3T3-L1 preadipocytes [American Type Culture Collection (ATCC), Manassas, VA, USA] were maintained in Dulbecco’s medified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Biomedia, Boussens, France), 100 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen, Life technologies, Carlsbad, CA, USA) at 37°C in 5% CO₂. The 3T3-L1 cells that were stably integrated into either the pcDNA3.1Myc/HisB-C10orf116 plasmid or the empty vector were established as previously described (3). To induce differentiation, 2-day post-confluent 3T3-L1 preadipocytes (day 0) were exposed to differentiation cocktail (100 μM methylisobutylxanthine, 0.25 μM dexamethasone and 1 μg/ml insulin). Two days later (day 2), cells were switched to medium containing 1 μg/ml insulin for 2 days (day 4). The cells were then switched back to DMEM containing only 10% FCS until day 8. Cultures were replenished every 2 days.

Adipocytes (2×10² cells/well) were seeded in 96-well culture plates and maintained in serum-free DMEM for 24 h until they were adherent. The cells were then cultured in DMEM supplemented with 10% FCS. Cell growth was monitored for 7 days successively using the Cell Proliferation MTT kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Following this, 10 μl of MTT labeling reagent were added to each well and incubated for 4 h at 37°C. Solubilization solution (100 μl) was then added to each well and cells were incubated overnight. The absorbance values at 560 and 660 nm were recorded by an ELISA reader and the difference between these values was recorded as the optical density (OD).

Cells were incubated at a density of 2×10⁶ cells/750 mm² in DMEM supplemented with 10% FCS, then washed with PBS and starved in serum-free DMEM for 24 h for synchronization. The cell cycle was initiated by replacement of the starvation medium with full medium (DMEM with 10% FCS) at various time points (0, 6, 12, 18, 24 h) following serum deprivation. Cultured cells were harvested using trypsin/EDTA and washed twice with PBS. Aliquots of 2×10⁶ cells were centrifuged, fixed in 70% ethanol and stained with 500 μl propidium iodide (PI) solution (100 μg/ml RNase and 50 μg/ml PI in 1X PBS). Labeled cells were analyzed using a BD FACScan (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using CellQuest software (BD Biosciences).

Cells were cultured in FCS-free DMEM for 24 h to induce apoptosis. Cells were then harvested using trypsin/EDTA, washed with PBS, resuspended in 1 ml of binding buffer and stained with 10 μl fluorescein isothiocyanate (FITC)-labeled Annexin V and 10 μl PI at room temperature for 5 min (BioVision, Mountain View, CA, USA). The fluorescence of FITC and PI was analyzed by flow cytometry.

Following the induction of apoptosis, cells were treated with the apoptosis Hoechst 33324 staining kit (Sigma, St. Louis, MO, USA) for 5–10 min according to the manufacturer’s instructions. The cells were incubated with phenol red-free Hanks’ balanced salt solution containing 3 μM of Hoechst 33342 and washed with PBS twice. Images were observed under a fluorescence microscope.

Following the induction of apoptosis, cells were collected and washed with PBS. The activity of caspase-3 and -8 was assayed using commercially available kits (Sigma) according to the manufacturer’s instructions. The measurements were based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). The reaction results in the release of the p-nitroaniline (pNA) moiety. The pNA moiety was detected spectrophotometrically at 405 nm. The concentration of pNA released from the substrate was calculated from a calibration curve prepared with pNA standards. In order to confirm the measurements, additional experiments using adequate inhibitors included in the kit were performed.

2-Deoxy-D-[³H]glucose (CIC, Beijing, China) uptake was assayed as previously described (4). The cells were cultured in 6-well plates and were serum starved in DMEM containing 0.5% FCS for 3 h prior to the experiments. The cells were then washed twice with PBS and incubated in KRP-HEPES buffer [30 mmol/l HEPES (pH 7.4), 10 mmol/l NaHCO₃, 120 mmol/l NaCl, 4 mmol/l KH₂PO₄, 1 mmol/l MgSO₄ and 1 mmol/l CaCl₂] in the presence or absence of 100 nmol/l insulin for 30 min at 37°C. Labeled 2-deoxy-D-[³H] glucose was added to a final concentration of 2 μCi/ml. After 10 min at 37°C, the reaction was terminated by washing with ice-cold PBS 3 times, supplemented with 10 mmol/l D-glucose. The cells were solubilized by adding 200 μl of 1 mol/l NaOH to each well and aliquots of the cell lysate were transferred to scintillation vials for radioactivity counting; the remainder was used for the protein assay.

