3T3-L1 and hASC Adipocyte Differentiation

Murine 3T3-L1 cells (ATCC, Manassas, VA, USA) were cultured in a growth media containing Dulbecco’s Modified Eagles’ Medium (DMEM), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY, USA), and 1% penicillin–streptomycin (Welgene, Daegu, Republic of Korea). The adipocyte differentiation was induced by changing the medium to DMEM supplemented with 10% fetal bovine serum (FBS) (Welgene, Daegu, Republic of Korea) added with a cocktail of hormones (MDI) that include 0.5 mM IBMX (M), 0.5 µM dexamethasone (D), and 5 µg/mL insulin (I) either with or without TAK-715 at the indicated concentrations. On day 2, the first differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 µg/mL insulin either with or without TAK-715 at the indicated doses for an additional 3 days. The cells were further fed with DMEM containing 10% FBS in the presence or absence of TAK-715 for an additional 3 days. On day 8, the preadipocytes became mature adipocytes that were rounded up and filled with lipid droplets.
The hASCs were isolated from abdominal subcutaneous adipose tissue of female patients admitted to Keimyung University Dongsan Hospital (KUDH), Daegu, Republic of Korea. The Ethics Committee of KUDH approved the study protocol (No. 2021-02-063-018), and the informed consent was obtained from the patients. The hASCs isolated were then cultured in growth media containing Dulbecco’s Modified Eagles’ Medium/ Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 0.25 μg/mL fungizone. The adipocyte differentiation of hASCs was induced by using adipocyte differentiation medium (Zenbio, DM-2) for 7 days, and then the medium was changed into adipocyte maintenance medium (Zenbio, AM-1) until 5 days in the absence or presence of TAK-715 at the indicated concentrations. On day 12, hASCs became mature adipocytes that rounded with large lipid droplets in the cytoplasm.