm) or DHT (10 nm) for various time points (0–24 h) and nuclear and cytoplasmic fractions were analyzed by Western blot analysis using anti-β catenin antibody. Bottom panel, Cells grown with none (control), T (100 nm) or T+ Bic (300 nm bicalutamide) for 24 h, nuclear and cytoplasmic fractions were analyzed by Western blot analysis using anti-β catenin antibody. C, Cells were treated as described in panel A, and localization of APC was detected by using anti-APC antibody and FITC conjugated secondary antibody. The data for androgen treatment groups are not shown. The arrows show the location of the nuclei. The cells were also counterstained with DAPI (blue).

Fig. 5

Colocalization and physical interaction of AR and TCF4 with β-catenin in DHT-treated cells. A, Double immunofluorescence: Cells were treated as described in Fig. 4 and double immunofluorescence experiments were performed using anti-AR and FITC-labeled secondary antibody for the detection of AR (green) and Texas Red-conjugated secondary antibody for the detection of β-catenin (red), and merging of the red and green pictures are shown as yellow. Cells were counterstained with DAPI (blue) to localize the position of nuclei in these cells. B, Immunoprecipitation: Cells were treated with either DHT (10 and 30 nm) alone or with bicalutamide (100 nm) for 24 h and immunoprecipitation was carried out using 500 μg of total cell lysates and 1–2 μg of respective primary antibodies (see Materials and Methods). The immunoprecipitated proteins were analyzed by Western blot analysis. Upper panel shows the detection of β-catenin, AR, and TCF4 bands after immunoprecipitation with anti-AR antibody, and lower panel shows the detection of β-catenin and TCF4 bands after immunoprecipitation with anti-TCF4 antibody.

Fig. 6

Inhibition of adipogenic differentiation in 3T3-L1 cells by testosterone is blocked by overexpression of TCF4 or a dominant negative TCF4 construct. 3T3-L1 cells were transfected either with full-length TCF4 or a dominant negative TCF4 (Dn-TCF4) cDNA construct or a control vector and were allowed to differentiate either in the presence (+T) or in the absence (−T) of testosterone (100 nm) for 12 d. Cells were fixed and stained with Oil Red O and quantitative image analysis was performed. A, Oil Red O staining: Representative photographs of Oil Red O-stained adipocytes are shown from different treatment groups (panel A). B, The graph shows the quantitative image analysis data obtained after averaging 20 fields from each treatment group, mock (Cont), TCF4 transfection (TCF4), and Dn-TCF4 transfection (Dn-TCF4) (P values vs. control: *, P < 0.05; IOD denotes integrated optical density; T denotes testosterone). C, Real-time quantitative RT-PCR analysis demonstrating the effects of testosterone on C/EBP-α and AP2 mRNA expression in TCF4 and Dn-TCF4 overexpressed 3T3-L1 cells 3T3-L1 cells were transfected with full-length TCF4 or Dn-TCF4 or a control vector plasmid and were allowed to differentiate in adipogenic medium (3 d) then GM with or without testosterone (100 nm) for 9 d as in panel A. Total cellular RNA was isolated and C/EBP-α and AP2 mRNA expression was analyzed by real-time RT-PCR. [*, P < 0.03 and **, P < 0.0005 denotes P values vs. control group (no T) in the upper panel; whereas #, P < 0.05, ##, P < 0.01 and ###, P < 0.002 denotes P values vs. control group (no T) in the lower panel.]

Fig. 7

Activation of Wnt target genes by testosterone (T). A, Effect of testosterone treatment on Wnt signaling pathway genes in 3T3-L1 cells by SuperArray Analysis. 3T3-L1 cells were allowed to differentiate into AM as described in Materials and Methods for 48 h either in presence or absence of testosterone (100 nm). Total RNA was isolated form these cells, and Wnt signaling pathway genes were analyzed by SuperArray as described. Circled spots represent some of the listed Wnt target genes, which are up-regulated by testosterone treatment. B, Verification of SuperArray data by quantitative real-time RT-PCR analysis. Total RNA isolated from 3T3-L1 cells after testosterone treatment were analyzed by real-time PCR as described in Materials and Methods using gene-specific primers Fst, CD44 and LEF-1 normalized with GAPDH (**, P < 0.0004).

Fig. 8

Effect of constitutive activation of AR on adipogenesis. A, Expression of C/EBPα mRNA and protein. Cells were transiently transfected either with pAct-AR or pARE₄-Luc using Lipofectamine 2000 and allowed to differentiate in adipogenic conditions for 9 d in absence of androgen. C/EBP-α protein and mRNA were analyzed by Western blot (right) and real-time RT-PCR (left), respectively. Data for real-time RT-PCR (done in quadruplicate for each treatment) and Western blot are from a single experiment representative of three separate experiments. B, Dual-luciferase reporter assay. Cells were transiently transfected with either pGL3, pARE₄-Luc alone or pAct-AR in combination with pARE₄-Luc and allowed to grow for 2 d in growth medium. Cells were also cotransfected with Renilla luciferase plasmid pRL-TK-Luc (50:1) using Lipofectamine 2000 and dual-luciferase assay was performed using standard protocols. The relative luciferase activity is presented in arbitrary units as mean ± sd from four separate experiments. C, Immunofluorescence analysis. Cells plated on two-well chamber slides were transiently transfected with pAct-AR, fixed with 2% paraformaldehyde, and immunofluorescence was performed using anti-β catenin antibody (Texas Red). D, Expression of p21 protein. Cells were transiently transfected either with pARE₄-Luc or pAct-AR as described in panel A and allowed to differentiate in adipogenic medium for another 2 d. Cells were lysed, and p21 expression was analyzed using anti-p21 antibody.