The 3T3-L1 preadipocytes were purchased from American Type Culture Collection (Manassas, VA, USA; cat. no. CL-173) and the cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Little Chalfont, UK) with 25 mM glucose, supplemented with 10% fetal bovine calf serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin/100 μg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were incubated at 37°C in a humidified atmosphere, containing 5% CO₂, and their subcultures were performed at <70% confluence. The cells in passage numbers P10 to P20 were used for subsequent experiments.

The 3T3-L1 preadipocytes were cultured until they reached 100% confluence under normal culture conditions. At 48 h following the attainment of confluence, the cells were cultured with DMEM containing 25 mM glucose and supplemented with 10% heat-inactivated FBS, 5 μg/ml insulin, 5 μM dexamethasone and 5 μM rosiglitazone (all from Sigma-Aldrich, St. Louis, MO, USA) for 48 h. Subsequently, the cells were incubated for 48 h with DMEM (25 mM glucose), supplemented with 10% FBS and 5 μg/ml insulin. The medium was subsequently exchanged with DMEM (25 mM glucose), supplemented with 10% FBS on every other day for 4 days. If necessary, DMEM containing a different concentration of glucose (5.5 mM) was used for the differentiation of 3T3-L1 preadipocytes into adipocytes.

SP was purchased from EMD Millipore (San Diego, CA, USA; cat. no. 05-23-0600), and was prepared with 5% acetic acid (Sigma-Aldrich). When required, SP was added to the 3T3-L1 cells at various concentrations whenever the medium was exchanged.

The 3T3-L1 cells were seeded onto fibronectin-coated cover-slips (1 μg/ml) in 24-well plates at a density of 4×10³ cells/well. The cells were initially incubated for 24 h with normal culture medium, prior to an incubation of 18–24 h duration under conditions of serum starvation. The cells were subsequently treated with SP for 48 h. For the final 6 h of the incubation period, 20 μM BrdU (Sigma-Aldrich) was added to the cells. The cells were subsequently prepared for the immunocytochemical analysis using BrdU by fixation in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) for 10 min on ice. The fixed cells were incubated with 2 N HCl for 15 min at room temperature and washed with PBS vigorously. Following permeabilization with 0.2% Triton X-100 (Affymetrix, Inc., Santa Clara, CA, USA), the cells were treated with blocking solution (5% non-fat milk in PBS with 0.1% Triton X-100) for 30 min at room temperature. Subsequently, the cells were incubated with primary mouse monoclonal anti-BrdU antibody (1:20, cat. no. #11-170-376-001; Roche Diagnostics GmbH, Mannheim, Germany) for 1.5 h at room temperature. Following three washes with 1% non-fat milk in PBS with 0.1% Triton X-100, the secondary antibody, Invitrogen Alexa-488 anti-mouse immunoglobulin G1 (Thermo Fisher Scientific, Inc.), was added, and the cells were incubated for a further 45 min at room temperature. Finally, the samples were mounted using Invitrogen ProLong^® Gold Antifade mounting solution with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Inc.), and left to dry overnight prior to observation. Images were captured using a fluorescence microscope (DMI4000; Leica, Solms, Germany), and the total number of cells and BrdU-positive cells were counted.

SP (0, 10, 100 or 300 nM) was added to the 3T3-L1 cells on the initial day of differentiation, and the total RNA was extracted from the cells on day 2 following the induction of adipogenesis using the Invitrogen TRIzol™ reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Aliquots of 5 μg total RNA were used for single-strand cDNA synthesis using the Invitrogen Superscript First-Strand cDNA Synthesis system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RT-qPCR was performed using the Invitrogen Power SYBR Green PCR Master mix (Thermo Fisher Scientific, Inc.). The ribosomal protein 36B4 gene from the mouse was used as an endogenous control. The following primers were used to detect the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), adipocyte protein 2 (aP2) and 36B4 protein: PPAR-γ, sense: 5′-CGC TGA TGC ACT GCC TAT GA-3′ and antisense: 5′-AGA CCT CCA CAG AGC TGA TTCC-3′; aP2, sense: 5′-CAT GGC CAA GCC CAA CAT-3′ and antisense: 5′-CGC CAA GTT TGA AGG AAA TC-3′; 36B4, sense: 5′-GAA CAT CTC CCC CTT CTC CTT-3′ and antisense: 5′-GCA GGG CCT GCT CTG TGAT-3′.

To assess adipogenesis in the 3T3-L1 cells, Oil Red O staining was performed on differentiated cells. Oil Red O solution (0.3%; Sigma-Aldrich) was prepared by dissolving Oil Red O in 60% isopropanol (Daejung Chemicals & Metals, Co., Ltd., Shiheung, Korea). The differentiated cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at room temperature, and subsequently washed with PBS. The fixed cells were incubated with Oil Red O solution for 30 min at room temperature. Images of the stained cells were captured using a light microscope (DMI4000; Leica, Solms, Germany). To further quantify the Oil-Red O-stained lipid drops, the stained cells were rinsed twice with 60% isopropanol and subsequently dried. The stains were eluted with 1 ml isopropanol for 10 min at room temperature and the optical density was measured at 490 nm using an absorbance plate reader (Spectramax190; Molecular Devices; Thermo Fisher Scientific, Inc.).

