The desalinated boiled tuna extract used in this study was prepared in Korea in 2014. First, boiled tuna extract was centrifuged to remove any suspended solids that may interfere with the desalting step. This process involves a change from 55 Brix, 13% salinity to 45 Brix, 12% salinity. We performed membrane filtration (membrane 2319/size 200 kDa) on the desalinated boiled tuna extract. We finally obtained a tuna extract of 30 Brix, 1% salinity, which was subjected to heat exchanger-type momentary sterilization (conditions: 110°C, 10 sec). The tuna extract sample was then divided into 1.5-ml tubes and stored at −70°C until use.

First, we reacted the desalinated boiled tuna extract with Tween-20 and -60 extraction buffer by overnight incubation at room temperature. The boiled tuna extract in extraction buffer was centrifuged (5,000 rpm, 10 min, 4°C) and the upper phase was collected and mixed with cold methanol and chloroform to separate the proteins. The mixture was centrifuged again (12,000 rpm, 5 min, 4°C), and the aqueous layer was removed and cold methanol was added, followed by further centrifugation (12,000 rpm, 10 min, 4°C). Finally, we removed the supernatant and dried the pellet.

We assessed the molecular weight of the tuna proteins by Coomassie blue staining. After subjecting the tuna extract to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands were observed on the gel using staining solution (7% acetic acid, 40% methanol, 0.1% Coomassie blue) and destaining solution (7% acetic acid, 20% methanol). Tuna extract proteins of ~10 kDa were selected for analysis by quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS). Each protein sample was subjected to in-gel trypsin digestion according to the method described in the study by Shevchenko et al (15). The digested peptide mixture was desalted and concentrated, then prepared using a C18 nano column and POROS R2 reverse-phase material (20–30-μm bead size; PerSeptive Biosystems, Framingham, MA, USA). MS/MS analysis was performed using a nano-ESI and MicrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany). Following their identification, 3 peptides were synthesized by Peptron (Daejeon, Korea): tuna-derived peptide, D-I-V-D-K-I-E-I (TP-D), tuna-derived peptide, I-D-T-I-I-E-T-I-M-E (TP-I) and tuna-derived peptide, N-I-N-E-D-P-Y-E-N-W-I-V (TP-N). Purification of the tuna-derived peptides was performed using a C18 column (Shiseido CAPCELL PAK; Shiseido, Tokyo, Japan) and a Shimadzu Prominence high-performance liquid chromatography (HPLC) apparatus in 0.1% trifluoroacetic acid (TFA)/water and a gradient of 0–90% acetonitrile in 0.1% TFA, with a flow rate of 1 ml/min and UV detection at 220 nm. The TP-D, TP-I and TP-N molecular weights were determined as 944, 1,177 and 1,505 kDa, respectively, by mass analysis (HP 1100 series LC/MSD).

The 3T3-L1 mouse fibroblasts (American Type Culture Collection, Manassas, VA, USA) were maintained at 37°C in a 5% CO₂, humidified atmosphere. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% bovine calf serum (BCS; HyClone, Logan, UT, USA) and 100 U/ml penicillin/100 mg/ml streptomycin. The 3T3-L1 cells were cultured to ~60–80% confluence in a 6-well plate, and upon confluence, were allowed to grow for 2–4 days in DMEM medium containing 10% fetal bovine serum (FBS; HyClone). Cell differentiation was initiated by treatment with MDI (0.5 mM IBMX, 0.25 μM dexamethasone and 10 mg/l insulin) for 48 h. The medium was then replaced with DMEM supplemented with 10 mg/l insulin and changed once every 2 days.

The 3T3-L1 preadipocytes were incubated with DMEM containing 10% BCS. Cell differentiation was induced by treatment with MDI in fresh DMEM containing 10% FBS. Following differentiation, the medium was changed to serum-free medium (SFM), and the cells were treated with TP-D, TP-I or TP-N at 500 ng/ml for 48 h before the glucose uptake assay. After collecting the cell culture medium, we confirmed glucose uptake using a kit according to the manufacturer’s instructions (Asan Pharmaceutical Co., Ltd., Gyeonggi, Korea). Enzyme solution was added to the culture medium and maintained at 37°C for 5 min in a 5% CO₂, humidified atmosphere. The absorbance at 500 nm was measured within 40 min. The absorbance of the solution in each well was measured at 490 nm using a microplate reader (Benchmark microplate reader; Bio-Rad Laboratories, Hercules, CA, USA).

