The cell culture reagents, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin, were all obtained from Life Technologies (Grand Island, NY, USA). Anti-PPARγ (#2430), anti-FABP4 (#2120), anti-p-AMPK (#2535), anti-AMPK (#2603), anti-FABP4 (#2120) and anti-p-SREBP1c (#9847) antibodies, and anti-mouse (#7076S)/anti-rabbit (#7074) secondary antibody were all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-C/EBPα (sc-61) and anti-β-actin (sc-47778) antibodies were all purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Anti-SREBP1c (557036) was purchased from BD science (Franklin Lakes, NJ, USA). AMPK small interfering (si)RNA and control siRNA were both purchased from Santa Cruz Biotechnology, Inc. Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). Shikonin, acetylshikonin, isobutyrylshikonin and β-HIVS were all purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

The 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and subcultured every 2 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 a differentiation-induction medium containing DMEM, supplemented with 1 µg/ml insulin, 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 10% FBS, for 2 days. The cells were then maintained in a differentiation-maintenance medium containing DMEM, supplemented with 1 µg/ml insulin and 10% FBS. The differentiation-maintenance medium was replaced every 2 days until the cells were harvested.

The 3T3-L1 cells were seeded in 12-well plates at a density of 7.5×10⁴ cells/well. After differentiation for 7 days, the cells were washed with phosphate-buffered saline (PBS), and incubated for 4 h in 4% paraformaldehyde (Sigma-Aldrich). The cells were then washed with distilled water and stained with 0.3% Oil Red O (Sigma-Aldrich) solution for 1 h. After washing them 3 times with distilled water, the cells were completely dried and observed under an inverted microscope (Olympus, Tokyo, Japan). Photographic images were captured and then 100% isopropanol (Sigma-Aldrich) was added to each well to extract the stained Oil Red O dye. The contents of the extracted dye were measured at 540 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Cell viability was determined using a CellCountEZ™ Cell Survival assay kit (purchased from Rockland Immunochemicals, Limerick, PA, USA), as previously described (15); viability was determined based on the ability of viable mammalian cells to convert hydroxyethyl disulfide into mercaptoethanol. The amount of mercaptoethanol produced from hydroxyethyl disulfide can be measured in the extracellular culture media since mercaptoethanol produced inside cells is extruded quickly by cells through an active transport mechanism.

The 3T3-L1 cells were seeded into 12-well plates at a density of 7.5×10⁴ cells/well, and, after differentiation, 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). qPCR was performed using the 7000 Real-Time PCR System (Applied Biosystems) in a final volume of 20 µl, which included the TaqMan Gene Expression Master Mix, 250 nM of TaqMan probe, an optimized concentration of each primer and 1 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 efficiency of RT-qPCR was approximately 100%. The Assays-on-Demand gene expression products (Applied Biosystems) were used to evaluate the mRNA levels of PPARγ (Mm00440945_m1), C/EBPα (Mm01265914_s1), SREBP-1c (Mm00550338_m1), FABP4 (Mm00445880_m1), FAS (Mm01253292_m1), ACC1 (Mm01304257_m1), SCD1 (Mm00772290_m1) as well as the level of 18S rRNA (Hs99999901_s1). 18S rRNA was used as an internal control, as previously described (16). For each sample, the mRNA level was normalized against the level of 18S rRNA, and the ratio of normalized mRNA in each sample to that in the control sample was determined using the comparative Ct method (17).

Cells were harvested using a cell scraper in ice-cold PBS, and lysed with 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) for 30 min at 4°C. The total cell lysates were then obtained after centrifuging 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). Twenty micrograms of protein were separated using 10% polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes at 180 mA for 1 h. The membranes were then blocked for 2 h at room temperature with Tris-buffered saline (TBS) containing 5% skim milk. Blocked nitrocellulose membranes were incubated with TBS containing 0.1% Tween-20 and a 1:1,000-dilution 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).

Two days after reaching confluence, 3T3-L1 cells were incubated in serum-free medium for 1 h and then transfected with 60 nM of AMPK siRNA or 60 nM of control siRNA using Lipofectamine RNAiMAX transfection reagent. Six hours later, the transfected cells were differentiated by replacing the medium with differentiation-induction medium. Total RNA and protein were extracted for RT-qPCR and western blot analysis, respectively.

