Ceramide mediates vascular dysfunction in diet-induced obesity by PP2A-mediated dephosphorylation of the eNOS-Akt complex

Abstract

Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.

FIG. 1.

Inhibiting de novo ceramide accumulation improves the systemic metabolic environment of C57Bl/6 mice with diet-induced obesity. C57Bl/6 mice that consumed CON or HF chow for 3 months were treated concurrently with vehicle (V) or myriocin (M). Vascular (aorta) (A) and liver (B) ceramide content, body mass (C), body composition (D), gonadal fat pad mass (E), area under the curve during a glucose tolerance test (GTT) (F) and insulin tolerance test (G). Fasting (6 h) serum triglycerides (H) and serum leptin (I) concentrations. Fasting (6 h) blood glucose (mg/dL) before the GTT in panel F was higher in HF-V (122 ± 8) vs. CON-V (95 ± 7) mice but was similar between HF-M (102 ± 5) and CON-M (96 ± 5) animals. *P < 0.05 CON vs. HF, #P < 0.05 M vs. V. Results represent mean ± SEM from 10 CON-V, 13 HF-V, 7 CON-M, and 13 HF-M mice. AU, arbitrary unit.

FIG. 2.

Inhibiting de novo ceramide accumulation in vivo normalizes endothelial dysfunction and hypertension in C57Bl/6 mice with diet-induced obesity. Systolic (A), mean (B), and diastolic (C) arterial blood pressure during light and dark cycles. Data are averaged from 4- × 24-h periods for 10 CON-V, 10 HF-V, 9 CON-M, and 9 HF-M mice. D: EDR. E: EIR. F: NR-mediated vasocontraction. G: Receptor (R)-mediated vasocontraction. Data are from two femoral artery segments from 18 CON-V, 23 HF-V, 15 CON-M, and 17 HF-M mice. A–H: *P < 0.05 HF-V vs. all. G: #P < 0.05 HF-M vs. all. Results represent mean ± SEM. H: Representative immunoblot and densitometry of the ratio of p-eNOS at serine (S)1177 to total eNOS from aorta/iliac arterial homogenates from 8 CON-V, 13 HF-V, 6 CON-M, and 8 HF-M mice. *P < 0.05 HF-V vs. all. V, vehicle; M, myriocin.

FIG. 3.

Arterial function in fat-fed mice with targeted disruption of des1. des1^+/− and des1^+/+ mice were placed on CON or HF diets for 3 months. Vascular ceramide content (A), body composition (B), and AUC during a glucose tolerance test (GTT) (C) in 9 HF des1^+/+, 11 HF des1^+/−, 12 CON des1^+/+, and 13 CON des1^+/− mice. Fasting (6 h) blood glucose (mg/dL) before the GTT in panel C was higher in HF (164 ± 10) vs. CON des1^+/+ (122 ± 11) mice and HF (182 ± 11) vs. CON des1^+/− (147 ± 9) animals. D and E: EDR and NR-mediated vasocontraction from HF vs. CON des1^+/+ mice. F and G: EDR and NR-mediated vasocontraction from HF vs. CON des1^+/− mice. H: EIR from all groups. Data are from two femoral artery segments from each of the des1^+/+ and des1^+/− mice. I: Representative immunoblot and densitometry of the ratio of p-eNOS Ser1177 to total eNOS from aorta/iliac arterial homogenates from des1^+/+ and des1^+/− mice. *P < 0.05 CON vs. HF; #P < 0.05 des1^+/+ vs. des1^+/−. Results represent mean ± SEM. AU, arbitrary unit.

FIG. 4.

Arterial function in isolated arteries from wild-type and des1^+/− mice. Aorta from 10 C57Bl/6 mice were incubated with vehicle (veh), palmitate (pal), or pal + myriocin (myr). A: Pal-induced ceramide accumulation. B: EDR assessed in four femoral artery segments from the same 10 mice. Of these four segments, two were incubated with pal for 3 h while two were incubated with pal + myr for 3 h. C: EIR post pal ± myr treatment. D: Ceramide content in aorta from 6 des1^+/+ and des1^+/− mice after treatment with veh or pal. E: EDR in vessels from des1^+/+ and des1^+/− before and after 3-h pal treatment. F: EIR post pal in both genotypes. G: p-eNOS Ser1177 to total eNOS in aorta from wild-type mice (lanes 1–3) and in aorta from des1^+/− mice (lanes 4–5) treated with or without pal (n = 5 segments per treatment). Representative blot (upper) and mean densitometry (lower). *P < 0.05 pre vs. post pal incubation. Results represent mean ± SEM.

