Tangeretin Improves Glucose Uptake in a Coculture of Hypertrophic Adipocytes and Macrophages by Attenuating Inflammatory Changes

We obtained 3T3-L1 preadipocyte cells and RAW 264.7 murine macrophage cells from the American Type Culture Collection (Rockville, MD, USA) and the Korea Cell Line Bank (Seoul, South Korea), respectively. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Gaithersburg, MD, USA), which contained 1 % penicillin/streptomycin (PS) (100 units penicillin/mL and 100 pg streptomycin/mL) and was supplemented with either 10% bovine calf serum (BCS, Gibco, NY, USA) or 10% fetal bovine serum (FBS, Gibco, NY, USA), at 37℃ in a 5% CO₂ atmosphere. To prepare hypertrophic adipocytes, post-confluent preadipocytes (designated as day 0) were cultured until confluent in differentiation medium [DMEM containing 1% PS, 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, and 1 µg/mL insulin] for 2 days. The cells were then cultured for 2 more days in DMEM containing 1% PS, 10% FBS, and 1 µg/mL insulin. Thereafter, the hypertrophic 3T3-L1 cells with larger lipid droplets were prepared in post-differentiation medium (DMEM containing 1% PS and 10% FBS) for 14 days, which was replaced every 2 days. Co-culturing of adipocytes and macrophages was performed in accordance with a previously described protocol (Jeon et al., 2015; Shin et al., 2016). RAW 264.7 macrophages were plated in culture dishes containing serum-starved DMEM. Then, they were plated onto a hypertrophic 3T3-L1 monolayer and the cocultured cells were incubated in serum-starved DMEM for 24 h. Equal numbers of RAW 264.7 macrophages and 3T3-L1 adipocytes were cultured separately as controls. After 24 h, tangeretin (Wako Pure Chemical Industries, Osaka, Japan) was added to the cocultured cells at various concentrations for an additional 24 h. The supernatants and cells were collected and stored at −20℃ until use. Cell cytotoxicity was determined by lactate dehydrogenase (LDH) release assay (Takara Shuzo Co., Otsu, Japan).

The amount of nitrite was determined by colorimetric assay. Briefly, 100 µL of cell culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 540 nm was recorded using a microplate reader (BioTek instrument Inc., Winooski, VT, USA). The nitrite concentration was determined by extrapolation from a sodium nitrite standard curve.

Cells were homogenized with lysis buffer [1×RIPA (Upstate Cell Signaling Solutions, Lake Placid, NY, USA), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 1 mM NaF, 1 µg/mL aprotinin, 1 µg/mL pepstatin, and 1 µg/mL leupeptin] and incubated on ice for 1 h. Cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. These membranes were then blocked with a solution of 0.05% Tween 20/Tris-buffered saline containing 5% non-fat dry milk for 1 h at room temperature. Antibody against iNOS was obtained from Calbiochem (San Diego, CA, USA), and antibody against COX-2 was obtained from BD (Mountain View, CA, USA). Anti-phospho-AKT and anti-insulin receptor substrate (IRS) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-IRS and AKT were obtained from Cell Signaling Technologies (Beverly, MA, USA). After incubation overnight at 4℃ with the indicated primary antibody, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Immunodetection was carried out using the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ, USA).

Total RNA was isolated from cells using Trizol reagent (Molecular Research Center Inc., Cincinnati, OH, USA). RNA concentrations were measured from the absorbance at 260 and 280 nm. Complementary DNA (cDNA) synthesis was performed using a Maxime RT PreMix Kit (Intron Biotechnology, Seoul, South Korea), in accordance with the manufacturer’s protocol. The following primers were used for real-time RT-PCR analyses: IL-6, 5′-GTA CTC CAG AAG ACC AGA GG-3′ and 5′-TGC TGG TGA CAA CCA CGG CC-3′; IL-1β, 5′-CAG GAT GAC ATG AGC ACC-3′ and 5′-CTC TGC AGA CTC AAA CTC CAC-3′; TNF-α, 5′-TTG ACC TCA GCG CTG AGT TG-3′ and 5′-CCT GTA GCC CAC GTC GTA GC-3′; and β-actin, 5′-AGG CTG TGC TGT CCC TGT AT-3′ and 5′-ACC CAA GAA GGA AGG CTG GA-3′. The levels of IL-1β, IL-6, TNF-α, and β-actin mRNAs were measured using a Chromo 4TM Real-Time PCR Detector (Bio-Rad Laboratories Inc., Hercules, CA, USA). For relative quantification, the reactions were performed using 2× iQTM SYBR Green Supermix (Bio-Rad). The PCR amplification was performed for 44 cycles, each lasting 20 s at 95℃ and 65℃, and 30 s at 72℃, with initial denaturation at 95℃ for 3 min. Cytokine mRNA levels were compared after correction using the β-actin mRNA level as an internal standard. Expression level was analyzed by gene expression analysis using the Chromo 4 instrument (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Glucose uptake was detected using fluorescent 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2- NBDG; Invitrogen, Carlsbad, CA, USA). Briefly, hypertrophic 3T3-L1 adipocytes grown in 12-well plates were washed twice with serum-free DMEM and incubated in DMEM containing 0.2% bovine serum albumin for 4 h. Then, RAW 264.7 cells were added to 3T3-L1 adipocytes and incubated for a further 24 h before treatment with tangeretin. After 24 h, the cells were washed with Krebs-Ringer-Hepes (KRH) buffer (Boston BioProducts, Inc., Ashland, MA, USA) and treated with tangeretin (100 µM), insulin (100 nM), and 2-NBDG (100 µM) for 15 min at room temperature in dark condition. Glucose uptake was terminated by washing the cells three times with cold PBS. The cells were then lysed and harvested using 1% Triton X-100. The fluorescence intensity was measured at an excitation of 485 nm and emission of 520 nm using a fluorescence micro-reader (Fluostat Optima; BMG Labtech, Germany).

All data are representative of at least three independent experiments. Results are expressed as means±standard deviation (SD). Statistical analyses were conducted using SPSS 12.0 (SPSS Inc., Chicago, IL, USA).