3h 2dg

The 3T3-L1 fibroblasts were seeded on coated Poly-L-lysine (PLL) (Sigma-Aldrich) dishes and differentiated. Mature 3T3-L1 adipocytes were washed twice with warm Krebs–Ringer–phosphate–HEPES (KRPH) buffer (20 mmol/L HEPES, 5 mmol/L KH₂PO₄, 1 mmol/L CaCl₂, 136 mmol/L NaCl, and 4.7 mmol/L KCl set at pH 7.4 with NaOH) containing either 1 or 0 mmol/L Mg²⁺. Cells were incubated without or with 10 nmol/L human insulin (Sigma-Aldrich) and 0 or 1 mmol/L Mg²⁺ for 0, 10, 20 or 30 minutes at 37°C in radioactive buffer containing 5 mmol/L 2-deoxyglucose (2DG) (Sigma-Aldrich) and 1 μCi ³H-2DG (PerkinElmer, Hoogvliet Rotterdam, the Netherlands). The incubation was stopped by washing with cold KRPH buffer and cells were lysed with 0.05% (w/v) SDS (MP Biomedicals) in dH₂O. Cell lysates were added to Opti-Fluor O scintillation liquid (PerkinElmer) and counted using the Hidex 600 SL. Radioactive counts were corrected for the background count in each of the incubation buffers. The assay was repeated without the addition of ³H-2DG to correct for protein concentrations using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Briefly, [³H]-2-deoxyglucose (³H-2DG, cat#NET328A; Perkin Elmer, Beaconsfield, UK) uptake was carried out using 1 × 10⁶ cells resuspended in 0.4 mL uptake medium. Each uptake for a biological replicate is performed in triplicate. ³H-2DG uptake was carried out in glucose free RPMI (cat#11879020, ThermoFisher Scientific, UK) containing ³H-2DG (1 μci/ml). 4 min uptake assays were carried out layered over 0.5 mL of 1:1 silicone oil (Dow Corning 550 (BDH silicone products; specific density, 1.07 g/mL: Cat#175633, Sigma-Aldrich, UK):dibutyl phthalate (Cat#524980, Sigma-Aldrich, UK). Cells were pelleted below the oil, the aqueous supernatant solution, followed by the silicone oil/dibutyl phthalate mixture was aspirated, and the cell pellet underneath resuspended in 200 μL NaOH (1 M) and β-radioactivity measured by liquid scintillation counting in a Beckman LS 6500 Multi-Purpose Scintillation Counter (scintillant Optiphase HiSafe 3, cat#1200.437; PerkinElmer, Beaconsfield, UK). Where indicated, 5 mM 2-deoxyglucose (2DG, cat#D6134; Sigma-Aldrich, UK), 5 mM 2-NBDG (cat#N13195, ThermoFisher Scientific, UK), 20 mM 4,6-O-ethylidene-α-d-glucose (4,6-O, cat#E32754; Sigma-Aldrich, UK) or 10 μM cytochalasin B (CytB, cat#C6762; Sigma-Aldrich, UK), were used respectively to inhibit radiolabelled ligand uptake.