Anti-obesity effects of the dual-active adenosine A2A/A3 receptor-ligand LJ-4378

Cell culture

Brown adipocytes were differentiated from immortalized preadipocytes obtained from interscapular BAT of mice as previously described [11]. Cells were cultured in Dulbecco’s modified Eagle’s medium (Welgene, LM001-07, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS, Gibco, 16000044, Waltham, MA, USA) and 1% Penicillin Streptomycin (Welgene, LS202-02, Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO2. When the cells reached about 90% confluency, the cells were exposed to a differentiation medium supplemented with 2.5 mM isobutyl methylxanthine (IBMX, Cayman, I5879, Ann Arbor, MI, USA), 0.125 mM indomethacin (Cayman, 70270, Ann Arbor, MI, USA), 1 μM dexamethasone (Cayman,11015, Ann Arbor, MI, USA), 1 μg/mL insulin (Sigma, I9278, St. Louis, MO, USA), and 1 nM triiodothyronine (T3, Cayman, 6028, Arbor, MI, USA) for 3 days for adipogenic differentiation. Then the cells were maintained in a growth media with 1 μg/mL insulin and 1 nM T3 (triiodothyronine) for 3 days. Intracellular cAMP levels were measured using the Direct cAMP ELISA Kit (Enzo Life Science, ADI-901-066A, New York, NY, USA) in accordance with the manufacturer’s protocol.

3T3-L1 cells obtained from ATCC (Manassas, VA, USA) were cultured in growth medium (Dulbecco’s Modified Eagle’s Medium (Welgene, LM001-07, St. Louis, MO, USA) supplemented with 10% FBS and 1% Penicillin Streptomycin)) at 37 °C in a humidified atmosphere with 5% CO2 [11]. Cells were then exposed to adipogenic differentiation medium (growth medium supplemented with 0.125 mM indomethacin (Cayman, 70270, Ann Arbor, MI, USA), 2.5 mM isobutyl methylxanthine (IBMX, Cayman, I5879, Ann Arbor, MI, USA), 1 µM dexamethasone (Cayman,11015, Ann Arbor, MI, USA), 1 μg/mL insulin (Sigma, I9278, St. Louis, MO, USA), and 1 nM triiodothyronine (T3, Cayman, 6028, Arbor, MI, USA)) for 3 days. For maintenance of differentiation, cells were exposed to a maintenance medium (growth medium supplemented with 1 µg/mL insulin and 1 nM triiodothyronine) for 3 days. For siRNA knockdown, fully differentiated 3T3L1 adipocytes were transfected with Negative Control siRNA (Bioneer, SN-1013, Daejeon, Republic of Korea) or A2AAR siRNA (Bioneer, 1211, Daejeon, Republic of Korea) using INTERFERin (Polyplus, 409-10, Florida, NY, USA) according to the manufacturer’s protocol. For in vitro experiments, cells were randomly assigned to each experimental group.

The LJ-4378, LJ-4433, and LJ-529 were synthesized as described previously [10, 12, 13]. CGS21680 (Cayman, 124431-80-7, Ann Arbor, MI, USA) was used for experiments.

Oxygen consumption rate (OCR)

XFp Analyzers were used to measure oxygen consumption rate (OCR). Cells were incubated in XF DMEM base medium (Agilent, 103575-100, pH 7.4, Cedar Creek, Texas, USA) supplemented with 4 mM L-glutamine (Sigma, G8540, St. Louis, MO, USA) and 25 mM D-glucose (Sigma, G7021, St. Louis, MO, USA) at 37 °C for the measurement. XFp Cell Mito Stress Test Kit (Agilent, 103010-100, Cedar Creek, Texas, USA) was sequentially prepared with the following optimal final concentrations: 2.5 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone/antimycin A, and basal, maximal, proton leak and were calculated previously described [14].

Immunocytochemistry

Cells were exposed to MitoTracker™ Red CMXRos (1:3000, Invitrogen, M7512, Waltham, MA, USA) for 15 min at 37 °C for mitochondria staining, then fixed with 4% paraformaldehyde (PFA, Sigma, 158127, St. Louis, MO, USA) as previously described [15]. Images were obtained using LSM800 confocal microscope (Zeiss, Germany) and analyzed with Zen software (version 3.0).

Cytotoxicity assay

EZ-CYTOX (DoGEN, EZ-3000, Seoul, Republic of Korea) was used to evaluate the cell viability, according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Thermo MULTISKAN GO, 8816-2015, Waltham, MA, USA).

