FIG. 2.

Mechanism of ceramide action on insulin-induced phosphorylation of PKB/Akt in 3T3-L1 adipocytes. A: Control 3T3-L1 adipocytes and KD-PKCζ–infected cells were preincubated with 100 μmol/l C₂-ceramide for 2 h, followed by 500 μmol/l OKA for the last 30 min. Then, 100 nmol/l insulin was added to the cells for 10 min before being lysed. Cell lysates were immunoblotted with antibodies against either native PKB/Akt, Ser473 PKB/Akt, Ser21/9 GSK3α/β, or PKCζ. Scanning densitometry was performed to quantify changes in Ser473 PKB/Akt abundance in cell lysates. Bars represent mean ± SEM. *Significant change P < 0.05 relative to the untreated control. Blots shown are representative of three separate experiments. B: Control, WT-PKCζ–, and KD-PKCζ–infected 3T3-L1 adipocytes were incubated with 100 nmol/l insulin for 10 min before being lysed. Cell lysates were immunoblotted with antibodies against either native PKB/Akt, Ser473 PKB/Akt, or PKCζ. Scanning densitometry was performed to quantify changes in Ser473 PKB/Akt abundance in cell lysates. Bars represent mean ± SEM. *Significant change P < 0.05 relative to the untreated control WT-PKCζ–expressing cells. Blots shown are representative of six separate experiments.