Adipoinducer reagent

Mouse 3T3-L1 adipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were plated in a 24-well plate and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% foetal bovine serum (FBS; Gibco™, Life Technologies, Rockville, MD, USA), 1% penicillin-streptomycin, 2 mM L-glutamic acid, 100 mM pyruvic acid, 4.5 g/L glucose, and 7.5% sodium carbonate at 37°C under an atmosphere of 5% CO₂. Induction of adipocyte differentiation was induced using Adipo Inducer Reagent (for animal cells, Takara Bio Inc., Shiga, Japan) containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. Cell viability assay was performed using the CellTiter96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Fitchburg, WI, USA).
To estimate the effect of nutrient stress, we cultured cells in the following condition mediums for 4 days: control, 4.5 g/L glucose and 10% FBS; Low 1, 1.0 g/L glucose and 10% FBS; Low 2, 1.0 g/L glucose and 5% FBS; Low 3, 1.0 g/L glucose and 1% FBS; Low 4, 4.5 g/L glucose and 1% FBS; Low 5, 4.5 g/L glucose and 0% FBS. To estimate the effect of restoration by returning normal nutritional condition, we cultured cells for 9 days in Low/CT groups.

Mouse embryo 3T3-L1 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). 3T3-L1 pre-adipocytes were cultured in Dulbecco’s modified Eagle medium (Wako) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO₂. AdipoInducer Reagent (Takara Bio, Otsu, Japan) was used for the differentiation of 3T3-L1 pre-adipocytes. For the differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, 3T3-L1 pre-adipocytes were induced with differentiation media (DMEM with low glucose content supplemented with 10% fetal bovine serum, 2.5 μM dexamethasone (DEX), 0.5 mM 3-Isobutyl 1-methylxanthine (IBMX), and 10 μg/mL insulin (days 0–2). On day 2, the medium was replenished with maturation media (DMEM with high glucose content supplemented with 10% fetal bovine serum and 10 μg/mL insulin) and maintained at a 37 °C and 5% CO₂ environment. This medium was changed every 2 days until day 8. At this time, the cells exhibited characteristics of mature adipocytes. At day 8, the medium was replenished, and 3T3-L1 mature adipocytes were treated with or without recombinant adiponectin (20 μg/mL, Prospec, Ness-Ziona, Israel). Samples were collected at 24 h to extract RNA after adiponectin treatment.

Overall, 3T3-L1 cells were purchased from ATCC and were cultured in accordance with the supplier's instruction manual. Briefly, 3T3-L1 cells were grown to confluence in 24-well plates containing Dulbecco's modified Eagle's medium (high-glucose; Nacalai Tesque, Kyoto, Japan) supplemented with 10% calf serum (CS; Gibco, NY). Differentiation was initiated by culturing cells in differentiation medium containing 10% CS, 0.5 mmol/L isobutylmethylxanthine, 2.5 μmol/L dexamethasone, and 10 μg/mL insulin (AdipoInducer Reagent; Takara, Shiga, Japan). After 2 days of culture, media were replaced with adipocyte growth medium containing 10% CS and 10 μg/mL insulin and were exchanged every 2 or 3 days for an additional 8 days. Latanoprost free acid (LAT-A, 0.1 μmol/L), PGF_2α (0.1 μmol/L), from Cayman Chemical Co. (MI), or OMD (0.1, 1, 10, and 40 μmol/L) were added to adipocyte differentiation medium and growth medium (Table 1).

Pluripotent potential and expression of specific surface markers were identified. We validated pluripotency as an osteogenic/adipogenic differentiation assay by using Osteoblast-Inducer Reagent and Adipo-Inducer Reagent (Takara Biotechnology, Shiga, Japan; S1 Fig.). The surface markers of MSCs were stained; positive for CD29, CD44, CD73, CD105, CD106, and Sca-1 and negative for CD11b and CD45. All of the primary antibodies (Mouse Mesenchymal Stem Cell Marker Antibody Panel; R&D Systems, Spokane, WA) were diluted 1:100 in phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin and 0.3% Triton X-100 (Wako Pure Chemical Industries, Osaka, Japan). Cultured MSCs were respectively incubated with primary antibody solutions overnight at 4°C. Thereafter, they were incubated with a fluorophore-conjugated secondary antibody (Alexa Fluor488 conjugate anti-rat IgG antibody; Cell Signaling Technology, Danvers, MA or FITC-labeled rabbit anti-sheep IgG antibody, Pierce Biotechnology, Rockford, IL) at room temperature for 2 h. After counterstaining with DAPI (Vector Laboratories, Burlingame, CA), they were visualized using a BZ-9000 fluorescent microscope (Keyence Japan, Osaka, Japan).

Mouse 3T3-L1 (pre)adipocyte cells were purchased from the American Type Culture Collection (Manassas, VA, USA). They were maintained in Dulbecco's modified Eagle medium (DMEM) (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Gibco™, Life Technologies, Rockville, MD, USA), a 1% penicillin–streptomycin mixed solution, 200 mM L glutamic acid, 100 mM pyrubic acid, 4.5 g/L glucose, and 7.5% sodium carbonate at 37 °C under an atmosphere of 5% CO₂ and seeded on 24-well plates. The day after the cells reached confluence (<90%), their differentiation into adipocytes was induced by using the Adipo Inducer Reagent (for animal cells, TAKARA BIO INC., Shiga, Japan) that contains insulin, dexamethasone, and 3-isobutyl-1-methylxanthine.