The cells were rinsed twice with ice-cold PBS and lysed with 2X SDS loading buffer [100 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM β-mercaptoethanol]. Subsequently, the cell lysates were denatured at 92°C for 10 min. The denatured protein samples were separated using 10% SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany. Following blocking with 5% non-fat milk in 20 mM Tris buffer, containing 0.1% Tween-20 (TBS-T), the membranes were incubated with primary antibody diluted with TBS-T buffer, including 5% non-fat milk, overnight at 4°C. The following primary antibodies were used: Rabbit monoclonal anti-phosphorylated AMPK (1:1,000, cat. no. #2535; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-phosphorylated Akt (1:4,000, cat. no. #4060; Cell Signaling Technology, Inc.,), rabbit polyclonal anti-GLUT4 (1:500, cat. no. ab65976; Abcam, Cambridge, UK) and mouse monoclonal anti-α-tubulin antibody (1:5,000, cat. no. T5618; Sigma-Aldrich). Subsequently, the membranes were incubated with goat anti-rabbit (1,5,000; cat. no. 7074; Cell Signaling Technology, Inc.) or goat anti-mouse IgG (1:10,000; cat. no. 170-6516; Bio-Rad Laboratories, Inc., Hercules, CA, USA) horseradish peroxidase-conjugated secondary antibodies at room temperature for 30 min. The target proteins were visualized using an enhanced chemiluminescence detection kit (EMD Millipore). The band densities were measured using ImageJ software (NIH, Bethesda, MD, USA). If required, the stripping of the membranes was performed using Restore™ Western Blot Stripping buffer (Thermo Fisher Scientific, Inc.) for 15 min at room temperature. The membranes were reblotted using an antibody raised against α-tubulin.

The data are expressed as the mean ± standard deviation or the mean ± standard error of the mean. An unpaired Student's t-test was used to evaluate differences between the two groups. All statistical analyses were performed using GraphPad Prism version 5.01 software (GraphPad Software, Inc., San Diego, CA, USA; http://www.graphpad.com). P<0.05 was considered to indicate a statistically significant difference.

A previous study demonstrated that SP increases the cellular proliferation of mesenteric preadipocytes (35). Therefore, whether or not SP affected the proliferation of the 3T3-L1 preadipocytes was examined using a BrdU incorporation assay. The 3T3-L1 preadipocytes were revealed to express NK-1R, which is a receptor of SP (data not shown) (12). As shown in Fig. 1, SP caused no effect on the cellular proliferation of 3T3-L1 cells, irrespective of the concentration of SP.

Whether SP regulated the differentiation of 3T3-L1 preadipocytes into adipocytes was subsequently examined. The 3T3-L1 cells were incubated with the differentiation medium, which included 10% FBS, 5 μg/ml insulin, 5 μM dexamethasone and 5 μM rosiglitazone (36), for 2 days. SP failed to affect the expression level of PPAR-γ or aP2 (Fig. 2), which were previously used as markers for differentiated adipocytes (37). Therefore, it is possible that SP does not promote the differentiation of 3T3-L1 cells into adipocytes.

Although SP failed to promote the differentiation of the 3T3-L1 cells, it was hypothesized that SP may affect the accumulation of lipids in these cells. Therefore, the effects of SP on the accumulation of lipids in the 3T3-L1 adipocytes were analyzed using Oil Red O staining. SP was added to the 3T3-L1 cells every other day during the differentiation of the cells into adipocytes. Compared with the undifferentiated control cells, the accumulation of lipids increased markedly in the differentiated cells without SP treatment (Figs. 3A and B). Notably, the treatment with SP increased the accumulation of lipids in the 3T3-L1 adipocytes by ~0.3-fold compared with the untreated differentiated 3T3-L1 cells (Figs. 3A and B), even though SP failed to promote the differentiation of 3T3-L1 cells into adipocytes (Fig. 2). In addition, an SP-mediated increase in the accumulation of lipids was observed in the differentiated 3T3-L1 adipocytes, which were cultured with medium, containing a high concentration of glucose (25 mM), however, not in the cells which were cultured in medium containing a normal concentration of glucose (5 mM; Fig. 3C). These results suggested that SP may increase the accumulation of lipids in adipocytes in a manner which is dependent on the concentration of glucose.

It is well known that AMPK is a key regulator in the metabolism of glucose and fatty acids in various cell types, including adipocytes (27,38). Therefore, whether SP regulated the activity of AMPK in the 3T3-L1 cells was examined. Indeed, SP was revealed to induce the activation of AMPK in a dose-dependent manner (Fig. 4A). Notably, however, the activity of Akt was downregulated by SP (Fig. 4B). These results are in very good agreement with a previous report, which demonstrated that the regulation of AMPK activity is associated with the regulation of Akt activity (39). It is also known that AMPK promotes the translocalization of GLUT4 to the plasma membrane (28), and furthermore, that AMPK increases the expression level of GLUT4 (29,30). It is noteworthy that SP increased the expression level of GLUT4 in 3T3-L1 cells (Fig. 4C). Therefore, these results suggested that SP may modulate glucose uptake in 3T3-L1 adipocytes, and that this is associated with the activity of AMPK.