Fig. 3.
Ceramide recruits PTEN to membrane rafts and suppresses PDK1 activation, Akt membrane recruitment, and Akt activity. (A) Ceramide recruits PTEN to membrane rafts. Crude plasma membranes from 3T3 L1 adipocytes (in the presence or absence of 50 μM C2-ceramide) were solubilized and subjected to sucrose density gradient fractionation, followed by Western blotting with an anti-PTEN antibody. CTB was used as a raft marker. Anti-tubulin was used to ensure the separation of membrane proteins from cytosolic proteins. (B) Densitometric analysis indicated a statistically significant difference between the raft PTEN levels in the presence of C2-ceramide and those in its absence (**P < 0.01; n = 3). (C) Representative time courses showing that the response of Lyn-PARE (n = 7) in 3T3 L1 preadipocytes was abolished by preincubation with 50 μM C2-ceramide (n = 6). (D) Representative time courses showing that membrane translocation of Akt PH domain (n = 3) in 3T3 L1 preadipocytes was abolished by preincubation with 50 μM C2-ceramide (n = 3). (E) Representative time courses showing that the response of AktAR (n = 6) in 3T3 L1 preadipocytes was abolished by preincubation of 50 μM C2-ceramide (n = 5). (F) Ceramide-mediated suppression of insulin-induced glucose uptake in 3T3 L1 adipocytes. Preincubation of 50 μM C2-ceramide with 3T3 L1 adipocytes for 60 min inhibited insulin-induced glucose uptake in these cells (****P < 0.0001; n = 3).