In the present study, the cells were harvested using cell scrapers, and the 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). Twenty micrograms of nuclear protein were separated using 10% polyacrylamide gel electrophoresis and analyzed by western blot analysis using an anti-SREBP-1c antibody followed by secondary antibody.

All data are represented as the means ± standard deviation (SD) of at least three replicated experiments. Statistically significant differences between treated and untreated samples were detected using unpaired t-tests. All analyses were performed using SPSS v. 14 (SPSS, Inc., Chicago, IL, USA). A P-value <0.05 was considered to indicate a statistically significant difference.

It has previously been reported that Lithospermum erythrorhizon contains various shikonin compounds, including shikonin, acetylshikonin, isobutyrylshikonin and β-hydroxy-isovalerylshikonin (β-HIVS) (14). These shikonin compounds have the common basic structure of naphthoquinone, while the attached chemical groups are slightly different (Fig. 1A). To determine which compound exerted the greatest anti-adipogenic effect, the 3T3-L1 cells were treated with the four shikonin compounds at various concentrations for 7 days. Analysis of the effects on intracellular fat content revealed that isobutyrylshikonin and β-HIVS exerted a greater anti-adipogenic effect than shikonin and acetylshikonin (Fig. 1B). However, the intracellular fat content of 3T3-L1 cells treated with isobutyrylshikonin or β-HIVS for 7 days were similar. Thus, in the subsequent experiment, 3T3-L1 cells were treated with isobutyrylshikonin or β-HIVS on days 0–2, 3–4 and 4–7 of adipogenic differentiation (Fig. 2). The anti-adipogenic effect of β-HIVS was found to be slightly greater than that of isobutyrylshikonin (Fig. 2C and D). These data indicate that β-HIVS exerted the greatest anti-adipogenic effect among the shikonin compounds, and further studies were therefore conducted using β-HIVS.

In order to determine the most effective concentration of β-HIVS required for anti-adipogenic activity, the 3T3-L1 cells were treated with β-HIVS at various concentrations (0.25, 0.5, 1, 1.5 and 2 µM) for 7 days. The Oil Red O staining revealed that intracellular fat droplet formation was inhibited by β-HIVS in a dose-dependent manner, and that β-HIVS markedly inhibited fat droplet formation at a concentration of 2 µM, almost to the control level (Fig. 3A and B). Cytotoxicity was not observed when the 3T3-L1 cells were treated with β-HIVS at concentrations of up to 2 µM for 7 days, and cell viability was maintained at >90% of the untreated control cells (Fig. 3C), indicating that the inhibitory effects of β-HIVS on fat accumulation were not mediated by cytotoxicity. Further experiments were conducted with β-HIVS at a concentration of 2 µM.

During adipogenesis of the 3T3-L1 cells, the mRNA expression of the fat-forming enzymes, ACC1, FAS and SCD1, was upregulated, and this effect was significantly reduced by β-HIVS treatment (Fig. 4A–C). The major transcription factors of adipogenesis, PPARγ and C/EBPα, which are markedly upregulated during adipogenesis and are involved in the transcription of adipocyte marker genes including FABP4, were also significantly reduced by β-HIVS treatment (Fig. 4D–F).

It is noteworthy that ACC1, FAS and SCD1 have been reported to be transcribed by a transcription factor, SREBP-1c. It has also been reported that the mRNA expression of SCD1 is induced by the binding of nuclear SREBP-1c to its promoter (18), and SREBP-1c is the transcription factor that binds to a regulatory element in the enhancer of the FAS gene (19). The level of ACC1 mRNA is also controlled by the binding of SREBP-1c to the ACC1 promoter (19,20). It has also been reported that SREBP-1c induces the expression of PPARγ, which collaborates with C/EBPα to transcribe adipocyte marker genes such as FABP4 (21).

