Dy1119

To determine the level of secreted adiponectin in control or SEPT1-knockdown 3T3-L1 adipocytes, cells were washed with PBS, and 500 µl of serum-free IMDM containing 100 nM insulin and 1% penicillin/streptomycin were added per well in a 12-well plate. 3T3-L1 adipocytes were incubated for 24 h at 37°C. Then the medium was recovered and the remaining material was centrifuged for 5 min at 500 g at room temperature to remove cell debris. Adiponectin levels in the collected media were measured with an enzyme-linked immunosorbent assay (ELISA) (DY1119, R&D Systems GmbH) following the manufacturer's instructions. Cells were washed with PBS and lysed to determine protein concentration. For quantification, adiponectin levels were normalized to the respective protein concentration.

The serum Adiponectin and Leptin levels were examined using an enzyme-linked immunosorbent assay (ELISA) (DY1119 and DY498, R&D Systems, Minnesota, USA). The serum fasting insulin levels and Glycated Hemoglobin A1c (GHbA1c) levels were examined using an ELISA kit (E08141m, Cusabio, Wuhan, China). The serum creatinine, TG, TCHO, LDLC and HDLC levels were examined using commercial reagent kits (C011-2-1, A110-1-1, A111-2-1, A113-1-1, A112-1-1, Jiancheng, Nanjing, Jiangsu, China). Urinary hemoglobinuria test was used a Urine Hemoglobin Qualitative Detection Kit (ADS043TC0, MEIMIAN, Jiangsu, China).

The serum insulin (M1104, Morinaga Institute of Biological Science, Inc., Yokohama, Japan), tumor necrosis factor-α (TNF-α; BMS607HS, Invitrogen, Carlsbad, CA, USA), interleukin-6 (IL-6; BMS603HS, Invitrogen), adiponectin (DY1119, R&D Systems, Minneapolis, MN, USA), and leptin levels (DY498, R&D Systems) were determined using an enzyme-linked immunosorbent assays (ELISA) kit. The serum AST and ALT levels were measured using a Fuji Dri-Chem 3500i (Fujifilm, Tokyo, Japan). The serum glucose level was determined using the enzymatic method (Asan Pharmaceutical Co., Ltd., Seoul, Korea). The HOMA-IR was calculated using the following formula: insulin (µIU/mL) × glucose (mmol/L) / 22.5.