Cell culture reagents, including Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, calf serum and antibiotics were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). AMPKα1 small interfering (si)RNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. BBR, insulin, dexamethasone and 3-isobutyl-1-methylxanthine were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

3T3-L1 pre-adipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and seeded in 12-well plates at a density of 1.5×10⁵ cells/well with DMEM containing 10% calf serum at 37°C in 5% CO₂ atmosphere with relative humidity of 85–95%. When the cells were 100% confluent following 3 days of culture (taken as day 0), the cells were induced to differentiate by incubation in a differentiation induction medium consisting of DMEM, 10% fetal bovine serum, 1 µg/ml insulin, 0.25 µM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine for 2 days. The culture medium was then replaced with differentiation maintenance medium, consisting of DMEM, 1 µg/ml insulin and 10% fetal bovine serum on days 2 and 4, and the cells were harvested on day 7. To examine the effects of BBR on cell differentiation, BBR was dissolved in dimethylsulfoxide and diluted >1,000-fold in differentiation maintenance medium. In the present study, BBR was administered during the late phase of adipogenic differentiation (days 2–7) in order to analyze the effects of BBR on fat accumulation and avoid its effects on mitotic clonal expansion, as BBR was previously reported to inhibit the mitotic clonal expansion of 3T3-L1 cells (10). In dose-response experiments, cells were treated with BBR (0, 5, 10, 15 and 20 µM) at day 2 and day 4, when media were replaced, and harvested at day 7. In time-response experiments, cells were treated with 0 or 20 µM BBR at day 2 and day 4, when media were replaced, and harvested at days 3, 5 and 7. Cells harvested at day 3 were treated with 20 µM BBR at day 2 only. Control cells were treated with an equal volume of dimethylsulfoxide diluted in differentiation maintenance medium. Intracellular fat accumulation in 1.2×10⁶ cells was measured by 0.3% Oil red O staining solution at room temperature for 30 min followed by light microscopy with a magnification of ×400. Oil red O dye in intracellular fat droplets was extracted by adding 700 µl 60% isopropanol per well and shaking at 100 rpm for 2 h at room temperature. The extracted dye was measured by optical density at 510 nm using the methods described previously (11). Cell viability was determined according to the manufacturer's protocol using a CellCountEZ™ Cell Survival assay kit (Rockland Immunochemicals, Inc., Pottstown, PA, USA), which is based on the ability of viable mammalian cells to convert hydroxyethyl disulfide into mercaptoethanol (12). Cell number was determined using a hemocytometer following the harvesting of cells by trypsinization.

RT-qPCR experiments were performed as described previously (13). Cells (1.2×10⁶) were treated with BBR and harvested as described above. Total RNA was extracted using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. RNA (1 µg) was reverse-transcribed at 37°C using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed using a StepOne Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), in triplicate, in a final volume of 20 µl, which consisted of 10 µl TaqMan Gene Expression Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) containing Taq polymerase and dNTPs, 1 µl TaqMan Gene Expression assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) containing 20X TaqMan probe and primers, cDNA (1/10th that produced by RT) and distilled water. The reaction mixtures were preheated at 95°C for 10 min to activate the enzyme, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extension at 60°C for 1 min. TaqMan Gene Expression assays containing TagMan probe and primers were used to evaluate the mRNA levels of the following genes: peroxisome proliferator-activated receptor-γ (PPARγ; assay ID, Mm00440945_m1), CCAAT-enhancer-binding protein α (C/EBPα; assay ID, Mm01265914_s1), SREBP-1c (assay ID, Mm00550338_m1), lipoprotein lipase (LPL; assay ID, Mm00434764_m1), fatty acid synthase (FAS; assay ID, Mm01253292_m1) and acetyl-CoA carboxylase-1 (ACC-1; assay ID, Mm01304257_m1) and 18S ribosomal (r) RNA (assay ID, Hs99999901_s1). The 18S rRNA was used as an internal control, as previously described (11). For each sample, target gene mRNA levels were normalized against the level of 18S rRNA, and the ratio of normalized mRNA in each sample to that of the control sample was determined using the comparative C_q method (14).

