Rolipram

Male C57BL/6J mice (7 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and used for the serum hormone assay and RT-PCR. Male ICR mice were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan), and female PERIOD2::LUCIFERASE (PER2::LUC) knock-in mice (ICR background) bred at Waseda University were used for blood glucose measurements and in vivo imaging assays, respectively. The mice were all housed under conditions of controlled temperature (23 °C ± 3 °C), humidity (50% ± 20%), and lighting (lights on from 07:00 to 19:00 at Taisho Pharmaceutical Co., Ltd. or from 08:00 to 20:00 at Waseda University). In the 24 h cycle, Zeitgeber time 0 (ZT0) was designated as the time when the lights were turned on, and ZT12 was designated as the time when the lights were turned off. All the mice were given access to food and tap water ad libitum, and nobiletin (10–100 mg/kg; purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was administered by intraperitoneal (i.p.) injection in the vehicle (10 mL/kg body weight) containing 0.5% carboxymethyl cellulose (CMC). Rolipram (PDE4 inhibitor, FUJIFILM Wako Pure Chemical Corporation) and SR1078 (RORα/γ agonist, FUJIFILM Wako Pure Chemical Corporation) were also used for the experiment at 10 mg/kg in 0.5% CMC [12 (link)]. Mice were euthanized by rapid decapitation for whole blood samples.

PSB1115 was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Methylcellulose, ibudilast and rolipram were obtained from Wako Pure Chemical Industries (Osaka, Japan). Medetomidine chloride (Domitor^®) and butorphanol tartrate (Vetorphale^®) were obtained from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) and midazolam was purchased from SANDOZ (Tokyo, Japan). Istradefylline and theophylline were purchased from Sigma (St. Louis, MO). Cilostazol and aminophylline were obtained from LKT laboratories, Inc. (St Paul, MN). MRS-1754 and BAY60-6583 were from Tocris Bioscience (Bristol, UK) and 1,3-dipropyl-8-cyclopentylxanthine (DCPCX), ZM241385 and CGS21680 were from Abcam (Cambridge, UK). ICR mice (5- or 6-week-old males, 28–33 g), primiparous late pregnant Wistar female rats and normal male Wistar rats (4- or 5-week-old, 150–250 g) were obtained from Charles River Laboratories Japan (Yokohama, Japan). The animals were housed under conditions of controlled temperature (22–24 °C) and illumination (12-h light cycle) conditions for 1 or 2 weeks before experiments. The experiments and procedures described here were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and were approved by the Animal Care Committees of Keio University and St. Marianna University.

The PDE inhibition assay was performed using the PDE-Glo^TM Phosphodiesterase assay (Promega). Bovine brain-derived PDE, majority of which was PDE4 isozyme [26 (link)], was purchased from Sigma. One milliunit of PDE was pre-incubated with varying concentrations of rolipram (Wako Chemicals, Japan), resveratrol, T4HS or 4-PAP for 30 min at room temperature, and then 1 μM cAMP substrate was added and the reactions were incubated for a further 90 minutes at 37°C. Luminescence was measured using the Tecan Infinite 200 plate-reader.