Insulin

Human adipose-derived mesenchymal stem cells (hADMSCs) were purchased from CEFO (Seoul, Korea). The cells with biological passage number 4 were seeded into appropriate cell culture dishes at the density of 6000 cells/cm² and cultured in hADMSC growth medium (CEFO, Seoul, Korea) until cells were 80% confluent. Adipogenic differentiation was induced for 21 days in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Amarillo, TX, USA), containing 1% penicillin–streptomycin (Gibco, Amarillo, TX, USA), 10% fetal bovine serum (Alphabioregen, Boston, MA, USA), 1 μM dexamethasone (Sigma-Aldrich Chemicals, St. Louis, MO, USA), 100 μM indomethacin (Sigma-Aldrich Chemicals, St. Louis, MO, USA), 10 μg/mL insulin (Welgene, Daegu, Korea), and 500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Chemicals, St. Louis, MO, USA). Osteogenic differentiation was induced by culturing cells for 21 days in osteogenic differentiation medium containing low-glucose DMEM (Gibco, Amarillo, TX, USA), 1% penicillin–streptomycin (Gibco, Amarillo, TX, USA), 10% fetal bovine serum (Alphabioregen, Boston, MA, USA), 0.1 μM dexamethasone (Sigma-Aldrich Chemicals, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich Chemicals, St. Louis, MO, USA), and 50 μM L-ascorbic acid-2-phosphate (Sigma-Aldrich Chemicals, St. Louis, MO, USA).

A murine preadipocyte cell line, 3T3-L1, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Gyeongsan, Korea), supplemented with 10% bovine calf serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (P/S) (Invitrogen) in a 37 °C and 5% CO₂ environment. The cells were differentiated into adipocytes in DMEM, 10% fetal bovine serum (FBS) (Gibco), 1% P/S, 500 μM isobutylmethylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/mL insulin (Welgene) for 2 days. After the differentiation, the adipocytes were incubated in 10% FBS, 1% P/S, and 1 μg/mL insulin for 6 days. The cells were treated with 30, 50, or 100 μM NHDC or GNHDC for 8 days.

The human mesenchymal cell line, hADMSCs, were purchased from CEFO (Seoul, Korea) and cultured in hADMSC growth medium (CEFO, Seoul, Korea) until cells were 80% confluent. To induce adipogenic differentiation, hADMSCs of biological passage number 4 were used. Cells were detached using Accutase (Innovative Cell Technologies, San Diego, CA, USA) and then seeded into appropriate cell culture dishes at the density of 6000 cells/cm² and cultured until they become 80% confluent. Then, differentiation into adipocytes was induced by culturing the cells for 18 days in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Amarillo, TX, USA) containing 1% penicillin streptomycin (Gibco, Amarillo, TX, USA), 10% fetal bovine serum (Alphabioregen, Boston, MA, USA), 1 μM dexamethasone (Sigma-Aldrich Chemical, St. Louis, MO, USA), 100 μM indomethacin (Sigma-Aldrich Chemical, St. Louis, MO, USA), 10 μg/mL insulin (Welgene, Gyeongsan, Korea), and 500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Chemical, St. Louis, MO, USA). The differentiation medium was replaced every 3 days. Differentiated adipocytes were validated by the relative expression of mature adipocyte markers and the stained cellular lipid droplets in differentiated adipocytes were compared with hADMSCs (Figure S1).