Western blotting was performed using the methods described previously (13). Cells (1.2×10⁶) were treated with BBR and harvested as described above. Cells were lysed for 1 h in ice-cold radioimmunoprecipitation assay 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; Merck KGaA). The cell lysates were centrifuged at 19,000 × g for 20 min at 4°C to remove insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein extracts (50 µg) were resolved by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes at 150 mA for 1 h. The membranes were then blocked for 2 h at room temperature with PBS containing 5% skim milk and 0.1% Tween-20 and, after washing for 10 min three times with PBS containing 0.1% Tween 20, were incubated with primary antibodies (diluted 1:1,000 in the blocking solution) overnight at 4°C and subsequently with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (diluted 1:1,000 in the blocking solution) for 1 h at room temperature. Protein bands were visualized using Pierce ECL Western Blotting Substrate, according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). An anti-β-Actin antibody (diluted 1:1,000 in the blocking solution) was used as an endogenous control to confirm that equal amounts of proteins were loaded. Anti-phosphorylated (p)-AMPKα (cat. no. 2535), anti-AMPKα (cat. no. 2603), anti-p-SREBP-1c (cat. no. 9874) and anti-PPARγ (cat. no. 2430) primary antibodies, as well as anti-mouse IgG (cat. no. 7076S) and anti-rabbit IgG (cat. no. 7074) secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-SREBP-1c (cat. no. 557036) antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-C/EBPα (cat. no. sc-61), anti-TATA box-binding protein (TBP; cat. no. sc-204) and anti-β-actin (cat. no. sc-47778) primary antibodies were purchased from Santa Cruz Biotechnology, Inc.

Cells (1.2×10⁶) were treated with BBR and harvested as described above. Cells were harvested using cell scrapers, and nuclear lysates were prepared using a Nuclear Extract kit (Active Motif, Inc. Carlsbad, CA, USA) according to the manufacturer's protocol. The protein concentration of nuclear lysates was determined using a BCA protein assay kit. To determine nuclear SREBP-1c protein expression levels, 10 µg nuclear protein was separated by 10% SDS-PAGE, followed by western blotting using anti-SREBP-1c antibody followed by secondary antibody. The procedures and antibodies used for western blotting were identical to those described above.

Binding of nuclear SREBP-1c to the target DNA sequence, 5′-TCACCTGA-3′, which contains an E-box motif, was measured using a SREBP-1 Transcription Factor ELISA kit (cat. no. 10010854; Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, 10 µg protein from the nuclear lysates was added to wells of a 96-well plate that were provided as part of the ELISA kit. The wells included in the kit were already coated with the 5′-TCACCTGA-3′ oligonucleotide. Following incubation for 1 h at room temperature, unbound nuclear proteins were removed by washing five times with a wash buffer provided in the kit. Each well was incubated with 100 µl anti-SREBP-1 (1:100) primary antibody for 1 h at room temperature followed by 100 µl horseradish peroxidase-conjugated secondary antibody (1:100) for 1 h at room temperature. Optical density at 450 nm was measured following the addition of 100 µl developing solution followed by 100 µl stop solution provided in the kit.

The kinase activity of AMPK was measured by ELISA using an AMPK assay kit (cat. no. CY-1182; Cyclex Inc., Columbia, MD, USA). Cells (1.2×10⁶) were lysed in 0.2 ml lysis buffer containing 20 mM Tris HCl (pH 7.5) 250 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 0.2 mM PMSF, 1 µg/ml pepstatin, 0.5 µg/ml leupeptin, 5 mM NaF, 2 mM Na₃VO₄, 2 mM β-glycerophosphate and 1 mM dithiothreitol for 90 min at 4°C, followed by centrifugation at 22,000 × g for 10 min at 4°C. Cell lysates (10 µl) and 90 µl kinase reaction buffer with 50 µM ATP (included in the kit) were added to wells of a 96-well plate coated with peptides consisting of the sequence surrounding Ser789 of insulin receptor substrate (IRS)-1, which is efficiently phosphorylated by AMPK, and incubated at 30°C for 30 min. The quantity of phosphorylated substrate was measured by incubating with 100 µl anti-p-IRS-1 antibody at room temperature for 30 min followed by incubation a with 100 µl horseradish peroxidase-conjugated secondary antibody at room temperature for 30 min. The primary and secondary antibodies included in the ELISA kit were prediluted. Subsequently, 100 µl substrate reagent was added at room temperature for 10 min followed by 100 µl stop solution included in the kit. Optical density was measured at 450 nm.

