FIGURE 3.

Ablation of Des1 inhibits ceramide accumulation and protects insulin signaling. A, Akt Ser-473 phosphorylation (pAkt) was determined in C2C12 myotubes 48 h after transfection with Des1 siRNA or control. Cells were treated for 16 h with 0.75 mm PA followed by insulin stimulation (100 nm; 10 min). Des1 knockdown protected insulin signaling. pGSK3β, phospho-glycogen synthase kinase 3β; GSK3β, glycogen synthase kinase 3β. A and B, Des1 knockdown was confirmed by Western blot (A) and quantitative PCR (B). To confirm the quality of Des1 knockdown, levels of ceramides and dihydroceramides were determined and found to vary significantly between treatments. C and D, Des1 knockdown robustly inhibited ceramide accumulation (C) and induced a significant increase in dihydroceramides in response to 0.75 mm PA (D) when compared with control conditions. *, p < 0.05 for treatment versus BSA. +, p < 0.05 for dihydroceramides versus ceramides in BSA with Des1 siRNA (n = 4–5).