Following differentiation, 3T3-L1 adipocytes were co-cultured with RAW264.7 macrophages using the Transwell system (0.4 µm porous membrane; Corning). In the co-culture, differentiated 3T3-L1 cells (1 × 105) were cultured in the lower well; while, 5 × 104 RAW264.7 macrophages were cultured in inserts that constituted the upper chamber. Following the establishment of co-culture, RAW264.7 cells in the upper chamber were treated with MTC (5–20 µM) for 30 min, followed by stimulation with LPS (1 µg/mL) and IFNγ (10 ng/mL) for a further 24 h. Culture supernatants were collected and analysed for levels of NO, TNFα, IL-1β, IL-6 using similar protocols described in experiments on RAW264.7 macrophage mono-culture. Production of monocyte chemoattractant protein-1 (MCP-1/CCL2) was measured using a mouse CCL2 ELISA kit (Invitrogen); while, levels of RANTES in culture supernatants were detected with a mouse CCL5/RANTES ELISA kit (R and D Systems).
Glucose uptake was determined by incubating differentiated 3T3-L1 adipocytes in the lower chamber with 2‐deoxy‐2‐[(7‐nitro‐2, 1, 3‐benzoxadiazol‐4‐yl) amino]‐d ‐glucose (2‐NBDG) (100 µM) in glucose-free DMEM for a further 1 h after treatment. Thereafter, cells were washed with PBS and glucose uptake by fluorescence detection at an excitation/emission wavelength of 485 nm/535 nm.
Glucose uptake was determined by incubating differentiated 3T3-L1 adipocytes in the lower chamber with 2‐deoxy‐2‐[(7‐nitro‐2, 1, 3‐benzoxadiazol‐4‐yl) amino]‐