Kk ay mice

Male C57BL/6J, ddY and ICR mice (Tokyo Laboratory Animals Science, Tokyo, Japan) and KK-A^y mice (CLEA Japan, Tokyo, Japan) were acclimatized at the age of 9 weeks for 1 week on a LFD (D12450B; Research Diets, New Brunswick, NJ, U.S.A) and subsequently fed a LFD or HFD (D12492; Research Diets) for 2, 4 or 20 weeks ad libitum. The LFD contained 3.85 kcal/g, with percentages of carbohydrate, fat and protein of 70%, 10% and 20%, respectively. The HFD contained 5.24 kcal/g with percentages of 20%, 60% and 20%, respectively. The carbohydrate was a combination of cornstarch, maltodextrin and sucrose, while the fat was soybean oil and lard. A group of C57BL/6J mice were orally administered Wy-14,643 (Tokyo Chemical Industry, Tokyo, Japan) at 50 mg/kg, once a day for 2 weeks after acclimatization, and were maintained on a LFD. After an overnight fast, the mice were anesthetized by intraperitoneal injection of a mixture of medetomidine (0.75 mg/kg), midazolam (4 mg/kg) and butorphanol (5 mg/kg), and sacrificed by decapitation. The serum was collected, and interscapular BAT and other tissues were excised, snap-frozen and stored at −80 °C until analysis.

This study was conducted in accordance with the Guidelines of Hirosaki University for Animal Experimentation (permission number: A 17002). Female KK-A^y mice (3 years old) were purchased from CLEA Japan, Inc., Tokyo, Japan. The animals were housed in an air-controlled room (temperature, 23 ± 1 °C) with a 12/12 h light/dark cycle. The mice were provided ad libitum access to standard diet and tap water. They were individually housed in plastic cages. After acclimatization for 1 week, the mice were fed the AIN-93G-based high-fat diet and divided into the following three groups: the Control group without BCE supplementation (n = 9), the BCE1 group with dietary supplementation of 1% BCE powder (n = 8), and the BCE2 group with dietary supplementation of 3% BCE powder (n = 8). The BCE concentrations were decided on the basis of preliminary tests in mice, which were designed based on the results obtained for cell lines. The experimental diets were fed to the mice for 9 weeks. The food intake was measured during the experiment. At the end of the experiment, the animals were euthanized and the abdominal aorta was collected for evaluation.

C57BL/6 mice (male) and KKAy mice (male) were purchased from CLEA Japan, Inc. (Tokyo, Japan) for use as a nondiabetic normal control and a model of metabolic disorders with type 2 diabetes, respectively [9 (link)–11 (link)]. These mice were housed in a controlled environment with a 12 h light-dark cycle and were allowed free access to food and water. They were fed a standard diet (3.6 kcal/g; 13.3% energy as fat; Oriental MF, Oriental Yeast, Co., Ltd.). Male KKAy mice at 9 weeks of age were treated with the oral administration of olmesartan (3 mg/kg per day) in drinking water for 4 weeks, and body weight and food intake were measured. The KKAy mice treated with vehicle were previously described [12 (link)]. On the other hand, C57BL/6 control mice were treated with vehicle during the study period. Mice were sacrificed under anesthesia and the tissues were collected at the end of the experimental period. The protocol was reviewed and approved by the Animal Studies Committee of Yokohama City University and all experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals.