Insulin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), lipopolysaccharide (LPS) from Escherichia coli, Oil Red O, 2-mercaptoethanol, anti–β-actin, Folin-Ciocalteu reagent, and gallic acid (GA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against PPAR-γ (sc-7273), C/EBP-α (sc-61), iNOS (sc-650), nuclear factor-κB (NF-κB; sc-372) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and c-Jun NH2-terminal kinase (JNK; #9252), p38 mitogen-activated protein kinase (p38 MAPK; #8690), and signal transducers and activators of the transcription-1 (STAT-1; #9172) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was purchased from Invitrogen (Carlsbad, CA, USA). Sodium carbonate, methanol, and ethanol were purchased from DUKSAN (Ansan, Korea).

GC used in this study was collected from Jeollanam-do, South Korea. The taxonomic identity of seaweed was confirmed by Prof. Chan-Sun Park and the sample was preserved for reference in the herbarium of the Department of Nutraceutical Resources, Mokpo National University, South Korea. The GC extract was produced with subcritical water (SW) and several solvents (water, methanol, and ethanol) to investigate its potential. After collection, GC was washed thoroughly to remove salt. For the solvent extraction, 20 g of GC was dissolved in 200 mL of 100% EtOH; 20 g were dissolved in 100% MeOH; and 300 g were dissolved in 800 mL of water. The organic solvent extract was immersed in 100% MeOH and EtOH until the color faded, and water extract was performed at 100°C for 15 min. These extracts were filtered and evaporated under vacuum conditions and then freeze-dried for up to 72 h to yield 0.3 g of dry EtOH extract (GCE; 1.5% yield), 0.4 g of MeOH extract (GCM; 2% yield), 8 g of water extract powder (GCW; 2.67% yield). For the subcritical water extract (SWE) process, the reaction temperature for hydrolysis of raw GC was maintained at several temperatures (90, 150, and 210°C) and the extract was labeled as GCSW90, GCSW150, and GCSW210, respectively. Fresh GC (20 g) was soaked in an extraction cell with 200 mL of distilled water, and botanical extraction equipment (TPR-1, TAIATSU TECHNO, Osaka, Japan) maintained a constant pressure at 3 MPa. Then, the reaction temperature for hydrolysis was raised to 90, 150, and 210°C and the extraction was kept for 1 min. This extract was filtered and freeze-dried for up to 72 h to yield 13 g of GCSW90 (65% yield), 5.1 g of GCSW150 (25.5% yield), and 2.3 g of GCSW210 powder (11.5% yield).

The total phenolic content was determined for all each extract using the Folin-Ciocalteu method (Rumbaoa et al. 2009). Briefly, 200 μL of GC (1 mg mL⁻¹), 1.4 mL of distilled water, and 100 μL of Folin-Ciocateu reagent were mixed and allowed to stand for 30 s at 25°C. Then, 300 μL of 20% sodium carbonate solution was added to the reaction mixture and incubated at 25°C for 2 h. Finally, the absorbance of the reaction mixture was measured at 765 nm using a spectrophotometer (Immuno Mini NJ-2300; Shinshu-u, Tokyo, Japan). The standard solutions of GA (10 to100 ppm) were similarly treated to prepare the calibration curve. The results were expressed as mg gallic acid 100 g⁻¹ of dry sample.

3T3-L1 preadipocytes purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% newborn calf serum and 1% penicillin-streptomycin. To induce differentiation, 3T3-L1 pre-adipocytes were cultured on a 96- or 6-well plate until confluence was reached and the culture medium was replaced with a fresh medium containing 5 μg mL⁻¹ insulin, 0.5 mM IBMX, and 1 μM dexamethasone in DMEM with 10% fetal bovine serum (FBS) for 2 days. The medium was then replaced with a differentiation medium containing 5 μg mL⁻¹ insulin and DMEM medium containing 10% FBS and replaced every 2 days for 8 days until cells were harvested (Park et al. 2013). RAW 264.7 macrophages (ATCC) were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C with 5% CO₂ in a humidified incubator.

Following the methods of Shin et al. (2016), the co-culture model was used with modification in this study. In brief, RAW 264.7 macrophages were cocultured onto differentiated 3T3-L1 adipocytes with direct contact in culture well plates. After 24 h of incubation, co-culture cells were treated with DMSO or GCSW210 extract (125 and 250 μg mL⁻¹) for a further 24 h.

