Cell culture reagents were obtained from Life Technologies Inc. (Grand Island, NY, USA). Anti-PPARγ, anti-FABP4, anti-phospho-GSK3β and anti-GSK3β antibodies and secondary antibody were obtained from Cell Signaling (Beverly, MA, USA). Anti-C/EBPα, anti-DVL2, anti-β actin and anti-TBP antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-catenin antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA). All other chemicals including 1,25(OH)2D3 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and were subcultured every 2 or 3 days. The cells were seeded in 6-well plates at a density of 1.5×10⁵ cells/well. Two days after reaching confluence (day 0), the 3T3-L1 cells were differentiated in Dulbecco’s modified Eagle’s medium (DMEM) containing 1 μg/ml insulin, 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 10% fetal bovine serum (differentiation-induction medium) for 2 days. The cells were then maintained in DMEM containing 1 μg/ml insulin and 10% fetal bovine serum (differentiation maintenance medium). The differentiation maintenance medium was changed every 2 days until the cells were harvested. To test its effects on adipo-genesis, 1,25(OH)2D3 was added to the differentiation induction medium and differentiation maintenance medium until the cells were harvested. Lipid droplets in the cells were stained with Oil Red O as previously described (24).

Cells were harvested, and total RNA was extracted using an RNeasy kit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse-transcribed at 37°C using the cDNA Reverse Transcription kit (Applied Biosystems, Inc., Foster City, CA, USA). Briefly, the reaction was performed in a final volume of 20 μl, which included reverse transcriptase reaction buffer, 100 mM dNTP mix, random primers, MultiScribe™ Reverse Transcriptase, RNase inhibitor and total RNA. The reaction mixtures were heated at 25°C for 10 min, 37°C for 120 min and 85°C for 5 min. Real-time PCR was performed using the 7000 Real-Time PCR system (Applied Biosystems) in a final volume of 20 μl, which included TaqMan gene expression master mix, 250 nM of TaqMan probe, an optimized concentration of each primer, and 2 μl of the reverse transcription product containing cDNA. The reaction mixtures were preheated at 95°C for 10 min to activate the enzyme and then subjected to 40 cycles of melting at 95°C for 15 sec and annealing/extension at 60°C for 1 min. The assay-on-demand gene expression products (Applied Biosystems, Inc.) were used to evaluate the mRNA levels of PPARγ (Mm00440945_m1), C/EBPα (Mm01265914_ s1), FABP4 (Mm00445880_m1), LPL (Mm00434764_m1), WNT10B (Mm00442104_m1), LRP6 (Mm00999795_m1), DVL2 (Mm00432899_m1), GSK3β (Mm00444911_m1) as well as the level of 18S rRNA (Hs99999901_s1). The 18S rRNA was used as an internal control as previously described (25). For each sample, the mRNA level was normalized against the level of 18S rRNA, and the ratio of normalized mRNA to the preadipocytes (day 0) was determined using the comparative Ct method (26).

Cells were harvested using a cell scraper and lysed with ice-cold RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS and a protease inhibitor cocktail (Sigma-Aldrich) to obtain total cell lysates. The total cell lysates were then centrifuged at 20,000 × g for 20 min at 4°C to remove the insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Fifty micrograms of each protein extract was separated using 12% polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes at 150 mA for 1 h. The membranes were then blocked for 2 h at room temperature with phosphate buffered saline containing 5% skim milk and 0.1% Tween-20, and incubated with 1:1,000-dilutions of primary antibody overnight at 4°C followed by a 1:1,000-dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Peroxidase activity was visualized using an ECL kit (Pierce). β-actin was used as a loading control for cytosolic proteins.

Cells were harvested using a cell scraper and then nuclear extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Protein concentrations in the nuclear extracts were determined using a BCA protein assay kit (Pierce). Ten micrograms of nuclear protein were separated using 12% polyacrylamide gel electrophoresis and analyzed by western blotting using an anti-β-catenin antibody followed by a secondary antibody. TATA-box binding protein (TBP) was used as a loading control for nuclear proteins.

All data are expressed as means ± standard deviations from at least three replicate experiments. Statistically significant differences between treated and untreated samples were detected using an unpaired t-test. All analyses were performed using SPSS ver. 19 (SPSS, Chicago, IL, USA).

As shown in Fig. 1, treatments of nanomolar concentrations of 1,25(OH)2D3 during the adipogenesis of 3T3-L1 cells effectively inhibited the adipogenesis of 3T3-L1 cells in a dose-dependent manner. At the concentration of 10 nM, 1,25(OH)2D3 completely inhibited lipid droplet formation (Fig. 1A) as well as the expressions of adipocyte maker protein, FABP4, PPARγ and C/EBPα (Fig. 1B). Because 10 nM is the optimal anti-adipogenic concentration of 1,25(OH)2D3, all further experiments were conducted at this concentration. In time-course experiments, 1,25(OH)2D3 treatment significantly suppressed the mRNA expression of major transcription factors of adipogenesis, PPARγ and C/EBPα, as well as the proteins involved in lipid metabolism, FABP4 and LPL (Fig. 2A). The protein expression was also suppressed by 1,25(OH)2D3 showing similar patterns with mRNA expression (Fig. 2B).

To investigate the effects of 1,25(OH)2D3 on the WNT signaling, we measured the expressions of the genes involved in the WNT signaling in 3T3-L1 cells differentiated for 0, 2, 4 and 6 days in the presence or absence of 1,25(OH)2D3. The mRNA level of WNT10B was markedly downregulated during adipogenesis of untreated cells, but the treatment of 1,25(OH)2D3 maintained its expression levels (Fig. 3A). The mRNA level of LRP6 was mildly decreased during adipogenesis of untreated cells, but 1,25(OH)2D3 maintained its level (Fig. 3B). DVL2 is reported to be activated by WNT signaling and prevent the formation of the APC-GSK3β-AXIN destruction complex which degrade β-catenin (27). The mRNA and protein levels of DVL2 were markedly downregulated during adipogenesis of untreated cells, but 1,25(OH)2D3 maintained their mRNA and protein levels during adipogenesis (Fig. 3C and D). During adipogenesis of 3T3-L1 cells, treated or untreated, the mRNA and protein levels of GSK3β were not significantly changed (Fig. 3E and F, lower panel), but its phosphorylation on serine 9 residue was significantly decreased in untreated cells (Fig. 3F, upper panel). 1,25(OH)2D3 efficiently elevated GSK3β phosphorylation even though it has no effects on total GSK3β protein levels as well as its mRNA levels. GSK3β is a component of APC-GSK3β-AXIN destruction complex which is involved in the proteolytic degradation of β-catenin, and it is also well-known that GSK3β is inactivated by its phosphorylation (15,19). We found that GSK3β was activated by its dephosphorylation during adipogenesis of untreated 3T3-L1 cells, but the treatment of 1,25(OH)2D3 efficiently elevated its phosphorylation levels resulting in its inactivation.

β-catenin is a transcriptional cofactor which plays a central role in the WNT/β-catenin pathway (20). The nuclear level of β-catenin protein was significantly reduced during adipogenesis of untreated cells, but 1,25(OH)2D3 treatment maintained the nuclear β-catenin levels (Fig. 4A). TBP protein was used as an endogenous control for the loading of equal amounts of the nuclear protein. The levels of the β-catenin mRNA, which were significantly downregulated during the adipogenesis of untreated cells, were also maintained by 1,25(OH)2D3 treatment (Fig. 4B).