Animals and experimental design

Male wild-type (WT) mice and Ppara-null (KO) mice (age, 13 weeks; body weight, 24–30 g) were used in the present study. WT and KO mice were on a C57BL6 background, as previously described [23]. All procedures were performed in accordance with the guidelines of Shinshu University, the National Institutes of Health and the Association for Assessment and Accreditation of Laboratory Animal Care.

WT and KO mice were divided into three groups: a control group (vehicle- [0.1% methylcellulose, MC] treated group, n = 10 and n = 6 respectively), Los-treated group (n = 10 and n = 6 respectively) and Irbe-treated group (n = 10 and n=6 respectively) (50 bw/day of each ARB for 14 days). The chemicals were administered via oral gavage. After 14 days, the mice were killed.

WT and KO mice were administered a daily intra-peritoneal bolus injection of 0.3 g of FFA-BSA in sterile saline for 4 days, and the dose was increased (0.35 g for 3 days and 0.4 g for 7 days) to induce PON [21,22]. The following WT and KO mice groups were used: vehicle- (0.1% MC) treated mice without PON (Con, n = 10 and n = 6 respectively), vehicle-treated mice with PON (Veh-PON, n = 6 and n = 6 respectively), Los-treated mice with PON (Los-PON, n = 6 and n = 6 respectively), Irbe-treated mice with PON (Irbe-PON, n = 6 and n = 6 respectively), and Irbe and MK886 (PPARα antagonist)-treated mice with PON (Irbe-MKPON, n = 6, WT only). The vehicle or respective ARB (50 mg/kg bw/day) and PPARα antagonist (MK886, 1 mg/kg, intraperitoneally) were administered every day from Day 1 to Day 20. BSA was injected intraperitoneally every day from Day 7 to Day 20. All groups of WT mice were killed at Day 20. The procedures related to the preliminary experiments for the determination of the appropriate doses of Irbe and Los, as well as to the effect of MK886 on blood pressure, are described in the Supplementary Methods.