PPARδ activation protects endothelial function in diabetic mice

Abstract

Recent evidence highlights the therapeutic potential of peroxisome proliferator-activated receptor-δ (PPARδ) agonists to increase insulin sensitivity in diabetes. However, the role of PPARδ in regulating vascular function is incompletely characterized. We investigate whether PPARδ activation improves endothelial function in diabetic and obese mice. PPARδ knockout (KO) and wild-type (WT) mice fed with high-fat diet and db/db mice were used as diabetic mouse models, compared with PPARδ KO and WT mice on normal diet and db/m(+) mice. Endothelium-dependent relaxation (EDR) was measured by wire myograph. Flow-mediated vasodilatation (FMD) was measured by pressure myograph. Nitric oxide (NO) production was examined in primary endothelial cells from mouse aortae. PPARδ agonist GW1516 restored EDRs in mouse aortae under high-glucose conditions or in db/db mouse aortae ex vivo. After oral treatment with GW1516, EDRs in aortae and FMDs in mesenteric resistance arteries were improved in obese mice in a PPARδ-specific manner. The effects of GW1516 on endothelial function were mediated through phosphatidylinositol 3-kinase (PI3K) and Akt with a subsequent increase of endothelial nitric oxide synthase (eNOS) activity and NO production. The current study demonstrates an endothelial-protective effect of PPARδ agonists in diabetic mice through PI3K/Akt/eNOS signaling, suggesting the therapeutic potential of PPARδ agonists for diabetic vasculopathy.

FIG. 1.

Effects of PPARδ, PI3K, or Akt inhibitors on EDRs in mouse aortae. A: HG impairs EDRs of aortae from C57BL/6J mice, an effect that is reversed by GW0742 or GW1516 (PPARδ agonist, 0.1 µmol/L, 36 h). B: The beneficial effect of GW1516 is reversed by GSK0660 (PPARδ antagonist, 1 µmol/L) or Akt inhibitor V (5 µmol/L). C: The beneficial effect of GW1516 is reversed by LY294002 (PI3K inhibitor, 5 µmol/L) or wortmannin (PI3K inhibitor, 0.1 µmol/L). D: Western blot showing that HG reduces eNOS phosphorylation at Ser¹¹⁷⁷, and reduces Akt phosphorylation at Thr³⁰⁸ and Ser⁴⁷³. These effects are reversed by GW1516 (0.1 µmol/L). The beneficial effects of GW1516 are abrogated by LY294002 (PI3K inhibitor, 5 µmol/L), wortmannin (PI3K inhibitor, 0.1 µmol/L), or GSK0660 (PPARδ antagonist, 1 µmol/L). E: Densitometry of Western blots showing the effect of GW1516, and the antagonists of its action on eNOS and Akt phosphorylations in primary MAECs treated with HG (30 mmol/L, 36 h). eNOS, 140 kDa; Akt, 60 kDa. Results are means ± SEM of six mice. *P < 0.05 vs. NG; #P < 0.05 vs. HG; †P < 0.05 vs. HG+GW1516.

FIG. 2.

The effect of GW1516 on NO production in endothelial cells (MAECs). A: Representative images of DAF-FM DA fluorescence signal in MAECs in response to Ca²⁺ ionophore A23187 (0.1 µmol/L) under a confocal microscope, analyzed by comparing fluorescence intensity before (F0) and after (F1) the addition of A23187. B–D: Summarized results showing the levels of NO production in MAECs treated with GW1516 (0.1 µmol/L) or GW0742 (0.1 µmol/L), in the presence or absence of GSK0660 (1 µmol/L), LY294002 (5 µmol/L), wortmannin (0.1 µmol/L), or Akt inhibitor V (5 µmol/L) in MAECs exposed to NG or HG. E: Summarized data using the area under the curve (AUC) starting from the addition of A23187 for 120 s of B–D. Results are means ± SEM of six to eight experiments. *P < 0.05 vs. NG; #P < 0.05 vs. HG; †P < 0.05 vs. HG+GW1516. Bar, 200 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

