FIGURE 7.

ORM improves energy metabolism in adipocytes. A, 3T3-L1 adipocytes were incubated with low (5.5 mm) or high (25 mm) glucose (Gluc) media in the presence or absence of ORM (250 μg/ml) for 24 h. The levels of the accumulated ROS in adipocytes were measured by 5-(and -6)-chloromethyl-2′m7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) fluorescence staining. B, basal and insulin-dependent glucose uptake activity assay. 3T3-L1 adipocytes were incubated with low (5.5 mm) or high (25 mm) glucose media in the presence or absence of ORM (250 μg/ml) for 24 h. Glucose uptake activity was measured at 30 min after the insulin treatment. NS, not significant. C, suppression of Orm1 expression via siRNA impairs insulin sensitivity in adipocytes. 3T3-L1 cells stably expressing empty pSuper.retro (mock) or pSuper.retro-Orm1siRNA were differentiated into adipocytes. Mature adipocytes expressing mock or Orm1 siRNA were incubated with or without ORM (250 μg/ml) for 24 h. Glucose uptake activity was measured at 30 min after insulin treatment. D, ORM potentiates insulin signaling. 3T3-L1 adipocytes were serum-starved for 16 h in hyperglycemic condition (25 mm) and then treated with ORM (250 μg/ml) for 6 h. After 15 min of stimulation with insulin (10 nm), the cells were subjected to Western blot analysis. Ctl, control. E and F, 3T3-L1 adipocytes were pretreated with ORM (250 μg/ml, black bar) or sodium salicylate (5 mm, gray bar) in DMEM supplemented with 0.2% bovine serum albumin and then stimulated with TNFα (10 ng/ml) for a further 48 h. mRNA levels of perilipin were measured by Q-PCR analysis (E). Levels of lipolysis were determined by measuring glycerol concentration in the adipocyte-conditioned media (F). *, p < 0.05; **, p < 0.01. n = 3.