At one day prior to reaching confluence (taken as day-1), 3T3-L1 cells were incubated in serum-free medium for 1 h and then transfected with 12.5, 25, 50, or 75 nM AMPKα1 siRNA, or 50 or 75 nM control siRNA using Lipofectamine RNAiMAX transfection reagent according to the manufacturer's protocol. The following day (on day 0), the transfected cells were induced to differentiate by replacing the medium with differentiation-induction medium. Following 7 days of differentiation into adipocytes, cells were harvested for Oil red O staining, western blotting, determination of SREBP-1c binding to target DNA or RT-qPCR analysis using the aforementioned methods.

Data are presented as the mean ± standard deviation of four replicate experiments. Differences among multiple groups were determined using one-way analysis of variance followed by post hoc analysis using Duncan's multiple range test, and comparisons between two groups were analyzed using an unpaired Student's t-test. Statistical tests were performed using SPSS software (version, 14.0; SPSS, Inc., Chicago, IL, USA).

SREBP-1c is synthesized as a 125-kDa precursor, which is processed by proteolytic cleavage into a mature 68-kDa form that translocates into the nucleus. Phosphorylation of the SREBP-1c precursor suppresses its proteolytic maturation (15). In the present study BBR activated AMPK in a dose-dependent manner (Fig. 1A), which was associated with elevation of p-AMPK and p-SREBP-1c levels (Fig. 1B). The increase in p-SREBP-1c levels was accompanied by reduced proteolytic processing of the 125-kDa SREBP-1c precursor into the 68-kDa mature form (Fig. 1B). Western blotting of total SREBP-1c revealed gradual proteolytic degradation of the 125-kDa precursor form into the 68-kDa mature form (Fig. 1B). The level of the mature 68-kDa SREBP-1c isoform was decreased in the total cell lysate and nuclear lysate in a dose-dependent manner following treatment with BBR, as proteolytic maturation is required for the nuclear translocation of SREBP-1c (Fig. 1B). Similarly, the binding of nuclear SREBP-1c to its E-box motif-containing target DNA sequence was reduced in a dose-dependent manner following BBR treatment (Fig. 1C).

SREBP-1c is a transcription factor that binds to the promoter region of numerous lipogenesis-associated genes (16–21). As a result of decreased binding of nuclear SREBP-1c to target DNA, the mRNA expression of lipogenic genes, including FAS, LPL, SREBP-1c, PPARγ, C/EBPα and ACC-1 were reduced by BBR treatment in a dose-dependent manner (Fig. 2). This is consistent with the dose-dependent reduction in PPARγ and C/EBPα protein expression levels demonstrated in Fig. 1B. Intracellular fat accumulation, which is mediated by the collective actions of lipogenic genes, was reduced by BBR treatment in a dose-dependent manner (Fig. 3A). BBR exhibited no significant effect on cell viability, which was maintained at >90% of untreated adipocytes (Fig. 3B). This indicates that the BBR-induced suppression of nuclear SREBP-1c binding to its target DNA, as well as the reduction in lipogenic gene expression and intracellular fat accumulation, were not caused by increased cytotoxicity. Previously, BBR was reported to inhibit the mitotic clonal expansion of 3T3-L1 cells when treated during the early phase of differentiation (10) and the present study aimed to elucidate the molecular mechanism of BBR for reducing fat accumulation without its effects on mitotic clonal expansion. In the present study, BBR was administered during the late phase of adipogenic differentiation (from days 2–7) in order to avoid its effects on mitotic clonal expansion, which occurs during the early phase of differentiation. As demonstrated in Fig. 3C, increasing concentrations of BBR demonstrated no significant effects on the number of cells, demonstrating that mitotic clonal expansion was not affected in this experimental condition (Fig. 3C).

