Fig. 7.

The depletion of Cav 1 in 3T3-L1 adipocytes mistargeted Kv1.3 lipid raft localization as well as impaired insulin-dependent phosphorylation of the channel. Lipid rafts were isolated from 3T3-L1 pre-adipocytes and adipocytes. A sucrose gradient, from low (1)- to high (12)-density fractions was applied, and the expression of Kv1.3, clathrin (non-raft marker) and caveolin (lipid raft marker) was analyzed. In addition, the 3T3-L1 Cav 1⁻ cell line, with a silenced expression of Cav 1, was used. a Low expression of Kv1.3 in lipid rafts from 3T3-L1 wild type (WT) pre-adipocytes. b Increased expression of Kv1.3 targeting to rafts in 3T3-L1 WT adipocytes. Note the exacerbated augmentation of Cav 1 due to adipocyte differentiation. c Low expression of Kv1.3 in lipid rafts from 3T3-L1 Cav1⁻ pre-adipocytes. d A minor increase of Kv1.3 is concomitant with reduced channel localization in rafts in 3T3-L1 Cav 1⁻ adipocytes. Note the limited augmentation of Cav 1 due to adipocyte differentiation in this cell line. e Increase in Kv1.3 expression during adipocyte differentiation in 3T3-L1 WT and Cav 1⁻ cells. *p < 0.05 vs pre-adipocytes (Student’s t test, n = 3-5). f Percentage of Kv1.3 in lipid rafts. Kv1.3 expression in caveolin-positive floating fractions was analyzed and relativized to the total amount of Kv1.3. *p < 0.05, ***p < 0.001 (Student’s t test, n = 3–5). White columns represent WT, black columns represent Cav 1⁻ cells. g The reduction of Cav 1 in Cav 1⁻ 3T3-L1 cell line impairs the insulin-dependent phosphorylation of Kv1.3. WT and Cav 1⁻ cells were incubated with (+) or without (−) insulin, as described in methods. Total cell lysates were immunoprecipitated (IP) against phosphotyrosines (P-Tyr) and immunoblotted (IB) against the insulin receptor (InsR) and Kv1.3. Note an impairment in the phosphorylation of InsR and Kv1.3 in Cav 1⁻ cells in the presence (+) of insulin