AMPK is known to suppress adipogenesis when it is activated by phosphorylation (22). To elucidate the association between β-HIVS and the activatation of AMPK, we determined whether β-HIVS induces AMPK phosphorylation during adipogenesis, and it was found that the p-AMPK level was markedly upregulated in the β-HIVS-treated cells compared with the untreated cells. The total AMPK protein level was almost unchanged, demonstrating that β-HIVS induced the phosphorylation of AMPK without affecting its total protein level (Fig. 5A). 5-Aminoimidazole-4-carbox-amide-1-β-dribofuranoside (AICAR) is the most well-known activator of AMPK, which activates AMPK by increasing its phosphorylation, and it was thus necessary to compare the AMPK-activating effect of β-HIVS with AICAR (23). The levels of p-AMPK and p-precursor-SREBP-1c, which is one of the products made by p-AMPK, were measured in order to compare the AMPK-activating effect of AICAR and β-HIVS at various concentrations (AICAR: 25, 150, 250 and 500 mM vs. β-HIVS: 0.1, 0.5, 1 and 2 µM). The results revealed that the levels of p-AMPK and p-precursor-SREBP-1c were increased by AICAR and β-HIVS (Fig. 5B). However, β-HIVS increased the levels of p-AMPK and p-precursor-SREBP-1c at a much lower concentration, <1/100,000, compared with AICAR, demonstrating that β-HIVS is a much more effective AMPK activator compared with AICAR.

Li et al previously reported that phosphorylated/activated AMPK directly phosphorylates precursor SREBP-1c at the Ser372 residue, which prevents the proteolytic maturation of precuser SREBP-1c into mature SREBP-1c and the translocation of mature SREBP-1c into the nucleus (11). When the 3T3-L1 cells were treated with β-HIVS for 6, 12, 24 and 48 h, the level of p-precursor-SREBP-1c was increased similarly to the p-AMPK level. As a result, the level of precursor SREBP-1c (unphosphorylated) and the proteolytic cleavage of precurser SREBP-1c (unphosphorylated) to mature-SREBP-1c was decreased by β-HIVS treatment. Furthermore, the level of nuclear mature SREBP-1c was also decreased, as the phosphorylation of Ser372 prevented nuclear translocation of SREBP-1c (Fig. 5C). When the 3T3-L1 cells were treated with β-HIVS for longer periods of time, of 2, 4 and 7 days, similar patterns of change were observed in the phosphorylation, proteolytic maturation and nuclear translocation of SREBP-1c. The level of precursor SREBP-1c (unphosphorylated) and its proteolytic cleavage into mature SREBP-1c, followed by its nuclear translocation, were reduced by the increase of p-precursor SREBP-1c in the β-HIVS-treated cells compared with the untreated cells (Fig. 5D).

To confirm the essential role which AMPK plays in the anti-adipogenic mechanism of β-HIVS, we performed siRNA-mediated knockdown of AMPK in 3T3-L1 cells in the presence or absence of β-HIVS. The siRNA-mediated knockdown of AMPK reduced the levels of AMPK and p-AMPK. We noted that p-precursor-SREBP-1c, which is a product made by p-AMPK, was also reduced by AMPK knockdown. As a result, levels of precursor SREBP-1c (unphosphorylated), mature SREBP-1c and nuclear mature SREBP-1c were increased by AMPK knockdown (Fig. 6A). Mature SREBP-1c is a transcription factor required for the expression of the fat-forming enzymes ACC1, FAS and SCD1, the levels of which were all significantly increased by AMPK siRNA compared to transfection with control siRNA (Fig. 6B–D). SREBP-1c is also known to induce self mRNA transcription by binding to its own promoter (24,25). In the present study, it was also found that the SREBP-1c mRNA level was increased by AMPK knockdown in β-HIVS-treated cells, possibly through the reduction in SREBP-1c phosphorylation and increase in mature SREBP-1c level (data not shown).

We noted that as a result of the effects of AMPK knockdown on the expression of the fat-forming enzymes, intracellular fat accumulation, which had been suppressed by the anti-adipogenic activity of β-HIVS, was significantly recovered by AMPK siRNA compared to transfection with the control siRNA (Fig. 7A and B). AMPK knockdown also reduced the effect of β-HIVS on the expression level of the major adipogenic transcription factors, PPARγ and C/EBPα, and an adipocyte marker gene, FABP4, transcribed by them. The mRNA and protein levels of PPARγ, C/EBPα and FABP4, which had been suppressed by β-HIVS, were significantly increased by AMPK knockdown (Fig. 7C–E).

The results of the present study demonstrated that AMPK is involved in the anti-adipogenic mechanism of β-HIVS through the modulation of SREBP-1c phosphorylation, maturation and nuclear translocation as well as the transcription of downstream fat-forming enzymes and adipogenic transcription factors. A possible molecular mechanism for the anti-adipogenic activity of β-HIVS and the effects of AMPK knockdown are described in Fig. 8.