Total RNA was extracted from 3T3-L1 adipocytes using TRIzol reagent (Invitrogen Life Technologies) and the extracted RNA was quantified by spectrophotometry at 260 nm. cDNA was synthesized from 1 μg of total RNA by using an AMV Reverse Transcriptase kit (Promega A3500; Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions. Real-time RT-PCR was performed using the TaqMan Sequence Detection System and the DNA double-strand-specific SYBR-Green I dye (Roche Diagnostics GmbH) according to the manufacturer’s instructions. The samples were briefly incubated at 95°C for 10 min for an initial denaturation, followed by 40 PCR cycles. Each cycle consisted of incubation at 95°C for 15 sec and 60°C for 1 min. We used β-actin as the reference in a comparative CT method and obtained the relative changes in the target samples. We used the following primers for the PCR analyses: GLUT4 homologous genes in mouse forward, 5′-ATT GGA CGC TCT CTC TCC AA-3′ and reverse, 5′-GAT TCT GCT GCC CTT CTGTC-3′; and β-actin forward, 5′-ATC TGG CAC CAC ACC TTC-3′ and reverse, 5′-AGC CAG GTC CAG ACG CA-3′.

All data are expressed as the means ± SEM. Statistical analysis was performed using the paired Student’s t-test using the SPSS 10.0 statistical software package (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

As indicated by MTT assay (Fig. 1), the overexpression of C10orf116 in 3T3-L1 preadipocytes resulted in a significantly higher rate of proliferation compared with the cells transfected with the empty vector at each time point. C10orf116 also had an effect on the cell cycle. The percentage of 3T3-L1 cells overexpressing C10orf116 in the S phase was significantly higher compared to the control cells following the replacement of the starvation medium with full medium at 12, 18 and 24 h and following serum deprivation, as shown by flow cytometry (Fig. 2).

Annexin V was shown to interact strongly and specifically with phosphatidylserine and may be used to detect the early stages of apoptosis by targeting the loss of plasma membrane asymmetry (5). To examine the effects of C10orf116 on cell apoptosis, cells were cultured in FCS-free DMEM for 24 h to induce apoptosis and the apoptotic index was analyzed. As shown in Fig. 3, C10orf116 protects 3T3-L1 preadipocytes from serum deprivation-induced apoptosis. In addition, the results of the Hoechst 33342 staining demonstrated that the 3T3-L1 cells transfected with the pcDNA.3.1 vector exhibited more significant apoptotic morphological changes, including chromatin condensation and the formation of apoptotic bodies (Fig. 4C) compared with the cells transfected with the C10orf116-pcDNA3.1 vector (Fig. 4D). However, no significant differences were observed in the control cells (Fig. 4A) and the cells overexpressing C10orf116 without the induction of apoptosis (Fig. 4B). Caspase-3 and -8 activity was also determined. The data (Fig. 5) also showed that 3T3-L1 preadipocytes transfected with the C10orf116-pcDNA3.1 vector had a lower caspase-3 and -8 activity compared with the cells transfected with the pcDNA.3.1 vector. These results indicate that C10orf116 inhibits apoptosis in preadipocytes induced by serum deprivation.

The transfected 3T3-L1 preadipocytes were induced to differentiate as described in Materials and methods. Glucose uptake was then assayed in the 3T3-L1 adipocytes with or without C10orf116 overexpression. As shown in Fig. 6, the insulin-stimulated glucose uptake was significantly enhanced in the adipocytes overexpressing C10orf116; however, the basal glucose uptake was similar compared with the control cells. In the adipocytes, insulin-stimulated glucose uptake is dependent on the expression, the activity or the translocation of the insulin-responsive glucose transporter GLUT4 (6–10). Therefore, we further examined the effects of C10orf116 on GLUT4 expression during the differentiation of 3T3-L1 preadipocytes. The results demonstrated that C10orf116 overexpression significantly increased GLUT4 expression on days 4 and 8 of differentiation (Fig. 7).