The 3T3-L1 preadipocytes were incubated with DMEM containing 10% BCS. Cell differentiation was induced by MDI treatment in fresh DMEM containing 10% FBS. After differentiation, the medium was changed to SFM supplemented with 500 ng/ml TP-D, TP-I and TP-N for 48 h prior to the TG assay. We performed the TG assay according to the kit protocol (Cleantech TG-S kit; Asan Pharm Co., Ltd., Seoul, Korea). The enzyme solution was added to the cell lysate and maintained at 37°C for 10 min in a 5% CO₂, humidified atmosphere. The absorbance at 550 nm was measured within 60 min. The absorbance of the solution in each well was measured at 490 nm using a microplate reader (Benchmark microplate reader; Bio-Rad Laboratories).

The 3T3-L1 cells were washed carefully with phosphate-buffered saline (PBS) and then fixed with 10% formalin for 5 min. The formalin was then refreshed followed by incubation for 1 h. After removal of the formalin, 60% isopropanol was added to each well and dried. Subsequently, 60% Oil Red O staining solution was added to each well for 1 h. In the final step, the wells were washed 3 times with PBS, and the cell morphology and staining of the lipid droplets were observed using an inverted microscope (ECLIPSE TS100-F; Nikon, Tokyo, Japan).

The 3T3-L1 preadipocytes were seeded into 6-well plates at 2×10⁴ cells/well in 2 ml medium. Cell differentiation was induced by treatment with MDI in fresh DMEM containing 10% FBS. Following differentiation, the medium was changed to SFM supplemented with 500 or 1,000 ng/ml TP-D for 48 h. The cells were then treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the extracted RNA was used as a template for cDNA synthesis using an oligo(dT) primer (Intron Co., Gyeonggi, Korea). The synthesized cDNA was mixed with 2X TOPsimple DyeMIX-nTaq (Enzynomics Inc., Daejeon, Korea) and primers (Table I) in 0.1% diethylpyrocarbonate (DEPC)-treated water for PCR. Using a 1% agarose gel, the PCR products were separated and stained with Red safe nucleic acid staining solution (Intron Co.).

The 3T3-L1 preadipocytes were seeded into 6-well plates at 2×10⁴ cells/well in 2 ml medium. Cell differentiation was induced by treatment with MDI in fresh DMEM containing 10% FBS. Following differentiation, the medium was changed to SFM supplemented with 500 or 1,000 ng/ml TP-D for 48 h. The collected cells were washed with PBS, and extraction lysis buffer (20 mM Tris-base, pH 8, 150 mM NaCl, 100 μM sodium vanadate, 100 μM ammonium molybdate, 10% glycerol, 0.1% Nonidet P-40, 0.1% SDS, 1 mM glycerophosphate, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A and 1 mM PMSF) was added. The proteins were separated on 7–15% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked at room temperature with 1% bovine serum albumin in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20) and then incubated with primary antibodies: anti-C/EBP-α (1:1,000; sc-9314), anti-PPAR-γ (1:1,000; sc-1984) and anti-GAPDH (1:1,000; sc-25778) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The secondary antibody was a peroxidase-conjugated goat (sc-2420), mouse, or rabbit antibody (sc-358920;f 1:10,000; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The signal was developed using SuperSignal West Pico Stable Peroxide Solution and the SuperSignal West Pico Luminol/Enhancer solution (Thermo Fisher Scientific, Rockford, IL, USA) prior to exposure to X-ray film (Kodak, Rochester, NY, USA).

The results are expressed as the means ± SD. SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA) was used. Comparisons were made using ANOVA and Duncan’s multiple range test. The level of significance was set at P<0.05.

