Cellular ceramide biosynthesis impairs ins-stimulated p-eNOS in a PP2A-dependent manner. BAECs were incubated for 3 h ± pal
± myr or ± pal ± OA. For the last 10 min, BAECs were treated with veh or ins. p-eNOS(S)1177 (A) and NOx production (B) in cells incubated with pal, myr, and OA as shown (n = 6–33 per treatment). C: Representative immunoblot showing impact of PP2A gene silencing on ins-mediated eNOS phosphorylation ± pal. D: The densitometry of six independent experiments are shown. E–G: BAECs were incubated ± pal ± myr. After eNOS immunoprecipitation (IP), a Western blot (IB) for PP2A, Akt, Hsp90, and eNOS
was performed. E: Pal promotes PP2A association with eNOS in a ceramide-dependent manner (n = 9–11 per treatment). A reciprocal IP is shown in Supplementary Fig. 9A. *P < 0.05 vs. (−) ins (−) pal; #P < 0.05 vs. (+) ins (−) pal. F and G: A trend (P = 0.05) existed for pal to decrease Akt and Hsp90 association with eNOS. Pal prevented ins-stimulated Akt and Hsp90 association
with eNOS in a ceramide-dependent manner (n = 5–8 per treatment). A reciprocal IP for Akt is shown in Supplementary Fig. 9B. *P < 0.05 vs. respective (−) ins, (−) pal treatment. H: In arterial homogenates from CON and HF des1+/+ and des1+/− mice, an IP for eNOS was performed followed by an IB for PP2A (n = 5–6 per treatment). *P < 0.05 vs. CON, veh; #P < 0.05 vs. (+ ins) (− pal) (F and G) or vs. des1+/+ (H). Results represent mean ± SEM. Pal, palmitate; myr, myriocin; veh, vehicle; ins, insulin.