FIGURE 6.

ORM reduces interaction between adipocytes and macrophages. A, ORM suppresses the effect of adipocyte-conditioned media on adhesion of THP-1 monocytes to the bottom of culture dishes. Adipocyte-conditioned media were obtained from mock or Orm1 siRNA overexpressing 3T3-L1 adipocytes in the presence or absence of ORM (250 μg/ml) or TNFα (10 ng/ml) for 24 h. The adhesion of the THP-1 monocytes onto the bottom of the culture dish was photographed after incubating the cells in the adipocyte-conditioned media and washing them three times with PBS. B, ORM suppresses the recruitment of THP-1 monocytes to adipocytes. 3T3-L1 adipocytes and THP-1 monocytes were pretreated with or without ORM (250 μg/ml) or sodium salicylate (5 mm) for 24 h, and then the THP-1 monocytes were added to the upper chamber of the Transwell plates that contained the adipocytes and stimulated with TNFα. The recruitment of THP-1 cells to adipocytes was measured by immunostaining monocytes with an anti-CD68 antibody (red). C, ORM binds to macrophages in the adipose tissue. Epididymal adipose tissues from the lean (control) or DIO (fed HFD for 3 months) mice were immunostained with fluorescein isothiocyanate-conjugated BODIPY (green; adipocytes) or antibodies against ORM (red) or F4/80 (blue; macrophages) and whole-mounted for multiphoton fluorescence confocal microscopy.