Glucose uptake assay

Adipocyte glucose uptake was measured using the Glucose Uptake-Glo Assay (Promega) according to the manufacturer’s protocol. In brief, 3T3-L1 cells, stably transduced with retroviral vectors (_WTMCPIP1, _D141NMCPIP1 and control cells), were seeded in a 96-well white plate at the density of 2 × 10³ cells per well. After reaching confluence, adipocyte differentiation was induced with DMI medium. At day 7 of differentiation, cells were starved overnight in serum-free medium (DMEM with 4.5 g/l glucose). Next, cells were starved for glucose by preincubating with 2% bovine albumin serum (BSA; Bioshop) in Krebs-Ringer-Phosphate-HEPES (KRPH; 20 mM HEPES, 5 mM KH₂PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 136 mM NaCl, 4.7 mM KCl, pH 7.4) buffer for 40 min. Then, cells were stimulated with 1 μM insulin for 20 min, following by 1 mM 2-deoxyglucose (2-DG) incubation for 20 min. 2DG6P Detection Reagent was incubated for 1 h at room temperature. To measure glucose uptake, the luminescence was recorded with 1000 ms integration time using Tecan Spectra Fluor Plus Microplate Reader (Thermo Fisher Scientific) with Tecan iControl 1.5. Software.