The effect of Akt activity inhibition on the EDRs in mouse aortae and NO production in MAECs. Effects of Ad-GFP (A), Ad-DN-Akt (B), Ad-CA-PI3K (C), and Ad-CA-Akt (D) on EDRs in mouse aortae treated with GW1516 (0.1 µmol/L) and/or HG (30 mmol/L, 36 h). Mouse aortae were exposed to adenovirus for 4 h in serum-free DMEM, and then changed to DMEM with 10% FBS, before vasoreactivity was measured in the myograph. Effects of CA-Akt (E) or DN-Akt (F) transfection on NO production in MAECs coincubated with GW1516 (0.1 µmol/L) and/or HG (30 mmol/L, 36 h). MAECs were transiently transfected with CA-Akt or DN-Akt plasmid by electroporation. G: Summarized data using the area under the curve (AUC) starting from the addition of A23187 for 120 s of E and F. H: Representative Western blot to show the effect of CA-Akt and DN-Akt on Akt and eNOS phosphorylation in MAECs. p-eNOS (Ser¹¹⁷⁷) and eNOS, 140 kDa; p-Akt (Thr³⁰⁸ and Ser⁴⁷³) and Akt, 60 kDa. Experiments were repeated five times using MAECs from different mice. Results are means ± SEM of five to six experiments. *P < 0.05 vs. NG from each group; #P < 0.05 vs. HG from each group.

FIG. 4.

The effect of GW1516 treatment on endothelial function in db/db mice. A: PPARδ agonist GW0742 (0.1 µmol/L, 24 h) or GW1516 (0.1 µmol/L, 24 h) improved EDRs in aortae from db/db mice. The beneficial effect of GW1516 was abrogated by coincubation with GSK0660 (PPARδ antagonist, 1 µmol/L). B: The beneficial effect of GW1516 in aortae from db/db mice is abrogated by LY294002 (5 µmol/L), wortmannin (0.1 µmol/L), or Akt inhibitor V (5 µmol/L). C: GW1516 treatment (5 mg/kg/day, 7–10 days) improved EDRs in aortae from db/db mice, without affecting endothelium-independent relaxations to SNP (D). E and F: GW1516 treatment increased phosphorylations of eNOS and Akt in db/db mouse aortae. Results are means ± SEM of n mice (n specified in Supplementary Table 1). *P < 0.05 vs. db/m⁺; #P < 0.05 vs. db/db.

FIG. 5.

GW1516 treatment improves FMD and insulin-induced vasodilatation in pressurized mesenteric arteries of diet-induced obese mice. GW1516 was administered by oral gavage (5 mg/kg/day, 7–10 days) to DIO C57BL/6 and db/db mice. A: Representative image of mesenteric resistance arteries from control C57BL/6 mice pressurized at 100 mmHg. B: Representative trace of FMD from each group. Pressure rise was induced in stepwise order (20–100 mmHg). Phe (10 μmol/L) was added after arteries stabilized at 100 mmHg to induce contraction before FMD was examined. C: Summarized FMD response in arteries from all the groups. D: Summarized data of insulin-induced vasodilatation in mesenteric resistance arteries from C57BL/6 control and DIO mice. Results are means ± SEM of four to five mice. *P < 0.05 vs. control or db/m⁺; #P < 0.05 vs. vehicle.

FIG. 6.

GW1516 treatment in vivo improved endothelial function in aortae from PPARδ WT mice but not in PPARδ KO mice after high-fat diet (DIO). GW1516 was administered by oral gavage (5 mg/kg/day, 7–10 days) to DIO PPARδ WT and age-matched PPARδ KO fed on high-fat diet for 3 months. Effects of GW1516 treatment on EDRs in aortae from PPARδ WT (A) and PPARδ KO (B) after high-fat diet–induced obesity compared with control mice on normal diet. Endothelium-independent relaxations to SNP in aortae from PPARδ WT (C) and PPARδ KO (D). E and F: Phosphorylation of eNOS and Akt in aortae from DIO PPARδ WT mice increased after GW1516 treatment, an effect that was not observed in those from DIO PPARδ KO mice. Results are means ± SEM of n mice (n specified in Supplementary Table 1). *P < 0.05 vs. control (normal diet) from each group; #P < 0.05 vs. DIO from each genotype.