Insulin

Brown preadipocytes were isolated from the interscapular BAT of 4-week-old male Sprague Dawley rats (Orient Bio Inc., Seongnam, Korea) and differentiated as described previously [9 (link)11 (link)]. Isolated brown preadipocytes were incubated in high glucose Dulbecco's Modified Eagle's Medium containing a 1% antibiotic solution and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37℃ in a humidified atmosphere with 5% CO₂. For differentiation, the immortalized brown adipocytes were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum, 1 nM T3, and 20 nM insulin (Boehringer-Mannheim, Mannheim, Germany) until 70% confluent; thereafter, it was called differentiation medium (DM). Then, the cells were cultured in DM supplemented with 0.5 mM isobutylmethylxanthine, 0.125 µM indomethacin (Sigma-Aldrich Inc., St. Louis, MO, USA), and 0.5 µM dexamethasone (Sigma-Aldrich Inc.) for 2 days; thereafter, it was called induction medium (IM). Next, the cells were cultured in DM until they exhibited a fully differentiated phenotype with multiple multilobular lipid droplets in the cytoplasm. For the in vitro study, RSV (Sigma-Aldrich Inc.) was provided during the IM supplementation period and maintained thereafter. RSV was dissolved in dimethyl sulfoxide (Sigma-Aldrich Inc.) for stock solution with concentration of 100 mM.

Fibroblasts were incubated for 10 days in a differentiation medium of serum-free DMEM supplemented with 17 µM pantothenic acid, 33 µM biotin, 10 µg/mL transferrin, 1 µM insulin (Boehringer-Mannheim, Mannheim, Germany), 0.2 nM T3, and 0.2 µM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), according to a previously published protocol.¹⁶ (link) For the first 4 days of cell growth, the differentiation medium included 10 µM dexamethasone and 0.1 mM isobutylmethylxanthine (IBMX). From day 5 onward, dexamethasone and IBMX were excluded. Starting on the first day of differentiation, rosiglitazone (10 µM; Cayman, Ann Arbor, MI, USA), a PPARγ agonist, or 10 ng/mL IL-1β or both were added for heightened adipogenesis stimulation. To assess the influence of GS on adipogenesis, cells were treated with GS (5, 10, or 25 µM) for the entire differentiation period of 10 days, with media changes every 2 to 3 days.

Orbital pre-adipocyte fibroblasts were differentiated into adipocytes for 14 days using previously described methods.¹⁷ (link)^,18 (link) To assess the effect of silencing adipsin on adipogenesis, cells were transfected with si-adipsin or si-con for 48 hours before 14-day differentiation period. Briefly, cells grown in 6-well plates were stimulated with adipogenic solutions; 10% FBS DMEM, 33 µM biotin, 17 µM pantothenic acid, 10 µg/mL transferrin, 0.2 nM T3, 1 µM insulin (Boehringer-Mannheim, Mannheim, Germany), 0.2 µM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), and 10 µM rosiglitazone (Cayman, Ann Arbor, MI, USA). For the first 4 days, 10 µM dexamethasone and 0.1 mM isobutylmethylxanthine (IBMX) were added in the media and the media was replaced every 2 to 3 days.

Fibroblasts were induced to differentiate into adipocytes following a previously published protocol.¹³ (link)^,14 (link) Briefly, cells were cultured in serum-free DMEM supplemented with T3, insulin (Boehringer-Mannheim, Mannheim, Germany), carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), and dexamethasone, along with a peroxisome proliferator activator gamma (PPARγ) agonist, rosiglitazone (10 µM; Cayman Chemical, Ann Arbor, MI, USA) for 10 days.