Figure 4. Membrane-embedded DHA and EPA acylate and anchor PDK1 and AKT2 to membrane.

(A) Dose-dependent accumulation of DHA and EPA to the cell membrane. Various levels of DHA or EPA were added to the culture media for 1 h. The membrane and cytosolic levels of DHA and EPA were detected by GC-MS (n = 3, mean ± SEM).

(B) Time course accumulation of DHA and EPA to the cell membrane. 100 μM DHA or EPA were used to treat cells; the membrane and cytosolic levels of DHA and EPA were detected by GC-MS (n = 3, mean ± SEM).

(C) PUFA phospholipid formation affected DHA and EPA efficacies. The activation of PI3K-AKT signaling by DHA and EPA was detected in 3T3-L1 cells when phospholipid formation and hydrolyzation were inhibited by chlorpromazine (CPZ) and quinacrine (QA), respectively.

(D) DHA and EPA acylation promote PDK1 and AKT2 enrichment on the cell membrane in vivo. 129/C57BL6 mice were starved for 6 h before gavage with normal saline (control), DHA, DHA/tryptophan, EPA, or EPA/tryptophan. The distribution of PDK1 and AKT2 in the skeletal muscle of treated mice was analyzed by immunofluorescence following sacrifice after 4 h of treatment. Scale bar, 50 μm.

(E) The sites and the acylation levels of PDK1 and AKT2 in cells before and after DHA or EPA treatment. Targeted proteomic analysis was used to search for possible acylated PDK1 and AKT2 in 3T3-L1 adipocytes prior to and after being treated with DHA or EPA. Levels of acylation were estimated by employing synthetic peptides of known concentrations as internal controls (n = 3, mean ± SEM).

(F) DHA and EPA promote AKT2, AKT2^W334L, PDK1, PDK1^W266/347L, but not the AKT2^W414L, PDK1^W448/543L enrichment on the cell membrane. The localization of AKT2, AKT2^W334L, AKT2^W414L, PDK1, PDK1^W266/347L, and PDK1^W448/543L were monitored in confocal live-cell images after the cells were treated with DHA and EPA. Results of representative time points are presented (left). Scale bar, 20 μm. The fraction of responded cells were quantified (right) (n = 50, mean ± SEM).