Analysis of glycerol and free fatty acid (FFA) assay

Glycerol and free fatty acid (FFA) levels in media were measured using glycerol reagent (Sigma-Aldrich, F6428, St. Louis, MO, USA) and NEFA reagents (WAKO, 436-91693, Osaka, Japan) following the manufacturer’s product protocol as previously described [16].

Animals

Animal experiments were performed following approved protocols by Institutional Animal Care and Use Committees of Seoul National University (SNU-201107-1, SNU-201221-3). C57BL/6 male mice and CIDEA reporter mice [17] were used (8-week-old, male). Mice were housed at 12 h-light/12 h-dark cycle condition with free access to a normal chow diet (NCD, Purina Lab, 38057, protein: 24.52% calories, carbohydrates: 63.07% calories, fat: 12.41% calories, Seongnam, Republic of Korea) and water at 22 ± 1 °C. For the diet-induced obesity model, 8-week-old male mice were fed a HFD (Research Diets, D12492, protein: 20% kcal, carbohydrate: 20% kcal, fat: 60% kcal, New Brunswick, NJ, USA) for 8 weeks. Mice were treated either with LJ-4378 (1 mg kg−1 day−1) or vehicle intraperitoneally for 10 days [8]. LJ-4378 was dissolved in dimethylsulfoxide (DMSO) and diluted in sterile 0.9% saline (0.2% DMSO as the final concentration).

Indirect calorimetry was performed to measure expenditure (EE), VO2, VCO2, activity and food intake using PhenoMaster (TSE Systems, Bad Homburg, Germany). Body composition was measured by nuclear magnetic resonance scanning EchoMRI-700 (Echo Medical Systems, India).

Mice were randomly assigned to experimental groups, and experimental groups of mice were not blinded.

Western blot analysis and quantitative PCR

Western blot analysis and qPCR were conducted, as described previously [17]. Primers used for the qPCR analyses are listed in Supplementary Table S1. Primary antibodies used for western blot are summarized in Supplementary Table S2. Anti-rabbit horseradish peroxidase antibodies (1:3000, Thermo Fisher, 31460, Waltham, MA, USA) and anti-mouse horseradish peroxidase antibodies (1:3000, Jackson, 115-035-174, USA) were used for secondary antibodies. All antibodies were diluted in blocking buffer (5% skim milk or 5% bovine serum albumin in TBST). Western blot images were acquired using the Fusion Solo chemiluminescence imaging system (Vilber Lourmat, France) and analyzed with EvolutionCapt software (version 17.03). Images of the whole western blot membrane are provided in Supplementary Data. NIH ImageJ software was used for the quantification.

Histology

Adipose tissues were fixed using 10% formalin (Sigma, St. Louis, MO, USA) and then embedded in paraffin blocks. The paraffin sections were stained with hematoxylin/eosin (H&E) (BBC biochemical, McKinney, TX, USA) as described previously [14].

Glucose tolerance test

For the glucose tolerance test, mice were intraperitoneally injected with 20% D-Glucose (1 g kg−1, Sigma-Aldrich, G7021, St. Louis, MO, USA) as described previously [18]. A glucose meter (Gluco Dr. Top, allmedicus, AGM-4100, Anyang, Republic of Korea) was used to measure glucose concentration.

TTC analysis

Ex vivo electron transport activity related to mitochondrial oxidative phosphorylation was evaluated by monitoring the reduction of 0.1% triphenyltetrazolium chloride (TTC, Sigma, T8877, St. Louis, MO, USA) as described previously [16].

Bioluminescence and fluorescence imaging

In vivo bioluminescence was detected as previously described using an optical imaging device (Ami-X, Spectral Instruments Imaging) [17]. To detect in vivo bioluminescence signal, mice were i.p. injected with D-luciferin (150 mg kg−1, Goldbio, St. Louis, MO, USA). Aura Software (Spectral Instruments Imaging, Version 2.2.1.1) was used for quantification. Also, ex vivo imaging, tissues were collected from the CIDEA reporter mice treated with D-luciferin (150 mg kg−1, Goldbio, St. Louis, MO, USA). During imaging, the isolated tissues were maintained in 12 well plates containing D-luciferin (300 μg/ml).

Statistical analysis

Sample sizes were determined based on reproducibility and statistical significance. Prism 7 software (GraphPad Software, USA) was used for statistical analysis. Data were shown as mean ± SEM. An unpaired t-test was used between two groups to measure statistical significance. Data were normally distributed with equal variances between sample groups. No data were excluded from in vitro and in vivo experiments.

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