In order to examine the effect of BBR exposure over time, 3T3-L1 adipocytes were treated with 0 or 20 µM BBR from days 2–7, and were analyzed on days 3, 5 and 7 of adipogenic differentiation. Under these conditions, the protein expression levels of p-AMPK and p-SREBP-1c were increased in BBR-treated cells relative to the untreated cells (Fig. 4A). The protein expression levels of the 125-kDa SREBP-1c precursor and its processed 68-kDa mature form were reduced in BBR-treated cells when compared with in untreated cells (Fig. 4A). In addition, the nuclear levels of mature 68-kDa SREBP-1c were reduced in BBR-treated cells when compared with untreated cells, as proteolysis is required for nuclear translocation (Fig. 4A). The binding of nuclear SREBP-1c to its target DNA sequence was elevated in a time-dependent manner in untreated cells, which was significantly suppressed in BBR-treated cells, in a similar manner to nuclear SREBP-1c levels (Fig. 4B). Intracellular fat accumulation was reduced in BBR-treated cells when compared with the untreated cells (Fig. 4C). Consistent with these observations, the mRNA levels of lipogenesis-associated genes, including SREBP-1c, were significantly suppressed in BBR-treated cells when compared with the untreated cells (Fig. 5).

In order to confirm the essential role of AMPK in BBR-induced SREBP-1c phosphorylation and processing, 3T3-L1 cells were transfected with different concentrations of control siRNA or AMPKα1 siRNA. siRNA sequences targeting the α1 isoform of AMPK, rather than α2, were employed for knockdown experiments in the present study, as α1 is the predominant isoform expressed in adipocytes (2). Levels of p-AMPK and p-SREBP-1c were elevated in adipocytes treated with BBR alone when compared with untreated adipocytes (Fig. 6A). Levels of p-SREBP-1c were reduced in a dose-dependent manner following transfection with increasing concentrations of AMPKα1 siRNA, whereas, this effect was not observed following transfection with control siRNA (Fig. 6A). This confirmed that SREBP-1c is a substrate of AMPK. Conversely, the level of total SREBP-1c, including the 125-kDa precursor form and the mature 68-kDa form, were reduced in BBR-treated cells when compared with in untreated cells (Fig. 6A). These levels increased in a dose-dependent manner in AMPKα1 siRNA-transfected cells, however, not in control siRNA-transfected cells (Fig. 6A). The nuclear expression levels of the 68-kDa SREBP-1c form demonstrated a similar pattern to that observed in the total cell lysate. The binding of nuclear SREBP-1c to its target DNA sequence, which was suppressed by BBR, was rescued by AMPKα1 siRNA transfection in a dose-dependent manner (Fig. 6B). This effect was not observed following transfection with control siRNA. Intracellular fat accumulation, which was suppressed by BBR exposure, was rescued by AMPKα1 siRNA transfection, whereas transfection with control siRNA demonstrated no effect (Fig. 6C). In addition, the mRNA levels of lipogenesis-associated genes, including FAS, LPL, SREBP-1c, PPARγ, C/EBPα and ACC-1, which were suppressed by BBR treatment, were rescued AMPKα1 siRNA transfection in a dose-dependent manner, whereas the control siRNA sequences demonstrated no significant effect (Fig. 7). Similar to the protein expression levels of PPARγ and C/EBPα (Fig. 6A), the mRNA expression levels of these factors were rescued by AMPKα1 siRNA transfection in a dose-dependent manner (Fig. 7). Therefore, the results of the present study demonstrate that AMPK serves an essential role in the BBR-induced phosphorylation of SREBP-1c and the suppression of proteolytic processing, nuclear translocation and target DNA binding of SREBP-1c, which leads to the downregulation of lipogenesis-associated gene expression and decreased intracellular fat accumulation (Fig. 8).