FIG. 5.

Endogenous ceramide biosynthesis impairs NO generation. BAECs were incubated in the presence (+) or absence (−) of palmitate (pal) ± myriocin (myr). A: Pal-induced ceramide accrual (n = 6 per treatment). In some experiments, BAECs were treated for the last 10 min with vehicle (veh), insulin (ins), or VEGF. B and C: Basal (i.e., veh), ins-, or VEGF-stimulated p-eNOS Ser1177 to total eNOS (n = 10–32 per treatment). D: p-eNOS Ser617 (n = 15–19 per treatment). E: p-eNOS Thr495 to total eNOS (n = 10 per treatment). F and G: Ins- or VEGF-stimulated increases in the eNOS dimer to monomer ratio (n = 7–12 per treatment). H: NOS activity (n = 6–7 per treatment). I: Ins-mediated NO_x production (n = 10–15 per treatment). *P < 0.05 vs. respective veh treatment; #P < 0.05 vs. (+) ins or (+) VEGF and (−) pal. Results represent mean ± SEM.

FIG. 6.

Ceramide-associated changes in kinase signaling and O₂^•−-mediated peroxynitrite formation. BAECs incubated ± pal ± myr for 3 h were treated for the last 10 min ± veh or ins. p-Akt Ser473 and Thr308 to total Akt (n = 12–37 per treatment) (A), p-AMPK T172 to total AMPK (n = 12–15 per treatment) (B), and p-ERK 1/2 to total ERK 1/2 (n = 13–27 per treatment) were not altered by pal (C). D: ESR indicated that 500 μmol/L pal increased cellular O₂^•− production and that responses could be negated by myr and the O₂^•− scavengers SOD, PEG-SOD, and tiron (n = 4–18 per treatment). E: Nitrotyrosine formation did not occur in response to 500 μmol/L pal (n = 8–16 per treatment). F: Pal-induced suppression of ins-stimulated p-eNOS to total eNOS could be restored by tiron + myr but not by tiron alone (n = 12–28 per treatment). *P < 0.05 vs. respective veh, #P < 0.05 vs. (+) ins (−) pal. Results represent mean ± SEM. Pal, palmitate; myr, myriocin; veh, vehicle; ins, insulin; PEG, polyethylene glycol.

FIG. 7.

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 NO_x 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.

FIG. 8.

PP2A promotes dephosphorylation of Akt that associates with eNOS. BAECs were incubated ± pal ± OA. For the last 10 min, cells were treated with veh or ins (A–D). After eNOS immunoprecipitation (IP), Western blot (IB) for PP2A, Akt, p-Akt, and eNOS was performed. A: PP2A association with eNOS (n = 12–34 per treatment). B: Akt association with eNOS (n = 13–35 per treatment). C: p-Akt Ser473 and p-Akt Thr308 association with eNOS (n = 6–8 per treatment). D: p-eNOS Ser1177 to total eNOS (n = 12–18 per treatment). *P < 0.05 vs. veh, #P < 0.05 vs. (−) pal (+) ins (−) OA. E: BAECs were incubated ± pal ± myr. After PP2A IP, IB for I2PP2A and PP2A was performed. I2PP2A that coimmunoprecipitated with PP2A was decreased by pal in a ceramide-dependent manner (n = 15–17 per treatment). A reciprocal IP is shown in Supplementary Fig. 9C. *P < 0.05 vs. (−) myr (−) pal; #P < 0.05 vs. (−) myr (+) pal. Results represent mean ± SEM. Pal, palmitate; myr, myriocin; veh, vehicle; ins, insulin.

FIG. 9.

Working model. HF feeding leads to arterial ceramide accumulation in vivo. Palmitate incubation elevates ceramide in isolated arteries and endothelial cells in vitro. Ceramide increases the association of PP2A with eNOS and decreases the association between eNOS and Akt and between eNOS and Hsp90. PP2A promotes the dephosphorylation of Akt that colocalizes with eNOS and/or decreases eNOS phosphorylation at Ser1177 and Ser617 directly. This impairs NO bioavailability and leads to vascular dysfunction.