The MTT assay was performed to evaluate the cytotoxicity of the GC extract in adipocytes and macrophages. 3T3-L1 preadipocytes were seeded into 96-well plates at a density of 1 × 10⁴ cells per well. Differentiated adipocytes were exposed to the different concentrations of GC extracts prepared with SWE at different temperatures (90, 150, and 210°C) and different solvents (GCW, GCM, and GCE) at the concentrations of 125 to 500 μg mL⁻¹ for 24 h at 37°C with 5% CO₂.

RAW 264.7 macrophages (1 × 10⁴ cells per well) were seeded in 96-well plates and incubated with GCSW210 (125 or 250 μg mL⁻¹), for 24 h. The MTT solution (1 mg mL⁻¹) was added to each well. After 3 h of incubation, the MTT solution was removed and replaced by DMSO to dissolve the purple crystals. Absorbance was measured at a wavelength of 540 nm (Immuno Mini NJ-2300).

Differentiated adipocytes were washed twice with phosphate-buffered saline (PBS) and then fixed for 30 min at 25°C with 10% formalin, prepared in PBS. Then, cells were washed twice with PBS, and stained with a filtered Oil Red O solution (0.5% in 60% isopropanol) for 1 h. Finally, cells were washed twice with distilled water, and the fat droplets in 3T3-L1 adipocytes were observed via phase-contrast microscopy.

NO production was determined according to the previously described methods by Sharma and Rhyu (2014) with modifications. RAW 264.7 macrophages were seeded in 96-well plates and incubated with or without 1 μg mL⁻¹ LPS in the presence or absence of the GCSW210 extract. After 24 h of incubation, the supernatant was collected and incubated with Griess reagent containing a 1 : 1 mixture of 0.1% N-(1-naphthyl)-ethylene diamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid at 25°C for 5 min. Absorbance was measured at a wavelength of 540 nm (Immuno Mini NJ-2300). NO production was measured as the concentration of nitrite by comparing it to a standard curve generated using NaNO₂.

Cell lysates containing 20 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose (NC) membrane in a western blot apparatus (Bio-Rad, Hercules, CA, USA). The NC membrane was blocked with 5% skim milk for 1 h, rinsed by Tris-buffered-saline (TBS) with Tween-20 (TBS-T buffer; 1 mol L⁻¹ Tris, 5 mol L⁻¹ NaCl, and 0.1% Tween-20), and incubated for overnight with anti-PPAR-γ (1 : 250), C/EBP-α (1 : 250), iNOS (1 : 500), NF-κB (1 : 1,000), JNK (1 : 1,000), p38 (1 : 1,000), and STAT (1 : 1,000) primary antibodies. The NC membrane was again washed with TBS-T buffer followed by incubation with secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse and rabbit IgG (1 : 1,000) for 1 h at 25°C. Finally, the expressed proteins were measured by analyzing the signal captured in NC using a chemiluminescent substrate and Vision-Works TMLS (Analysis Software, Upland, CA, USA).

RNA was isolated using TRIzol reagent (Molecular Research Center, Cincinnati, OH, USA). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using Diastar 2X One-Step RT-PCR Premix (SolGent, Daejeon, Korea). RNA primers were purchased from Integrated DNA Technologies (Coralville, IA, USA) and the sequences of the oligonucleotide primers were as follows: PPAR-γ forward, 5′-ACC ACAGTTGATTTCTCCAG-3′ and reverse, 5′-TGTTGTAGAGCTGGGTCTTT-3′; FAS forward, 5′-CTGCGGAAACTTCAGGAAATG-3′ and reverse, 5′-GGTTGCTCGGAATGCTATCCAGG-3′; and sterol regulatory element-binding protein-1c (SREBP-1c) forward, 5′-CACTTCTGGAGACATCGCAAAC-3′ and reverse, 5′-TGGTAGACAACAGCCGCATC-3′. The PCR conditions were 50°C for 30 min, 95°C for 15 min and 45 cycles of denaturation at 95°C (10 s), annealing at 60°C (10 s) and extension at 72°C (20 s). PCR products were analyzed on a Red-Safe-stained 1.5% agarose gel (iNtRON Biotechnology, Seoul, Korea). Band intensities were analyzed with UVP image acquisition and analysis software (VisionWorks, San Antonio, LX, USA).

Fully differentiated 3T3-L1 adipocytes cocultured with RAW 264.7 macrophages for 24 h, were treated with GCSW210 for a further 24 h and followed by treatment with 2-NBDG, dissolved in PBS and incubated for 30 min. After incubation, cells were washed twice with PBS. Fluorescence was quantified using a fluorescence microplate reader (Perkin Elmer, Waltham, MA, USA) at excitation and emission wavelengths of 485 and 535 nm, respectively.