In this study, we performed a desalting process before ultimately extracting the tuna peptides. We confirmed the molecular weight of the tuna proteins by Coomassie blue staining. We observed a separated protein of boiled tuna extract at ~10 kDa and performed protein extraction using Tween-60 extraction buffer, as it yielded larger quantities of protein than did Tween-20 buffer (Fig. 1). We then characterized the ~10-kDa protein band by Q-TOF MS/MS. As shown in Fig. 2, we determined 3 peptide peaks and sequenced these tuna-derived peptides. These samples were termed tuna-derived peptide, D-I-V-D-K-I-E-I (TP-D), tuna peptide, I-D-T-I-I-E-T-I-M-E (TP-I) and tuna peptide, N-I-N-E-D-P-YE-N-W-I-V (TP-N); their molecular weights were determined to be 944, 1,177 and 1,505 kDa, respectively (Table II).

Glucose consumption is a prerequisite for 3T3-L1 cell differentiation. In this study, we compared glucose consumption, measured by a glucose uptake assay, between the differentiated cell group (MDI treatment) and the groups of cells treated with TP-D, TP-I or TP-N. All 3 tuna peptides were applied at concentrations of 500 and 1,000 ng/ml for 48 h. The TP-D-treated cells showed decreased glucose uptake compared with the MDI-treated group. TP-D significantly attenuated glucose uptake following treatment for 48 h at both 500 and 1,000 ng/ml with a maximal effect observed at the dose of 1,000 ng/ml. However, TP-I and TP-N had no significant effect following treatment for 48 h at either of the concentrations used (Fig. 3).

In order to evaluate the effects of TP-D, TP-N and TP-I on TG levels in differentiating 3T3-L1 cells, the cells were treated with the tuna-derived peptides at the dose of 500 and 1,000 ng/ml. Glucose consumption induced the active differentiation of the 3T3-L1 cells and the accumulation of TG. In the glucose uptake assay, we observed decreased glucose levels following treatment with TP-D. Thus, we performed a TG assay under the same TP-D, TP-N and TP-I treatment conditions. The TG levels in the 3T3-L1 cells treated with TP-D (at 500 or 1,000 ng/ml), were significantly deceased in a dose-dependent manner (Fig. 4). However, the TG levels in the cells treated with TP-N and TP-I were not altered. Thus, all further experiments were performed using treatment with TP-D at concentrations of 500 and 1,000 ng/ml for 48 h.

In the previous assays, we observed a decrease in the glucose and TG levels by TP-D in a dose-dependent manner. Thus, we then wished to confirm the reduced lipid accumulation by Oil Red O staining, which targets lipid droplets and allows the visualization of lipids (16). To determine the effects of TP-D on lipid accumulation in differentiating 3T3-L1 cells, the cells were treated with 500 and 1,000 ng/ml of TP-D. The cells were stained with Oil Red O solution and examined under a microscope. The number of stained lipid droplets detected decreased following treatment with TP-D in a dose-dependent manner compared with the MDI-treated group (Fig. 5).

The apparent morphological changes included changes in the number of lipid droplets and lipid accumulation during the differentiation of the cells into adipocytes. In addition, biochemical changes were observed in terms of the expression of adipocyte-specific protein markers. Generally, there is a significantly increased expression of transcription factors and the secretion of adipokines during adipocyte differentiation (17,18). Among these transcription factors, C/EBP-α and PPAR-γ are considered important (19), with increased expression levels observed in differentiated adipocytes. Their expression is induced during early differentiation, and during the later stages of differentiation, they induce the expression of various adipogenic genes. In our previous experiment, we confirmed the inhibition of lipid accumulation and cellular differentiation by treatment with TP-D. Thus, we then measured adipocyte-related factors at the protein and mRNA level by western blot analysis and RT-PCR, respectively, and observed the inhibition of the activation of C/EBP-α and PPAR-γ proteins. In turn, the controlled inhibition of C/EBP-α and PPAR-γ by TP-D inhibited adipocyte formation in a dose-dependent manner (Fig. 6).