Figure 1.

Assay. 3T3-L1 fibroblasts stably expressing HA–Glut4/GFP were differentiated into adipocytes in 96-well plates. The cells develop a three-dimensional co-culture of adipocytes embedded in a collagen matrix, with a monolayer of fibroblasts on the plastic. The cultures were incubated in serum-free media for 2 h (basal) at 37 °C. Insulin was added, and cells were incubated for 45 min. Compound was added, and cells were incubated for 1 h. Cells were placed on ice, and surface Glut4 was labeled with AF647-conjugated anti-HA antibody. Cells were detached from the plate with collagenase and analyzed by flow cytometry. A, insulin dose response. B, time course. C, pilot screen. D, identification of hits. For activators (green), relative geometric mean fluorescence (rGMF) was ≥1.5, and for inhibitors (red), rGMF was ≤0.62. 64 activators and 65 inhibitors were verified by rescreening, and representative compounds were confirmed with pure compound and validated by concentration response, SAR, and time course (Table S1).