Data are expressed as the means ± standard error of the mean. All statistical analyses were performed using one-way analysis of variance and Dunnett’s test using GraphPad Prism version 5.03 (GraphPad Software, Inc., San Diego, CA, USA). Significant differences were defined for p < 0.05.

Phenolic compounds are important plant constituents with redox properties responsible for anti-oxidant activity (Shahidi and Ambigaipalan 2015). We used Folin-Ciocalteu reagent to measure the total phenol content of each extract of GC. The results were derived from a calibration curve of GA and expressed in mg GA 100 g⁻¹ dry extract weight. SWE was shown to extract phenolic compounds more effectively than the organic solvent extract. Among them, GCSW210 showed the highest phenolic content of 170 mg equivalent of GA 100 g⁻¹ of the extract (Fig. 1).

All GC extracts did not show cytotoxicity when treated up to 500 μg mL⁻¹ concentration in 3T3-L1 adipocytes (Fig. 2A). A concentration of 250 μg mL⁻¹ was used to measure the adipogenic effect of the GC extracts. GCSW210 inhibited lipid accumulation and triglyceride content most distinctly compared to other extracts (Fig. 2B & C). In addition, anti-adipogenic effects of GCSW210 were expressed in a dose-dependent manner (Fig. 3B & C). Because GCSW210 showed strong effects on lipid accumulation, further experiments were performed.

PPAR-γ and C/EBP-α are key activators of adipogenesis, and SREBP-1c control genes involved in lipid metabolism including FAS (Sharma et al. 2019). In this study, the expression of lipid regulators was found to be downregulated in a dose-dependent manner when treated with GCSW210. Compared with the control group, the GCSW210-treated groups showed a lower expression for both regulators (Fig. 4A & B). Also, GCSW210 significantly alleviated mRNA expression of PPAR-γ, SREBP-1c, and FAS in mature 3T3-L1 adipocytes (Fig. 5).

GCSW210 significantly inhibited the inflammatory response in LPS-treated RAW 264.7 macrophages. In Fig. 6, the induction of LPS in RAW 264.7 macrophages highly elevated NO production and nitric oxide synthase 2 (NOS2) expression, as a marker of the inflammatory response, whereas the cell viability was reduced by about 20% compared to the normal group. Treatment of GCSW210 in RAW 264.7 macrophages effectively inhibited NO production and NOS2 protein expression induced by LPS in a dose-dependent manner, enhancing cell viability.

The activation of NF-κB, MAPK, and STAT-1 pathways causes the transcription of inflammatory cytokines and mediators such as iNOS and tumor necrosis factor-α (TNF-α) (Bagaev et al. 2019). LPS significantly stimulated the translocation of NF-κB p65 subunit from the cytosol to the nucleus compared with no treatment in RAW 264.7 macrophages, but treatment of GCSW210 highly suppressed the translocation of NF-κB p65 in a dose-dependent manner (Fig. 7). Additionally, LPS-stimulated MAPK and STAT-1 pathways significantly inhibited by treatment of GCSW210, and there were restored almost like normal cells at the concentration of 250 μg mL⁻¹ (Fig. 8).

A prolonged state of obesity can increase the systemic levels of pro-inflammatory cytokines, such as IL-6 (Makki et al. 2013). In co-culture of 3T3-L1 adipocytes and RAW 264.7 macrophages, GCSW210 showed no cytotoxicity up to 250 μg mL⁻¹ (Fig. 9A). The pro-inflammatory cytokine IL-6 and NO level as biomarker of inflammatory response significantly enhanced in cocultured adipocytes compared to adipocytes (Fig. 9B & C). However, the addition of GCSW210 effectively reduced IL-6 and NO production in adipocyte-macrophage co-culture system such as obesity-induced inflammation. To investigate the regulatory activity of GCSW210 on glucose metabolism in a co-culture model, glucose uptake was measured with or without insulin (Fig. 9D). In 3T3-L1 adipocytes, the treatment of insulin (100 nmol L⁻¹) or GCSW210 (250 μg mL⁻¹) significantly increased 2-NBDG uptake compared with no treatment. However, 2-NBDG uptake significantly decreased in obesity-induced inflammation condition of adipocyte-macrophage co-culture compared with adipocytes, suggesting the induction of IR. Nevertheless, the reduced 2-NBDG uptake in obesity-related IR condition of co-culture system was almost restored to normal by treatment of insulin or GCSW210, respectively. In addition, when insulin and GCSW210 were treated together in adipocyte-macrophage co-culture system, 2-NBDG uptake was higher than that of the single treatment. This indicates that GCSW210 could improve glucose metabolism by regulating obesity-stimulated inflammation and IR in adipocyte-macrophage co-culture system.