Recombinant human insulin

Primary BAT SVF cells were isolated from C57BL/6J background mice as described (Shan et al., 2016 (link)). Briefly, the interscapular brown fat was dissected from 8-week-old mice. Next, the brown fat was cut to fine pieces and digested with 1.5 mg/ml collagenase type I (catalog number SCR103, Sigma-Aldrich) at 37°C water bath for 50 min. The tissue was vortexed every 10 min. Then, the digestion was filtered through 100-μm and 70-μm cell strainers. The digestion was centrifuged at 400 g for 6 min to enrich SVF cells. The cells were seeded into a cell culture plate with growth medium (DMEM and 10% fetal bovine serum). To induce adipogenic differentiation, the growth medium was supplemented with 2.85 mM recombinant human insulin (catalog number I8830, Solarbio), 0.3 mM dexamethasone (catalog number D8040, Solarbio), and 0.63 mM 3-isobutyl-methylxanthine (catalog number I7018, Sigma-Aldrich). After 96 h, the medium was switched to growth medium, supplemented with 10 nM triiodothyronine (catalog number T6397, Sigma-Aldrich), and 200 nM insulin to induce mature adipocytes. For Oil red O staining, cells were fixed with 10% formaldehyde for 5 min and stained with staining solutions (catalog number G1262, Solarbio) according to the instructions.

Fatty acid-free bovine serum albumin (BSA) and recombinant human insulin were purchased from Solarbio Life Science (Beijing, China). Chemical compounds including leucine, isoleucine, valine, alanine, phenylalanine, serine, threonine, glutamic acid, glutamine, arginine, glycine, ornithine, tyrosine, proline, lysine, 3-hydroxybutyric acid (BHB), palmitic acid (PA), linoleic acid (LOA), stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). U0126-EtOH was purchased from MedChemExpress (Monmouth Junction, NJ, USA). The antibodies of ERK1/2, phosphor-ERK1/2 (Thr202/Tyr204) were purchased from Affinity Biosciences (USA) and c-Myc, cyclin D1, and alpha-tubulin were purchased from Proteintech (Chicago, IL, USA). Palbociclib and SCH772984 were purchased from Jiangsu Aikang Biomedicine Research Co., Ltd. (Nanjing, China). Isophane Protamine Biosynthetic Human Insulin Injection (Pre-mixed 30R) was purchased from Novo Nordisk (China) Pharmaceuticals Co., Ltd.