Fig. 4.

Overexpression of uncoupling protein (UCP)-1 or manganese superoxide dismutase (MnSOD) does not prevent the development of high-glucose/insulin-mediated insulin resistance. A: 3T3-L1 adipocytes were infected with adenovirus expressing UCP-1 or MnSOD or with empty vector as described in research design and methods. Controls not treated with adenovirus (NA) were also assayed. After infection (5 days), cells were preincubated for 18 h in 5 mM glucose (open bars) or in 25 mM glucose with 0.6 nM insulin (filled bars) and then deprived of serum and insulin for 2 h before acute stimulation for 15 min with 100 nM insulin followed by measurement of glucose transport [assessed as the uptake of 2-deoxyglucose (2-DOG) over 3 min]. Cells that were not stimulated with insulin acutely are indicated as “Basal”. Overexpression of UCP-1 stimulated glucose transport in the basal state (*P < 0.05-P < 0.01), and both the overexpression of UCP-1 and MnSOD enhanced stimulation of glucose transport by insulin in cells that were preincubated in 5 mM glucose (*P < 0.01). However, neither UCP-1 nor MnSOD prevented the marked impairment in insulin-stimulated glucose transport in cells that had been preincubated in high glucose + insulin (filled bars); n = 4 for basal and 8 for “Acute Insulin” from 4 independent experiments. B: Western blots of cells expressing UCP-1 or MnSOD or no adenovirus (NA), incubated for 18 h with 5 mM glucose (5) or 25 mM glucose + 0.6 nM insulin (25I), and then stimulated acutely with 100 nM insulin. The blots are developed with an antibody against phosphorylated Akt (p-Ser). C: quantitative analysis of data in B; n = 4 from 2 experiments. D: Western blots of cell lysates obtained under conditions as in B but in the basal state and developed with antibodies against UCP-1 and MnSOD.