Figure 4.

Time course of insulin-mediated inhibition of IRS-2 promoter activity in transfected rat hepatocytes. Primary rat hepatocytes were isolated and plated as described in Materials and Methods. Hepatocytes were cotransfected with pCMV-SEAP plasmids and either pIRS2-Luciferase(wt) (▴) or pIRS2-Luciferase(mut-3bp) (●) as described in the legend to Fig. 3. Six hours after transfection (zero time for the experiment), the serum-free RPMI medium 1640 in all dishes was supplemented with 5 μM dexamethasone, and the dishes were incubated an additional 24 h before harvest. Insulin (100 nM) was added to duplicate dishes at the indicated time before harvest. Luciferase and alkaline phosphatase activities were measured as described in the legend to Fig. 3. The value for wild-type pIRS2-Luciferase activity in the absence of insulin was arbitrarily set as 1. Each value represents the average of duplicate incubations. This experiment was repeated three times with similar results.